CN101528768A - Genes and polypeptides relating to breast cancers - Google Patents

Abstract

The present application provides novel human genes A7322, whose expressio n is markedly elevated in breast cancer. The present application also provid es human genes F3374 whose expression is markedly elevated in breast cancer. These genes and polypeptides encoded thereby can be used, for example, in the diagnosis of breast cancer, and as target molecules for developing drugs against breast cancer. The invention features methods of screening for modul ators of the kinase activity of PBK/TOPK. The invention further provides met hods of screening for agents to prevent or treat cancer, such as breast cancer.

Description

Genes and polypeptides associated with breast cancer

The present application claims the benefit of U.S. provisional patent application serial No. 60/837,428 filed on 10.8.2006, U.S. provisional patent application serial No. 60/840,250 filed on 25.8.2006, and U.S. provisional patent application serial No. 60/915,022 filed on 30.4.2007, which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to the field of biological science, in particular the present invention relates to the field of cancer research. In particular, the present invention relates to cancer-associated genes A7322, F3374 and PBK/TOPK, which are involved in the proliferation mechanism of breast cancer, and polypeptides encoded by said genes. The gene and the polypeptide of the invention can be used for example in the prognosis and diagnosis of breast cancer and as target molecules for the development of anti-breast cancer drugs.

Background

Breast cancer is a genetically heterogeneous disease (genetic cancer) which is the most common malignancy in women. The presumed new cases reported worldwide each year are about 800,000 (Parkin DM, et al, (1999). CA Cancer J Clin 49: 33-64). Mastectomy is still the treatment of choice for the medical treatment of this disease. However, even when the primary tumor is surgically removed, recurrence may still occur at local or distant sites due to undetectable micrometastases at the time of diagnosis (Saphner T, et al, 1996, J Clin Oncol, 14, 2738-46). Cytotoxic agents are usually administered post-operatively as an adjuvant therapy in order to kill those residual or precancerous cells. Treatment with traditional chemotherapeutic agents often relies on experience, which is based primarily on histological tumor parameters. Without knowledge of the specific mechanisms, targeted drugs are increasingly becoming the primary treatment for breast cancer. Tamoxifen (tamoxifen) and aromatase inhibitors are 2 representatives of this class of drugs, which, administered as an adjuvant or chemopreventive agent to patients with metastatic breast Cancer, achieve a good response (Fisher B, et al, (1998) J Natl Cancer Inst, 90, 1371-88; Cuzick J (2002) Lancet 360, 817-824). However, the disadvantages are: only patients expressing estrogen receptors are sensitive to these drugs. In addition, there has recently been increased concern about side effects such as endometrial cancer due to long-term tamoxifen therapy and fractures due to aromatase therapy in postmenopausal women (Coleman RE (2004). oncology.18(5 Suppl 3), 16-20).

Despite recent advances in diagnostic and therapeutic strategies, the prognosis of patients with advanced cancer remains poor. Although molecular studies have revealed alterations in tumor suppressor genes and/or oncogenes involved in carcinogenesis, the precise mechanism remains to be elucidated.

cDNA microarray technology has allowed the construction of comprehensive profiles of gene expression in normal and malignant cells and comparison of gene expression in malignant and corresponding normal cells (Okabe et al, Cancer Res 61: 2129-37 (2001); Kitahara et al, Cancer Res 61: 3544-9 (2001); Lin et al, Oncogene 21: 4120-8 (2002); Hasegawa et al, Cancer Res 62: 7012-7 (2002)). This approach helps understand the complex nature of cancer cells and helps elucidate the mechanisms of carcinogenesis. Identification of genes that are down-regulated in tumors enables more accurate and precise diagnosis of individual cancers and the development of new therapeutic targets (Bienz and Clevels, Cell 103: 311-20 (2000)). In order to reveal the mechanism of tumors from a genome-wide perspective and to find target molecules for diagnosis and for the development of novel therapeutic agents, the present inventors analyzed the gene expression pattern of tumor cells using a cDNA microarray of 23,040 genes (Okabe et al, Cancer Res 61: 2129-37 (2001); Kitahara et al, Cancer Res 61: 3544-9 (2001); Lin et al, Oncogene 21: 4120-8 (2002); Hasegawa et al, Cancer Res 62: 7012-7 (2002)).

Studies aimed at revealing the mechanisms of carcinogenesis have facilitated the identification of molecular targets for anti-tumor agents. For example, farnesyltransferase inhibitors (FTIs) -which were originally developed for the purpose of inhibiting activation of the Ras-associated growth signaling pathway dependent on posttranslational farnesylation-have been shown to be effective in treating Ras-dependent tumors in animal models (Sun J, et al, oncogene.1998; 16: 1467-73). Clinical trials in humans with a combination of an anti-Cancer drug and the anti-HER 2 monoclonal antibody trastuzumab (trastuzumab) with the aim of antagonizing the proto-oncogene receptor HER2/neu have resulted in improved clinical response and overall survival in breast Cancer patients (Molina MA, et al, Cancer Res.2001; 61: 4744-9). The tyrosine kinase inhibitor STI-571, which selectively inactivates bcr-abl fusion proteins, has been developed to treat chronic myelogenous leukemia (chronic myelogenous leukemia) in which constitutive activation of bcr-abl tyrosine kinase plays a crucial role in leukocyte transformation. These classes of drugs are designed to inhibit the oncogenic activity of specific gene products (O' Dwyer ME & Druker BJ, Curr Opin Oncol.2000; 12: 594-7). Therefore, gene products that are commonly up-regulated in cancer cells may serve as potential targets for the development of novel anti-cancer drugs.

For example, a novel method of cancer treatment using gene-specific siRNA has been attempted in clinical trials (Bumcroot D et al, Nat Chem Biol 2006 Dec, 2 (12): 711-9). RNAi appears to have a place among the major technology platforms (Putral LN et al, Drug News Perspectrum 2006Jul-Aug, 19 (6): 317-24; Frantz S, Nat Rev Drug Discov 2006Jul, 5 (7): 528-9; Dykxhoorn DM et al, Gene Ther 2006 Mar, 13 (6): 541-52). However, several challenges need to be faced before RNAi can be used clinically. These challenges include poor RNA stability in vivo (HallAH et al, Nucleic Acids Res 2004 Nov 15, 32 (20): 5991-. There is a possible toxicity associated with partially homologous gene silencing or the induction of general gene suppression by activation of the interferon response, a well-known fact (Judge AD et AL, Nat Biotechnol 2005 Apr, 23 (4): 457-62, Epub 2005 Mar 20; Jackson AL & Linsley PS, Trends Genet 2004 Nov, 20 (11): 521-4). Therefore, there is a need for double-stranded molecules targeting cancer-specific genes without adverse side effects for the development of anticancer drugs.

In addition, it has been confirmed that CD8+ Cytotoxic T Lymphocytes (CTLs) recognize epitope peptides derived from Tumor Associated Antigens (TAAs) presented on MHC class I molecules and lyse tumor cells. Since the first instance of TAA, the MAGE family, many other TAAs have been discovered immunologically (Boon, IntJ Cancer 54: 177-80 (1993); Boon and van der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al, Science 254: 1643-7 (1991); Brichard et al, J ExpMed 178: 489-95 (1993); Kawakami et al, J Exp Med 180: 347-52 (1994)). Currently, some newly discovered TAAs are being targeted for immunotherapy in the clinical development stage. TAAs found to date include MAGE (van der Bruggen et al, Science 254: 1643-7(1991)), gp100(Kawakami et al, J Exp Med 180: 347-52(1994)), SART (Shichijo et al, J Exp Med 187: 277-88(1998)), and NY-ESO-1(Chen et al, Proc Natl Acad Sci USA 94: 1914-8 (1997)). On the other hand, gene products that have been demonstrated to be specifically overexpressed in tumor cells have been shown to be recognized as targets for inducing cellular immune responses. Such gene products include p53(Umano et al, Brit J Cancer 84: 1052-7(2001)), HER2/neu (Tanaka et al, Brit J Cancer 84: 94-9(2001)), CEA (Nukaya et al, IntJ Cancer 80: 92-7(1999)), and the like.

Although significant progress has been made in basic and clinical studies relating to TAAs (Rosenberg et al, Nature Med 4: 321-7 (1998); Mukherji et al, Proc Natl Acadsi USA 92: 8078-82 (1995); Hu et al, Cancer Res 56: 2479-83(1996)), the number of candidate TAAs currently available for the treatment of adenocarcinomas, including breast Cancer, is still limited. TAAs, which are abundantly expressed in cancer cells and at the same time their expression is restricted to cancer cells, are considered as ideal candidate immunotherapeutic targets. Furthermore, the identification of novel TAAs that induce effective and specific anti-tumor immune responses is expected to encourage the use of peptide vaccination strategies in the clinic against many types of Cancer (Boon and van der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al, Science 254: 1643-7 (1991); Brichard et al, J Exp Med 178: 489-95 (1993); Kawakami et al, J Exp Med 180: 347-52 (1994); Shichijo et al, J Exp Med 187: 277-88 (1998); Chen et al, Proc Natl Acad Sci USA 94: 1914-8 (1997); Harris, J Natl Inst 88: 1442-55 (1996); Butterer et al, Cancer Res 59: 3134-42 Res et al, Visser et al, J Natl Inst 88: 1442-55 (1996); Cancer 1999; Cancel et al, J40: 3309-54; Jimmka et al, 1996), cancer Res 57: 4465-8 (1997); fujie et al, Int J Cancer 80: 169-72 (1999); kikuchi et al, Int J Cancer 81: 459-66 (1999); oiso et al, Int J Cancer 81: 387-94(1999)).

There are a number of reports in the literature that peptide-stimulated Peripheral Blood Mononuclear Cells (PBMC) from certain healthy donors produce significant levels of IFN- γ in response to the peptide, but in the absence of such a peptide⁵¹Cr-release tests rarely exhibit cytotoxicity against tumor cells in an HLA-A24 or A0201-restricted manner (Kawano et al, Cancer Res 60: 3550-8 (2000); Nishizaka et al, Cancer Res 60: 4830-7 (2000); Tamura et al, Jpn J Cancer Res 92: 762-7 (2001)). However, as in the Caucasian population, both HLA-A24 and HLA-A0201 are common HLA alleles in Japan (Date et al, Tissue antibodies 47: 93-101 (1996); Kondo et al, J Immunol 155: 4307-12 (1995); Kubo et al, J Immunol 152: 3913-24 (1994); Imanishi et al, proceedings of the eleven International Histocompatibility Workshop and conference Oxford University Press, Oxford, 1065 (1992); Williams et al, Tissue antibody 49: 129 (1997)). Therefore, cancer antigen peptides presented by these HLAs may be particularly useful for treating cancers in japanese and caucasian populations. In addition, it is known that low affinity CTLs can be induced in vitro using high concentrations of peptides, while high levels of specific peptide/MHC complexes are produced on Antigen Presenting Cells (APC), which complexes will effectively activate these CTLs (Alexander-Miller et al, Proc Natl Acad Sci USA 93: 4102-7 (1996)).

To determine the mechanism of breast carcinogenesis and to identify new diagnostic markers and/or therapeutic drug targets for these tumors, the present inventors analyzed the expression pattern of genes in breast carcinogenesis using a whole genome cDNA microarray containing 27,648 genes. From a pharmacological point of view, inhibition of oncogenic signals is in practice much easier than activation of tumor suppressive effects. Thus, the present inventors searched for genes that are up-regulated during breast cancer development.

Because cytotoxic drugs often cause severe adverse reactions, careful selection of novel target molecules based on a relatively clear mechanism of action will promote the development of effective anticancer drugs with minimal risk of side effects. To achieve this goal, the present inventors previously performed expression pattern analysis on 81 cases of breast cancer (Nishidate T et al, Int J Oncol2004, 25: 797-.

The PBK (PDZ-binding kinase)/TOPK (protein kinase derived from T-LAK cells) gene is one of these genes, which was found to be significantly overexpressed in most of the breast cancer cases examined (the PBK/TOPK gene is referred to as "A7870" in WO 05/028676). In addition, the present inventors demonstrated that small interfering RNA (siRNA) designed to reduce expression of the PBK/TOPK gene has a growth inhibitory effect on breast cancer cells expressing the gene.

PBK/TOPK is a member of the serine/threonine kinase family and was originally identified as a Dlg 1-interacting protein by yeast two-hybrid screening (yeast two-hybrid screening) and characterized as a mitotic kinase with a PDZ-binding motif at the C-terminus (Gaudet S et al, Proc Natl Acad SciUSA 2000, 97: 5167-72). Another group also proposed that PBK/TOPK is a MAPKK (mitogen-activated protein kinase) -like protein kinase that phosphorylates p38 protein (Abe Y et al, JBiol Chem 2000, 275: 21525-31). In addition, analysis by yeast two-hybrid screening revealed possible interactions between Raf and PBK/TOPK (Yuryev A et al, Genomics 2003, 81: 112-25). These two findings suggest that PBK/TOPK may be involved in the MAPK pathway.

Post-translational modifications of the N-terminal part of histone H3, including acetylation, methylation, and phosphorylation, have been previously described (Martin C & Zhang Y, Nat Rev Mol Cell Biol 2005, 6: 838-49; Nowak SJ et al, Trends Genet 2004, 214-20; Prigent C & Dimitov S, J Cell Sci2003, 116: 3677-85). Among them, it is known that phosphorylation at Ser10 of histone H3 is involved in the initiation of chromosome condensation (an important event in Cell mitosis) in mammals (Prigent C & Dimitov S, J Cell Sci2003, 116: 3677-85; Van Hooser A et al, J Cell Sci 1998, 111: 3497-. According to the "ready to produce marker (ready to produce label)" model, phosphorylation at Ser10 of histone H3 reaches a maximum level in the middle stage as an indicator of ready segregation of chromosomes, and Ser10 is then dephosphorylated with concomitant mid/late transition (Hans F and Dimitov S, Oncogene 2001, 20: 3021-7). Interestingly, previous reports indicated that okadaic acid ("OA") induces Ser10 phosphorylation of histone H3 by inhibiting Protein Phosphatases (PPs) (Murnion ME et al, J Biol Chem 2001, 276: 26656-65; Eyers PA et al, Curr Biol2003, 13: 691-7). For example, Aurora-A is known to be inactivated by the protein phosphatase 2A (PP2A), but reactivated by autophosphorylation via binding to the TPX2 (target protein of Xenopus kinesin-like protein 2) protein, which blocks PP2A activity (Eyers PA et al, Curr Biol2003, 13: 691-7).

Activation of CDK 1-cyclin B1 kinase, which targets a number of substrates, triggers entry into mitosis in mammalian cells to induce a subsequent mitotic process (Nigg ea., Nat Rev Mol CellBiol 2: 21-32 (2001)). Those substrates, which are also involved in the late stages of mitosis in cells by phosphorylation of the CDK 1-cyclin B1 complex, are: APC (anaphase promoting complex) ubiquitin ligase, which is activated to initiate mitotic withdrawal (Kraft C et al, EMBO J22: 6598-609(2003)) and acquisition of conformational proteins at the PLK1 docking site, INCENP (internal centromere protein, Goto H et al, Nat Cell Biol 8: 180-7(2006)) and PRC1 (cytokinin regulator 1, Neef R et al, Nat Cell Biol 9: 436-44 (2007)). Furthermore, because recent work reported the activity of protein phosphatase 1 (PP1 α has an inactive phosphorylation site (Thr320) targeted by CDK 1-cyclin B1 kinase (Kwon YG et al, Proc Natl Acad Sci U S A94: 2168-73(1997)), this suggests a tight cooperation between protein kinase and phosphatase to promote mitosis in cells. although it has been reported that CDK 1-cyclin B1 can phosphorylate PBK/TOPK at Thr9, it is still poorly understood how PBK/TOPK is activated by CDK 1-cyclin B1 complex mitotic cells and its function in cell proliferation and cancer progression.

Disclosure of Invention

Accordingly, the present invention aims to provide novel proteins involved in the proliferation mechanism of breast cancer cells and genes encoding the proteins, as well as methods for their production and use in diagnosis and treatment of breast cancer.

Among the transcripts that are normally up-regulated in breast cancer, the human genes PBK/TOPK, A7322 and F3374, respectively, were identified. In addition, through the transfection of PBK/TOPK, A7322 and F3374 specific small interfering RNA reduced their expression, inhibited the breast cancer cell growth. Many anticancer drugs, such as inhibitors of DNA and/or RNA synthesis, metabolic inhibitors, and DNA intercalators, are toxic not only to cancer cells, but also to normally growing cells. However, agents that inhibit expression of PBK/TOPK, a7322, or F3374 do not adversely affect other organs because expression of these genes is restricted in normal organs, e.g., a7322 is restricted to the brain, while F3374 is restricted to the testis and thymus, placenta and bone marrow.

Thus, the present invention provides isolated nucleic acid molecules comprising the PBK/TOPK, F3374 and A7322(SEQ ID NO: 79) genes. These nucleic acid molecules are candidate cancer prognostic and diagnostic markers and are promising potential targets for developing new diagnostic strategies and effective therapeutic anti-cancer drugs. In addition, the invention provides polypeptides encoded by these genes, as well as their production and use.

The invention also provides a method of producing a protein by transfecting or transforming a host cell with a polynucleotide sequence encoding at least one of the PBK/TOPK, F3374 or a7322 proteins and expressing the polynucleotide sequence. In addition, the invention provides vectors comprising a nucleotide sequence encoding at least one of the PBK/TOPK, F3374 or a7322 proteins, as well as host cells carrying the polynucleotides encoding the a7322 proteins. Such vectors and host cells are useful for the production of PBK/TOPK, F3374 and A7322 proteins.

The present application also provides antibodies or non-antibody binding proteins that recognize the PBK/TOPK, F3374, or a7322 proteins. In part, inhibitory polynucleotides of the PBK/TOPK, F3374 or A7322 genes, such as antisense DNA, ribozymes, and siRNA (small interfering RNA), are also provided.

The present invention further provides a method of diagnosing breast cancer, the method comprising the steps of: determining the expression level of a7322 or F3374V1 gene in the biological sample of the specimen and comparing the expression level of a7322 or F3374V1 gene with the expression level in a normal sample; wherein a high expression level of the a7322 or F3374V1 gene in the sample is indicative of breast cancer.

The invention further provides methods of screening for compounds useful in the treatment of breast cancer. The method comprises contacting a test compound with an a7322 or F3374V1 polypeptide, and selecting a test compound that binds to an a7322 or F3374V1 polypeptide.

The invention further provides a method of screening for a compound useful in the treatment of breast cancer, wherein the method comprises the steps of: contacting a test compound with an a7322 or F3374V1 polypeptide; and selecting a test compound that inhibits the biological activity of the a7322 or F3374V1 polypeptide.

The present application also provides pharmaceutical compositions useful in the treatment of breast cancer. The pharmaceutical composition may for example be an anti-cancer agent. The pharmaceutical composition comprises the amino acid sequences shown and described in SEQ ID NOs: 34. 35, 37, 38, 67 or 68, F3374V1 or AURKB.

The invention further provides methods of treating breast cancer using the pharmaceutical compositions provided by the invention.

In addition, the present invention provides a method of treating or preventing breast cancer comprising the step of administering an a7322 or F3374V1 polypeptide. Inducing anti-tumor immunity by administering the a7322 or F3374V1 polypeptide. Accordingly, the present invention also provides a method of inducing anti-tumor immunity, comprising the step of administering an a7322 or F3374V1 polypeptide, and a pharmaceutical composition for treating or preventing breast cancer, comprising the a7322 or F3374V1 polypeptide.

These and other objects and features of the present invention will become more apparent upon reading the following detailed description in conjunction with the accompanying drawings and embodiments. It is to be understood, however, that both the foregoing summary of the invention and the following detailed description are of preferred embodiments and are not restrictive of the invention or other alternative embodiments of the invention.

The present invention is also based, at least in part, on the discovery of a novel mechanism by which PBK/TOPK phosphorylates histone H3 at Ser10 in vitro and in vivo. Since PBK/TOPK is a cancer/testis antigen, its kinase function may be associated with its oncogenic activity, this protein is also a promising molecular target for breast cancer therapy.

In particular, the invention provides a method of screening for agents that induce mitosis in breast cancer cells. Screening can also be performed by: contacting the PBK/TOPK polypeptide with a substrate that is phosphorylated by the PBK/TOPK polypeptide and an agent under conditions that permit phosphorylation of the substrate; detecting the phosphorylation level of the substrate; comparing the level of phosphorylation of the substrate to the level of phosphorylation of the substrate detected in the absence of the agent; and selecting an agent that reduces the phosphorylation level of the polypeptide. According to this method, a histone or a fragment of histone comprising at least its phosphorylation site (e.g. Ser10 of histone H3) can be used as a substrate.

The identified agents screened by the above method induce mitosis in breast cancer cells. Thus, the agent screened is a candidate for treating or preventing breast cancer. Thus, the invention also provides a method of screening for an agent useful in the treatment or prevention of breast cancer by preventing or inhibiting phosphorylation of Ser10 of H3 by PBK/TOPK.

The invention further provides a method of screening for a compound useful in the treatment of breast cancer, wherein the method comprises the steps of: contacting CDK1, cyclin B1, and test compound with PBK/TOPK; and selecting a compound that inhibits the phosphorylation level of the PBK/TOPK polypeptide.

The present invention also relates to a method for the treatment and/or prevention of breast cancer, comprising the steps of: administering an inhibitory polypeptide comprising MEGISNFKTPSKLSEKKK (SEQ ID NO: 98); or a polynucleotide encoding it. Furthermore, the invention relates to the use of a polypeptide according to the invention or of a nucleotide coding therefor for the production of a pharmaceutical preparation for the treatment and/or prophylaxis of breast cancer.

The invention further provides a method of screening for a compound useful in the treatment of breast cancer, wherein the method comprises the steps of: contacting a test compound with a cell expressing protein phosphatase 1 alpha (PP1 alpha) and a PBK/TOP polypeptide; and selecting a test compound that inhibits the level of phosphorylation of the PBK/TOPK polypeptide.

The invention further provides a method of screening for a compound useful in the treatment of breast cancer, wherein the method comprises the steps of: contacting a PBK/TOPK polypeptide with a p47 polypeptide, a p97 polypeptide, and a test compound; and selecting a test compound that inhibits the level of binding between PBK/TOPK and p47 or phosphorylation of the p97 polypeptide.

The invention further provides a method of screening for a compound useful in the treatment of breast cancer, wherein the method comprises the steps of: contacting a test compound with a cell expressing a PBK/TOP polypeptide; and selecting a test compound that causes an intercellular bridge (intercellular bridge) to grow and/or increases the G2/M population of cells.

Drawings

FIG. 1 expression of A7322 and F3374 in breast cancer and normal tissues

(A) A7322 expression in tumor cells from 12 breast cancer patients (3T, 31T, 149T, 175T, 431T, 453T, 491T, 554T, 571T, 709T, 772T, and 781T), F3374 expression in tumor cells from breast cancer cases (16, 102, 247, 252, 302, 473, 478, 502, 552, 646, 769, and 779), and PBK/TOPK expression in tumor cells from breast cancer cases (#4, 5, 13, 86, 110, 214, 327, 411, 623, 624, 631, and 869) were obtained by semi-quantitative RT-PCR.

(B) Expression of F3374 in 9 breast cancer cell lines (HBC4, HBC5, HBL100, HCC1937, MCF7, MDA-MB-231, SKBR3, T47D, YMB1) and normal human tissues (breast, lung, heart, liver, kidney and brain) was obtained by semi-quantitative RT-PCR.

(C) Northern blot analysis of various human tissues A7322, F3374 and PBK/TOPK. MTN membranes comprise the following human normal tissues: 1: heart, 2: brain, 3: placenta, 4: lung, 5: liver, 6: skeletal muscle, 7: kidney, 8: pancreas, 9: spleen, 10: thymus, 11: prostate, 12: testis, 13: ovary, 14: small intestine, 15: colon, 16: peripheral blood leukocytes, 17: stomach, 18: thyroid, 19: spinal cord, 20: lymph node, 21: trachea, 22: adrenal gland, and 23: bone marrow.

(D) Northern blot analysis, for A7322, 22 breast cancer cell lines (HBC4, HBC5, HBL100, HCC1937, MCF7, MDA-MB-231, MDA-MB 435S, SKBR3, T47D, YMB1, BSY-1, BT-549, HCC1935, MDA-MB-157, BT-20, MDA-MB-453, ZR75-1, BT474, HCC1143, HCC1500, HCC1599, OCUB-F) and normal human tissues (breast, lung, heart, liver, kidney and brain); breast cancer cell lines and normal human tissues including thymus, lung, heart, liver, kidney and bone marrow for F3374; breast cancer cell lines (HBC4, HBC5, HBL100, HCC1937, NCF-7, MDA-MB-231MDA-MB-435S, SKBR3, T47D and YBB-1) and normal human tissues (breast, lung, heart, liver, kidney and bone marrow) were used for PBK/TOPK.

(E) Genome structure of F3374V 1.

(F) Expression pattern of F3374V1 in breast cancer cell lines and normal tissues, obtained by semi-quantitative RT-PCR.

(G) Expression of the exogenous A7322 protein in BT-549 cells was obtained by Western blot analysis.

FIG. 2. endogenous expression of A7322 in breast cancer cell lines and tissue sections

(A) Expression of endogenous a7322 protein in SK-BR-3 breast cancer cells was obtained by Western blot analysis using an anti-a 7322 polyclonal antibody.

(B) Subcellular localization of endogenous A7322 protein in SK-BR-3 breast cancer cells. Immunocytochemical staining was performed using affinity purified anti-a 7322 polyclonal antibody (green) and DAPI (blue) to differentiate nuclei. Endogenous a7322 was shown to localize in the cytoplasm.

(C) - (E) immunohistochemical staining analysis using affinity purified anti-a 7322 polyclonal antibody. The cytoplasm of the cancer cell was strongly stained in (C) papillary tubular carcinoma (sample numbers 240 and 241),

(D) solid tubular carcinomas (sample numbers 238, 242 and 290),

(E) and in normal breast tissue (sample No. 453).

(F) Immunohistochemical staining of a7322 in normal human tissues (heart, lung and liver). The expression of a7322 protein was hardly detected in the heart, lung and liver.

FIG. 3 immunocytochemistry and immunohistochemistry

(A) Expression of endogenous F3374 protein in breast cancer cell lines and HMECs was obtained by western blot analysis using anti-F3374 antibody.

(B) Lambda phosphatase experiments were performed with full-length F3374 protein for exogenous expression.

(C) Typical scheme for F3374 deletion constructs used to determine the phosphorylated region.

(D) Lambda phosphatase experiments were performed with various F3374 deletion constructs (. DELTA.1,. DELTA.2 and. DELTA.3) for exogenous expression.

(E) Subcellular localization of endogenous PRC1 protein in the cell cycle in breast cancer cells. HBC5 cells were immunocytochemically stained using affinity purified anti-F3374 polyclonal antibody (red) and DAPI (blue) to differentiate nuclei (see "materials and methods"). White arrows indicate intermediates where F3374 localizes to terminal cells.

(F) Immunohistochemical staining results of breast cancer and normal breast tissue sections. Endogenous F3374 protein was stained with anti-F3374 antibody. Expression was barely detectable from normal breast tissue (10441N), but cancer cells were strongly stained in all cancer tissues studied, including papillary tubular carcinomas (10005T and 00317T), hard carcinomas (10069T and 10571T), and solid tubular carcinomas (10164T and 10185T). Typical images are from microscopic observations at original magnification x 200. Representative image of immunohistochemical staining of F3374 in normal human tissue sections (heart, lung, kidney, liver and testis). Endogenous F3374 protein was stained with anti-F3374 antibody. Original magnification x 50.

FIG. 4 expression of PBK/TOPK protein in breast cancer cell lines and tissue sections

(A) Expression of endogenous PBK/TOPK proteins in breast cancer cell lines and HMEC was obtained by western blot analysis using anti-PBK/TOPK monoclonal antibodies.

(B) Subcellular localization of endogenous PBK/TOPK proteins in breast cancer cell lines T47D, BT-20 and HBC5, which were immunocytochemically stained with anti-PBK/TOPK monoclonal antibodies (red) and DAPI (blue) to differentiate nuclei. The endogenous PBK/TOPK protein was stained in the cytoplasm.

(C) Immunohistochemical staining of breast cancer (1-3) and normal breast (4) tissue sections. Endogenous PBK/TOPK proteins were stained with anti-PBK/TOPK monoclonal antibodies. Protein expression was barely detectable in normal breast tissue (4), but the cytoplasm of cancer cells was strongly stained in all cancer tissues studied, including intraductal carcinoma (1), tubular carcinoma of the nipple (2) and dural carcinoma (3). The drawing depicts a typical micrograph, original magnification, left drawing: x100, right drawing: x 200.

(D) Expression pattern of PBK/TOPK protein in normal human tissues. Tissues of heart (1), lung (2), liver (3), kidney (4) and testis (5) were examined using anti-PBK/TOPK monoclonal antibodies. As a result, the expressed PBK/TOPK protein was barely detectable in 4 vital organs (1-4), but strongly stained in testis, only in the outer layer of seminiferous tubules (5). These immunohistochemical staining results correlated well with the results for MTN (fig. 1C). The drawing depicts a typical micrograph, original magnification, left drawing: x100, right drawing: x 200.

FIG. 5 growth inhibition of small interfering RNAs (siRNAs) designed to reduce A7322 expression in breast cancer cells

(A) Semi-quantitative RT-PCR, shown to inhibit endogenous expression of a7322 in breast cancer cell lines (BT-549 cells). Beta 2MG was used as an internal control.

(B) MTT assay, showing that knocking down a7322 in BT-549 cells reduced colony numbers.

(C) Colony formation assay, showing that knocking down a7322 in BT-549 cells reduces colony numbers.

(D) Semi-quantitative RT-PCR, shown to inhibit endogenous expression of a7322 in BT-549 cells. Designed not to reduce the knock-down effect of siRNAs-mismatches expressed by a 7322.

(E) MTT assay, showing that knocking down a7322 in BT-549 cells reduced colony numbers.

(F) Colony formation assay, showing that knocking down a7322 in BT-549 cells reduces colony numbers.

(G) Designed not to reduce the knockdown effect of a7322 expressed siRNA mismatches in breast cancer cell line (BT-474 cells), as determined by semi-quantitative RT-PCR.

(H) MTT assay, showed a reduction in colony numbers in BT-474 cells by A7322-mismatched siRNA (mis- # 3; originally designed from si- # 3).

(I) FACS analysis showed an increase in apoptotic cell population (expressed as percentage of sub-G1) by inhibiting endogenous expression of a7322 in BT-474 breast cancer cells. The next day after neomycin selection, a total of 10,000 cells from mock and si- #3 transfected BT-474 cells were counted identically.

FIG. 6. Small interfering RNA (siRNAs) designed to reduce the growth inhibitory effect of F3374 expressed in breast cancer cells

(A) Semi-quantitative RT-PCR, shown to inhibit endogenous expression of F3374 in breast cancer cell line (T47D cells). Beta 2MG was used as an internal control.

(B) Colony formation assay, showing that knocking down F3374 in BT-549 cells reduces colony numbers.

(C) MTT assay, showing that knocking down F3374 in T47D cells reduced colony numbers.

(D) Semi-quantitative RT-PCR, showing that endogenous expression of F3374 in breast cancer cell lines (HBC4) was inhibited by F3374-specific sirnas (si #1 and si # 4). β 2MG served as loading control.

(E) Colony formation assay, showing that knocking down F3374 in HBC4 cells reduced colony numbers.

(F) MTT assay showed that knocking down F3374 in HBC4 cells reduced colony numbers (si #1 and si # 4: p < 0.001, respectively; unpaired t-test).

(G) Silencing of endogenous F3374 expression by siRNA was confirmed by western blot analysis. Beta-actin served as loading control.

(H) Morphological changes of HBC4 cells transfected with siF3374 seen microscopically. siEGFP was used as control siRNA. Arrows indicate two isolated cells (right panel).

FIG. 7 growth inhibition of breast cancer cell lines by PBK/TOPK-siRNA

(A) Results of semi-quantitative RT-PCR (B) showed PBK/TOPK silencing 11 days after neomycin selection. GAPDH was used as an internal control. MTT assays were performed on day 11 to evaluate cell viability, and normalized results were plotted, with the simulated results taken as 1.0. 3 weeks after selection, colony formation assays were performed (see "materials and methods"). Two siRNA constructs (si- #2 and #3) showed knockdown effects against internal PBK/TOPK expression and inhibited cell growth of both cell lines T47D (A) and BT-20 (B). The simulation was used as a negative control.

(C) 2 weeks after- (D) neomycin selection, phenotypic differences between mock control (C) and si- #3 induced T47D cells (D) were studied by microscopic observation. Irregular shapes of PBK/TOPK-deleted cells were observed: prolonged intermediates, disappearance and uncontrolled cytokinesis (D).

(E) Western blot results are shown confirming silencing of internal PBK/TOPK expression.

(F) FACS results are depicted showing a larger population of apoptotic cells (expressed as percentage of sub-G1) in si- # 3-induced T47D cells than in mock-transfected cells. A total of 10,000 cells were similarly counted from T47D cells transfected with mock and si- # 3.

FIG. 8 identification of PHB2/REA as an interacting protein of A7322

(A) Silver staining of SDS-PAGE gels containing immunoprecipitated proteins. BT-549 cells were transfected with A7322(A7322-FLAG lane) either mock (mock lane) or FLAG-tagged (FLAG-tagged). The difference band appearing in lane A7322 was analyzed by mass spectrometry, and a band appearing near 33kDa was identified as PHB 2/REA. The right panel shows Western blot analysis of immunoprecipitated samples. The expression of FLAG-tagged a7322 was detected using an anti-FLAG M2 monoclonal antibody.

(B) Semi-quantitative RT-PCR results of PHB2/REA and A7322 transcripts in breast cancer clinical samples (4T, 13T, 86T, 138T, 327T, 341T, 411T, 631T, 818T and 846T) and in the mammary gland. beta.2-MG served as an internal control. Results of semi-quantitative RT-PCR of PHB2/REA and A7322 transcripts in breast cancer cell lines (HBC4, HBC5, HBL100, HCC1937, MCF-7, MDA-MB-231, MDA-MB 435S, SK-BR-3, T-47D, YMB-1, BSY-1, BT-549, HCC1935, MDA-MB-157, BT-20, MDA-MB-453, ZR-75-1, BT474, HCC1143, HCC1500, HCC1599, OCUB-F), HMEC, and breast. beta.2-MG served as an internal control.

(C) Interaction of A7322 with PHB2/REA protein. COS-7 cells were transfected with a combination of FLAG tag mimic, FLAG tag A7322, HA tag mimic and HA tag PHB2/REA, immunoprecipitated with anti-FLAG M2 agarose, and immunoblotted with an anti-HA high affinity (3F10) rat monoclonal antibody. The fourth lane transfected with FLAG tag A7322 and HA tag PHB2/REA shows direct binding of the two proteins. The right panel shows confirmation of the interaction of a7322 and PHB2/REA proteins by immunoprecipitation with anti-HA agarose conjugates and immunoblotting with anti-FLAG M2 monoclonal antibody. The fourth lane transfected with FLAG tag A7322 and HA tag PHB2/REA shows direct binding of the two proteins.

(D) Endogenous expression of PHB2/REA in breast cancer cells. Immunocytochemical staining was performed in SK-BR-3 breast cancer cells using polyclonal antibodies against PHB2/REA (green) and DAPI (blue) to differentiate nuclei. Endogenous PHB2/REA was shown to localize mainly in the cytoplasm, but some cells also showed localization in the nucleus (arrows).

FIG. 9.A7322 shows that no direct binding to ER α protein occurs

(A) Confirmation that a7322 and era protein do not interact. COS-7 cells were transfected with a combination of HA-tag mimic (mock-HA), HA-tag A7322(A7322-HA), FLAG-tag mimic (mock-FLAG), and FLAG-tag ER α (ER α -FLAG), immunoprecipitated with anti-FLAG M2 agarose, and immunoblotted with anti-HA high affinity (3F10) rat monoclonal antibody. The fourth lane transfected with A7322-HA and ER α -FLAG shows that the two proteins do not bind directly. The right panel shows confirmation of the interaction of a7322 and ER α proteins by immunoprecipitation with anti-HA agarose conjugates and immunoblotting with anti-FLAG M2 monoclonal antibody. The fourth lane transfected with A7322-HA and ER α -FLAG shows that the two proteins do not bind directly.

(B) A7322 and era in subcellular localization under estradiol treatment. MCF-7(ER +) cells were transfected with A7322-HA (green) and ER α -FLAG (red) for 24 hours and treated with DMSO (-E2; left panel) or 1 μ M E2(+ E2; right panel) for 24 hours. A7322 remains in the cytoplasm under E2. The same experiment was performed using SK-BR-3(ER-) cells. (C) Showing that a7322 does not move under E2.

FIG. 10A 7322 inhibition of nuclear transport of PHB2/REA

(A) Subcellular localization of PHB2/REA in the presence of A7322. MCF-7(ER +) cells were transfected with HA tag PHB2/REA (Green), FLAG tag ER α (Red), and either FLAG tag mimic (-A7322; left panel) or FLAG tag A7322 (Red) (+ A7322; right panel) for 24 h and treated with 1 μ M E2 for an additional 24 h. The arrow in the left panel shows nuclear transport of PHB2/REA in the absence of A7322, while the arrow in the right panel shows that PHB2/REA remains in the cytoplasm in the presence of A7322.

(B) The same experiment was performed using SK-BR-3(ER-) cells, showing that the presence of A7322 inhibits the nuclear transport of PHB 2/REA.

(C) The expression of a722, ER α and PHB2 was knocked down at the protein level using siRNA oligonucleotides. si-EGFP was used as a control siRNA. ACTB served as a loading control for western blot analysis.

(D) Subcellular localization of endogenous PHB2/REA in the absence of A7322. MCF-7(ER +) cells were treated with si-A7322 or si-EGFP as controls. 24 hours after siRNA treatment, cells were treated with E2 for 48 hours and then analyzed by immunocytochemical staining.

FIG. 11 enhancement of ER transcriptional Activity by inhibition of Nuclear transport of endogenous PHB2/REA

(A) Expression of exogenous A7322 and endogenous PHB2/REA proteins in MCF-7 and SK-BR-3 cells.

(B) SEAP assay determines the transcriptional activity of ER α. MCF-7(ER +) or SK-BR-3(ER-) cells were co-transfected with either a FLAG tag A7322(FLAG-A7322) construct and an estrogen response reporter (pERE-TA-SEAP) construct or a mock control and pERE-TA-SEAP reporter construct, respectively.

(C) A summary of the inhibition of PHB2/REA nuclear transport by A7322 is shown. In the absence of A7322 (-A7322), PHB2/REA translocated to the nucleus with ER α, inhibiting the transcriptional activity of ER α in combination with estradiol (left panel). On the other hand, in the presence of A7322 (+ A7322), PHB2/REA binds to A7322 in the cytoplasm, inhibiting nuclear transport of PHB2/REA, and promoting enhancement of the transcriptional activity of ER α (right panel).

FIG. 12 cell cycle dependent expression of F3374

(A) FACS analysis showed a population of T47D cells collected every 3 hours from 0-12 hours after synchronization.

(B) Western blot analysis of F3374 during mitosis in T47D cells. Notably, the expression of F3374 was highest 0-3 hours after release from cell cycle arrest (G1/S phase), and its phosphorylation became apparent between 9-12 hours after release from cell cycle arrest (G2/M phase).

FIG. 13F 3374 protein expression regulated by AURKB

(A) The deduced amino acid sequence of the C-terminal end of the F3374 protein (amino acids 591-730). The 3 putative consensus phosphorylation sites of Aurora kinase ([ R/K ] X [ T/S ] and [ R/K ] X [ T/S ] [ I/L/V ] are underlined.

(B) Semi-quantitative RT-PCR experiments of F3374 and AURKB transcripts in 11 breast cancer cell lines (BT-20, BT549, HBC4, HBC5, HCC1937, MCF-7, MDA-MB-231, SK-BR-3, T47D and YMB-1), human mammary epithelial cell line (HMEC) and normal mammary glands. FDFT1 was used as a quantitative control.

(C) Co-immunoprecipitation of F3374 and AURKB protein. Cell lysates from HEK293T cells transfected with HA-tagged F3374 and Flag-tagged AURKB proteins were immunoprecipitated with mouse anti-Flag or normal rat IgG. Immunoprecipitates were immunoblotted with mouse anti-HA antibody. W.c.l represents whole cell lysate.

(D) In vitro kinase assays were performed with purified F3374C terminal recombinant protein (36kDa, including histidine tag). The F3374 recombinant protein was added to the reaction mixture containing ARUKB (see text). Arrows indicate phosphorylated F3374.

(E) Elimination of AURKB endogenous expression by treatment with AURKB-specific siRNA results in a decrease in the total amount of F3374 protein and phosphorylation. Beta-actin served as a quantitative control for proteins.

(F) T47D cells were immunocytochemically stained using affinity purified anti-F3374 and AURKB polyclonal antibodies (green) and DAPI (blue) to differentiate nuclei (see "materials and methods"). Arrows indicate AURKB and F3374 proteins, respectively, in the cytokinesis of T47D cells.

FIG. 14 phosphorylation of PBK/TOPK protein during mitosis

(A) Results of FACS analysis are depicted, showing cell populations collected every 3 hours from 0-15 hours after synchronization.

(B) The results of Western blots examining PBK/TOPK expression are depicted. Notably, PBK/TOPK was phosphorylated and upregulated 6-12 hours after cell cycle release (which represents the G2/M phase as shown in (A)).

(C) Typical immunocytochemical staining is shown 12 hours after cell cycle release. Strong staining (indicated by arrows) of endogenous PBK/TOPK was detected near the pre-or metaphase condensed chromosomes.

(D) The results of PBK/TOPK phosphorylation during mitosis are depicted. Treatment with 0.3ug/mL nocodazole (nocodazole) for 6 hours, 12 hours and 18 hours showed an increased intensity of time-dependent increase in phosphorylated PBK/TOPK (left panel). Further incubation of the cell lysate with/without 1U lambda phosphatase at 30 ℃ for 2 hours showed a slow migrating band indicated by the arrow as phosphorylated PBK/TOPK protein (right panel).

(E) Results of FACS analysis are depicted showing an increased proportion of cells (arrows) in the G2/M phase 6-18 hours after treatment with nocodazole.

FIG. 15 phosphorylation of the PBK/TOPK protein by Ser 10-Histone H3 in vitro and in vivo

(A) Depicted are the results of in vitro kinase assays with purified PBK/TOPK recombinant protein (40kDa, including histidine tag). In addition to PBK/TOPK, a histone mixture or histone H3 was added as substrate. Autophosphorylation of phosphorylated histones H3 and PBK/TOPK was indicated by arrows and asterisks, respectively.

(B) T47D cells were transfected with wild type and kinase-inactivating (kinase-dead) mutant (K64-65A) followed by treatment with 100nM Okadaic Acid (OA) for 6 h. OA treatment caused phosphorylation of both PBK/TOPK proteins (arrows), but only the wild-type protein induced phosphorylation of H3 detected by phosphorylation of Ser 10-specific antibodies.

(C) siRNA (si- #3) is shown to silence the internal expression of PBK/TOPK in T47D cells 2 weeks after transfection and neomycin selection. As a result, elimination of PBK/TOPK was accompanied by a decrease in phosphorylation of histone H3 at Ser 10. Beta-actin and total H3 were also examined as loading controls.

(D) Results of immunocytochemical staining analysis of PBK/TOPK and histone H3 are depicted. The results show that PBK/TOPK (red) pools with histone H3 (green) phosphorylated at Ser10 on condensed chromosomes (blue) of mitotic cells (pre-phase) in breast cancer cell lines T47D and HBC 5.

(E) The subcellular localization of histone H3 phosphorylated at PBK/TOPK and serine 10 in the metaphase of T47D cells is shown.

(F) It was shown that PBK/TOPK expression and histone H3 phosphorylation were reduced in late cells (open arrows). Solid arrows indicate cells at interphase.

FIG. 16 CDK 1-cyclin B1 phosphorylated PBK/TOPK protein in mitotic cells

(A) Nuclear transport of endogenous PBK/TOPK, CDK1 and cyclin B1 in mitotic cells of breast cancer cell lines (T47D cells). Arrows indicate nuclear transport of PBK/TOPK (top panel), CDK1 (middle panel) and cyclin B1 (bottom panel) in mitotic cells.

(B) PBK/TOPK was directly phosphorylated in vitro by CDK 1-cyclin B1. The wild-type-PBK/TOPK (wt) recombinant protein was phosphorylated by CDK 1-cyclin B1 recombinant protein, but the alanine substituted mutant at Thr9-PBK/TOPK (T9A) was not phosphorylated by CDK 1-cyclin B1 recombinant protein.

(C) The pp1-18 peptide was used to inhibit phosphorylation of PBK/TOPK at Thr9 by CDK 1-cyclin B1. The efficacy of this peptide in blocking CDK 1-cyclin B1-induced TOPK phosphorylation was examined by an in vitro kinase assay. The recombinant proteins of TOPK and CDK 1-cyclin B1 were incubated with permeant peptides added at concentrations of 0, 5. mu.M, 10. mu.M and 20. mu.M, respectively. Phosphorylated proteins were observed after SDS-PAGE and autoradiography.

(D) pp1-18 peptide treatment significantly dose-dependently inhibited PBK/TOPK expressing T47D cell growth (P ═ 0.0096, Student's T test). On the other hand, pp1-18 peptide did not affect the growth of PBK/TOPK-negative HMEC cells. The number of viable cells was determined by the MTT assay.

(E) Influence of pp1-18 peptide treatment on the cell cycle of T47D cells. T47D cells were treated with nocodazole (0.3. mu.g/mL), followed by the addition of pp1-18 peptide (10. mu.M) for 18 or 24 hours before collection, followed by western blot analysis and FACS analysis using anti-PBK/TOPK antibodies.

(F) Morphological changes of T47D cells treated with 50. mu.M pp1-18 peptide as seen by microscope. Arrows indicate long intercellular bridges of pp1-18 peptide-treated cells during cytokinesis.

FIG. 17 autophosphorylation of PBK/TOPK protein or PP1 alpha regulated PBK/TOPK phosphorylation in mitotic cells and CDK1 activation of PBK/TOPK or inactivation of PP1 alpha

(A) PBK/TOPK is phosphorylated in mitotic cells. T47D cells were treated with nocodazole for 18 hours, followed by FACS analysis and lambda phosphatase assay.

(B) Autophosphorylation of PBK/TOPK in mitotic cells. T47D cells were transfected with wild-type TOPK (WT), the alanine-substituted mutant at Thr9 (T9A), kinase inactivated (KD) and double mutant (T9A/KD), respectively, and subjected to western blot analysis using anti-HA monoclonal antibodies. WT and T9A were phosphorylated, but KD and T9A/KD were not phosphorylated.

(C) Treatment with Okadiac Acid (OA) induced phosphorylation of PBK/TOPK. T47D cells were treated with 100nM Okadaic Acid (OA) and cells were harvested at 1, 3 and 9 hours post treatment. A phosphorylated band appeared after 9 hours of treatment with OA, as confirmed by the lambda PPase assay.

(D) Interaction of PBK/TOPK and PP1 alpha. COS-7 cells were co-transfected with GST-fused PP1 α (GST-PP1 α) and HA-tagged PBK/TOPK (HA-PBK/TOPK), and the cells were either pulled down (pull-down) with equilibrated Glutathione Sepharose 4B beads or immunoprecipitated with anti-HA monoclonal antibodies, followed by western blot analysis using anti-GST or HA monoclonal antibodies.

(E) TOPK was dephosphorylated in mitotic cells by treatment with PP1 α and λ PPase.

(F) T47D cells were treated with nocodazole for 16 hours, followed by 0-4 hour incubation with 25nM CDK1 inhibitor before collection and FACS analysis.

(G) The time points (0 hour, 0.5 hour, 1 hour, 2 hours and 4 hours) after treatment with the CDK1 inhibitor were plotted against the population (%) of each cell cycle.

(H) Respectively using anti-TOPK monoclonal antibody and anti-phosphate-

(Thr320) polyclonal antibody, anti-total-PP 1 a polyclonal antibody, anti-phospho-Rb (Ser807/811) polyclonal antibody and anti-total-Rb monoclonal antibody were immunoblotted against equal amounts of total protein.

FIG. 18 siRNA-induced PBK/TOPK abrogation leading to mitotic disorders and G1 arrest

(A) Western blot analysis of si-TOPK- #3 knockdown PBK/TOPK expression at the protein level. PBK/TOPK expression was significantly inhibited in si-TOPK- #3 treated T47D cells compared to in siEGFP treated cells. Beta-actin served as a control for western blot analysis.

(B) Cell morphology was observed with a phase contrast microscope 2 days after transfection with si-TOPK- #3 or siEGFP (upper panel). Cell morphology was also investigated by immunocytochemical staining 2 days after transfection with si-TOPK- #3 or siEGFP (lower panel). To elucidate the morphology of the cells, actin structures were stained with Alexa Fluor 594 phalloidin (phariodin) and nuclei were counterstained with DAPI.

(C) T47D cells were transfected with si-TOPK- #3 or siEGFP. 2 days after transfection, cells were treated with 0.3. mu.g/mL nocodazole and incubated for an additional 24 hours. Cell morphology and cell cycle were studied by phase contrast microscopy and FACS analysis, respectively.

(D) T47D cells were transfected with si-EGFP as a control and the duration of cell mitosis was determined by Time-difference microscopy.

(E) T47D cells were transfected with TOPK- #3 and the duration of cell mitosis was determined by differential microscopy.

(F) T47D cells were transfected with Wild Type (WT) or kinase-inactivated HA-tagged TOPK expression vectors, followed by si-EGFP or si-TOPK- #3, respectively. At 48 hours after transfection of each siRNA, we performed immunocytochemical staining. The exogenously expressed TOPK protein was immunofluorescent stained with an anti-HA monoclonal antibody. Actin constructs were stained with diluted Alexa Fluor 594 phalloidin (pharloidin) and nuclei were counterstained with DAPI.

FIG. 19 phosphorylation of p97/VCP protein by PBK/TOPK in vitro and in vivo

(A) Interaction of PBK/TOPK with p47 protein. COS-7 cells were transfected with the HA-tagged PBK/TOPK (HA-PBK/TOPK) construct and then lysed with lysis buffer. Next, the cell lysate was mixed with GST-labeled p47(GST-p47) recombinant protein and then pulled down with GST-beads. Immunoblotting of the pellet with anti-HA antibody showed GST-p47 co-precipitated with HA-PBK/TOPK.

(B) Co-localization of exogenously expressed P47 and endogenous PBK-TOPK in T47D cells with or without nocodazole (colocalization).

(C) Expression patterns of p97 and PBK/TOPK proteins in breast cancer cell lines. Equal amounts of total protein were prepared from breast cancer cell lines (BT-549, HBC5, HCC1937, MCF-7, MDA-MB-231, MDA-MB 435S, T47D and ZR75-1) and HBL100, as well as a human mammalian epithelial cell line (HMEC). After SDS-PAGE and membrane transfer, the proteins were immunoblotted with anti-TOPK monoclonal antibodies or anti-p 97 polyclonal antibodies. Beta-actin served as a control for western blot analysis.

(D) Interaction of PBK/TOPK and p97 proteins as shown in co-IP experiments. We co-transfected the HA-PBK/TOPK and myc-tagged 97(myc-p97) constructs into COS7 cells, followed by co-immunoprecipitation with HA-tagged antibody. HA-PBK/TOPK does not interact directly with myc-p 97.

(E) PBK/TOPK binds to the p47/p97 complex via the p47 protein as an adaptor. COS-7 cells were triple transfected (tri-transfect) with GST-fused p47, myc-tagged p97, or HA-tagged TOPK constructs. Complexes in those proteins were immunoprecipitated using anti-GST antibody or anti-myc monoclonal antibody, followed by western blotting with anti-HA or anti-myc monoclonal antibody, respectively. After 5 washes with lysis buffer and SDS-PAGE, those interactions between proteins were studied as described above.

(F) In vitro kinase assay of p 97. Immunoprecipitated p97 protein was incubated with recombinant TOPK protein at 30 ℃ for 30 minutes.

(G) T47D cells were transfected with 100pmol each of siRNA duplexes of si-EGFP and si-p 97.

(H) Two days after transfection with siRNA, cell morphology was observed with a phase contrast microscope.

Detailed description of the invention

SUMMARY

To understand the oncogenic mechanisms associated with cancer and identify potential targets for the development of novel anti-cancer drugs, gene expression patterns in purified populations of breast cancer cells were analyzed on a large scale using cDNA microarrays representing 27,648 genes. More specifically, in order to isolate a new molecular target for breast cancer therapy, cDNA microarrays were used in combination with laser beam microdissection, and the precise genome-wide expression pattern of 81 breast tumors was examined.

In up-regulating genes, the present inventors focused on a7322, which is expressed up-regulated in many breast cancer specimens. Subsequent semi-quantitative RT-PCR and Northern blot confirmed that a7322 was up-regulated in clinical breast cancer specimens and breast cancer cell lines, but not expressed in normal organs (except brain). Since the assembled a7322 cDNA sequence in NCBI database was shorter than about 15kb transcript from northern blot analysis, the present inventors performed exon ligation and 5' RACE experiments to obtain full-length a7322 mRNA. Finally, a cDNA sequence of 14,763 nucleotides was obtained (Genbank accession AB252196), containing an open reading frame of 6534 nucleotides (172-6702 of SEQ ID NO: 79), which encodes a protein of 2,177 amino acids. The simple modular architecture search tool (SMART) program showed that the predicted a7322 protein contained a Sec7 region between codons 586 and 798, which may be necessary for the protein to be transported correctly through the transport golgi.

Furthermore, the present inventors identified PHB2/REA (GenBank accession No.: NM-007273) as an A7322-interacting protein. A7322 and PHB2/REA co-localize in the cytoplasm of breast cancer cells. A7322 acts in breast carcinogenesis by inhibiting nuclear transport of PHB2/REA protein to reactivate ER α.

In up-regulating genes, the inventors also focused on identifying a full-length cDNA sequence of F3374V1 (GenBank accession No.: NM-016448) comprising 4,221 nucleotides, containing an open reading frame of 2,193 nucleotides, encoding a 730 amino acid polypeptide. The F3374V1 gene has 15 exons. RT-PCR showed that F3374V1(1,296bp) was significantly overexpressed in breast cancer cells compared to normal human tissue. Subsequent semi-quantitative RT-PCR and Northern blot analysis confirmed that F3374 was overexpressed in 10 of 12 breast cancer specimens and in all breast cancer cell lines tested, compared to normal human tissue (except testis, thymus, placenta, and bone marrow). Immunohistochemical staining analysis using an anti-F3374 polyclonal antibody detecting endogenous F3374 showed cell cycle dependent localization in breast cancer cells.

Treatment of breast cancer cells with small interfering rnas (sirnas) effectively inhibited the expression of a7322 and F3374, and inhibited the cell/tumor growth of breast cancer cell lines BT-549 and BT-474 (for a7322), or inhibited the cell/tumor growth of cell lines T47D and HBC4 (for F3374), suggesting that these genes play a key role in cell growth proliferation. These findings, consistent with the conclusion that a7322 and F3374 overexpression are associated with breast tumorigenesis, provide promising strategies for patient-specific treatment of breast cancer.

In addition, the present inventors have discovered that the F3374 protein interacts with and is phosphorylated by the mitotic kinase Aurora-B (AURKB). It was demonstrated that elimination of mitotic kinase AURKB expression in breast cancer cells with siRNA reduced phosphorylation of F3374 protein and decreased stability of F3374 protein.

Thus, a7322 and F3374 genes that were significantly overexpressed in breast cancer cells were isolated. The expression patterns of a7322 and F3374 in breast cancer cells were confirmed to be specifically overexpressed by semi-quantitative RT-PCR and Northern blot analysis. ESTs of A7322 and F3374 were previously reported to be upregulated in both bladder cancer and non-small cell lung cancer. However, the association of these genes with breast cancer has not been known before. In addition, the present invention provides for the first time the full-length nucleotide sequences of these genes.

Among the genes detected as being overexpressed in breast cancer but not expressed in normal human tissues (except testis and thymus) using the cDNA microarray technique, the present inventors focused on the PBK/TOPK gene. Immunohistochemical analysis also supported high levels of endogenous PBK/TOPK expression, consistent with the results of Northern blot analysis. In addition, knockdown of endogenous PBK/TOPK expression by siRNA technology resulted in growth inhibition of breast cancer cell lines (fig. 5A and 5B), demonstrating the carcinogenic role of PBK/TOPK genes in breast cancer cells.

In addition to the important role of PBK/TOPK in testis reported so far, the present inventors found its subcellular transport during M, suggesting its critical role in cancer cell mitosis. Furthermore, it was demonstrated that knock down of PBK/TOPK expression with specific sirnas caused cytokinesis, followed by cancer cell apoptosis (fig. 5C-F). These results are consistent with the conclusion that PBK/TOPK plays an important role in cell division and cytokinesis. Notably, microscopic and FACS observations of the effect of siRNA on PBK/TOPK are very similar to the effect of siRNA on Annexin (Annexin)11, which is required for completion of cytokinesis: annexin 11 knockdown produces a narrow cytoplasmic bridge, increasing the Cell population at sub-G1 (Tomas A et al, J Cell Biol 2004, 165: 813-22).

Since PBK/TOPK contains a kinase domain, the inventors treated cells with several stimuli including OA (okadiac acid), PMA (phorbol 12-myrisitate 13-acetate, phorbol (12-) myristate (-13-) acetate), β -estradiol and nocodazole to investigate their association with estrogen receptors and cell mitotic signals, respectively (data not shown). Among these stimuli, OA was found to cause phosphorylation of PBK/TOPK, which is a specific inhibitor of serine/threonine protein phosphatase, causing mitolike processes in interphase cells, chromosome condensation and entry into mitosis in a Cdc 2-independent manner (Ajiro K et al, J Biol Chem 1996, 271: 13197-201; Gowdy PM et al, J Cell Sci 1998, 111: 3401-10).

In contrast to the prediction that PBK/TOPK is an upstream kinase of p38(Abe Y et al, J Biol Chem 2000, 275: 21525-31) and p42/ERK2 (which is usually up-regulated in breast cancer cell lines), the in vitro kinase assay did not show phosphorylation of these proteins (data not shown). Whereas highly selective histone H3 phosphorylation was observed for PBK/TOPK as first reported in the present invention. Interestingly, phosphorylation at the N-terminus of histone H3 (Ser10) suggests that this phosphorylation step is an early mitotic event with chromosome condensation following OA treatment (Ajiro K et al, J Biol Chem 1996, 271: 13197-.

In addition, since immunostaining experiments using breast cancer cells showed that PBK/TOPK was sub-cellularly localized around the chromosome in the cells at mitosis (especially at pre-and metaphase) (fig. 8C), PBK/TOPK was examined to determine whether it phosphorylates histone H3 at serine 10 in vivo. Comparing the wild type and kinase inactivated (K64-65A mutant: lysine 64 and 65 in SEQ ID NO: 92 become alanine mutants) PBK/TOPK proteins with or without OA stimulation, it was demonstrated that PBK/TOPK phosphorylates serine 10 of histone H3 (FIG. 9B) and that endogenous PBK/TOPK proteins are well confluent with phosphorylated histone H3 in mitotic cells (FIG. 9D).

The cell cycle dependent Ser10 phosphorylation of histone H3 correlated with PBK/TOPK expression levels and localization, particularly early in mitosis (fig. 9D and 9E). Thus, the PBK/TOPK-histone H3 pathway promotes mitotic events, thereby enhancing cancer cell proliferation, similar to Pak1(Li F et al, EMBO Rep 2002, 3: 767-73), which has been indicated by humans to have an important role in breast cancer cells. However, morphological changes in PBK/TOPK siRNA-knocked-down cells suggested the presence of other substances involved in cytokinesis (FIG. 5).

The present invention is based, in part, on the discovery that PBK/TOPK is overexpressed in breast cancer and its kinase activity plays an important role in breast cancer development, including breast cancer cell growth. Furthermore, the fact that PBK/TOPK acts as an expression pattern for cancer/testis antigens demonstrates that PBK/TOPK holds great promise as a molecular target for the treatment of breast cancer by cancer vaccine-mediated immunotherapy and/or inhibition of PBK/TOPK-specific kinase function. Therefore, the use of PBK/TOPK kinase activity as an index provides a strategy for the development of anti-cancer drugs.

Definition of

The words "a" or "an" or "the" as used in this specification mean "at least one" or "at least one", unless stated otherwise.

Genes differentially expressed in breast cancer ("BC") are referred to herein collectively as "BC gene", "BC nucleic acid" or "BC polynucleotide", and the corresponding encoded polypeptide is referred to as "BC polypeptide" or "BC protein". The BC gene is selected from the group consisting of: a7332, F3374V1, PHB2/REA and PBK/TOPK genes.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Nucleotide, polypeptide, vector and host cell

The present invention includes a human gene a7322 comprising the amino acid sequence set forth as SEQ ID NO: 79, and concatemers and mutants thereof (limited to those encoding a7322 protein), including the polynucleotide sequences set forth in SEQ ID NOs: 80 or a functional equivalent thereof. Examples of polypeptides functionally equivalent to a7322 include, for example, homologous proteins of other organisms corresponding to the human a7322 protein, and mutants of the human a7322 protein.

The invention also includes a novel human gene F3374V1 comprising SEQ ID NO: 81, and concatemers and mutants thereof (limited to those encoding F3374V1 protein), including the polynucleotide sequences set forth in SEQ ID NOs: 82 or a functional equivalent thereof. Examples of polypeptides functionally equivalent to F3374V1 include, for example, homologous proteins of other organisms corresponding to the human F3374V1 protein, and mutants of the human F3374V1 protein. However, the above mutants retain a phosphorylated region, such as, but not limited to, amino acids 591-730 of F3374V 1.

The nucleotide sequence of the human PHB2/REA gene is shown in SEQ ID NO: 89, also available from GenBank accession No. NM _ 007273.3. The amino acid sequence of the gene encoding human PHB2/REA is shown in SEQ ID NO: 90, also available from GenBank accession No. NP _ 009204. In the present invention, the polypeptide encoded by PHB2/REA gene is referred to as "PHB 2/REA", and sometimes as "PHB 2/REA polypeptide" or "PHB 2/REA protein".

The nucleotide sequence of the human AURKB gene is shown in SEQ ID NO: 87, also available from GenBank accession No. NM _ 004217. The amino acid sequence encoding the human AURKB gene is shown in SEQ ID NO: 88. in the present invention, the polypeptide encoded by the AURKB gene is referred to as "AURKB", and is sometimes referred to as "AURKB polypeptide" or "AURKB protein".

The nucleotide sequence of the human PBK/TOPK gene is shown in SEQ ID NO: 91, also available from GenBank accession No. AF 237709. In this specification, the phrase "PBK/TOPK gene" includes PBK/TOPK genes from humans as well as other animals including, but not limited to, non-human primates, mice, rats, dogs, cats, horses, and cattle, and allelic variants corresponding to the PBK/TOPK gene and genes found in other animals. The amino acid sequence encoding the human PBK/TOPK gene is shown in SEQ ID NO: 92, also available from GenBank Accession No. aaf 71521.1. In the present invention, the polypeptide encoded by the PBK/TOPK gene is referred to as "PBK/TOPK", sometimes referred to as "PBK/TOPK polypeptide" or "PBK/TOPK protein".

The nucleotide sequence of the human CDK1 gene is shown in SEQ ID NO: 94, also available from GenBank accession No. NM _ 001786. In the present specification, the phrase "CDK 1 gene" includes CDK1 genes of humans as well as other animals including, but not limited to, non-human primates, mice, rats, dogs, cats, horses and cattle, allelic variants corresponding to the CDK1 gene, and genes found in other animals. The amino acid sequence encoding the human CDK1 gene is shown in SEQ ID NO: 95, the polypeptide encoded by the CDK1 gene is referred to as "CDK 1", sometimes as "CDK 1 polypeptide" or "CDK 1 protein".

The nucleotide sequence of the human cyclin B1(cyclin B1) gene is shown in SEQ ID NO: 96, also available from GenBank accession No. NM _ 031966. In this specification, the phrase "cyclin B gene" includes the cyclin B1 gene of humans as well as other animals including, but not limited to, non-human primates, mice, rats, dogs, cats, horses, and cattle, allelic variants corresponding to the cyclin B1 gene, and genes found in other animals. The amino acid sequence of the gene encoding human cyclin B1 is shown in SEQ ID NO: 97. in the present invention, the polypeptide encoded by the cyclin B1 gene is referred to as "cyclin B1", and sometimes as "cyclin B1 polypeptide" or "cyclin B1 protein".

Human protein phosphatase 1-alpha (

The nucleotide sequence of the gene is shown in SEQ ID NO: 115, also available from GenBank accession No. NM _ 002708. In this specification, the phrase "PP 1 a gene" includes the PP1 a gene of humans as well as other animals including, but not limited to, non-human primates, mice, rats, dogs, cats, horses and cattle, and allelic mutants corresponding to the PP1 a gene and genes found in other animals. The amino acid sequence encoding the human PP1 alpha gene is shown in SEQ ID NO: 116. in the inventionIn (3), the polypeptide encoded by the PP1 α gene is referred to as "PP 1 α", and is sometimes referred to as "PP 1 α polypeptide" or "PP 1 α protein".

The nucleotide sequence of the human p47 gene is shown in SEQ ID NO: 117, also available from GenBank accession No. NM _ 016143. In this specification, the phrase "p 47 gene" includes the p47 gene of humans as well as other animals including, but not limited to, non-human primates, mice, rats, dogs, cats, horses and cattle, and allelic mutants corresponding to the p47 gene and genes found in other animals. The amino acid sequence encoding the human p47 gene is shown in SEQ ID NO: 118. in the present invention, the polypeptide encoded by the p47 gene is referred to as "p 47", and sometimes as "p 47 polypeptide" or "p 47 protein".

The nucleotide sequence of the human p97 gene is shown in SEQ ID NO: 119, also available from GenBank accession No. NM _ 007126. In this specification, the phrase "p 97 gene" includes the p97 gene of humans as well as other animals including, but not limited to, non-human primates, mice, rats, dogs, cats, horses and cattle, and allelic mutants corresponding to the p97 gene and genes found in other animals. The amino acid sequence encoding the human p97 gene is shown in SEQ ID NO: 120. in the present invention, the polypeptide encoded by the p97 gene is referred to as "p 97", and sometimes as "p 97 polypeptide" or "p 97 protein".

Methods for preparing polypeptides functionally equivalent to a given protein are well known to those skilled in the art and include well known methods for introducing mutations into proteins. For example, one skilled in the art can prepare polypeptides functionally equivalent to either of the human BC proteins or AURKB by introducing appropriate mutations into the amino acid sequences of these proteins by site-directed mutagenesis (Hashimoto-Gotoh et al, Gene 152: 271-5 (1995); Zoller and Smith, Methods Enzymol 100: 468-500 (1983); Kramer et al, Nucleic Acids Res.12: 9441-9456 (1984); Kramer and Fritz, Methods Enzymol 154: 350-67 (1987); Kunkel, Proc Natl Acad Sci USA 82: 488-92 (1985); Kunkel, Methods Enzymol 125-204: 1991)). Amino acid variations may also occur naturally. The polypeptide of the present invention also includes a protein having an amino acid sequence of human BC protein or AURKB in which one or more amino acid residues are mutated, as long as the resulting mutant polypeptide is functionally equivalent to the human BC protein or AURKB. The number of amino acids mutated in the mutant is usually 10 amino acids or less, preferably 6 amino acids or less, more preferably 3 amino acids or less.

The terms "polypeptide", "peptide" and "protein" are used interchangeably in this specification to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, to naturally occurring amino acid polymers, to amino acid polymers containing modified residues, and to non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline (hydroxyproline), γ -carboxyglutamate (γ -carboxyglutamate), and O-phosphoserine (O-phosphoserine). Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid (e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine (homoserine), norleucine (norleucine), methionine sulfoxide (methionine sulfoxide), methionine methyl sulfonium. The analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

In the present specification, an amino acid may be represented by 3 letter symbols commonly known thereto, or by one letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission (International Union of theory and applied chemistry-International Commission on the Nomenclature of biochemistry). Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

Unless specifically stated otherwise, the terms "gene", "polynucleotide", "oligonucleotide", "nucleotide" and "nucleic acid" are used interchangeably throughout this specification, analogous to the amino acids being represented by their commonly accepted single letter codes. The term applies to nucleic acid (nucleotide) polymers in which one or more nucleic acids are linked by ester bonds. The polynucleotide, oligonucleotide, nucleotide or nucleic acid may be comprised of DNA, RNA or a combination thereof.

The term "double-stranded molecule" as used herein refers to nucleic acid molecules that inhibit expression of a target gene, including, for example, short interfering RNAs (siRNAs: e.g., double-stranded ribonucleic acids (dsRNA) or small hairpin RNAs (shRNAs)) and short interfering DNAs/RNAs (siD/R-NA: e.g., a double-stranded chimera of DNA and RNA (dsD/R-NA) or a small hairpin chimera of DNA and RNA (shD/R-NA)).

In the present invention, the term "functionally equivalent" means that the subject polypeptide has an activity of promoting cell proliferation and an activity of imparting oncogenic activity to cancer cells, similar to the BC protein. Assays for determining such activity are well known in the art. For example, the DNA encoding the polypeptide of interest is introduced into cells expressing the polypeptide of interest, and the cell growth promoting effect or the improvement in colony forming activity is examined to determine whether or not the polypeptide has cell growth activity. Such cells include, for example, COS7 and NIH3T3 cells.

In certain embodiments of the invention, the region of Sec7 in the functional equivalent of a7332 is conserved in order to maintain the biological activity of a7332 polypeptide. The Sec7 region of the a7332 polypeptide corresponds to seq id NO: 80 from position 139(Ala) to 209(Val) of the amino acid sequence of SEQ ID NO: 79, and codons 586 and 798. In the present invention, the biological activity of a7332 or F3374V1 polypeptide includes cell proliferative activity. Thus, the functional equivalents of the invention have cell proliferative activity.

In certain embodiments of the invention, functional equivalents are also included in a7322 polypeptide. Here, a "functional equivalent" of a protein is a polypeptide having biological activity, in particular having binding activity to PHB2/REA and having nuclear transport activity of the PHB2/REA protein. That is, any polypeptide that retains the PHB2/REA binding domain of the A7322 protein may be used as the functional equivalent in the present invention. Such functional equivalents include those in which one or several amino acids are substituted, deleted, added or inserted in the naturally occurring amino acid sequence of the a7322 protein.

In addition, functional equivalents are also included in PHB2/REA polypeptides. Herein, a "functional equivalent" of a protein is a polypeptide having equivalent biological activity to said protein, in particular a7322 binding activity. That is, any polypeptide that retains the A7322 binding domain of PHB2/REA protein may be used as the functional equivalent in the present invention. Such functional equivalents include those in which one or several amino acids are substituted, deleted, added or inserted in the naturally occurring amino acid sequence of the PHB2/REA protein.

In a preferred embodiment of the invention, functional equivalents are also included in the F3374V1 polypeptide. Herein, a "functional equivalent" of a protein is a polypeptide having biological activity, in particular binding activity to AURKB, and which is phosphorylated by AURKB. That is, any polypeptide that retains the binding domain and phosphorylation site of the F3374V1 protein may be used as the functional equivalent in the present invention. Such functional equivalents include those in which one or several amino acids are substituted, deleted, added or inserted in the naturally occurring amino acid sequence of the F3374V1 protein.

Additionally, functional equivalents are also included in the AURKB polypeptide. Herein, a "functional equivalent" of a protein is a polypeptide having biological activity equivalent to that of the protein, in particular binding and phosphorylation activity against F3374V 1. That is, any polypeptide that retains the binding and phosphorylation activity of AURKB protein against F3374V1 may be used as the functional equivalent in the present invention. Such functional equivalents include those in which one or more amino acids are substituted, deleted, added or inserted in the naturally occurring amino acid sequence of the AURKB protein.

In a preferred embodiment of the invention, the PBK/TOPK polypeptide also includes functional equivalents. Herein, a "functional equivalent" of a protein is a polypeptide having biological activity, in particular phosphorylation activity, equivalent to that of the protein. That is, any polypeptide that retains the phosphorylation activity of the PBK/TOPK protein can be used as the functional equivalent in the present invention. Such functional equivalents include those in which one or more amino acids are substituted, deleted, added or inserted in the naturally occurring amino acid sequence of the PBK/TOPK protein.

It is known that mutated or modified proteins having an amino acid sequence modified by substitution, deletion, insertion and/or addition of one or more amino acid residues in a specific amino acid sequence retain the original biological activity (Mark et al, Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10: 6487-500 (1982); Dalbadie-Famcland et al, Proc Natl Acad Sci USA 79: 6409-13 (1982)).

The amino acid residue to be mutated is preferably mutated to other amino acids that retain the properties of the amino acid side chain (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following common functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl-containing side chains (S, T, Y); sulfur atom containing side chains (C, M); carboxylic acid and amide containing side chains (D, N, E, Q); a base-containing (base) side chain (R, K, H) and an aromatic-containing side chain (H, F, Y, W). It should be noted that the letters in parentheses represent one-letter symbols of amino acids.

Conservative substitution tables giving functionally similar amino acids are well known in the art. In addition to such conservatively modified variants, polymorphic variants, interspecies homologs, and alleles (allels) of the invention are also included and are not excluded. For example, the following 8 groups each contain amino acids that constitute conservative substitutions for one another:

1) alanine (a), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); and

8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)).

Such conservatively modified polypeptides are included in the BC and AURKB proteins of the invention. However, the present invention is not limited thereto, and the BC protein and the AURKB protein also include non-conservative modifications as long as they retain the phosphorylation activities of the BC protein and AURKB. The number of amino acids to be mutated in the modified protein is usually 10 amino acids or less, preferably 6 amino acids or less, more preferably 3 amino acids or less.

Examples of polypeptides obtained by adding one or more amino acid residues to the amino acid sequence of the human BC protein or AURKB protein are fusion proteins containing the human BC protein or AURKB protein. The present invention includes fusions of human BC protein or AURKB protein with other peptides or proteins, i.e., fusion proteins. Fusion proteins can be prepared by techniques well known to those skilled in the art, for example, by linking the DNA encoding the human BC protein or AURKB protein of the invention to DNA encoding other peptides or proteins so that the reading frames are in frame, then inserting the fusion DNA into an expression vector and expressing the fusion DNA in a host. There is no limitation on the peptide or protein fused to the protein of the present invention.

Known peptides that can be used as peptides fused to the protein of the invention include, for example, FLAG (Hopp et al, Biotechnology 6: 1204-10(1988)), 6 XHis containing 6 His (histidine) residues, 10 XHis, influenza virus lectin (HA), human C-myc fragments, VSP-GP fragments, p18HIV fragments, T7-tags, HSV-tags, E-tags, SV40T antigen fragments, lck tags, beta-tubulin fragments, B-tags, protein C fragments, and the like. Examples of proteins that can be fused with the protein of the present invention include GST (glutathione-S-transferase), influenza virus lectin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and the like.

A fusion protein can be prepared by fusing commercially available DNA encoding the above-mentioned fusion peptide or protein with DNA encoding the polypeptide of the present invention and expressing the prepared fusion DNA.

Alternative methods for isolating functionally equivalent polypeptides are known in the art, for example, methods using hybridization techniques (Sambrook et al, Molecular Cloning 2nd ed.9.47-9.58, Cold spring harbor Lab. Press (1989)). One skilled in the art can readily isolate a DNA having high homology to all or part of the DNA sequence encoding the human BC protein or AURKB protein (i.e., SEQ ID NO: 80, 82, 90, 92 or 88), and isolate a polypeptide functionally equivalent to the human BC protein or AURKB protein from the isolated DNA. The polypeptide of the present invention includes a polypeptide functionally equivalent to the human BC protein or AURKB protein encoded by a DNA hybridizing to all or part of the DNA sequence encoding the human BC protein or AURKB protein. These polypeptides include mammalian homologs corresponding to human-derived proteins (e.g., polypeptides encoded by monkey, rat, rabbit, and bovine genes). For example, in isolating a cDNA highly homologous to a DNA encoding a human a7322 protein from an animal, it is particularly preferable to use a tissue from a testis or a breast cancer cell line. Alternatively, when a cDNA highly homologous to a DNA encoding human F3374V1 protein is isolated from an animal, it is particularly preferable to use a tissue from a breast cancer cell line.

The skilled person can routinely select for isolating the function of the encoded and human BC protein or AURKB proteinHybridization conditions for DNA of the above equivalent protein. The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, usually in a complex mixture of nucleic acids, but not to other sequences. Stringent conditions are sequence dependent and will be different under different circumstances. Longer sequences hybridize specifically at higher temperatures. A comprehensive guide to nucleic acid Hybridization can be found in Tijssen, Techniques in Biochemistry and Molecular Biology- -Hybridization with nucleic Probes, "Overview of principles of Hybridization and the strategy of nucleic acid assays" (1993). Generally, specific sequence stringency conditions are selected to be specific thermal melting temperature (T) for a defined ionic strength at pH_m) About 5-10 deg.c lower. T is_mProbes that are 50% complementary to the target are in equilibrium (at T due to the presence of excess target sequence)_m50% of the probe is occupied) of the target sequence (at defined ionic strength, pH and nucleic acid concentration). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least twice background, preferably 10 times background hybridization.

For example, hybridization can be carried out as follows: prehybridization was performed at 68 ℃ for 30 minutes or more using "Rapid-hyb buffer" (AmershamLIFE SCIENCE), a labeled probe was added, and the mixture was incubated at 68 ℃ for 1 hour or more. The subsequent washing step may, for example, be carried out under low stringency conditions. The low stringency conditions are, for example, 42 ℃, 2 XSSC, 0.1% SDS, or preferably 50 ℃, 2 XSSC, 0.1% SDS. More preferably, high stringency conditions are used. Examples of high stringency conditions include 3 washes with 2 XSSC, 0.01% SDS at room temperature for 20 minutes each, followed by 3 washes with 1 XSSC, 0.1% SDS at 37 ℃ for 20 minutes each, followed by 2 washes with 1 XSSC, 0.1% SDS at 50 ℃ for 20 minutes each. However, several factors, such as temperature and salt concentration, will influence the stringency of hybridization, and one skilled in the art can appropriately select these factors to achieve the desired stringency.

DNA encoding a polypeptide functionally equivalent to the human BC protein or AURKB protein can be isolated using a gene amplification method using primers synthesized based on sequence information (SEQ ID NO: 79, 81, 89, 91, or 87) of the DNA encoding the protein, such as a Polymerase Chain Reaction (PCR) method, instead of hybridization.

The polypeptide functionally equivalent to the human BC protein or AURKB protein encoded by the DNA isolated by the above hybridization technique or gene amplification technique generally has a high homology with the amino acid sequence of the human BC protein or AURKB protein. "high homology" or "high sequence identity" are used interchangeably and refer to homology (sequence identity) of 40% or greater, preferably 60% or greater, more preferably 80% or greater, and more preferably 95% or greater. The homology of the polypeptides can be determined, for example, by "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30(1983) ". Other examples of algorithms suitable for determining percent sequence identity are described in this specification.

The polypeptides of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains or forms, and the like, depending on the cell or host used to produce the polypeptide or the purification method employed. In any case, it is within the scope of the present invention as long as the polypeptide has a function equivalent to the human BC protein or AURKB protein of the present invention.

The present invention also includes the use of a partial peptide of the BC protein or the AURKB protein. The partial peptide has an amino acid sequence specific to the protein of BC or AURKB and consists of less than about 400, usually less than about 200, often less than about 100, and at least about 7, preferably about 8 or more, more preferably about 9 or more amino acids. The partial peptide of the present invention can be produced by genetic engineering, known peptide synthesis methods, or by digesting the polypeptide of the present invention with an appropriate peptidase. For example, peptide synthesis can be performed by solid phase synthesis or liquid phase synthesis.

A partial peptide for screening of the present invention suitably comprises at least the PHB2/REA binding site or the active center of the A7322 protein nuclear transport activity. The phrase "functional equivalent" of the a7322 protein also includes the partial peptide.

A partial peptide for screening of the invention suitably comprises at least the A7322 binding site of the PHB2/REA protein. The phrase "functional equivalent" of PHB2/REA protein also includes such partial peptides.

A partial peptide for screening of the present invention suitably comprises at least the AURKB binding site of the F3374V1 protein or the site of phosphorylation of the F3374V1 protein by AURKB protein (amino acids 591-730 of SEQ ID NO: 88). The phrase "functional equivalent" of the F3374V1 protein also includes such partial peptides.

A partial peptide for screening of the present invention suitably comprises at least the F3374V1 protein binding site of AURKB protein or the catalytic domain of AURKB protein. The phrase "functional equivalent" of the PBK/TOPK protein also includes such partial peptides.

The partial peptides used in the screening of the present invention using the kinase activity level of PBK/TOPK as an index suitably comprise at least the kinase domain (amino acids 32-318 of SEQ ID NO: 92), in particular retaining the catalytic sites of the PBK/TOPK protein (Lys 64 and Lys65 of SEQ ID NO: 92). The phrase "functional equivalent" of the PBK/TOPK protein also includes such partial peptides. Furthermore, the partial peptide used for the screening of the present invention using the phosphorylation level of PBK/TOPK as an index suitably contains at least the phosphorylation site of PBK/TOPK protein (Thr 9 of SEQ ID NO: 92). The phrase "functional equivalent" of the PBK/TOPK protein also includes such partial peptides.

The partial peptide may be produced by genetic engineering, known peptide synthesis methods, or by digesting the native BC protein with an appropriate peptidase. For example, peptide synthesis can be solid phase synthesis or liquid phase synthesis. Conventional peptide synthesis methods that can be used for the synthesis include:

1)Peptide Synthesis，Interscience，New York，1966；

2)The Proteins，Vol.2，Academic Press，New York，1976；

3) peptide Synthesis (japanese), Maruzen co., 1975;

4) basics and experience of Peptide Synthesis (japanese), Maruzen co., 1985;

5) development of Pharmaceuticals (volume II) (Japanese), Vol.14 (peptesynthesis), Hirokawa, 1991;

6) WO 99/67288; and

7)Barany G.& Merrifield R.B.，Peptides Vol.2，“Solid Phase PeptideSynthesis”，Academic Press，New York，1980，100-118。

the polypeptide or fragment thereof may be further linked to other substances, so long as the polypeptide or fragment retains its original ability to biologically active, e.g., phosphorylate a substance or be phosphorylated by a kinase. Useful materials include: peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These kinds of modifications may be made to impart additional functions or to stabilize polypeptides and fragments.

The polypeptide of the present invention can be prepared as a recombinant protein or a natural protein by a method known to those skilled in the art. The term "recombinant" when used in reference to, for example, a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene not found in the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise abnormally expressed, under expressed, or not expressed at all.

The term "recombinant nucleic acid" in this specification refers to a nucleic acid that is initially formed in vitro in a form that is not normally found in nature, typically by manipulation of the nucleic acid (e.g., using polymerases and endonucleases). In this way, it is possible to operably link different sequences. Thus, nucleic acids isolated in linear form, or expression vectors formed by ligating DNA molecules that are not normally joined in vitro, are considered recombinant for the present invention. It will be understood that once a recombinant nucleic acid is made and reinserted into a host cell or organism, it will replicate non-recombinantly, i.e., using the host cell's in vivo cellular machinery rather than by in vitro manipulation; however, once produced recombinantly, the nucleic acid is considered recombinant for the purposes of the present invention even if it is subsequently replicated non-recombinantly.

Likewise, a "recombinant protein" is a protein made using recombinant techniques, i.e., a protein made by expressing a recombinant nucleic acid as described above. Thus, a recombinant protein can be produced by: a DNA encoding the polypeptide of the present invention (e.g., a DNA having a nucleotide sequence of SEQ ID NO: 79, 81, 89, 91 or 87) is inserted into an appropriate expression vector, the vector is introduced into an appropriate host cell to obtain an extract, and then the extract is subjected to chromatography such as ion exchange chromatography, reverse phase chromatography, gel filtration or affinity chromatography using a chromatography column to which an antibody against the protein of the present invention is immobilized, or a combination of more than one column described above to purify the polypeptide.

In addition, when the polypeptide of the present invention is expressed as a fusion protein with glutathione S-transferase protein or a recombinant protein to which a plurality of histidines are added in a host cell (e.g., animal cells and E.coli), the expressed recombinant protein may be purified using a glutathione column or a nickel column. Alternatively, where the polypeptide of the invention is expressed as a protein tagged with c-myc, multiple histidines, or FLAG, detection and purification may be performed using antibodies directed against c-myc, His, or FLAG, respectively.

After purification of the fusion protein, it is also possible to remove regions other than the polypeptide of interest by cleaving the fusion protein with thrombin (thrombin) or factor Xa as necessary.

The native protein may be isolated by methods known to those skilled in the art, for example, by contacting an affinity column to which an antibody that binds to the BC protein described below is bound with an extract from a tissue or cell expressing a polypeptide of the present invention. The antibody may be a polyclonal antibody or a monoclonal antibody.

In addition, the present invention provides polynucleotides encoding the polypeptides of the present invention. The polynucleotides of the invention may be used for the in vivo or in vitro production of the polypeptides of the invention described above. Any form of the polynucleotide of the present invention may be used, including mRNA, RNA, cDNA, genomic DNA, chemically synthesized polynucleotides, so long as they encode the polypeptide of the present invention. The polynucleotide of the present invention includes a DNA comprising a given nucleotide sequence and a degenerate sequence thereof, so long as the resulting DNA encodes the polypeptide of the present invention.

The polynucleotides of the present invention can be prepared by methods known to those skilled in the art. For example, a polynucleotide of the present invention may be derived from a cDNA library of cells expressing a polypeptide of the present invention, and hybridized by using a partial sequence of a DNA of the present invention (e.g., SEQ ID NO: 79, 81, 89, 91, or 87) as a probe. cDNA libraries can be prepared, for example, by the methods described in Sambrook et al, Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press (2001); alternatively, commercially available cDNA libraries can be used. The following method can also be used to prepare a cDNA library: RNA is extracted from cells expressing the polypeptide of the present invention, an oligo DNA is synthesized based on the sequence of the DNA of the present invention (e.g., SEQ ID NO: 79, 81, 89, 91, or 87), and PCR is performed using the oligo DNA as a primer to amplify cDNA encoding the protein of the present invention.

In addition, by sequencing the nucleotides of the resulting cDNA, the translated region encoded by the cDNA can be determined routinely, and thus the amino acid sequence of the polypeptide of the present invention can be easily obtained. Furthermore, by screening a genomic DNA library using the obtained cDNA or a part thereof as a probe, genomic DNA can be isolated.

More specifically, mRNA can be first prepared from cells, tissues, organs (e.g., a brain or breast cancer cell line for A7322; a testis or breast cancer cell line for F3374V 1; a breast cancer cell line for PHB 2/REA; and a testis or breast cancer cell line for PBK/TOPK) expressing the subject polypeptide of the present invention. mRNA can be isolated by a known method; for example, total RNA can be prepared by guanidine ultracentrifugation (Chirgwin et al, Biochemistry 18: 5294-9(1979)) or AGPC (Chomczynski and Sacchi, Anal Biochem 162: 156-9 (1987)). In addition, mRNA can be purified from total RNA using an mRNA purification kit (Pharmacia) or the like, or mRNA can be directly purified using a QuickPrep mRNA purification kit (Pharmacia).

The obtained mRNA was used to synthesize cDNA using reverse transcriptase. The cDNA can be synthesized using a commercially available kit, such as AMV reverse transcriptase first strand cDNA Synthesis kit (Seikagaku Kogyo). Alternatively, cDNA can be synthesized and amplified using the primers and the like described in the present specification, 5 '-Ampli FINDER RACE kit (Clontech) and Polymerase Chain Reaction (PCR) using the 5' -RACE method (Frohman et al, Proc Natl Acad Sci USA 85: 8998-9002 (1988); Belyavsky et al, Nucleic acids sRs 17: 2919-32 (1989)).

The desired DNA fragment is prepared from the PCR product and ligated with the vector DNA. Coli and the like are transformed with the recombinant vector, and the desired recombinant vector is prepared from the selected colony. The nucleotide sequence of the desired DNA can be verified by a conventional method such as the dideoxynucleotide chain termination method.

The nucleotide sequence of the polynucleotide of the present invention may be designed to enable more efficient expression, taking into account the frequency of codon usage to be used in the expression host (Grantham et al, Nucleic Acids Res 9: 43-74 (1981)). In addition, the sequence of the polynucleotide of the present invention can be altered using commercially available kits or conventional methods. For example, the sequence may be altered by digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate polynucleotide fragments, addition of linkers or insertion of an initiation codon (ATG) and/or a stop codon (TAA, TGA or TAG).

In a particularly preferred embodiment, the polynucleotide of the invention comprises a polynucleotide comprising the sequence of SEQ ID NO: 79. 81, 89, 91 or 87.

Furthermore, the invention provides methods of producing a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 79. 81, 89, 91 or 87 and encodes a polypeptide functionally equivalent to the BC protein or AURKB protein of the invention as described above. As described above, stringent conditions can be appropriately selected by those skilled in the art. For example, low stringency conditions can be used. Preferably, high stringency conditions are used. These conditions are as described above. The hybrid DNA is preferably cDNA or chromosomal DNA.

The present invention also provides a vector into which the polynucleotide of the present invention is inserted. The vectors of the invention may be used to maintain a polynucleotide, particularly a DNA, of the invention in a host cell for expression of a polypeptide of the invention.

When the host cell is selected as Escherichia coli, and the vector is amplified and prepared in large amounts in Escherichia coli (e.g., JM109, DH 5. alpha., HB101 or XL1Blue), the vector should have "ori" for amplification in Escherichia coli and a marker gene (e.g., a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol, etc.) for selection of transformed Escherichia coli. For example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, and the like can be used. In addition, pGEM-T, pDIRECT and pT7 can also be used for subcloning and extracting cDNA, as with the vectors described above. Expression vectors are particularly useful when the vectors are used to prepare the proteins of the invention.

For example, an expression vector for expression in E.coli must have the above characteristics in order to be amplified in E.coli. When Escherichia coli, such as JM109, DH 5. alpha., HB101 or XL1Blue, is used as the host cell, the vector should have a promoter capable of efficiently expressing the desired gene in Escherichia coli, for example, lacZ promoter (Ward et al, Nature 341: 544-6 (1989); FASEB J6: 2422-7(1992)), araB promoter (Better et al, Science 240: 1041-3(1988)), T7 promoter, or the like. In this connection, for example, pGEX-5X-1(Pharmacia), "QIAexpress System" (Qiagen), pEGFP and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase) may be used in place of the above-mentioned vector. In addition, the vector may also contain a signal sequence for secretion of the polypeptide. An exemplary signal sequence directing secretion of the polypeptide into the periplasm of E.coli is the pelB signal sequence (Lei et al, J Bacteriol 169: 4379-83 (1987)). Methods for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.

In addition to E.coli, the following vectors may be used to prepare the polypeptides of the invention: for example, expression vectors derived from mammalian cells (e.g., pcDNA3(Invitrogen) and pEGF-BOS (Mizushima S., Nucleic Acids Res 18 (17): 5322(1990)), pEF, pCDM8), expression vectors derived from insect cells (e.g., "Bac-to-BAC baculovirus expression System" (GIBCO BRL), pBACPAK8), expression vectors derived from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g., pZIpNEO), expression vectors derived from yeast (e.g., "Pichia expression kit" (Invitrogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (e.g., pppKP 608, pKP 50).

For expression of the vector in animal cells such as CHO, COS or NIH3T3 cells, the vector should have a promoter necessary for expression in the cells, such as SV40 promoter (Mullingan et al, Nature 277: 108-14(1979)), MMLV-LTR promoter, EF1 alpha promoter (Mizushima et al, Nucleic Acids Res 18: 5322(1990)), CMV promoter, etc., and preferably a marker gene for selection of transformants (e.g., drug resistance gene selected by drugs such as neomycin, G418). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP 13.

In addition, a method can be used in which a gene is stably expressed and the copy number of the gene in a cell is amplified. For example, a vector containing a complementary (complementary) DHFR gene (e.g., pCHOI) can be introduced into CHO cells deficient in the nucleic acid synthesis pathway, and the vector can be amplified using Methotrexate (MTX). Further, when the gene is transiently expressed, the following method may be employed: COS cells containing the SV40T antigen-expressing gene on their chromosome were transformed with a vector containing the SV40 origin of replication (pcD, etc.).

The polypeptide of the present invention obtained as described above may be isolated from the inside or outside (e.g., culture medium) of the host cell and purified as a substantially pure homogeneous polypeptide. The term "substantially pure" when used in this specification to refer to a given polypeptide means that the polypeptide is substantially free of other biological macromolecules. A substantially pure polypeptide is at least 75% (e.g., at least 80%, 85%, 95%, or 99%) pure, expressed on a dry weight basis. Purity can be determined by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis or HPLC analysis. The method for isolating and purifying the polypeptide is not limited to any particular method, and any standard method may be used.

For example, separation and purification of a polypeptide can be carried out by appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization and the like.

Examples of chromatography include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography and the like (stratgies for Protein Purification and chromatography: A Laboratory Course Manual. Ed. Daniel R. Marshak et al, Cold Spring harbor Laboratory Press (1996)). These chromatographies can be performed by liquid chromatography, such as HPLC and FPLC. Accordingly, the present invention provides a high purity polypeptide prepared by the above method.

In the context of the present invention, "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window (compare window), wherein the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) as compared to a reference sequence (e.g., a polypeptide of the present invention) that does not contain additions or deletions, in order to optimally align the two sequences. The percentage is calculated by the following method: the percentage of sequence identity is determined by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of alignment, and multiplying the result by 100.

The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more identical sequences or subsequences. Two sequences are "substantially identical" if they satisfy the following condition: two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity, over a specified region, or, when not specified, over the entire sequence) when compared and aligned for maximum correspondence over a comparison window or over a specified region, as determined using any of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region of at least about 50 nucleotides in length, or more preferably over a region of 100-500 or 1000 or more nucleotides in length, or over the full-length amino acid or nucleic acid sequence.

For sequence comparison, one sequence is typically used as a reference sequence to which test sequences are compared. When using the sequence comparison algorithm, the test sequence and the reference sequence are input into the computer, subsequence coordinates are set if necessary, and sequence algorithm program parameters are set. Default program parameters may be used or parameters may be set otherwise. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, "contrast window" may refer to a segment having any number of consecutive positions selected from the group consisting of: 20-600, typically about 50-200, more typically about 100-150, and after optimally aligning a sequence with a reference sequence having the same number of consecutive positions, the two sequences can be compared in the field. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by Smith and Waterman (1981) adv.appl.math.2: 482-9, Needleman and Wunsch (1970) J.mol.biol.48: 443-53, Pearson and Lipman (1988) proc.nat' l.acad.sci.usa 85: 2444-8, Computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA, in Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or manual alignment and visualization (see, e.g., Ausubel et al, Current Protocols in molecular Biology (1995).

Two algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1977) Nuc.acids Res.25: 3389 402 and Altschul et al (1990) J.mol.biol.215: 403-10. Software for BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence (query sequence) that either match or meet some positive-valued threshold T when aligned with a word of the same length in a database sequence. T is referred to as the neighbor score threshold (Altschul et al, supra). These initial neighborhood hits (word hits) are used as seeds to begin the search for longer HSPs containing them. Hits extend in both directions in each sequence, as long as the cumulative alignment score can be increased. Calculation of cumulative score for nucleotide sequences the parameters M (reward score for matching residues; always > 0) and N (penalty score for mismatching residues; always < 0) were used. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the hit in each direction stops when: the cumulative alignment score decreases from its maximum value obtained by an amount X; (ii) an accumulated value of 0 or less due to accumulation of one or more negative-scoring residue alignments; or to the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) is set to default values: word length (W) 11, expectation (E) 10, M5, N-4, and the two chains are compared. For amino acid sequences, the BLASTP program is set to default values: word length 3, expected value (E) 10, BLOSUM62 score matrix (see Henikoff and Henikoff (1989) proc. natl. acad. sci. usa 89: 10915) compare (B) 50, expected value (E) 10, M5, N-4, and compare both strands.

The BLAST algorithm can also perform statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)). it provides an indication of the probability by which a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest aggregate probability in comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The polypeptides of the invention may optionally be modified or partially deleted by treatment with a suitable protein modifying enzyme before or after purification. Useful protein modifying enzymes include, but are not limited to, trypsin (trypsin), chymotrypsin (chymotrypsin), lysylendopeptidase (lysylendopeptidase), protein kinase (protease), glucosidase (glucosidase), and the like.

Antibodies and non-antibody binding proteins

The invention also provides antibodies and non-antibody binding proteins that specifically bind to the polypeptides of the invention. The antibodies and non-antibody binding proteins of the present invention may be used in any form, including monoclonal or polyclonal antibodies, and include antisera obtained by immunizing an animal such as a rabbit with the polypeptide of the present invention, all kinds of polyclonal and monoclonal antibodies, human antibodies, and humanized antibodies prepared by gene recombination.

The terms "specifically binds to" or "attached to" as they appear in the context of an antibody or non-antibody binding protein, refer to: preferential binding of agents or ligands to target epitopes (e.g. A7322, F3374 or PBK/TOPK) in whole or in partThe ligand binds to a7322, F3374 or PBK/TOPK expressed in or on the cell or tissue, or competes with another agent or ligand for binding to a7322, F3374 or PBK/TOPK expressed in or on the cell or tissue. Of course, it is recognized that some degree of non-specific interaction may occur between the antibody and the non-target epitope. Nevertheless, specific binding can still be distinguished from it, since it is mediated by specific recognition of the epitope of interest. Specific binding typically results in a much stronger binding between the delivered molecule and the entity bearing the target epitope (e.g., test well or cell) than between the bound antibody and the entity lacking the target epitope (e.g., test well or cell). Specific binding typically results in an increase in the amount of agent or ligand (per unit time) bound to cells and tissues containing the epitope of interest (i.e., REG4) that is at least about 2-fold, preferably greater than about 10-fold, and most preferably greater than about 100-fold over background, as compared to cells or tissues lacking the epitope of interest. Specific binding between two entities typically means at least 10 ⁶M^-1The affinity of (a). Greater than 10⁸M^-1Or higher affinity is preferred. Specific binding of nucleic acids as well as protein agents and ligands can be determined. Specific binding can be determined for a nucleic acid agent using any assay known in the art, including but not limited to Northern blotting, gel shift assays, and in situ hybridization. Specific binding can be determined for the protein agent and ligand using any binding assay known in the art, including but not limited to gel electrophoresis, Western blotting, ELISA, flow cytometry, and immunohistochemistry.

Antibodies

The term "antibody" as used herein includes natural and non-natural antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional and humanized antibodies and antigen-binding fragments thereof (e.g., Fab ', F (ab')₂Fab, Fv and rIgG). Reference may also be made to Pierce Catalog and handbook, 1994- > 1995(Pierce Chemical Co., Rockford, IL). Reference may also be made to, for example, Kuby，J.，Immunology，3rd Ed.，W.H.Freeman&Co., New York (1998). Such non-natural antibodies can be constructed by solid phase peptide synthesis, can be produced recombinantly, or can be produced as described by Huse et al, Science 246: 1275-81(1989), which is incorporated herein by reference, by screening combinatorial libraries composed of variable light and variable heavy chains. For example, the above-described and other methods of making chimeric, humanized, CDR-grafted, single chain and bifunctional Antibodies are well known to those skilled in the art (see Winter and Harris, Immunol. today 14: 243-6(1993), Ward et al, Nature 341: 544-6(1989), Harlow and Lane, Antibodies, 511-52, Cold spring harbor Laboratory publications, New York, 1988; Hilyard et al, protein engineering: A practical approach (IRL Press 1992); Borreliack, antibody engineering, 2 d. (Oxford University Press 1995)).

The term "antibody" includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate (e.g., bifunctional antibodies) and the like. The term also refers to recombinant single chain Fv fragments (scFv). The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies (triabodies), and tetrabodies (tetrabodies). Bivalent and bispecific molecules are described, for example, in the following documents: kostelny et al (1992) J Immunol 148: 1547-53; pack and Pluckthun (1992) Biochemistry 31: 1579-84; holliger et al (1993) Proc Natl Acad Sci U S A.90: 6444-8; gruber et al (1994) J Immunol: 5368-74; zhu et al (1997) ProteinSci 6: 781-8; hu et al (1997) Cancer Res.56: 3055-61; adams et al (1993) Cancer Res.53: 4026; and McCartney, et al (1995) Protein Eng.8: 301-14.

Typically, antibodies have a heavy chain and a light chain. The heavy and light chains comprise constant and variable regions (these "regions" are also referred to as "domains"), respectively. The heavy and light chain variable regions comprise 4 "framework regions" separated by 3 hypervariable regions (also known as "complementarity determining regions" or "CDRs"). The framework regions and the CDR ranges have been determined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combination of the framework regions of the light and heavy chains that make up the antibody, aid in the positioning and arrangement of the CDRs in three-dimensional space.

The CDRs primarily function to bind to an epitope of the antigen. The CDRs of each chain are generally numbered sequentially from the N-terminus as CDR1, CDR2, and CDR3, and are generally identified by the chain in which the particular CDR is located. Thus, the VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is present, while the VL CDR1 is the CDR1 of the variable region of the light chain of the antibody in which it is present.

The polypeptide of the invention used as an antigen to obtain an antibody may be derived from any animal species, but is preferably derived from a mammal, such as a human, mouse or rat, more preferably from a human. The human polypeptide can be obtained from the nucleotide or amino acid sequence disclosed by the invention.

The polypeptides used as immunizing antigens according to the invention may be intact proteins or partial peptides of proteins. The partial peptide may comprise, for example, an amino (N) -terminal fragment or a carboxy (C) -terminal fragment of a polypeptide of the invention.

The gene encoding the polypeptide of the present invention or a fragment thereof may be inserted into a known expression vector, and then the host cell as described in the present specification may be transformed with the vector. The desired polypeptide or fragment thereof can be recovered from the host cell either intracellularly or extracellularly by any standard method and can then be used as an antigen. In addition, whole cells expressing the polypeptide or a lysate thereof, or chemically synthesized polypeptide can be used as the antigen.

Any mammal may be immunized with the antigen, but compatibility with the parent cell for cell fusion is preferably considered. Animals of the order Rodentia (Rodentia), Lagomorpha (Lagomorpha) or Primates (Primates) are generally used. Animals of the order rodentia include, for example, mice, rats, and hamsters (hamster). Animals of the order lagomorpha include, for example, rabbits. Primates include, for example, gibbons (Catarrhini) (eastern hemisphere monkeys) such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (rhesus monkey), baboons (sacred baboon), and chimpanzees (chimpanzee).

Methods of immunizing animals with antigens are known in the art. For example, intraperitoneal or subcutaneous injection of antigen is the standard method of immunization of mammals. More specifically, the antigen may be diluted and suspended in an appropriate amount of Phosphate Buffered Saline (PBS), physiological saline, or the like. If desired, the antigen suspension is mixed with an appropriate amount of standard adjuvant such as Freund's complete adjuvant, made into an emulsion, and then administered to the mammal. Preferably, the antigen is administered several times every 4 to 21 days thereafter in admixture with an appropriate amount of incomplete Freund's adjuvant. Immunization can also be carried out using a suitable carrier. Following immunization as described above, the serum is tested for an increase in the amount of the desired antibody using standard methods.

Polyclonal antibodies against the polypeptides of the invention can be prepared as follows: blood is collected from the immunized mammal whose serum is tested for an increase in the desired antibodies, and serum is isolated from the blood by any conventional method. Polyclonal antibodies include sera containing polyclonal antibodies, and fractions containing the polyclonal antibodies can be isolated from the sera. Immunoglobulin G or M can be prepared from a fraction that recognizes only the polypeptide of the present invention as follows: this fraction is further purified using, for example, an affinity column coupled to a polypeptide of the invention, and a protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from a mammal immunized with an antigen, the serum is examined for elevated levels of the desired antibody as described above, and used for cell fusion. The immune cells used for cell fusion are preferably obtained from the spleen. Other preferred parental cells for fusion with the above-described immune cells include, for example, mammalian myeloma cells, more preferably myeloma cells that have acquired properties useful for drug selection of fused cells.

The above immune cells and myeloma cells can be fused according to known Methods, for example, the method of Milstein et al (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).

Hybridomas resulting from cell fusion can be selected by culturing in standard selection medium such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium). Typically, cell culture is continued in HAT medium for several days to several weeks for a period of time sufficient to allow all other cells (non-fused cells) except the desired hybridoma cells to die. Then, standard limiting dilution (standard limiting dilution) screening and cloning of hybridoma cells producing the desired antibody is performed.

In addition to the above-described method for producing hybridomas by immunizing non-human animals with antigens, human lymphocytes such as EB virus-infected lymphocytes may be immunized with a polypeptide, cells expressing the polypeptide, or a lysate thereof in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells capable of dividing indefinitely, such as U266, and hybridoma cells producing a desired human antibody capable of binding to the polypeptide can be obtained (Japanese patent laid-open No. 63-17688).

The obtained hybridoma cells were then transplanted into the abdominal cavity of a mouse, and ascites was extracted. The obtained monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the polypeptide of the present invention is coupled. The antibody of the present invention can be used not only for purifying and detecting the polypeptide of the present invention, but also as a candidate for agonists and antagonists of the polypeptide of the present invention. In addition, the antibody can be used for antibody therapy of diseases related to the polypeptide of the present invention. When the obtained antibody is administered to a human body (antibody therapy), it is preferable to use a human antibody or a humanized antibody to reduce immunogenicity.

For example, transgenic animals having a human antibody gene bank (reporty) can be immunized with an antigen selected from the group consisting of a polypeptide, a cell expressing a polypeptide, or a lysate thereof. Then, antibody-producing cells are collected from the animal, and fused with myeloma cells to obtain a hybridoma, from which a human antibody against the polypeptide can be prepared (see WO92-03918, WO94-02602, WO94-25585, WO96-33735, and WO 96-34096).

Alternatively, antibody-producing immune cells, such as immunized lymphocytes, can be immortalized by oncogenes and used to produce monoclonal antibodies.

The Monoclonal Antibodies thus obtained can also be recombinantly produced by means of genetic engineering techniques (see, for example, Borebaeck and Larrick, (1990) Therapeutic Monoclonal Antibodies, MacMillan publishers LTD, published in the United kingdom). For example, recombinant antibodies can be prepared by cloning DNA encoding the antibody from immune cells, such as antibody-producing hybridomas or immunized lymphocytes, inserting the DNA into a suitable vector, and introducing into host cells. The present invention also provides recombinant antibodies prepared as described above.

Furthermore, the antibody of the invention may be an antibody fragment or a modified antibody, as long as it binds to one or more polypeptides of the invention. For example, the antibody fragment may be Fab, F (ab') ₂Fv or single chain Fv (scFv) in which Fv fragments derived from H chain and L chain are linked by an appropriate linker (Huston et al, (1988) Proc Natl Acad Sci USA 85: 5879-83). More specifically, the antibody may be treated with an enzyme such as papain or pepsin to produce antibody fragments. Alternatively, the gene encoding the antibody fragment can also be constructed, inserted into a suitable expression vector, and expressed in a suitable host cell (see, e.g., Co et al, (1994) J Immunol 152: 2968-76; Better and Horwitz, (1989) Methods Enzymol 178: 476-96; Pluckthun and Skerra, (1989) Methods Enzymol 178: 497-515; Lamoyi, (1986) Methods Enzymol 121: 652-63; Rousseaux et al, (1986) Methods Enzymol 121: 663-9; Bird and Walker, (1991) trends Biotechnol 9: 132-7).

Reference to "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, including the heavy chain of Fv, scFv or Fab. Reference to "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab.

"Single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy and light chains of a conventional diabody are joined to form one chain. Generally, a linker peptide is inserted between the two chains, enabling proper folding and formation of an active binding site.

A "chimeric antibody" is an immunoglobulin molecule that has (a) a constant region or a portion thereof altered, substituted, or exchanged such that the antigen binding site (variable region) binds to a constant region of a different or altered class (class), effector function, and/or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) an immunoglobulin molecule in which the variable region or a portion thereof is altered, substituted or exchanged to have a variable region with a different or altered antigen specificity.

A "humanized antibody" is an immunoglobulin molecule that contains minimal non-human immunoglobulin-derived sequences. Humanized antibodies comprise human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (e.g., mouse, rat or rabbit) having the desired specificity, affinity and capacity (capacity) (donor antibody). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may contain residues that are found neither in the recipient antibody nor in the introduced CDR or framework sequences. In general, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions have human immunoglobulin consensus sequences. The humanized antibody also preferably comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (see Jones et al, (1986) Nature.; 321: 522-5; Riechmann et al, (1988) Nature.; 332: 323-7; Presta, (1992) Curr Opin Struct biol. 2: 593-6).

Humanization can be achieved essentially by the following method: rodent CDRs or CDR sequences are substituted for the corresponding sequences of a human antibody according to Winter's method with its co-investigators (Jones et al, Nature 321: 522-5 (1986); Riechmann et al, Nature 332: 323-7 (1988); Verhoeyen et al, Science 239: 1534-6 (1988)). Thus, such humanized antibodies are chimeric antibodies in which regions substantially shorter than the full length of the human variable region are replaced with corresponding sequences from non-human species (U.S. Pat. No. 4,816,567).

The terms "epitope" and "antigenic determinant" refer to the site on an antigen to which an antibody binds. The epitope may be formed of adjacent amino acids or may be formed of non-adjacent amino acids juxtaposed by three-dimensional folding of the protein. In general, epitopes formed by adjacent amino acids can be retained even by denaturing solvent treatment, while epitopes formed by three-dimensional folding disappear by solvent treatment. Epitopes are typically at least 3, more typically at least 5 or at least 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., epipope Mapping Protocols in Methods in Molecular Biology, Vol.66, Glenn E.Morris, Ed (1996).

Antibodies can be modified by conjugation to various molecules, such as polyethylene glycol (PEG). The present invention provides such modified antibodies. The modified antibody can be obtained by chemically modifying the antibody. These modification methods are conventional in the art.

Alternatively, the antibody of the present invention may be obtained as a chimeric antibody between a variable region derived from a non-human antibody and a constant region derived from a human antibody, or as a humanized antibody containing Complementarity Determining Regions (CDRs) derived from a non-human antibody, and a Framework Region (FR) and a constant region derived from a human antibody. The antibodies can be prepared using known techniques.

Fully human antibodies comprising human variable regions in addition to human framework and constant regions are also useful. Such antibodies can be prepared using various techniques known in the art. For example, in vitro methods include the use of recombinant libraries of human antibody fragments displayed on phage (e.g., Hoogenboom & Winter, (1992) J.mol.biol.227: 381-8). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which endogenous immunoglobulin genes are partially or completely inactivated. Such methods are described, for example, in U.S. patent nos.6,150,584; 5,545,807, respectively; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,661,016.

Non-antibody binding proteins

The invention also includes antigen-binding or non-antibody binding proteins (e.g., ligands) that specifically bind to the polypeptides of the invention. Non-antibody ligands include antibody mimetics using non-immunoglobulin albumin scaffolds, including Adnectins, Avimers, single chain polypeptide binding molecules, and antibody-like binding peptide mimetics, as discussed in more detail below.

Other substances have been developed that target and bind to targets in a manner similar to antibodies. Some such "antibody mimetics" use non-immunoglobulin protein scaffolds as alternative protein frameworks for antibody variable regions.

For example, Ladner et al (U.S. Pat. No.5,260,203) describe single polypeptide chain binding molecules with binding specificity similar to that of aggregated, but molecularly separated, antibody light and heavy chain variable regions. Single chain binding molecules contain antigen binding sites for the variable regions of the heavy and light chains of the antibody linked by peptide linkers and will fold into a structure similar to that of a bi-peptide antibody. Single chain binding molecules exhibit several advantages over conventional antibodies, including smaller size, better stability, and easier modification.

Ku et al (Proc. Natl. Acad. Sci. U.S.A.92 (14): 6552-6556(1995)) disclose antibody alternatives based on cytochrome b 562. Ku et al (1995) made a library in which two loops of cytochrome b562 were randomized and selected for binding to bovine serum albumin. Mutant individuals were found to bind selectively to BSA, as were anti-BSA antibodies.

Lipovsek et al (U.S. Pat. Nos.6,818,418 and 7,115,396) disclose an antibody mimetic characterized by a fibronectin or fibronectin-like protein scaffold and at least one variable loop. These fibronectin-based antibody mimics, known as Adnectins, exhibit many of the same characteristics as natural or engineered antibodies, including high affinity and specificity for any target ligand. Any technique for developing new or improved binding proteins can be used for these antibody mimetics.

The structure of these fibronectin based antibody mimetics is similar to that of the IgG heavy chain variable region. Thus, these mimetics exhibit antigen binding properties and affinities that are substantially similar to the native antibody. In addition, these fibronectin-based antibody mimetics exhibit certain benefits over antibodies and antibody fragments. For example, the natural folding stability of these antibody mimetics is not dependent on disulfide bonds, and thus these antibody mimetics are stable under conditions that would normally destroy the antibody. Furthermore, because the structure of these fibronectin based antibody mimetics is similar to the IgG heavy chain, methods for loop randomization and shuffling can be employed in vitro, which is similar to the process of antibody affinity maturation in vivo.

Beste et al (Proc. Natl. Acad. Sci. U.S.A.96 (5): 1898-1903(1999)) disclose a lipocalin scaffold-based antibody mimetic (A. a. lipocalin scaffold)

). Lipocalins consist of a beta-barrel with four hyper-variable loops at the ends of the protein. Beste (1999) performed random mutagenesis of the loop and selected for binding to, for example, fluorescein. Three variants showed specific binding to fluorescein, with one variant showing similar binding to the anti-fluorescein antibody. Further analysis revealed that all randomized positions were variable, indicating

Will be suitable for use as an alternative to antibodies.

Are small single chain peptides, typically 160-180 residues, which offer several advantages over antibodies, including reduced production costs, increased storage stability, and reduced immune responses.

Hamilton et al (U.S. patent No.5,770,380) disclose a synthetic antibody mimetic in which a rigid, non-peptidic organic scaffold, calixarene, is used to which are attached multiple variable peptide loops that serve as binding sites. The peptide loops all geometrically protrude from the same side of the calixarene with respect to each other. Due to this geometric conformation, all loops are available for binding, increasing the binding affinity to the ligand. However, in comparison to other antibody mimetics, the calixarene-based antibody mimetic is not composed entirely of peptides, and thus it is less susceptible to attack by proteases. The scaffold is also not composed purely of peptides, DNA or RNA, meaning that such antibody mimetics are relatively stable and long-lived under extreme environmental conditions. In addition, because the calixarene-based antibody mimetics are relatively small, they are less likely to produce an immune response.

Murali et al (cell. mol. biol.49 (2): 209-216(2003)) discuss methodologies for reducing antibodies to smaller peptidomimetics, which they call "antibody-like binding peptidomimetics" (ABiP), and may also be used as antibody substitutes.

Silverman et al (nat. Biotechnol. (2005), 23: 1556-1561) disclose fusion proteins which are single chain polypeptides comprising multiple domains referred to as "avimers". Avimers, developed from the human extracellular receptor domain by in vitro exon shuffling and phage display, are a class of binding proteins that resemble antibodies to some extent in their affinity and specificity for a variety of target molecules. The resulting multidomain protein may comprise a plurality of independent binding domains that exhibit improved affinity (in some cases sub-nanomolar) and specificity compared to single epitope binding proteins. Additional details regarding the methods of construction and the use of avimers are disclosed in, for example, U.S. patent application publication nos. 20040175756, 20050048512, 20050053973, 20050089932, and 20050221384.

In addition to non-immunoglobulin albumin frameworks, antibody properties are also mimicked in compounds comprising RNA molecules and non-natural oligomers (e.g., protease inhibitors, benzodiazepines, purine derivatives, and β -turn mimetics), all of which are suitable for use in the present invention.

The antibody obtained as above may be purified to homogeneity. For example, the separation and purification of the antibody can be performed according to separation and purification methods used for general proteins. For example, Antibodies can be isolated by appropriately selecting and combining column chromatography such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing (Antibodies: A Laboratory Manual. ed Harbor and David Lane, (1988) Cold Spring Harbor Laboratory), but not limited thereto. Protein a columns and protein G columns can be used as affinity columns. Exemplary protein A columns that can be used include, for example, HyperD, POROS and Sepharose F.F (Pharmacia).

Exemplary chromatographies, in addition to affinity chromatography, include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and the like (stratgies for Protein Purification and chromatography: A Laboratory Course Manual. Ed Daniel R. Marshak et al, (1996) Gold Spring Harbor Laboratory Press). The chromatographic procedure can be performed by liquid chromatography, such as HPLC and FPLC.

For example, the antigen binding activity of the antibody of the present invention can be determined using absorbance assays, enzyme linked immunosorbent assays (ELISAs), Enzyme Immunoassays (EIAs), Radioimmunoassays (RIA), and/or immunofluorescence assays. In ELISA, an antibody or non-antibody-binding protein of the present invention is immobilized on a plate, a polypeptide of the present invention is applied to the plate, and a sample containing a desired antibody, such as a culture supernatant of antibody-producing cells or purified antibody, is applied. A second antibody, labeled with an enzyme (e.g., alkaline phosphatase), recognizing the first antibody is then applied and the plate is incubated. Next, after washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, and the absorbance is measured to evaluate the antigen binding activity of the sample. Fragments of the polypeptide, such as C-terminal or N-terminal fragments, can be used as antigens to evaluate the binding activity of the antibody. BIAcore (pharmacia) can be used to evaluate the activity of the antibody of the present invention.

The above methods allow for the detection or determination of a polypeptide of the invention by exposing an antibody or non-antibody binding protein of the invention to a sample putatively containing a polypeptide of the invention and detecting or determining the immune complex formed by the antibody and polypeptide.

Since the method for detecting or determining a polypeptide according to the present invention can specifically detect or determine a polypeptide, the method can be applied to various experiments using polypeptides.

Antisense oligonucleotides

As described above, the present invention also provides a polynucleotide which hybridizes to the polynucleotide encoding the human A7322 or F3374V1 protein (SEQ ID NO: 79 or 81) or the complementary strand thereof and which comprises at least 15 nucleotides. Preferred are, for example, antisense oligonucleotides that hybridize to a sequence selected from the group consisting of SEQ id nos: 79(A7322) are complementary to each other at positions 1 to 384. In general, nucleotide sequences comprising an initiation codon are preferred for designing effective antisense oligonucleotides. The start codon (172-174) of A7322 is located in SEQ ID NO: 79(A7322) at positions 1 to 384. The polynucleotide of the present invention is preferably a polynucleotide which specifically hybridizes with a DNA encoding the polypeptide of the present invention.

The term "specifically hybridize" as used herein means that no significant cross-hybridization (cross-hybridization) occurs with DNA encoding other proteins under normal hybridization conditions, preferably under stringent hybridization conditions. Such polynucleotides include probes, primers, nucleotides and nucleotide derivatives (e.g., antisense oligonucleotides and ribozymes) that specifically hybridize to DNA encoding a polypeptide of the invention or its complementary strand. In addition, such polynucleotides can be used to prepare DNA chips.

The term "antisense nucleic acid" as used in the present specification refers not only to antisense nucleic acids in which the nucleotides corresponding to those constituting a certain region of DNA or mRNA are completely complementary, but also to nucleic acids having one or more nucleotide mismatches, as long as DNA or mRNA and antisense oligonucleotide are capable of hybridizing with the nucleotide sequence of SEQ ID NO: 79 or 81, respectively.

Included are polynucleotides that have a homology of at least about 70% or greater, preferably at least about 80% or greater, more preferably about at least 90% or greater, and even more preferably at least about 95% or greater, over "at least 15 contiguous nucleotide sequence regions". The homology can be determined using the algorithm described in this specification. Such polynucleotides may be used as probes for isolating or detecting DNA encoding a polypeptide of the present invention, or as primers for amplification, as described in one embodiment below.

Derivatives or modified products of antisense oligonucleotides can be used as antisense oligonucleotides of the invention. Examples of such modified products include lower alkyl phosphonate modifications, such as methyl-phosphonate type or ethyl-phosphonate type, phosphorothioate modifications and phosphoramidate modifications.

The antisense oligonucleotide derivative of the present invention acts on cells producing the polypeptide of the present invention by: binds to DNA or mRNA encoding the polypeptide of the present invention, inhibits its transcription or translation, promotes mRNA degradation, and inhibits the expression of the polypeptide of the present invention, thereby inhibiting the function of the polypeptide.

The antisense oligonucleotide derivative of the present invention can be mixed with a base inactive to the derivative to prepare an external preparation such as liniment or cataplasm.

Further, such derivatives can be formulated into, for example, tablets, powders, granules, capsules, liposome capsules, injections, solutions, nasal drops and freeze-drying agents by adding excipients, isotonic agents (isotonics agents), solubilizers, stabilizers, preservatives, analgesics and the like as necessary. These formulations may be prepared by conventional methods.

The antisense oligonucleotide derivative is administered to the patient by directly applying it to the affected part (ailing site), or by infusing it into a blood vessel so that it reaches the affected part. Antisense-mounting media (antisense-mounting medium) can also be used to increase persistence and membrane permeability. Examples of encapsulation vehicles include liposomes, poly-L-lysine, lipids, cholesterol, lipofectamine (lipofectamine), or derivatives thereof.

The dose of the antisense oligonucleotide derivative of the present invention can be suitably adjusted depending on the condition (condition) of the patient and used in a desired amount. For example, a dosage range of 0.1-100mg/kg, preferably 0.1-50mg/kg, can be administered.

siRNA

The term "siRNA" means a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques for introducing siRNA into cells are used, including those in which RNA is transcribed using DNA as a template. The siRNA of the present invention comprises a sense nucleic acid sequence and an antisense nucleic acid sequence of a polynucleotide encoding a human A7322, F3374V1, PBK/TOPK or AURKB protein (SEQ ID NO: 79, 81, 92 or 88). The siRNA can be constructed such that a single transcript (double-stranded RNA) has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin. The siRNA may be dsRNA or shRNA.

As used herein, the term "dsRNA" refers to a construct of two RNA molecules that comprise sequences that are complementary to each other and anneal together by the complementary sequences to form a double-stranded RNA molecule. The nucleotide sequences of both strands may comprise not only "sense" or "antisense" RNA of a protein coding sequence selected from the target gene sequence, but also RNA molecules having a nucleotide sequence selected from the non-coding region of the target gene.

The term "shRNA" as used herein refers to an siRNA having a stem-loop structure comprising a first region and a second region complementary to each other, i.e., a sense strand and an antisense strand. The degree and orientation of complementarity of the two regions is sufficient to allow base pairing to occur between the two regions, the first and second regions being joined by a loop region that results from the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of the shRNA is a single-stranded region between the sense strand and the antisense strand, and may also be referred to as an "intervening single-strand".

As used herein, the term "siD/R-NA" refers to a double-stranded polynucleotide molecule composed of both RNA and DNA, including hybrids and chimeras of RNA and DNA, which prevents translation of the target mRNA. Herein, a hybrid means a molecule in which a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize to each other to form a double-stranded molecule; and chimeras indicate that one or both of the strands making up the double-stranded molecule may contain both RNA and DNA. Standard techniques for introducing siD/R-NA into cells were used. siD/R-NA includes a CX sense nucleic acid sequence (also referred to as "sense strand") and/or a CX antisense nucleic acid sequence (also referred to as "antisense strand"). siD/R-NA can be constructed such that a single transcript has both a sense nucleic acid sequence and a complementary antisense nucleic acid sequence from the target gene, e.g., a hairpin. siD/R-NA can be dsD/R-NA or shD/R-NA.

As used herein, the term "dsD/R-NA" refers to a construct of two molecules that comprise sequences that are complementary to each other and that have annealed together by the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequences of both strands may not only comprise "sense" or "antisense" polynucleotide sequences selected from the protein coding sequences of the target gene sequence, but may also comprise polynucleotides having nucleotide sequences selected from non-coding regions of the target gene. One or both of the two molecules that make up dsD/R-NA are composed of both RNA and DNA (chimeric molecules), or one molecule is composed of RNA and the other is composed of DNA (heteroduplexes).

The term "shD/R-NA" as used herein refers to siD/R-NA having a stem-loop structure comprising a first region and a second region that are complementary to each other, i.e., a sense strand and an antisense strand. The regions are sufficiently complementary and oriented to allow base pairing to occur between them, and the first and second regions are joined by a loop region that results from the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The shD/R-NA loop region is a single-stranded region between the sense and antisense strands, and may also be referred to as an "intervening single strand".

siRNAs to A7322, F3374V1, PBK/TOPK or AURKB are directed to a single target of A7322, F3374V1, PBK/TOPK or AURKB gene sequences, respectively. Alternatively, the siRNA is directed against multiple targets of A7322, F3374V1, PBK/TOPK, or AURKB sequences. For example, the compositions comprise sirnas to a7322, F3374V1, PBK/TOPK, or AURKB to two, three, four, or five or more target sequences of a7322, F3374V1, PBK/TOPK, or AURKB, respectively. By "target sequence of A7322, F3374V1, PBK/TOPK or AURKB" is meant a nucleotide sequence identical to a portion of an A7322, F3374V1, PBK/TOPK or AURKB gene.

The target sequence may comprise the 5 'Untranslated (UT), Open Reading Frame (ORF), or 3' untranslated region of the human A7322, F3374V1, PBK/TOPK, or AURKB genes. siRNAs to A7322, F3374V1, PBK/TOPK or AURKB that hybridize to a target mRNA bind to mRNA transcripts that are single stranded under normal conditions, thereby interfering with translation and thereby suppressing expression of the protein, thereby reducing or inhibiting production of the A7322, F3374V1, PBK/TOPK or AURKB polypeptide product encoded by the A7322, F3374V1, PBK/TOPK or AURKB gene. Thus, the siRNA molecules of the present invention may be defined by their ability to specifically hybridize to mRNA or cDNA from the A7322, F3374V1, PBK/TOPK or AURKB genes under stringent conditions.

Binding of the siRNA to the transcript corresponding to A7322, F3374V1, PBK/TOPK or AURKB in the target cell causes a decrease in production of the protein by the cell. The length of the oligonucleotide is at least 10 nucleotides, and may be as long as the naturally occurring transcript. Preferably, the oligonucleotide is less than 100, 75, 50, 25 nucleotides in length. More preferably, the oligonucleotide is 19 to 25 nucleotides in length. Examples of siRNA oligonucleotides A7322, F3374V1, PBK/TOPK or AURKB that inhibit the growth of cancer cells include those comprising SEQ ID NO: 34 or 35 (for a 7322), comprising the sequence of seq id NO: 37. 38 or 67 (for F3374V 1), a target sequence comprising SEQ ID NO: 39 or 40 (for PBK/TOPK), or a target sequence comprising SEQ ID NO: 68 (for AURKB).

In addition, to enhance the inhibitory activity of siRNA, nucleotide "u" may be added to the 3' end of the antisense strand of the target sequence. The number of "u" added is at least about 2, usually from about 2 to about 10, preferably from about 2 to about 5. The added "u" forms a single strand at the 3' end of the siRNA antisense strand.

siRNAs to A7322, F3374V1, PBK/TOPK, or AURKB may be introduced directly into cells in a form capable of binding to mRNA transcripts. In these embodiments, the siRNA molecules of the invention are generally modified as described above for antisense molecules. Other modifications are also possible, for example siRNA conjugated to cholesterol show improved pharmacological properties (Song et al Nature Med.9: 347-51 (2003)). Alternatively, DNA encoding siRNA of A7322, F3374V1, PBK/TOPK, or AURKB may be contained in a vector.

Vectors are generated, for example, by cloning the A7322, F3374V1, PBK/TOPK or AURKB target sequences into expression vectors, operably linked to regulatory sequences flanking the A7322, F3374V1, PBK/TOPK or AURKB sequences (by transcription of DNA molecules) in a manner that allows expression of both strands (Lee, N.S. et al, Nature Biotechnology 20: 500-5.). Antisense RNA molecules to mRNA of A7322, F3374V1, PBK/TOPK, or AURKB are transcribed by a first promoter (e.g., a promoter sequence located 3 'of the cloned DNA), while sense RNA molecules to mRNA of A7322, F3374V1, PBK/TOPK, or AURKB are transcribed by a second promoter (e.g., a promoter sequence located 5' of the cloned DNA). The sense and antisense strands hybridize in vivo to produce siRNA constructs useful for silencing A7322, F3374V1, PBK/TOPK, or AURKB genes. Alternatively, two constructs can be used to generate the sense and antisense strands of the siRNA construct. Clones a7322, F3374V1, PBK/TOPK or AURKB may encode constructs with secondary structures such as hairpins, where a single transcript has both the sense and complementary antisense sequences from the target gene.

In addition, in order to form a hairpin loop structure, a loop sequence (loop sequence) composed of an arbitrary nucleotide sequence may be placed between the sense sequence and the antisense sequence. Accordingly, the present invention also provides sirnas having the general formula 5 ' - [ a ] - [ B ] - [ a ' ] -3 ', wherein [ a ] is a ribonucleotide sequence, which corresponds to SEQ id no: 34. 35, 37, 38, 39, 40, 67 or 68 nucleotides, [ B ] is a ribonucleotide sequence consisting of 3-23 nucleotides, and [ a' ] is a ribonucleotide sequence consisting of the complement of [ a ]. The loop sequence may consist of any sequence of preferably 3-23 nucleotides in length. The loop sequence may for example be selected from the group consisting of http:// www.ambion.com/techlib/tb/tb _506. html. In the siRNA of the present invention, in order to enhance the inhibitory activity of the siRNA, a nucleotide "u" may be added to the 3 'end of [ A' ]. The number of "u" s added is at least 2, usually 2 to 10, preferably 2 to 5. In addition, a loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J. -M., et al, (2002) Nature 418: 435-8.):

CCC, CCACC or CCACACC: jacque, j. -m., et al, Nature 418: 435-8 (2002);

UUCG: lee, n.s., et al, (2002) Nature Biotechnology 20: 500-5.Fruscoloni, p., et al, proc.natl.acad.sci.usa 100 (4): 1639-44 (2003); and

UUCAAGAGA: dykxhoorn, d.m., et al, Nature Reviews Molecular cell biology 4: 457-67(2003).

For example, preferred sirnas having hairpin structures of the invention are shown below. In the structures below, the loop sequences may be selected from the group consisting of: CCC, UUCG, CCACC, CCACACC and UUCAAGAGA. One preferred ring structure is UUCAAGAGA ("ttcaagaga" in DNA).

aagaaagcaucgcagucucag- [ B ] -cugagacugcgaugcuuucuu (against the target sequence SEQ ID NO: 34)

aagaugcguucucugccacac- [ B ] -guguggcagagaacgcaucuu (targeting sequence SEQ ID NO: 35)

gaucaugucuccgagaaaa- [ B ] -uuuucucggagacaugauc (against the target sequence SEQ ID NO: 37)

ggaagccauagaauugcuc- [ B ] -gagcaauucuauggcuucc (for the target sequence SEQ ID NO: 38)

cuggaugaaucauaccaga- [ B ] -ucugguaugauucauccag (against the target sequence SEQ ID NO: 39)

guguggcuugcguaaauaa- [ B ] -uuauuuacgcaagccacac (for the target sequence SEQ ID NO: 92)

acuccuacguucucuauua- [ B ] -uaauagagaacguaggagu (for the target sequence SEQ ID NO: 67)

aaggugauggagaauagcagu- [ B ] -acugcuauucuccaucaccuu (targeting sequence SEQ ID NO: 68)

The regulatory sequences flanking the A7322, F3374V1, PBK/TOPK or AURKB sequences are identical or different such that their expression can be regulated independently, or in a temporal or spatial manner. siRNA is transcribed intracellularly by cloning the A7322, F3374V1, PBK/TOPK, or AURKB gene templates onto a vector containing, for example, an RNA polymerase III transcription unit from a small nuclear RNA (snRNA) U6 or human H1 RNA promoter. For introducing the vector into the cell, a transfection-enhancing agent (transfection-enhancing agent) can be used. FuGENE (Roche diagnostics), Lipofectamine 2000(Invitrogen), Oligofectamine (Invitrogen) and nucleofector (Wako pure chemical) can be used as transfection enhancers.

The nucleotide sequence of siRNA can be designed using an siRNA design computer program accessible from the Ambion website on the world Wide Web (http:// www.ambion.com/techlib/misc/siRNA _ finder. html). The computer program selects the nucleotide sequence of the siRNA based on the following protocol:

Selection of siRNA target sites:

1. the AA dinucleotide sequence is scanned downstream starting from the AUG start codon of the target transcript, looking for AA dinucleotide sequences. The occurrence of each AA and its 3' adjacent 19 nucleotides were recorded as potential siRNA target sites. Tuschl et al in Genes Dev 13 (24): 3191-7(1999), it is not recommended to design siRNAs against 5 'and 3' untranslated regions (UTRs) and regions adjacent to the start codon (within 75 bases) because these regions may be more rich in regulatory protein binding sites. UTR binding proteins and/or translation initiation complexes can interfere with binding of siRNA endonuclease complexes.

2. The potential target sites are compared to the human genome database, and any target sequences that are significantly homologous to other coding sequences are excluded from consideration. Homology searches can be performed using BLAST (Altschul SF, et al., Nucleic Acids Res.1997; 25 (17): 3389-: www.ncbi.nlm.nih.gov/BLAST/.

3. Eligible target sequences were selected for synthesis. In Ambion, several target sequences are preferably selected along the length of the gene to be evaluated.

Their ability to reduce the production of A7322, F3374V1, PBK/TOPK or AURKB in tumor cells was tested in vitro using standard methods for oligonucleotides and oligonucleotides complementary to different portions of A7322, F3374V1, PBK/TOPK or AURKB mRNA (e.g., BT-549, BT-474 using breast cancer cell lines such as A7322, D47T and HBC4 for F3374, and T47D and BT-20 for PBK/TOPK). A cell contacted with a candidate siRNA composition can be tested for a reduction in A7322, F3374V1, PBK/TOPK or AURKB gene product as compared to a cell cultured in the absence of the candidate siRNA composition using A7322, F3374V1, PBK/TOPK or AURKB specific antibodies or using other detection strategies. For sequences that reduce the production of A7322, F3374V1, PBK/TOPK or AURKB in an in vitro cell assay or a cell-free assay, their inhibitory effect on cell growth can be further determined. For sequences that inhibit cell growth in an in vitro cell assay, in vivo assays were performed in rats or mice to confirm the reduction in A7322, F3374V1, PBK/TOPK or AURKB production and the reduction in tumor cell growth in animals with malignant neoplasms.

The invention also includes double-stranded molecules comprising a nucleic acid sequence of a target sequence, such as SEQ ID NO: 34. 35, 37, 38, 39, 67, or 68. In the present invention, the double stranded molecule comprises a sense strand and an antisense strand, wherein said sense strand comprises a sequence corresponding to SEQ ID NO: 34. 35, 37, 38, 39, 67 or 68 and the antisense strand comprises a ribonucleotide sequence that is complementary to the sense strand, wherein the sense strand and the antisense strand hybridize to each other to form the double-stranded molecule, and wherein the double-stranded molecule, when introduced into a cell expressing an a7322, F3374V1, or AURKB gene, inhibits expression of the gene.

In the present invention, when the isolated nucleic acid is RNA or a derivative thereof, the base "t" should be replaced with "u" in the nucleotide sequence. The term "complementary" as used herein refers to the formation of Watson-Crick or Hoogsteen base pairing between the nucleotide units of a nucleic acid molecule. And the term "binding" refers to a physical or chemical interaction between two polypeptides or compounds, or related (associated) polypeptides or compounds, or a combination thereof. When the polynucleotide comprises modified nucleotides and/or non-phosphodiester linkages, these nucleic acids may also be bound to each other in the same manner.

Complementary nucleic acid sequences hybridize under appropriate conditions to form a stable duplex with little or no mismatch. In addition, the sense and antisense strands of the isolated nucleotides of the invention may form a double-stranded nucleotide or hairpin loop structure by hybridization. In a preferred embodiment, such duplexes contain on average no more than 1 mismatch per 10 matches. In a particularly preferred embodiment, the strands of the duplex are fully complementary, such that the duplex contains no mismatches.

For example, the nucleic acid molecule is shorter than 500, 200, 75 nucleotides in length. The invention also includes vectors comprising one or more of the nucleic acids described in the specification, and cells comprising the vectors. The isolated nucleic acids of the invention are useful for siRNA against a7322 or F3374V1 or DNA encoding these sirnas. When these nucleic acids are used for siRNA or DNA encoding the same, the sense strand is preferably longer than 19 nucleotides, more preferably longer than 21 nucleotides.

The double-stranded molecules of the invention may comprise one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art can increase the stability, availability and/or cellular uptake of the double-stranded molecule. Those skilled in the art will appreciate that other types of chemical modifications of the present molecules may be introduced (WO 03/070744; WO 2005/045037). In one embodiment, the modification may be used to provide better resistance to degradation or better uptake. Examples of such modifications include phosphorothioate linkages, 2 '-O-methyl ribonucleotides (particularly on the sense strand of double-stranded molecules), 2' -deoxy-fluoro ribonucleotides, 2 '-deoxy-ribonucleotides, "universal base" nucleotides, 5' -C-methyl nucleotides and the introduction of inverted deoxyabasic residues (US 20060122137).

In another embodiment, the modification can be used to enhance the stability of the double-stranded molecule or increase the targeting efficiency. Modifications include chemical cross-linking between the two complementary strands of a double-stranded molecule, chemical modifications at the 3 ' or 5 ' end of one strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2 ' -deoxyribonucleotides (WO 2004/029212). In another embodiment, the modification may be used to increase or decrease the affinity for complementary nucleotides in the target mRNA and/or in the strand of the complementary double-stranded molecule (WO 2005/044976). For example, an unmodified pyrimidine nucleotide may be substituted with a 2-thio, 5-alkynyl (5-alkinyl), 5-methyl or 5-propynyl (5-propylnyl) pyrimidine. In addition, unmodified purines may be substituted with 7-deaza (7-deaza), 7-alkyl or 7-alkenyl purines. In another embodiment, when the double stranded molecule is a double stranded molecule having a 3 'overhang, the overhanging nucleotide of the 3' -terminal nucleotide may be replaced by a deoxyribonucleotide (Elbashir SM et al, Genes Dev 2001 Jan15, 15 (2): 188-. For further details, publications such as US20060234970 may be utilized. The present invention is not limited to these examples, and any known chemical modification may be applied to the double-stranded molecule of the present invention as long as the resulting molecule retains the ability to inhibit expression of a target gene.

Furthermore, the double-stranded molecules of the invention may comprise both DNA and RNA, for example dsD/R-NA or shD/R-NA. Specifically, hybrid polynucleotides formed of one DNA strand and one RNA strand or DNA-RNA chimeric polynucleotides show improved stability. A mixture of DNA and RNA, that is, a hybrid type double-stranded molecule composed of one DNA strand (polynucleotide) and one RNA strand (polynucleotide), or a chimeric type double-stranded molecule comprising both DNA and RNA on either single strand (polynucleotide) or both single strands (polynucleotides), or the like, may be formed to enhance the stability of the double-stranded molecule. The hybrid of the DNA strand and the RNA strand may be a hybrid in which the sense strand is DNA and the antisense strand is RNA, or vice versa, so long as the hybrid has an activity of inhibiting the expression of a target gene when introduced into a cell expressing the gene. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimeric double-stranded molecule may be one in which both the sense strand and the antisense strand are composed of DNA and RNA, or either the sense strand or the antisense strand is composed of DNA and RNA, as long as the double-stranded molecule has an activity of inhibiting the expression of a target gene when introduced into a cell expressing the gene.

To enhance the stability of the double-stranded molecule, the molecule preferably comprises as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule must be RNA within a certain range to induce sufficient inhibition of expression. A preferred example of a chimeric double-stranded molecule is where the upstream region of the double-stranded molecule (i.e., the flanking region of the target sequence or its complement within the sense or antisense strand) is RNA. Preferably, the upstream part region represents the 5 '-side (5' -end) of the sense strand and the 3 '-side (3' -end) of the antisense strand. That is, in a preferred embodiment, the 3 ' flanking region of the antisense strand consists of RNA, or both the 5 ' flanking region of the sense strand and the 3 ' flanking region of the antisense strand consist of RNA. For example, the chimeric or hybrid double-stranded molecules of the invention comprise the following combinations.

Sense strand: 5 '- [ DNA ] -3'

3 '- (RNA) - [ DNA ] -5': the antisense strand of the nucleic acid sequence is,

sense strand: 5 '- (RNA) - [ DNA ] -3'

3 '- (RNA) - [ DNA ] -5': an antisense strand, and

sense strand: 5 '- (RNA) - [ DNA ] -3'

3 '- (RNA) -5': the antisense strand.

The upstream part region is preferably a domain consisting of 9 to 13 nucleotides, counted from the end of the target sequence or its complement within the sense strand or antisense strand of the double-stranded molecule. Further, preferred examples of such chimeric double-stranded molecules include those having a chain length of 19 to 21 nucleotides, wherein at least the upstream half region (5 '-side region for sense strand and 3' -side region for antisense strand) of the polynucleotide is RNA and the other half is DNA. In such chimeric double-stranded molecules, the effect of suppressing the expression of a target gene is much stronger than when the antisense strand is RNA as a whole (US 20050004064).

In the present invention, the double-stranded molecule may form a hairpin, such as short hairpin RNA (shRNA) and short hairpin consisting of DNA and RNA (shD/R-NA). shRNA or shD/R-NA is a sequence of RNA or a mixture of RNA and DNA that forms a tight hairpin turn that can be used to silence gene expression through RNA interference. The shRNA or shD/R-NA comprises a sense target sequence and an antisense target sequence on a single strand, wherein the sequences are separated by a loop sequence. Typically, the hairpin structure is cleaved by cellular machinery into either dsRNA or dsD/R-NA, which is thereafter bound by an RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that matches the target sequence of the dsRNA or dsD/R-NA.

Diagnosis of breast cancer

The inhibitory polynucleotides of the invention (e.g., antisense oligonucleotides or sirnas) inhibit the expression of the polypeptides of the invention and are therefore useful for inhibiting the biological activity of the polypeptides of the invention. In addition, an expression inhibitor containing the antisense oligonucleotide or siRNA of the present invention is useful for inhibiting the biological activity of the polypeptide of the present invention. Thus, compositions comprising one or more inhibitory polynucleotides of the invention (e.g., antisense oligonucleotides or sirnas) are useful for the treatment of breast cancer. The present invention also provides a method for prognostically predicting, diagnosing, detecting or examining breast cancer, which uses the expression level of the polypeptide of the present invention as a marker for prognosis and/or diagnosis.

The diagnostic method of the present invention comprises the steps of:

(a) detecting the expression level of the a7322 or F3374V1 gene of the invention; and

(b) an increase in the level of expression (e.g., transcription and/or translation) of a7322 and/or F3374V1 is correlated with the diagnosis or prognosis of breast cancer.

The expression level of the a7322 gene or F3374V1 gene in a particular sample can be assessed by quantifying the mRNA corresponding to the a7322 gene or F3374V1 gene or the protein encoded by the a7322 or F3374V1 gene. methods for quantifying mRNA are well known to those skilled in the art. For example, the level of mRNA corresponding to the a7322 or F3374V1 gene can be assessed by Northern blot analysis or RT-PCR (e.g., using quantitative PCR or real-time PCR). The full-length nucleotide sequence of the a7322 or F3374V1 gene is set forth in SEQ ID NO: 79 or 81, and thus any person skilled in the art can design the nucleotide sequence of a probe or primer for quantifying the a7322 or F3374V1 gene.

In addition, the expression level of the gene can also be analyzed based on the activity or amount (quality) of the protein encoded by the a7322 or F3374V1 gene. One method for determining the amount of a7322 or F3374V1 protein is shown below. For example, immunoassays are very effective for the determination of proteins in biological materials. Any biological material can be used as the biological sample for the determination of the protein or its activity. For example, analysis of blood samples can be used to assess the proteins encoded by serum markers. On the other hand, the activity of the protein encoded by the a7322 or F3374V1 gene can be determined by selecting an appropriate method according to the activity of the protein to be analyzed.

As another method for detecting the expression level thereof based on the translation product of a7322 or F3374V1 gene, the intensity of staining can be observed by immunohistochemical analysis using an antibody against a7322 protein or F3374V1 protein. That is, if strong staining is observed, it shows that the presence amount of a7322 protein or F3374V1 protein is increased, and that the expression level of a7322 or F3374V1 gene is high. Breast cancer tissue is preferably used as the test material for immunohistochemical analysis.

According to the method of the present invention, the expression level of a7322 or F3374V1 gene in a sample (test sample) is evaluated and compared with the expression level in a normal sample. When such comparison shows that the expression level of the target gene is higher than that in the normal sample, the subject is judged to have (treated with) breast cancer. The expression level of a7322 or F3374V1 gene in a sample from a normal sample and a subject can be determined simultaneously. Alternatively, a normal range of expression level can be determined statistically based on the results obtained by analyzing the expression level of the gene in a sample collected in advance from the control group. Comparing the results obtained from the subject sample to a normal range; when the result does not fall within the normal range, the subject is judged to have breast cancer. The expression level of the a7322 or F3374V1 gene in the sample (test sample) may also be compared to the expression level in one or more breast cancer samples. The breast cancer sample may represent various disease stages of breast cancer. When such comparison shows that the expression level of the target gene is substantially equal to the expression level in the breast cancer sample, the subject is determined to have breast cancer. Furthermore, by comparison with breast cancer samples at various disease stages, prognostic prediction and/or diagnosis relating to the degree of disease progression in a biological sample is made possible.

According to the present invention, an intermediate result for examining the condition of a subject can be obtained. Combining such intermediate results with additional information can provide assistance to a physician, caregiver, or other technician in diagnosing the subject as having the disease. Alternatively, the invention may also be used to detect cancerous cells in tissue from which a subject is derived, as well as to provide useful information to a physician in diagnosing that a subject has the disease.

In the present invention, a diagnostic agent for diagnosing breast cancer is also provided. The diagnostic reagent of the present invention comprises a compound that binds to the polynucleotide or polypeptide of the present invention. Preferably, oligonucleotides that hybridize to the polynucleotides of the invention, or antibodies or non-antibody binding proteins that bind to the polypeptides of the invention, are useful as such compounds.

For example, an oligonucleotide comprising 15 consecutive nucleotide bases selected from the nucleotide sequence of the a7322 or F3374V1 gene, or the complement thereof, can be used as a preferred diagnostic reagent of the present invention. Such oligonucleotides are useful as probes for isolating or detecting the a7322 or F3374V1 gene. Alternatively, an antibody or non-antibody binding protein that specifically recognizes the polypeptide encoded by the a7322 or F3374V1 gene is also useful as a diagnostic reagent of the present invention.

Monitoring of breast cancer treatment

The expression level of the a7322 or F3374V1 gene also makes it possible to monitor the course of breast cancer treatment. In the present method, a population of test cells is provided from a subject undergoing treatment for breast cancer. If desired, test cell populations are obtained from the subject at various time points before, during and/or after treatment. Expression of one or more of the a7322 or F3374V1 genes in the test cell population is then determined and compared to a reference cell population comprising cells of known breast cancer status. In the context of the present invention, the reference cell should not receive a relevant treatment.

When the reference cell population does not comprise breast cancer cells, expression of one or more of the a7322 or F3374V1 genes in the test cell population and the reference cell population are similar, indicating that the treatment of the subject under investigation is effective. The expression of these genes is different between the test population and the normal control reference cell population, indicating that the clinical outcome or prognosis is not very good. Similarly, when the reference cell population comprises breast cancer cells, the expression of one or more of the a7322 or F3374V1 genes differs between the test cell population and the reference cell population, indicating that the treatment of the subject under consideration is effective. In contrast, similar gene expression in the test population and the reference cell population indicates that the clinical outcome or prognosis is not very good.

Furthermore, the expression level of the gene of the present invention determined in a subject-derived biological sample obtained after treatment (i.e., post-treatment level) can also be compared with the expression level of one or more of the a7322 or F3374V1 genes determined in a subject-derived biological sample obtained before the start of treatment (i.e., pre-treatment level). A decrease in expression levels in the sample after treatment indicates that the treatment of the subject under investigation is effective; the increase or maintenance of the expression level in the treated sample indicates that the clinical outcome or prognosis is not very good.

The term "effective" as used in this specification means that the treatment causes a decrease in the expression of a pathologically up-regulated gene, an increase in the expression of a pathologically down-regulated gene, or a decrease in the size, prevalence (prevalence), or metastatic potential of a breast cancer (e.g., ductal carcinoma of the breast) in a subject. The term "effective" when used in the context of prophylactic administration indicates that the treatment delays or prevents the formation of breast tumors, or delays, prevents or reduces the symptoms of clinical breast cancer. Evaluation of breast tumors can be performed using standard clinical protocols.

Furthermore, the effectiveness of the treatment is determined in combination with any known method of diagnosing or treating breast cancer. For example, breast cancer can be diagnosed by determining symptomatic abnormalities (e.g., weight loss, abdominal pain, back pain, loss of appetite, nausea, vomiting, and general feelings of fatigue, weakness, and jaundice).

Screening method

(1) Test compounds for screening

In the context of the present invention, the agent identified by the present screening method may be any compound or composition comprising a plurality of compounds. Furthermore, the test agent exposed to a cell or protein according to the screening method of the present invention may be a single compound or a combination of compounds. When a combination of compounds is used in the method, the compounds may be contacted sequentially or simultaneously.

Any agent tested, such as cell extracts, cell culture supernatants, products of fermenting microorganisms, marine extracts, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic small molecule compounds (including nucleic acid constructs, such as antisense RNA, siRNA, ribozymes, etc.), and natural compounds, can be used in the screening methods of the invention. The test agents of the present invention can be obtained using any of a variety of combinatorial library methods known in the art, including:

(1) a biological library of a biological sample,

(2) spatially addressable parallel solid phase or solution phase libraries,

(3) A synthetic library approach requiring deconvolution (deconvolution),

(4) "one bead one compound" library method, and

(5) synthetic library methods using affinity chromatography selection.

The biological library method using affinity chromatography selection is limited to peptide libraries, while the other four methods are applicable to peptide, non-peptide oligomer or compound small molecule libraries (Lam (1997) Anticancer Drug Des.12: 145). Examples of methods for synthesizing libraries of molecules are found in the art (DeWitt et al (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al (1994) J. Med. chem.37: 2678-85; Cho et al (1993) Science 261: 1303-5; Carell et al (1994) Angew. chem.int. Ed. Engl. 33: 2059; Carell et al (1994) Angew. chem.Int. Engl. 33: 2061; Gallop et al (1994) J. Med. chem.37: 1233-51). The compound library may be present in solution (see Houghten (1992) Bio/Techniques 13: 412-21) or on beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. No. 5,571,698; U.S. Pat. No. 5,403,484 and 5,223,409), plasmids (Cull et al (1992) Proc. Natl.Acad.Sci.USA 89: 1865-9) or phages (Scott and Smith (1990) Science 249: 386-90; Delvin (1990) Science: 404-6; Cwirla et al (1990) Proc.Natl.Acad.Sci.USA 87: 6378-82; Felici (1991) J.mol.Biol.222: 301-10; U.S. patent application No. 249: 2002103360).

Further, the agent obtained by the screening method of the present invention also includes a compound converted by adding, deleting and/or substituting a part of the structure in a compound screened by any of the screening methods of the present invention.

Furthermore, when the test agent to be screened is a protein, in order to obtain a DNA encoding the protein, the whole amino acid sequence of the protein may be determined to presume the nucleic acid sequence encoding the protein. Alternatively, a partial amino acid sequence of the resulting protein may be analyzed, an oligo-DNA may be prepared based on the sequence as a probe, and a cDNA library may be screened using the probe to obtain a DNA encoding the protein. The obtained DNA can be used for preparing candidate test agents for treating or preventing cancers.

The agent to be tested useful for the screening described in the present specification may be an antibody or a non-antibody-binding protein that specifically binds to a BC protein or a partial BC peptide lacking the binding activity to a partner or lacking the activity of phosphorylating a substrate in vivo or phosphorylating a kinase. Such partial proteins or antibodies can be prepared as described in the present specification (with reference to the "nucleotides, polypeptides, vectors and host cells" or "antibodies") and can be tested for their ability to block phosphorylation of the BC protein, or the binding activity of the protein (e.g., A7322, F3374V1 or PBK/TOPK) to its binding partner.

(2) General screening methods

To screen for compounds that bind to the BC protein, these antibodies or non-antibody binding proteins are added to cell lysates prepared with suitable detergents in an immunoprecipitation method to form an immune complex. The immune complex consists of a polypeptide, a polypeptide having the ability to bind to the polypeptide, and an antibody or non-antibody binding protein. In addition to using antibodies against the above epitopes, immunoprecipitation can also be performed using antibodies against the polypeptides, which can be prepared as described above (see "antibody" item).

When the antibody is a mouse IgG antibody, the immune complex can be precipitated by, for example, protein a Sepharose or protein G Sepharose. If the polypeptide of the present invention is prepared as a fusion protein with an epitope such as GST, an immune complex can be formed in the same manner as that using an antibody against the polypeptide using a substance specifically binding to these epitopes, for example, glutathione-Sepharose 4B.

Immunoprecipitation can be carried out according to or following, for example, the procedures described in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

Immunoprecipitated proteins are typically analyzed using SDS-PAGE, and bound proteins can be analyzed by their molecular weight using a gel of appropriate concentration. Since it is difficult to detect a protein bound to a polypeptide using a conventional staining method such as Coomassie (Coomassie) staining or silver staining, the detection sensitivity of the protein can be improved by: in the presence of a radioisotope ³⁵S-methionine or³⁵Culture of S-cysteineCulturing the cells in nutrient medium, labeling the protein in the cells, and detecting the protein. When the molecular weight of the protein is known, the target protein can be directly purified from SDS-polyacrylamide gel and the sequence thereof can be determined.

As a method for screening proteins binding to the BC polypeptide using the polypeptide, for example, West-Western blot analysis (Skolnik et al, Cell 65: 83-90(1991)) can be employed. Specifically, a protein capable of binding to the BC polypeptide can be obtained by: preparing a cDNA library from cells, tissues, organs (reference to "nucleotide, polypeptide, vector and host cell" or "antibody" items) or cultured cells that are desired to express a protein that binds to a BC polypeptide using a phage vector (e.g., ZAP); the protein is expressed on LB-agarose (LB-agarose), the expressed protein is immobilized on a filter, the BC polypeptide purified and labeled is reacted with the filter, and plaques expressing the protein bound to the BC polypeptide are detected based on the label. The BC polypeptide can be labeled using a binding between biotin and avidin, or using an antibody that specifically binds to the BC polypeptide or a peptide or polypeptide (e.g., GST) fused to the BC polypeptide. Methods using radioisotopes, fluorescence, or the like may also be employed. In the present specification, the terms "label" and "detectable label" are used to refer to any composition that is capable of being detected using spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels include biotin, magnetic beads (e.g., DYNABEADS) for staining with labeled chains and conjugates of elements ^TM) Fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, Green fluorescent protein, etc.), radioactive labels (e.g., fluorescent dye, etc.)³H、¹²⁵I、³⁵S、¹⁴C or³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and labels for colorimetric quantification such as colloidal gold or beads made of colored glass or plastic (e.g., styrene, polypropylene, latex, etc.), and the like. Patents teaching the use of such labels include U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,275,149; and 4,366,241. Detecting such a targetMethods of note-up are well known to those skilled in the art. Thus, for example, the radioactive label may be detected using a photographic negative or scintillation detector; the fluorescent label may be detected using a light detector for detecting the emitted light. Enzyme labels are typically detected as follows: administering a substrate to an enzyme, and detecting a reaction product produced by the enzyme acting on the substrate; colorimetric quantitative markers can be detected by simple visualization of the chromogenic marker.

Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system (two-hybrid system) using cells ("MATCHMAKER two-hybrid system", "mammalian MATCHMAKER two-hybrid assay kit", "MATCHMAKER one-hybrid system" (Clontech); "HybriZAP two-hybrid vector system" (Stratagene); reference "Dalton and dTresman, Cell 68: 597-.

In a two-hybrid system, the polypeptide of the invention is fused to an SRF-binding region or GAL 4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express a protein that binds to the polypeptide of the present invention, and this library is fused with VP16 or GAL4 transcriptional activation region (transcriptional activation region) upon expression. Then, the cDNA library is introduced into the above-mentioned yeast cells, and cDNA derived from the library is isolated from the clones detected as positive (when a protein capable of binding to the polypeptide of the present invention is expressed in the yeast cells, the binding of the two activates the reporter gene, so that positive clones can be detected). The protein encoded by the cDNA can be prepared by introducing the isolated cDNA into E.coli and expressing the protein.

As the reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene, etc. can be used in addition to the HIS3 gene.

Compounds that bind to the BC polypeptide can also be screened using affinity chromatography. For example, the BC polypeptide can be immobilized on a support of an affinity column and a test compound comprising a protein capable of binding the BC polypeptide can be applied to the column. The test compound in the present specification may be, for example, a cell extract, a cell lysate, or the like. After loading the test compound, the chromatography column is washed and compounds that have bound to the BC polypeptide can be prepared.

When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and a cDNA library is screened using the oligo DNA as a probe, thereby obtaining a DNA encoding the protein.

In the present invention, a biosensor (biosensor) using a surface plasmon resonance phenomenon (surface plasmon resonance) can be used as a means for detecting or quantifying a bound compound. When such biosensors are used, and with minimal amounts of polypeptide and no labeling, the interaction between the BC polypeptide and the test compound, which is manifested as a surface plasmon resonance signal, can be observed in real time (e.g. BIAcore, Pharmacia). Thus, a biosensor such as BIAcore may be used to assess binding between the BC polypeptide and the test compound.

As a method for screening compounds that inhibit the binding between the BC protein (e.g., A7322, F3374V1 or PBK/TOPK) and its binding partner, various methods known to those skilled in the art can be employed. For example, screening can be performed using in vitro assay systems such as cell systems and the like. More specifically, the BC protein or one of its binding partners is first bound to the support, and the other protein is then added thereto along with the test compound. The mixture is then incubated and washed, and the detection or assay of another protein bound to the support is performed.

In the context of the present invention, "inhibition of binding" between 2 proteins means: at least to reduce binding between proteins. Thus, in some cases, the percent of binding pairs in the presence of the test agent in a sample is reduced compared to an appropriate control (e.g., untreated with the test compound, or from a non-cancerous sample, or from a cancerous sample). The reduction in the amount of protein bound can be, e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 25%, less than 10%, less than 5%, less than 1%, or less (e.g., 0%) relative to the binding pair in the control sample.

Examples of supports (supports) that can be used for binding proteins include, for example, insoluble polysaccharides such as agarose, cellulose, and dextran; and synthetic resins such as polyacrylamide, polystyrene, and silicon; commercially available beads and plates (e.g., multiwell plates, biosensor chips, etc.) prepared from the above materials can be preferably used. When beads are used, they can be packed into columns. Alternatively, the use of magnetic beads is also well known in the art, and the method allows proteins bound to the beads to be easily separated by magnetic force.

The binding of the protein to the support can be carried out according to conventional methods, such as chemical binding or physical adsorption. Alternatively, the protein may be bound to the support by an antibody that specifically recognizes it. In addition, avidin and biotin may also be used to bind the polypeptide to the support.

Methods for screening molecules that bind to immobilized polypeptides when exposed to synthetic compounds, natural substance libraries, or random phage peptide display libraries, as well as screening methods for isolating proteins and protein-binding compounds, including agonists and antagonists, based on combinatorial chemistry techniques using high throughput (Wright et al, Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9(1996)) are well known to those skilled in the art.

Furthermore, the phosphorylation level of the polypeptide or functional equivalent thereof can be detected according to any method known in the art. For example, a test compound is contacted with a cell expressing a polypeptide, the cell is incubated for a sufficient time to allow phosphorylation of the polypeptide, and then the amount of phosphorylated polypeptide can be detected. Alternatively, the test substance is contacted with the polypeptide in vitro, the polypeptide is incubated under conditions which allow phosphorylation of the polypeptide, and the amount of phosphorylated polypeptide is then measured (see "(17) kinase assay in vitro and in vivo items).

In the present invention, conditions suitable for phosphorylation may be provided by incubating the substrate and enzyme protein in the presence of a phosphate donor, such as ATP. Conditions suitable for phosphorylation also include conditions in the culture of the cell expressing the polypeptide. For example, the cell is a transformed cell with an expression vector comprising a polynucleotide encoding a BC polypeptide (see "nucleotides, polypeptides, vectors and host cells"). Following incubation, the level of phosphorylation of the substrate can be detected, for example, using an antibody that recognizes the phosphorylated substrate, or by detecting labeled gamma phosphate transferred by the ATP phosphate donor. The substrate may be separated from other components or from the cell lysate of the transformed cell prior to detection of the phosphorylated substrate. For example, gel electrophoresis may be used for separation of substrates. Alternatively, the substrate may be captured by contacting a carrier with an antibody directed against the substrate.

SDS-PAGE or immunoprecipitation can be used to detect phosphorylated proteins. Furthermore, antibodies that recognize phosphorylated residues or transferred labeled phosphate can be used to detect phosphorylated protein levels. Any immunological method using an antibody recognizing the phosphorylated polypeptide may be used for the detection. ELISA or immunoblotting using antibodies recognizing phosphorylated polypeptides may be used in the present invention. When a labeled phosphate donor is used, the phosphorylation level of the substrate can be detected using a tracking label. For example, radiolabeled ATP may be used (e.g., as ³²P-ATP) as a phosphate donor, when the radioactivity of the isolated substrate correlates with the level of phosphorylation of the substrate. Alternatively, antibodies that specifically recognize phosphorylated substrates from unphosphorylated substrates can be used for detection of phosphorylated substrates.

If the amount of phosphorylated BC polypeptide contacted with the test compound is decreased as compared with the amount detected in the absence of the test compound, the test compound is judged to inhibit polypeptide phosphorylation of BC protein, and therefore, it is judged that the test compound has breast cancer inhibitory ability. In the present specification, a "reduction" may be judged if the phosphorylation level is reduced by, for example, 10%, 25% or 50%, or by at least 0.1-fold, at least 0.2-fold, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold or more, compared to the phosphorylation level detected in a cell not contacted with the test agent. Student's t-test, Mann-WhitneyU-test or ANOVA can be used for statistical analysis.

Furthermore, the expression level of the polypeptide or functional equivalent thereof may be detected according to any method known in the art. For example, reporter assays may be employed. Suitable reporter genes and host cells are well known in the art. Reporter constructs necessary for the screening can be prepared by using the transcriptional regulatory region of the BC gene or genes downstream thereof. Reporter constructs can be prepared using existing sequence information when the transcriptional regulatory regions of a gene are well known to those skilled in the art. When the transcriptional regulatory region has not been identified, a nucleotide segment comprising the transcriptional regulatory region can be isolated from a genomic library based on the nucleotide sequence information of the gene. In the present specification, the transcriptional regulatory region of a gene refers to a region from the initiation codon to at least 500bp upstream, preferably 1000bp, more preferably 5000 or 10000bp upstream. The nucleotide segment comprising the transcriptional regulatory region may be isolated from a genomic library or amplified using PCR. Methods and assay protocols for identifying transcriptional regulatory regions are well known (Molecular Cloning third edition receiver 17, 2001, Cold Springs harbor laboratory Press).

Various low-throughput and high-throughput enzymatic assay formats are well known in the art and can be readily adapted for the detection or determination of the phosphorylation level of a BC polypeptide. For high throughput screening, the substrate may suitably be immobilized on a solid support. After the reaction, the phosphorylated substrate on the solid support can be detected by the above-described method. Alternatively, the contacting step may be performed in solution, and the substrate may then be immobilized on a solid support and the phosphorylated substrate detected. To facilitate such an assay, the solid support may be coated with streptavidin and the substrate labeled with biotin, or an antibody against the substrate. One skilled in the art can determine the appropriate assay format based on the desired throughput capability for the screening.

The assays of the invention are also suitable for automated procedures that facilitate high throughput screening. A variety of known robotic systems have been developed for solution phase chemistry. These systems include automated workstations, such as automated synthesis equipment developed by Takeda Chemical Industries, Inc. (Osaka, Japan), and a number of robotic systems using robotic arms (Zymate II, Zymark, Hopkinton, Mass.; Orca, Hewlett Packard, Palo Alto, Calif.), which simulate manual synthesis operations by Chemical technicians. Any of the above devices is suitable for use in the present invention. The nature and implementation of modifications, if any, to these devices to enable them to function as described herein will be apparent to those skilled in the relevant art. In addition, many combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

(3) Screening Using binding Activity against A3722 or F3374 as an index

The present invention also provides methods of using the polypeptides of the invention to screen for compounds useful in the treatment of breast cancer. The present invention also provides a method for screening a compound having a binding ability to the protein of the present invention. One embodiment of such a screening method comprises the steps of:

(a) contacting a test compound with a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a polypeptide having an amino acid sequence as set forth in SEQ ID NO,

(2) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, or a pharmaceutically acceptable salt thereof

(3) And a polypeptide comprising SEQ ID NO: 80 or 82, and a polypeptide having at least about 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide of the amino acid sequence of SEQ ID NO: 80 or 82, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 80 or 82, or a polypeptide comprising an amino acid sequence as set forth in seq id no;

(b) Detecting the binding activity between the test compound and the polypeptide; and

(c) selecting a test compound that binds to the polypeptide.

The polypeptide used for screening may be a recombinant polypeptide or a protein derived from a natural source, or a partial peptide thereof. Any of the foregoing test compounds can be used for screening.

For example, as a method for screening for a protein that binds to a polypeptide using the a3722 polypeptide or the F3374V1 polypeptide (or their functional equivalents, see "nucleotides, polypeptides, vectors and host cells"), various methods known to those skilled in the art can be employed. This screening can be carried out using immunoprecipitation, West-Western blot analysis (Skolnik et al, Cell 65: 83-90(1991)), two-hybrid system using cells ("MATCHMAKER two-hybrid system", "mammalian MATCHMAKER two-hybrid assay kit", "MATCHMAKER one-hybrid system" (Clontech), "HybriZAP two-hybrid vector system" (Stratagene), "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sterngland, Trends Genet 10: 286-92 (1994)"), affinity chromatography and biosensors using the phenomenon of surface plasmon resonance (see item "(2), general screening method".

Any of the aforementioned test compounds can be used (see item (1) "test compound for screening"). (4) Screening Using expression level of A3722 or F3374 as an index

In addition, the screening method of the present invention may comprise the steps of:

(a) contacting a cell into which a vector comprising a transcriptional regulatory region of the a7322 gene or the F3374V1 gene and a reporter gene expressed under the control of the transcriptional regulatory region with a candidate compound;

(b) determining the level of expression or activity of the reporter gene; and

(c) selecting a compound that: the compound reduces the level of expression or activity of the reporter gene as compared to the level of expression or activity of the reporter gene detected in the absence of the test compound.

Suitable reporter genes and host cells are well known in the art. The reporter constructs necessary for the screening can be prepared as described above (see section "(2) general screening methods").

The compound isolated by this screening is a candidate for an agent that inhibits the activity of the a7322 polypeptide or the F3374V1 polypeptide, and is useful for the treatment and prevention of breast cancer. The compounds obtained by the screening method of the present invention also include compounds obtained by adding, deleting and/or substituting a part of the structure of the compound having an activity of binding to the a7322 polypeptide or the F3374V1 polypeptide, which is obtained by the screening method.

In a still further embodiment, the present invention provides a method of screening for a candidate compound as a target in the treatment of breast cancer. As discussed in detail above, by modulating the expression level of the a7322 protein or the F3374V1 protein, the development and progression of breast cancer can be controlled. Therefore, screening using the expression level and activity of a7322 or F3374V1 as indicators enables identification of candidate compounds as therapeutic targets for breast cancer. In the context of the present invention, such screening may comprise, for example, the following steps:

(a) contacting a candidate compound with a cell expressing an a7322 protein or an F3374V1 protein; and

(b) selecting a compound that: the compound reduces the expression level of a7322 or F3374V1 compared to the expression level detected in the absence of the test compound.

Cells expressing at least one of the a7322 protein or the F3374V1 protein include, for example, cell lines established from breast cancer, and such cells can be used in the above screening of the present invention. Expression levels can be assessed using methods well known to those skilled in the art. In the screening method, a compound that reduces the expression level of at least one of a7322 or F3374V1 may be selected as a candidate compound.

In another embodiment of the method of screening for a compound useful in the treatment of breast cancer of the present invention, the method uses the biological activity of the polypeptide of the present invention as an index. The a7322 protein or the F3374V1 protein has an activity of promoting cell proliferation, and therefore a compound that inhibits the activity of one of these proteins of the present invention can be screened using this activity as an index.

Any polypeptides can be used for screening as long as they contain the biological activity of the a7322 protein or the F3374V1 protein (e.g., binding to PHB2/REA or AURKB, respectively, promotes cell proliferation). Such biological activities include cell proliferative activity of the human a7322 protein or the F3374V1 protein. For example, human A7322 protein or F3374V1 protein may be used, and polypeptides functionally equivalent to these proteins may also be used. Such polypeptides may be expressed endogenously or exogenously by the cell.

(5) Screening Using A7322 and PHB2/REA binding as an index

In the present invention, it was confirmed that the A7322 protein interacted with the PHB2/REA protein to inhibit nuclear transfer of the PHB2/REA protein (FIG. 10A). Furthermore, inhibition of reactivation of era in the presence of a7322 protein was also confirmed (fig. 11A and B). PHB2/REA is a known selective co-regulator of estrogen receptor alpha (ER α) that inhibits the transcriptional activity of Er α with estradiol as a ligand. Thus, the present inventors revealed that a7322 activates the transcriptional activity of ER α by inhibiting the interaction between ER α and PHB2/REA (fig. 11C). Therefore, compounds that inhibit the binding between a7322 protein and PHB2/REA can be screened by using the binding between such a7322 protein and PHB2/REA, the cellular localization of PHB2/REA protein, or the transcriptional activity of era as an index. Accordingly, the present invention also provides a method for screening a compound for inhibiting the binding between a7322 protein and PHB2/REA, which method enables screening using as an index the binding between such a7322 protein and PHB2/REA, the cellular localization of PHB2/REA protein, or the transcriptional activity of era. Furthermore, the present invention also provides a method for screening a compound for treating or preventing breast cancer. The method is particularly suitable for screening for agents that may be used in the treatment or prevention of breast cancer. More specifically, the method comprises the steps of:

(a) Contacting an a7322 polypeptide or a functional equivalent thereof with a PHB2/REA polypeptide or a functional equivalent thereof in the presence of a test compound;

(b) detecting binding between the polypeptides of step (a); and

(c) selecting a test compound that inhibits binding between the a7322 polypeptide and the PHB2/REA polypeptide.

In the context of the present invention, a A7322 polypeptide or a functional equivalent of a PHB2/REA polypeptide refers to a polypeptide having equivalent biological activity to the A7322 polypeptide (SEQ ID NO: 79) or the PHB2/REA polypeptide (SEQ ID NO: 90), respectively (see "nucleotides, polypeptides, vectors and host cells").

As a method for screening a compound that inhibits the binding between a7322 and PHB2/REA, various methods known to those skilled in the art can be used.

The polypeptide used for screening may be a recombinant polypeptide or a protein derived from a natural source, or a partial peptide thereof. Any of the foregoing test compounds can be used for screening.

As a method for screening, for example, a protein binding to a polypeptide using the A3722 polypeptide or PHB2/REA polypeptide (or functional equivalents thereof, see "nucleotides, polypeptides, vectors and host cells"), various methods known to those skilled in the art can be employed. Such screening can be carried out using, for example, immunoprecipitation, West-Western blot analysis (Skolnik et al, Cell 65: 83-90(1991)), two-hybrid system using cells ("MATCHMAKER two-hybrid system", "mammalian MATCHMAKER two-hybrid assay kit", "MATCHMAKER one-hybrid system" (Clontech), "HybriZAP two-hybrid vector system" (Stratagene), "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)"), affinity chromatography and biosensors utilizing the surface plasmon resonance phenomenon (see item "(2) general screening method").

Any of the aforementioned test compounds can be used (see item (1) "test compound for screening").

Further, the present invention provides a method for producing a compound that inhibits the interaction between A7322 and PHB2/REA, using the cellular localization of PHB2/REA as an indicator. More specifically, the method comprises the steps of:

(a) contacting a candidate compound with a cell expressing an a7322 protein and a PHB2/REA protein;

(b) detecting the subcellular localization of PHB2/REA protein; and

(c) selecting a compound that: the compound reduces the level of PHB2/REA protein in the nucleus compared to the level of the protein detected in the absence of the test compound.

In certain embodiments, the method further comprises the step of detecting binding of the candidate compound to a7322 or PHB2/REA, or the step of detecting the level of binding between a7322 and PHB 2/REA. Cells expressing the a7322 protein and PHB2/REA protein include, for example, cell lines established from breast cancer, and such cells can be used for the above screening of the present invention as long as they express these 2 genes. Alternatively, for cells, expression vectors for A7322 and/or PHB2/REA can be transfected to express these 2 genes. Subcellular localization of PHB2/REA protein can be detected by immunocytochemical staining with anti-PHB 2/REA antibodies (see item "(8) immunocytochemical staining), fractionation in combination with Western blotting or in combination with PHB2/REA protein using isotopes or fluorescent labels (see item" nucleotides, polypeptides, vectors and host cells ").

In another embodiment, the present invention provides a method of inhibiting the interaction between a7322 and PHB2/REA using the transcriptional activity of ER α as an indicator. More specifically, the method comprises the steps of:

(a) contacting the candidate compound with a cell expressing the a7322 protein, the PHB2/REA protein and the era protein under treatment with E2 and introduced with a vector comprising an estrogen-responsive transcriptional regulatory region and a reporter gene expressed under the control of the transcriptional regulatory region;

(b) determining the level of expression or activity of the reporter gene; and

(c) selecting a compound that: the compound reduces the level of expression or activity of the reporter gene as compared to the level of expression or activity of the reporter gene detected in the absence of the test compound.

Cells expressing the A7322 protein, PHB2/REA protein and ER α protein include, for example, cell lines established from breast cancer, and such cells can be used for the above screening of the present invention as long as they express these 3 genes. Alternatively, for cells, each or any of the expression vectors for A7322, PHB2/REA, and ER α (each or other) can be transfected to allow expression of these 3 genes. Suitable reporter genes and host cells are well known in the art. The reporter constructs required for screening can be prepared according to the methods described above or below (see items "(2) general screening methods" and "(19) Estrogen Response Element (ERE) reporter assay").

(6) Screening Using phosphorylation level of F3374V1 as an index

In the present invention, it was confirmed that the F3374V1 protein was modified by phosphorylation at the C-terminal region (amino acids 591-730). Therefore, compounds that inhibit phosphorylation of the F3374V1 protein can be screened using such modifications as an index. Accordingly, the present invention also provides a screening method for a compound for inhibiting phosphorylation of F3374V1 protein. Furthermore, the present invention provides a method of screening for a compound for treating or preventing breast cancer. The method is particularly suitable for screening for agents that may be used in the treatment or prevention of breast cancer. More specifically, the method comprises the steps of:

(a) contacting a test compound with a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 82, or a pharmaceutically acceptable salt thereof,

(3) and a polypeptide comprising SEQ ID NO: 82, and a polypeptide having at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide of amino acid sequence of seq id NO: 82, and

(4) A polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the phosphorylation level of the polypeptide;

(c) comparing the phosphorylation level of the polypeptide to the phosphorylation level of the polypeptide detected in the absence of the test compound;

(d) selecting a compound that reduces the phosphorylation level of the polypeptide as an inhibitor of the phosphorylation level of the polypeptide or a compound for treating or preventing breast cancer.

In the present specification, any cell may be used as long as it expresses the F3374V1 polypeptide or a functional equivalent thereof (see items "nucleotides, polypeptides, vectors and host cells"). The cells used in the present screen may be cells which naturally express the F3374V1 polypeptide, including, for example, cells derived from breast cancer and testis, and cell lines established therefrom. Cell lines of breast cancer such as HBC4, HBC5, HBL100, HCC1937, MCF-7, MDA-MB-231, MDA-MB-435S, SKBR3, T47D, and YMB1 can be used.

Alternatively, the cell used in the screening may be one which does not naturally express the F3374V1 polypeptide, but which is transfected with a vector expressing the F3374V1 polypeptide or a functional equivalent of F3374V 1. Such recombinant cells can be obtained according to well-known genetic engineering Methods such as those described above (see, for example, the items "nucleotides, polypeptides, vectors and host cells"; for example, Morrison DA., JBacteriolology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in enzymology (Wu et al eds.) 1983, 101: 347-62).

Any of the test compounds described above may be used in the screening of the present invention. However, it is preferred to select compounds that are permeable to the inside of the cell. Alternatively, where the test compound is a polypeptide, the cells in the screen can be contacted with the test agent by transfecting a vector comprising a nucleotide sequence encoding the test agent into the cells, expressing the test agent in the cells.

In another embodiment, conditions suitable for phosphorylation of the F3374V1 polypeptide or a functional equivalent of F3374V1 may be provided in vitro. The screening method comprises the following steps:

(a) contacting a test compound with a polypeptide of the present invention or a fragment thereof (e.g., a C-terminal region (amino acids 591-730));

(b) detecting phosphorylation of the polypeptide of step (a);

(c) selecting a compound that: the compound inhibits phosphorylation of the polypeptide as compared to the biological activity detected in the absence of the test compound.

In the present invention, as described above, the biological activity of the F3374V1 protein is preferably phosphorylation activity. The level of phosphorylation can be assessed by one skilled in the art as described above (see item "(2) general screening methods").

Thus, in these embodiments, the present invention provides a method of screening for an agent for inhibiting phosphorylation of F3374V1 or for preventing or treating breast cancer, the method comprising the steps of:

(a) Contacting a test compound with a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 82, or a pharmaceutically acceptable salt thereof,

(3) and a polypeptide comprising SEQ ID NO: 82, wherein the polypeptide has at least about 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide of the amino acid sequence of seq id NO: 82, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81 or a fragment thereof comprising a phosphorylation site, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the level of phosphorylation of the polypeptide or fragment thereof;

(c) comparing the level of phosphorylation of the substrate to the level of phosphorylation of the polypeptide detected in the absence of the test compound;

(d) Selecting a compound that reduces the phosphorylation level of the polypeptide as a compound for inhibiting phosphorylation of the polypeptide or preventing or treating breast cancer.

In these embodiments, the conditions that allow phosphorylation of the F3374V1 polypeptide can be provided by incubating the polypeptide with a kinase appropriate for phosphorylation of the F3374V1 polypeptide and ATP. In some embodiments, the F3374V1 polypeptide is also contacted with an AURKB polypeptide. Furthermore, in a preferred embodiment, a substance that enhances phosphorylation of the F3374V1 polypeptide may be added to the screened reaction mixture. If the phosphorylation of the polypeptide is enhanced by the addition of the substance, the phosphorylation level can be determined with higher sensitivity.

The phosphorylation level of the F3374V1 polypeptide or functional equivalent thereof can be detected by any method known in the art (see section "(2) general screening methods").

Furthermore, the present inventors revealed that F3374V1 interacts with AURKB in breast cancer cells (FIG. 13). Thus, the interaction of two polypeptides can be considered to play a crucial role in carcinogenesis or cell proliferation, particularly breast cancer cell proliferation. Based on this, it is desired to screen compounds that inhibit the interaction between the F3374V1 polypeptide and the AURKB polypeptide or the reverse interaction (vice versa interaction) thereof and are useful in the treatment or prevention of breast cancer. Accordingly, the present invention provides methods for screening compounds that inhibit the interaction of a F3374V1 polypeptide with an AURKB polypeptide. Furthermore, the present invention provides a method of screening for a compound for treating or preventing breast cancer. The method comprises the following steps:

(a) Contacting an AURKB polypeptide or functional equivalent thereof with an F3374V1 polypeptide or functional equivalent thereof in the presence of a test compound;

(b) detecting binding between the polypeptides of step (a);

(c) selecting a test compound that inhibits binding between the AURKB polypeptide and the F3374V1 polypeptide.

In the context of the present invention, a functional equivalent of a F3374V1 polypeptide or an AURKB polypeptide refers to a polypeptide having biological activity equivalent to a F3374V1 polypeptide (SEQ ID NO: 82) or an AURKB polypeptide (SEQ ID NO: 88), respectively (see items "nucleotides, polypeptides, vectors and host cells").

As a method for screening a compound that inhibits AURKB-induced phosphorylation of F3374V1, various methods known to those skilled in the art can be used. For example, the screening may be performed in the form of an in vitro assay system such as a cell system.

The invention is also based on the recognition that: AURKB has kinase activity against F3374V 1. For example, the site of AURKB-induced phosphorylation of F3374V1 is located in the C-terminal portion (amino acids 591-730) of the F3374 protein (SEQ ID NO: 122). These insights suggest: phosphorylation of F3374V1 by AURKB plays an important role in tumor cell proliferation, and inhibition of phosphorylation of F3374V1 by AURKB can be a promising target for anticancer drug development. For this purpose, one aspect of the invention relates to the identification of test compounds that modulate AURKB-mediated phosphorylation of F3374V 1. Accordingly, the present invention provides a method of screening for compounds useful for inhibiting AURKB-mediated phosphorylation of F3374V 1. Furthermore, the present invention provides a method of screening a compound for treating or preventing breast cancer. The method comprises the following steps:

(a) Incubating F3374V1 and AURKB together in the presence of a test compound under conditions suitable for phosphorylation of F3374V1 by AURKB, wherein the F3374V1 is a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82 (F3374V 1);

(2) SEQ ID NO: 82, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 82, or a pharmaceutically acceptable salt thereof;

(3) and a polypeptide comprising SEQ ID NO: 82, wherein the polypeptide has at least about 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide of the amino acid sequence of seq id NO: 82, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the phosphorylation level of F3374V 1;

(c) comparing the phosphorylation level of F3374V1 to a control level; and

(d) Selecting a compound that: the compound reduces the phosphorylation level of F3374V1 compared to a control level detected in the absence of the test compound.

Here, the method for screening a compound inhibiting AURKB-mediated phosphorylation of F3374V1 or a compound for treating or preventing breast cancer includes detection of the phosphorylation level of F3374V1 at the C-terminal F3374 protein (amino acids 591-730) (SEQ ID NO: 122) or at a homologous position of the polypeptide.

In another aspect of the invention, kits are also provided for screening compounds that inhibit AURKB-mediated phosphorylation of F3374V1 or for use in the treatment or prevention of breast cancer. The kit comprises the following components:

(a) a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82 (F3374V1),

(2) SEQ ID NO: 82, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 82, or a protein equivalent biological activity,

(3) and a polypeptide comprising SEQ ID NO: 82, wherein the polypeptide has at least about 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide of the amino acid sequence of seq id NO: 82, and

(4) A polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 88 (AURKB),

(2) SEQ ID NO: 88, with the proviso that said polypeptide has an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 88, or a protein equivalent biological activity,

(3) and a polypeptide comprising SEQ ID NO: 88, wherein the polypeptide has at least about 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to a polypeptide of the amino acid sequence of seq id NO: 88, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 87, provided that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 88, or a polypeptide consisting of the amino acid sequence shown in seq id no; and

(c) A reagent for detecting the phosphorylation level of F3374V 1.

Furthermore, the present invention also provides a kit for screening a compound for treating or preventing breast cancer. The kit comprises the following components:

(a) a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82(F3374V1),

(2) SEQ ID NO: 82, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 82, or a protein equivalent biological activity,

(3) and a polypeptide comprising SEQ ID NO: 82, wherein the polypeptide has at least about 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polypeptide of the amino acid sequence of seq id NO: 82, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no; and

(b) a reagent for detecting the phosphorylation level of F3374V 1.

Furthermore, the kit for screening a compound that inhibits AURKB-mediated phosphorylation of F3374V1 or a compound for treating or preventing breast cancer comprises a cell that further expresses a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 88(AURKB),

(b) SEQ ID NO: 88, with the proviso that said polypeptide has an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 88, or a protein equivalent biological activity,

(c) and a polypeptide comprising SEQ ID NO: 88, wherein the polypeptide has at least about 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to a polypeptide of the amino acid sequence of seq id NO: 88, and

(d) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 88, or a polypeptide consisting of the amino acid sequence shown in seq id no.

In another aspect, the cells used in the kit are breast cancer cells.

In the present invention, a phosphate donor may be further contained in the kit. The kit can also comprise an antibody for recognizing phosphorylated C-terminal F3374 protein (amino acids 591-730) (SEQ ID NO: 122) as a reagent for detecting phosphorylated F3374V 1. Accordingly, the present invention also provides a kit for screening a compound for treating or preventing breast cancer, wherein the reagent for detecting the phosphorylation level of F3374V1 is an antibody recognizing phosphorylation occurring at amino acids 591-730 (SEQ ID NO: 122) of the C-terminal F3374 protein. According to the present invention, it can be determined whether or not a protein of interest is a target of phosphorylation. For example, kinase activity against F3374V1 can be determined by: the level of phosphorylated F3374V1 was detected by incubating the polypeptide under conditions suitable for phosphorylation of F3374V 1. For example, the site phosphorylated by AURKB on F3374V1 is the C-terminal F3374 protein (amino acids 591-730) (SEQ ID NO: 122).

In the present invention, conditions suitable for phosphorylation of F3374V1 by AURKB can be provided by incubating F3374V1 and AURKB in the presence of a phosphate donor, such as ATP. The conditions suitable for phosphorylation of F3374V1 by AURKB also include conditions in the culture of cells expressing the polypeptide (see section "(2) general screening methods").

Methods for preparing polypeptides functionally equivalent to a given protein are well known to those skilled in the art, and include well-known methods for introducing mutations into proteins such as those described above (see "nucleotides, polypeptides, vectors and host cells").

In some preferred embodiments, functional equivalents of F3374V1 may include amino acid sequences corresponding to the AURKB binding domain, such as SEQ ID NO: 122. Similarly, the amino acid sequence corresponding to the F3374V1 binding domain may be included in a functional equivalent of AURKB.

As described above, inhibition of binding between F3374V1 and AURKB resulted in inhibition of cell proliferation. Furthermore, inhibition of phosphorylation of F3374V1 by AURKB also results in inhibition of cell proliferation. Therefore, compounds that inhibit this binding or phosphorylation may serve as drugs for the treatment or prevention of breast cancer. The F3374V1 polypeptide and AURKB polypeptide used in the screening methods of the present invention may be recombinant polypeptides, may be naturally derived proteins, or may be partial peptides thereof, as long as they retain the binding activity or phosphorylation activity of the full-length protein. Such partial peptides retaining binding ability or phosphorylation activity are referred to as "functional equivalents" in the present specification. The F3374V1 polypeptide and AURKB polypeptide to be used in the screening method may be, for example, purified polypeptides, soluble proteins, forms bound to a carrier, or fusion proteins with other polypeptides.

As a method for screening a compound that inhibits the binding between F3374V1 and AURKB, various methods known to those skilled in the art can be used. The binding between proteins is preferably performed in a buffer, examples of which include, but are not limited to, phosphate buffer and Tris buffer. However, the buffer chosen must not inhibit binding between proteins. Any of the aforementioned detection methods can be used for the present screening (see item "(2) general screening method"). Furthermore, any of the aforementioned test compounds can be used in the present screening (see item "(1) test compound for screening").

The compound isolated according to the screening method of the present invention is a candidate for a drug inhibiting the activity of F3374V1 or AURKB for use in the treatment or prevention of a disease attributed to, for example, a cell proliferative disease, such as breast cancer. The compounds obtained by converting a partial structure of the compounds obtained by the screening method according to the present invention by addition, deletion and/or substitution are also included in the compounds obtained by the screening method according to the present invention. Compounds effective in inhibiting the expression of the overexpressed genes, i.e., the F3374V1 gene and the AURKB gene, may be considered clinically valuable and may be further tested for their ability to reduce or prevent cancer cell proliferation in animal models or subjects.

The invention may also include the screening of proteins that bind to the F3374V1 polypeptide or the AURKB polypeptide, thereby inhibiting their interaction. For this purpose, various methods known to those skilled in the art can be employed. Such screening can be performed, for example, by immunoprecipitation assay using a method known in the art. The proteins of the invention can be produced recombinantly using standard procedures as described above (see item "(2) general screening methods"). Compounds that bind to the F3374V1 polypeptide or the AURKB polypeptide may also be screened using affinity chromatography as described above (see item "(1) test Compound for screening").

(7) Screening Using phosphorylation level of PBK/TOPK as an index

The present invention provides methods of screening for agents that induce apoptosis or cell cycle arrest in breast cancer cells. Agents that induce apoptosis or cell cycle arrest in cells expressing TOPK, such as breast cancer cells, are believed to be useful for the treatment or prevention of breast cancer. Accordingly, the present invention also provides methods for screening for agents that treat or prevent breast cancer. The method is particularly useful for screening agents that can be used for the treatment or prevention of invasive ductal carcinoma of the breast (IDC).

More specifically, the method comprises the steps of:

(a) contacting a cell expressing a PBK/TOPK polypeptide or functional equivalent thereof with an agent;

(b) detecting the phosphorylation level of the PBK/TOPK polypeptide;

(c) comparing the phosphorylation level of the polypeptide to a phosphorylation level of the polypeptide detected in the absence of the agent; and

(d) selecting an agent that reduces the level of phosphorylation of the polypeptide as an agent that induces apoptosis or cell cycle arrest in cells expressing TOPK, such as breast cancer cells, or as an agent for treating or preventing breast cancer.

In another embodiment, the method comprises the steps of:

(a) contacting a cell expressing a PP1 alpha polypeptide and a PBK/TOPK polypeptide or functional equivalents thereof with an agent;

(b) detecting the phosphorylation level of the PBK/TOPK polypeptide;

(c) comparing the phosphorylation level of the polypeptide to a phosphorylation level of the polypeptide detected in the absence of the agent; and

(d) selecting an agent that reduces the level of phosphorylation of the polypeptide as an agent that induces apoptosis or cell cycle arrest in breast cancer cells, or as an agent for treating or preventing cells expressing TOPK, such as breast cancer.

Here, any cell may be used as long as it expresses the PBK/TOPK polypeptide or a functional equivalent thereof. The cells used in the present screen may be cells that naturally express the PBK/TOPK polypeptide, including, for example, cells from breast cancer (e.g., IDC), thymus, and testis, as well as cell lines established therefrom. Breast cancer cell lines HBC4, HBC5, HBL100, HCC1937, MCF-7, MDA-MB-231, MDA-MB-435S, SKBR3, T47D, and YMB1 can be used.

Alternatively, the cells used in the screening may be cells which do not naturally express the PBK/TOPK polypeptide and PP1 α, but which are transfected with a vector expressing the PBK/TOPK polypeptide or a functional equivalent of PBK/TOPK, or a vector expressing PP1 α. Such recombinant cells can be obtained according to well-known genetic engineering Methods such as those described above (e.g., Morrison DA., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al) 1983, 101: 347-62) (see "nucleotides, polypeptides, vectors and host cells").

Any of the aforementioned test agents can be used in the present screen (see item "(1) test compound for screening"). However, it is preferable to select an agent that is permeable into cells. Alternatively, where the agent is a polypeptide, the cell in the screen may be contacted with the agent by transfecting a vector comprising a nucleotide sequence encoding the agent into the cell, expressing the agent in the cell.

In the present invention, a substance that enhances phosphorylation of the PBK/TOPK polypeptide may be added to the reaction mixture screened. If the phosphorylation of the polypeptide is enhanced by the addition of the substance, the phosphorylation level can be determined with higher sensitivity.

The level of phosphorylation of the PBK/TOPK polypeptide or functional equivalent thereof can be detected by any method known in the art (see section "(2) general screening methods").

Alternatively, the level of phosphorylation of the PBK/TOPK polypeptide or functional equivalent thereof can also be detected by measuring the cell cycle of the cell. Specifically, the cell cycle of the cells can be determined by conventional methods known in the art including FACS and the like. When the cell cycle of the cell is to be examined in order to determine the phosphorylation level of the polypeptide, it is preferred that the cell is incubated for a sufficient time, e.g., 12 hours or more, after the cell is contacted with the test agent until normal cells have passed the G2/M phase. According to this protocol, when cell cycle arrest at the G2/M phase is detected, the test substance can be determined to have the ability to induce apoptosis of breast cancer cells.

In another embodiment, the method comprises the steps of:

(a) contacting CDK1, cyclin B1 and PBK/TOPK polypeptide or functional equivalents thereof with a substrate capable of being phosphorylated by the polypeptide and an agent under conditions permitting phosphorylation of the substrate;

(b) Detecting the phosphorylation level of the PBK/TOPK polypeptide;

(c) comparing the phosphorylation level of the PBK/TOPK polypeptide to the phosphorylation level detected in the absence of the agent; and

(d) selecting an agent that reduces the level of phosphorylation of the PBK/TOPK polypeptide as an agent for inducing apoptosis of breast cancer cells, or as an agent for inhibiting phosphorylation of the PBK/TOPK polypeptide or for treating or preventing breast cancer.

Here, CDK1, cyclin B1 and PBK/TOPK polypeptides or functional equivalents thereof used in the screening may be prepared as recombinant proteins or as native proteins using methods well known to those skilled in the art. Polypeptides can be obtained according to any of the well-known genetic engineering Methods for producing polypeptides such as those described above (e.g., Morrison, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al) 1983, 101: 347-62) (see "nucleotides, polypeptides, vectors and host cells").

Furthermore, a protein complex of CDK1 and cyclin B1 may also be used in the present invention, as long as it retains kinase activity against the PBK/TOPK protein. Such partial peptides can be produced by known methods of genetic engineering, peptide synthesis or by digestion of the native CDK1 and cyclin B1 proteins with appropriate peptidases (see "nucleotides, polypeptides, vectors and host cells").

The PBK/TOPK polypeptide or functional equivalent thereof contacted with the protein complex of CDK1 and cyclin B1 may be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused to other polypeptides.

In these embodiments, conditions which allow for kinase activity with CDK1 and cyclin B1 polypeptides may be achieved by incubating CDK1 and cyclin B1 polypeptides with PBK/TOPK polypeptides for phosphorylating the PBK/TOPK polypeptides and ATP. Furthermore, in the present invention, a substance that enhances phosphorylation of the PBK/TOPK polypeptide may be added to the reaction mixture screened. If phosphorylation of the PBK/TOPK polypeptide is enhanced by the addition of the substance, the level of phosphorylation of the PBK/TOPK polypeptide can be determined with greater sensitivity.

The contacting of CDK1, cyclin B1, and PBK/TOPK polypeptide or functional equivalents thereof with the agent may be performed in vivo or in vitro. In vitro screening can be performed in buffers, examples of which include, but are not limited to, phosphate buffer and Tris buffer, so long as the buffers do not inhibit phosphorylation of the PBK/TOPK polypeptide or a functional equivalent thereof.

According to an aspect of the present invention, the components necessary for the screening method may be provided as a kit for screening an agent for inducing apoptosis or cell cycle arrest of breast cancer cells or an agent for treating or preventing breast cancer. The kit of the invention may comprise, for example, cells expressing a PBK/TOPK polypeptide or a functional equivalent thereof, or a PBK/TOPK polypeptide and/or PP1 alpha or a functional equivalent thereof, or polypeptides of PBK/TOPK, CDK1 and cyclin B1 or functional equivalents thereof. Furthermore, the kit may also contain control reagents (positive and/or negative), detectable labels, cell culture media or buffers, containers necessary for screening, instructions for carrying out the method (e.g., written documents, magnetic tape, VCR, CD-ROM, others), and the like. The components and reagents may be packaged separately in different containers.

(8) Screening Using kinase Activity of PBK/TOPK on substrate as an index

According to another aspect of the invention, the phosphorylation level of a PBK/TOPK substrate can be used as an indicator to screen for agents that induce apoptosis of breast cancer cells or can be used to treat or prevent breast cancer (e.g., IDC). Specifically, the method comprises the following steps:

(a) contacting the PBK/TOPK polypeptide or functional equivalent thereof with a substrate phosphorylated by the polypeptide and an agent under conditions permitting phosphorylation of the substrate;

(b) detecting the phosphorylation level of the substrate;

(c) comparing the phosphorylation level of the substrate to a phosphorylation level of the substrate detected in the absence of the agent; and

(d) selecting an agent that reduces the phosphorylation level of a substrate as an agent that inhibits kinase activity of the PBK/TOPK polypeptide, induces apoptosis of breast cancer cells, or as an agent for treating or preventing breast cancer.

In some embodiments, the substrate is a histone H3 polypeptide. The PBK/TOPK polypeptide or functional equivalent thereof used in the screening may be prepared in the form of a recombinant protein or a native protein using methods well known to those skilled in the art. Polypeptides can be obtained according to any of the well-known genetic engineering Methods for producing polypeptides such as those described above (e.g., Morrison, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al) 1983, 101: 347-62) (see "nucleotides, polypeptides, vectors and host cells").

Furthermore, partial peptides of the PBK/TOPK protein may also be used in the present invention, as long as they retain the kinase activity of the protein. Such partial peptides can be produced by known methods of genetic engineering, peptide synthesis or digestion of the native PBK/TOPK protein with appropriate peptidases (see "nucleotides, polypeptides, vectors and host cells").

The PBK/TOPK polypeptide or functional equivalent thereof contacted with the test substance and substrate may be, for example, a purified polypeptide, a soluble protein, or a fusion protein fused with other polypeptides.

The substrate is any compound capable of accepting a phosphate group, such as a protein, nucleic acid (RNA or DNA), or small molecule. For example, the substrate may be a histone or a histone fragment containing a phosphorylation site. It was confirmed that Ser10 of histone H3 can be phosphorylated by the PBK/TOPK protein. Thus, histone H3, or a fragment of histone H3 comprising Ser10, is useful as a substrate.

Similarly to the PBK/TOPK polypeptide, histone H3 used in this screen can be prepared as a recombinant protein or a native protein. Furthermore, like the PBK/TOPK polypeptide, histone H3 can also be prepared in the form of a fusion protein, as long as the resulting fusion protein can be phosphorylated by the PBK/TOPK polypeptide. The nucleotide sequence of histone H3 is well known in the art. Furthermore, histone H3 is also commercially available (e.g., product of Roche).

In these embodiments, the conditions that allow phosphorylation of the histone H3 polypeptide can be achieved by incubating the histone H3 polypeptide with a PBK/TOPK polypeptide for phosphorylating the histone H3 polypeptide and ATP. Furthermore, in the present invention, a substance that enhances the kinase activity of the PBK/TOPK polypeptide may be added to the reaction mixture screened. If the phosphorylation of the substrate is enhanced by the addition of the substance, the phosphorylation level of the substrate can be determined with higher sensitivity.

The contacting of the PBK/TOPK polypeptide or functional equivalent thereof, its substrate and the agent to be tested may be performed in vivo or in vitro. In vitro screening may be performed in buffers, examples of which include, but are not limited to, phosphate buffer and Tris buffer, so long as the buffer does not inhibit phosphorylation of the substrate by the PBK/TOPK polypeptide or a functional equivalent thereof.

In the present invention, the phosphorylation level of a substrate can be determined by a method known in the art (see item "(2) general screening method").

(9) Screening Using the binding between PBK/TOPK and p47 or phosphorylation of p97 as an indicator

In the present invention, it was confirmed that: the PBK/TOPK protein interacts with the p97 protein via the p47 protein as an adaptor (adapter), thereby inhibiting cell division. Therefore, using such binding between PBK/TOPK protein and p47 or phosphorylation level of p97 as an index, compounds that inhibit binding between PBK/TOPK protein and p47 or phosphorylation of p97 can be screened. Therefore, the present invention also provides a method of screening for compounds that inhibit the binding between PBK/TOPK and p47, or reduce the phosphorylation of p 97. Furthermore, the present invention provides a method of screening a compound for treating or preventing breast cancer. The method is particularly suitable for screening for agents that may be used to treat or prevent breast cancer. More specifically, the method comprises the steps of:

(a) Contacting a PBK/TOPK polypeptide or functional equivalent thereof with a p47 polypeptide or functional equivalent thereof and a p97 polypeptide or functional equivalent thereof in the presence of a test compound;

(b) detecting binding between the PBK/TOPK polypeptide and the p47 polypeptide or phosphorylation of p 97; and

(c) test compounds are selected that inhibit binding between the PBK/TOPK polypeptide and the p47 polypeptide, or that reduce phosphorylation of p 97.

In the context of the present invention, functional equivalents of PBK/TOPK or p47 or p97 polypeptides refer to polypeptides having equivalent biological activity to PBK/TOPK (SEQ ID NO: 92) or p47(SEQ ID NO: 118) or p97(SEQ ID NO: 120), respectively (see "nucleotides, polypeptides, vectors and host cells").

As a method for screening for a compound that inhibits the binding of a PBK/TOPK polypeptide to a p47 polypeptide, various methods known to those skilled in the art can be employed.

The polypeptide used for screening may be a recombinant polypeptide or a naturally derived protein, or a partial peptide thereof. Any of the foregoing test compounds can be used for screening.

As a method of screening for proteins, e.g.binding to a polypeptide, using a PBK/TOPK polypeptide and a p47 polypeptide (or their functional equivalents, see "nucleotides, polypeptides, vectors and host cells"), various methods known to the person skilled in the art may be used. Such screening can be carried out using, for example, immunoprecipitation, West-Western blot analysis (Skolnik et al, Cell 65: 83-90(1991)), two-hybrid system using cells ("MATCHMAKER two-hybrid system", "mammalian MATCHMAKER two-hybrid assay kit", "MATCHMAKER one-hybrid system" (Clontech), "HybriZAP two-hybrid vector system" (Stratagene), "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)"), affinity chromatography, and biosensors utilizing the surface plasmon resonance phenomenon (see item "(2) general screening method").

Any of the aforementioned test compounds can be used (see item (1) "test compound for screening").

In the present invention, the screening method can detect phosphorylation of p97 peptide. Thus, conditions that allow phosphorylation of the p97 polypeptide can be achieved by incubating the p97 polypeptide with the PBK/TOPK polypeptide and the p47 polypeptide for phosphorylating the p97 polypeptide, and ATP. Furthermore, in the present invention, a substance that enhances the kinase activity of the PBK/TOPK polypeptide or the phosphorylation of the p97 polypeptide may be added to the reaction mixture screened. If phosphorylation of p97 is enhanced by the addition of this substance, the phosphorylation level of p97 can be determined with greater sensitivity.

The contacting of the PBK/TOPK polypeptide or functional equivalent thereof, p97 or functional equivalent thereof and the agent to be tested may be performed in vivo or in vitro. In vitro screening may be performed in buffers, examples of which include, but are not limited to, phosphate buffer and Tris buffer, so long as the buffer does not inhibit phosphorylation of the substrate by the PBK/TOPK polypeptide or a functional equivalent thereof.

In the present invention, the phosphorylation level of a substrate can be determined by a method known in the art (see item "(2) general screening method").

(10) Screening Using cell cycle composition (Stracture) of PBK/TOPK-expressing cells and G2/M population as indices

The present invention provides a method for screening for agents that induce cell cycle arrest in breast cancer cells. We believe that agents that induce cell cycle arrest in breast cancer cells are useful for the treatment or prevention of breast cancer. Accordingly, the present invention also provides a method of screening for an agent for treating or preventing breast cancer. The method is particularly useful for screening agents useful for the treatment or prevention of invasive ductal carcinoma of the breast (IDC).

More specifically, the method comprises the steps of:

(a) contacting a candidate agent with a cell expressing a PBK/TOPK polypeptide or a functional equivalent thereof;

(b) observing the G2/M population on the cell structure and/or cell cycle; and

(c) selecting a compound that changes intercellular junctions to long intercellular bridges, and/or a compound that increases the G2/M population of cells.

Here, any cell may be used as long as it expresses the PBK/TOPK polypeptide or a functional equivalent thereof. The cells used in the present screen may be cells that naturally express the PBK/TOPK polypeptide, including, for example, cells from breast cancer (e.g., IDC), thymus, and testis, as well as cell lines established therefrom. Breast cancer cell lines HBC4, HBC5, HBL100, HCC1937, MCF-7, MDA-MB-231, MDA-MB-435S, SKBR3, T47D, and YMB1 can be used.

Alternatively, the cells used in the screening may be cells which do not naturally express the PBK/TOPK polypeptide and the PP1 alpha polypeptide, but which are transfected with a vector which expresses the PBK/TOPK polypeptide or a functional equivalent of PBK/TOPK, or which expresses PP1 alpha. Such recombinant cells can be obtained according to well-known genetic engineering Methods such as those described above (e.g., Morrison DA., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al) 1983, 101: 347-62) (see "nucleotides, polypeptides, vectors and host cells").

Any of the aforementioned test compounds can be used for the screening of the present invention (see item (1) "test compound for screening"). However, it is preferable to select an agent that is permeable into cells. Alternatively, where the agent is a polypeptide, the cell in the screen may be contacted with the agent by transforming the cell with a vector comprising a nucleotide sequence encoding the agent, and expressing the agent in the cell.

In the present invention, a substance that facilitates cell observation, for example, DAPI, an anti-cell membrane protein antibody, or the like, may be added to the reaction mixture to be screened. After 2 days of contact with the test agent, the cell structure can be observed by phase-contrast microscopy or time-lapse (time-lapse) microscopy.

The cell cycle of the cells can be determined by conventional methods known in the art including FACS and the like. When detecting the cell cycle of a cell, it is preferred to incubate the cell for a sufficient time, e.g., 12 hours or more, after contacting the cell with the test agent until normal cells have passed the G2/M phase. Based on this protocol, when cell cycle arrest at the G2/M phase is detected, the test agent can be determined to have the ability to inhibit proliferation of breast cancer cells.

According to one aspect of the present invention, the components necessary for the screening method may be provided as a kit for screening an agent for inducing apoptosis or cell cycle arrest of breast cancer cells or an agent for treating or preventing breast cancer. The present kit may comprise, for example, cells expressing a7322 or F3374V1 or PBK/TOPK and/or PP1 α polypeptide or functional equivalents thereof, or a7322 or AURKB or F3374V1 or PHB2/REA or era or PBK/TOPK or histone H3 or CDK1 or cyclin B1 or p47 or p97 polypeptide or functional equivalents thereof. Furthermore, the kit may also contain control reagents (positive and/or negative), detectable labels, cell culture media, containers necessary for the screening, instructions for carrying out the method (e.g., written document, magnetic tape, VCR, CD-ROM, others), and the like. The composition and the reagents may be packaged separately in different containers.

The compound isolated according to the screening method of the present invention is a candidate for a drug inhibiting the expression or activity of F3374V1, F3374V1, PBK/TOPK or AURKB, for use in the treatment or prevention of a disease attributable to, for example, a cell proliferative disease such as breast cancer.

The compounds isolated according to this screening method are candidates for antagonists to the polypeptide of the present invention. Likewise, the term "antagonist" refers to a molecule that inhibits the function of a polypeptide of the invention by binding to the polypeptide. Moreover, compounds isolated by this screen are candidates for compounds that inhibit in vivo interactions of the polypeptides of the invention with molecules (including DNA and proteins).

When the biological activity to be detected in the present invention is cell proliferation, it can be detected, for example, by: cells expressing the polypeptide of the present invention are prepared, cultured in the presence of a test compound, and the rate of cell proliferation, cell cycle, and the like are measured, as well as by measuring colony forming activity as described in the examples.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Isolated compounds and pharmaceutical compositions

The compounds isolated by the above screening are candidates for inhibiting the activity of the BC polypeptide of the present invention, and are useful in the treatment of breast cancer. More specifically, when the biological activity of the BC protein is taken as an index, the compound screened according to the present method is a candidate for a drug for the treatment of breast cancer. For example, the present invention provides a composition for treating or preventing breast cancer, the composition comprising a pharmaceutically effective amount of an inhibitor having at least 1 function selected from the group consisting of:

(a) inhibit binding between A7322 and PHB2/REA, between F3374V1 and AURKB, or between PBK/TOPK and histone H3;

(b) inhibiting phosphorylation of F3374V1 by AURKB or histone H3 by PBK/TOPK;

(c) inhibiting the expression of a gene selected from a7322 or F3374; and

(d) inhibit nuclear transfer of PHB2/REA protein.

A "pharmaceutically effective amount" of a compound refers to an amount sufficient to treat or alleviate cancer in an individual. An example of a pharmaceutically effective amount includes an amount required to reduce the expression or biological activity of a7322 or F3374V1 when administered to an animal. The reduction may be, for example, a change in expression of at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 99% or 100%.

Such an active ingredient (c) that inhibits the expression of any one gene selected from the BC gene and AURKB may also be an inhibitory oligonucleotide (e.g., antisense oligonucleotide, siRNA or ribozyme) directed against the gene, or a derivative of the antisense oligonucleotide such as an expression vector, siRNA or ribozyme, as described above (see "antisense oligonucleotide", "siRNA" items). Alternatively, the active ingredient (b) which inhibits AURKB-induced phosphorylation of F3374V1 may be, for example, F3374V1 or a dominant negative mutant of PBK/TOPK. Furthermore, an antagonist against F3374V1 may be used as the active ingredient that inhibits the binding between F3374V1 and AURKB, or an antagonist against PBK/TOPK may be used as the active ingredient (a) that inhibits the binding between PBK/TOPK and histone H3. Alternatively, such active ingredients may be selected according to a screening method such as described above (see item "screening method").

In addition, the compounds obtainable by the screening method of the present invention also include compounds in which a part of the structure of a compound inhibiting the activity of one of the BC proteins is converted by addition, deletion and/or substitution.

The agents isolated by any of the methods of the invention may be administered as a medicament or may be used to prepare pharmaceutical (therapeutic or prophylactic) compositions for use in humans and other mammals, such as mice, rats, guinea pigs (guinea-pig), rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons (baboon), chimpanzees (chimpanzee)) for the treatment or prevention of breast cancer. Preferred cancers to be treated or prevented using the agents selected by the method of the present invention include invasive ductal carcinoma of breast (IDC) and the like.

The isolated compounds may be administered directly or may be formulated into dosage forms using known pharmaceutical manufacturing methods. Pharmaceutical formulations may include those suitable for oral, rectal, nasal, topical (including buccal or sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, as well as those suitable for administration by inhalation or insufflation (inhalation). For example, the agents may be administered orally as sugar-coated tablets, capsules, elixirs and microcapsules, as desired; or as a sterile solution or suspension in water or any other pharmaceutically acceptable liquid for parenteral administration in the form of injections. For example, the agents may be combined with a pharmacologically acceptable carrier or vehicle, such as sterile water, saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients (excipients), vehicles (vehicles), preservatives, binders, and the like, in unit dosage forms as required by generally accepted pharmaceutical formulation regimes (drug administration). The amount of active ingredient in these preparations constitutes (make) the appropriate dosage within the specified range available.

The phrase "pharmaceutically acceptable carrier" refers to an inert substance used as a diluent or vehicle for a drug.

Examples of additives that can be incorporated into tablets and capsules are: binders such as gelatin, corn starch, gum tragacanth (tragacanth gum) and acacia; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin and alginic acid (alginic acid); lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; flavoring agents such as peppermint (peppermint), Gaultheria adenothrix oil and cherry. When the unit dosage form is a capsule, a liquid carrier, such as an oil, may also be included in the above ingredients. Sterile compositions for injection can be formulated in standard pharmaceutical formulations using vehicles such as distilled water for injection.

Physiological saline, glucose and other isotonic liquids containing excipients (adjuvants) such as D-sorbitol, D-mannose, D-mannitol and sodium chloride may be used as aqueous solutions for injection. These may be used in combination with suitable solubilizing agents such as alcohols, particularly ethanol, polyols such as propylene glycol and polyethylene glycol, nonionic surfactants such as Polysorbate 80(TM) and HCO-50.

Sesame oil or soybean oil may be used as the oily liquid, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent, and may also be used with a buffer such as a phosphate buffer and a sodium acetate buffer; analgesics, such as procaine hydrochloride; stabilizers, such as benzyl alcohol, phenol; and an antioxidant. The prepared injection can be filled into a suitable ampoule (ampoule).

Pharmaceutical formulations suitable for oral administration may be inexpensively provided as discrete units such as: capsules, cachets (cachets) or tablets, each unit containing a predetermined amount of active ingredient; as a powder or granules; either as a solution, suspension or emulsion. The active ingredient may also be presented in the form of a bolus (bolus), electuary or paste (paste), as well as in pure form, i.e., without a carrier. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. Tablets may be prepared by compression or molding, optionally containing one or more formulation ingredients. Compressed tablets may be prepared by the following process: the active ingredient is mixed in a free-flowing form (such as a powder or granules), optionally with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant, and compacted in a suitable machine. Molded tablets may be prepared by the following method: the mixture of powdered compounds moistened with an inert liquid diluent is molded in a suitable machine. The tablets may be coated according to methods known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. The liquid preparation may contain conventional additives such as suspending agents, emulsifiers, non-aqueous vehicles (which may include edible oils) or preservatives. The tablets may optionally be formulated so as to provide sustained or controlled release of the active ingredient therein.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats (bacteriostats) and solutes which render the formulation isotonic with the blood of the intended recipient (isotonics); and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials; it can also be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, e.g., saline, water for injection, immediately prior to use. Alternatively, the formulation may be provided for continuous infusion (continuous infusion). Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type previously described.

Formulations suitable for rectal administration may be presented as suppositories (suspensions) with conventional carriers such as cocoa butter or polyethylene glycols. Formulations for topical administration in the mouth, for example buccal (buccal) or sublingual administration, include lozenges comprising the active ingredient in a flavoured base, such as sucrose and acacia (acacia) or tragacanth, and pastilles comprising the active ingredient in a base, such as gelatin and glycerol or sucrose and acacia (pastilles). For intranasal administration, the compounds obtained by the present invention may be used as a liquid spray or dispersible powder, or in the form of drops (drop). Drops may be formulated with an aqueous or non-aqueous base also containing one or more dispersing, solubilising or suspending agents. Liquid sprays are inexpensively delivered from pressurized packages.

For administration by inhalation, the compounds may be conveniently delivered by an insufflator, nebulizer, pressurized pack (pressurized pack), or other convenient means of delivering an aerosol spray. The pressurized pack may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount (metered dose).

Alternatively, where administration is by inhalation or insufflation, the compounds may be in the form of a dry powder composition, for example a powder mix of the compound with a suitable powder base such as lactose or starch. The powder compositions may be provided in unit dosage forms such as capsules, cartridges, gels or blisters which may be administered by means of an inhaler or insufflator.

If desired, the above-described formulations adapted for sustained release of the active ingredient may be employed. The pharmaceutical compositions may also contain other active ingredients such as antimicrobial agents, immunosuppressive agents or preservatives.

Preferred unit dosage forms contain an effective amount, or a suitable fraction of the active ingredient, as set forth below.

The pharmaceutical compounds of the invention may be administered to a patient by methods well known to those skilled in the art, for example, by intraarterial, intravenous, intradermal injection, and as intranasal, transbronchial, intramuscular, or oral administration. The dosage and method of administration vary depending on the weight and age of the patient, and the method of administration, but they can be routinely selected by those skilled in the art. If the compound is encoded by DNA, the DNA may be inserted into a vector for gene therapy and the vector administered to effect therapy. The administration dose and the administration method vary depending on the body weight, age and symptoms of the patient, but those skilled in the art can appropriately select them.

For example, when orally administered to a standard adult (60 kg body weight), the dose of the compound that binds to the polypeptide of the present invention and modulates its activity is about 0.1mg to about 100mg per day, preferably about 1.0mg to about 50mg per day, more preferably about 1.0mg to about 20mg per day, although there are some differences according to the symptoms.

When administered parenterally in the form of injection to a standard adult (body weight 60kg), a dose of about 0.01mg to about 30mg per day, preferably about 0.1mg to about 20mg per day, more preferably about 0.1mg to about 10mg per day is inexpensively injected intravenously, although there are some differences depending on the patient, target organ, symptom and administration method. In the case of other animals, the administration amount may be converted to 60kg body weight.

The agent is preferably administered orally or by injection (intravenous or subcutaneous), the exact amount administered to the patient will be determined by the attending physician within the scope of his or her authority taking into account a number of factors including the age and sex of the patient, the exact condition being treated and its severity. Alternatively, the route of administration may vary depending on the condition or severity thereof.

Furthermore, the present invention provides methods of treating or preventing breast cancer using antibodies to the polypeptides of the invention. According to the method, a pharmaceutically effective amount of an antibody directed against a polypeptide of the invention is administered. Since expression of BC proteins is up-regulated in cancer cells and inhibition of expression of these proteins results in decreased cell proliferative activity, we expect that breast cancer can be treated or prevented by binding of antibodies to these proteins. Thus, an antibody directed against a polypeptide of the present invention may be administered in an amount sufficient to reduce the activity of the protein of the present invention, such amount being in the range of 0.1 to about 250mg/kg per day. The dosage for adults will generally range from about 5mg to about 17.5 g/day, preferably from about 5mg to about 10 g/day, more preferably from about 100mg to about 3 g/day.

Generally, the effective (effective) or effective (effective) amount of one or more BC protein inhibitors is determined by: a low or low amount of the BC protein inhibitor is administered first, followed by a gradual increase in the dose or doses administered, and/or a second BC protein inhibitor is added as needed, until the desired effect, i.e., suppression or prevention of breast cancer, is observed in the subject being treated with minimal or no toxic side effects. Methods suitable for determining The appropriate dosage and schedule of administration of The pharmaceutical compositions of The present invention are described, for example, in Goodman and Gilman's The pharmacological basis of Therapeutics, 11th Ed., Brunton, et al, eds., McGraw-Hill (2006), and inRemington: the Science and Practice of Pharmacy, 21st Ed., University of The sciences in Philadelphia (USIP), Lippincott Williams & Wilkins (2005), all of which are incorporated herein by reference.

The agents screened by the present invention may also be used to treat or prevent breast cancer, such as Invasive Ductal Carcinoma (IDC), in a subject. Can be administered prophylactically or therapeutically to a subject at risk for (or susceptible to) a condition associated with aberrant phosphorylation activity of a BC protein or having the condition. The method includes reducing the function of the BC protein in breast cancer cells. The function can be inhibited by applying the agent obtained by the screening method of the present invention.

As used herein, the term "preventing" refers to prophylactic administration of an agent in order to delay or prevent the formation of a tumor, or to delay, inhibit or ameliorate at least one clinical symptom of cancer. The condition of a tumor in a subject can be assessed using standard clinical protocols.

Alternatively, antibodies that bind to tumor cell specific cell surface markers can be used as a vehicle for drug delivery. For example, an antibody conjugated with a cytotoxic agent is administered at a dose sufficient to damage tumor cells.

Method for inducing anti-tumor immunity and tumor vaccine

The present invention also relates to a method of inducing anti-tumor immunity, the method comprising the step of administering an a7322 or F3374V1 protein, or an immunologically active fragment thereof, or a polynucleotide encoding the protein or fragment thereof. The a7322 or F3374V1 protein or immunologically active fragments thereof are useful as vaccines against breast cancer. In some cases, the protein or fragment thereof may be administered in a form that binds to a T Cell Receptor (TCR) or is presented by an Antigen Presenting Cell (APC), such as a macrophage, Dendritic Cell (DC), or B cell. DC is most preferably used among APC because of its strong antigen presenting ability.

In the present invention, a vaccine against breast cancer refers to a substance having the ability to induce anti-tumor immunity when inoculated to an animal. In general, anti-tumor immunity includes immune responses such as:

-inducing cytotoxic lymphocytes against breast cancer,

-inducing antibodies that recognize breast cancer, and

-inducing the production of anti-breast cancer cytokines.

Therefore, when a protein induces any one of the immune responses when inoculated into an animal, the protein is considered to have an anti-tumor immunity-inducing effect. The anti-tumor immunity induced by a protein can be detected by observing the response of the immune system in a host against the protein in vivo or in vitro.

For example, methods for detecting induction of cytotoxic T lymphocytes are well known. Specifically, foreign substances entering a living body are presented to T cells and B cells by the action of Antigen Presenting Cells (APCs). T cells that respond to an antigen presented by APC in an antigen-specific manner differentiate into cytotoxic T cells (or cytotoxic T lymphocytes; CTL) by stimulation with the antigen, and then proliferate (this is called T cell activation). Therefore, CTL induction by a certain peptide can be evaluated by presenting the peptide to T cells via APC, and detecting CTL induction. In addition, APC has the effect of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils (eosinophil) and NK cells. Since CD4+ T cells and CD8+ T cells are also important in antitumor immunity, the antitumor immunity-inducing effect of peptides can be evaluated using the activation effect of these cells as an index.

Methods for evaluating CTL induction using Dendritic Cells (DCs) as APCs are well known in the art. Among APCs, DC is a representative APC having the strongest CTL induction. In this method, the subject polypeptide is first contacted with a DC, and the DC is then contacted with a T cell. When a T cell having a cytotoxic effect against a target cell is detected after contact with DC, it indicates that the test polypeptide has an inducerLeads to cytotoxic T cell activity. The antitumor activity of CTL can be, for example, employed⁵¹Lysis of Cr-labeled tumor cells (lysis) was detected as an index. Or, adopt³Methods for evaluating the degree of tumor cell damage using H-thymidine uptake activity or LDH (lactose dehydrogenase) release as an index are also well known.

In addition to DCs, Peripheral Blood Mononuclear Cells (PBMCs) can also be used as APCs. It has been reported that CTL induction is enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of Keyhole Limpet Hemocyanin (KLH) and IL-7.

The test polypeptide determined to have CTL inducing activity by these methods is a polypeptide having DC activating effect and subsequent CTL inducing activity. Therefore, a polypeptide that induces CTLs against tumor cells can be used as an anti-tumor vaccine. In addition, APC that has acquired the ability to induce CTLs against tumors by contacting with the polypeptide can be used as an anti-tumor vaccine. In addition, CTLs that have been rendered cytotoxic by presentation of a polypeptide antigen by APC can also be used as vaccines against tumors. These tumor treatment methods using anti-tumor immunity by APC and CTL are called cellular immunotherapy.

In general, when a polypeptide is used for cellular immunotherapy, it is known that the efficiency of CTL induction can be increased by combining a plurality of polypeptides having different structures and contacting them with DCs. Therefore, when stimulating DCs with protein fragments, it is advantageous to use a mixture of multiple types of fragments.

Alternatively, the induction of immunity of the polypeptide against a tumor can be confirmed by observing the induction of antibody production against the tumor. For example, when antibodies against a polypeptide are induced in an experimental animal immunized with the polypeptide and the antibodies inhibit the growth of tumor cells, the polypeptide can be determined to have the ability to induce anti-tumor immunity.

Administration of the vaccine of the invention induces anti-tumor immunity, and the induction of anti-tumor immunity enables one to treat and prevent BLC. Anti-cancer treatment or prevention of the onset of cancer may comprise any of the following steps: such as inhibiting the growth of cancerous cells, causing the regression (invocations) of cancer, and inhibiting the onset of cancer. Treatment or prevention of cancer also includes reducing mortality in individuals with cancer, reducing tumor markers in the blood, alleviating detectable symptoms associated with cancer, and the like. Such therapeutic and prophylactic effects are preferably statistically significant. For example, in observations at a significance level of 5% or less, where the therapeutic or prophylactic effect of the vaccine against breast cancer was compared to a control without vaccine administration. For example, Student's t-test, Mann-Whitney U-test or ANOVA can be used for statistical analysis.

The above-mentioned protein having an immunological activity or a vector encoding the protein may be combined with an adjuvant. An adjuvant refers to a compound that, when co-administered (or sequentially) with an immunologically active protein, enhances the immune response against the protein. Examples of adjuvants include, but are not limited to, cholera toxin (cholera toxin), salmonella toxin (salmonella toxin), alum (alum), and the like. Alternatively, the vaccine of the present invention may be suitably combined with a pharmaceutically acceptable carrier. Examples of such carriers include sterile water, physiological saline, phosphate buffer, and culture medium. Alternatively, the vaccine may contain a stabilizer, a suspending agent, a preservative, a surfactant, etc., as required. The vaccine may be administered systemically or locally. Vaccine administration may be by single administration, or may be enhanced by multiple administrations (boost).

When APCs or CTLs are used as the vaccine of the present invention, tumors can be treated or prevented, for example, by an ex vivo method. More specifically, PBMCs from a subject receiving treatment or prophylaxis are collected, and after contacting the cells with the polypeptide ex vivo to induce APC or CTL, the cells can be administered to the subject. APC can also be induced by introducing a vector encoding the polypeptide into PBMC under ex vivo conditions. The in vitro induced APC or CTL may be cloned prior to administration. By cloning and culturing cells having high target cell destruction activity, cellular immunotherapy can be performed more efficiently. Alternatively, the APC and CTL isolated in this manner can be used not only for cellular immunotherapy against a subject that provides cells, but also for cellular immunotherapy against similar types of tumors from other individuals.

Furthermore, the present invention provides a pharmaceutical composition for treating or preventing breast cancer, comprising a pharmaceutically effective amount of the polypeptide of the present invention. The pharmaceutical composition can be used for stimulating (raise) anti-tumor immunity. In normal tissues, expression of a7322 is restricted to the brain; while the expression of F3374V1 in normal organs was restricted to testis, thymus, placenta and bone marrow. Therefore, it is considered that inhibition of these genes does not adversely affect other organs. Therefore, the a7322 and F3374V1 polypeptides are preferred for the treatment of breast cancer. In the present invention, the polypeptide or fragment thereof is administered in a dose range of 0.1mg to 10mg, preferably 0.3mg to 5mg, more preferably 0.8mg to 1.5mg, sufficient to induce anti-tumor immunity. The application is repeated. For example, to induce anti-tumor immunity, 1mg of the peptide or fragment thereof may be administered 4 times every 2 weeks.

Inhibitory dominant negative protein

The present invention relates to inhibitory polypeptides comprising MEGISNFKTPSKLSEKKK (SEQ ID NO: 98). In some preferred embodiments, the inhibitory polypeptide comprises MEGISNFKTPSKLSEKKK (SEQ ID NO: 98); a polypeptide functionally equivalent to the polypeptide; or polynucleotides encoding these polypeptides; wherein the polypeptide lacks a polypeptide consisting of SEQ ID NO: 92, respectively, in a biological function. SEQ ID NO: 92 is disclosed in WO 2006/028676. It is known that cancer cell proliferation can be regulated by inhibiting the expression of the amino acid sequence. However, the present inventors have demonstrated a novel finding that a fragment comprising a sequence having a specific mutation in the above amino acid sequence can inhibit cancer cell proliferation.

The polypeptide comprising a selected amino acid sequence of the invention can be of any length, so long as the polypeptide inhibits cancer cell proliferation. In particular, the amino acid sequence may range in length from 8 to 70 residues, such as 8 to 50, preferably 8 to 30, more particularly 8 to 20, still more particularly 8 to 16 residues.

The polypeptides of the invention may comprise two or more "selected amino acid sequences". The two or more "selected amino acid sequences" may be the same or different amino acid sequences. Alternatively, the "selected amino acid sequences" may be directly linked. Alternatively, any intervening sequence may be arranged between them.

In addition, the present invention relates to methods similar to MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 polypeptides are homologous (i.e., have sequence identity). In the present invention, the nucleotide sequence similar to MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 is a polypeptide comprising any mutation selected from the group consisting of addition, deletion, substitution and insertion of one or several amino acid residues and having homology to MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 polypeptides are functionally equivalent. The phrase "binds to MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 polypeptide is functionally equivalent "meaning having the function of inhibiting the binding of CDK1 and cyclin B1 complex to PBK/TOPK. At a composition corresponding to MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 polypeptide is preferably conserved MEGISNFKTPSKLSEKKK/SEQ ID NO: 98, and (b) a sequence. Thus, in the present invention, the amino acid sequence of MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 peptide a functionally equivalent polypeptide is preferably represented by MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 sequence with amino acid mutations in positions outside the sequence. In the present invention, the amino acid sequence shown in MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 peptide the amino acid sequence of the functionally equivalent polypeptide is conserved MEGISNFKTPSKLSEKKK/SEQ ID NO: 98, and has a homology of 60% or more, usually 70% or more, preferably 80% or more, more preferably 90% or more, or 95% or more, and more preferably 98% or more with the "selected amino acid sequence". Amino acid homology can be determined using algorithms well known in the art, for example BLAST or ALIGN set to their default settings.

Alternatively, the number of amino acids that can be mutated is not particularly limited as long as MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 peptide activity. Typically, up to about 10 amino acids, more preferably up to about 3 amino acids, can be mutated. Likewise, the site of mutation is not specifically limited, as long as the mutation does not result in a mutation of MEGISNFKTPSKLSEKKK/SEQ ID NO: disruption of 98 peptide activity.

In a preferred embodiment, MEGISNFKTPSKLSEKKK/SEQ ID NO: the activity of the 98 peptide comprises the effect of inducing cell cycle arrest in PBK/TOPK expressing cells (i.e., breast cancer cells). Cell cycle arrest refers to the arrest at the checkpoint of DNA replication and mitosis (checkpoint). Methods for detecting cell cycle arrest are well known. For example, FACS (flow cytometry) can be used to confirm cell cycle arrest.

In another embodiment, MEGISNFKTPSKLSEKKK/SEQ ID NO: the activity of the 98 peptide comprises the effect of inducing apoptosis in PBK/TOPK expressing cells (i.e., breast cancer cells). Apoptosis means cell death caused by the cell itself, sometimes referred to as programmed cell death. Aggregation of nuclear chromosomes, fragmentation of nuclei, or concentration of cytoplasm was observed in cells undergoing apoptosis. Methods for detecting apoptosis are well known. For example, apoptosis can be confirmed by TUNEL staining (terminaldeoxynecetyltransferase Transferase Biotin-dUTP Nick End Labeling); Gavrili et al (1992) J.cell biol.119: 493-. Alternatively, DNA ladder assays (DNA ladder assay), Annexin V staining (Annexin V staining), caspase assays, electron microscopy or observation of conformational changes in the nucleus or cell membrane may be used to detect apoptosis. Any commercially available kit can be used to detect these apoptosis-induced manifestations in cells (behavior). For example, these apoptosis detection kits are commercially available from the following suppliers:

LabChem Inc.，

Promega，

BD Biosciences Pharmingen，

Calbiochem，

Takara Bio Company(CLONTECH Inc.)，

CHEMICON International，Inc，

Medical & Biological Laboratories co., ltd.

The polypeptides of the invention can be based on the selected amino acid sequence from any position chemical synthesis. The methods used in general peptide chemistry can be applied to methods for synthesizing polypeptides. Specifically, the methods include those described in the following documents and japanese patent publications:

peptide Synthesis, Interscience, New York, 1966; the Proteins, Vol.2, Academic Press Inc., New York, 1976;

peputido gousei (peptide synthesis), Maruzen (Inc.), 1975;

peputido gousei no kiso to jikken (basis and experiment for peptide synthesis), Maruzen (Inc.), 1985;

iyakuhin no kai hatsu (drug development), sequal, vol.14: peputido gousei (peptide synthesis), Hirokawa Shoten, 1991;

international patent publication WO 99/67288.

Alternatively, the polypeptides of the invention may be synthesized by known genetic engineering techniques. An example of genetic engineering techniques is as follows. Specifically, a DNA encoding a desired peptide is introduced into an appropriate host cell to prepare a transformed cell. The polypeptide of the present invention can be obtained by recovering the polypeptide produced by such transformed cells. Alternatively, the desired polypeptide may be synthesized using an in vitro translation system that recombines the elements necessary for polypeptide synthesis in vitro.

When using genetic engineering techniques, the polypeptides of the invention may be expressed as fusion proteins with peptides having different amino acid sequences. A vector for expressing a desired fusion protein can be obtained by ligating a polynucleotide encoding a polypeptide of the present invention and polynucleotides encoding different peptides so that they are in the same reading frame, and then introducing the resulting nucleotides into an expression vector. The fusion protein is expressed by transforming an appropriate host with the resulting vector. Different peptides used in forming fusion proteins include the following peptides:

FLAG (Hopp et al, (1988) Biotechnology 6, 1204-10),

6XHis and 10XHis consisting of six His residues,

an influenza Hemagglutinin (HA) which is capable of inhibiting the growth of influenza virus,

a fragment of human c-myc,

(ii) a VSV-GP fragment,

a p18HIV fragment of a human,

the T7-label is a label,

an HSV-tag which is a label for a human,

the E-label is used for carrying out the labeling,

a fragment of the SV40T antigen,

the lck label is a label that is,

a fragment of alpha-tubulin, which is,

b-the label is added with a label,

(ii) a fragment of protein C,

GST (glutathione-S-transferase),

HA (influenza hemagglutinin) is a peptide of the human body,

an immunoglobulin constant region which is selected from the group consisting of,

beta-galactosidase, and

MBP (maltose binding protein).

The polypeptide of the present invention can be obtained by treating the thus-produced fusion protein with an appropriate protease and then recovering the desired polypeptide. To purify the polypeptide, the fusion protein is captured in advance by affinity chromatography bound to the fusion protein, and then the captured fusion protein may be treated with a protease. The desired polypeptide is separated from the affinity chromatography using protease treatment and recovered in high purity.

The polypeptides of the invention include modified polypeptides. In the present invention, the term "modified" means, for example, binding to other substances. Therefore, in the present invention, the polypeptide of the present invention may further comprise other substances such as a cell membrane permeable substance. Such other substances include organic compounds such as peptides, lipids, sugars, and various naturally occurring or synthetic polymers. The polypeptides of the invention may have any modification as long as the polypeptide retains the desired activity of inhibiting the binding of CDK1 and cyclin B1 complex to PBK/TOPK. In some embodiments, the inhibitory polypeptide is capable of directly competing with PBK/TOPK for binding to CDK1 and cyclin B1 complex. Modifications may also confer additional functions on the polypeptides of the invention. Examples of additional functionality include targeting ability, delivery ability, and stabilization.

Preferred examples of modification in the present invention include, for example, introduction of a cell membrane permeable substance. Typically, the cell membrane isolates the intracellular structures from the environment. Therefore, it is difficult to efficiently introduce an extracellular substance into cells. The polypeptide of the present invention can be imparted with cell membrane permeability by modifying the polypeptide with a cell membrane-permeable substance. Thus, by contacting a cell with a polypeptide of the present invention, the polypeptide can be delivered into the cell to act on the cell.

"cell membrane permeable substance" refers to a substance that is capable of penetrating a mammalian cell membrane into the cytoplasm. For example, certain liposomes fuse with the cell membrane, thereby releasing the contents into the cell. At the same time, certain types of polypeptides penetrate the plasma membrane of mammalian cells into the interior of the cell. As the polypeptide having such a cell-entering activity, a cytoplasmic membrane or the like in the present invention is preferable as the substance. Specifically, the invention includes polypeptides having the following general formula.

[R]-[D]；

Wherein,

[ R ] represents a cell membrane-permeable substance; [D] represents a polypeptide comprising MEGISNFKTPSKLSEKKK/SEQ ID NO: 98, or a fragment thereof. In the above formula, [ R ] and [ D ] may be directly connected or indirectly connected through a linker. A peptide, a compound having a plurality of functional groups, or the like may be used as the linker. Specifically, an amino acid sequence containing-G-can be used as a linker. Alternatively, a cell membrane permeable substance and a polypeptide comprising a selected sequence can be bound to the surface of the microparticle. [ R ] may be bonded to any position of [ D ]. Specifically, [ R ] may be bonded to the N-terminus or C-terminus of [ D ], or to the side chain of the amino acid constituting [ D ]. Alternatively, more than one [ R ] molecule may be combined with one [ D ] molecule. The [ R ] molecule can be introduced at different positions on the [ D ] molecule. Alternatively, [ D ] may be modified with a plurality of [ R ] linked together.

For example, a variety of naturally occurring or synthetic polypeptides with Cell membrane permeability have been reported (Joliot A. and Prochiantz A., Nat Cell biol.2004; 6: 189-96). All of these known cell membrane permeable substances can be used in the present invention to modify polypeptides. In the present invention, for example, any substance selected from the group consisting of:

poly-arginine; matsushita et al, (2003) j.neurosci; 21, 6000-7.

[ Tat/RKKRRQRRR ] (SEQ ID NO: 47) Frankel et al, (1988) Cell55, 1189-93.

Green and Loewenstein (1988) Cell55, 1179-88.

[Penetratin/RQIKIWFQNRRMKWKK](SEQ ID NO：101)

Derossi et al, (1994) J.biol.chem.269, 10444-50.

[Buforin II/TRSSRAGLQFPVGRVHRLLRK](SEQ ID NO：102)

Park et al, (2000) Proc.Natl Acad.Sci.USA 97, 8245-50.

[Transportan/GWTLNSAGYLLGKINLKALAALAKKIL](SEQ ID NO：103)

Pooga et al, (1998) FASEB J.12, 67-77.

[ MAP (patterned amphiprotic peptide)/KLALKLALKALKAALKLA ] (SEQ ID NO: 104)

Oehlkke et al, (1998) Biochim. Biophys. acta.1414, 127-39.

[K-FGF/AAVALLPAVLLALLAP](SEQ ID NO：105)

Lin et al, (1995) J.biol.chem.270, 14255-8.

[Ku70/VPMLK](SEQ ID NO：106)

Sawada et al, (2003) Nature Cell biol.5, 352-7.

[Ku70/PMLKE](SEQ ID NO：114)

Sawada et al, (2003) Nature Cell biol.5, 352-7.

[ prion/MANLGYWLLALFVTMWTDVGLCKKRPKP ] (SEQ ID NO: 107)

Lundberg et al, (2002) biochem. biophysis. res. commun.299, 85-90.

[pVEC/LLIILRRRIRKQAHAHSK](SEQ ID NO：108)

Elmquist et al, (2001) exp.cell Res.269, 237-44.

[Pep-1/KETWWETWWTEWSQPKKKRKV](SEQ ID NO：109)

Morris et al, (2001) Nature Biotechnol.19, 1173-6.

[SynB1/RGGRLSYSRRRFSTSTGR](SEQ ID NO：110)

Roussell et al, (2000) mol. Pharmacol.57, 679-86.

[Pep-7/SDLWEMMMVSLACQY](SEQ ID NO：111)

Gao et al, (2002) bioorg.Med.chem.10, 4057-65.

[HN-1/TSPLNIHNGQKL](SEQ ID NO：112)

Hong and Clayman (2000) Cancer Res.60, 6551-6.

In the present invention, the polyarginines listed above as examples of cell membrane permeable substances consist of any number of arginine residues. Specifically, for example, it consists of 5 to 20 consecutive arginine residues. The preferred number of arginine residues is 11 (SEQ ID NO: 113).

Comprising MEGISNFKTPSKLSEKKK/SEQ ID NO: 98 pharmaceutical composition

The polypeptide of the present invention inhibits the proliferation of cancer cells. Accordingly, the present invention provides an agent for the treatment and/or prevention of cancer, comprising as an active ingredient an agent comprising MEGISNFKTPSKLSEKKK/SEQ ID NO: 98; or a polynucleotide encoding the polypeptide. Alternatively, the present invention relates to a method for the treatment and/or prevention of cancer comprising the step of administering a polypeptide of the present invention. Alternatively, the present invention relates to the use of a polypeptide of the invention for the manufacture of a pharmaceutical composition for the treatment and/or prevention of cancer. The cancer that can be treated or prevented by the present invention is not limited as long as the expression of PBK/TOPK is up-regulated in cancer cells. For example, the polypeptides of the invention may be used to treat breast cancer.

Alternatively, the inhibitory polypeptides of the invention may be used to induce cell cycle arrest in cancer cells. Accordingly, the present invention provides an agent for inducing cell cycle arrest of a cell, comprising as an active ingredient an agent comprising MEGISNFKTPSKLSEKKK/SEQ ID NO: 98; or a polynucleotide encoding the polypeptide. The agent inducing cell cycle arrest of the present invention can be used for the treatment of cell proliferative diseases such as cancer. The cancer that can be treated or prevented by the present invention is not limited as long as the expression of PBK/TOPK is up-regulated in cancer cells. For example, the polypeptides of the invention may be used to treat breast cancer. Alternatively, the present invention relates to a method of inducing apoptosis comprising the step of administering a polypeptide of the present invention. Furthermore, the present invention relates to the use of a polypeptide of the invention for the manufacture of a pharmaceutical composition for inducing cell cycle arrest in a cell.

The inhibitory polypeptides of the invention induce cell cycle arrest in PBK/TOPK expressing cells such as breast cancer. At the same time, PBK/TOPK expression was not observed in most normal organs. In some normal organs, the expression level of PBK/TOPK is relatively low compared to cancer tissues. Thus, the polypeptides of the invention can specifically induce cell cycle arrest in cancer cells.

When a polypeptide of the invention is administered (as a manufactured drug) to treat cancer or induce cell cycle arrest in cells, to humans and other mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, monkeys, baboons, and chimpanzees, the isolated compound can be administered directly or formulated into an appropriate dosage form using methods known for the manufacture of medicaments. For example, if desired, the medicaments may be administered orally as sugar-coated tablets, capsules, elixirs and microcapsules, or parenterally in the form of injections, which are sterile solutions or suspensions containing water or any other pharmaceutically acceptable liquid. For example, the compounds may be mixed with a pharmacologically acceptable carrier or vehicle, in particular, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, corrigents (correct), excipients, vehicles, preservatives and binders, in a unit dosage form (unit dose form) necessary for the manufacture of a generally accepted medicament. Depending on the amount of active ingredient in these formulations, a suitable dosage within the specified range may be determined.

Examples of additives that may be mixed in tablets and capsules are binders such as gelatin, corn starch, tragacanth and acacia; media such as crystalline cellulose; bulking agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose or saccharin; and corrective drugs such as peppermint, oil of wintergreen, and cherry. When the unit dosage form is a capsule, a liquid carrier such as an oil may be further included in the above ingredients. Sterile mixtures for injection can be formulated using vehicles such as distilled water for injection in accordance with conventional pharmaceutical knowledge.

Physiological saline, dextrose, and other isotonic solutions containing adjuvants (adjuvants) such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as the aqueous injection solution. They may be used in combination with suitable solubilizers, for example alcohols, in particular ethanol and polyols such as propylene glycol and polyethylene glycol, nonionic surfactants such as Polysorbate 80. TM. and HCO-50.

Sesame oil or soybean oil may be used as the oily liquid, and may also be used together with benzyl benzoate or benzyl alcohol as a solubilizing agent. Alternatively, they may be formulated further together with: buffers such as phosphate buffer and sodium acetate buffer; analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol and phenol; and an antioxidant. The injection thus prepared may be filled into a suitable ampoule.

Methods well known to those skilled in the art may be used to administer the pharmaceutical compounds of the invention to a patient, for example, by intra-arterial, intravenous or subcutaneous injection, and similarly by intranasal, transtracheal, intramuscular or oral administration. The dosage and administration method vary depending on the weight and age of the patient and the administration method. However, those skilled in the art can select them as usual. The DNA encoding the polypeptide of the present invention may be inserted into a vector for gene therapy, and the vector may be administered for therapy. Although the dose and administration method vary depending on the body weight, age and symptoms of the patient, those skilled in the art can appropriately select them. For example, when administered orally to a normal adult (60 kg body weight), the dose of the compound that binds to the polypeptide of the present invention to thereby modulate their activity is about 0.1mg to about 100mg per day, preferably about 1.0mg to about 50mg per day, more preferably about 1.0mg to about 20mg, although there is little variation depending on the symptoms.

When the compound is administered parenterally in the form of injection to a normal adult (body weight 60kg), it is convenient to administer intravenously at a dose of about 0.01mg to about 30mg per day, preferably about 0.1mg to about 20mg per day, more preferably about 0.1mg to about 10mg per day, although there is little variation depending on the patient, the target organ, the symptom and the administration method. Similarly, the compounds can be administered to other animals in amounts converted from the dosage used for 60kg body weight.

The present invention will be described in more detail below with reference to examples. The following materials, methods, and examples, however, are illustrative of aspects of the invention only and are not limiting upon the scope of the invention. Thus, methods and materials similar or equivalent to those described herein can be used in the practice or research of the present invention.

Examples

From the above disclosure, it can be seen that the present invention has a wide range of applications. Accordingly, the following examples are provided for illustrative purposes and are not to be construed as limiting the invention in any way. Those skilled in the art will readily recognize a wide variety of noncritical parameters which may be varied or modified to achieve substantially similar results.

The present invention is illustrated in detail by the following examples, but the present invention is not limited to these examples.

Example 1 materials and methods

(1) Cell lines and clinical material

Human breast cancer cell lines HBL100, HCC1937, MCF-7, MDA-MB-435S, SKBR3, T47D, BT-549, YMB1, ZR-75-1, OCUB-F, MDA-MB-453, MDA-MB-157, HCC1599, HCC1500, HCC1395, HCC1143, BT-474, BT-20, and human embryonic kidney cell line HEK293T cells, BTL100, and COS7 were purchased from American type culture Collection (ATCC, Rockville, Md.). HBC4, HBC5, BSY-1 and MDA-MB-231 cell lines were gifted by Dr. Yamori, Provisional society for Cancer Chemotherapy Center, molecular pharmacology, Division of molecular pharmacology, Cancer Chemotherapy Center, Japan Foundation for Cancer research. All cells were cultured according to the recommendations of their respective depositors, i.e., for HBC4, HBC5, BT-483, SKBR3, BT-549, HCC1143, HCC1599, HCC1500, HCC1395, T47D, YMB1, HC DC1937, BSY-1 and ZR-75-1 using RPMI-1640(Sigma-Aldrich, St. Louis, Mo.) (2 mM L-glutamine added); dulbecco's modified Eagle Medium (Invitrogen, Carlsbad, Calif.) was used for HBL100 BT-474 and OCUB-F; EMEM (Sigma-Aldrich) containing 0.1mM essential amino acids (Roche, Basel, Switzerland), 1mM sodium pyruvate (Roche), 0.01mg/ml insulin (Sigma-Aldrich) was used for MCF-7 and BT-20; for MDA-MB-231 and MDA-MB-435S, MDA-MB-453 and MDA-MB-157 use L-15 (Roche). Each medium was supplemented with 10% fetal bovine serum (Cansera) and 1% antibiotic/antifungal solution (Sigma-Aldrich). At 37 ℃ in the absence of CO ₂The MDA-MB-231 and MDA-MB-435s cells were maintained in the humid air of (1). At 37 ℃ in a 5% CO atmosphere₂The other cell lines were maintained in the humidified air. After written informed consent, tissue samples were obtained from surgically excised breast cancers, along with their corresponding clinical information.

(2) Semi-quantitative RT-PCR analysis

The present inventors extracted total RNA from each breast cancer clinical sample. The present inventors extracted total RNA from microdissected cells as previously described, followed by T7-based amplification and reverse transcription (Nishidate T et al Int J Oncol 2004; 25: 797-. The present inventors prepared suitable dilutions of each single stranded cDNA for subsequent PCR by monitoring glyceraldehyde-3-phosphate dehydrogenase (. beta.2MG), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and farnesyl diphosphate farnesyl transferase 1(FDFT1) as quantitative internal controls. The PCR primer sequences were as follows:

for β 2 MG: 5'-AACTTAGAGGTGGGAGCAG-3' (SEQ ID NO: 1) and 5'-CACAACCATGCCTTACTTTATC-3' (SEQ ID NO: 2);

for a 7322: 5'-CTTGACAAGGCCTTTGGAGT-3' (SEQ ID NO: 3) and 5'-CAATATGCTTTTCCCGCTTT-3' (SEQ ID NO: 4);

for F3374: 5'-AACCAAGCACACCATAGCCTTA-3' (SEQ ID NO: 5) and 5'-GGAGATGGGTAGGGATACAAAC-3' (SEQ ID NO: 6),

For AURKB: 5'-GGGAGAGCTGAAGATTGCTG-3' (SEQ ID NO: 7) and 5'-GACAGATTGAAGGGCAGAGG-3' (SEQ ID NO: 8);

for GAPDH: 5'-CGACCACTTTGTCAAGCTCA-3' (SEQ ID NO: 9) and 5'-GGTTGAGCACAGGGTACTTTATT-3' (SEQ ID NO: 10);

for FDFT 1: 5'-AGTGAAATGCAGGTGAGAAGAAC-3' (SEQ ID NO: 11) and 5'-TCATTCTAGCCAGGATCATACTAAG-3' (SEQ ID NO: 12);

for PBK/TOPK: 5'-AGACCCTAAAGATCGTCCTTCTG-3' (SEQ ID NO: 13) and 5'-GTGTTTTAAGTCAGCATGAGCAG-3' (SEQ ID NO: 14); and

for PHB 2/REA: 5'-GCTGACAACCTTGTGCTGAA-3' (SEQ ID NO: 15) and 5'-TGAGAAATCACGCACTGTCC-3' (SEQ ID NO: 16).

(3) Northern blot analysis

Total RNA was extracted from all breast cancer cell lines using RNeasy kit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. After treatment with DNase I (Nippon Gene, Osaka, Japan), mRNA was isolated using the mRNA purification kit (GE Healthcare, Buckinghamshire, United kingdom) according to the manufacturer's instructions. Aliquots of 1 μ g of each mRNA isolated from normal adult breast (Biochain, Hayward, CA), lung, heart, liver, kidney, and bone marrow (BD Biosciences, San Jose, CA) were separated on 1% denaturing agarose gels and transferred to nylon membranes (breast cancer Northern blots). Human multi-tissue Northern blots (BD Biosciences)) were combined with [ α ] prepared by RT-PCR (see below) ³²P]-dCTP labeling of A7322 PCR product for hybridization. Prehybridization, hybridization and washing were performed as recommended by the supplier. The blot was autoradiographed using an intensifying screen (intensifying screen) at-80 ℃ for 14 days. Specific probes for A7322(459bp) and F3374 were prepared by RT-PCR using the following primer set and radiolabeled with the megaprime DNA labeling system (GE Healthcare)

Within the 3' UTR of a 7322: 5'-CAAGCTTGCTTACAGAGACCTG-3' (SEQ ID NO: 17) and 5'-GGGCCAAACCTACCAAAGTT-3' (SEQ ID NO: 18);

for F3374: 5'-GCAATCTGCTATGTCAGCCAAC-3' (SEQ ID NO: 19) and 5'-CAGGATCAGCTCAAAGTCTGACA-3' (SEQ ID NO: 20);

5'-AGACCCTAAAGATCGTCCTTCTG-3' (SEQ ID NO: 13) and 5'-GTGTTTTAAGTCAGCATGAGCAG-3' (SEQ ID NO: 14).

(4) cDNA end 5 'Rapid amplification (5' RACE)

The 5 'RACE experiment was performed using the SMART RACE cDNA amplification kit (TakaraClontech) according to the manufacturer's instructions. For amplification of the 5' portion of the a7322 cDNA, gene-specific primers were as follows:

5'-GCCTCCTTCTGCAGCTTCCTCAGGATTT-3' (SEQ ID NO: 21) and the universal primer mixture provided in the kit was used. A cDNA template was synthesized from mRNA extracted and purified from MDA-MB-453 breast cancer cells using Superscript III reverse transcriptase (Invitrogen). PCR products were cloned using TA cloning kit (Invitrogen) and the sequence was determined by DNA sequencing (ABI 3700; PE Applied Biosystems, Foster, Calif.).

(5) Construction of expression vectors

To construct the A7322, PHB2/REA or F3374 expression vectors, the entire coding sequence of A7322 or cDNA was amplified by PCR using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan). The primer group is

A 7322-forward:

5’-CGGAATTCATGGAAGAAATCCTGAGGAAGC-3' (SEQ ID NO: 22) (EcoRI site underlined) and

a 7322-reverse:

5’-ATAGTTTAGCGGCCGCACAATGATGTCATAGACACGG-3' (SEQ ID NO: 23) (NotI site underlined))；

PHB 2/REA-Forward

：5’-CGGAATTCCAGACCGTGCATCATGGCCCAGAACTTGAAGGA-3' (SEQ ID NO: 24) (EcoRI site underlined) and

PHB 2/REA-Reversal:

5’-CCGCTCGAGTTTCTTACCCTTGATGAGGCTGT-3' (SEQ ID NO: 25) (the XhoI site is underlined);

ER α -forward:

5’-CGGAATTCATGACCATGACCCTCCACACCAAAGCATCC-3' (SEQ ID NO: 26) and

ER α -reverse: 5' -CCGCTCGAGGACCGTGGCAGGGAAACCCTCT-3' (SEQ ID NO: 27) (recognition sites for restriction enzymes are underlined);

f3374 — forward:

5’-AAGGAAAAAAGCGGCCGCGATGCTCTTCAATTCGGTGCT-3' (SEQ ID NO: 28) (not I site underlined) and

f3374-reverse:

5’-CCGCTCGAGTAATTCTGTTGAGTGTTCAGGACC-3' (SEQ ID NO: 29) (the XhoI site is underlined).

The PCR products were inserted into the EocRI and NotI sites (for A7322), EocRI and XhoI sites (for PHB2/REA), EocRI and XhoI sites (for ER α) or NotI and XhoI sites (for B3374) of the pCAGGS-nH3F expression vector and were co-read with an N-terminal HA-tag and a C-terminal Flag-tag. The construct was confirmed by DNA sequencing (ABI3700, PE Applied Biosystems, Foster, Calif.).

(6) Preparation of anti-A7322 polyclonal antibody and anti-F3374 polyclonal antibody

Plasmids were designed to express two A7322 fragments (codons 459-. Two recombinant peptides were expressed in E.coli BL21 codon-plus strain (Stratagene, La Jolla, Calif.) respectively, and purified using Ni-NTA resin agarose (QIAGEN) according to the supplier's protocol. The purified recombinant proteins were mixed together and then used to inoculate rabbits (Medical and Biological Laboratories, Nagoya, Japan). The immune sera were then purified on an antigen affinity column using Affigel 15 gel (Bio-Rad Laboratories, Hercules, Calif.) according to the supplier's instructions. The inventors confirmed that the antibody can specifically recognize the endogenous a7322 protein in breast cancer cell lines (SK-BR-3 cells). An affinity purified anti-a 7322 antibody was used for Western blot, immunocytochemical staining and immunohistochemical staining analyses described below.

A plasmid designed to express a portion of C-terminally His-tagged F3374 was prepared using the pET21 vector (Merck, Novagen, Madison, Wis.). The recombinant peptide (36kDa) was expressed in E.coli BL21 codon-plus (Stratagene, La Jolla, Calif.) and purified using Ni-NTA resin (Qiagen) according to the supplier's protocol. To remove E.coli proteins as contaminants, F3374 fragment proteins were excised from SDS-PAGE gels and extracted using an electroelution apparatus (Bio-Rad, Hercules, Calif.). The extracted proteins were inoculated into rabbits, and then immune sera were purified on an antigen affinity column using Affigel 15 gel (Bio-Rad) according to the supplier's instructions. The affinity purified anti-F3374 antibody was used for Western blot, immunohistochemistry, and immunocytochemistry analyses described below.

(7) Cloning and mutagenesis

To construct the PBK/TOPK expression vector, the complete coding sequence of the PBK/TOPK cDNA was amplified by PCR using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan). The primer group for wild PBK/TOPK is

5’-CCGGAATTCATGGAAGGGATCAGTAATTTC-3' (SEQ ID NO: 30) and

5’-CCGCTCGAGTCAGACATCTGTTTCCAGAGCTTC-3' (SEQ ID NO: 31) (underlinedRecognition sites for restriction enzymes). The PCR product was inserted into the EcoRI site and XhoI site of the pCAGGS-nHA expression vector. Two-step mutagenic PCR was performed to generate kinase-inactive (dead) mutants (K64-65A) in which Lys64 and Lys65 were substituted with alanine as previously described (Gaudet S, et al, ProcNatl Acad Sci USA 2000, 97: 5167-72). The primer group for the mutant K64-65A is

5’-CATTCTCCTTGGGCTGTAGCAGCGATTAATCCTATATGTAATG-3' (SEQ ID NO: 32) and

5’-CATTACATATAGGATTAATCGCTGCTACAGCCCAAGGAGAATG-3' (SEQ ID NO: 33) (underlined are nucleotides substituted from wild type). All constructs were confirmed by DNA sequencing (ABI3700, PE Applied Biosystems, Foster, Calif.).

(8) Immunocytochemical staining

To investigate the subcellular localization of the endogenous a7322 protein in breast cancer cells, 1x10 was used⁵Each well was seeded with SK-BR-3 cells (Lab-Tek II cell Slide System; Nalge Nunc International, Naperville, Ill.). After 24 hours of incubation, cells were fixed with 4% paraformaldehyde (paraformaldehyde) in PBS (-) for 30 minutes at 4 ℃ and treated with 0.1% TritonX-100 in PBS (-) for 2 minutes at 4 ℃ to make the cells permeable. Next, the cells were covered with 3% BSA in PBS (-) for 1 hour to block non-specific hybridization, followed by 1 hour incubation with 1: 250 dilution of anti-A7322 polyclonal antibody. After washing with PBS (-), cells were stained with Alexa 488-conjugated anti-rabbit secondary antibody (Molecular Probe, Eugene, OR) diluted 1: 1000 for 1 hour. Nuclei were counterstained with 4 ', 6 ' -diamidino-2 ' -phenylindole Dihydrochloride (DAPI). Fluorescence images were acquired under a TCSSP2 AOBS microscope (Leica, Tokyo, Japan).

To examine the subcellular localization of F3374, 5X10⁴Each well was seeded with HBC5 cells. Then, the cells were fixed with 4% paraformaldehyde (paraformaldehyde) in PBS for 20 minutes, and then treated with 0.1% Triton X-100 in PBS at room temperatureTreatment was performed for 2 minutes to make the cells permeable. Next, the cells were covered with 3% BSA in PBS for 1 hour at room temperature to block non-specific hybridization. Next, the cells were incubated with a 1: 100 dilution of rabbit anti-F3374 antibody. After washing with PBS, cells were stained with Alexa 488-conjugated anti-rabbit secondary antibody (Molecular Probe) diluted 1: 1000. Nuclei were counterstained with 4 ', 6 ' -diamidino-2 ' -phenylindole Dihydrochloride (DAPI). Fluorescence images were acquired under a TCS SP2AOBS microscope (Leica, Tokyo, Japan). To investigate the subcellular localization of endogenous F3374 and AURKB proteins, 1X10 was used⁵Each cell was seeded with T47D cells. Cell fixation, blocking reactions and staining procedures were performed under the above conditions except that 1: 100 dilution of anti-F3374 antibody or 1: 500 dilution of anti-AURKB antibody (Abcam, Cambridge, MA) was used.

To investigate the subcellular localization of endogenous PBK/TOPK proteins in the breast cancer cell lines T47D, BT-20 and HBC5, at 2X10 ⁵Each well was seeded with cells (Lab-Tek II chamber slide, Nalgen Nunc International, Naperville, Ill.). After 48 hours of incubation, the cells were fixed with 4% paraformaldehyde (Paraformaldehyde) in PBS (-) for 15 minutes and treated with 0.1% Triton X-100 in PBS (-) at 4 ℃ for 2.5 minutes to render the cells permeable. Next, the cells were covered with 3% BSA in PBS (-) for 12 hours at 4 ℃ to block non-specific hybridization, followed by incubation with a 1: 100 dilution of mouse anti-PBK/TOPK monoclonal antibody (BD Biosciences). After washing with PBS (-), cells were stained with Alexa 594-conjugated anti-mouse secondary antibody (molecular Probe, Eugene, OR) diluted 1: 1000. Nuclei were counterstained with 4 ', 6 ' -diamidino-2 ' -phenylindole Dihydrochloride (DAPI). Fluorescence images were acquired under a TCS SP2 AOBS microscope (Leica, Tokyo, Japan). To investigate histone H3 phosphorylated at Ser10, the protein was detected with phospho-histone H3(Ser10) -specific rabbit polyclonal antibody (Cell Signaling Technologies, Berverly, MA).

(9) Western blot analysis

To detect the exogenous A7322 protein, pCAGGSnHsF-A7322 expression vector plasmid (20. mu.g) was transfected into BT-549 cells using FuGene 6 (Roche). After 24 hours, cells were lysed in lysis buffer (50mM Tris-HCl, pH8.0/150mM NaCl/0.1% NP-40, 0.5% CHAPS) containing 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, Calif.). The amount of total protein was estimated using a protein detection kit (Bio-Rad, Hercules, Calif.), and then the protein was mixed with SDS sample buffer, boiled and loaded on a 6% SDS-PAGE gel. After electrophoresis, the proteins were blotted onto nitrocellulose membranes (GE Healthcare). The exogenous a7322 protein was detected by blocking the protein-containing membrane with blocking solution and incubating it with anti-Flag M2 monoclonal antibody. Finally, the membrane was incubated with HRP conjugated secondary antibody and protein bands were visualized using ECL detection reagent (GE Healthcare).

To examine the expression of endogenous A7322 protein in SK-BR-3 cells, the cells were lysed with lysis buffer (50mM Tris-HCl, pH8.0, 150mM NaCl, 0.1% NP-40 and 0.5% CHAPS) containing 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, Calif.). After homogenization, the cell lysate was incubated on ice for 30 minutes and centrifuged at 14,000rpm for 5 minutes to separate the supernatant from the cell debris. The amount of total protein was determined using a protein detection kit (Bio-Rad), and then the protein was mixed with SDS sample buffer, boiled for 5 minutes and loaded on a 7.5% SDS-PAGE gel. After electrophoresis, the proteins were blotted onto nitrocellulose membranes (GE Healthcare). Endogenous a7322 protein was detected by blocking the protein-containing membrane with blocking solution (blocking solution) for 1 hour and incubating it with purified anti-a 7322 polyclonal antibody for an additional 1 hour. Finally, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour and protein bands were visualized using ECL detection reagent (GE Healthcare).

To detect endogenous F3374 and AURKB proteins in breast cancer cell lines (HBC4, BT-549, HBC5, HBL100, HCC1937, MCF-7, MDA-MB-231, MDA-MB-453, SKBR3 and T47D, ZR75-1) and human breast epithelial cells (HMEC), cells were lysed in lysis buffer (50mM Tris-HCl, pH 8.0/150mM NaCl/0.5% NP-40) containing 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, Calif.). The amount of total protein was estimated using a protein detection kit (Bio-Rad, Hercules, Calif.), and then the protein was mixed with SDS sample buffer, boiled and loaded on a 10% SDS-PAGE gel. After electrophoresis, the protein was blotted on nitrocellulose membrane (GEHealthcare). After blocking with blocking solution (4% BlockAce; Dainippon pharmaceutical. Co., Ltd, Osaka, Japan), the Western blot membrane was incubated with either 1: 100 dilution of anti-F3374 polyclonal antibody or 1: 100 dilution of anti-AURKB rabbit polyclonal antibody (abcam, Cambridge, UK) to detect endogenous F3374 or AURKB protein. Finally, the membrane was incubated with HRP conjugated secondary antibody and protein bands were visualized using ECL detection reagent (GE Healthcare). Beta-actin (Beta-actin) was also investigated as loading control.

To detect endogenous PBK/TOPK proteins in breast cancer cells (BT-20, HBC4, HBC5, HBL-100, MCF-7, MDA-MB-231, SKBR3 and T47D), the cells were lysed in lysis buffer (50mM Tris-HCl, pH 8.0/150mM NaCl/0.5% NP-40) containing 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, Calif.). After homogenization, the cell lysate was incubated on ice for 30 minutes and centrifuged at 14,000rpm for 15 minutes to separate only the supernatant and the cell debris. The amount of total protein was estimated using a protein detection kit (Bio-Rad, Hercules, Calif.), and then the protein was mixed with SDS sample buffer, boiled and loaded on a 10% SDS-PAGE gel. After electrophoresis, the proteins were blotted onto nitrocellulose membranes (GE Healthcare). The western blotted membranes were blocked with blocking solution (blocking solution) and incubated with anti-PBK/TOPK monoclonal antibodies (BDBiosciences) to detect endogenous PBK/TOPK proteins. Finally, the membrane was incubated with HRP conjugated secondary antibody and protein bands were visualized using ECL detection reagent (GE Healthcare). Beta-actin was also examined as a loading control.

Wild type and kinase-inactivated (kinase-dead) PBK/TOPK proteins were exogenously expressed by transfecting T47D cells with the pCAGGS-nHA expression vector. Whole cell lysates were collected 48 hours after transfection. Cells were lysed in the cell lysis buffer described above. The latter steps are the same as described above except that the antibody reaction uses anti-HA rat high affinity antibody (Roche). In addition, for endogenous detection of activated PBK/TOPK protein, T47D cells were treated with 100nM Okadaic Acid (OA) (Calbiochem) or 0.3 μ g/mL nocodazole (nocodazole) (Sigma-Aldrich) for 6 or 18 hours, respectively, prior to collection (see text). The latter steps are also performed as described above. Phosphorylated proteins were confirmed by treatment with 1U of lambda protein phosphatase (New England Biolabs, Ipshich, Mass.) for 2 hours at 30 ℃.

(10) Lambda phosphatase assay

To examine the phosphorylation state of the F3374 protein in breast cancer cells, the present inventors treated cell extracts from T47D cells with lambda phosphatase (New England Biolabs, Beverly, MA). The cells were lysed with NP-40 lysis buffer (50mM Tris-HCl (pH8.0), 150mM NaCl, 0.5% NP-40) in 50mM Tris-HCl (pH7.5), 0.1mM Na₂EDTA, 5mM dithiothreitol, 2mM MgCL₂And 0.01% Brij-35 in phosphatase buffer the cell lysates were treated with 400 units of protein phosphatase (New England Biolabs) at 30 ℃ for 2 hours. In addition, to clarify the phosphorylation site(s) of F7433 protein, 2X10 was applied per 10cm dish⁶Individual cells were seeded with HEK293T cells. After 24 hours, the inventors transiently transfected HEK293T cells with 8. mu.g of pCAGGS-F3374-. DELTA.1-HA,. DELTA.2 and. DELTA.3 using the FuGENE 6 transfection reagent (Roche) according to the manufacturer's instructions. The resulting mixture was washed with NP-40 buffer (0.5% NP-40, 150mM NaCl, 50mM Tris-HCl (pH7.5)), phosphatase buffer (50mM Tris-HCl, pH7.5, 0.1mM Na)₂EDTA, 5mM dithiothreitol, 2mM MgCl₂And 0.01% Brij-35) lysed cells. 48 hours after transfection, cells were lysed with NP-40 lysis buffer. The lysed cells were then treated with 400 units of protein phosphatase (P0753S New England Biolabs) for 2 hours at 30 ℃.

To investigate the phosphorylation of PBK/TOPK protein, 10ng of active PBK/TOPK protein and 15. mu.g and total mitotic cell lysate were incubated with 2 units of lambda PPase and PP1 alpha recombinant protein according to the manufacturer's instructions. After incubation at 30 ℃ for 2 hours, the reaction was stopped by adding SDS sample buffer and boiling. Finally the protein samples were electrophoresed and immunoblotted as described above.

(11) Construction of A7322, F3374V1 or PBK/TOPK specific siRNA expression vectors

The present inventors established a vector-based RNAi (RNA interference) expression system using the psiU6BX3.0siRNA expression vector as described previously (Taniuchi K et al Cancer Res., 65: 105-112.2005.). siRNA expression vectors for A7322(psiU6BX3.0-A7322), F3374V1(psiU6BX3.0-F3374V1), EGFP (psiU6BX3.0-EGFP), scrambled (scramble) (psiU6BX3.0-SCR), and Mock (Mock) (psiU6BX3.0-Mock) were prepared by cloning double-stranded oligonucleotides into the BbsI site in the psiU6BX3.0 vector. The target sequences of the synthetic oligonucleotides for siRNA are as follows:

for a 7322: si- # 2; 5'-AAGAAAGCATCGCAGTCTCAG-3' (SEQ ID NO: 34),

si- # 3; 5'-AAGATGCGTTCTCTGCCACAC-3' (SEQ ID NO: 35) and

si-#m3；5’AATATTCGATCTCTGCCACAC-3' (SEQ ID NO: 36) (the sequence of the mismatch to si- #3 is underlined);

For F3374: si- # 1; 5'-GATCATGTCTCCGAGAAAA-3' (SEQ ID NO: 37),

si-#4；5’-GGAAGCCATAGAATTGCTC-3’(SEQ ID NO：38)；

for PBK/TOPK: si- # 2; 5'-CTGGATGAATCATACCAGA-3' (SEQ ID NO: 39),

si-#3；5’-GTGTGGCTTGCGTAAATAA-3’(SEQ ID NO：40)；

for the control: si-out of order; 5'-GCGCGCTTTGTAGGATTCG-3' (SEQ ID NO: 41) and

si-EGFP；5’-GAAGCAGCACGACTTCTTC-3’(SEQ ID NO：42)。

all constructs were also confirmed by DNA sequencing.

Regarding the effect of cell growth on siRNA against p97, at 1X10 per 60 mm dish⁵Individual cells were seeded with T47D cells. After 2 days of incubation, 100pmol each of the siRNA duplexes of si-EGFP and si-p97 (5'-AAGUAGGGUAUGAUGACAUUG-3': SEQ ID NO: 121; WLou jcik C et al, JCelSci 117; 281-. After 2 days of siRNA transfection, cell morphology was observed using a phase contrast microscope (phase contrast microscopy). Cells were then harvested and equal amounts of total protein were immunoblotted with an anti-TOPK monoclonal antibody (1: 3,000) anti- β -actin monoclonal antibody (1: 10,000).

TABLE 1

(12) Gene silencing effects of A7322, F3374V1, AURKB or PBK/TOPK

Human breast cancer cell lines BT-549 and BT-474 (for A7322) and T47D and HBC4 (for F3374) were plated on 10-cm dishes (2X 10) ⁶Individual cells/dish), 8 μ g each of psiu6bx3.0-Mock (no insert), psiu6bx3.0-a7322(#2, #3, and #3 mismatch constructs containing 3 base substitutions (m #3)), psiu6bx3.0-F3374V1(#1, and #4), psiu6bx3.0-EGFP, psiu6bx3.0-SCR, were transfected as described above using FuGENE6 reagent (Roche). The present inventors selected psi U6BX3.0-introduced BT-549, BT-474, T47D and HBC4 using a medium containing 0.2mg/ml or 1mg/ml neomycin (Geneticin, Gibco BRL, Carlsbad, Calif.), respectively. 48 hours after treatment with geneticin (geneticin), cells were reseeded for colony formation assay (2X 10)⁶Individual cells/10 cm dish), RT-PCR (2X 10)⁶Cell/10 cm dish) And MTT assay (2X 10)⁵Individual cells/well). To evaluate the effect of siRNA, total RNA was extracted from cells on day 4 of neomycin incubation, and then the gene knockdown (knock down) effect of siRNA was investigated by semi-quantitative RT-PCR using the following specific primer sets:

for β 2MG as internal control: 5'-AACTTAGAGGTGGGAGCAG-3' (SEQ ID NO: 1) and

5'-CACAACCATGCCTTACTTTATC-3' (SEQ ID NO: 2) and

for a 7322: 5'-GCCCTTGAAGCCAATATTCC-3' (SEQ ID NO: 61) and

5’-AGATGGTTTCAGTGGGCTTG-3’(SEQ ID NO：62)；

For F3374V 1: 5'-GCAATCTGCTATGTCAGCCAAC-3' (SEQ ID NO: 19) and

5’-CAGGATCAGCTCAAAGTCTGACA-3’(SEQ ID NO：20)。

transfectants expressing siRNA were grown in neomycin-containing selection medium for 4 weeks, then fixed with 4% paraformaldehyde for 15 minutes, followed by staining with Giemsa solution (Giemsa's solution) (Merck, Whitehouse Station, NJ) to assess colony numbers. To quantify cell viability, MTT assays were performed 4 days post-transfection using the cell counting kit-8 (Wako, Osaka, Japan) according to the manufacturer's recommendations. The absorbance at a wavelength of 570nm was measured by a Microplate Reader 550 (Bio-Rad). These experiments were performed in triplicate.

In addition, since siRNA oligonucleotides (Sigma Aldrich Japan KK, Tokyo, Japan) have high transfection efficiency, the present inventors used it to further verify the subcellular localization of PHB2/REA protein in cells where the a7322 gene had been knocked down by siRNA. The sequence or simulation of targeting a7322 is as follows:

si-A7322；5’-GAUGCGUUCUCUGCCACACUU-3’(SEQ ID NO：63)，

siEGFP (control); 5'-GCAGCACGACUUCUUCAAG-3' (SEQ ID NO: 64).

MCF-7 cells (2.5X 10 in 10cm dishes) were transfected with those siRNAs in Optimem (Invitrogen) medium using Lipofectamin RNAImax (Invitrogen, Carlsbad, Calif.) following the manufacturer's instructions ⁵Individual cells for FACS analysis). 48 hours after transfection, cells were treated with 1 μ M ME2 (. beta. -estradiol); Sigma-Aldrich), and immunocytochemical staining and western blot analysis were performed as described in the immunocytochemical staining analysis section using anti-PHB 2/REA polyclonal antibody (abcam, Cambridge, UK), anti-A7322 antibody, and anti-ER α monoclonal antibody (LAB VISION, Fremount, Calif.). Fluorescence images were acquired under a TCS SP2 AOBS microscope.

Alternatively, the present inventors used siRNA oligonucleotides (Sigma Aldrich Japan KK, Tokyo, Japan) to further verify the knockdown effect of F3374 and AURKB in cell morphology due to their high transfection efficiency. The sequences targeting each gene are as follows:

siF 3374: 5'-ACUCCUACGUUCUCUAUUA-3' (SEQ ID NO: 65),

for siAURKB: 5'-AAGGUGAUGGAGAAUAGCAGU-3' (SEQ ID NO: 66),

for siefp (control): 5'-GCAGCACGACUUCUUCAAG-3' (SEQ ID NO: 64).

T47D or HBC4 cells (2.5X 10 in 10cm dishes) were transfected with those siRNAs using Lipofectamin RNAImax (Invitrogen) in Optimem medium (Invitrogen) according to the manufacturer's instructions⁵Individual cells for FACS analysis). At 48 hours post-transfection, morphological changes of HBC4 cells were examined using Alexa Fluor 594Phalloidin (Molecular Probe) by microscopy and immunocytochemical staining analysis.

Human breast cancer cell lines T47D and BT-20 were plated on 15-cm dishes (4X 10)⁶Individual cells/dish) were transfected with psiu6bx3.0-Mock (without insert) and psiu6bx3.0-PBK/TOPK (#2 and #3, table 1) each at 16 μ g using FuGENE6 reagent (Roche) according to the manufacturer's instructions. 24 hours after transfection, cells were reseeded for colony formation assay (2X 10)⁶Individual cells/10 cm dish), RT-PCR (2X 10)⁶Individual cells/10 cm dish) and MTT assay (1X 10)⁵Individual cells/well). T47D or BT-20 cells introduced with psiU6BX3.0 were selected using a medium containing 0.7mg/ml or 0.6mg/ml neomycin (Geneticin, Invitrogen, Gibco BRL, Carlsbad, Calif.), respectively. The medium was changed twice a week. To evaluate the effect of siRNA, total RNA was extracted from cells on day 11 of neomycin incubation, and then the knock-down (knock down) effect of siRNA was investigated by semi-quantitative RT-PCR using the following specific primer sets:

for the internal control GAPDH: 5'-ATGGAAATCCCATCACCATCT-3' (SEQ ID NO: 70) and

5'-GGTTGAGCACAGGGTACTTTATT-3' (SEQ ID NO: 10), and

for the PBK/TOPK gene: 5'-GCCTTCATCATCCAAACATT-3' (SEQ ID NO: 71) and

5’-GGCAAATATGTCTGCCTTGT-3’(SEQ ID NO：72)。

transfectants expressing siRNA were grown in selection medium containing neomycin for 3 weeks, then fixed with 4% paraformaldehyde for 15 minutes, and then stained with Giemsa solution (Merck, Whitehouse Station, NJ) to evaluate colony numbers. To quantify cell viability, MTT assays were performed using the cell counting kit-8 (Wako, Osaka, Japan) according to the manufacturer's recommendations. The absorbance at a wavelength of 570nm was measured by a Microplate Reader 550 (Bio-Rad). These experiments were performed in triplicate.

(13) Construction of truncated F3374V1 protein Using pCAGGS-HA vector

To determine the phosphorylated region of the F3374V1 protein, deletion constructs were prepared using the following primer sets: dF3374V 1-F703-NotI;

5’-AAGGAAAAAAGCGGCCGCGCTGTGGATGGGATAATCAAA-3' (SEQ ID NO: 73) and dF3374V1-R721-Xho 1;

5’-CCGCTCGAGTTTGATTATCCCATCCACAGC-3' (SEQ ID NO: 74) for the Δ -1 construct (first underlined to indicate NotI site, second underlined to indicate XhoI site), dF3374V 1-F1162-NotI;

5’-AAGGAAAAAAGCGGCCGCTGGCGCTTGAATAGAGGC-3' (SEQ ID NO: 75) and dF3374V1-R1203-Xho 1;

5’-CCGCTCGAGATCACCTCCTGGTTTCTCCTC-3' (SEQ ID NO: 76) for the Δ -2 construct (first underlined to indicate a NotI site, second underlined to indicate an XhoI site), dF3374V 1-F1729-NotI;

5’-AAGGAAAAAAGCGGCCGCCTTGATGGCCAAGTTGAAAAT-3' (SEQ ID NO: 77) and dF3374V 1-R1770-XhoI;

5’-CCGCTCGAGGCAGCACAGATCCAAATGAAG-3' (SEQ ID NO: 78) was used for the delta-3 construct (first underlined for NotI site and second underlined for XhoI site). The construct was confirmed by DNA sequencing (ABI3700, PE Applied Biosystems, Foster, Calif.).

(14) Immunohistochemical staining

To examine the expression of a7322 protein in breast cancer and normal tissues, the present inventors prepared slides of paraffin-embedded breast cancer tissue sections (sample numbers 240, 241, 238, 242, and 290), normal breast tissue sections (sample number 453), and other commercial normal human tissues (lung, heart, and liver) (BioChain). The specimens were deparaffinized by xylene and ethanol treatment, then subjected to antigen retrieval by high pressure steam at 108 ℃ for 15 minutes in a high pH antigen retrieval solution (DAKO cytometry, Glostrup, Denmark) and treated with a peroxidase blocking agent (DAKO cytometry) for 1 hour. Tissue sections were incubated with a 1: 150 dilution of anti-A7322 polyclonal antibody for 1 hour followed by a horseradish peroxidase-conjugated secondary antibody (DAKO cytotoxicity) for 30 minutes. Specific immunostaining was visualized with the peroxidase substrate (3, 3 '-diaminobenzidine tetrahydrochloride, 3, 3' -diaminobenzidine tetrahydrochloride) (DAKO liquid DAB + chromogen; DAKO Cytomation). Finally, the tissue specimens were stained with hematoxylin (hematoxylin) to distinguish the nuclei from the cytoplasm.

The expression pattern of the F3374V1 protein in breast Cancer and normal human tissues was examined as described previously (Togashi A et al, Cancer Res 2005, 65: 4817-26). Slides of paraffin-embedded breast cancer (10005T, 10317T, 10069T, 10571T, 10164T, and 10185T) specimens, normal breast tissue (10441N) specimens, and normal human tissue (lung, heart, liver, kidney, colon, pancreas, skeletal muscle, small intestine, and testis) specimens were treated with xylene and ethanol to remove paraffin. Antigen retrieval was performed by autoclaving at 121 ℃ for 15 minutes in a targetrieval Solution High pH (DAKO, Carpinteria, Calif.). F3374 was detected using ENVISION + Kit/HRP (Dakocytomation, Kyoto, Japan); after blocking reaction with endogenous peroxidase and protein, affinity purified rabbit anti-F3374 pAb diluted 1: 50 was added as primary antibody and the mixture was treated with HRP-labeled anti-rabbit IgG. Finally, matrix-chromogen (substrate-chromogen) was added and the tissue specimens were counterstained with hematoxylin to distinguish the nuclei from the cytoplasm.

The expression pattern of the PBK/TOPK protein in breast Cancer as well as in normal tissues was investigated using anti-PBK/TOPK mouse monoclonal antibodies (BD Biosciences) as described previously (Togashi A et al, Cancer Res 2005, 65: 4817-26). For the study of normal organs, commercial tissue sections of heart, lung, liver, kidney and testis (Biochain) were purchased. Specifically, paraffin-embedded specimens were treated with xylene and ethanol and blocked with a protein blocking reagent (Dako Cytomation, Carpinteria, CA). An antibody dilution solution (1: 50) containing a monoclonal antibody was added, and staining was performed with a substrate-chromogen (DAKO liquid DAB chromogen, DakoCytomation). Finally, the tissue specimens were stained with hematoxylin to distinguish the nuclei from the cytoplasm.

(15) Fluorescence Activated Cell Sorting (FACS) analysis

BT-474 breast cancer cells, on which siRNA experiments were performed as described above, were harvested after 2 days of incubation in selection medium containing 1.0mg/ml neomycin. Cells were collected and fixed with cold 70% ethanol and maintained at 4 ℃ prior to use. Cells were incubated at 37 ℃ in PBS (-) containing 10mg/ml RNase I for 30 minutes and stained with 50. mu.g Propidium Iodide (PI) for 30 minutes at room temperature. The DNA content of the cell suspension was analyzed using a flow cytometer (FACS calibur; Becton Dickinson, San Diego, Calif.). Data were analyzed using CELLQuest software (BD Biosciences). The assays were performed independently in triplicate.

Cell cycle synchronization of cultured T47D breast cancer cells was synchronized by treatment with 2. mu.g/ml aphidiclin (Sigma-Aldrich) for 24 hours. Next, cells were washed 5 times with PBS (-) and fresh media was added to relieve cell cycle arrest. After cell cycle arrest was released, cells were harvested, fixed with 70% ethanol, and then kept at 4 ℃ until use. The cells were incubated at 37 ℃ in PBS (-) containing 10mg/ml RNase I for 30 minutes and stained with 50. mu.g propidium iodide at room temperature for 30 minutes. Cell suspensions at each time point were analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ). In addition, to examine the expression level of the endogenous F3374 protein, the present inventors performed western blotting using an anti-F3374 polyclonal antibody on cells collected every 3 hours, as described in the western blotting analysis section.

For PBK/TOPK, cultured T47D breast cancer cells were synchronized in their cell cycle by treatment with African forest (Sigma-Aldrich) for 16 hours, washed 5 times with PBS (-) and fresh media was added to relieve cell cycle arrest. At 15 hours (every 3 hours) after release, cells were harvested, fixed with 70% ethanol, and then kept at 4 ℃ until use. The cells were incubated with 10mg/ml RNase I in PBS (-) at 37 ℃ for 30 minutes and stained with 50. mu.g propidium iodide at room temperature for 30 minutes. Cell suspensions at each time point were analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ).

To collect G2/M arrested cells, the medium was treated with 0.3. mu.g/mL nocodazole (nocodazole) (Sigma-Aldrich) for the last 16 hours prior to collection.

(16) Co-immunoprecipitation and immunoblot analysis

To determine the interaction protein of the A7322 protein, BT-549 human breast cancer cells were plated on 15cm dishes (1X 10)⁷Individual cells/dish) were transfected with 20 μ g of pCAGGSnH3F-Mock (without insert) and pCAGGSnH3F-a7322, respectively, using FuGENE6 reagent (Roche) according to the manufacturer's instructions. The inventors transfected 6 dishes for each construct. After 48 hours, cells were lysed with 0.1% NP-40 lysis buffer as described in the Western blot analysis section. Cell lysates were pre-cleaned with normal mouse IgG and rec-Protein G Sepharose 4B (Zymed, San Francisco, Calif.) at 4 ℃ for 1 hour. Subsequently, the lysates were incubated overnight at 4 ℃ with anti-FLAG M2 agarose (Sigma-Aldrich). After washing 5 times with lysis buffer, the proteins on the beads were eluted by boiling the SDS sample buffer for 5 minutes. Eluted protein samples were separated by SDS-PAGE using NuPAGE 4-12% Bis-Tris gels (Invitrogen). The proteins in the polyacrylamide gels were Silver stained using the SilverQuest Silver Staining Kit (Invitrogen) according to the manufacturer's instructions. Differential bands between the simulated and A7322 transfected lanes were excised with a clean, sharp scalpel and PMF (Peptide Mass fingerprinting) analysis was performed using MALDITOF-MS (Shimadzu Biotech, Tsukuba, Japan).

COS-7 cells were transiently transfected with pCAGGSn3FC-A7322, pCAGGSnHC-PHB2/REA, either alone or in combination. 48 hours after transfection, cells were lysed with 0.1% NP-40 lysis buffer as described in the Western blot analysis section. Cell lysates were precleaned at 4 ℃ for 1 hour, followed by incubation of the cell lysates with anti-FLAG M2 agarose (Sigma-Aldrich) or monoclonal anti-HA agarose conjugate (Sigma-Aldrich) overnight at 4 ℃. The beads were washed and the proteins eluted as before. Finally, the inventors performed Western blot analysis using either anti-HA high affinity (3F10) rat monoclonal antibody (Roche) or anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) to detect exogenously expressed PHB2/REA or A7322 proteins, respectively.

HEK293T cells were plated on 5 dishes (8X 10)⁶Cells/15 cm dish), 8. mu.g of plasmid expressing F3374(pCAGGSn-F3374-HA) and AURKB (pCAGGSn-AURKB-3F) were co-transfected. The pCAGGSn-AURKB-3F plasmid was previously prepared (unpublished data; Daigo Y and Nakamura Y). 48 hours after transfection, immunoprecipitation buffer (50mM Tris-HCl (pH 7.5), 150mM NaCL, 0.5% NP-40, 50mM NaF, 1mM NaVO₃And 1mM dithiothreitol) in the presence of a protease inhibitor (Calbiochem). Cell lysates were pre-clarified by incubation with 3.75 μ G normal mouse IgG for 3 hours at 4 ℃ in 200 μ l protein G-Sepharose beads (Zymed Laboratories, South San Francisco, Calif.). After centrifugation at 14,000rpm for 1 minute at 4 ℃, the supernatant was incubated with 30. mu.g of anti-Flag M2 or mouse normal IgG for 1 hour at 4 ℃ and then 100. mu.l of protein G-sepharose beads were added. After collecting the beads from each sample by centrifugation at 14,000rpm for 1 minute and washing 5 times with 1ml of immunoprecipitation buffer, they were eluted with 30. mu.l of Laemmli sample buffer and boiled for 5 minutes. Proteins were separated on an 8% SDS-PAGE gel (Bio-Rad). After electrophoresis, the proteins were detected by western blot analysis as described in the western blot analysis section.

To investigate the interaction of endogenous F3374V1 and AURKB proteins, 6X10 was used⁶Each cell/15 cm dish was seeded with T47D cells. Two days later, in immunoprecipitation buffer (50mM Tris-HCL (pH7.5), 150mM NaCL, 0.5% NP-40, 50mM NaF, 1mM NaVO₃And 1mM DTT) in the presence of protease inhibitor (Calbiochem). Cell lysates were pre-clarified by incubation with 7.5. mu.g normal mouse IgG for 3 hours at 4 ℃ in 750. mu.l protein G-Sepharose beads. The supernatant was incubated with 30. mu.g of anti-F3374V 1 antibody or rabbit normal IgG for 1 hour at 4 ℃ and then 100. mu.l of protein G-Sepharose beads were added. The beads were collected from each sample by centrifugation at 14,000rpm for 1 minute, and after washing 5 times with 1ml of immunoprecipitation buffer, they were washed with 30. mu.l of Laemmli sample buffer was eluted and boiled for 5 minutes. Proteins were separated on an 8% SDS-PAGE gel (Bio-Rad). After electrophoresis, the proteins were detected by western blot analysis as described in the western blot analysis section.

For detection of PBK/TOPK (WT, T9A, KD, T9A/KD), p47, p97 or

Expression of the protein was immunoblotted as previously reported (Park et al, 2006). Briefly, after lysis of cells in lysis buffer (50mM Tris-HCl, pH 8.0/150mM NaCl/0.5% NP-40), homogenization and incubation on ice for 30 minutes, the soluble fraction was separated only from the cell debris by centrifugation. After SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), the proteins were blotted onto nitrocellulose membranes (GEHealthcare, Buckinghamshire, United Kingdom) and incubated with the corresponding antibodies, and visualized using the ECL detection kit (GE Healthcare). To collect mitotic cells (mitotic cells), the present inventors used the "mitotic shake-off" method previously described (Dechat T et al, EMBO J17: 4887-902 (1998)). After co-transfection of COS-7 cells with the construct of interest described above using FuGene6 reagent (Roche), protein interactions were investigated by immunoprecipitation as described previously (Shimo A et al, Cancer Sci 98: 174-81(2007)), except that an anti-6 XHis antibody was used for the precipitated p97/VCP-myc-6XHis protein (Santa Cruz Biotechnology). After washing 5 times with lysis buffer, the immune complexes were loaded onto SDS-PAGE gels and immunoblotted as described above.

(17) In vitro and in vivo kinase assays

To examine phosphorylation of F3374V1 by AURKB, the present inventors performed in vitro kinase assays using the C-terminal recombinant protein of F3374 (amino acid 437-730) and the full-length recombinant protein of AURKB (Upstate, Temecula, Calif.). According to the preparation of the anti-F3374 polyclonal antibodyThe F3374 recombinant protein was prepared by the method of (1). Briefly, 1. mu.g of AURKB was added to 20. mu.l of kinase assay buffer (50mM Tris-HCL (pH 7.5), 10mM MgCL₂2mM dithiothreitol, 1mM EGTA, 0.01% Brij35, 500. mu.M ATP) and then 5. mu. Ci [ alpha ], [ solution ]³²P-α]Atp (ge healthcare). As a substrate, the present inventors added 1. mu. g F3374 recombinant protein to the reaction solution. After incubation at 30 ℃ for 10 minutes, the reaction was stopped by adding SDS sample buffer. Protein samples were boiled, run on 10% SDS gels, and then autoradiographed.

To assess the kinase activity of PBK/TOPK, an in vitro kinase assay was performed using the full-length recombinant PBK/TOPK protein (Invitrogen, Carlsbad, CA). Specifically, 1. mu.g of PBK/TOPK protein was assayed in 30. mu.l of kinase assay buffer (50mM Tris-HCl, pH 7.5/150mM NaCl/10mM MgCl)₂/10mM NaF/1mM Na₃VO₄Incubation in/1 mM EDTA/1mM DTT/50uMATP) followed by addition of 5. mu. Ci of ( ³²P-gamma) -ATP (GE healthcare). Mu.g of histone mixture or 2.5. mu.g of histone H3(Roche) was added to the reaction solution as a substrate. After incubation at 30 ℃ for 30 minutes, the reaction was stopped by adding SDS sample buffer. Protein samples were boiled and electrophoresed on 10-20% gradient gels (Bio-Rad) followed by autoradiography. To further investigate whether PBK/TOPK phosphorylates histone H3(Ser10), the wild-type protein and kinase-inactivating (kinase-dead) mutant (K64-65A) were transfected into T47D cells. After 48 hours of culture, the cells were treated with 100nMOA for 6 hours to activate the PBK/TOPK protein. The total amount of H3 protein and its degree of phosphorylation were examined with anti-histone H3(Abcam, Cambridge, UK) and anti-phospho-H3 (Ser10) antibodies (Cell Signaling Technologies), respectively.

For in vitro kinase assays, 5. mu. Ci was added³²P-. gamma. -ATP (GE healthcare) in 30. mu.l reaction buffer (50mM Tris-HCl, pH 7.5/10mM MgCl)₂2mM dithiothreitol/1 mM MEGTA/0.01% Brij 35/50. mu.M cold ATP) 0.5. mu.g of non-activated recombinant PBK/TOPK protein prepared with an E.coli expression system was incubated with 0.5 units CDK 1-cyclin B1(New England Biolabs). Incubation at 30 ℃After 30 minutes, the reaction was stopped by adding SDS sample buffer. Protein samples were boiled, electrophoresed, and autoradiographed.

To investigate that p97 is a substrate for PBK/TOPK kinase, we performed an in vitro kinase assay. Briefly, the precipitated p97 protein was reacted with 1. mu.g of recombinant TOPK protein as described above.

(18) Cell culture and transfection in the absence of Estrogen

MCF-7 or SK-BR-3 cells were cultured in the following media, respectively: phenol-free red D-MEM/F-12 or RPMI-1640(Invitrogen), supplemented with 10% FBS (Cansera International) and 1% antibiotic/antifungal solution (Sigma-Aldrich) filtered with a minisart-plus (Sartorius AG, Goettingen, Germany). The cells were maintained at 37 ℃ in a 5% CO atmosphere₂Is in an atmosphere of humid air. Transfection was performed with phenol-free Opti-MEM (Invitrogen) using FuGENE6 transfection reagent (Roche) according to the manufacturer's instructions. 24 hours after transfection, the medium was replaced with Opti-MEM without phenol red containing 1. mu. M E2 (. beta. -estradiol; Sigma-Aldrich) and incubated for an additional 24 hours. Immunocytochemical staining was performed using 1: 500 dilution of anti-HA high affinity (3F10) rat monoclonal antibody (Roche) and anti-FLAG rabbit polyclonal antibody (Sigma-Aldrich), respectively, and 1: 1000 dilution of Alexa 488-conjugated anti-rat mouse secondary antibody and Alexa 594-conjugated anti-rabbit OR anti-rat secondary antibody (Molecular Probe, Eugene, OR), respectively.

(19) Estrogen Response Element (ERE) reporter gene assay

ERE reporter construction plasmids and fluorescent SEAP detection kits were purchased from Clontech (Takara, Kyoto, Japan). MCF-7(ER +) or SK-BR-3(ER-) cells were co-transfected with FLAG-tagged A7322(FLAG-A7322) construct and estrogen-responsive reporter (pERE-TA-SEAP) construct or mock control and pERE-TA-SEAP reporter construct, respectively, using FUGENE transfection reagent (Roche). 48 hours after transfection, cells were treated with 1. mu. M E2 (. beta. -estradiol; Sigma-Aldrich) and incubated for additional 48 hours and 72 hours for SEAP assay and western blot analysis, respectively. SEAP reporter gene assays were performed using SEAP assay kit (Clontech) according to the supplier's recommendations.

(20) Statistical analysis

Statistical significance (Statistical significance) was calculated by Student's t test (Student's t test) using Statview 5.0 software (SAS Institute, Cary, NC). Differences of P < 0.05 were considered statistically significant.

(21) Proteins, constructs, antibodies and reagents

Active recombinant PBK/TOPK proteins recombinant proteins from Invitrogen (Carlsbad, CA), Histone H3 and protein phosphatase 1-alpha (PP 1. alpha.) were obtained from Upstate Biotechnology (Lake Placity, NY). Cyclin-dependent kinase 1 (CDK 1 kinase) (cdc 2/Cyclin B1) and lambda protein phosphatase (lambda PPase) were from New England Biolabs (Ipswich, MA). The Glutathione S-transferase (GST) -tagged PBK/TOPK (GST-PBK/TOPK) protein was expressed in E.coli and purified using Glutathione Sepharose 4B beads (GE healthcare care, Buckinghamshire, United kingdom) as previously reported (Lin et al, 2007). According to the previous report (Park et al, 2006), N-terminal HA-tagged PBK/TOPK (HA-PBK/TOPK) and N-terminal GST-tagged PP1 alpha (GST-

) And a C-terminal GST-tagged p47(p47-GST) protein. An N-terminal HA-tagged mutant of alanine substitution at Thr9(T9A), kinase-inactivated (KD) and double mutant (T9A/KD) proteins were constructed using pCAGGSnHA. The C-terminal myc-6 XHis-tagged p97/VCP-myc-6XHis (p97/VCP-myc-His) was also constructed using the pCDNA3.1-myc-His vector (Invitrogen). Monoclonal antibodies to PBK/TOPK, beta-actin (beta-actin) and HA were purchased from BDbiosciences (San Jose, CA), Sigma-Aldrich (St. Louis, MO) and Roche (Basel, Switzerland), respectively. Polyclonal antibodies against Total PP1 α, phospho-PP 1 α (T320) were purchased from CellSignaling technologyes (Berverly, MA), Total Rb, phospho-Rb (Ser807/811) and p97/VCP were from Santa Cruz Biotechnology (Santa. Cruz, Calif.). Okadaic Acid (OA), CDK1 inhibitor (CGP74514A) and protease inhibitor cocktail III were purchased from Calbiochem (San Diego, CA).

(22) Immunocytochemical staining of PBK/TOPK, CDK 1-cyclin B1, p47, p97 and PP1 alpha

T47D cells at 5X10⁴Individual cells were seeded in 35mm dishes (IWAKI) with col-I coated glass. After two days of incubation, cells were studied for mitosis by treatment with 0.3. mu.g/mL nocodazole (nocodazole) (Sigma-Aldrich) for an additional 18 hours. After fixation and blocking, cells were immunostained with either 1: 100 diluted anti-TOPK monoclonal antibody (BD Biosciences) or 1: 300 diluted anti-CDK 1 monoclonal antibody (BD Biosciences) and cyclin B1 monoclonal antibody (Santa Cruz). Finally, cells were stained with Alexa594(PBK/TOPK) or 488-conjugated (CDK1 and cyclin B1) anti-mouse secondary antibodies (Molecular probes) diluted 1: 1000. Nuclei were counterstained with 4 ', 6 ' -diamidino-2 ' -phenylindole Dihydrochloride (DAPI). Fluorescence images were acquired under a confocal microscope (Leica).

For investigating exogenous expression of p47, p97 or

Subcellular localization of proteins, we performed 1X10 on 6-well plates with col-1 coated slides⁵Each cell was seeded with T47D cells. 48 hours after transfection, cells were fixed with 4% paraformaldehyde (Paraformaldehyde) in PBS (-) for 15 minutes and treated with 0.1% Triton X-100 in PBS (-) at 4 ℃ for 2.5 minutes to permeabilize the cells. Next, the cells were covered with 3% BSA in PBS (-) at 4 ℃ for 12 hours to block non-specific hybridization, followed by 1 hour incubation with each of the first and second fluorescently labeled antibodies diluted with 3% BSA in PBS (-) (Park et al, 2006).

(23) Cell-permeable peptide treatment and inhibition of phosphorylation of PBK/TOPK at Thr9

To inhibit phosphorylation of PBK/TOPK by CDK 1-cyclin B1 at Thr9, we designed a permeable peptide identical to the N-terminus of PBK/TOPK (RRRRRRRRRRR-G-MEGISNFKTPSKLSEKKK: SEQ ID NO: 99) (pp1-18), synthesized by Sigma-Aldrich. The potency of the peptides to block PBK/TOPK phosphorylation by CDK 1-cyclin B1 was estimated by in vitro kinase assays as described in the in vitro kinase section. Inactive PBK/TOPK and CDK 1-cyclin B1 recombinant proteins were incubated as described above, except that the pp1-18 peptide was added at various concentrations of 0. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M and 20. mu.M, respectively. Phosphorylation of PBK/TOPK and cyclin B1 was observed by SDS-PAGE and autoradiography. For cell proliferation assay, at 1.3 × 10 ⁴Individual cells were seeded with T47D and HMEC cells in 12-well plates, respectively. The following day, each concentration of pp1-18 peptide (0 μ M, 2.5 μ M, 5 μ M and 10 μ M) was treated daily (treated every day), and cell viability was determined by MTT assay (cell viability) on day 5. The significance of the pp1-18 peptide in inhibiting the growth of T47D cells was statistically estimated by the Student's T test. To inhibit phosphorylation of PBK/TOPK in mitotic cells by using pp1-18 peptide, T47D cells were plated at 1X10⁵Individual cells were seeded in 60mm dishes. Two days after incubation, collected after 18 or 24 hours of additional treatment with nocodazole (0.3. mu.g/mL) and permeable peptide (10. mu.M), and then the phosphorylation of PBK/TOPK was studied by western blotting and FACS analysis using anti-PBK/TOPK monoclonal antibodies. On day 5 post-treatment, the cell morphology of T47D cells treated with 50. mu.M pp1-18 peptide was observed using phase contrast microscopy.

(24) GST pull-down test

GST-tagged PP1 alpha and p47 proteins were co-expressed with/without HA-tagged PBK/TOPK proteins in COS-7 cells. Each cell lysate was prepared using lysis buffer as described in the section above. Total protein was incubated with equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 1 hour at 4 ℃. After 5 washes with lysis buffer, the final pellet beads were maintained at-20 ℃ before use in SDS-PAGE.

(25) Observation of changes in cell architecture

Cell morphology was observed with a phase contrast microscope (Olympus) 2 days after transfection with siRNA.

In RNAi-rescue experiments, T47D cells were transfected with the pCAGGS-PBK/TOPK-HA construct 24 hours prior to transfection with each siRNA as described previously (Zhu C et al, Proc Natl Acad Sci U S A103: 6196-cell 201 (2006)). At 1x10⁵Individual cells were seeded in 60mm dishes with T47D cells. After two days of incubation, cells were transfected with 100pmol si-EGFP or siPBK/TOPK- #3 and the duration of mitosis was measured using a time-shift microscope (Sanyo).

Example 2-A7322

(1) Identification of a7322 as a gene up-regulated in breast cancer

To identify molecules that could be used as targets for novel therapeutic drugs, the present inventors previously established a genome-wide gene expression pattern for 81 breast cancer patients using cDNA microarrays representing 27,648 cDNAs (Nishidate T et al Int J Oncol 2004; 25: 797-. In up-regulating genes, the present inventors focused on a7322, whose expression is up-regulated in most breast cancer specimens. Subsequent semi-quantitative RT-PCR and northern blot analysis confirmed that a7322 was significantly upregulated in breast cancer specimens (fig. 1A) and breast cancer cell lines (14/22), but not expressed in normal organs (except brain) (fig. 1B and fig. 1D).

As shown in fig. 1D, the assembled cDNA sequence of a7322 in the NCBI database was shorter compared to the approximately 15kb transcript from northern blot analysis, so the inventors performed exon ligation and 5' RACE experiments to obtain full-length a7322 mRNA. The inventors finally obtained a cDNA sequence of 14,763 nucleotides (Genbank accession AB252196) (SEQ ID NO: 23) containing an open reading frame of 6534 nucleotides encoding a 2,177 amino acid protein (SEQ ID NO: 24). The simple modular architecture search tool (SMART) program shows that the predicted A7322 protein contains a Sec7 domain between codons 586 and 798, which may be necessary for proper transport of the protein across the Golgi (Chardin P et al Nature 1996; 384: 481-4; Jackson CL et al Trends Cell Biol 2000; 10: 60-7; ShinHW et al J Biochem (Tokyo) 2004; 136: 761-7).

To investigate exogenous expression of a7322, the present inventors transfected a7322 expression vector into BT549 breast cancer cells, and then performed western blot analysis 24 hours after transfection. Figure 1G shows that approximately 250kDa a7322 was detected in BT549 24 hours post-transfection.

(2) Immunocytochemistry and immunohistochemistry analysis of A7322

To investigate the endogenous a7322 protein, the inventors prepared anti-a 7322 polyclonal antibodies (see (6) preparation of anti-a 7322 polyclonal antibodies and anti-F3374 polyclonal antibodies). The inventors first confirmed that the purified a7322 polyclonal antibody can recognize an endogenous a7322 protein of approximately 250-kDa in a breast cancer cell line (SK-BR-3) without producing any non-specific band (fig. 2A). The inventors performed immunocytochemical staining analysis using SK-BR-3 breast cancer cells with an anti-a 7322 polyclonal antibody, which was found to detect a strong signal of the endogenous a7322 protein in the cytoplasm (fig. 2B). Although the inventors counterstained mitochondria or golgi using MitoTracker or anti-golgi monoclonal antibodies, a7322 was not co-localized in both organelles (fig. 2C and 2D).

In addition, the present inventors performed immunohistochemical staining experiments using breast cancer and normal tissue sections. The inventors have shown that breast cancer is present in two different histological subtypes: strong staining was observed in the cytoplasm of two papillary tubular carcinomas (papillotubular carcinomas) and three solid tubular carcinomas (solid-tubular carcinomas) (FIG. 2E). However, no staining was observed in the normal breast ducts (fig. 2E) or in the heart, lungs and liver (fig. 2E).

(3) Growth inhibition by A7322-directed siRNA

To evaluate the growth promoting effect of a7322, the present inventors knocked down the expression of endogenous a7322 in breast cancer cell lines BT-549 and BT-474 that have shown a7322 overexpression, by mammalian vector-based RNA interference (RNAi) techniques (see (11) construction of a7322, F3374V1, or PBK/TOPK-specific siRNA expression vectors and (12) gene silencing effects of a7322, F3374V1, AURKB, or PBK/TOPK).

The present inventors investigated the expression level of a7322 by semi-quantitative RT-PCR analysis. The a 7322-specific sirnas (si- #2 and si- #3) significantly inhibited the expression of each gene compared to the control siRNA construct (psiU6 BX-Mock) (fig. 5A, 5D, and 5G). To confirm the cell growth inhibition of a 7322-specific siRNA, the inventors performed colony formation (fig. 5C and 5F) and MTT assays (fig. 5B, 5E and 5H), respectively. The inventors also generated sirnas containing 3bp substitutions in si- #3 (si-a 7322-mismatch (m #3), see "materials and methods") and found that this had no inhibitory effect on the expression of a7322 or the growth of BT-549 and BT-474 cells (fig. 5D, fig. 5E, fig. 5F, fig. 5G and fig. 5H), suggesting that the si- #2 construct had a specific knockdown effect on a 7322. Introduction of si- #2 and si- #3 constructs inhibited the growth of BT-549 and BT-474 cells, consistent with the above reduction in expression. Each result was validated by 3 independent experiments. These findings suggest that a7322 has an important function in the cell growth of breast cancer cells.

Alternatively, the inventors investigated the relationship of apoptosis since depletion of a7322 resulted in a significant decrease in colony number and cell viability. The present inventors performed Fluorescence Activated Cell Sorting (FACS) analysis to detect the proportion of apoptotic cell populations. The results show a significant increase in the population of apoptotic (sub-G1) cells caused by si- #3 compared to the mock (fig. 5I), indicating that inhibition of a7322 expression causes apoptosis.

(4) Identification of PHB2/REA as an interacting protein of A7322

Since the biological function of a7322 is completely unknown, the present inventors searched for proteins that interact with a7322 by immunoprecipitation and mass spectrometry analysis (see (16) co-immunoprecipitation and immunoblot analysis) to investigate the biological function of a7322 protein in breast cancer cells. Lysates of BT-549 cells transfected with pCAGGSnH3F-A7322 vector or pCAGGSnH3F-Mock (Mock control) were extracted and immunoprecipitated with anti-FLAG M2 monoclonal antibody (see (16) co-immunoprecipitation and immunoblot analysis). The protein complexes were silver stained on SDS-PAGE gels. An approximately 30-kDa protein was extracted, which was visible in the immunoprecipitates of cell lysates transfected with FLAG-tagged a7322 plasmid, but not in the immunoprecipitates of cell lysates transfected with mock control plasmid, whose peptide sequence was determined by mass spectrometry analysis (fig. 8A). This method identified inhibin 2 (probibitin)/inhibitor of estrogen receptor activity (PHB2/REA) as a candidate for an a7322 interacting protein (fig. 8A). Subsequent semi-quantitative RT-PCR confirmed PHB2/REA expression in 9 of the 10 breast cancer clinical samples examined and 16 of the 22 breast cancer cell lines, similar to the expression of A7322 (FIG. 8B). To investigate the interaction between A7322 and PHB2/REA proteins, the present inventors constructed plasmids designed to express FLAG-tagged A7322(A7322-FLAG) and HA-tagged PHB2/REA (PHB2/REA-HA) (see (5) construction of expression vectors). These plasmids were co-transfected into COS-7 cells, and the proteins were then immunoprecipitated with anti-FLAG antibody. Immunoblotting of the pellet with anti-HA antibody showed that A7322-FLAG co-precipitated with PHB2/REA-HA (FIG. 8C: left panel). In contrast, the inventors immunoprecipitated using anti-HA antibody, and then immunoblotted the precipitate with anti-FLAG antibody. The results showed that PHB2/REA-HA was co-precipitated with A7322-FLAG (FIG. 8C: right panel). Alternatively, to examine the localization of endogenous PHB2/REA and A7322 proteins in breast cancer cell lines (SK-BR-3), the inventors performed immunocytochemical staining analysis using polyclonal antibodies against PHB2/REA (see (8) immunocytochemical staining). The inventors observed that PHB2/REA and a7322 were predominantly localized in the cytoplasm in most cells (fig. 8D), but their expression was observed in both cytoplasm and nucleus in a small number of cells (fig. 8D, arrows), suggesting that those proteins may be partially co-localized at the cytoplasm in breast cancer cells.

PHB2/REA is a protein that is recruited to the estrogen receptor alpha (ER α) occupied by hormones (Osborne CK. Breast Cancer Res Treat 1998; 51: 227-38), which reportedly selectively inhibits the transcriptional activity of ER α through interactions in the nucleus (Montano MM, et al ProcNatl Acad Sci USA 1999; 96: 6947-52; Delage-Mourroux R, et al J Biol Chem 2000; 275: 35848-56), so the inventors investigated the possibility that A7322 binds to ER α proteins in addition to PHB 2/REA. To investigate the interaction between a7322 and ER α proteins, the inventors constructed plasmids designed to express FLAG-tagged ER α and HA-tagged a7322 (see (5) construction of expression vectors). These plasmids were co-transfected into COS-7 cells, and the proteins were then immunoprecipitated with anti-FLAG antibody. Immunoblotting of the pellet with anti-HA antibody showed that the two proteins were not co-immunoprecipitated (FIG. 9A: left panel). When the inventors immunoprecipitated with anti-HA antibody and immunoblotted with anti-FLAG antibody, the inventors did not yet observe the interaction of these proteins (FIG. 9A: right panel). Furthermore, immunocytochemistry analysis showed that a7322 was expressed in the cytoplasm and era in the nucleus in the presence or absence of estradiol (E2), supporting no interaction of the two proteins (fig. 9B). In addition, similar results were observed when the inventors used SK-BR-3 cells showing no ER expression (ER-) (FIG. 9C). In conclusion, the inventors concluded that a7322 binds directly to PHB2/REA, whereas it showed no direct binding to era protein, regardless of the ER status of breast cancer cells.

(5) A7322 inhibiting PHB2/REA nuclear transport

PHB2/REA was reported to localize predominantly in the cytoplasm and to migrate to the nucleus in the presence of E2 and ER α (Kasashima K, et al J Biol Chem 2006; 281: 36401-10). Since the inventors observed that a7322 localized to the cytoplasm regardless of the presence or absence of E2, the inventors hypothesized that a7322 might interact with PHB2/REA and interfere with PHB2/REA nuclear transport in the cytoplasm. To investigate this hypothesis, the inventors investigated the subcellular distribution of PHB2/REA protein in the presence or absence of a7322 expression. The present inventors transfected constructs of HA-tagged PHB2/REA (HA-PHB2/REA), FLAG-tagged ER α (FLAG-ER α), and FLAG-tagged A7322(FLAG-A7322) or mock controls into MCF-7(ER +) cells, followed by immunocytochemical staining (see (5) construction of expression vectors and (8) immunocytochemical staining).

The results showed that PHB2/REA and ER α were translocated to the nucleus in the absence of A7322 (FIG. 10A: left panel, arrow), while remaining cytoplasmic in the presence of A7322 treated with E2 (FIG. 10A: right panel). A difference in PHB2/REA subcellular localization with or without A7322 was also observed in SK-BR-3(ER-) cells (FIG. 10B). Furthermore, the inventors investigated the subcellular localization of endogenous PHB2/REA in A7322-knocked-down MCF-7 cells. FIG. 10C shows confirmation of knockdown of A7322 expression in MCF-7 cells. As expected by the present inventors, PHB2/REA was observed in the nucleus 48 hours after E2 treatment in a 7322-knockdown cells, but PHB2/REA was still observed in the cytoplasm in control siRNA (si-EGFP) -treated cells (fig. 10D). Thus, these results suggest that binding of A7322 to PHB2/REA inhibits nuclear transport of PHB2/REA, reduces ER α -PHB2/REA interactions, and may lead to enhanced ER α transcriptional activity.

(6) Enhancement of ER transcriptional activity by inhibition of nuclear transport of endogenous PHB2/REA

As described above, the present inventors observed that a7322 interferes with the nuclear transport of PHB2/REA by interacting with PHB2/REA in the cytoplasm, so the present inventors hypothesized that the a7322 protein enhances ER transcriptional activity by inhibiting the nuclear transport of PHB2/REA in breast cancer cells. To test this hypothesis, the inventors co-transfected the FLAG-tagged a7322(FLAG-a7322) construct and the estrogen response reporter (pERE-TA-SEAP) construct or mock control and the pERE-TA-SEAP reporter construct, respectively, into MCF-7(ER +) or SK-BR-3(ER-) cells, and then performed reporter gene assays using the SEAP assay kit (see (19) Estrogen Response Element (ERE) reporter gene assay).

The inventors determined the expression of the exogenous a7322 and endogenous PHB2/REA proteins in those cells by western blot analysis (fig. 11A). Surprisingly, the introduction of a7322 protein significantly enhanced ER transcriptional activity in MCF7(ER +) cells 48 hours after E2 treatment, but not in SK-BR-3(ER-) cells (fig. 11B). These findings suggest that the a7322 protein might enhance ER transcriptional activity by inhibiting nuclear transport of PHB2/REA in breast cancer cells.

Discussion of the related Art

The identification and characterization of cancer-associated genes and their products has prompted the development of molecularly targeted drugs for cancer therapy over the past 20 years. The proportion of patients showing an effective response to currently available treatments is still not high (Taniuchi K, et al Cancer Res 2005; 65: 3092-9). Therefore, there is an urgent need to develop novel anticancer drugs having high specificity to malignant cells, with minimal or no adverse effects. Herein, through an accurate analysis of the expression pattern of breast cancer, the present inventors identified a7322, which is significantly overexpressed in most breast cancer cases and breast cancer cell lines. Furthermore, northern blot analysis showed that expression of a7322 could hardly be detected in any normal human tissues examined (except brain). Immunohistochemical staining experiments using anti-a 7322 polyclonal antibodies clearly indicated up-regulation of a7322 expression in breast cancer cells, but not in surrounding normal cells or vital organs.

The present inventors have also characterized some biological functions of the a7322 protein, suggesting that it would be an excellent candidate molecular target for breast cancer therapy. The inventors have demonstrated, by siRNA technology, that the knockdown of endogenous a7322 expression leads to significant growth inhibition of breast cancer cells. Furthermore, the present inventors found by our cDNA microarray analysis that a7322 is generally up-regulated in almost all cancers, including bladder cancer (blast cancer), colon cancer (colon cancer), non-small cell lung cancer (non-small cell lung cancer), prostate cancer (prostate cancer), and breast cancer (breast cancer). These results indicate that this gene will serve as a valuable target for the development of anti-cancer agents for various types of clinical cancer.

To find clues on the biological significance of a7322 in breast cancer cells, the present inventors searched for proteins that might interact with a7322 by immunoprecipitation and mass spectrometry, and identified PHB2/REA as an a 7322-interacting protein. The present inventors demonstrated in vivo interaction and co-localization of cytoplasmic A7322 and PHB2/REA in breast cancer cells. PHB2/REA is known to be an estrogen receptor alpha (ER α) -selective regulator inhibiting the transcriptional activity of ER α with estradiol as a ligand (Kasashimak, J Biol Chem 2006; 281: 36401-10). Thus, the inventors hypothesized that a7322 activates the transcriptional activity of ER α by inhibiting the interaction of ER α and PHB2/REA (fig. 11C), leading to the possible activation of ER-downstream genes.

In summary, our findings clearly suggest that a7322 is overexpressed in both breast cancer specimens and cancer cell lines, and that its interaction with PHB2/REA may play a significant role in promoting breast cancer cell growth. Recent strategies for developing anticancer drugs have focused on target molecules that are critically involved in oncogenic pathways, such as imatinib mesylate (imatinib mesylate) and trastuzumab (trastuzumab). The inventors found that a7322 down-regulation by siRNA treatment significantly inhibited cell growth of breast cancer, suggesting a key role in breast cancer proliferation and tumor formation. In particular, the present inventors proposed the possibility that a7322 could act by inhibiting nuclear transport of PHB2/REA protein to reactivate ER α in the case of mammary carcinogenesis. These data would facilitate a better understanding of breast cancer development and suggest that a7322 is a promising molecular target for breast cancer therapy.

Example 3F 3374

(1) Identification of F3374 as a gene upregulated in breast cancer

To identify molecules that could be used as targets for novel therapeutic drugs, the present inventors previously established a genome-wide gene expression pattern for 81 breast cancer patients using cDNA microarrays representing 27,648 cDNAs (Nishidate T et al Int J Oncol 2004; 25: 797-. Among the up-regulated genes, the present inventors focused on the expression of up-regulated F3374 in many breast cancer specimens. Subsequent semi-quantitative RT-PCR and northern blot analysis confirmed that F3374 was significantly upregulated in 10 of 12 breast cancer specimens (fig. 1A) and all breast cancer cell lines (fig. 1B), but not expressed in normal organs (except testis and thymus, placenta, bone marrow) (fig. 1C and fig. 1D).

The full-length cDNA sequence of F3374V1 included 4,221 nucleotides, containing an open reading frame of 2,193 nucleotides encoding a 730 amino acid polypeptide (fig. 1E). Next, to determine the expression pattern of F3374V1 in breast cancer cell lines and normal human tissues, the inventors performed semi-quantitative RT-PCR using a primer set that recognizes F3374V 1. The results of RT-PCR showed that F3374V1(1,296bp) was predominantly overexpressed in breast cancer cells compared to normal human tissue, while the other variants were not expressed in breast cancer cells. Thus, the inventors focused on the F3374V1 transcript for further analysis (fig. 1F).

To examine the expression pattern of the endogenous F3374 protein, the present inventors first developed a specific polyclonal antibody against the F3374 protein. Western blot analysis was then performed using cell lysates from breast cancer cell lines HBC4, HBC5, HBL100, HCC1937, MCF-7, MDA-MB-231, SKBR3, T47D and YMB1 and HMEC (Human mammalian epithelial cells) cells. Figure 1D shows that a strong band was clearly detected in most cell lines, but hardly in HMEC cells.

Interestingly, BT-549, MCF-7 and MDA-MB231 showed no expression of F3374 protein or a shortened F3374 protein, although F3374mRNA was overexpressed in these cell lines (FIG. 3A). This suggests that there may be some mutations that result in truncated proteins due to alternative splicing in these cell lines, but that sequence analysis is necessary.

Furthermore, the western blot showed one additional band and one 79.5 kDa-band, consistent with the expected size of the F3374 protein (FIG. 3A). To examine whether this additional band represents a form of the phosphorylation-modified F3374 protein, the present inventors treated cell extracts from T47D cells with lambda-phosphatase, followed by immunoblotting. Since the additional band did not appear after lambda-phosphatase treatment, the inventors judged that F3374 was phosphorylated in breast cancer cells (fig. 3B). To determine the phosphorylated region of F3374, the inventors designed a 3-fragment of F3374 (fig. 3C). The results show that the extra band disappeared after phosphatase treatment when transiently expressed with the delta-3 construct, but did not change when expressed with the other constructs (fig. 3D). These findings indicate that the C-terminal region (amino acids 591-730) is phosphorylated in the cells.

(2) Immunocytochemical and immunohistochemical analysis of F3374V1

To examine the subcellular localization of the endogenous F3374V1 protein in the breast cancer cell line HBC5, the inventors performed immunocytochemical staining analysis using an anti-F3374 polyclonal antibody. Interestingly, endogenous F3374V1 showed a cell cycle dependent localization (fig. 3E). At interphase, it localizes in the nucleus, and then on chromosomes at the previous phase. In the later stages, F3374V1 focused into the late spindle center (midzone) of the cell as a series of narrow bands (fig. 3E). Finally, the protein is accumulated to a terminal intermediate in all breast cancer cells. As the cell progresses along the mitotic process, F3374V1 undergoes significant redistribution. These findings suggest that F3374V1 may play an important role in the cell cycle, particularly during cytokinesis, of breast cancer cells.

To further study the expression of F3374 in breast cancer and normal tissue sections, the inventors performed immunohistochemical staining with anti-F3374 antibody. The present inventors determined that three different histological subtypes of breast cancer, namely, papillary tubular carcinoma (papillo-tubular carcinoma), solid small tubular carcinoma (solid tubular carcinoma) and hard carcinoma (scarrhous carcinoma), are highly expressed in the nucleus and cytoplasm, but their expression is barely detectable in normal breast duct cells (fig. 3F, left panel). Furthermore, the present inventors performed microarray analysis of breast cancer tissues, verifying that F3374V1 was staining positive in 33 of 39 cases of invasive ductal carcinoma (inflicting ductal carcinomas), while no staining was observed in 5 cases of normal breast tissues including ductal cells (data not shown). Among the 9 normal tissues examined by the present inventors, expression was detected in testis in accordance with the results of northern blot analysis, but was hardly detected in heart, liver, kidney, lung, colon, pancreas, skeletal muscle, and small intestine (fig. 3F, right panel). These results suggest that the F3374V1 protein is overexpressed in breast cancer cells in vivo.

(3) Cell cycle dependent expression of F3374

To examine the expression of the F3374 protein at various stages of the cell cycle, the present inventors performed FACS analysis and western blot analysis using T47D cells after synchronizing the cell cycle with African forest treatment. The expression of the F3374 protein was higher in the transition phase from G1 phase to S phase (0-6 hours), with the highest point just after release from cell cycle arrest (fig. 12A and 12B). On the other hand, its expression was significantly reduced at the 9-12 hour point (when most of the cells were in the G2/M phase). Interestingly, most of the F3374 protein was non-phosphorylated at G1/S phase, but was changed to the phosphorylated form at G2/M phase (9-12 hours) (fig. 12B) in a significant proportion, suggesting that endogenous F3374 protein showed cell cycle dependent localization and modification, possibly playing an important role in cell cycle progression in breast cancer cells.

(4) Growth inhibition by siRNA against F3374V1

To evaluate the growth-promoting effect of F3374V1, the inventors knocked down the expression of endogenous F3374V1 in breast cancer cell lines T47D and HBC4 that have shown F3374V1 overexpression, by mammalian vector-based RNA interference (RNAi) techniques (see materials and methods). The present inventors examined the expression level of F3374V1 by semi-quantitative RT-PCR analysis. Of the two siRNA constructs for each gene examined, F3374V 1-specific siRNA (si- #1 and si- #4) significantly and moderately inhibited the expression of each gene compared to the control siRNA constructs (psiU6BX-EGFP (siEGFP) and psiU6BX-SCR (siSCR)) (FIG. 6A and FIG. 6D). To determine the cell growth inhibitory effect of F3374V 1-specific siRNA, the inventors performed colony formation (fig. 6B and 6E) and MTT assays (fig. 6C and 6F), respectively. Introduction of the F3374V1(Si- #1 and Si- #4) constructs significantly inhibited growth of T47D and HBC4 cells, consistent with the above results of reduced expression. Each result was validated by 3 independent experiments. These findings suggest that F3374V1 has an important function in cell growth of breast cancer cells.

Furthermore, the present inventors examined the morphological changes of HBC4 cells transfected with F3374 specific siRNA oligonucleotide (siF3374) (see (12) gene silencing effects of A7322, F3374V1, AURKB or PBK/TOPK), determining a significant knockdown effect at the protein level (FIG. 6G). Interestingly, the inventors observed that its knockdown resulted in the appearance of an intercellular bridge between two isolated cells (arrows in the picture of FIG. 6H: siF3374), indicating dysfunction late in the cytokinesis process. The inventors also observed an increase in the size of cells transfected with siF3374 compared to those transfected with control siefp (fig. 6H). Similar results were obtained in T47D cells (data not shown), indicating dysfunction of the cytokinesis process. These findings indicate that the lack of F3374 causes cytokinesis, resulting in arrest of cells in the G2/M phase, followed by cell death.

(5) Aurora kinase-B regulates F3374 protein

It has been mentioned above that F3374 is phosphorylated and concentrated in the contraction loop (contraction) when the cells are in the terminal and cytokinesis phases in breast cancer cells. Aurora-B kinase (AURKB) is known as a chromosomal passenger protein (chromosome passager protein) that moves from the central body to the central zone (midzone) spindle in the mid-late phase of HeLa cells, to the intermediate body and in the late phase and cytokinesis (Terada y. Cell Struct Funct 2001; 26: 653-7; Adams RR, et al trends Cell Biol 2001; 11: 49-54; Carmena M, et al Nat Rev Mol Cell Biol 2003; 4: 842-54), so the inventors considered their similar subcellular localization at certain Cell cycle stages. Furthermore, as shown in FIG. 13A, the present inventors found 3 consensus Aurora kinase-B phosphorylation sites ([ R/K ] X [ T/S ] and [ R/K ] X [ T/S ] [ I/L/V ]; Cheeseman IM, et al, Cell 2002; 111: 163-72; Ohashi S, et al, Oncogene 2006; 25: 7691-702) in the phosphorylated C-terminal region (amino acids 591-730) observed in F3374 (FIG. 2D). Therefore, the possible interaction of the F3374 protein with AURKB in breast cancer cells was investigated.

The present inventors first compared the expression patterns of F3374 and AURKB by semi-quantitative RT-PCR analysis, and confirmed that F3374 and AURKB are almost all up-regulated in all 10 breast cancer cell lines examined (FIG. 13B). To investigate the interaction between F3374 and AURKB proteins, the present inventors constructed plasmids designed to express HA-tag F3374(HA-F3374) and Flag-tag AURKB (Flag-AURKB) (see (5) construction of expression vectors). These plasmids were co-transfected into HEK293T cells, and then proteins were immunoprecipitated with anti-Flag antibodies. Immunoblotting of the pellet with anti-HA antibody showed that Flag-AURKB co-precipitated with HA-F3374 (FIG. 13C). In addition, immunocytochemical staining experiments confirmed that both proteins accumulated to intermediates in T47D cell cytokinesis (fig. 13F).

To further investigate whether F3374 was phosphorylated by AURKB, the present inventors performed in vitro kinase assays (see (17) in vitro and in vivo kinase assays) using purified C-terminal F3374 (amino acid 437-730) recombinant protein and full-length AURKB recombinant protein, and found that F3374 protein was phosphorylated by AURKB in vitro (FIG. 13D). To further investigate the interaction between F3374 and AURBK protein and what role it might play by phosphorylation of AURKB, siRNA-AURKB (siAURKB) was transfected into T47D cells, followed by western blot analysis. A significant reduction in total F3374 protein as well as phosphorylated F3374 protein was observed in T47D cells transfected with siAURKB compared to those transfected with control siEGFP (fig. 13E), suggesting the possibility that phosphorylation of F3374 by AURKB stabilizes F3374 late in mitosis (fig. 13F).

Discussion of the related Art

Over the past 20 years, molecular targeted drugs for cancer therapy have been developed through the identification and characterization of cancer-associated genes and their products, but the proportion of patients that could benefit from currently available therapies is still very limited (Navolanic PM, et al Int J Oncol 2005; 27: 1341-4; Bange J, et al Nat Med 2001; 7: 548-52). Therefore, there is an urgent need to develop novel anticancer drugs with high specificity to malignant cells with minimal risk of adverse reactions. In this study, the inventors identified F3374, by detailed expression pattern analysis of breast cancer, which was significantly overexpressed in clinical breast cancer cases and breast cancer cell lines, but barely detectable in any normal human tissues examined, except for low-level expression in a few organs. Subsequent northern blot and immunohistochemical staining analyses clearly indicated that F3374 expression was up-regulated at the transcriptional level and protein level in breast cancer cells, but not in surrounding normal cells.

The F3374 gene encodes a putative 730 amino acid protein comprising 6 highly conserved WD 40-repeat domains at its N-terminus and a consensus nuclear localization signal. Our results also demonstrate that the F3374 protein is localized mainly to the nucleus of interphase cells, where the central spindle zone (midzone) accumulates as a series of narrow bands, eventually being concentrated in the contractile loops at the end stage and cytokinesis. These findings suggest an important role for the protein in cell cycle progression.

The present inventors demonstrated by siRNA technology that the knockdown of endogenous F3374 expression significantly inhibited cell growth of breast cancer cell lines (T47D and HBC4) due to abnormal cell division and subsequent cell death, both of which may be due to dysfunction in the process of cell kinetics. The inventors also demonstrated an increase in the proportion of cells of larger size in siF3374 transfected cells, but the inventors did not find an increase in multinucleated cells. Since inactivation of F3374 in unstressed HeLa cells was reported to result in stabilization of p53 (Bank D, et al CellCycle 2006; 5: 1719-29), the accumulation of G2/M cells by knockdown of F3374 in the breast cancer cell line HBC4 was probably due to activation of the checkpoint system (checkpoint system) by p 53.

Due to their similarity in subcellular localization at certain cell cycle stages in breast cancer cells and their co-expression in breast cancer cells, the present inventors focused on Aurora-b (aurkb) serine-threonine kinase as a F3374 interacting protein candidate. The present inventors confirmed in vivo interaction with AURKB, and its phosphorylation by AURKB in vitro, and possible stabilization of AURKB by the same, by RNAi experiments. In addition, it has been reported that knockdown of AURKB also inhibits growth of HeLa cells due to cytokinesis defects (Goto H, et al J Biol Chem 2003; 278: 8526-30; Severson AF, et al Curr Biol 2000; 10: 1162-71) similar to depletion of F3374. In summary, the inventors herein demonstrate for the first time that the interaction of F3374 and AURKB may play an important role in cytokinesis. Furthermore, it has been reported that F3374 is required as a component of the CUL4-DDB1 ubiquitin E3 ligase complex (SansamCL, et al Genes Dev 2006; 20: 3117-29; Higa LA, et al Cell Cycle 2006; 5: 1675-80; Higa LA, et al Nat Cell Biol 2006; 8: 1277-83) in the initiation of the radiation-induced G2/M checkpoint (checkpoint), suggesting multiple roles for F3374 in Cell Cycle progression.

Therefore, inhibitors of their binding would be a potentially valuable target for the development of agents directed against breast cancer, as inhibition of their binding may lead to cytokinesis in breast cancer cells, and subsequently cell death.

In summary, our results have shown that the interaction of F3374 with AURK and its phosphorylation by AURKB may play an important role in the cytokinesis of breast cancer cells. The inventors also found that down-regulation of F3374 with siRNA significantly inhibited the growth of breast cancer cells, suggesting an important role in breast cancer cell proliferation. Our data would facilitate a better understanding of breast cancer development, suggesting that F3374 may be a promising molecular target for breast cancer therapy. Furthermore, it is noteworthy that our cDNA microarray data established that F3374 is commonly upregulated in many types of clinical cancers, including bladder (blader cancer), cholangiocarcinoma (cholangiocarcinoma), lung (lung) and renal cell (renal cell carcinoma) and breast (breast cancer) (data not shown). These results show that this gene can serve as a valuable target for the development of anticancer agents for a wide range of human cancers.

Example 4 PBK/TOPK

(1) Up-regulation of PBK/TOPK in breast cancer cells

The present inventors previously performed genome-wide expression profiling of 81 breast cancer cases using cDNA microarrays (Nishidate T et al, Int J Oncol 2004, 25: 797-. Among genes up-regulated in breast cancer, genes encoding proteins containing kinase domain were searched for, either based on reported information or according to prediction by protein-motif (SMART) program (II)http://smart.embl-heidelberg.de) (Schultz J et al, Proc Natl Acad Sci USA 1998, 95: 5857-64; letunic I et al, Nucleic Acids Res 2004, 32: d142-4). Among the genes sought, the inventors focused on the PBK/TOPK gene, whose high level of transactivation (transactivation) could be confirmed in most breast cancer cells (fig. 1A). Northern blot analysis of 10 breast cancer cell lines and 6 normal organs further confirmed that PBK/TOPK was specifically upregulated in all 10 breast cancer cell lines examined, but its expression was barely detectable in lung, heart, liver, kidney, bone marrow and breast (fig. 1D).

To further examine the expression pattern of the PBK/TOPK gene in each normal tissue, Northern blot analysis was performed using mRNAs from 23 tissues and two transcripts unique (exclusive) in testis and thymus were identified (FIG. 1C). According to the NCBI database, two representative transcripts of 1,899 nucleotides (GenBank Accession No. NM-018492) and 1,548 nucleotides (# AF189722) appeared to correspond to those two bands observed in Northern analysis. Both transcripts share the same open reading frame encoding a 322 amino acid polypeptide.

(2) Immunocytochemistry and immunohistochemical analysis of PBK/TOPK

Cell lysates from breast cancer Cell lines BT-20, HBC4, HBC5, HBL-100, MCF-7, MDA-MB-231, SKBR3, and T47D were examined for endogenous PBK/TOPK protein expression by Western blot analysis using HMEC (Human Mammali Epithelial Cell, Human Mammalian Epithelial Cell) as an experimental control (FIG. 4A). All breast cancer cell lines showed high levels of PBK/TOPK expression, whereas the normal breast epithelial cell line HMEC cells showed no expression. Subsequent immunocytochemical analysis of the breast cancer cell lines T47D, BT-20, and HBC5 using anti-PBK/TOPK monoclonal antibodies revealed that endogenous PBK/TOPK was predominantly localized in the cytoplasm (FIG. 4B).

To further study PBK/TOPK expression in breast cancer and normal tissue sections, immunohistochemical staining was performed using anti-PBK/TOPK monoclonal antibodies. Strong staining was detected in the cytoplasm of three different histological subtypes of breast cancer, intraductal carcinoma (intraductal carcinoma), papillary-tubular carcinoma (papillo-tubalarcinoma) and scleroma (scarrhous carcinoma) (fig. 4C (1) - (3)), but its expression was barely detectable in normal breast tissue (fig. 4C (4)). Furthermore, consistent with the results of Northern blot analysis, strong staining of PBK/TOPK protein was detected in the outer cell layer of testicular tubules, while no expression was observed in heart, lung, liver or kidney (fig. 4D (1) - (4)).

(3) Knock-down effect of endogenous PBK/TOPK

To investigate the growth promoting effect of the PBK/TOPK gene in breast cancer cells, the expression of endogenous PBK/TOPK was knocked down in two breast cancer cells (T47D and BT-20) by RNA interference (RNAi) technique (FIGS. 7A and 7B). A semi-quantitative RT-PCR experiment detected significant knockdown effects of PBK/TOPK in cells transfected with PBK/TOPK-si- #2 and si- #3, but not in cells transfected with control siRNA (mock). Consistent with the knock-down effect shown, colony formation and MTT assays clearly revealed that two siRNAs (PBK/TOPK-si- #2 and si- #3) inhibited breast cancer cell growth compared to two siRNAs that did not show a knock-down effect, used to rule out the possibility of off-target effect (off-target effect) of PBK/TOPK-siRNA (si- #3) (FIGS. 7A and 7B). These results suggest that PBK/TOPK plays a key role in breast cancer cell growth.

In addition, phenotypic changes (phenotypic isomers) were observed for cells transfected with sirnas showing significant knockdown effects. In T47D cells in which PBK/TOPK expression was inhibited, prolonged intermediates and erroneous cell division due to abnormal cytokinesis were observed (fig. 7C and 7D). Western blot and FACS analysis also identified an increase in the population of apoptotic (sub-G1) cells in PBK/TOPK siRNA-treated cells, but no phenotypic change or increase in the sub-G1 population was observed in those transfected with mock constructs (FIGS. 7E and 7F), suggesting an essential role for PBK/TOPK in proliferation and mitosis and/or cytokinesis of breast cancer cells.

(4) Cell cycle dependent expression of PBK/TOPK

It has been reported that PBK/TOPK is probably a mitotic kinase (Gaudet S et al, Proc NatlAcad Sci USA 2000, 97: 5167-72), so the inventors investigated its relationship to cell cycle progression. After cell cycle synchronization with African, expression of PBK/TOPK protein in T47D cells was examined. FACS analysis showed a significant increase in the proportion of cells in the G2/M phase 6 hours after release from cell cycle arrest (figure 14A). Interestingly, after 9-12 hours, Western blot analysis detected an additional band of high molecular weight PBK/TOPK when most cells were in the G2/M phase. At the 15 hour time point, the strength of the high molecular weight tape decreased (fig. 14B). Immunochemical analysis also showed that the subcellular localization of the PBK/TOPK protein in cells at mitosis (especially at pre-and metaphase) is located around condensed chromosomes (FIG. 14C).

To further investigate the role of high molecular weight PBK/TOPK in cell cycle progression, T47D breast cancer cells were treated with nocodazole (nocodazole) for Western blotting and FACS analysis. As expected, the intensity of the additional high molecular weight band of endogenous PBK/TOPK in T47D cells increased in a time-dependent manner (6-18 hours) after treatment with nocodazole (nocodazole) (fig. 14D, left panel), and the band disappeared after treatment with lambda phosphatase (fig. 14D, right panel). In addition, FACS analysis showed that 6-18 hours after the release of cell cycle arrest, the proportion of cells in the G2/M phase increased (FIG. 14E), indicating an important role for phosphorylated PBK/TOPK in mitosis.

(5) PBK/TOPK phosphorylates histone H3(Ser10) in vitro and in vivo

The PBK/TOPK protein is predominantly localized around the chromosomal surface in mitotic cells (especially in the early and metaphase), so the inventors have focused on histone proteins as candidate substrates for PBK/TOPK proteins. In vitro kinase assays using purified recombinant PBK/TOPK and mixed histones (H2a, H2b, H3 and H4) detected an approximately 15kDa phosphorylated protein (lane 2), suggesting that the PBK/TOPK protein may phosphorylate the histone H3 protein based on its molecular size (FIG. 15A, left panel). Further, in vitro kinase assays were performed using histone-H3 recombinant protein to determine PBK/TOPK protein phosphorylation histone H3 (fig. 15A, right panel). In addition, autophosphorylated PBK/TOPK of approximately 40kDa was detected by in vitro kinase assay as shown in FIG. 15A (indicated by asterisks).

The localization of PBK/TOPK around the chromosome and its increased early phosphorylation at mitosis suggest a physiological role for the phosphorylation of histone H3 by PBK/TOPK in breast cancer cells. Thus, wild-type or kinase-inactivated (K64-65A) PBK/TOPK were first transfected into T47D cells, which were then stimulated by treatment with Okadaic Acid (OA), which is known to induce premature mitosis (Gaudet S et al, Proc Natl Acad Sci USA 2000, 97: 5167-72). () By Western blot analysis using anti-HA rat antibodies, it was detected that wild-type PBK/TOPK and kinase-inactivated PBK/TOPK were phosphorylated at the same level after OA treatment. However, compared to the kinase-inactivated mutant protein, phosphorylation at Ser10 was increased in the histone H3 of the wild-type protein (fig. 15B). It was also determined that phosphorylation of histone H3 Ser10 was specifically reduced in PBK/TOPK-depleted T47D cells by siRNA (si- #3) compared to mock-siRNA transfected cells (fig. 15C).

In addition, the localization of endogenous PBK/TOPK protein and phosphorylated histone H3 was examined. Specifically, T47D and HBC5 cells were synchronized with afillin, and then immunocytochemical staining was performed using anti-PBK/TOPK and anti-phospho-Ser 10H 3 antibodies. As shown in fig. 15D, it was observed that PBK/TOPK partially overlapped with phosphorylated histone H3 around the condensed chromosome in the early cells, both proteins overlapped in the metaphase cells (fig. 15E), and both disappeared in the later stage (fig. 15F). Combining these results, it was determined that endogenous PBK/TOPK is capable of specifically phosphorylating histone H3 at Ser10 during mitosis (especially pre-to metaphase) in breast cancer cells.

(6) Phosphorylation of Thr9 is important for cell proliferation

PBK/TOPK has previously been shown to be up-regulated in breast Cancer and to migrate from the cytoplasm to the nucleus during mitosis in breast Cancer cells (Park JH et al, Cancer Res 66: 9186-95 (2006)). In addition, nuclear transport of the CDK 1-cyclin B1 complex protein has also been reported to occur in mitotic cells. Thus, the inventors first performed immunocytochemistry to determine the subcellular localization of PBK/TOPK, CDK1 and cyclin B1, respectively, in breast cancer cells. Similar nuclear transport of those proteins during mitosis was observed in T47D breast cancer cells, suggesting possible signaling between the PBK/TOPK and CDK 1-cyclin B1 complex in breast cancer cells (fig. 16A). Although it was reported that PBK/TOPK could be phosphorylated at Thr9 by CDK 1-cyclin B1 as determined by immune complex kinase using immunoprecipitates of CDK1, it was not yet explained whether its phosphorylation was direct (Matsumoto et al, Biochem Biophys ResCommun 325: 997-1004 (2004)). Wild-type inactive recombinant protein of PBK/TOPK and T9A mutant were prepared using E.coli expression system. It was demonstrated that the wild-type PBK/TOPK recombinant protein was phosphorylated by the CDK 1-cyclin B1 recombinant protein complex, whereas the mutant in which Thr9(T9A) of the PBK/TOPK recombinant protein was replaced by alanine was not phosphorylated by the CDK 1-cyclin B1 recombinant protein complex (fig. 16B), suggesting that PBK/TOPK was directly phosphorylated by the CDK 1-cyclin B1 complex at Thr9 in vitro.

To investigate the biological significance of phosphorylation at PBK/TOPK Thr9 in breast carcinogenesis, attempts were made to inhibit its phosphorylation by using synthetic peptides. The present inventors designed the N-terminus (pp1-18) of the PBK/TOPK peptide; SEQ ID NO: 98, coupled to an arginine (R) -repeat to promote cell permeability. FIG. 16C shows that the addition of the pp1-18 peptide reduced the phosphorylation of the recombinant PBK/TOPK protein by the CDK 1-cyclin B1 recombinant protein in a dose-dependent manner. Further, whether the peptide can inhibit the growth of cancer cells was examined by treating cancer and normal Human Mammary Epithelial Cells (HMECs) with the peptide. pp1-18 peptide treatment significantly inhibited growth of T47D breast cancer cells in a dose-dependent manner, but showed no effect on HMEC cell growth (5 μmol/mL, T-test); p ═ 0.0096), thereby excluding the possibility of off-target effect (off-target effect) of the peptide (fig. 16D). Next, we further investigated whether this peptide inhibits PBK/TOPK phosphorylation in mitotic cells. T47D cells were treated with nocodazole, followed by the addition of pp1-18 peptide to T47D cells. FIG. 16E shows that PBK/TOPK phosphorylation in mitotic cells is significantly time-dependently reduced by 10 μ M pp1-18 peptide treatment. In addition, 24 hours after nocodazole treatment, treatment with this peptide blocked the transition of the cell cycle to the G2/M phase (fig. 16E, lower panel). In addition, prolonged intermediates due to abnormal cytokinesis were observed in pp1-18 treated (50 μ M) -T47D cells (FIG. 16F), as well as in PBK/TOPK knockdown T47D cells as previously described (Park JH et al, Cancer Res 66: 9186-95 (2006)). Taken together, these findings suggest that phosphorylation of PBK/TOPK by CDK 1-cyclin B1 at Thr9 may play a key role in breast cancer cell growth, although this peptide may inhibit possible interactions with other PBK/TOPK interaction partner proteins (interacting partners) by its N-terminal region.

(7) Autophosphorylation of PBK/TOPK in mitotic cells

To investigate the phosphorylation of PBK/TOPK during mitosis, the inventors isolated mitotic cells by the "mitotic shake-off" method (see example 1-material and method (16) co-immunoprecipitation and immunoblot analysis) (fig. 17A, upper panel) and performed immunoblot analysis using mitotic cell lysates. The high molecular weight band was completely shifted after treatment with lambda phosphatase, so that the protein was said to be highly phosphorylated during cell mitosis in breast cancer cells (FIG. 17A). To further investigate whether this hyperphosphorylation of PBK/TOPK occurred only at its Thr9 due to the CDK 1-cyclin B1 complex in mitotic cells, T9A, kinase-inactive (KD) and dual (T9A/KD) mutant constructs, as well as wild-type PBK/TOPK constructs, were transfected into T47D breast cancer cells, respectively. Interestingly, the phosphorylation band of T9A protein remained after nocodazole treatment, while the phosphorylation band in KD and the double (T9A/KD) mutant as well as in cells treated with lambda phosphatase completely disappeared (fig. 17B). These results strongly suggest that in mitotic cells, the PBK/TOPK protein may be autophosphorylated by itself.

(8) PP1 alpha regulates phosphorylation of PBK/TOPK

It has also been previously shown that treatment with Okadaic Acid (OA), a strong inhibitor of Ser/Thr protein phosphatase, in addition to nocodazole treatment, also results in phosphorylation of PBK/TOPK protein in T47D breast Cancer cells (Park JH et al, Cancer Res 66: 9186-95 (2006)). However, how treatment with OA leads to its phosphorylation in breast cancer cells is still unknown. Because of the reportedly protein phosphatases

Has a relatively high IC50 value for OA and is inactivated during cell mitosis (Kwon YG et al, Proc Natl Acad Sci U S A94: 2168-73 (1997); Ammosova T et al, Retrovirology 2: 47(2005)), prompting the present inventors to focus on human protein phosphatasesPP1 α acts as a potential modulator of PBK/TOPK phosphorylation. First, the inventors treated T47D cells with high (100nM) or low (less than 100nM) OA and found that treatment with 100nM OA for 9 hours resulted in PBK/TOPK phosphorylation (FIG. 17C), but treatment with low concentrations did not result in PBK/TOPK phosphorylation (data not shown). Next, to investigate the interaction of PBK/TOPK with PP1 α, GST-tagged PP1 α (PP1 α -GST) and HA-tagged PBK/TOPK (HA-PBK/TOPK) constructs were co-expressed into COS-7 cells, followed by GST pull-down (GST pulldown) assay. FIG. 17D shows that PP1 α -GST was significantly pulled down with HA-PBK/TOPK (upper panel), while in turn HA-PBK/TOPK co-immunoprecipitated with PP1 α -GST (lower panel), indicating an interaction between the two proteins. It was further investigated whether the PP1 alpha protein dephosphorylates PBK/TOPK proteins directly. Following incubation with recombinant PP1 α protein and lambda phosphatase, the activated recombinant PBK/TOPK protein was dephosphorylated (FIG. 17E, top panel). In addition, endogenous PBK/TOPK proteins in mitotic cell lysates from T47D cells were also dephosphorylated by PP1 α protein treatment (fig. 17E, lower panel). These findings suggest that PP1 α may regulate PBK/TOPK autophosphorylation through their interaction during mitosis.

(9) CDK 1-cyclin B1 activates PBK/TOPK by PP1 alpha inactivation

The above description indicates that in mitotic cells, PBK/TOPK is phosphorylated by the CDK 1-cyclin B1 kinase complex and its phosphorylation is regulated by PP1 α. In addition, PP1 α is known to be inactivated in mitotic cells by its phosphorylation by the CDK 1-cyclin B1 complex (Kwon YG et al, Proc Natl Acad Sci U S A94: 2168-73 (1997)). Thus, it was examined in further detail how PBK/TOPK is regulated by CDK 1-cyclin B1 or PP1 α during mitosis. Treatment with nocodazole for 16 hours synchronized T47D cells in G2/M phase (61-70%), followed by incubation of T47D cells with CDK1 inhibitor for up to 4 hours (fig. 17F and 17G). Next, the phosphorylation levels of PBK/TOPK or PP1 α were examined by immunoblot analysis. It was found that the phosphorylation of PBK/TOPK caused by G2/M block (0H) decreased in a time-dependent manner after treatment with CDK1 inhibitor (0-4H) (FIG. 17H, panel 1). Meanwhile, the present inventors found that in addition to the results in the previous studies, PP1 α was less phosphorylated at Thr320, which is known to be phosphorylated and inactivated by CDK 1-cyclin B1 complex (fig. 17H, panel 3). CDK1 inactivation was confirmed by the decreased phosphorylation of the Rb protein at Ser807 and 811 (fig. 17H, panel 4). Taken together, these findings suggest that PBK/TOPK is activated in mitotic cells by its direct autophosphorylation and inactivation of PP1 α (which inhibits PBK/TOPK autophosphorylation); whereas inactivation of PP1 α is caused by CDK 1-cyclin B1 being maintained at a steady level prior to the onset of mitosis.

(10) Depletion of PBK/TOPK by siRNA leads to mitotic disorders and G1 arrest

Since defects in cytokinesis and delays caused by PBK/TOPK silencing were previously observed in breast cancer cells, the biological effects of PBK/TOPK in mitotic cells were examined in more detail by RNAi experiments. Knockdown of PBK/TOPK protein expression was confirmed in PBK/TOPK-specific siRNA treated cells 2 days after treatment with PBK/TOPK-specific siRNA or si-EGFP oligonucleotide as a control (FIG. 18A). As shown in FIG. 18B, the inventors observed longer intercellular bridges in PBK/TOPK-siRNA treated cells but not in si-EGFP treated cells using microscopy and immunocytochemical staining with phalloidin fluoroscein, indicating a cytoplasmic division defect due to depletion of PBK/TOPK expression (FIG. 18B, white arrows). In addition, to examine the effect of knockdown of PBK/TOPK expression on cell cycle, FACS analysis was performed on cells treated with PBK/TOPK-specific siRNA or si-EGFP oligonucleotides. siEGFP-treated cells showed a clear shift from the G1 peak to the G2/M peak. On the other hand, treatment of siPBK/TOPK-treated cells with nocodazole induced a lag in mitotic arrest and did not show a shift from the G1 peak to the G2/M peak (fig. 18C), suggesting that knockdown of PBK/TOPK may occur with G1-arrest. To classify in further detail the cytokinesis defects in PBK/TOPK depleted cells, the inventors examined real-Time images of breast cancer T47D cells in the absence of PBK/TOPK with Time-lapse microscopy (Time-microscopy). FIG. 18D shows that cell division was photographed from late to late (was taken for) for 1.5 min in EGFP-transfected cells (as indicated by white arrows). On the other hand, in PBK/TOPK-depleted cells, cell division was photographed for 4.5 minutes from late to late, particularly 5 minutes at the cytokinesis step, and then finally cleaved (as shown by the arrow in FIG. 18E).

Furthermore, to verify the results, the present inventors conducted RNAi-rescue experiments by introducing wild-type PBK/TOPK and kinase-inactivated forms, respectively. FIG. 18F shows that the introduction of wild-type PBK/TOPK protein restores the cytokinetic barrier due to PBK/TOPK depletion (indicated by the arrow), whereas the introduction of kinase-inactive form is not able to restore (indicated by the arrow), supporting that the kinase activity of PBK/TOPK is essential for cytokinesis.

(11) PBK/TOPK phosphorylates p97/VCP protein in vitro by p47 as adapter protein (adaptor protein)

Since the kinase activity of PBK/TOPK is important for the cytokinesis of breast cancer cells, the present inventors attempted to identify PBK/TOPK-specific substrates using GST fusion PBK/TOPK (GST-PBK/TOPK) recombinant proteins and GST proteins as controls in vitro protein pull-down (pull-down) assays. Comparison of the silver staining of SDS-PAGE gels containing the pulled down proteins identified an approximately 47kDa protein that appeared specifically in the corresponding lane of the protein co-immunoprecipitated with GST-PBK/TOPK protein, but not in the lane with GST as a control (data not shown). MALDI-TOF analysis determined that the 47-kDa protein is p47 protein, which is an adaptor protein of p97/VCP (valosin-binding protein), and p97/VCP belongs to AAA ATPase family involved in cell mitosis. The expression pattern of p47 at the transcriptional level in breast cancer cell lines was examined using semi-quantitative RT-PCR and p47 was found to be expressed in all breast cancer cells examined (data not shown).

To verify the interaction between PBK/TOPK and P47 proteins, a pull-down in vitro assay was performed. The HA-tagged PBK/TOPK (HA-PBK/TOPK) construct was transfected into COS-7 cells, and the cells were then lysed with lysis buffer. Next, the cell lysate was mixed with GST-tagged p47(GST-p47) recombinant protein and then pulled down with GST-beads. Immunoblotting of the pellet with anti-HA antibody showed that GST-p47 co-precipitated with HA-PBK/TOPK (FIG. 19A). In addition, the inventors performed immunocytochemical staining and observed that endogenous PBK/TOPK and exogenously expressed FLAG-tagged p47 protein co-localized at the cytoplasm in T47D cells. After nocodazole treatment, the inventors found that they co-localized in the nucleus of cells (particularly mitotic cells), suggesting that PBK/TOPK protein interacts with p47 (fig. 19B). Since p47 is known to form a tight complex with p97 protein, the inventors believe that PBK/TOPK might bind to the p47/p97 protein complex. The expression of endogenous p97 protein in breast cancer cell lines as well as in HBL-100 and HMEC was first investigated by western blot analysis using anti-p 97 antibody, and endogenous p97 protein was found to be expressed in all breast cancer cell lines investigated as well as in HBL-100 and HMEC (fig. 19C). Next, whether PBK/TOPK and p97/p47 complex interact or not was examined by co-IP experiments. The HA-PBK/TOPK and myc-tagged p97(myc-p97) constructs were co-transfected into COS7 cells and then co-immunoprecipitated with HA-tagged antibodies. The results show that HA-PBK/TOPK does not interact directly with myc-p97 (FIG. 19D). On the other hand, co-immunoprecipitation of HA-PBK-TOPK with GST-p47/myc-97 complex was detected when myc-p97, GST-p47 and HA-PBK/TOPK constructs were co-transfected into COS-7 cells, then immunoprecipitated with myc-tagged antibody and western blots with each tagged antibody separately (FIG. 19E). Taken together, these findings suggest that PBK-TOPK interacts with the p97 protein through p47 as an adaptor.

In addition, phosphorylation of p97 by PBK/TOPK was investigated by immune complex kinase assay using recombinant activated PBK/TOPK proteins. FIG. 19F shows that the PBK/TOPK recombinant protein phosphorylates p97 immunoprecipitated in breast cancer cells. To further investigate the effect of p97 on cytokinesis, the expression of p97 was knocked down in T47D cells by using PBK-TOPK-siRNA (FIG. 19G). The results showed that, like the depletion of PBK/TOPK, depletion of p97 also resulted in a disruption of cytokinesis (depletion of p97 occured to cytokinesis defects as well as the depletion of PBK/TOPK) (FIG. 19H). It has been reported that p97/VCP (valosin-binding protein, a protein containing valosin) belongs to the AAA ATPase family, such as the regeneration of Golgi apparatus (Golgiapapratus) which has been disrupted and reassembled at the end of the cycle (Uchiyama K et al, J Biochem (Tokyo) 137: 115-9(2005)), and the microtubule dynamics at the end of mitosis (Cao K et al, Cell 115: 355-67 (2003)). These findings suggest that p97/VCP may regulate cellular morphogenesis, possibly acting during the M to G1 transition in cytokinesis (Cao K et al, Cell Cycle 3: 422-4 (2004)). Therefore, in summary, we conclude that PBK/TOPK regulates cytokinesis (in particular mitotic withdrawal (exit)) in cancer cells, probably through phosphorylation of p97/p 47.

Discussion of the related Art

The present inventors previously reported that PBK/TOPK (PDZ-binding kinase/protein kinase derived from T-LAK cells is significantly upregulated and phosphorylated during mitosis and is involved in cell growth in breast cancer. however, the biological role of PBK/TOPK in mitosis and its pathophysiological role in breast cancer development remain unknown.

Industrial applicability

The expression of the novel human genes a7322 and F3374V1 was significantly enhanced in breast cancer compared to non-cancerous human tissues. Thus, these genes may serve as diagnostic markers for cancer, and the proteins encoded thereby may be used in diagnostic assays for cancer.

It is shown in this specification that expression of the novel proteins a7322 and F3374V1 promotes cell growth, while antisense oligonucleotides or small interfering RNAs corresponding to the a7322 and F3374V1 genes inhibit cell growth. These findings suggest that each of the a7322 and F3374V1 proteins can stimulate oncogenic activity. Thus, each of these novel oncoproteins is a useful target for the development of anti-cancer drugs. For example, agents that block the expression of or inhibit the activity of a7322 and F3374V1 may have therapeutic utility as anticancer agents, particularly for the treatment of breast cancer. Examples of such agents include antisense oligonucleotides, small interfering RNAs, and antibodies that recognize a7322 and F3374V 1.

All publications, databases, Genbank sequences, patents, and patent applications mentioned in this specification are herein incorporated by reference into the specification.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof, as defined in the appended claims.

The present invention is based on the discovery of a novel mechanism for phosphorylation of histone H3 at Ser10 by PBK/TOPK in vitro and in vivo. Since PBK/TOPK is a cancer/testis antigen, its kinase function is likely to be associated with its oncogenic activity, this protein is considered a promising molecular target for breast cancer therapy.

Since the substance screened by the above method has a high possibility of causing apoptosis in breast cancer cells, the screened substance serves as a candidate for the treatment or prevention of breast cancer.

All patents, patent applications, and publications mentioned in this application are incorporated by reference into this application in their entirety.

Additionally, while the invention has been described in detail and with reference to specific embodiments thereof, it will be understood that the foregoing description is illustrative and explanatory in nature and is intended to illustrate the invention and preferred embodiments thereof. One skilled in the art will readily recognize through routine experimentation that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. Accordingly, the invention is not intended to be limited by the foregoing description, but is to be defined by the following claims and their equivalents.

Sequence listing

<110> Oncotherapy SCIENCE incorporated (ONCOTHERAPY SCIENCE, INC.)

<120>GENES AND POLYPEPTIDES RELATING TO BREAST CANCERS

<130>ONC-A0608P

<150>US 60/837,428

<151>2006-08-10

<150>US 60/840,250

<151>2006-08-25

<150>US 60/915,022

<151>2007-04-30

<160>122

<170>PatentIn version 3.4

<210>1

<211>19

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>1

aacttagagg tgggagcag 19

<210>2

<211>22

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>2

cacaaccatg ccttacttta tc 22

<210>3

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>3

cttgacaagg cctttggagt 20

<210>4

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>4

caatatgctt ttcccgcttt 20

<210>5

<211>22

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>5

aaccaagcac accatagcct ta 22

<210>6

<211>22

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>6

ggagatgggt agggatacaa ac 22

<210>7

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>7

gggagagctg aagattgctg 20

<210>8

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>8

gacagattga agggcagagg 20

<210>9

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>9

cgaccacttt gtcaagctca 20

<210>10

<211>23

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>10

ggttgagcac agggtacttt att 23

<210>11

<211>23

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>11

agtgaaatgc aggtgagaag aac 23

<210>12

<211>25

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>12

tcattctagc caggatcata ctaag 25

<210>13

<211>23

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>13

agaccctaaa gatcgtcctt ctg 23

<210>14

<211>23

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>14

gtgttttaag tcagcatgag cag 23

<210>15

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>15

gctgacaacc ttgtgctgaa 20

<210>16

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>16

tgagaaatca cgcactgtcc 20

<210>17

<211>22

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>17

caagcttgct tacagagacc tg 22

<210>18

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>18

gggccaaacc taccaaagtt 20

<210>19

<211>22

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>19

gcaatctgct atgtcagcca ac 22

<210>20

<211>23

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>20

caggatcagc tcaaagtctg aca 23

<210>21

<211>28

<212>DNA

<213> Artificial

<220>

<223> synthetic primer for 5' RACE

<400>21

gcctccttct gcagcttcct caggattt 28

<210>22

<211>30

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>22

cggaattcat ggaagaaatc ctgaggaagc 30

<210>23

<211>37

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>23

atagtttagc ggccgcacaa tgatgtcata gacacgg 37

<210>24

<211>41

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>24

cggaattcca gaccgtgcat catggcccag aacttgaagg a 41

<210>25

<211>32

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>25

ccgctcgagt ttcttaccct tgatgaggct gt 32

<210>26

<211>38

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>26

cggaattcat gaccatgacc ctccacacca aagcatcc 38

<210>27

<211>31

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>27

ccgctcgagg accgtggcag ggaaaccctc t 31

<210>28

<211>39

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>28

aaggaaaaaa gcggccgcga tgctcttcaa ttcggtgct 39

<210>29

<211>33

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>29

ccgctcgagt aattctgttg agtgttcagg acc 33

<210>30

<211>30

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>30

ccggaattca tggaagggat cagtaatttc 30

<210>31

<211>33

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>31

ccgctcgagt cagacatctg tttccagagc ttc 33

<210>32

<211>43

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>32

cattctcctt gggctgtagc agcgattaat cctatatgta atg 43

<210>33

<211>43

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>33

cattacatat aggattaatc gctgctacag cccaaggaga atg 43

<210>34

<211>21

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>34

aagaaagcat cgcagtctca g 21

<210>35

<211>21

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>35

aagatgcgtt ctctgccaca c 21

<210>36

<211>21

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>36

aatattcgat ctctgccaca c 21

<210>37

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>37

gatcatgtct ccgagaaaa 19

<210>38

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>38

ggaagccata gaattgctc 19

<210>39

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>39

ctggatgaat cataccaga 19

<210>40

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>40

gtgtggcttg cgtaaataa 19

<210>41

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>41

gcgcgctttg taggattcg 19

<210>42

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>42

gaagcagcac gacttcttc 19

<210>43

<211>55

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>43

caccaagaaa gcatcgcagt ctcagttcaa gagactgaga ctgcgatgct ttctt 55

<210>44

<211>55

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>44

aaaaaagaaa gcatcgcagt ctcagtctct tgaactgaga ctgcgatgct ttctt 55

<210>45

<211>51

<212>DNA

<213> Artificial

<220>

<223> siRNA hairpin design

<400>45

aagaaagcat cgcagtctca gttcaagaga ctgagactgc gatgctttct t 51

<210>46

<211>55

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>46

caccaagatg cgttctctgc cacacttcaa gagagtgtgg cagagaacgc atctt 55

<210>47

<211>55

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>47

aaaaaagatg cgttctctgc cacactctct tgaagtgtgg cagagaacgc atctt 55

<210>48

<211>51

<212>DNA

<213> Artificial

<220>

<223> siRNA hairpin design

<400>48

aagatgcgtt ctctgccaca cttcaagaga gtgtggcaga gaacgcatct t 51

<210>49

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>49

caccgatcat gtctccgaga aaattcaaga gattttctcg gagacatgat c 51

<210>50

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>50

aaaagatcat gtctccgaga aaatctcttg aattttctcg gagacatgat c 51

<210>51

<211>47

<212>DNA

<213> Artificial

<220>

<223> siRNA hairpin design

<400>51

gatcatgtct ccgagaaaat tcaagagatt ttctcggaga catgatc 47

<210>52

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>52

caccggaagc catagaattg ctcttcaaga gagagcaatt ctatggcttc c 51

<210>53

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>53

aaaaggaagc catagaattg ctctctcttg aagagcaatt ctatggcttc c 51

<210>54

<211>47

<212>DNA

<213> Artificial

<220>

<223> siRNA hairpin design

<400>54

ggaagccata gaattgctct tcaagagaga gcaattctat ggcttcc 47

<210>55

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>55

caccctggat gaatcatacc agattcaaga gatctggtat gattcatcca g 51

<210>56

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>56

aaaactggat gaatcatacc agatctcttg aatctggtat gattcatcca g 51

<210>57

<211>47

<212>DNA

<213> Artificial

<220>

<223> siRNA hairpin design

<400>57

ctggatgaat cataccagat tcaagagatc tggtatgatt catccag 47

<210>58

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>58

caccgtgtgg cttgcgtaaa taattcaaga gattatttac gcaagccaca c 51

<210>59

<211>51

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>59

aaaagtgtgg cttgcgtaaa taatctcttg aattatttac gcaagccaca c 51

<210>60

<211>47

<212>DNA

<213> Artificial

<220>

<223> siRNA hairpin design

<400>60

gtgtggcttg cgtaaataat tcaagagatt atttacgcaa gccacac 47

<210>61

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>61

gcccttgaag ccaatattcc 20

<210>62

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>62

agatggtttc agtgggcttg 20

<210>63

<211>21

<212>RNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>63

gaugcguucu cugccacacu u 21

<210>64

<211>19

<212>RNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>64

gcagcacgac uucuucaag 19

<210>65

<211>19

<212>RNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>65

acuccuacgu ucucuauua 19

<210>66

<211>21

<212>RNA

<213> Artificial

<220>

<223> Artificial Synthesis oligonucleotide for siRNA

<400>66

aaggugaugg agaauagcag u 21

<210>67

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>67

actcctacgt tctctatta 19

<210>68

<211>21

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>68

aaggtgatgg agaatagcag t 21

<210>69

<211>19

<212>DNA

<213> Artificial

<220>

<223> for siRNA target sequence

<400>69

gcagcacgac ttcttcaag 19

<210>70

<211>21

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>70

atggaaatcc catcaccatc t 21

<210>71

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>71

gccttcatca tccaaacatt 20

<210>72

<211>20

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for RT-PCR

<400>72

ggcaaatatg tctgccttgt 20

<210>73

<211>39

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>73

aaggaaaaaa gcggccgcgc tgtggatggg ataatcaaa 39

<210>74

<211>30

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>74

ccgctcgagt ttgattatcc catccacagc 30

<210>75

<211>36

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>75

aaggaaaaaa gcggccgctg gcgcttgaat agaggc 36

<210>76

<211>30

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>76

ccgctcgaga tcacctcctg gtttctcctc 30

<210>77

<211>39

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>77

aaggaaaaaa gcggccgcct tgatggccaa gttgaaaat 39

<210>78

<211>30

<212>DNA

<213> Artificial

<220>

<223> Artificial Synthesis of primers for PCR

<400>78

ccgctcgagg cagcacagat ccaaatgaag 30

<210>79

<211>14852

<212>DNA

<213> human (Homo sapiens)

<400>79

gtggcccgcg gcatggagcg ggcgtgattc atcagcatcc gcgccggggc ggcatggggg 60

cgcgcgcggc ggccgcctag gcgcccaggg ccaggcagcg gcggcttccc cggcccggct 120

cgcccgcgct tctctccctg tgggcggcgg cccggcgcct ggaaggtcaa gatggaagaa 180

atcctgagga agctgcagaa ggaggcgtcc gggagcaagt acaaagccat caaggagagc 240

tgcacctggg ccctggaaac tctaggtggt ctggatacca ttgtcaagat ccctccacat 300

gtactgaggg agaaatgcct gctgcctctc cagttggctt tggaatccaa gaatgtgaag 360

ctggcccaac atgctttggc agggatgcag aagcttctgt cggaagagag gtttgtatcc 420

atggaaacag attctgatga gaagcagctg ctcaatcaga tactgaatgc cgtgaaagtg 480

acgccttcgc tcaacgagga cctgcaggtg gaagtgatga aggttttact atgcatcacc 540

tacacgccaa catttgatct gaatgggagt gccgtgctga agatcgcgga ggtgtgcatt 600

gagacgtaca taagcagctg tcaccagcgt agcataaaca ctgctgtgcg ggcaactctc 660

agtcaaatgc tgagtgactt gactttacag ttacgacaga ggcaggagaa tacgataatt 720

gaaaacccag atgtcccaca ggatttcggg aatcaagggt caacagtaga gtccctctgt 780

gatgatgttg tctctgtact caccgtcctg tgtgagaagc tgcaagccgc cataaatgac 840

agccagcagc tgcagcttct ctacctggag tgcatcctgt ctgtgctcag cagctcctcc 900

tcctccatgc acctgcacag gcgcttcacg gacctgatct ggaaaaacct ctgccctgct 960

ctcatcgtga tcttggggaa tccaattcat gacaaaacca tcacctctgc tcacaccagc 1020

agcaccagta ccagcctgga gtcggactct gcgtctccgg gagtgtctga ccacggccga 1080

ggatcaggct gctcctgcac tgcgccggcc ctgagcggac ctgtggctcg gactatctat 1140

tacatcgcag ccgagctggt ccggctggtg gggtctgtgg actccatgaa gcccgtgctc 1200

cagtccctct accaccgagt gctgctctac cccccacccc agcaccgggt ggaagccatc 1260

aaaataatga aagagatact tgggagccca cagcgtctct gtgacttggc aggacccagc 1320

tccactgaat cagagtccag aaaaagatca atttcaaaaa gaaagtctca tctggatctc 1380

ctcaaactca tcatggatgg catgaccgaa gcatgcatca agggtggcat cgaagcttgc 1440

tatgcagccg tgtcctgtgt ctgcaccttg ctgggtgccc tggatgagct cagccagggg 1500

aagggcttga gcgaaggtca ggtgcaactg ctgcttctgc gccttgagga gctgaaggat 1560

ggggctgagt ggagccgaga ttccatggag atcaatgagg ctgacttccg ctggcagcgg 1620

cgagtgctgt cctcagaaca cacgccgtgg gagtcaggga acgagaggag ccttgacatc 1680

agcatcagtg tcaccacaga cacaggccag accactctcg agggagagtt gggtcagact 1740

acacccgagg accattcggg aaaccacaag aacagtctca agtcgccagc catcccagag 1800

ggtaaggaga cgctgagcaa agtattggaa acagaggcgg tagaccagcc agatgtcgtg 1860

cagagaagcc acacggtccc ttaccctgac ataactaact tcctgtcagt agactgcagg 1920

acaaggtcct atggatctag gtatagtgag agcaatttta gcgttgatga ccaagacctt 1980

tctaggacag agtttgattc ctgtgatcag tactctatgg cagcagaaaa ggactcgggc 2040

aggtccgacg tgtcagacat tgggtcggac aactgttcac tagccgatga agagcagaca 2100

ccccgggact gcctaggcca ccggtccctg cgaactgccg ccctgtctct aaaactgctg 2160

aagaaccagg aggcggatca gcacagcgcc aggctgttca tacagtccct ggaaggcctc 2220

ctccctcggc tcctgtctct ctccaatgta gaggaggtgg acaccgctct gcagaacttt 2280

gcctctactt tctgctcagg catgatgcac tctcctggct ttgacgggaa tagcagcctc 2340

agcttccaga tgctgatgaa cgcagacagc ctctacacag ctgcacactg cgccctgctc 2400

ctcaacctga agctctccca cggtgactac tacaggaagc ggccgaccct ggcgccaggc 2460

gtgatgaagg acttcatgaa gcaggtgcag accagcggcg tgctgatggt cttctctcag 2520

gcctggattg aggagctcta ccatcaggtg ctcgacagga acatgcttgg agaggctggc 2580

tattggggca gcccagaaga taacagcctt cccctcatca caatgctgac cgatattgac 2640

ggcttagaga gcagtgccat tggtggccag ctgatggcct cggctgctac agagtctcct 2700

ttcgcccaga gcaggagaat tgatgactcc acagtggcag gcgtggcatt tgctcgctat 2760

attctggtgg gctgctggaa gaacttgatc gatactttat caaccccact gactggtcga 2820

atggcgggga gctccaaagg gctggccttc attctgggag ctgaaggcat caaagagcag 2880

aaccagaagg agcgggacgc catctgcatg agcctcgacg ggctgcggaa agccgcacgg 2940

ctgagctgcg ctctaggcgt tgctgctaac tgcgcctcag cccttgccca gatggcagct 3000

gcctcctgtg tccaagaaga aaaagaagag agggaggccc aagaacccag tgatgccatc 3060

acacaagtga aactaaaagt ggagcagaaa ctggagcaga ttgggaaggt gcagggggtg 3120

tggctgcaca ctgcccacgt cttgtgcatg gaggccatcc tcagcgtagg cctggagatg 3180

ggaagccaca acccggactg ctggccacac gtgttcaggg tgtgtgaata cgtgggcacc 3240

ctggagcaca accacttcag cgatggtgcc tcgcagcccc ctctgaccat cagccagccc 3300

cagaaggcca ctggaagcgc tggcctcctt ggggaccccg agtgtgaggg ctcgcccccc 3360

gagcacagcc cggagcaggg gcgctccctg agcacggccc ctgtcgtcca gcccctgtcc 3420

atccaggacc tcgtccggga aggcagccgg ggtcgggcct ccgacttccg cggcgggagc 3480

ctcatgagcg ggagcagcgc ggccaaggtg gtgctcaccc tctccacgca agccgacagg 3540

ctctttgaag atgctacgga taagttgaac ctcatggcct tgggaggttt tctttaccag 3600

ctgaagaaag catcgcagtc tcagcttttc cattctgtta cagatacagt tgattactct 3660

ctggcaatgc caggagaagt taaatccact caagaccgaa aaagcgccct ccacctgttc 3720

cgcctgggga atgccatgct gaggattgtg cggagcaaag cacggcccct gctccacgtg 3780

atgcgctgct ggagccttgt ggccccacac ctggtggagg ctgcttgcca taaggaaaga 3840

catgtgtctc agaaggctgt ttccttcatc catgacatac tgacagaagt cctcactgac 3900

tggaatgagc cacctcattt tcacttcaat gaagcactct tccgaccttt cgagcgcatt 3960

atgcagctgg aattgtgtga tgaggacgtc caagaccagg ttgtcacatc cattggtgag 4020

ctggttgaag tgtgttccac gcagatccag tcgggatgga gacccttgtt cagtgccctg 4080

gaaacagtgc atggcgggaa caagtcagag atgaaggagt acctggttgg tgactactcc 4140

atgggaaaag gccaagctcc agtgtttgat gtatttgaag cttttctcaa tactgacaac 4200

atccaggtct ttgctaatgc agccactagc tacatcatgt gccttatgaa gtttgtcaaa 4260

ggactggggg aggtggactg taaagagatt ggagactgtg ccccagcacc cggagccccg 4320

tccacagacc tgtgcctccc ggccctggat tacctcaggc gctgctctca gttattggcc 4380

aaaatctaca aaatgccctt gaagccaata ttccttagtg ggagacttgc cggcttgcct 4440

cgaagacttc aggaacagtc agccagcagt gaggatggaa ttgaatcagt cctgtctgat 4500

tttgatgatg acaccggtct gatagaagtc tggataatcc tgctggagca gctgacagcg 4560

gctgtgtcca attgtccacg gcagcaccaa ccaccaactc tggatttact ctttgagctg 4620

ttgagagatg tgacgaaaac accaggacca gggtttggta tctatgcagt ggttcacctc 4680

ctccttcctg tgatgtccgt ttggctccgc cggagccata aagaccattc ctactgggat 4740

atggcctctg ccaatttcaa gcacgctatt ggtctgtcct gtgagctggt ggtggagcac 4800

attcaaagct ttctacattc agatatcagg tacgagagca tgatcaatac catgctgaag 4860

gacctctttg agttgctggt cgcctgtgtg gccaagccca ctgaaaccat ctccagagtg 4920

ggctgctcct gtattagata cgtccttgtg acagcgggcc ctgtgttcac tgaggagatg 4980

tggaggcttg cctgctgtgc cctgcaagat gcgttctctg ccacactcaa gccagtgaag 5040

gacctgctgg gctgcttcca cagcggcacg gagagcttca gcggggaagg ctgccaggtg 5100

cgagtggcgg ccccgtcctc ctccccaagt gccgaggccg agtactggcg catccgagcc 5160

atggcccagc aggtgtttat gctggacacc cagtgctcac caaagacacc aaacaacttt 5220

gaccacgctc agtcctgcca gctcattatt gagctgcctc ctgatgaaaa accaaatgga 5280

cacaccaaga aaagcgtgtc tttcagggaa attgtggtga gcctgctgtc tcatcaggtg 5340

ttactccaga acttatatga catcttgtta gaagagtttg tcaaaggccc ctctcctgga 5400

gaggaaaaga cgatacaagt gccagaagcc aagctggctg gcttcctcag atacatctct 5460

atgcagaact tggcagtcat attcgacctg ctgctggact cttataggac tgccagggag 5520

tttgacacca gccccgggct gaagtgcctg ctgaagaaag tgtctggcat cgggggcgcc 5580

gccaacctct accgccagtc tgcgatgagc tttaacattt atttccacgc cctggtgtgt 5640

gctgttctca ccaatcaaga aaccatcacg gccgagcaag tgaagaaggt cctttttgag 5700

gacgacgaga gaagcacgga ttcttcccag cagtgttcat ctgaggatga agacatcttt 5760

gaggaaaccg cccaggtcag ccccccgaga ggcaaggaga agagacagtg gcgggcacgg 5820

atgcccttgc tcagcgtcca gcctgtcagc aacgcagatt gggtgtggct ggtcaagagg 5880

ctgcacaagc tgtgcatgga actgtgcaac aactacatcc agatgcactt ggacctggag 5940

aactgtatgg aggagcctcc catcttcaag ggcgacccgt tcttcatcct gccctccttc 6000

cagtccgagt catccacccc atccaccggg ggcttctctg ggaaagaaac cccttccgag 6060

gatgacagaa gccagtcccg ggagcacatg ggcgagtccc tgagcctgaa ggccggtggt 6120

ggggacctgc tgctgccccc cagccccaaa gtggagaaga aggatcccag ccggaagaag 6180

gagtggtggg agaatgcggg gaacaaaatc tacaccatgg cagccgacaa gaccatttca 6240

aagttgatga ccgaatacaa aaagaggaaa cagcagcaca acctgtccgc gttccccaaa 6300

gaggtcaaag tggagaagaa aggagagcca ctgggtccca ggggccagga ctccccgctg 6360

cttcagcgtc cccagcactt gatggaccaa gggcaaatgc ggcattcctt cagcgcaggc 6420

cccgagctgc tgcgacagga caagaggccc cgctcaggct ccaccgggag ctccctcagt 6480

gtctcggtga gagacgcaga agcacagatc caggcatgga ccaacatggt gctaacagtt 6540

ctcaatcaga ttcagattct cccagaccag accttcacgg ccctccagcc cgcagtgttc 6600

ccgtgcatca gtcagctgac ctgtcacgtg accgacatca gagttcgcca ggctgtgagg 6660

gagtggctgg gcagggtggg ccgtgtctat gacatcattg tgtagccgac tcctgttcta 6720

ctctcccacc aaataacagt agtgagggtt agagtcctgc caatacagct gttgcatttt 6780

ccccaccact agccccactt aaactactac tactgtctca gagaacagtg tttcctaatg 6840

taaaaagcct ttccaaccac tgatcagcat tggggccata ctaaggtttg tatctagatg 6900

acacaaacga tattctgatt ttgcacatta ttatagaaga atctataatc cttgatatgt 6960

ttctaactct tgaagtatat ttcccagtgc ttttgcttac agtgttgtcc ccaaatgggt 7020

cattttcaag gattactcat ttgaaaacac tatattgatc catttgatcc atcatttaaa 7080

aaataaatac aattcctaag gcaatatctg ctggtaagtc aagctgataa acactcagac 7140

atctagtacc agggattatt aattggagga agatttatgg ttatgggtct ggctgggaag 7200

aagacaacta taaatacata ttcttgggtg tcataatcaa gaaagaggtg acttctgttg 7260

taaaataatc cagaacactt caaaattatt cctaaatcat taagattttc aggtattcac 7320

caatttcccc atgtaaggta ctgtgttgta cctttatttc tgtatttcta aaagaagaaa 7380

gttctttcct agcagggttt gaagtctgtg gcttatcagc ctgtgacaca gagtacccag 7440

tgaaagtggc tggtacgtag attgtcaaga gacataagac cgaccagcca ccctggctgt 7500

tcttgtggtg tttgtttcca tccccaaggc aaacaaggaa aggaaaggaa agaagaaaag 7560

gtgccttagt cctttgttgc acttccattt ccatgcccca caattgtctg aacataaggt 7620

atagcatttg gtttttaaga aaacaaaaca ttaagacgca actcatttta tatcaacacg 7680

cttggaggaa agggactcag ggaagggagc agggagtgtg gggtggggat ggattatgat 7740

gaaatcattt tcaatcttaa aatataatac aacaatcttg caaaattatg gtgtcagtta 7800

cacaagctct agtctcaaaa tgaaagtaat ggagaaagac actgaaattt agaaaatttt 7860

gtcgatttaa aatatttctc ctatctacca agtaaagtta ccctatgttt gatgtctttg 7920

cattcagacc aatatttcag gtggatattt ctaagtatta ctagaaaata cgtttgaaag 7980

ctttatctta ttatttacag tatttttata tttcttacat tatcctaatg attgaaaact 8040

cctcaatcaa gcttacttac acacattcta cagagttatt taaggcatac attataatct 8100

cccagcccca ttcataatga ataagtcacc ctttaaatat aagacacaaa ttctacagta 8160

ttgaaataag gatttaaagg ggtatttgta aactttgccc tccttgagaa atatggaact 8220

accttagagg ttaagaggaa ggcagtgttc tgacttcttt aggtgatctg aaaaaaacac 8280

ccttatcatc cagtgtacca tctagagatc accacagaat ccattttttt cccagttcca 8340

caaaacactc tgtttgcctt cagtttttac tcactagaca ataattcaag tttagaaaca 8400

ggtaatcagc tatttgatct taaaaggcaa tgaattgttg ggatatcagt gaactatgtt 8460

gtatactttt gaatttttac attttataaa tggaattgaa agttggataa ctgctttttt 8520

taaattttcc aacagaagta acaccacagt tgctttgttt ctttttatag cttacctgag 8580

gttcagttct tctttgtgaa cctgtgagta ctccacagtt tactggggga aaaggcttca 8640

gtaaagcaga ggctagaatt acagtattta tacatagcaa cttttcataa agtagaaaaa 8700

ttcaaaggaa gctgtctcaa tttgagaata ccagctgggc acggtggctc acgcctgtaa 8760

tcccagcact tactttggga ggccaaggtg ggcagataac ctgcggtcag gagtttgaga 8820

ccaggctgga caacatggtg aaacctcgtc tctactaaaa atacaaaaat tagccaggtg 8880

tggtaggatg cacctgtaat cccagctact taggaggccg agacaggaga atcgctcgaa 8940

cccaggaggc ggacgttgca gtgagccaag attgcaccat tgcactccag actgggtgac 9000

aagagtgaaa ctccatctaa aaaaaaaaaa aaaaaaaagt gaatactgta tcccaaagta 9060

tgttagttgt ttgtttggaa atcagcattc tccccgatgc tctattatgg gatccaaaat 9120

tcttgaacat aagtttaccc tgtactgtgt ccaaacactg ttctagttct agcctgatta 9180

tgggtcccaa gaataaaagg atgagtaggt gtacagagct cttgacctac aattttttaa 9240

gagtgttttg gtaccttccc attgtcttct ctataactca gtcctaacat actctgcact 9300

cgagttacca gccatccaca ctgacatcag atttcaacca gaaccatcac tgagtgacag 9360

cagtacttct cagaggtatt tgcagcttga tgcaaagtag tctctaatga gtaggcattc 9420

aggtggttct tcccagcagg tggagaagaa agggaggaga tgaagaacac tgagagggga 9480

gtggcacctt cccaggctgc ccagctcagt ctcttgccct gttcctgtga ctcagctgcc 9540

cactccccca actttgtttc cctccctccc agtctctgaa agtgtcaggt gtttctctcc 9600

tcacagtctc ttttgcagca acagtaagac aaaattcaag gcagcctttt aaagttacga 9660

acagttatta gcatgtattt acagacctaa gcagaatgag agtttataca ttgtttttag 9720

ttgcctgtat ttatagccaa aagtatatta ccttaaagtt gagatctttc tcttcttttc 9780

ctaaattttg gtaaagtgtg cttcatgaaa caaacatctg gaaaactcca agtataagag 9840

accctggact gatgatggcc cagccaagta tatggaggga cagagttctc tctgtcatta 9900

atgaggacat cggttttcac aattgaacct catgcactgt ccacagcatc tcacctagct 9960

cctgtatctc ctgatctgct tttaaaaata gttagttagg ctgccttttt acaccacctt 10020

ctctctctcc ccttgtggta attttccagc cttccccata gatataaaac tagaacacct 10080

ttatgatttg gggtctatgt aatgactgac cgataagaac ccaggcagat gctaacatac 10140

ttaacagctc gcattaaaat actttaaatc aggcgtgatg gctcattcct gtaatctcaa 10200

gcactttggg aggctaaggt gggtggatct cttgaggtca ggagttcgag accaacctgg 10260

ccaacgtggt gaaaccccgt ctctactaaa aatacaaaat tagccgggca tggtggcagc 10320

tgcctgtaat cccagctact cgggaagctg aggcaggaga attgcttgaa cctgggaggt 10380

ggggattgca gtcagccaag attgttctgc agcatgggtg acaaagtgag acttcgtctc 10440

aagtaaataa aactaaaatt tttaaatcaa acatgacaaa aatgttaata taattcagaa 10500

gtaccttgaa attgaaacat atttgtgcaa tgatcattag gctttttgtc cttgttgttt 10560

taaaatgagg cttatacaga gtgagttgag agtcaagtag ccttcgctgt gagacggtaa 10620

tgcagttata taatagatac ccttgacttt gccagattca tcacaatact gcttatacag 10680

gaaagttttc tcagaaagga aaatccatta gtatcagtcc catcaagcca aacagaatga 10740

agacctttga tagtaatagc aagaggttac aaatagcagg gaggaggcga gtagtgaatg 10800

tcactgtgat tgcaaaccct tacctgtatt atcacacgta gtcctcacaa caaccttgtg 10860

agacaagtgt tgtgttcctc attttttcag aggggaacac agacccagag aggttaagaa 10920

atttgcccaa gataacaagt aaaaggcaaa gttggttgca aaagaggtgt ttctgaattc 10980

aagggccata ctctctctct gacaacatgc tctaagtcca tagagtaagc actctagtat 11040

gaaaaaaagt ttcaaggaac gaggccatga aaatgagact atttgacatc tcagatctgt 11100

ctgggatgtt atggaggttt ttaaaaataa agttgaaaaa agaaaatgaa tcatgtttat 11160

acataaaaaa atcacatgta acacatttca agtgtttgaa aataaaacca aaatctaaac 11220

tttagtcttc aagcagacat tcagtgttac tttagaaaac tcactgaatt aggtggaaat 11280

gatggaataa tactattcat ggccagctat taacacagaa gaacatggca gtgtgtgtct 11340

ggaacggcat gcacaatttg taaacctttt tcaaatatca tttaatcaac tcagaataaa 11400

gtgccctgta gccaacagtg cctctttact tgcttctctg ggaaatacat ggtactaaat 11460

tagtagcaca aagtttggga atatgcaaaa taatggataa ccatttttca aaatgtacat 11520

tctctgaaga ggaagcagct ggttggacag gatttcttga agagccaggt gctaagggca 11580

tcaggtcgac atccatagta accatgtgcc ataacatcta cacatttcca cttgttttac 11640

agacaaggta acaggcagaa ggaaaatcca gagtcttgca gtaagcagat gacaaaactt 11700

caatatgctt gggcaccact taggtgaccc cagggagatt tagtgtggcc ttaggaaagc 11760

aaaagagcac tttttattgg aaatatgagc ttgtcactgg gaaagatttg taaaattgat 11820

caagaacttg atttataatt atgcctcaaa aaaaaaagtt ctcatttagt agtggagcaa 11880

tctagaaaac ataccttttt tgtttgtttg gaagatcctc tttccctggc tgtattgtag 11940

tgtttgctat ttgatgtgga aataactaat aacttaagat tttggaacag aacacccttt 12000

agatttccaa aacacaattc ttatttcagg gaagacagac caaaaatatc tcctgagatc 12060

attggtttct ttataaattg tggtaccact ccatcattga agagaaacca ctaccacacc 12120

actagcacca tacagaacct tttctctgta tctttgtaca atactacaaa ggggtaccag 12180

ggaggagaga gtggctgacc actttagtga caaaacagca ctccactgct ggtgaatccc 12240

atctaattat ggtccttcca cccttttcaa ccaccaacaa ctgttcgtac tgttaattcc 12300

tatcctgaag gtttaaccag tggttgtcta gtatcttctg tctttagaac agtggttctc 12360

aaactttagt acacatcagc atcacctgga gggccttttt ttaaaataag acacagattg 12420

ctgggctcat ggtcagagtt cccagttaag taaatcagga aatttgtatt tctaacaagt 12480

ttataggtga ggccaatact gctgttttgg gaactatgct ttgagaacca ctgccttgaa 12540

aaaatttcca acttctacct ttaagatcag cctgacttat caaacgctag agaaaaactg 12600

aatctaccct tgggcagatg acttgggatt ggattctata cagcagtctt gctcaatctt 12660

cccagtttcc agttttatta taccaacaat tggtttttac aagctagaag acaatgaatg 12720

tataagttct atggaacagt gagataaatc taagcttctt gtctttgtat ttagaaacat 12780

tgattctatg gatgatcatt tgtatcatgt tgaccctttg acttgtactg aaggtgattt 12840

taaatttaag tatgtagtgt ttgaatttct tccatccatg tcgttttaat gagatgtttc 12900

catgtcagct cctttacagc cttggctcct ggcttacaga tttttgaata gttgtttgct 12960

tgccagttgt tttacatctt tcattggcca ccaaaatatt agccatttga gatgagatga 13020

gactacttgt tgtaccttca tctttcattt aattttctgg cgtaaattaa cattttaatt 13080

tcatatatat ctgtaaagag tctacccaaa ggcttcacgg aaatttgcaa aatgaactaa 13140

ttccctttta agcagcaggt gtgcctgttt ttgacttttc agtaaatatg ttgtttgtgc 13200

acatatctac atggtggaga ccatattcat tatttcatct tccaaataat gggaaaaata 13260

taaaagtgaa tcagtgtgct ttgggaattc agtgaaatca tgttaactca tatagagggg 13320

gccttagttt atctcttctt tactgaatta attagttttg gaaattcttt taccattaaa 13380

aaaaattaag gaccatacag agaatgattt aagaaaaaac aagtcactta aaaatcatca 13440

cctatttata aactgtatta attacacata atgcttattg attcaatgag gtttctctaa 13500

agacttctgc ttaataaata tgctgacttc atttaaatta gtttagacta ttgtaggaat 13560

ggaaggaaat gattatattt actagaatta gtgagatcag aaagcatatc agaatgttga 13620

tgatatcaag gagacaatct acagagtttt tgcctctgtg gatggaaata agggtgtttt 13680

tttttggttt tttttttact ttagtttccc ataatttttg gaaattatgt gtgcatttag 13740

ttcttttagt aacactgatt ttaaaattaa atttcaaaag tcaatctcta agagtaattt 13800

atttttgttt taccaaccag tgccaaaaag gagaggaggg aatccaaaag ccaatctttt 13860

gaaccaatgt gtaaaagatt atgttttttc ttaaagttag ggaggctcgg gccctgacac 13920

tgccagcccc agtgagcatc cctggctacc tcgggattat gtgcaagctg ctttgtccta 13980

catttctttc atctggttct tattgggagt gcttctctct aataaaaatt gatttcccac 14040

aaaataggca aagctgaaca aagatgaatg cttttgataa gttgggtttc acttcagttg 14100

aaacaatgtg atagaatatc caggtgtggc atgatggggc aggaggaggt gcctagaggg 14160

aaaagttatt tttgtttctt agtgttgtgt tgtggggatg ggacagataa gaataagatg 14220

tttattgccc taatcatgct aagagactat tattcaatat gcttttcccg cttttctaag 14280

aggaataaac ttagacaaat tacattataa acagttcccc tactactatc tcccactcta 14340

gataaagcca gtgggtggta tgggtccttt tattccttat agtattatgc caaagaatca 14400

acttattttc attgaagatt ataaataaat gaagcttgtt atagccataa tgatttgagt 14460

cagtatacca ttttacctat aaaatgcaaa attcatcctt gcaaccccat tcaccaggag 14520

ccttgaagca ttttgtttac tccaaaggcc ttgtcaagga agcataattt tttgttttgc 14580

cttcttattt agtcagtttg gtcatattta cttaaaaaaa caaactgaaa atcacactcc 14640

tttatatgtt gatataactg attttataga atctgtctgt tctttgttta acaggtctct 14700

gtaagcaagc ttgcaagtgt attttgtgta cattttatct gaggtggaaa tgaaaattct 14760

aaagagaaaa tattttaaaa gatattgtat ttatgttgct tgtgttgtag aataaagatt 14820

caaatgcatt aaaaatctgg tacatgaaac aa 14852

<210>80

<211>2177

<212>PRT

<213> human (Homo sapiens)

<400>80

Met Glu Glu Ile Leu Arg Lys Leu Gln Lys Glu Ala Ser Gly Ser Lys

1 5 10 15

Tyr Lys Ala Ile Lys Glu Ser Cys Thr Trp Ala Leu Glu Thr Leu Gly

20 25 30

Gly Leu Asp Thr Ile Val Lys Ile Pro Pro His Val Leu Arg Glu Lys

35 40 45

Cys Leu Leu Pro Leu Gln Leu Ala Leu Glu Ser Lys Asn Val Lys Leu

50 55 60

Ala Gln His Ala Leu Ala Gly Met Gln Lys Leu Leu Ser Glu Glu Arg

65 70 75 80

Phe Val Ser Met Glu Thr Asp Ser Asp Glu Lys Gln Leu Leu Asn Gln

85 90 95

Ile Leu Asn Ala Val Lys Val Thr Pro Ser Leu Asn Glu Asp Leu Gln

100 105 110

Val Glu Val Met Lys Val Leu Leu Cys Ile Thr Tyr Thr Pro Thr Phe

115 120 125

Asp Leu Asn Gly Ser Ala Val Leu Lys Ile Ala Glu Val Cys Ile Glu

130 135 140

Thr Tyr Ile Ser Ser Cys His Gln Arg Ser Ile Asn Thr Ala Val Arg

145 150 155 160

Ala Thr Leu Ser Gln Met Leu Ser Asp Leu Thr Leu Gln Leu Arg Gln

165 170 175

Arg Gln Glu Asn Thr Ile Ile Glu Asn Pro Asp Val Pro Gln Asp Phe

180 185 190

Gly Asn Gln Gly Ser Thr Val Glu Ser Leu Cys Asp Asp Val Val Ser

195 200 205

Val Leu Thr Val Leu Cys Glu Lys Leu Gln Ala Ala Ile Asn Asp Ser

210 215 220

Gln Gln Leu Gln Leu Leu Tyr Leu Glu Cys Ile Leu Ser Val Leu Ser

225 230 235 240

Ser Ser Ser Ser Ser Met His Leu His Arg Arg Phe Thr Asp Leu Ile

245 250 255

Trp Lys Asn Leu Cys Pro Ala Leu Ile Val Ile Leu Gly Asn Pro Ile

260 265 270

His Asp Lys Thr Ile Thr Ser Ala His Thr Ser Ser Thr Ser Thr Ser

275 280 285

Leu Glu Ser Asp Ser Ala Ser Pro Gly Val Ser Asp His Gly Arg Gly

290 295 300

Ser Gly Cys Ser Cys Thr Ala Pro Ala Leu Ser Gly Pro Val Ala Arg

305 310 315 320

Thr Ile Tyr Tyr Ile Ala Ala Glu Leu Val Arg Leu Val Gly Ser Val

325 330 335

Asp Ser Met Lys Pro Val Leu Gln Ser Leu Tyr His Arg Val Leu Leu

340 345 350

Tyr Pro Pro Pro Gln His Arg Val Glu Ala Ile Lys Ile Met Lys Glu

355 360 365

Ile Leu Gly Ser Pro Gln Arg Leu Cys Asp Leu Ala Gly Pro Ser Ser

370 375 380

Thr Glu Ser Glu Ser Arg Lys Arg Ser Ile Ser Lys Arg Lys Ser His

385 390 395 400

Leu Asp Leu Leu Lys Leu Ile Met Asp Gly Met Thr Glu Ala Cys Ile

405 410 415

Lys Gly Gly Ile Glu Ala Cys Tyr Ala Ala Val Ser Cys Val Cys Thr

420 425 430

Leu Leu Gly Ala Leu Asp Glu Leu Ser Gln Gly Lys Gly Leu Ser Glu

435 440 445

Gly Gln Val Gln Leu Leu Leu Leu Arg Leu Glu Glu Leu Lys Asp Gly

450 455 460

Ala Glu Trp Ser Arg Asp Ser Met Glu Ile Asn Glu Ala Asp Phe Arg

465 470 475 480

Trp Gln Arg Arg Val Leu Ser Ser Glu His Thr Pro Trp Glu Ser Gly

485 490 495

Asn Glu Arg Ser Leu Asp Ile Ser Ile Ser Val Thr Thr Asp Thr Gly

500 505 510

Gln Thr Thr Leu Glu Gly Glu Leu Gly Gln Thr Thr Pro Glu Asp His

515 520 525

Ser Gly Asn His Lys Asn Ser Leu Lys Ser Pro Ala Ile Pro Glu Gly

530 535 540

Lys Glu Thr Leu Ser Lys Val Leu Glu Thr Glu Ala Val Asp Gln Pro

545 550 555 560

Asp Val Val Gln Arg Ser His Thr Val Pro Tyr Pro Asp Ile Thr Asn

565 570 575

Phe Leu Ser Val Asp Cys Arg Thr Arg Ser Tyr Gly Ser Arg Tyr Ser

580 585 590

Glu Ser Asn Phe Ser Val Asp Asp Gln Asp Leu Ser Arg Thr Glu Phe

595 600 605

Asp Ser Cys Asp Gln Tyr Ser Met Ala Ala Glu Lys Asp Ser Gly Arg

610 615 620

Ser Asp Val Ser Asp Ile Gly Ser Asp Asn Cys Ser Leu Ala Asp Glu

625 630 635 640

Glu Gln Thr Pro Arg Asp Cys Leu Gly His Arg Ser Leu Arg Thr Ala

645 650 655

Ala Leu Ser Leu Lys Leu Leu Lys Asn Gln Glu Ala Asp Gln His Ser

660 665 670

Ala Arg Leu Phe Il e Gln SerLeu Glu Gly Leu Leu Pro Arg Leu Leu

675 680 685

Ser Leu Ser Asn Val Glu Glu Val Asp Thr Ala Leu Gln Asn Phe Ala

690 695 700

Ser Thr Phe Cys Ser Gly Met Met His Ser Pro Gly Phe Asp Gly Asn

705 710 715 720

Ser Ser Leu Ser Phe Gln Met Leu Met Asn Ala Asp Ser Leu Tyr Thr

725 730 735

Ala Ala His Cys Ala Leu Leu Leu Asn Leu Lys Leu Ser His Gly Asp

740 745 750

Tyr Tyr Arg Lys Arg Pro Thr Leu Ala Pro Gly Val Met Lys Asp Phe

755 760 765

Met Lys Gln Val Gln Thr Ser Gly Val Leu Met Val Phe Ser Gln Ala

770 775 780

Trp Ile Glu Glu Leu Tyr His Gln Val Leu Asp Arg Asn Met Leu Gly

785 790 795 800

Glu Ala Gly Tyr Trp Gly Ser Pro Glu Asp Asn Ser Leu Pro Leu Ile

805 810 815

Thr Met Leu Thr Asp Ile Asp Gly Leu Glu Ser Ser Ala Ile Gly Gly

820 825 830

Gln Leu Met Ala Ser Ala Ala Thr Glu Ser Pro Phe Ala Gln Ser Arg

835 840 845

Arg Ile Asp Asp Ser Thr Val Ala Gly Val Ala Phe Ala Arg Tyr Ile

850 855 860

Leu Val Gly Cys Trp Lys Asn Leu Ile Asp Thr Leu Ser Thr Pro Leu

865 870 875 880

Thr Gly Arg Met Ala Gly Ser Ser Lys Gly Leu Ala Phe Ile Leu Gly

885 890 895

Ala Glu Gly Ile Lys Glu Gln Asn Gln Lys Glu Arg Asp Ala Ile Cys

900 905 910

Met Ser Leu Asp Gly Leu Arg Lys Ala Ala Arg Leu Ser Cys Ala Leu

915 920 925

Gly Val Ala Ala Asn Cys Ala Ser Ala Leu Ala Gln Met Ala Ala Ala

930 935 940

Ser Cys Val Gln Glu Glu Lys Glu Glu Arg Glu Ala Gln Glu Pro Ser

945 950 955 960

Asp Ala Ile Thr Gln Val Lys Leu Lys Val Glu Gln Lys Leu Glu Gln

965 970 975

Ile Gly Lys Val Gln Gly Val Trp Leu His Thr Ala His Val Leu Cys

980 985 990

Met Glu Ala Ile Leu Ser Val Gly Leu Glu Met Gly Ser His Asn Pro

995 1000 1005

Asp Cys Trp Pro His Val Phe Arg Val Cys Glu Tyr Val Gly Thr

1010 1015 1020

Leu Glu His Asn His Phe Ser Asp Gly Ala Ser Gln Pro Pro Leu

1025 1030 1035

Thr Ile Ser Gln Pro Gln Lys Ala Thr Gly Ser Ala Gly Leu Leu

1040 1045 1050

Gly Asp Pro Glu Cys Glu Gly Ser Pro Pro Glu His Ser Pro Glu

1055 1060 1065

Gln Gly Arg Ser Leu Ser Thr Ala Pro Val Val Gln Pro Leu Ser

1070 1075 1080

Ile Gln Asp Leu Val Arg Glu Gly Ser Arg Gly Arg Ala Ser Asp

1085 1090 1095

Phe Arg Gly Gly Ser Leu Met Ser Gly Ser Ser Ala Ala Lys Val

1100 1105 1110

Val Leu Thr Leu Ser Thr Gln Ala Asp Arg Leu Phe Glu Asp Ala

1115 1120 1125

Thr Asp Lys Leu Asn Leu Met Ala Leu Gly Gly Phe Leu Tyr Gln

1130 1135 1140

Leu Lys Lys Ala Ser Gln Ser Gln Leu Phe His Ser Val Thr Asp

1145 1150 1155

Thr Val Asp Tyr Ser Leu Ala Met Pro Gly Glu Val Lys Ser Thr

1160 1165 1170

Gln Asp Arg Lys Ser Ala Leu His Leu Phe Arg Leu Gly Asn Ala

1175 1180 1185

Met Leu Arg Ile Val Arg Ser Lys Ala Arg Pro Leu Leu His Val

1190 1195 1200

Met Arg Cys Trp Ser Leu Val Ala Pro His Leu Val Glu Ala Ala

1205 1210 1215

Cys His Lys Glu Arg His Val Ser Gln Lys Ala Val Ser Phe Ile

1220 1225 1230

His Asp Ile Leu Thr Glu Val Leu Thr Asp Trp Asn Glu Pro Pro

1235 1240 1245

His Phe His Phe Asn Glu Ala Leu Phe Arg Pro Phe Glu Arg Ile

1250 1255 1260

Met Gln Leu Glu Leu Cys Asp Glu Asp Val Gln Asp Gln Val Val

1265 1270 1275

Thr Ser Ile Gly Glu Leu Val Glu Val Cys Ser Thr Gln Ile Gln

1280 1285 1290

Ser Gly Trp Arg Pro Leu Phe Ser Ala Leu Glu Thr Val His Gly

1295 1300 1305

Gly Asn Lys Ser Glu Met Lys Glu Tyr Leu Val Gly Asp Tyr Ser

1310 1315 1320

Met Gly Lys Gly Gln Ala Pro Val Phe Asp Val Phe Glu Ala Phe

1325 1330 1335

Leu Asn Thr Asp Asn Ile Gln Val Phe Ala Asn Ala Ala Thr Ser

1340 1345 1350

Tyr Ile Met Cys Leu Met Lys Phe Val Lys Gly Leu Gly Glu Val

1355 1360 1365

Asp Cys Lys Glu Ile Gly Asp Cys Ala Pro Ala Pro Gly Ala Pro

1370 1375 1380

Ser Thr Asp Leu Cys Leu Pro Ala Leu Asp Tyr Leu Arg Arg Cys

1385 1390 1395

Ser Gln Leu Leu Ala Lys Ile Tyr Lys Met Pro Leu Lys Pro Ile

1400 1405 1410

Phe Leu Ser Gly Arg Leu Ala Gly Leu Pro Arg Arg Leu Gln Glu

1415 1420 1425

Gln Ser Ala Ser Ser Glu Asp Gly Ile Glu Ser Val Leu Ser Asp

1430 1435 1440

Phe Asp Asp Asp Thr Gly Leu Ile Glu Val Trp Ile Ile Leu Leu

1445 1450 1455

Glu Gln Leu Thr Ala Ala Val Ser Asn Cys Pro Arg Gln His Gln

1460 1465 1470

Pro Pro Thr Leu Asp Leu Leu Phe Glu Leu Leu Arg Asp Val Thr

1475 1480 1485

Lys Thr Pro Gly Pro Gly Phe Gly Ile Tyr Ala Val Val His Leu

1490 1495 1500

Leu Leu Pro Val Met Ser Val Trp Leu Arg Arg Ser His Lys Asp

1505 1510 1515

His Ser Tyr Trp Asp Met Ala Ser Ala Asn Phe Lys His Ala Ile

1520 1525 1530

Gly Leu Ser Cys Glu Leu Val Val Glu His Ile Gln Ser Phe Leu

1535 1540 1545

His Ser Asp Ile Arg Tyr Glu Ser Met Ile Asn Thr Met Leu Lys

1550 1555 1560

Asp Leu Phe Glu Leu Leu Val Ala Cys Val Ala Lys Pro Thr Glu

1565 1570 1575

Thr Ile Ser Arg Val Gly Cys Ser Cys Ile Arg Tyr Val Leu Val

1580 1585 1590

Thr Ala Gly Pro Val Phe Thr Glu Glu Met Trp Arg Leu Ala Cys

1595 1600 1605

Cys Ala Leu Gln Asp Ala Phe Ser Ala Thr Leu Lys Pro Val Lys

1610 1615 1620

Asp Leu Leu Gly Cys Phe His Ser Gly Thr Glu Ser Phe Ser Gly

1625 1630 1635

Glu Gly Cys Gln Val Arg Val Ala Ala Pro Ser Ser Ser Pro Ser

1640 1645 1650

Ala Glu Ala Glu Tyr Trp Arg Ile Arg Ala Met Ala Gln Gln Val

1655 1660 1665

Phe Met Leu Asp Thr Gln Cys Ser Pro Lys Thr Pro Asn Asn Phe

1670 1675 1680

Asp His Ala Gln Ser Cys Gln Leu Ile Ile Glu Leu Pro Pro Asp

1685 1690 1695

Glu Lys Pro Asn Gly His Thr Lys Lys Ser Val Ser Phe Arg Glu

1700 1705 1710

Ile Val Val Ser Leu Leu Ser His Gln Val Leu Leu Gln Asn Leu

1715 1720 1725

Tyr Asp Ile Leu Leu Glu Glu Phe Val Lys Gly Pro Ser Pro Gly

1730 1735 1740

Glu Glu Lys Thr Ile Gln Val Pro Glu Ala Lys Leu Ala Gly Phe

1745 1750 1755

Leu Arg Tyr Ile Ser Met Gln Asn Leu Ala Val Ile Phe Asp Leu

1760 1765 1770

Leu Leu Asp Ser Tyr Arg Thr Ala Arg Glu Phe Asp Thr Ser Pro

1775 1780 1785

Gly Leu Lys Cys Leu Leu Lys Lys Val Ser Gly Ile Gly Gly Ala

1790 1795 1800

Ala Asn Leu Tyr Arg Gln Ser Ala Met Ser Phe Asn Ile Tyr Phe

1805 1810 1815

His Ala Leu Val Cys Ala Val Leu Thr Asn Gln Glu Thr Ile Thr

1820 1825 1830

Ala Glu Gln Val Lys Lys Val Leu Phe Glu Asp Asp Glu Arg Ser

1835 1840 1845

Thr Asp Ser Ser Gln Gln Cys Ser Ser Glu Asp Glu Asp Ile Phe

1850 1855 1860

Glu Glu Thr Ala Gln Val Ser Pro Pro Arg Gly Lys Glu Lys Arg

1865 1870 1875

Gln Trp Arg Ala Arg Met Pro Leu Leu Ser Val Gln Pro Val Ser

1880 1885 1890

Asn Ala Asp Trp Val Trp Leu Val Lys Arg Leu His Lys Leu Cys

1895 1900 1905

Met Glu Leu Cys Asn Asn Tyr Ile Gln Met His Leu Asp Leu Glu

1910 1915 1920

Asn Cys Met Glu Glu Pro Pro Ile Phe Lys Gly Asp Pro Phe Phe

1925 1930 1935

Ile Leu Pro Ser Phe Gln Ser Glu Ser Ser Thr Pro Ser Thr Gly

1940 1945 1950

Gly Phe Ser Gly Lys Glu Thr Pro Ser Glu Asp Asp Arg Ser Gln

1955 1960 1965

Ser Arg Glu His Met Gly Glu Ser Leu Ser Leu Lys Ala Gly Gly

1970 1975 1980

Gly Asp Leu Leu Leu Pro Pro Ser Pro Lys Val Glu Lys Lys Asp

1985 1990 1995

Pro Ser Arg Lys Lys Glu Trp Trp Glu Asn Ala Gly Asn Lys Ile

2000 2005 2010

Tyr Thr Met Ala Ala Asp Lys Thr Ile Ser Lys Leu Met Thr Glu

2015 2020 2025

Tyr Lys Lys Arg Lys Gln Gln His Asn Leu Ser Ala Phe Pro Lys

2030 2035 2040

Glu Val Lys Val Glu Lys Lys Gly Glu Pro Leu Gly Pro Arg Gly

2045 2050 2055

Gln Asp Ser Pro Leu Leu Gln Arg Pro Gln His Leu Met Asp Gln

2060 2065 2070

Gly Gln Met Arg His Ser Phe Ser Ala Gly Pro Glu Leu Leu Arg

2075 2080 2085

Gln Asp Lys Arg Pro Arg Ser Gly Ser Thr Gly Ser Ser Leu Ser

2090 2095 2100

Val Ser Val Arg Asp Ala Glu Ala Gln Ile Gln Ala Trp Thr Asn

2105 2110 2115

Met Val Leu Thr Val Leu Asn Gln Ile Gln Ile Leu Pro Asp Gln

2120 2125 2130

Thr Phe Thr Ala Leu Gln Pro Ala Val Phe Pro Cys Ile Ser Gln

2135 2140 2145

Leu Thr Cys His Val Thr Asp Ile Arg Val Arg Gln Ala Val Arg

2150 2155 2160

Glu Trp Leu Gly Arg Val Gly Arg Val Tyr Asp Ile Ile Val

2165 2170 2175

<210>81

<211>4221

<212>DNA

<213> human (Homo sapiens)

<400>81

cgataacgat ttgtgttgtg agaggcgcaa gctgcgattt ctgctgaact tggaggcatt 60

tctacgactt ttctctcagc tgaggctttt cctccgaccc tgatgctctt caattcggtg 120

ctccgccagc cccagcttgg cgtcctgaga aatggatggt cttcacaata ccctcttcaa 180

tcccttctga ctggttatca gtgcagtggt aatgatgaac acacttctta tggagaaaca 240

ggagtcccag ttcctccttt tggatgtacc ttctcttctg ctcccaatat ggaacatgta 300

ctagcagttg ccaatgaaga aggctttgtt cgattgtata acacagaatc acaaagtttc 360

agaaagaagt gcttcaaaga atggatggct cactggaatg ccgtctttga cctggcctgg 420

gttcctggtg aacttaaact tgttacagca gcaggtgatc aaacagccaa attttgggac 480

gtaaaagctg gtgagctgat tggaacatgc aaaggtcatc aatgcagcct caagtcagtt 540

gccttttcta agtttgagaa agctgtattc tgtacgggtg gaagagatgg caacattatg 600

gtctgggata ccaggtgcaa caaaaaagat gggttttata ggcaagtgaa tcaaatcagt 660

ggagctcaca atacctcaga caagcaaacc ccttcaaaac ccaagaagaa acagaattca 720

aaaggacttg ctccttctgt ggatttccag caaagtgtta ctgtggtcct ctttcaagac 780

gagaatacct tagtctcagc aggagctgtg gatgggataa tcaaagtatg ggatttacgt 840

aagaattata ctgcttatcg acaagaaccc atagcatcca agtctttcct gtacccaggt 900

agcagcactc gaaaacttgg atattcaagt ctgattttgg attccactgg ctctacttta 960

tttgctaatt gcacagacga taacatctac atgtttaata tgactgggtt gaagacttct 1020

ccagtggcta ttttcaatgg acaccagaac tctacctttt atgtaaaatc cagccttagt 1080

ccagatgacc agtttttagt cagtggctca agtgatgaag ctgcctacat atggaaggtc 1140

tccacaccct ggcaacctcc tactgtgctc ctgggtcatt ctcaagaggt cacgtctgtg 1200

tgctggtgtc catctgactt cacaaagatt gctacctgtt ctgatgacaa tacactaaaa 1260

atctggcgct tgaatagagg cttagaggag aaaccaggag gtgataaact ttccacggtg 1320

ggttgggcct ctcagaagaa aaaagagtca agacctggcc tagtaacagt aacgagtagc 1380

cagagtactc ctgccaaagc ccccagggta aagtgcaatc catccaattc ttccccgtca 1440

tccgcagctt gtgccccaag ctgtgctgga gacctccctc ttccttcaaa tactcctacg 1500

ttctctatta aaacctctcc tgccaaggcc cggtctccca tcaacagaag aggctctgtc 1560

tcctccgtct ctcccaagcc accttcatct ttcaagatgt cgattagaaa ctgggtgacc 1620

cgaacacctt cctcatcacc acccatcact ccacctgctt cggagaccaa gatcatgtct 1680

ccgagaaaag cccttattcc tgtgagccag aagtcatccc aagcagaggc ttgctctgag 1740

tctagaaata gagtaaagag gaggctagac tcaagctgtc tggagagtgt gaaacaaaag 1800

tgtgtgaaga gttgtaactg tgtgactgag cttgatggcc aagttgaaaa tcttcatttg 1860

gatctgtgct gccttgctgg taaccaggaa gaccttagta aggactctct aggtcctacc 1920

aaatcaagca aaattgaagg agctggtacc agtatctcag agcctccgtc tcctatcagt 1980

ccgtatgctt cagaaagctg tggaacgcta cctcttcctt tgagaccttg tggagaaggg 2040

tctgaaatgg taggcaaaga gaatagttcc ccagagaata aaaactggtt gttggccatg 2100

gcagccaaac ggaaggctga gaatccatct ccacgaagtc cgtcatccca gacacccaat 2160

tccaggagac agagcggaaa gacattgcca agcccggtca ccatcacgcc cagctccatg 2220

aggaaaatct gcacatactt ccatagaaag tcccaggagg acttctgtgg tcctgaacac 2280

tcaacagaat tatagattct aatctgagtg agttactgag ctttggtcca ctaaaacaag 2340

ctgagctttg gtccactaaa acaagatgaa aaatacaaga gtgactctat aactctggtc 2400

tttaagaaag ctgccttttc atttttagac aaaatctttt caacgctgaa atgtacctaa 2460

tctggttcta ctaccataat gtatatgcag cttcccgagg atgaatgctg tgtttaaatt 2520

tcataaagta aatttgtcac tctagcattt tgaatgaata gtcttcactt tttaaattat 2580

tcatcttctc tataataatg acatcccagt tcatggaggc aaaaaacaag tttcttgtta 2640

tcctgaaact ttctatgctc agtggaaagt atctgccagc cacagcatga ggcctgtgaa 2700

ggctgactga gaaatcctct gctgaagacc cctggttctg ttctgcctcc aacatgtata 2760

attttatttg aaatacataa tcttttcact atgcttttgt ggggtttttt ttaagtatgt 2820

gtaaaaatgt gatgctcaga taagtacatt tatatcagtt cagtgttaaa atgcagtctc 2880

ttgagttaaa gtcatcttta ttttaaatgc agtgataaat gtcaactctt cggagaaact 2940

aggagaacaa caacagaaag ctgtgtttgt cttttttctc tcaaatatat ctcccgtatg 3000

agatttcagg tccccatgtt ttcaccaagc aatctgctat gtcagccaac ccaacatcac 3060

tttctacagg aggttatgat ttttgccatt tactagagga agatgtttta tgaaatcaat 3120

ttggggtttg aattcaggtg cagtcatcag ttctttaggg gctgcaatgt tttaaaaaaa 3180

ataagtcatc agattttaag aaaaaagtga tgatttctta ttgatatttt tgtaacagaa 3240

tatagctctt aactgaaaat ccagaaccag aaacataaat cttgagtttc ttttcatgta 3300

cataaaaagc aatagccttt tagtatagat agccctgagc caaaaagtaa tagaattttc 3360

tctagatatt taatacagag agtgtataga ctgactctaa gttaataatg tgcaaaatat 3420

cttaaacatc cctcccctta ttcaacaatt atgtatcagt gatcttgaac cattgtttta 3480

tatttttcac ctttgtaacc tcatggaaag aggctttaca tactttctat gtactattta 3540

cttagaaggg agcccccttc cagtcatgaa acttcatttg ttttatccat atccctgagg 3600

actgtgtaga ctttatgtca gttctgtgta gactttatgt cagtttttgt cattatttga 3660

aaatctattc tgacaacttt ttaattcctt tgatcttata agttaaagct gtaacaactg 3720

aaattgcatg gatcaagtaa gcatagtttt atccagggag aaaaataaaa ggaagccata 3780

gaattgctct ggtcaaaacc aagcacacca tagccttaac tgaatattta ggaaatctgc 3840

ctaatctgct tatatttggt gtttgttttt tgactgttgg gctttgggaa gatgttattt 3900

atgaccaata tctgccagta acgctgttta tctcacttgc tttgaaagcc aatgggggaa 3960

aaaaatccat gaaaaaaaaa agattgataa agtagatgat tttgtttgta tccctaccca 4020

tctcctggca gccctactga gtgaaattgg gatacatttg gctgtcagaa attataccga 4080

gtctactggg tataacatgt ctcacttgga aagctagtac ttttaaatgg gtgccaaagg 4140

tcaactgtaa tgagataatt atccctgcct gtgtccatgt cagactttga gctgatcctg 4200

aataataaag ccttttacct t 4221

<210>82

<211>730

<212>PRT

<213> human (Homo sapiens)

<400>82

Met Leu Phe Asn Ser Val Leu Arg Gln Pro Gln Leu Gly Val Leu Arg

1 5 10 15

Asn Gly Trp Ser Ser Gln Tyr Pro Leu Gln Ser Leu Leu Thr Gly Tyr

20 25 30

Gln Cys Ser Gly Asn Asp Glu His Thr Ser Tyr Gly Glu Thr Gly Val

35 40 45

Pro Val Pro Pro Phe Gly Cys Thr Phe Ser Ser Ala Pro Asn Met Glu

50 55 60

His Val Leu Ala Val Ala Asn Glu Glu Gly Phe Val Arg Leu Tyr Asn

65 70 75 80

Thr Glu Ser Gln Ser Phe Arg Lys Lys Cys Phe Lys Glu Trp Met Ala

85 90 95

His Trp Asn Ala Val Phe Asp Leu Ala Trp Val Pro Gly Glu Leu Lys

100 105 110

Leu Val Thr Ala Ala Gly Asp Gln Thr Ala Lys Phe Trp Asp Val Lys

115 120 125

Ala Gly Glu Leu Ile Gly Thr Cys Lys Gly His Gln Cys Ser Leu Lys

130 135 140

Ser Val Ala Phe Ser Lys Phe Glu Lys Ala Val Phe Cys Thr Gly Gly

145 150 155 160

Arg Asp Gly Asn Ile Met Val Trp Asp Thr Arg Cys Asn Lys Lys Asp

165 170 175

Gly Phe Tyr Arg Gln Val Asn Gln Ile Ser Gly Ala His Asn Thr Ser

180 185 190

Asp Lys Gln Thr Pro Ser Lys Pro Lys Lys Lys Gln Asn Ser Lys Gly

195 200 205

Leu Ala Pro Ser Val Asp Phe Gln Gln Ser Val Thr Val Val Leu Phe

210 215 220

Gln Asp Glu Asn Thr Leu Val Ser Ala Gly Ala Val Asp Gly Ile Ile

225 230 235 240

Lys Val Trp Asp Leu Arg Lys Asn Tyr Thr Ala Tyr Arg Gln Glu Pro

245 250 255

Ile Ala Ser Lys Ser Phe Leu Tyr Pro Gly Ser Ser Thr Arg Lys Leu

260 265 270

Gly Tyr Ser Ser Leu Ile Leu Asp Ser Thr Gly Ser Thr Leu Phe Ala

275 280 285

Asn Cys Thr Asp Asp Asn Ile Tyr Met Phe Asn Met Thr Gly Leu Lys

290 295 300

Thr Ser Pro Val Ala Ile Phe Asn Gly His Gln Asn Ser Thr Phe Tyr

305 310 315 320

Val Lys Ser Ser Leu Ser Pro Asp Asp Gln Phe Leu Val Ser Gly Ser

325 330 335

Ser Asp Glu Ala Ala Tyr Ile Trp Lys Val Ser Thr Pro Trp Gln Pro

340 345 350

Pro Thr Val Leu Leu Gly His Ser Gln Glu Val Thr Ser Val Cys Trp

355 360 365

Cys Pro Ser Asp Phe Thr Lys Ile Ala Thr Cys Ser Asp Asp Asn Thr

370 375 380

Leu Lys Ile Trp Arg Leu Asn Arg Gly Leu Glu Glu Lys Pro Gly Gly

385 390 395 400

Asp Lys Leu Ser Thr Val Gly Trp Ala Ser Gln Lys Lys Lys Glu Ser

405 410 415

Arg Pro Gly Leu Val Thr Val Thr Ser Ser Gln Ser Thr Pro Ala Lys

420 425 430

Ala Pro Arg Val Lys Cys Asn Pro Ser Asn Ser Ser Pro Ser Ser Ala

435 440 445

Ala Cys Ala Pro Ser Cys Ala Gly Asp Leu Pro Leu Pro Ser Asn Thr

450 455 460

Pro Thr Phe Ser Ile Lys Thr Ser Pro Ala Lys Ala Arg Ser Pro Ile

465 470 475 480

Asn Arg Arg Gly Ser Val Ser Ser Val Ser Pro Lys Pro Pro Ser Ser

485 490 495

Phe Lys Met Ser Ile Arg Asn Trp Val Thr Arg Thr Pro Ser Ser Ser

500 505 510

Pro Pro Ile Thr Pro Pro Ala Ser Glu Thr Lys Ile Met Ser Pro Arg

515 520 525

Lys Ala Leu Ile Pro Val Ser Gln Lys Ser Ser Gln Ala Glu Ala Cys

530 535 540

Ser Glu Ser Arg Asn Arg Val Lys Arg Arg Leu Asp Ser Ser Cys Leu

545 550 555 560

Glu Ser Val Lys Gln Lys Cys Val Lys Ser Cys Asn Cys Val Thr Glu

565 570 575

Leu Asp Gly Gln Val Glu Asn Leu His Leu Asp Leu Cys Cys Leu Ala

580 585 590

Gly Asn Gln Glu Asp Leu Ser Lys Asp Ser Leu Gly Pro Thr Lys Ser

595 600 605

Ser Lys Ile Glu Gly Ala Gly Thr Ser Ile Ser Glu Pro Pro Ser Pro

610 615 620

Ile Ser Pro Tyr Ala Ser Glu Ser Cys Gly Thr Leu Pro Leu Pro Leu

625 630 635 640

Arg Pro Cys Gly Glu Gly Ser Glu Met Val Gly Lys Glu Asn Ser Ser

645 650 655

Pro Glu Asn Lys Asn Trp Leu Leu Ala Met Ala Ala Lys Arg Lys Ala

660 665 670

Glu Asn Pro Ser Pro Arg Ser Pro Ser Ser Gln Thr Pro Asn Ser Arg

675 680 685

Arg Gln Ser Gly Lys Thr Leu Pro Ser Pro Val Thr Ile Thr Pro Ser

690 695 700

Ser Met Arg Lys Ile Cys Thr Tyr Phe His Arg Lys Ser Gln Glu Asp

705 710 715 720

Phe Cys Gly Pro Glu His Ser Thr Glu Leu

725 730

<210>83

<211>1193

<212>DNA

<213> human (Homo sapiens)

<400>83

atgctcttca attcggtgct ccgccagccc cagcttggcg tcctgagaaa tggatggtct 60

tcacaatacc ctcttcaatc ccttctgact ggttatcagt gcagtggtaa tgatgaacac 120

acttcttatg gagaaacagg agtcccagtt cctccttttg gatgtacctt ctcttctgct 180

cccaatatgg aacatgtact agcagttgcc aatgaagaag gctttgttcg attgtataac 240

acagaatcac aaagtttcag aaagaagtgc ttcaaagaat ggatggctca ctggaatgcc 300

gtttttgacc tggcctgggt tcctggtgaa cttaaacttg ttacagcagc aggtgatcaa 360

acagccaaat tttgggacgt aaaagctggt gagctgattg gaacatgcaa aggtcatcaa 420

tgcagcctca agtcagttgc cttttctaag tttgagaaag ctgtattctg tacgggtgga 480

agagatggca acattatggt ctgggatacc aggtgcaaca aaaaagatgg gttttatagg 540

caagtgaatc aaatcagtgg agctcacaat acctcagaca agcaaacccc ttcaaaaccc 600

aagaagaaac agaattcaaa aggacttgct ccttctgtgg atttccagca aagtgttact 660

gtggtcctct ttcaagacga gaatacctta gtctcagcag gagctgtgga tgggataatc 720

aaagtatggg atttacgtaa gaattatact gcttatcgac aagaacccat agcatccaag 780

tctttcctgt acccaggtag cagcactcga aaacttggat attcaagtct gattttggat 840

tccactggct ctactttatt tgctaattgc acagacgata acatctacat gtttaatatg 900

actgggttga agacttctcc agtggctatt ttcaatggac accagaacgc tacctcttcc 960

tttgagacct tgtggagaat aaaaactggt tgttggccat ggcagccaaa cggaaggctg 1020

agaatccatc tccacgaagt ccgtcatccc agacacccaa ttccaggaga cagagcggaa 1080

agacattgcc aagcccggtc accatcacgc ccagctccat gaggaaaatc tgcacatact 1140

tccatagaaa gtcccaggag gacttctgtg gtcctgaaca ctcaacagaa tta 1193

<210>84

<211>373

<212>PRT

<213> human (Homo sapiens)

<220>

<221>misc_feature

<222>(362)..(362)

<223> Xaa can be any naturally occurring amino acid

<400>84

Met Leu Phe Asn Ser Val Leu Arg Gln Pro Gln Leu Gly Val Leu Arg

1 5 10 15

Asn Gly Trp Ser Ser Gln Tyr Pro Leu Gln Ser Leu Leu Thr Gly Tyr

20 25 30

Gln Cys Ser Gly Asn Asp Glu His Thr Ser Tyr Gly Glu Thr Gly Val

35 40 45

Pro Val Pro Pro Phe Gly Cys Thr Phe Ser Ser Ala Pro Asn Met Glu

50 55 60

His Val Leu Ala Val Ala Asn Glu Glu Gly Phe Val Arg Leu Tyr Asn

65 70 75 80

Thr Glu Ser Gln Ser Phe Arg Lys Lys Cys Phe Lys Glu Trp Met Ala

85 90 95

His Trp Asn Ala Val Phe Asp Leu Ala Trp Val Pro Gly Glu Leu Lys

100 105 110

Leu Val Thr Ala Ala Gly Asp Gln Thr Ala Lys Phe Trp Asp Val Lys

115 120 125

Ala Gly Glu Leu Ile Gly Thr Cys Lys Gly His Gln Cys Ser Leu Lys

130 135 140

Ser Val Ala Phe Ser Lys Phe Glu Lys Ala Val Phe Cys Thr Gly Gly

145 150 155 160

Arg Asp Gly Asn Ile Met Val Trp Asp Thr Arg Cys Asn Lys Lys Asp

165 170 175

Gly Phe Tyr Arg Gln Val Asn Gln Ile Ser Gly Ala His Asn Thr Ser

180 185 190

Asp Lys Gln Thr Pro Ser Lys Pro Lys Lys Lys Gln Asn Ser Lys Gly

195 200 205

Leu Ala Pro Ser Val Asp Phe Gln Gln Ser Val Thr Val Val Leu Phe

210 215 220

Gln Asp Glu Asn Thr Leu Val Ser Ala Gly Ala Val Asp Gly Ile Ile

225 230 235 240

Lys Val Trp Asp Leu Arg Lys Asn Tyr Thr Ala Tyr Arg Gln Glu Pro

245 250 255

Ile Ala Ser Lys Ser Phe Leu Tyr Pro Gly Ser Ser Thr Arg Lys Leu

260 265 270

Gly Tyr Ser Ser Leu Ile Leu Asp Ser Thr Gly Ser Thr Leu Phe Ala

275 280 285

Asn Cys Thr Asp Asp Asn Ile Tyr Met Phe Asn Met Thr Gly Leu Lys

290 295 300

Thr Ser Pro Val Ala Ile Phe Asn Gly His Gln Asn Ala Thr Ser Ser

305 310 315 320

Phe Glu Thr Leu Trp Arg Ile Lys Thr Gly Cys Trp Pro Trp Gln Pro

325 330 335

Asn Gly Arg Leu Arg Ile His Leu His Glu Val Arg His Pro Arg His

340 345 350

Pro Ile Pro Gly Asp Arg Ala Glu Arg Xaa Cys Gln Ala Arg Ser Pro

355 360 365

Ser Arg Pro Ala Pro

370

<210>85

<211>1357

<212>DNA

<213> human (Homo sapiens)

<400>85

atgctcttca attcggtgct ccgccagccc cagcttggcg tcctgagaaa tggatggtct 60

tcacaatacc ctcttcaatc ccttctgact ggttatcagt gcagtggtaa tgatgaacac 120

acttcttatg gagaaacagg agtcccagtt cctccttttg gatgtacctt ctcttctgct 180

cccaatatgg aacatgtact agcagttgcc aatgaagaag gctttgttcg attgtataac 240

acagaatcac aaagtttcag aaagaagtgc ttcaaagaat ggatggctca ctggaatgcc 300

gtctttgacc tggcctgggt tcctggtgaa cttaaacttg ttacagcagc aggtgatcaa 360

acagccaaat tttgggacgt aaaagctggt gagctgattg gaacatgcaa aggtcatcaa 420

tgcagcctca agtcagttgc cttttctaag tttgagaaag ctgtattctg tacgggtgga 480

agagatggca acattatggt ctgggatacc aggtgcaaca aaaaagatgg gttttatagg 540

caagtgaatc aaatcagtgg agctcacaat acctcagaca agcaaacccc ttcaaaaccc 600

aagaagaaac agaattcaaa aggacttgct ccttctgtgg atttccagca aagtgttact 660

gtggtcctct ttcaagacga gaatacctta gtctcagcag gagctgtgga tgggataatc 720

aaagtatggg atttacgtaa gaattatact gcttatcgac aagaacccat agcatccaag 780

tctttcctgt acccaggtag cagcactcga aaacttggat attcaagtct gattttggat 840

tccactggct ctactttatt tgctaattgc acagacgata acatctacat gtttaatatg 900

actgggttga agacttctcc agtggctatt ttcaatggac accagaactc taccttttat 960

gtaaaatcca gccttagtcc agatgaccag tttttagtca gtggctcaag tgatgaagct 1020

gcctacatat ggaaggtctc cacaccctgg caacctccta ctgtgctcct gggtcattct 1080

caagaggtca cgtctgtgtg ctggtgtcca tctgacttca caaagattgc tacctgttct 1140

gatgacaata cactaaaaat ctggcgcttg aatagaggct tagaggagaa accaggaggt 1200

gataaacttt ccacggtggg ttgggcctct cagaagaaaa aagagtcaag acctggccta 1260

ggtcaccatc acgcccagct ccatgaggaa aatctgcaca tacttccata gaaagtccca 1320

ggaggacttc tgtggtcctg aacactcaac agaatta 1357

<210>86

<211>436

<212>PRT

<213> human (Homo sapiens)

<400>86

Met Leu Phe Asn Ser Val Leu Arg Gln Pro Gln Leu Gly Val Leu Arg

1 5 10 15

Asn Gly Trp Ser Ser Gln Tyr Pro Leu Gln Ser Leu Leu Thr Gly Tyr

20 25 30

Gln Cys Ser Gly Asn Asp Glu His Thr Ser Tyr Gly Glu Thr Gly Val

35 40 45

Pro Val Pro Pro Phe Gly Cys Thr Phe Ser Ser Ala Pro Asn Met Glu

50 55 60

His Val Leu Ala Val Ala Asn Glu Glu Gly Phe Val Arg Leu Tyr Asn

65 70 75 80

Thr Glu Ser Gln Ser Phe Arg Lys Lys Cys Phe Lys Glu Trp Met Ala

85 90 95

His Trp Asn Ala Val Phe Asp Leu Ala Trp Val Pro Gly Glu Leu Lys

100 105 110

Leu Val Thr Ala Ala Gly Asp Gln Thr Ala Lys Phe Trp Asp Val Lys

115 120 125

Ala Gly Glu Leu Ile Gly Thr Cys Lys Gly His Gln Cys Ser Leu Lys

130 135 140

Ser Val Ala Phe Ser Lys Phe Glu Lys Ala Val Phe Cys Thr Gly Gly

145 150 155 160

Arg Asp Gly Asn Ile Met Val Trp Asp Thr Arg Cys Asn Lys Lys Asp

165 170 175

Gly Phe Tyr Arg Gln Val Asn Gln Ile Ser Gly Ala His Asn Thr Ser

180 185 190

Asp Lys Gln Thr Pro Ser Lys Pro Lys Lys Lys Gln Asn Ser Lys Gly

195 200 205

Leu Ala Pro Ser Val Asp Phe Gln Gln Ser Val Thr Val Val Leu Phe

210 215 220

Gln Asp Glu Asn Thr Leu Val Ser Ala Gly Ala Val Asp Gly Ile Ile

225 230 235 240

Lys Val Trp Asp Leu Arg Lys Asn Tyr Thr Ala Tyr Arg Gln Glu Pro

245 250 255

Ile Ala Ser Lys Ser Phe Leu Tyr Pro Gly Ser Ser Thr Arg Lys Leu

260 265 270

Gly Tyr Ser Ser Leu Ile Leu Asp Ser Thr Gly Ser Thr Leu Phe Ala

275 280 285

Asn Cys Thr Asp Asp Asn Ile Tyr Met Phe Asn Met Thr Gly Leu Lys

290 295 300

Thr Ser Pro Val Ala Ile Phe Asn Gly His Gln Asn Ser Thr Phe Tyr

305 310 315 320

Val Lys Ser Ser Leu Ser Pro Asp Asp Gln Phe Leu Val Ser Gly Ser

325 330 335

Ser Asp Glu Ala Ala Tyr Ile Trp Lys Val Ser Thr Pro Trp Gln Pro

340 345 350

Pro Thr Val Leu Leu Gly His Ser Gln Glu Val Thr Ser Val Cys Trp

355 360 365

Cys Pro Ser Asp Phe Thr Lys Ile Ala Thr Cys Ser Asp Asp Asn Thr

370 375 380

Leu Lys Ile Trp Arg Leu Asn Arg Gly Leu Glu Glu Lys Pro Gly Gly

385 390 395 400

Asp Lys Leu Ser Thr Val Gly Trp Ala Ser Gln Lys Lys Lys Glu Ser

405 410 415

Arg Pro Gly Leu Gly His His His Ala Gln Leu His Glu Glu Asn Leu

420 425 430

His Ile Leu Pro

435

<210>87

<211>1253

<212>DNA

<213> human (Homo sapiens)

<400>87

gggcggccgg gagagtagca gtgccttgga ccccagctct cctccccctt tctctctaag 60

gatggcccag aaggagaact cctacccctg gccctacggc cgacagacgg ctccatctgg 120

cctgagcacc ctgccccagc gagtcctccg gaaagagcct gtcaccccat ctgcacttgt 180

cctcatgagc cgctccaatg tccagcccac agctgcccct ggccagaagg tgatggagaa 240

tagcagtggg acacccgaca tcttaacgcg gcacttcaca attgatgact ttgagattgg 300

gcgtcctctg ggcaaaggca agtttggaaa cgtgtacttg gctcgggaga agaaaagcca 360

tttcatcgtg gcgctcaagg tcctcttcaa gtcccagata gagaaggagg gcgtggagca 420

tcagctgcgc agagagatcg aaatccaggc ccacctgcac catcccaaca tcctgcgtct 480

ctacaactat ttttatgacc ggaggaggat ctacttgatt ctagagtatg ccccccgcgg 540

ggagctctac aaggagctgc agaagagctg cacatttgac gagcagcgaa cagccacgat 600

catggaggag ttggcagatg ctctaatgta ctgccatggg aagaaggtga ttcacagaga 660

cataaagcca gaaaatctgc tcttagggct caagggagag ctgaagattg ctgacttcgg 720

ctggtctgtg catgcgccct ccctgaggag gaagacaatg tgtggcaccc tggactacct 780

gcccccagag atgattgagg ggcgcatgca caatgagaag gtggatctgt ggtgcattgg 840

agtgctttgc tatgagctgc tggtggggaa cccacccttt gagagtgcat cacacaacga 900

gacctatcgc cgcatcgtca aggtggacct aaagttcccc gcttccgtgc ccatgggagc 960

ccaggacctc atctccaaac tgctcaggca taacccctcg gaacggctgc ccctggccca 1020

ggtctcagcc cacccttggg tccgggccaa ctctcggagg gtgctgcctc cctctgccct 1080

tcaatctgtc gcctgatggt ccctgtcatt cactcgggtg cgtgtgtttg tatgtctgtg 1140

tatgtatagg ggaaagaagg gatccctaac tgttccctta tctgttttct acctcctcct 1200

ttgtttaata aaggctgaag ctttttgtac tcatgaaaaa aaaaaaaaaa aaa 1253

<210>88

<211>344

<212>PRT

<213> human (Homo sapiens)

<400>88

Met Ala Gln Lys Glu Asn Ser Tyr Pro Trp Pro Tyr Gly Arg Gln Thr

1 5 10 15

Ala Pro Ser Gly Leu Ser Thr Leu Pro Gln Arg Val Leu Arg Lys Glu

20 25 30

Pro Val Thr Pro Ser Ala Leu Val Leu Met Ser Arg Ser Asn Val Gln

35 40 45

Pro Thr Ala Ala Pro Gly Gln Lys Val Met Glu Asn Ser Ser Gly Thr

50 55 60

Pro Asp Ile Leu Thr Arg His Phe Thr Ile Asp Asp Phe Glu Ile Gly

65 70 75 80

Arg Pro Leu Gly Lys Gly Lys Phe Gly Asn Val Tyr Leu Ala Arg Glu

85 90 95

Lys Lys Ser His Phe Ile Val Ala Leu Lys Val Leu Phe Lys Ser Gln

100 105 110

Ile Glu Lys Glu Gly Val Glu His Gln Leu Arg Arg Glu Ile Glu Ile

115 120 125

Gln Ala His Leu His His Pro Asn Ile Leu Arg Leu Tyr Asn Tyr Phe

130 135 140

Tyr Asp Arg Arg Arg Ile Tyr Leu Ile Leu Glu Tyr Ala Pro Arg Gly

145 150 155 160

Glu Leu Tyr Lys Glu Leu Gln Lys Ser Cys Thr Phe Asp Glu Gln Arg

165 170 175

Thr Ala Thr Ile Met Glu Glu Leu Ala Asp Ala Leu Met Tyr Cys His

180 185 190

Gly Lys Lys Val Ile His Arg Asp Ile Lys Pro Glu Asn Leu Leu Leu

195 200 205

Gly Leu Lys Gly Glu Leu Lys Ile Ala Asp Phe Gly Trp Ser Val His

210 215 220

Ala Pro Ser Leu Arg Arg Lys Thr Met Cys Gly Thr Leu Asp Tyr Leu

225 230 235 240

Pro Pro Glu Met Ile Glu Gly Arg Met His Asn Glu Lys Val Asp Leu

245 250 255

Trp Cys Ile Gly Val Leu Cys Tyr Glu Leu Leu Val Gly Asn Pro Pro

260 265 270

Phe Glu Ser Ala Ser His Asn Glu Thr Tyr Arg Arg Ile Val Lys Val

275 280 285

Asp Leu Lys Phe Pro Ala Ser Val Pro Met Gly Ala Gln Asp Leu Ile

290 295 300

Ser Lys Leu Leu Arg His Asn Pro Ser Glu Arg Leu Pro Leu Ala Gln

305 310 315 320

Val Ser Ala His Pro Trp Val Arg Ala Asn Ser Arg Arg Val Leu Pro

325 330 335

Pro Ser Ala Leu Gln Ser Val Ala

340

<210>89

<211>1416

<212>DNA

<213> human (Homo sapiens)

<400>89

aagttcgggt ccgtagtggg ctaaggggga gggtttcaaa gggagcgcac ttccgctgcc 60

ctttctttcg ccagccttac gggcccgaac cctcgtgtga agggtgcagt acctaagccg 120

gagcggggta gaggcgggcc ggcaccccct tctgacctcc agtgccgccg gcctcaagat 180

cagacatggc ccagaacttg aaggacttgg cgggacggct gcccgccggg ccccggggca 240

tgggcacggc cctgaagctg ttgctggggg ccggcgccgt ggcctacggt gtgcgcgaat 300

ctgtgttcac cgtggaaggc gggcacagag ccatcttctt caatcggatc ggtggagtgc 360

agcaggacac tatcctggcc gagggccttc acttcaggat cccttggttc cagtacccca 420

ttatctatga cattcgggcc agacctcgaa aaatctcctc ccctacaggc tccaaagacc 480

tacagatggt gaatatctcc ctgcgagtgt tgtctcgacc caatgctcag gagcttccta 540

gcatgtacca gcgcctaggg ctggactacg aggaacgagt gttgccgtcc attgtcaacg 600

aggtgctcaa gagtgtggtg gccaagttca atgcctcaca gctgatcacc cagcgggccc 660

aggtatccct gttgatccgc cgggagctga cagagagggc caaggacttc agcctcatcc 720

tggatgatgt ggccatcaca gagctgagct ttagccgaga gtacacagct gctgtagaag 780

ccaaacaagt ggcccagcag gaggcccagc gggcccaatt cttggtagaa aaagcaaagc 840

aggaacagcg gcagaaaatt gtgcaggccg agggtgaggc cgaggctgcc aagatgcttg 900

gagaagcact gagcaagaac cctggctaca tcaaacttcg caagattcga gcagcccaga 960

atatctccaa gacgatcgcc acatcacaga atcgtatcta tctcacagct gacaaccttg 1020

tgctgaacct acaggatgaa agtttcacca ggggaagtga cagcctcatc aagggtaaga 1080

aatgagccta gtcaccaaga actccacccc cagaggaagt ggatctgctt ctccagtttt 1140

tgaggagcca gccaggggtc cagcacagcc ctaccccgcc ccagtatcat gcgatggtcc 1200

cccacaccgg ttccctgaac ccctcttgga ttaaggaaga ctgaagacta gccccttttc 1260

tgggaaatta ctttcctcct ccctgtgtta actggggctg ttggggacag tgcgtgattt 1320

ctcagtgatt tcctacagtg ttgttccctc cctcaaggct gggaggagat aaacaccaac 1380

ccaggaattc tcaataaatt tttattactt aacctg 1416

<210>90

<211>299

<212>PRT

<213> human (Homo sapiens)

<400>90

Met Ala Gln Asn Leu Lys Asp Leu Ala Gly Arg Leu Pro Ala Gly Pro

1 5 10 15

Arg Gly Met Gly Thr Ala Leu Lys Leu Leu Leu Gly Ala Gly Ala Val

20 25 30

Ala Tyr Gly Val Arg Glu Ser Val Phe Thr Val Glu Gly Gly His Arg

35 40 45

Ala Ile Phe Phe Asn Arg Ile Gly Gly Val Gln Gln Asp Thr Ile Leu

50 55 60

Ala Glu Gly Leu His Phe Arg Ile Pro Trp Phe Gln Tyr Pro Ile Ile

65 70 75 80

Tyr Asp Ile Arg Ala Arg Pro Arg Lys Ile Ser Ser Pro Thr Gly Ser

85 90 95

Lys Asp Leu Gln Met Val Asn Ile Ser Leu Arg Val Leu Ser Arg Pro

100 105 110

Asn Ala Gln Glu Leu Pro Ser Met Tyr Gln Arg Leu Gly Leu Asp Tyr

115 120 125

Glu Glu Arg Val Leu Pro Ser Ile Val Asn Glu Val Leu Lys Ser Val

130 135 140

Val Ala Lys Phe Asn Ala Ser Gln Leu Ile Thr Gln Arg Ala Gln Val

145 150 155 160

Ser Leu Leu Ile Arg Arg Glu Leu Thr Glu Arg Ala Lys Asp Phe Ser

165 170 175

Leu Ile Leu Asp Asp Val Ala Ile Thr Glu Leu Ser Phe Ser Arg Glu

180 185 190

Tyr Thr Ala Ala Val Glu Ala Lys Gln Val Ala Gln Gln Glu Ala Gln

195 200 205

Arg Ala Gln Phe Leu Val Glu Lys Ala Lys Gln Glu Gln Arg Gln Lys

210 215 220

Ile Val Gln Ala Glu Gly Glu Ala Glu Ala Ala Lys Met Leu Gly Glu

225 230 235 240

Ala Leu Ser Lys Asn Pro Gly Tyr Ile Lys Leu Arg Lys Ile Arg Ala

245 250 255

Ala Gln Asn Ile Ser Lys Thr Ile Ala Thr Ser Gln Asn Arg Ile Tyr

260 265 270

Leu Thr Ala Asp Asn Leu Val Leu Asn Leu Gln Asp Glu Ser Phe Thr

275 280 285

Arg Gly Ser Asp Ser Leu Ile Lys Gly Lys Lys

290 295

<210>91

<211>1899

<212>DNA

<213> human (Homo sapiens)

<400>91

agcgcgcgac tttttgaaag ccaggagggt tcgaattgca acggcagctg ccgggcgtat 60

gtgttggtgc tagaggcagc tgcagggtct cgctgggggc cgctcgggac caattttgaa 120

gaggtacttg gccacgactt attttcacct ccgacctttc cttccaggcg gtgagactct 180

ggactgagag tggctttcac aatggaaggg atcagtaatt tcaagacacc aagcaaatta 240

tcagaaaaaa agaaatctgt attatgttca actccaacta taaatatccc ggcctctccg 300

tttatgcaga agcttggctt tggtactggg gtaaatgtgt acctaatgaa aagatctcca 360

agaggtttgt ctcattctcc ttgggctgta aaaaagatta atcctatatg taatgatcat 420

tatcgaagtg tgtatcaaaa gagactaatg gatgaagcta agattttgaa aagccttcat 480

catccaaaca ttgttggtta tcgtgctttt actgaagcca atgatggcag tctgtgtctt 540

gctatggaat atggaggtga aaagtctcta aatgacttaa tagaagaacg atataaagcc 600

agccaagatc cttttccagc agccataatt ttaaaagttg ctttgaatat ggcaagaggg 660

ttaaagtatc tgcaccaaga aaagaaactg cttcatggag acataaagtc ttcaaatgtt 720

gtaattaaag gcgattttga aacaattaaa atctgtgatg taggagtctc tctaccactg 780

gatgaaaata tgactgtgac tgaccctgag gcttgttaca ttggcacaga gccatggaaa 840

cccaaagaag ctgtggagga gaatggtgtt attactgaca aggcagacat atttgccttt 900

ggccttactt tgtgggaaat gatgacttta tcgattccac acattaatct ttcaaatgat 960

gatgatgatg aagataaaac ttttgatgaa agtgattttg atgatgaagc atactatgca 1020

gcgttgggaa ctaggccacc tattaatatg gaagaactgg atgaatcata ccagaaagta 1080

attgaactct tctctgtatg cactaatgaa gaccctaaag atcgtccttc tgctgcacac 1140

attgttgaag ctctggaaac agatgtctag tgatcatctc agctgaagtg tggcttgcgt 1200

aaataactgt ttattccaaa atatttacat agttactatc agtagttatt agactctaaa 1260

attggcatat ttgaggacca tagtttcttg ttaacatatg gataactatt tctaatatga 1320

aatatgctta tattggctat aagcacttgg aattgtactg ggttttctgt aaagttttag 1380

aaactagcta cataagtact ttgatactgc tcatgctgac ttaaaacact agcagtaaaa 1440

cgctgtaaac tgtaacatta aattgaatga ccattacttt tattaatgat ctttcttaaa 1500

tattctatat tttaatggat ctactgacat tagcactttg tacagtacaa aataaagtct 1560

acatttgttt aaaacactga accttttgct gatgtgttta tcaaatgata actggaagct 1620

gaggagaata tgcctcaaaa agagtagctc cttggatact tcagactctg gttacagatt 1680

gtcttgatct cttggatctc ctcagatctt tggtttttgc tttaatttat taaatgtatt 1740

ttccatactg agtttaaaat ttattaattt gtaccttaag catttcccag ctgtgtaaaa 1800

acaataaaac tcaaatagga tgataaagaa taaaggacac tttgggtacc agaaaaaaaa 1860

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1899

<210>92

<211>322

<212>PRT

<213> human (Homo sapiens)

<400>92

Met Glu Gly Ile Ser Asn Phe Lys Thr Pro Ser Lys Leu Ser Glu Lys

1 5 10 15

Lys Lys Ser Val Leu Cys Ser Thr Pro Thr Ile Asn Ile Pro Ala Ser

20 25 30

Pro Phe Met Gln Lys Leu Gly Phe Gly Thr Gly Val Asn Val Tyr Leu

35 40 45

Met Lys Arg Ser Pro Arg Gly Leu Ser His Ser Pro Trp Ala Val Lys

50 55 60

Lys Ile Asn Pro Ile Cys Asn Asp His Tyr Arg Ser Val Tyr Gln Lys

65 70 75 80

Arg Leu Met Asp Glu Ala Lys Ile Leu Lys Ser Leu His His Pro Asn

85 90 95

Ile Val Gly Tyr Arg Ala Phe Thr Glu Ala Asn Asp Gly Ser Leu Cys

100 105 110

Leu Ala Met Glu Tyr Gly Gly Glu Lys Ser Leu Asn Asp Leu Ile Glu

115 120 125

Glu Arg Tyr Lys Ala Ser Gln Asp Pro Phe Pro Ala Ala Ile Ile Leu

130 135 140

Lys Val Ala Leu Asn Met Ala Arg Gly Leu Lys Tyr Leu His Gln Glu

145 150 155 160

Lys Lys Leu Leu His Gly Asp Ile Lys Ser Ser Asn Val Val Ile Lys

165 170 175

Gly Asp Phe Glu Thr Ile Lys Ile Cys Asp Val Gly Val Ser Leu Pro

180 185 190

Leu Asp Glu Asn Met Thr Val Thr Asp Pro Glu Ala Cys Tyr Ile Gly

195 200 205

Thr Glu Pro Trp Lys Pro Lys Glu Ala Val Glu Glu Asn Gly Val Ile

210 215 220

Thr Asp Lys Ala Asp Ile Phe Ala Phe Gly Leu Thr Leu Trp Glu Met

225 230 235 240

Met Thr Leu Ser Ile Pro His Ile Asn Leu Ser Asn Asp Asp Asp Asp

245 250 255

Glu Asp Lys Thr Phe Asp Glu Ser Asp Phe Asp Asp Glu Ala Tyr Tyr

260 265 270

Ala Ala Leu Gly Thr Arg Pro Pro Ile Asn Met Glu Glu Leu Asp Glu

275 280 285

Ser Tyr Gln Lys Val Ile Glu Leu Phe Ser Val Cys Thr Asn Glu Asp

290 295 300

Pro Lys Asp Arg Pro Ser Ala Ala His Ile Val Glu Ala Leu Glu Thr

305 310 315 320

Asp Val

<210>93

<211>294

<212>PRT

<213> human (Homo sapiens)

<400>93

Lys Cys Asn Pro Ser Asn Ser Ser Pro Ser Ser Ala Ala Cys Ala Pro

1 5 10 15

Ser Cys Ala Gly Asp Leu Pro Leu Pro Ser Asn Thr Pro Thr Phe Ser

20 25 30

Ile Lys Thr Ser Pro Ala Lys Ala Arg Ser Pro Ile Asn Arg Arg Gly

35 40 45

Ser Val Ser Ser Val Ser Pro Lys Pro Pro Ser Ser Phe Lys Met Ser

50 55 60

Ile Arg Asn Trp Val Thr Arg Thr Pro Ser Ser Ser Pro Pro Ile Thr

65 70 75 80

Pro Pro Ala Ser Glu Thr Lys Ile Met Ser Pro Arg Lys Ala Leu Ile

85 90 95

Pro Val Ser Gln Lys Ser Ser Gln Ala Glu Ala Cys Ser Glu Ser Arg

100 105 110

Asn Arg Val Lys Arg Arg Leu Asp Ser Ser Cys Leu Glu Ser Val Lys

115 120 125

Gln Lys Cys Val Lys Ser Cys Asn Cys Val Thr Glu Leu Asp Gly Gln

130 135 140

Val Glu Asn Leu His Leu Asp Leu Cys Cys Leu Ala Gly Asn Gln Glu

145 150 155 160

Asp Leu Ser Lys Asp Ser Leu Gly Pro Thr Lys Ser Ser Lys Ile Glu

165 170 175

Gly Ala Gly Thr Ser Ile Ser Glu Pro Pro Ser Pro Ile Ser Pro Tyr

180 185 190

Ala Ser Glu Ser Cys Gly Thr Leu Pro Leu Pro Leu Arg Pro Cys Gly

195 200 205

Glu Gly Ser Glu Met Val Gly Lys Glu Asn Ser Ser Pro Glu Asn Lys

210 215 220

Asn Trp Leu Leu Ala Met Ala Ala Lys Arg Lys Ala Glu Asn Pro Ser

225 230 235 240

Pro Arg Ser Pro Ser Ser Gln Thr Pro Asn Ser Arg Arg Gln Ser Gly

245 250 255

Lys Thr Leu Pro Ser Pro Val Thr Ile Thr Pro Ser Ser Met Arg Lys

260 265 270

Ile Cys Thr Tyr Phe His Arg Lys Ser Gln Glu Asp Phe Cys Gly Pro

275 280 285

Glu His Ser Thr Glu Leu

290

<210>94

<211>1235

<212>DNA

<213> human (Homo sapiens)

<400>94

gggggggggg ggcacttggc ttcaaagctg gctcttggaa attgagcgga gagcgacgcg 60

gttgttgtag ctgccgctgc ggccgccgcg gaataataag ccgggatcta ccatacccat 120

tgactaacta tggaagatta taccaaaata gagaaaattg gagaaggtac ctatggagtt 180

gtgtataagg gtagacacaa aactacaggt caagtggtag ccatgaaaaa aatcagacta 240

gaaagtgaag aggaaggggt tcctagtact gcaattcggg aaatttctct attaaaggaa 300

cttcgtcatc caaatatagt cagtcttcag gatgtgctta tgcaggattc caggttatat 360

ctcatctttg agtttctttc catggatctg aagaaatact tggattctat ccctcctggt 420

cagtacatgg attcttcact tgttaagagt tatttatacc aaatcctaca ggggattgtg 480

ttttgtcact ctagaagagt tcttcacaga gacttaaaac ctcaaaatct cttgattgat 540

gacaaaggaa caattaaact ggctgatttt ggccttgcca gagcttttgg aatacctatc 600

agagtatata cacatgaggt agtaacactc tggtacagat ctccagaagt attgctgggg 660

tcagctcgtt actcaactcc agttgacatt tggagtatag gcaccatatt tgctgaacta 720

gcaactaaga aaccactttt ccatggggat tcagaaattg atcaactctt caggattttc 780

agagctttgg gcactcccaa taatgaagtg tggccagaag tggaatcttt acaggactat 840

aagaatacat ttcccaaatg gaaaccagga agcctagcat cccatgtcaa aaacttggat 900

gaaaatggct tggatttgct ctcgaaaatg ttaatctatg atccagccaa acgaatttct 960

ggcaaaatgg cactgaatca tccatatttt aatgatttgg acaatcagat taagaagatg 1020

tagctttctg acaaaaagtt tccatatgtt atgtcaacag atagttgtgt ttttattgtt 1080

aactcttgtc tatttttgtc ttatatatat ttctttgtta tcaaacttca gctgtacttc 1140

gtcttctaat ttcaaaaata taacttaaaa atgtaaatat tctatatgaa tttaaatata 1200

attctgtaaa tgtgaaaaaa aaaaaaaaaa aaaaa 1235

<210>95

<211>297

<212>PRT

<213> human (Homo sapiens)

<400>95

Met Glu Asp Tyr Thr Lys Ile Glu Lys Ile Gly Glu Gly Thr Tyr Gly

1 5 10 15

Val Val Tyr Lys Gly Arg His Lys Thr Thr Gly Gln Val Val Ala Met

20 25 30

Lys Lys Ile Arg Leu Glu Ser Glu Glu Glu Gly Val Pro Ser Thr Ala

35 40 45

Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Arg His Pro Asn Ile Val

50 55 60

Ser Leu Gln Asp Val Leu Met Gln Asp Ser Arg Leu Tyr Leu Ile Phe

65 70 75 80

Glu Phe Leu Ser Met Asp Leu Lys Lys Tyr Leu Asp Ser Ile Pro Pro

85 90 95

Gly Gln Tyr Met Asp Ser Ser Leu Val Lys Ser Tyr Leu Tyr Gln Ile

100 105 110

Leu Gln Gly Ile Val Phe CysHi s Ser Arg Arg Val Leu His Arg Asp

115 120 125

Leu Lys Pro Gln Asn Leu Leu Ile Asp Asp Lys Gly Thr Ile Lys Leu

130 135 140

Ala Asp Phe Gly Leu Ala Arg Ala Phe Gly Ile Pro Ile Arg Val Tyr

145 150 155 160

Thr His Glu Val Val Thr Leu Trp Tyr Arg Ser Pro Glu Val Leu Leu

165 170 175

Gly Ser Ala Arg Tyr Ser Thr Pro Val Asp Ile Trp Ser Ile Gly Thr

180 185 190

Ile Phe Ala Glu Leu Ala Thr Lys Lys Pro Leu Phe His Gly Asp Ser

195 200 205

Glu Ile Asp Gln Leu Phe Arg Ile Phe Arg Ala Leu Gly Thr Pro Asn

210 215 220

Asn Glu Val Trp Pro Glu Val Glu Ser Leu Gln Asp Tyr Lys Asn Thr

225 230 235 240

Phe Pro Lys Trp Lys Pro Gly Ser Leu Ala Ser His Val Lys Asn Leu

245 250 255

Asp Glu Asn Gly Leu Asp Leu Leu Ser Lys Met Leu Ile Tyr Asp Pro

260 265 270

Ala Lys Arg Ile Ser Gly Lys Met Ala Leu Asn His Pro Tyr Phe Asn

275 280 285

Asp Leu Asp Asn Gln Ile Lys Lys Met

290 295

<210>96

<211>2101

<212>DNA

<213> human (Homo sapiens)

<400>96

acgaacaggc caataaggag ggagcagtgc ggggtttaaa tctgaggcta ggctggctct 60

tctcggcgtg ctgcggcgga acggctgttg gtttctgctg ggtgtaggtc cttggctggt 120

cgggcctccg gtgttctgct tctccccgct gagctgctgc ctggtgaaga ggaagccatg 180

gcgctccgag tcaccaggaa ctcgaaaatt aatgctgaaa ataaggcgaa gatcaacatg 240

gcaggcgcaa agcgcgttcc tacggcccct gctgcaacct ccaagcccgg actgaggcca 300

agaacagctc ttggggacat tggtaacaaa gtcagtgaac aactgcaggc caaaatgcct 360

atgaagaagg aagcaaaacc ttcagctact ggaaaagtca ttgataaaaa actaccaaaa 420

cctcttgaaa aggtacctat gctggtgcca gtgccagtgt ctgagccagt gccagagcca 480

gaacctgagc cagaacctga gcctgttaaa gaagaaaaac tttcgcctga gcctattttg 540

gttgatactg cctctccaag cccaatggaa acatctggat gtgcccctgc agaagaagac 600

ctgtgtcagg ctttctctga tgtaattctt gcagtaaatg atgtggatgc agaagatgga 660

gctgatccaa acctttgtag tgaatatgtg aaagatattt atgcttatct gagacaactt 720

gaggaagagc aagcagtcag accaaaatac ctactgggtc gggaagtcac tggaaacatg 780

agagccatcc taattgactg gctagtacag gttcaaatga aattcaggtt gttgcaggag 840

accatgtaca tgactgtctc cattattgat cggttcatgc agaataattg tgtgcccaag 900

aagatgctgc agctggttgg tgtcactgcc atgtttattg caagcaaata tgaagaaatg 960

taccctccag aaattggtga ctttgctttt gtgactgaca acacttatac taagcaccaa 1020

atcagacaga tggaaatgaa gattctaaga gctttaaact ttggtctggg tcggcctcta 1080

cctttgcact tccttcggag agcatctaag attggagagg ttgatgtcga gcaacatact 1140

ttggccaaat acctgatgga actaactatg ttggactatg acatggtgca ctttcctcct 1200

tctcaaattg cagcaggagc tttttgctta gcactgaaaa ttctggataa tggtgaatgg 1260

acaccaactc tacaacatta cctgtcatat actgaagaat ctcttcttcc agttatgcag 1320

cacctggcta agaatgtagt catggtaaat caaggactta caaagcacat gactgtcaag 1380

aacaagtatg ccacatcgaa gcatgctaag atcagcactc taccacagct gaattctgca 1440

ctagttcaag atttagccaa ggctgtggca aaggtgtaac ttgtaaactt gagttggagt 1500

actatattta caaataaaat tggcaccatg tgccatctgt acatattact gttgcattta 1560

cttttaataa agcttgtggc cccttttact tttttatagc ttaactaatt tgaatgtggt 1620

tacttcctac tgtagggtag cggaaaagtt gtcttaaaag gtatggtggg gatattttta 1680

aaaactcctt ttggtttacc tggggatcca attgatgtat atgtttatat actgggttct 1740

tgttttatat acctggcttt tactttatta atatgagtta ctgaaggtga tggaggtatt 1800

tgaaaatttt acttccatag gacatactgc atgtaagcca agtcatggag aatctgctgc 1860

atagctctat tttaaagtaa aagtctacca ccgaatccct agtccccctg ttttctgttt 1920

cttcttgtga ttgctgccat aattctaagt tatttacttt taccactatt taagttatca 1980

actttagcta gtatcttcaa actttcactt tgaaaaatga gaattttata ttctaagcca 2040

gttttcattt tggttttgtg ttttggttaa taaaacaata ctcaaataca aaaaaaaaaa 2100

a 2101

<210>97

<211>433

<212>PRT

<213> human (Homo sapiens)

<400>97

Met Ala Leu Arg Val Thr Arg Asn Ser Lys Ile Asn Ala Glu Asn Lys

1 5 10 15

Ala Lys Ile Asn Met Ala Gly Ala Lys Arg Val Pro Thr Ala Pro Ala

20 25 30

Ala Thr Ser Lys Pro Gly Leu Arg Pro Arg Thr Ala Leu Gly Asp Ile

35 40 45

Gly Asn Lys Val Ser Glu Gln Leu Gln Ala Lys Met Pro Met Lys Lys

50 55 60

Glu Ala Lys Pro Ser Ala Thr Gly Lys Val Ile Asp Lys Lys Leu Pro

65 70 75 80

Lys Pro Leu Glu Lys Val Pro Met Leu Val Pro Val Pro Val Ser Glu

85 90 95

Pro Val Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Val Lys Glu

100 105 110

Glu Lys Leu Ser Pro Glu Pro Ile Leu Val Asp Thr Ala Ser Pro Ser

115 120 125

Pro Met Glu Thr Ser Gly Cys Ala Pro Ala Glu Glu Asp Leu Cys Gln

130 135 140

Ala Phe Ser Asp Val Ile Leu Ala Val Asn Asp Val Asp Ala Glu Asp

145 150 155 160

Gly Ala Asp Pro Asn Leu Cys Ser Glu Tyr Val Lys Asp Ile Tyr Ala

165 170 175

Tyr Leu Arg Gln Leu Glu Glu Glu Gln Ala Val Arg Pro Lys Tyr Leu

180 185 190

Leu Gly Arg Glu Val Thr Gly Asn Met Arg Ala Ile Leu Ile Asp Trp

195 200 205

Leu Val Gln Val Gln Met Lys Phe Arg Leu Leu Gln Glu Thr Met Tyr

210 215 220

Met Thr Val Ser Ile Ile Asp Arg Phe Met Gln Asn Asn Cys Val Pro

225 230 235 240

Lys Lys Met Leu Gln Leu Val Gly Val Thr Ala Met Phe Ile Ala Ser

245 250 255

Lys Tyr Glu Glu Met Tyr Pro Pro Glu Ile Gly Asp Phe Ala Phe Val

260 265 270

Thr Asp Asn Thr Tyr Thr Lys His Gln Ile Arg Gln Met Glu Met Lys

275 280 285

Ile Leu Arg Ala Leu Asn Phe Gly Leu Gly Arg Pro Leu Pro Leu His

290 295 300

Phe Leu Arg Arg Ala Ser Lys Ile Gly Glu Val Asp Val Glu Gln His

305 310 315 320

Thr Leu Ala Lys Tyr Leu Met Glu Leu Thr Met Leu Asp Tyr Asp Met

325 330 335

Val His Phe Pro Pro Ser Gln Ile Ala Ala Gly Ala Phe Cys Leu Ala

340 345 350

Leu Lys Ile Leu Asp Asn Gly Glu Trp Thr Pro Thr Leu Gln His Tyr

355 360 365

Leu Ser Tyr Thr Glu Glu Ser Leu Leu Pro Val Met Gln His Leu Ala

370 375 380

Lys Asn Val Val Met Val Asn Gln Gly Leu Thr Lys His Met Thr Val

385 390 395 400

Lys Asn Lys Tyr Ala Thr Ser Lys His Ala Lys Ile Ser Thr Leu Pro

405 410 415

Gln Leu Asn Ser Ala Leu Val Gln Asp Leu Ala Lys Ala Val Ala Lys

420 425 430

Val

<210>98

<211>18

<212>PRT

<213> Artificial

<220>

<223> artificially designed dominant negative control

<400>98

Met Glu Gly Ile Ser Asn Phe Lys Thr Pro Ser Lys Leu Ser Glu Lys

1 5 10 15

Lys Lys

<210>99

<211>30

<212>PRT

<213> Artificial

<220>

<223> artificially designed dominant negative control

<400>99

Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Met Glu Gly Ile

1 5 10 15

Ser Asn Phe Lys Thr Pro Ser Lys Leu Ser Glu Lys Lys Lys

20 25 30

<210>100

<211>9

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Tat sequence

<400>100

Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5

<210>101

<211>16

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Pennetratin sequence

<400>101

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>102

<211>21

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Buforin II sequence

<400>102

Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His

1 5 10 15

Arg Leu Leu Arg Lys

20

<210>103

<211>27

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Transportani sequence

<400>103

Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu

1 5 10 15

Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu

20 25

<210>104

<211>18

<212>PRT

<213> Artificial

<220>

<223> synthetic MAP (model amphipathic peptide) sequence

<400>104

Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys

1 5 10 15

Leu Ala

<210>105

<211>16

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized K-FGF sequence

<400>105

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

<210>106

<211>5

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Ku70 sequence

<400>106

Val Pro Met Leu Lys

1 5

<210>107

<211>28

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Prion sequence

<400>107

Met Ala Asn Leu Gly Tyr Trp Leu Leu Ala Leu Phe Val Thr Met Trp

1 5 10 15

Thr Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro

20 25

<210>108

<211>18

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized pVEC sequence

<400>108

Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His

1 5 10 15

Ser Lys

<210>109

<211>21

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Pep-1 sequence

<400>109

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val

20

<210>110

<211>18

<212>PRT

<213> Artificial

<220>

<223> synthetic SynB1 sequence

<400>110

Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Thr Ser Thr

1 5 10 15

Gly Arg

<210>111

<211>15

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Pep-7 sequence

<400>111

Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Gln Tyr

1 5 10 15

<210>112

<211>12

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized HN-1 sequence

<400>112

Thr Ser Pro Leu Asn Ile His Asn Gly Gln Lys Leu

1 5 10

<210>113

<211>11

<212>PRT

<213> Artificial

<220>

<223> synthetic arginine polymer sequence

<400>113

Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg

1 5 10

<210>114

<211>5

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized Ku70 sequence

<400>114

Pro Met Leu Lys Glu

1 5

<210>115

<211>1488

<212>DNA

<213> human (Homo sapiens)

<400>115

gcggggccgc gggccggggg cggactgggg cgggcggaag gagagccagg ccggaaggag 60

gctgccggag ggcgggaggc aggagcgggc caggagctgc tgggctggag cggcggcgcc 120

gccatgtccg acagcgagaa gctcaacctg gactcgatca tcgggcgcct gctggaagtg 180

cagggctcgc ggcctggcaa gaatgtacag ctgacagaga acgagatccg cggtctgtgc 240

ctgaaatccc gggagatttt tctgagccag cccattcttc tggagctgga ggcacccctc 300

aagatctgcg gtgacataca cggccagtac tacgaccttc tgcgactatt tgagtatggc 360

ggtttccctc ccgagagcaa ctacctcttt ctgggggact atgtggacag gggcaagcag 420

tccttggaga ccatctgcct gctgctggcc tataagatca agtaccccga gaacttcttc 480

ctgctccgtg ggaaccacga gtgtgccagc atcaaccgca tctatggttt ctacgatgag 540

tgcaagagac gctacaacat caaactgtgg aaaaccttca ctgactgctt caactgcctg 600

cccatcgcgg ccatagtgga cgaaaagatc ttctgctgcc acggaggcct gtccccggac 660

ctgcagtcta tggagcagat tcggcggatc atgcggccca cagatgtgcc tgaccagggc 720

ctgctgtgtg acctgctgtg gtctgaccct gacaaggacg tgcagggctg gggcgagaac 780

gaccgtggcg tctcttttac ctttggagcc gaggtggtgg ccaagttcct ccacaagcac 840

gacttggacc tcatctgccg agcacaccag gtggtagaag acggctacga gttctttgcc 900

aagcggcagc tggtgacact tttctcagct cccaactact gtggcgagtt tgacaatgct 960

ggcgccatga tgagtgtgga cgagaccctc atgtgctctt tccagatcct caagcccgcc 1020

gacaagaaca aggggaagta cgggcagttc agtggcctga accctggagg ccgacccatc 1080

accccacccc gcaattccgc caaagccaag aaatagcccc cgcacaccac cctgtgcccc 1140

agatgatgga ttgattgtac agaaatcatg ctgccatgct gggggggggt caccccgacc 1200

cctcaggccc acctgtcacg gggaacatgg agccttggtg tatttttctt ttcttttttt 1260

aatgaatcaa tagcagcgtc cagtccccca gggctgcttc ctgcctgcac ctgcggtgac 1320

tgtgagcagg atcctggggc cgaggctgca gctcagggca acggcaggcc aggtcgtggg 1380

tctccagccg tgcttggcct cagggctggc agccggatcc tggggcaacc catctggtct 1440

cttgaataaa ggtcaaagct ggattctcgc aaaaaaaaaa aaaaaaaa 1488

<210>116

<211>330

<212>PRT

<213> human (Homo sapiens)

<400>116

Met Ser Asp Ser Glu Lys Leu Asn Leu Asp Ser Ile Ile Gly Arg Leu

1 5 10 15

Leu Glu Val Gln Gly Ser Arg Pro Gly Lys Asn Val Gln Leu Thr Glu

20 25 30

Asn Glu Ile Arg Gly Leu Cys Leu Lys Ser Arg Glu Ile Phe Leu Ser

35 40 45

Gln Pro Ile Leu Leu Glu Leu Glu Ala Pro Leu Lys Ile Cys Gly Asp

50 55 60

Ile His Gly Gln Tyr Tyr Asp Leu Leu Arg Leu Phe Glu Tyr Gly Gly

65 70 75 80

Phe Pro Pro Glu Ser Asn Tyr Leu Phe Leu Gly Asp Tyr Val Asp Arg

85 90 95

Gly Lys Gln Ser Leu Glu Thr Ile Cys Leu Leu Leu Ala Tyr Lys Ile

100 105 110

Lys Tyr Pro Glu Asn Phe Phe Leu Leu Arg Gly Asn His Glu Cys Ala

115 120 125

Ser Ile Asn Arg Ile Tyr Gly Phe Tyr Asp Glu Cys Lys Arg Arg Tyr

130 135 140

Asn Ile Lys Leu Trp Lys Thr Phe Thr Asp Cys Phe Asn Cys Leu Pro

145 150 155 160

Ile Ala Ala Ile Val Asp Glu Lys Ile Phe Cys Cys His Gly Gly Leu

165 170 175

Ser Pro Asp Leu Gln Ser Met Glu Gln Ile Arg Arg Ile Met Arg Pro

180 185 190

Thr Asp Val Pro Asp Gln Gly Leu Leu Cys Asp Leu Leu Trp Ser Asp

195 200 205

Pro Asp Lys Asp Val Gln Gly Trp Gly Glu Asn Asp Arg Gly Val Ser

210 215 220

Phe Thr Phe Gly Ala Glu Val Val Ala Lys Phe Leu His Lys His Asp

225 230 235 240

Leu Asp Leu Ile Cys Arg Ala His Gln Val Val Glu Asp Gly Tyr Glu

245 250 255

Phe Phe Ala Lys Arg Gln Leu Val Thr Leu Phe Ser Ala Pro Asn Tyr

260 265 270

Cys Gly Glu Phe Asp Asn Ala Gly Ala Met Met Ser Val Asp Glu Thr

275 280 285

Leu Met Cys Ser Phe Gln Ile Leu Lys Pro Ala Asp Lys Asn Lys Gly

290 295 300

Lys Tyr Gly Gln Phe Ser Gly Leu Asn Pro Gly Gly Arg Pro Ile Thr

305 310 315 320

Pro Pro Arg Asn Ser Ala Lys Ala Lys Lys

325 330

<210>117

<211>3644

<212>DNA

<213> human (Homo sapiens)

<400>117

aactaggggt aaggaggtac aaagaacaag gaagttaggc tttgaaaata gagaacaaag 60

aacaagtaag ctgaacaagt aagttcctct ttggagaggc aagagggcaa agaagctgag 120

gacccagaag acaggattgt gcctaagaaa gtggccttgg agcagaggcc caaagaaagc 180

ggggagagcg ccttgcccat cggtaaggga aaggtgctcc aggcacagaa gcagctaatg 240

caacggccct gaggcaaatg caagataact ggcaagttcc aggaacagca aggagggcaa 300

ggcgcctgga gcagaggaag ctagcggatc ccttgatgag agggaggaac tggcccatct 360

ctcatgggtc ctgcaggcag gccatgagac tgtcttttat gcggggcgag ataacgaggc 420

atggacgtgt tgtgagcgga ggcctagcct gttcaaggtc tgaattttta cccctagaat 480

gtaagaagca tgggagcagg gactttgttt tgctccctgc catatccaca gccctagaaa 540

aacgtcgggc cattgatagc tctcagtaaa tgcttgacaa gtaagtgaat gaacgaatgc 600

acaattaatg aaatgagggc tgcgtcaatg aataactggg aatctgactc ctgcagggag 660

ccttcagccc acaggtgtat gtaagacccg ccccgcctct catccttcgg gctcccaccc 720

ccgccgttag gctgcggttc gcgccgcggt cgccagaggg cgcggagcgg cggggcttcc 780

cgcacggagg gctttgcgtg aggcaccgcg tggggcgggg ctgcgggcgg gctcccagct 840

gctgggccct catcggctgg gcctcgtcga ccggcaagcg gaacgcggca gcggggctgg 900

gcctgtgcgg cggccgccgg agcgctttgg aaggcgcacg gggcgaagat ggcggcggag 960

cgacaggagg cgctgaggga gttcgtggcg gtgacgggcg ccgaggagga ccgggcccgc 1020

ttctttctcg agtcggccgg ctgggacttg cagatcgcgc tagcgagctt ttatgaggac 1080

ggaggggatg aagacattgt gaccatttcg caggcaaccc ccagttcagt gtccagaggc 1140

acagccccca gtgataatag agtgacatcc ttcagagacc tcattcatga ccaagatgaa 1200

gatgaggagg aagaggaagg ccagaggttt tatgctgggg gctcagagag aagtggacag 1260

cagattgttg gccctcccag gaagaaaagt cccaacgagc tggtggatga tctctttaaa 1320

ggtgccaaag agcatggagc tgtagctgtg gagcgagtga ccaagagccc tggagagacc 1380

agtaaaccga gaccatttgc aggaggtggc taccgccttg gggcagcacc agaggaagag 1440

tctgcctatg tggcaggaga aaagaggcag cattccagcc aagatgttca tgtagtattg 1500

aaactctgga agagtggatt cagcctggat aatggagaac tcagaagcta ccaagaccca 1560

tccaatgccc agtttctgga gtctatccgc agaggggagg tgccagcaga gcttcggagg 1620

ctagctcacg gtggacaggt gaacttggat atggaggacc atcgggacga ggactttgtg 1680

aagcccaaag gagccttcaa agccttcact ggcgagggtc agaaactggg cagcactgcc 1740

ccccaggtgt tgagtaccag ctctccagcc caacaggcag aaaatgaagc caaagccagc 1800

tcttccatct taatcgacga atcagagcct accacaaaca tccaaattcg gcttgcagac 1860

ggcgggaggc tggtgcagaa atttaaccac agccacagga tcagcgacat ccgactcttc 1920

atcgtggatg cccggccagc catggctgcc accagcttta tcctcatgac tactttcccg 1980

aacaaagagc tggctgatga gagccagacc ctgaaggaag ccaacctgct caatgctgtc 2040

atcgtgcagc ggttaacata accgcccagc cagctgcctg gcctccctcc tgtgtttccc 2100

atggccagtg gccatgcccc atggggatcg cccctcctgc ccccttgtgc acacccagca 2160

gtccagtgca acgtctcctc catagctctg ggttcttaga tcttggttgg acgtttgttt 2220

tctccttagt tgcatttcct gggtttttgt gatgatcaat ggactttaat gaaaaaaaaa 2280

ataaaaacaa ccaaaaaaat tgaaggaata tcaccagcat gttgtacgga aactctccca 2340

ctgaagcagg ctttaattgc tttaaaatta tatttatctt ggggcctgtg ggaggaaact 2400

tccttccatc ttctctgcat aaaaacttgt ggcacacaat gcttattcac tagtgtgtcc 2460

cacccgccag ccccacagat gactggagga aggaggggaa atgtgtagaa agaggcttcg 2520

ccaccacttg ttcccacgag aatatgtcac ttgcccagat aaaactgggc ggcagccaga 2580

gttccctgaa gtgggaagtc agagctccat gcacacagtg tcttcagaag gtgaaaataa 2640

atatttccct gtgctccttt tactcaaccc ctggggtatc taatcttgcc aggtcttggc 2700

cagttgagat tctgttccac ctgcctgcct ggccctttcc tccattacca tccagactgc 2760

tcgcctcctg gggattctca ggggctccat tatggcttga tttactccac gtgcagaagt 2820

cttgagtgga cctaggaggt aggtgggata ttttttttca ctaggataca gctcatgcca 2880

acccatccta agtgagttca gaatcagggt atcttgccct ataagataaa cagtcaaaat 2940

gccaccgagc tgttcactag tgatgtgtgg caaatcaaat caactgttga agaaggggtg 3000

agttttctgt gctacaagca cctgtcactg ttggtacttg caggaggctt ctgctgggta 3060

tgttttggaa gtgagtgtca ctacttggct ttgcttagca ggttctgctt cacacttgtt 3120

ctttgacctg ctgacttgtg acttgcagaa acataggcag tagtcctagc ctggtaaaga 3180

ccctccacca cccctataag tttgattgct atgcaggttt gggagaggag gcctattggg 3240

ctcttggatg gaaccctttc ccgtattaaa caaaccagag acagaatcag tgctgactca 3300

ggatctcctg gtttggaatc gtaatgtgcc tcaatcctct ttccaagcag gcctcaccag 3360

tctctttctc tttcctgctt cacccctgca atgagccaag aaccaacact acatccacct 3420

agaactgcag aagggcttgt ggtttcaacc aagacccatc ctgagcaagg gacttggctt 3480

ggtgcttttg atcccaaagt tcccacaccg gcagtggcct gctggggcaa tggcatctgt 3540

cacggtgttt tctccagcag gtggagatta tggaacctac atatgggtct ggaaaaactg 3600

tacactgttg tcaccttgac cattaaaaac cagaatgagg acaa 3644

<210>118

<211>370

<212>PRT

<213> human (Homo sapiens)

<400>118

Met Ala Ala Glu Arg Gln Glu Ala Leu Arg Glu Phe Val Ala Val Thr

1 5 10 15

Gly Ala Glu Glu Asp Arg Ala Arg Phe Phe Leu Glu Ser Ala Gly Trp

20 25 30

Asp Leu Gln Ile Ala Leu Ala Ser Phe Tyr Glu Asp Gly Gly Asp Glu

35 40 45

Asp Ile Val Thr Ile Ser Gln Ala Thr Pro Ser Ser Val Ser Arg Gly

50 55 60

Thr Ala Pro Ser Asp Asn Arg Val Thr Ser Phe Arg Asp Leu Ile His

65 70 75 80

Asp Gln Asp Glu Asp Glu Glu Glu Glu Glu Gly Gln Arg Phe Tyr Ala

85 90 95

Gly Gly Ser Glu Arg Ser Gly Gln Gln Ile Val Gly Pro Pro Arg Lys

100 105 110

Lys Ser Pro Asn Glu Leu Val Asp Asp Leu Phe Lys Gly Ala Lys Glu

115 120 125

His Gly Ala Val Ala Val Glu Arg Val Thr Lys Ser Pro Gly Glu Thr

130 135 140

Ser Lys Pro Arg Pro Phe Ala Gly Gly Gly Tyr Arg Leu Gly Ala Ala

145 150 155 160

Pro Glu Glu Glu Ser Ala Tyr Val Ala Gly Glu Lys Arg Gln His Ser

165 170 175

Ser Gln Asp Val His Val Val Leu Lys Leu Trp Lys Ser Gly Phe Ser

180 185 190

Leu Asp Asn Gly Glu Leu Arg Ser Tyr Gln Asp Pro Ser Asn Ala Gln

195 200 205

Phe Leu Glu Ser Ile Arg Arg Gly Glu Val Pro Ala Glu Leu Arg Arg

210 215 220

Leu Ala His Gly Gly Gln Val Asn Leu Asp Met Glu Asp His Arg Asp

225 230 235 240

Glu Asp Phe Val Lys Pro Lys Gly Ala Phe Lys Ala Phe Thr Gly Glu

245 250 255

Gly Gln Lys Leu Gly Ser Thr Ala Pro Gln Val Leu Ser Thr Ser Ser

260 265 270

Pro Ala Gln Gln Ala Glu Asn Glu Ala Lys Ala Ser Ser Ser Ile Leu

275 280 285

Ile Asp Glu Ser Glu Pro Thr Thr Asn Ile Gln Ile Arg Leu Ala Asp

290 295 300

Gly Gly Arg Leu Val Gln Lys Phe Asn His Ser His Arg Ile Ser Asp

305 310 315 320

Ile Arg Leu Phe Ile Val Asp Ala Arg Pro Ala Met Ala Ala Thr Ser

325 330 335

Phe Ile Leu Met Thr Thr Phe Pro Asn Lys Glu Leu Ala Asp Glu Ser

340 345 350

Gln Thr Leu Lys Glu Ala Asn Leu Leu Asn Ala Val Ile Val Gln Arg

355 360 365

Leu Thr

370

<210>119

<211>3198

<212>DNA

<213> human (Homo sapiens)

<400>119

ctgccactgc cacctcgcgg atcaggagcc agcgttgttc gcccgacgcc tcgctgccgg 60

tgggaggaag cgagagggaa gccgcttggg ctcttgtcgc cgctgctcgc ccaccgcctg 120

gaagagccga gccccggcca gtcggtcgct tgccaccgct cgtagccgtt acccgcgggc 180

cgccacagcc gccggcggga gaggcgcgcg ccatggcttc tggagccgat tcaaaaggtg 240

atgacctatc aacagccatt ctcaaacaga agaaccgtcc caatcggtta attgttgatg 300

aagccatcaa tgaggacaac agtgtggtgt ccttgtccca gcccaagatg gatgaattgc 360

agttgttccg aggtgacaca gtgttgctga aaggaaagaa gagacgagaa gctgtttgca 420

tcgtcctttc tgatgatact tgttctgatg agaagattcg gatgaataga gttgttcgga 480

ataaccttcg tgtacgccta ggggatgtca tcagcatcca gccatgccct gatgtgaagt 540

acggcaaacg tatccatgtg ctgcccattg atgacacagt ggaaggcatt actggtaatc 600

tcttcgaggt ataccttaag ccgtacttcc tggaagcgta tcgacccatc cggaaaggag 660

acatttttct tgtccgtggt gggatgcgtg ctgtggagtt caaagtggtg gaaacagatc 720

ctagccctta ttgcattgtt gctccagaca cagtgatcca ctgcgaaggg gagcctatca 780

aacgagagga tgaggaagag tccttgaatg aagtagggta tgatgacatt ggtggctgca 840

ggaagcagct agctcagatc aaagagatgg tggaactgcc cctgagacat cctgccctct 900

ttaaggcaat tggtgtgaag cctcctagag gaatcctgct ttacggacct cctggaacag 960

gaaagaccct gattgctcga gctgtagcaa atgagactgg agccttcttc ttcttgatca 1020

atggtcctga gatcatgagc aaattggctg gtgagtctga gagcaacctt cgtaaagcct 1080

ttgaggaggc tgagaagaat gctcctgcca tcatcttcat tgatgagcta gatgccatcg 1140

ctcccaaaag agagaaaact catggcgagg tggagcggcg cattgtatca cagttgttga 1200

ccctcatgga tggcctaaag cagagggcac atgtgattgt tatggcagca accaacagac 1260

ccaacagcat tgacccagct ctacggcgat ttggtcgctt tgacagggag gtagatattg 1320

gaattcctga tgctacagga cgcttagaga ttcttcagat ccataccaag aacatgaagc 1380

tggcagatga tgtggacctg gaacaggtag ccaatgagac tcacgggcat gtgggtgctg 1440

acttagcagc cctgtgctca gaggctgctc tgcaagccat ccgcaagaag atggatctca 1500

ttgacctaga ggatgagacc attgatgccg aggtcatgaa ctctctagca gttactatgg 1560

atgacttccg gtgggccttg agccagagta acccatcagc actgcgggaa accgtggtag 1620

aggtgccaca ggtaacctgg gaagacatcg ggggcctaga ggatgtcaaa cgtgagctac 1680

aggagctggt ccagtatcct gtggagcacc cagacaaatt cctgaagttt ggcatgacac 1740

cttccaaggg agttctgttc tatggacctc ctggctgtgg gaaaactttg ttggccaaag 1800

ccattgctaa tgaatgccag gccaacttca tctccatcaa gggtcctgag ctgctcacca 1860

tgtggtttgg ggagtctgag gccaatgtca gagaaatctt tgacaaggcc cgccaagctg 1920

ccccctgtgt gctattcttt gatgagctgg attcgattgc caaggctcgt ggaggtaaca 1980

ttggagatgg tggtggggct gctgaccgag tcatcaacca gatcctgaca gaaatggatg 2040

gcatgtccac aaaaaaaaat gtgttcatca ttggcgctac caaccggcct gacatcattg 2100

atcctgccat cctcagacct ggccgtcttg atcagctcat ctacatccca cttcctgatg 2160

agaagtcccg tgttgccatc ctcaaggcta acctgcgcaa gtccccagtt gccaaggatg 2220

tggacttgga gttcctggct aaaatgacta atggcttctc tggagctgac ctgacagaga 2280

tttgccagcg tgcttgcaag ctggccatcc gtgaatccat cgagagtgag attaggcgag 2340

aacgagagag gcagacaaac ccatcagcca tggaggtaga agaggatgat ccagtgcctg 2400

agatccgtcg agatcacttt gaagaagcca tgcgctttgc gcgccgttct gtcagtgaca 2460

atgacattcg gaagtatgag atgtttgccc agacccttca gcagagtcgg ggctttggca 2520

gcttcagatt cccttcaggg aaccagggtg gagctggccc cagtcagggc agtggaggcg 2580

gcacaggtgg cagtgtatac acagaagaca atgatgatga cctgtatggc taagtggtgg 2640

tggccagcgt gcagtgagct ggcctgcctg gaccttgttc cctgggggtg gggcgcttgc 2700

ccaggagagg gaccaggggt gcgcccacag cctgctccat tctccagtct gaacagttca 2760

gctacagtct gactctggac agggggtttc tgttgcaaaa atacaaaaca aaagcgataa 2820

aataaaagcg attttcattt ggtaggcgga gagtgaatta ccaacaggga attgggcctt 2880

gggcctatgc catttctgtt gtagtttggg gcagtgcagg ggacctgtgt ggggtgtgaa 2940

ccaaggcact actgccacct gccacagtaa agcatctgca cttgactcaa tgctgcccga 3000

gccctccctt ccccctatcc aacctgggta ggtgggtagg ggccacagtt gctggatgtt 3060

tatatagaga gtaggttgat ttattttaca tgcttttgag ttaatgttgg aaaactaatc 3120

acaagcagtt tctaaaccaa aaaatgacat gttgtaaaag gacaataaac gttgggtcaa 3180

aatggaaaaa aaaaaaaa 3198

<210>120

<211>806

<212>PRT

<213> human (Homo sapiens)

<400>120

Met Ala Ser Gly Ala Asp Ser Lys Gly Asp Asp Leu Ser Thr Ala Ile

1 5 10 15

Leu Lys Gln Lys Asn Arg Pro Asn Arg Leu Ile Val Asp Glu Ala Ile

20 25 30

Asn Glu Asp Asn Ser Val Val Ser Leu Ser Gln Pro Lys Met Asp Glu

35 40 45

Leu Gln Leu Phe Arg Gly Asp Thr Val Leu Leu Lys Gly Lys Lys Arg

50 55 60

Arg Glu Ala Val Cys Ile Val Leu Ser Asp Asp Thr Cys Ser Asp Glu

65 70 75 80

Lys Ile Arg Met Asn Arg Val Val Arg Asn Asn Leu Arg Val Arg Leu

85 90 95

Gly Asp Val Ile Ser Ile Gln Pro Cys Pro Asp Val Lys Tyr Gly Lys

100 105 110

Arg Ile His Val Leu Pro Ile Asp Asp Thr Val Glu Gly Ile Thr Gly

115 120 125

Asn Leu Phe Glu Val Tyr Leu Lys Pro Tyr Phe Leu Glu Ala Tyr Arg

130 135 140

Pro Ile Arg Lys Gly Asp Ile Phe Leu Val Arg Gly Gly Met Arg Ala

145 150 155 160

Val Glu Phe Lys Val Val Glu Thr Asp Pro Ser Pro Tyr Cys Ile Val

165 170 175

Ala Pro Asp Thr Val Ile His Cys Glu Gly Glu Pro Ile Lys Arg Glu

180 185 190

Asp Glu Glu Glu Ser Leu Asn Glu Val Gly Tyr Asp Asp Ile Gly Gly

195 200 205

Cys Arg Lys Gln Leu Ala Gln Ile Lys Glu Met Val Glu Leu Pro Leu

210 215 220

Arg His Pro Ala Leu Phe Lys Ala Ile Gly Val Lys Pro Pro Arg Gly

225 230 235 240

Ile Leu Leu Tyr Gly Pro Pro Gly Thr Gly Lys Thr Leu Ile Ala Arg

245 250 255

Ala Val Ala Asn Glu Thr Gly Ala Phe Phe Phe Leu Ile Asn Gly Pro

260 265 270

Glu Ile Met Ser Lys Leu Ala Gly Glu Ser Glu Ser Asn Leu Arg Lys

275 280 285

Ala Phe Glu Glu Ala Glu Lys Asn Ala Pro Ala Ile Ile Phe Ile Asp

290 295 300

Glu Leu Asp Ala Ile Ala Pro Lys Arg Glu Lys Thr His Gly Glu Val

305 310 315 320

Glu Arg Arg Ile Val Ser Gln Leu Leu Thr Leu Met Asp Gly Leu Lys

325 330 335

Gln Arg Ala His Val Ile Val Met Ala Ala Thr Asn Arg Pro Asn Ser

340 345 350

Ile Asp Pro Ala Leu Arg Arg Phe Gly Arg Phe Asp Arg Glu Val Asp

355 360 365

Ile Gly Ile Pro Asp Ala Thr Gly Arg Leu Glu Ile Leu Gln Ile His

370 375 380

Thr Lys Asn Met Lys Leu Ala Asp Asp Val Asp Leu Glu Gln Val Ala

385 390 395 400

Asn Glu Thr His Gly His Val Gly Ala Asp Leu Ala Ala Leu Cys Ser

405 410 415

Glu Ala Ala Leu Gln Ala Ile Arg Lys Lys Met Asp Leu Ile Asp Leu

420 425 430

Glu Asp Glu Thr Ile Asp Ala Glu Val Met Asn Ser Leu Ala Val Thr

435 440 445

Met Asp Asp Phe Arg Trp Ala Leu Ser Gln Ser Asn Pro Ser Ala Leu

450 455 460

Arg Glu Thr Val Val Glu Val Pro Gln Val Thr Trp Glu Asp Ile Gly

465 470 475 480

Gly Leu Glu Asp Val Lys Arg Glu Leu Gln Glu Leu Val Gln Tyr Pro

485 490 495

Val Glu His Pro Asp Lys Phe Leu Lys Phe Gly Met Thr Pro Ser Lys

500 505 510

Gly Val Leu Phe Tyr Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala

515 520 525

Lys Ala Ile Ala Asn Glu Cys Gln Ala Asn Phe Ile Ser Ile Lys Gly

530 535 540

Pro Glu Leu Leu Thr Met Trp Phe Gly Glu Ser Glu Ala Asn Val Arg

545 550 555 560

Glu Ile Phe Asp Lys Ala Arg Gln Ala Ala Pro Cys Val Leu Phe Phe

565 570 575

Asp Glu Leu Asp Ser Ile Ala Lys Ala Arg Gly Gly Asn Ile Gly Asp

580 585 590

Gly Gly Gly Ala Ala Asp Arg Val Ile Asn Gln Ile Leu Thr Glu Met

595 600 605

Asp Gly Met Ser Thr Lys Lys Asn Val Phe Ile Ile Gly Ala Thr Asn

610 615 620

Arg Pro Asp Ile Ile Asp Pro Ala Ile Leu Arg Pro Gly Arg Leu Asp

625 630 635 640

Gln Leu Ile Tyr Ile Pro Leu Pro Asp Glu Lys Ser Arg Val Ala Ile

645 650 655

Leu Lys Ala Asn Leu Arg Lys Ser Pro Val Ala Lys Asp Val Asp Leu

660 665 670

Glu Phe Leu Ala Lys Met Thr Asn Gly Phe Ser Gly Ala Asp Leu Thr

675 680 685

Glu Ile Cys Gln Arg Ala Cys Lys Leu Ala Ile Arg Glu Ser Ile Glu

690 695 700

Ser Glu Ile Arg Arg Glu Arg Glu Arg Gln Thr Asn Pro Ser Ala Met

705 710 715 720

Glu Val Glu Glu Asp Asp Pro Val Pro Glu Ile Arg Arg Asp His Phe

725 730 735

Glu Glu Ala Met Arg Phe Ala Arg Arg Ser Val Ser Asp Asn Asp Ile

740 745 750

Arg Lys Tyr Glu Met Phe Ala Gln Thr Leu Gln Gln Ser Arg Gly Phe

755 760 765

Gly Ser Phe Arg Phe Pro Ser Gly Asn Gln Gly Gly Ala Gly Pro Ser

770 775 780

Gln Gly Ser Gly Gly Gly Thr Gly Gly Ser Val Tyr Thr Glu Asp Asn

785 790 795 800

Asp Asp Asp Leu Tyr Gly

805

<210>121

<211>21

<212>RNA

<213> Artificial

<220>

<223> sequences for siRNA Synthesis

<400>121

aaguagggua ugaugacauu g 21

<210>122

<211>138

<212>PRT

<213> human (Homo sapiens)

<400>122

Gly Asn Gln Glu Asp Leu Ser Lys Asp Ser Leu Gly Pro Thr Lys Ser

1 5 10 15

Ser Lys Ile Glu Gly Ala Gly Thr Ser Ile Ser Glu Pro Pro Ser Pro

20 25 30

Ile Ser Pro Tyr Ala Ser Glu Ser Cys Gly Thr Leu Pro Leu Pro Leu

35 40 45

Arg Pro Cys Gly Glu Gly Ser Glu Met Val Gly Lys Glu Asn Ser Ser

50 55 60

Pro Glu Asn Lys Asn Trp Leu Leu Ala Met Ala Ala Lys Arg Lys Ala

65 70 75 80

Glu Asn Pro Ser Pro Arg Ser Pro Ser Ser Gln Thr Pro Asn Ser Arg

85 90 95

Arg Gln Ser Gly Lys Thr Leu Pro Ser Pro Val Thr Ile Thr Pro Ser

100 105 110

Ser Met Arg Lys Ile Cys Thr Tyr Phe His Arg Lys Ser Gln Glu Asp

115 120 125

Phe Cys Gly Pro Glu His Ser Thr Glu Leu

130 135

Claims (109)

1. A substantially pure polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 80;

(b) SEQ id no: 80, and has an amino acid sequence identical to that shown by SEQ ID NO: 80, or a protein-equivalent biologically active polypeptide consisting of the amino acid sequence shown in seq id no; and

(c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in 80.

2. An isolated polynucleotide encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A host cell carrying the polynucleotide of claim 2 or a vector comprising the polynucleotide of claim 2.

5. A method for producing the polypeptide of claim 1, comprising the steps of:

(a) culturing a host cell carrying a polynucleotide encoding the polypeptide of claim 1 or carrying a vector comprising a polynucleotide encoding the polypeptide of claim 1;

(b) Allowing the host cell to express the polypeptide;

(c) collecting the expressed polypeptide.

6. An antibody that binds to the polypeptide of claim 1.

7. A polynucleotide which is complementary to the polynucleotide of claim 2 or its complementary strand and comprises at least 15 nucleotides.

8. An antisense polynucleotide or a small interfering RNA directed against the polynucleotide of claim 2.

9. The small interfering RNA of claim 8, having a sense strand selected from the group consisting of SEQ ID NO: 34 and 35.

10. A method of diagnosing breast cancer, the method comprising the steps of:

(a) detecting a nucleic acid sequence encoding SEQ ID NO: 80 or 82; and

(b) the increase in expression levels was correlated with breast cancer.

11. The method of claim 10, wherein the expression level is detected by any one method selected from the group consisting of:

(a) detecting a nucleic acid encoding SEQ ID NO: 80 or 82;

(b) detecting a polypeptide comprising SEQ ID NO: 80 or 82; and

(c) detecting a polypeptide comprising SEQ ID NO: 80 or 82, or a pharmaceutically acceptable salt thereof.

12. A method of screening for a compound for use in the treatment of breast cancer, the method comprising the steps of:

(a) Contacting a test compound with a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a polypeptide having an amino acid sequence as set forth in SEQ ID NO,

(2) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 80 or 82, or a polypeptide comprising an amino acid sequence as set forth in seq id no;

(b) detecting the binding activity between the test compound and the polypeptide; and

(c) selecting a test compound that binds to said polypeptide.

13. A method of screening for a compound for use in the treatment of breast cancer, the method comprising the steps of:

(a) allowing expression of a polypeptide comprising SEQ ID NO: 79 or 81 with a candidate compound; and

(b) selecting a compound that: the compound reduces the expression level of a polypeptide comprising SEQ ID NO: 79 or 81, or a pharmaceutically acceptable salt thereof.

14. A method of screening for a compound for use in the treatment of breast cancer, the method comprising the steps of:

(a) contacting a test compound with a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a polypeptide having an amino acid sequence as set forth in SEQ ID NO,

(2) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 80 or 82, or a polypeptide comprising an amino acid sequence as set forth in seq id no;

(b) detecting the biological activity of the polypeptide of step (a); and

(c) selecting a compound that inhibits the biological activity of the polypeptide compared to the biological activity detected in the absence of the test compound.

15. The method of claim 14, wherein the biological activity is a cell proliferative activity.

16. A method of screening for a compound for use in the treatment of breast cancer, the method comprising the steps of:

(a) Contacting a cell into which a vector comprising a transcriptional regulatory region of a7322 or F3374V1 gene and a reporter gene expressed under the control of the transcriptional regulatory region with a candidate compound;

(b) determining the level of expression or activity of the reporter gene; and

(c) selecting a compound that: the compound reduces the level of expression or activity of the reporter gene as compared to the level of expression or activity of the reporter gene detected in the absence of the test compound.

17. A method for screening for an inhibitor against the phosphorylation level of F3374V1 or a functional equivalent thereof, the method comprising the steps of:

(a) contacting a test compound with a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ id no: 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 82, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) Detecting the phosphorylation level of the polypeptide;

(c) comparing the phosphorylation level of the polypeptide to the phosphorylation level of the polypeptide detected in the absence of the compound;

(d) selecting a compound that reduces the phosphorylation level of the polypeptide as an inhibitor against the phosphorylation level of F3374V 1.

18. A method for screening for an inhibitor against the phosphorylation level of F3374V1 or a functional equivalent thereof, the method comprising the steps of:

(a) contacting a test compound with a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ id no: 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 82, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) Detecting the level of phosphorylation of the polypeptide or fragment thereof;

(c) comparing the level of phosphorylation of the substrate to the level of phosphorylation of the polypeptide detected in the absence of the test compound; and

(d) selecting a compound that reduces the phosphorylation level of said polypeptide as an inhibitor against the phosphorylation level of F3374V 1.

19. A method of screening for a compound for use in the treatment or prevention of breast cancer, the method comprising the steps of:

(a) contacting a test compound with a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ id no: 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 82, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the phosphorylation level of the polypeptide;

(c) comparing the level of phosphorylation of said polypeptide to the level of phosphorylation of said polypeptide detected in the absence of said test compound;

(d) Selecting a compound that reduces the level of phosphorylation of the polypeptide as a compound for treating or preventing breast cancer.

20. A method of screening for an agent useful for treating or preventing breast cancer, the method comprising the steps of:

(a) contacting a test compound with a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ id no: 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 82, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the level of phosphorylation of the polypeptide or fragment thereof;

(c) comparing the level of phosphorylation of the substrate to the level of phosphorylation of the polypeptide detected in the absence of the test compound;

(d) selecting a compound that reduces the level of phosphorylation of the polypeptide as a compound for treating or preventing breast cancer.

21. A composition for treating breast cancer, the composition comprising a pharmaceutically effective amount of an antisense polynucleotide or a small interfering RNA against a polynucleotide encoding a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a polypeptide having an amino acid sequence as set forth in SEQ ID NO,

(b) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, and

(c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 80 or 82, or a polypeptide consisting of the amino acid sequence shown in seq id no.

22. The composition of claim 21, wherein the small interfering RNA comprises a sequence corresponding to a sequence selected from SEQ ID NOs: 34. 35, 37, 38, 67 and 68 as target sequences.

23. The composition of claim 22, wherein the small interfering RNA has the general formula 5 ' - [ a ] - [ B ] - [ a ' ] -3 ', wherein [ a ] is a nucleotide sequence corresponding to SEQ ID NO: 34. 35, 37, 38, 67 or 68,

[B] Is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and

[ A' ] is a ribonucleotide sequence that is complementary to [ A ].

24. A composition for treating breast cancer, which comprises a pharmaceutically effective amount of an antibody against a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a polypeptide having an amino acid sequence as set forth in SEQ ID NO,

(b) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, and

(c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 80 or 82, or a polypeptide consisting of the amino acid sequence shown in seq id no.

25. A composition for treating breast cancer, the composition comprising a pharmaceutically effective amount of a peptide that inhibits a polypeptide consisting of seq id NO: 82 as an active ingredient, and a pharmaceutically acceptable carrier.

26. A method of treating breast cancer, the method comprising the steps of: administering a pharmaceutically effective amount of an antisense polynucleotide or a small interfering RNA against a polynucleotide encoding a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a polypeptide having an amino acid sequence as set forth in SEQ ID NO,

(b) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, and

(c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 80 or 82, or a polypeptide consisting of the amino acid sequence shown in seq id no.

27. The method of claim 26, wherein the small interfering RNA comprises a sequence corresponding to a sequence selected from SEQ ID NOs: 34. 35, 37, 38, 67 and 68 as target sequences.

28. The method of claim 27, wherein the small interfering RNA has the general formula 5 ' - [ a ] - [ B ] - [ a ' ] -3 ', wherein [ a ] is a nucleotide sequence corresponding to SEQ ID NO: 34. 35, 37, 38, 67 or 68,

[B] Is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and

[ A' ] is a ribonucleotide sequence that is complementary to [ A ].

29. A method of treating breast cancer, the method comprising the steps of: administering a pharmaceutically effective amount of an antibody against a polypeptide selected from the group consisting of:

(a) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a polypeptide having an amino acid sequence as set forth in SEQ ID NO,

(b) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, and

(c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 80 or 82, or a polypeptide consisting of the amino acid sequence shown in seq id no.

30. A method of treating breast cancer, the method comprising the steps of: administering a pharmaceutically effective amount of a polypeptide that inhibits the activity of a polypeptide represented by SEQ ID NO: 82.

31. A method of treating or preventing breast cancer, said method comprising the step of administering a pharmaceutically effective amount of a polypeptide selected from the group consisting of (a) - (c) or a polynucleotide encoding the polypeptide:

(a) Comprises the amino acid sequence of SEQ ID NO: 80 or 82 or a fragment thereof;

(b) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, or a fragment thereof;

(c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, or a fragment thereof, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80 or 82, or a polypeptide consisting of the amino acid sequence shown in seq id no.

32. A method for inducing anti-tumor immunity against breast cancer, the method comprising the step of contacting a polypeptide selected from the group consisting of (a) to (c) with an antigen presenting cell, or introducing a polynucleotide encoding the polypeptide or a vector comprising the polynucleotide into an antigen presenting cell:

(a) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a fragment thereof;

(b) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, or a fragment thereof;

(c) A polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, or a fragment thereof, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80 or 82, or a polypeptide consisting of the amino acid sequence shown in seq id no.

33. The method for inducing anti-tumor immunity according to claim 32, wherein the method further comprises the step of administering antigen-presenting cells to the subject.

34. A pharmaceutical composition for treating or preventing breast cancer, which comprises a pharmaceutically effective amount of a polypeptide selected from the group consisting of (a) to (c) or a polynucleotide encoding the polypeptide as an active ingredient, and a pharmaceutically acceptable carrier:

(a) comprises the amino acid sequence of SEQ ID NO: 80 or 82, or a fragment thereof;

(b) SEQ id no: 80 or 82, and has an amino acid sequence identical to that shown by SEQ ID NO: 80 or 82, or a fragment thereof;

(c) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 or 81, or a fragment thereof, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80 or 82, or a polypeptide consisting of the amino acid sequence shown in seq id no.

35. The pharmaceutical composition of claim 34, wherein the polynucleotide is incorporated into an expression vector.

36. A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a sequence corresponding to a sequence selected from SEQ ID NOs: 34. 35, 37, 38, 67 or 68, and wherein the antisense strand comprises a ribonucleotide sequence that is complementary to said sense strand, wherein said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule; and wherein the double-stranded molecule, when introduced into a cell expressing the A7322, F3374V1 or AURKB gene, inhibits expression of said gene.

37. The double-stranded molecule of claim 36, wherein the double-stranded molecule is an oligonucleotide of less than about 100 nucleotides in length.

38. The double-stranded molecule of claim 37, wherein the double-stranded molecule is an oligonucleotide of less than about 75 nucleotides in length.

39. The double-stranded molecule of claim 38, wherein the double-stranded molecule is an oligonucleotide of less than about 50 nucleotides in length.

40. The double-stranded molecule of claim 39, wherein the double-stranded molecule is an oligonucleotide of less than about 25 nucleotides in length.

41. The double stranded molecule of claim 36, wherein the target sequence comprises a sequence from SEQ id no: 79 or 81 from about 19 to about 25 contiguous nucleotides of the nucleotide sequence.

42. The double-stranded molecule of claim 41, wherein the double-stranded molecule is a single ribonucleotide transcript comprising the sense strand and the antisense strand connected by a single-stranded ribonucleotide sequence.

43. A vector encoding the double-stranded molecule of claim 36.

44. The vector of claim 43, wherein the vector encodes a transcript having secondary structure and comprises the sense strand and the antisense strand.

45. The vector of claim 43, wherein the transcript further comprises a single-stranded ribonucleotide sequence linking the sense strand and the antisense strand.

46. A vector that expresses a polynucleotide comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises SEQ ID NO: 34. 35, 37, 38, 67 or 68, the antisense strand nucleic acid consisting of a sequence complementary to the sense strand.

47. The vector of claim 46, wherein said polynucleotide has the general formula 5 ' - [ A ] - [ B ] - [ A ' ] -3 ', wherein [ A ] is SEQ ID NO: 34. 35, 37, 38, 67 or 68,

[B] is a nucleotide sequence consisting of 3-23 nucleotides, and

[ A' ] is a nucleotide sequence complementary to [ A ].

48. A method of screening for an inhibitor of the binding of F3374V1 to AURKB comprising the steps of:

(a) contacting an AURKB polypeptide or functional equivalent thereof with an F3374V1 polypeptide or functional equivalent thereof in the presence of a test compound;

(b) detecting binding between the polypeptides of step (a); and

(c) selecting a test compound that inhibits binding between the AURKB polypeptide and the F3374V1 polypeptide.

49. A method of screening for a compound for treating or preventing breast cancer, the method comprising the steps of:

(a) contacting an AURKB polypeptide or functional equivalent thereof with an F3374V1 polypeptide or functional equivalent thereof in the presence of a test compound;

(b) detecting binding between the polypeptides of step (a); and

(c) selecting a test compound that inhibits binding between the AURKB polypeptide and the F3374V1 polypeptide.

50. The method of claim 48 or 49, wherein said functional equivalent of the F3374V1 polypeptide comprises the amino acid sequence of an AURKB binding region.

51. The method of claim 50, wherein said functional equivalent of the F3374V1 polypeptide comprises the amino acid sequence of SEQ ID NO: 82 (amino acids 591-730) (SEQ ID NO: 122).

52. The method of claim 48 or 49, wherein said functional equivalent of an AURKB polypeptide comprises the amino acid sequence of a F3374V1 binding region.

53. A kit for screening a compound for treating or preventing breast cancer, the kit comprising the following components:

(a) AURKB polypeptide or functional equivalent thereof, and

(b) a F3374V1 polypeptide or a functional equivalent thereof.

54. A method of screening for an inhibitor of AURKB-mediated phosphorylation of F3374V1, the method comprising the steps of:

(a) incubating F3374V1 and AURKB under conditions suitable for AURKB to phosphorylate F3374V1 in the presence of a test compound, wherein said F3374V1 is a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82(F3374V1),

(2) SEQ ID NO: 82, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 82, or a polypeptide comprising the amino acid sequence shown in SEQ ID NO,

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has a sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the phosphorylation level of F3374V 1;

(c) Comparing the phosphorylation level of F3374V1 to a control level; and

(d) selecting a compound that: the compound reduces the phosphorylation level of F3374V1 compared to a control level detected in the absence of the test compound.

55. A method of screening for a compound for use in the treatment or prevention of breast cancer, the method comprising the steps of:

(a) incubating F3374V1 and AURKB under conditions suitable for AURKB to phosphorylate F3374V1 in the presence of a test compound, wherein said F3374V1 is a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82(F3374V1),

(2) SEQ ID NO: 82, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 82, or a polypeptide comprising the amino acid sequence shown in SEQ ID NO,

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has a sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the phosphorylation level of F3374V 1;

(c) Comparing the phosphorylation level of F3374V1 to a control level; and

(d) selecting a compound that: the compound reduces the phosphorylation level of F3374V1 compared to a control level.

56. The method of claim 54 or 55, wherein the phosphorylation level of F3374V1 is at the amino acid sequence set forth in SEQ ID NO: 82 (amino acids 591-730) (SEQ ID NO: 122) or the homologous position of the polypeptide.

57. A kit for screening a compound for treating or preventing breast cancer, the kit comprising the following components:

(a) a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82(F3374V1),

(2) SEQ ID NO: 82, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 82, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has a sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) A polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 88(AURKB),

(2) SEQ ID NO: 88, with the proviso that said polypeptide has an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 88, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 87, provided that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ id no: 88, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(c) a reagent for detecting the phosphorylation level of F3374V 1.

58. A kit for screening a compound for treating or preventing breast cancer, the kit comprising the following components:

(a) a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 82(F3374V1),

(2) SEQ ID NO: 82, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 82, and

(3) A polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 81, provided that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 82, or a polypeptide consisting of the amino acid sequence shown in seq id no; and

(b) a reagent for detecting the phosphorylation level of F3374V 1.

59. The kit of claim 58, wherein the cells further express a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 87(AURKB),

(2) SEQ ID NO: 88, with the proviso that said polypeptide has an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 88, and

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 87 and which has a sequence which hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 88, or a polypeptide consisting of the amino acid sequence shown in seq id no.

60. The kit of claim 58, wherein said cells are breast cancer cells.

61. The kit of claim 57 or 58, wherein the reagent for detecting the phosphorylation level of F3374V1 is a nucleic acid sequence identified in SEQ ID NO: 82 (amino acids 591-730) (SEQ ID NO: 122).

62. An antibody that recognizes an amino acid sequence set forth in SEQ ID NO: 82 (amino acids 437 to 730) (SEQ ID NO: 93).

63. A method for treating or preventing breast cancer in a subject, the method comprising the step of administering an inhibitor having at least any one function selected from the group consisting of:

(a) inhibiting the binding between F3374V1 and AURKB;

(b) inhibiting phosphorylation of F3374V1 by AURKB; and

(c) inhibiting the expression of any one gene selected from the group consisting of A7322, F3374V1, and AURKB.

64. A composition for preventing and treating breast cancer, which comprises a pharmaceutically effective amount of an inhibitor having at least any one function selected from the group consisting of:

(a) inhibiting the binding between F3374V1 and AURKB;

(b) inhibiting phosphorylation of F3374V1 by AURKB; and

(c) inhibiting the expression of any one gene selected from the group consisting of A7322, F3374V1, and AURKB.

65. A method for screening for an agent that induces apoptosis in a cell expressing TOPK, the method comprising the steps of:

(a) contacting a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the kinase activity of the polypeptide;

(c) comparing the kinase activity of the polypeptide to the kinase activity of the polypeptide detected in the absence of the agent; and

(d) selecting an agent that decreases kinase activity of the polypeptide as an agent that induces apoptosis of breast cancer cells.

66. A method of screening for an agent that induces apoptosis of breast cancer cells, the method comprising the steps of:

(a) contacting a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the phosphorylation level of the polypeptide;

(c) comparing the phosphorylation level of the polypeptide to a phosphorylation level of the polypeptide detected in the absence of the agent; and

(d) selecting an agent that reduces the level of phosphorylation of the polypeptide as an agent that induces apoptosis of the breast cancer cell.

67. A method of screening for an agent that induces apoptosis in cells expressing TOPK, the method comprising the steps of:

(a) contacting a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the phosphorylation level of the substrate;

(c) comparing the level of phosphorylation of the substrate to the level of phosphorylation of the polypeptide detected in the absence of the agent; and

(d) selecting an agent that reduces the level of phosphorylation of the substrate as an agent that induces apoptosis of the cell.

68. A method of screening for an agent that induces apoptosis of breast cancer cells, the method comprising the steps of:

(a) contacting a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the phosphorylation level of the substrate;

(c) comparing the level of phosphorylation of the substrate to a level of phosphorylation of the substrate detected in the absence of the agent; and

(d) selecting an agent that reduces the level of phosphorylation of the substrate as an agent that induces apoptosis of the breast cancer cell.

69. The method of claim 67 or 68, wherein the substrate is histone or a fragment of histone containing at least its phosphorylation site.

70. The method of claim 69, wherein said phosphorylation site is Ser10 of histone H3.

71. A method of screening for an agent that inhibits the kinase activity of TOPK, the method comprising the steps of:

(a) contacting a polypeptide selected from the group consisting of:

(1) Comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the phosphorylation level of the substrate;

(c) comparing the level of phosphorylation of the substrate to a level of phosphorylation of the substrate detected in the absence of the agent; and

(d) selecting as the inhibitor an agent that reduces the phosphorylation level of the substrate.

72. The method of claim 71, wherein said phosphorylation site is Ser10 of histone H3.

73. A method of screening for an agent useful for preventing or treating breast cancer, the method comprising the steps of:

(a) Contacting a cell expressing a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the phosphorylation level of the polypeptide;

(c) comparing the phosphorylation level of the polypeptide to a phosphorylation level of the polypeptide detected in the absence of the agent; and

(d) selecting an agent that reduces the phosphorylation level of the polypeptide as an agent for treating or preventing breast cancer.

74. A method of screening for an agent useful for preventing or treating breast cancer, the method comprising the steps of:

(a) Contacting a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the phosphorylation level of the substrate;

(c) comparing the level of phosphorylation of the substrate to a level of phosphorylation of the substrate detected in the absence of the agent; and

(d) selecting an agent that reduces the phosphorylation level of the substrate as an agent for treating or preventing breast cancer.

75. The method of claim 74, wherein the substrate is a histone or a fragment of histone containing at least its phosphorylation site.

76. The method of claim 75, wherein said phosphorylation site is Ser10 of histone H3.

77. A method of screening for a compound for use in the treatment or prevention of breast cancer, the method comprising the steps of:

(a) contacting a7322 with PHB2/REA or a functional equivalent thereof in the presence of a test compound, wherein said a7322 is a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 80(A7322),

(2) SEQ ID NO: 80, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO,

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 with the proviso that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting binding between the polypeptides of step (a); and

(c) selecting a test compound that inhibits binding between the a7322 polypeptide and a PHB2/REA polypeptide.

78. A method of screening for an inhibitor of the binding between a7322 and PHB2/REA as indicated by the cellular localization of PHB2/REA, the method comprising the steps of:

(a) Contacting a candidate compound with a cell expressing a7322 and PHB2/REA protein, wherein a7322 is a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 80(A7322),

(2) SEQ ID NO: 80, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO,

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 with the proviso that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in seq id no;

(b) detecting the subcellular localization of PHB2/REA protein; and

(c) selecting a compound that: the compound reduces the level of PHB2/REA protein in the nucleus compared to the level of that protein detected in the absence of the test compound.

79. A method of screening for an inhibitor of the binding between a7322 and PHB2/REA, wherein ER α transcriptional activity is used as an indicator, the method comprising the steps of:

(a) contacting the candidate compound with a cell expressing a7322 and PHB2/REA protein under treatment with E2, the cell having introduced therein a vector comprising an estrogen-responsive transcriptional regulatory region and a reporter gene expressed under the control of the transcriptional regulatory region;

(b) Determining the level of expression or activity of the reporter gene; and

(c) selecting a compound that: the compound reduces the level of expression or activity of the reporter gene as compared to the level of expression or activity of the reporter gene detected in the absence of the test compound.

80. A method of treating breast cancer comprising administering a pharmaceutically effective amount of a compound that inhibits binding between an a7322 polypeptide and a PHB2/REA polypeptide, wherein a7322 is a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 80(A7322),

(2) SEQ ID NO: 80, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO,

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 with the proviso that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in 80.

81. A method of treating breast cancer comprising administering a pharmaceutically effective amount of a compound that inhibits nuclear transfer of PHB2/REA protein, wherein a7322 is a polypeptide selected from the group consisting of:

(1) Comprises the amino acid sequence of SEQ ID NO: 80(A7322),

(2) SEQ ID NO: 80, provided that the polypeptide has an amino acid sequence identical to that set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in SEQ ID NO,

(3) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 79 with the proviso that the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 80, or a polypeptide consisting of the amino acid sequence shown in 80.

82. A method of screening for an agent that inhibits the phosphorylation level of TOPK, the method comprising the steps of:

(a) contacting a polypeptide selected from the group consisting of CDK1(SEQ ID NO: 95), cyclin B1(SEQ ID NO: 97), and an agent, under conditions that permit phosphorylation:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting (a) the level of phosphorylation at threonine 9 residue in the protein;

(c) comparing the level of phosphorylation of the threonine 9 residue in the protein to the level of phosphorylation of the threonine 9 residue in the protein detected in the absence of the agent; and

(d) selecting for reduction the amino acid sequence of SEQ ID NO: 92 as an agent for an inhibitor.

83. A method of screening for an agent useful for preventing or treating breast cancer, the method comprising the steps of:

(a) contacting a polypeptide selected from the group consisting of CDK1(SEQ ID NO: 95), cyclin B1(SEQ ID NO: 97), and an agent, under conditions that permit phosphorylation:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting (a) the level of phosphorylation at threonine 9 residue in the protein;

(c) comparing the level of phosphorylation of the threonine 9 residue in the protein to the level of phosphorylation of the threonine 9 residue in the protein detected in the absence of the agent; and

(d) selecting for reduction the amino acid sequence of SEQ ID NO: 92 as an agent for treating or preventing breast cancer.

84. A polypeptide comprising SEQ ID NO: 98, or a polypeptide functionally equivalent thereto, wherein said polypeptide inhibits the activity of a polypeptide consisting of SEQ ID NO: 92, respectively, in a biological function.

85. The polypeptide of claim 84, wherein the biological function is cell proliferative activity.

86. The polypeptide of claim 84, wherein said polypeptide consists of 8-50 residues.

87. The polypeptide of claim 84, wherein the polypeptide is modified by a cell membrane permeable substance.

88. The polypeptide of claim 84, having the following general formula:

[R]-[D]

wherein [ R ] represents a cell membrane-permeable substance; and [ D ] represents a polypeptide comprising SEQ ID NO: 98, or a fragment thereof; or a polypeptide functionally equivalent to a polypeptide comprising said fragment sequence, wherein [ R ] and [ D ] are directly linked or indirectly linked through a linker,

wherein the polypeptide inhibits the polypeptide encoded by SEQ ID NO: 92, respectively, in a biological function.

89. The polypeptide of claim 88, wherein the linker has the amino acid sequence of G.

90. The polypeptide of claim 88 wherein the cell membrane permeable substance is any one selected from the group consisting of:

poly-arginine;

Tat/RKKRRQRRR/SEQ ID NO：100；

Penetratin/RQIKIWFQNRRMKWKK/SEQ ID NO：101；

Buforin II/TRSSRAGLQFPVGRVHRLLRK/SEQ ID NO：102；

Transportan/GWTLNSAGYLLGKINLKALAALAKKIL/SEQ ID NO：103；

MAP (model amphipathic peptide)/KLALKLALKALKAALKLA/SEQ ID NO: 104;

K-FGF/AAVALLPAVLLALLAP/SEQ ID NO：105；

Ku70/VPMLK/SEQ ID NO：106

Prion/MANLGYWLLALFVTMWTDVGLCKKRPKP/SEQ ID NO：107；

pVEC/LLIILRRRIRKQAHAHSK/SEQ ID NO：108；

Pep-1/KETWWETWWTEWSQPKKKRKV/SEQ ID NO：109；

SynB1/RGGRLSYSRRRFSTSTGR/SEQ ID NO：110；

Pep-7/SDLWEMMMVSLACQY/SEQ ID NO：111；

HN-1/TSPLNIHNGQKL/SEQ ID NO: 112, a first electrode; and

Ku70/PMLKE/SEQ ID NO：114。

91. the polypeptide of claim 90, wherein the polyarginine is Arg11(SEQ ID NO: 113).

92. The polypeptide of claim 91, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 99.

93. an agent for treating and/or preventing breast cancer, which comprises

Comprises the amino acid sequence of SEQ ID NO: 98;

A polypeptide functionally equivalent to the polypeptide; or

Polynucleotides encoding these polypeptides

As an active ingredient, wherein the polypeptide inhibits a polypeptide consisting of SEQ ID NO: 92, respectively, and a pharmaceutically acceptable salt thereof.

94. The agent of claim 93 wherein the biological function is cell proliferative activity.

95. The agent of claim 93 wherein the polypeptide consists of 8 to 50 residues.

96. The agent of claim 93 wherein the active ingredient is the polypeptide and the polypeptide is modified by a cell membrane permeable substance.

97. The agent of claim 96 wherein the polypeptide has the general formula:

[R]-[D]

wherein [ R ] represents a cell membrane-permeable substance; and [ D ] represents a polypeptide comprising SEQ ID NO: 98, or a fragment thereof; or a polypeptide functionally equivalent to said polypeptide, wherein [ R ] and [ D ] are directly linked or indirectly linked through a linker,

wherein the polypeptide inhibits the polypeptide encoded by SEQ ID NO: 2, biological function of the peptide.

98. The agent of claim 97 wherein the linker has the amino acid sequence of G.

99. The polypeptide of claim 98, wherein the cell membrane permeable substance is any one selected from the group consisting of:

poly-arginine;

Tat/RKKRRQRRR/SEQ ID NO：100；

Penetratin/RQIKIWFQNRRMKWKK/SEQ ID NO：101；

Buforin II/TRSSRAGLQFPVGRVHRLLRK/SEQ ID NO：102；

Transportan/GWTLNSAGYLLGKINLKALAALAKKIL/SEQ ID NO：103；

MAP (model amphipathic peptide)/KLALKLALKALKAALKLA/SEQ ID NO: 104;

K-FGF/AAVALLPAVLLALLAP/SEQ ID NO：105；

Ku70/VPMLK/SEQ ID NO：106

Prion/MANLGYWLLALFVTMWTDVGLCKKRPKP/SEQ ID NO：107；

pVEC/LLIILRRRIRKQAHAHSK/SEQ ID NO：108；

Pep-1/KETWWETWWTEWSQPKKKRKV/SEQ ID NO：109；

SynB1/RGGRLSYSRRRFSTSTGR/SEQ ID NO：110；

Pep-7/SDLWEMMMVSLACQY/SEQ ID NO：111；

HN-1/TSPLNIHNGQKL/SEQ ID NO: 112, a first electrode; and

Ku70/PMLKE/SEQ ID NO：114。

100. the agent of claim 99, wherein the polyarginine is Arg11(SEQ ID NO: 113).

101. The agent of claim 100, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 99.

102. a method for treating and/or preventing breast cancer comprising administering

Comprises the amino acid sequence of SEQ ID NO: 98;

a polypeptide functionally equivalent to the polypeptide; or

Polynucleotides encoding these polypeptides

Wherein the polypeptide inhibits the polypeptide encoded by SEQ ID NO: 92, respectively, and a pharmaceutically acceptable salt thereof.

103. Comprises the amino acid sequence of SEQ ID NO: 98, a polypeptide,

A polypeptide functionally equivalent to the polypeptide, or

Polynucleotides encoding these polypeptides

Use of a polypeptide that inhibits a polypeptide consisting of SEQ ID NO: 92, respectively, and a pharmaceutically acceptable salt thereof.

104. A pharmaceutical composition comprising a peptide comprising SEQ ID NO: 98 or a polypeptide functionally equivalent thereto, and a pharmaceutically acceptable carrier, wherein the polypeptide inhibits a polypeptide consisting of SEQ ID NO: 92, respectively, and a pharmaceutically acceptable salt thereof.

105. A method of screening for an agent useful for preventing or treating breast cancer, the method comprising the steps of:

(a) contacting a candidate agent with a cell expressing protein phosphatase 1 alpha (SEQ ID NO: 116) and a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting the phosphorylation level of the protein of (a);

(c) comparing the phosphorylation level of the protein to a phosphorylation level of the protein detected in the absence of an agent; and

(d) selecting an agent that reduces the phosphorylation level of the protein as an agent for treating or preventing breast cancer.

106. A method of screening for an agent useful for preventing or treating breast cancer, comprising the steps of:

(a) contacting a polypeptide selected from the group consisting of p47(SEQ ID NO: 118), p97(SEQ ID NO: 120), and an agent:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) detecting binding between said polypeptides or phosphorylation of p97(SEQ ID NO: 120); and

(c) test compounds were selected that inhibited the binding between the polypeptides or phosphorylation of p97(SEQ ID NO: 120).

107. A method of screening for an agent useful for preventing or treating breast cancer, the method comprising the steps of:

(a) Contacting a candidate agent with a PBK/TOPK expressing cell;

(b) observing the G2/M population on the cell structure and/or cell cycle; and

(c) compounds are selected that alter intercellular junctions into long intercellular bridges and/or increase the G2/M population of cells.

108. A method of screening for an agent for enhancing phosphatase 1 α -mediated dephosphorylation of TOPK, the method comprising the steps of:

(a) contacting a candidate agent with a cell expressing protein phosphatase 1 alpha (SEQ ID NO: 116) and a polypeptide selected from the group consisting of:

(1) comprises the amino acid sequence of SEQ ID NO: 92, or a polypeptide having the amino acid sequence shown in SEQ ID NO,

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) Detecting the phosphorylation level of the protein of (a);

(c) comparing the phosphorylation level of the protein to a phosphorylation level of the protein detected in the absence of an agent; and

(d) selecting an agent that reduces the phosphorylation level of the protein as an agent for treating or preventing breast cancer.

109. A method of screening for an agent for inhibiting TOPK-mediated phosphorylation of p97, the method comprising the steps of:

(a) contacting a polypeptide selected from the group consisting of p47(SEQ ID NO: 118), p97(SEQ ID NO: 120), and an agent:

(1) comprises the amino acid sequence of SEQ ID NO: 92;

(2) SEQ ID NO: 92, and has an amino acid sequence identical to that shown by SEQ ID NO: 92, or a polypeptide equivalent to the polypeptide of the amino acid sequence shown in SEQ ID NO,

(3) comprises a nucleotide sequence substantially identical to SEQ ID NO: 92 having at least about 80% homology, and

(4) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a polypeptide encoded by SEQ ID NO: 91, wherein the polypeptide has an amino acid sequence that hybridizes to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 92, or a polypeptide comprising the amino acid sequence shown in seq id No. 92;

(b) Detecting binding between said polypeptides or phosphorylation of p97(SEQ ID NO: 120);

(c) test compounds were selected that inhibited binding between polypeptides or phosphorylation of p97(SEQ ID NO: 120).

Images