JP2005521421A - Methods for diagnosing and treating colorectal cancer - Google Patents

Abstract

The present application provides the human gene HELAD1, whose expression is significantly elevated in the majority of colorectal cancers compared to the corresponding non-cancerous tissue. Specifically, in examining the developmental mechanism of colorectal cancer, we searched for a gene regulated by a nematode-like multiple colon polyp gene product (APC) and identified the HELAD1 gene (helicase, APC downregulation 1). Recombinant polypeptides representing the cellular activity-related ATPase (AAA) domain of the HELAD1 product showed 3 ′ to 5 ′ helicase and exonuclease activity in vitro. HELAD1 was abundantly expressed in 16 out of 20 colon cancers examined, but was hardly detected in the corresponding non-cancerous mucosa. Treatment of colon cancer cells with antisense oligonucleotides suppressed their expression and induced apoptosis. This data demonstrates the importance of HELAD1 in colorectal cancer development and its usefulness as a diagnostic marker for colorectal cancer. Furthermore, suppression of HELAD1 suggests that it may be a promising therapeutic strategy in the treatment of colorectal cancer.

Description

  This application claims priority to US Provisional Application No. 60 / 370,604, filed Apr. 5, 2002.

TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of cancer research. More particularly, the invention relates to treating and diagnosing colorectal cancer using the HELAD1 (helicase, APC down-regulated 1) gene and corresponding proteins. The invention further provides a method of diagnosing colorectal tumors in a subject, a method of screening for therapeutic agents useful in the treatment of colorectal tumors, a method of treating colorectal tumors, and a method of inoculating a subject with a vaccine against colorectal tumors About.

Background of the Invention
Mutations in the APC gene are involved in sporadic colorectal tumors as well as familial nematoma-like multiple colon polyps (Nishisho et al., 1991; Kinzler et al., 1991), whose normal products function as tumor suppressors. When associated with β-catenin, wild-type APC acts as a negative regulator of the Wnt / wingless signaling pathway by regulating the cytoplasmic and nuclear levels of β-catenin (Munemitsu et al., 1995). Together with the scaffolding proteins axin / conductin, APC promotes phosphorylation of β-catenin by glycogen synthase kinase 3β (GSK-3β), leading to degradation of β-catenin via the ubiquitin-proteosome pathway (Behrens et al., 1998; Hamada et al., 1999; Hart et al., 1999; Nakamura et al., 1998). Abnormal accumulation of β-catenin as a result of mutations in the APC, β-catenin, or axin / conductin genes has been observed in a variety of human cancers including colorectal cancer and hepatocellular carcinoma (Miyoshi et al., 1998; Morin et al., 1997; Satoh et al., 2000). When β-catenin moves to the nucleus, it interacts with T cell factor / lymphocyte enhancing factor (Tcf / LEF) (Korinek et al., 1997; Morin et al., 1997), c-Myc (He et al., 1998), cyclinD1 (Tetsu and McCormick, 1999; Shtutman et al., 1999), c-jun, fra-1 (Mann et al., 1999), MMP7 (Brabletz et al., 1999; Crawford et al., 1999), PPARγ (He et al., 1999), NBL4 (Ishiguro et al., 2000), WISP1 (Xu et al., 2000), MDR1 (Yamada et al., 2000), AF17 (Lin et al., 2001), and ENC1 (Fujita et al., 2001) activate transcription of downstream genes.

  It is clear from the evidence that DNA helicases have an important role in biological processes including DNA replication, DNA repair, and transcription. For example, a common mechanism that controls the initiation of DNA replication in eukaryotic cells involves the minichromosome maintenance protein complex (MCM) that contains helicase activity. Along with DNA polymerase, the MCM complex unwinds double-stranded DNA at the origin of replication (Eisen and Lucchesi, 1998; Takisawa et al., 2000). XPB and XPD, helicases involved in nucleotide excision repair, form a complex with TFIIH and play a role in transcription as well as DNA repair (Guzder et al., 1994a; Guzder et al., 1994b). Consistent with its essential biological role, helicase inactivation is known as xeroderma pigmentosum (Weeda et al., 1990), trichothiodystrophy (Broughton et al., 1994), Bloom syndrome (Ellis et al., 1995), It has been implicated in human genetic disorders such as Werner syndrome (Yu et al., 1996) and Rothmund-Thomson syndrome (Kitao et al., 1999). On the other hand, overexpression of rck / p54, a protein belonging to the DEAD box protein / RNA family of helicases, was seen in B-cell lymphomas with t (11; 14) (q23: q32), and 50% of colorectal adenocarcinoma % (Nakagawa et al., 1999; Seto et al., 1995). Furthermore, the DEAD box gene DDX1, which encodes a putative RNA helicase, is co-amplified with N-myc in several retinoblastomas and neuroblastomas (Godbout et al., 1998). The exact role of helicase enhancement in human carcinogenesis remains unresolved.

  Here we describe the isolation and characterization of the human gene HELAD1, which encodes a protein with helicase activity, and records in detail its involvement in colorectal cancer development. Colorectal cancer is the leading cause of cancer death in developed countries. Specifically, more than 130,000 new cases of colorectal cancer are reported each year in the United States. Colorectal cancer accounts for about 15% of all cancers. Of these, approximately 5% are directly related to hereditary genetic defects. Many patients are diagnosed with precancerous colon or rectal polyps before the onset of cancer. Many small colorectal polyps are benign, but some types can progress to cancer.

  The most widely used colorectal cancer screening test is colonoscopy. This method is used to visualize suspicious growth and / or to take a tissue biopsy. Typically, a tissue biopsy is examined histologically and a diagnosis is made based on microscopic findings of the biopsied cells. However, this method is limited because it produces subjective results and cannot be used to detect precancerous conditions very early. The development of a sensitive, specific and simple diagnostic system for detecting early stage colorectal cancer or premalignant lesions is highly desirable because it can ultimately eradicate the disease.

SUMMARY OF THE INVENTION Accordingly, the present invention relates to the use of the HELAD1 gene and protein as a diagnostic marker for colorectal cancer.

  The present application provides the isolated gene HELAD1, which is overexpressed in colorectal cancer. The expression of the gene HELAD1 is often upregulated in colorectal cancer and appears to confer oncogenic activity to cancer cells through its helicase activity and suppression of apoptosis. Thus, HELAD1 serves as a novel molecular target for colorectal cancer.

  An object of the present invention is to provide a method of using the HELAD1 gene and the protein encoded thereby for diagnosing and treating colorectal cancer.

  The present inventors set out to discover and investigate the carcinogenic mechanism of colorectal cancer. Therefore, we analyzed the expression profile of colorectal cancer using a cDNA microarray. We report herein the identification of the human gene HELAD1, whose expression is significantly elevated in the majority of colorectal cancers compared to the corresponding non-cancerous tissue. In one embodiment, the HELAD1 cDNA comprises 10,646 nucleotides (sequences) comprising a 7,095 nucleotide open reading frame (ranging from residues 230 to 7324) encoding a putative protein of 2365 amino acids (SEQ ID NO: 2). Number: 1). In another embodiment, the HELAD1 cDNA comprises 10,939 nucleotides comprising a 7,287 nucleotide open reading frame (ranging from residues 331 to 7617) encoding a putative protein of 2429 amino acids (SEQ ID NO: 4). SEQ ID NO: 3). Both HELAD1 protein embodiments include both a CH (calponin homology) domain and an AAA (ATPase associated with various cellular activities) domain that exhibits helicase activity. Suppressing HELAD1 expression with antisense S-oligonucleotides consistently induced apoptosis in colorectal cell lines. These findings suggest that HELAD1 confers oncogenic activity to cancer cells and that inhibition of the activity of this protein can be a promising strategy for the treatment of colorectal cancer.

  The present invention therefore relates to the use of HELAD1 proteins involved in cell proliferation and the genes encoding them. More specifically, the present invention provides the following.

  The present application provides a method for diagnosing and treating colorectal cancer using the human protein HELAD1 or a functional equivalent thereof. In a preferred embodiment, the HELAD1 protein includes an AAA domain with helicase activity, which is encoded by the open reading frame of SEQ ID NO: 1 or SEQ ID NO: 3. The HELAD1 protein preferably includes the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in SEQ ID NO: 4. The application also encodes a polynucleotide sequence that is encoded from at least a portion of a HELAD1 polynucleotide sequence or that is at least 15%, more preferably at least 25% complementary to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 An isolated protein encoded by is provided.

  The present invention further provides a method of using the human gene HELAD1 for the diagnosis and treatment of colorectal cancer, wherein the expression is significantly elevated in the majority of colorectal cancer compared to the corresponding non-cancerous tissue. provide. The isolated HELAD1 gene includes the polynucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. The present invention further hybridizes to the polynucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 to the extent that it encodes a HELAD1 protein or functional equivalent thereof, and at least 15%, more preferably at least 25 Includes polynucleotides that are% complementary. An example of such a polynucleotide is the degenerate form of SEQ ID NO: 1 or SEQ ID NO: 3.

  As used herein, an “isolated HELAD1 gene” is a natural that spans more than three separate genes whose structure is not identical to the structure of any naturally occurring polynucleotide. A polynucleotide that is not identical to the structure of any fragment of the genomic polynucleotide present in Thus, this term includes, for example, (a) DNA having the sequence of part of a naturally occurring genomic DNA molecule in a genome that is naturally present in an organism; (b) whatever the resulting molecule is naturally occurring A polynucleotide that is incorporated into the vector or prokaryotic or eukaryotic genomic DNA so that it is not identical to the vector or genomic DNA; (c) cDNA, genomic fragments, fragments produced by polymerase chain reaction (PCR), or restriction fragments And (d) a hybrid gene, ie, a recombinant nucleotide sequence that is part of a gene encoding a fusion polypeptide. Polynucleotides of DNA molecules present in a mixture of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones, for example, they occur in a DNA library such as a cDNA or genomic DNA library Therefore, it is specifically excluded from this definition.

  Accordingly, in one aspect, the present invention utilizes an isolated polynucleotide that encodes a polypeptide described herein or a fragment thereof. Preferably, the isolated polypeptide includes a nucleotide sequence that is at least 60% identical to the nucleotide set forth in SEQ ID NO: 1 or SEQ ID NO: 3. More preferably, the isolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92 with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more are identical. For isolated polynucleotides that are longer than or equivalent to the length of a reference sequence, eg, SEQ ID NO: 1 or SEQ ID NO: 3, a comparison is made with the full length of the reference sequence. If the isolated polynucleotide is shorter than the reference sequence, eg shorter than SEQ ID NO: 1 or SEQ ID NO: 3, a comparison is made for segments of the reference sequence of the same length (excluding any loops necessary for homology calculation) To do).

  The application also provides therapeutic agents for treating cancer. The therapeutic agent can be described as at least part of an antisense S-oligonucleotide of the HELAD1 polynucleotide sequence shown and described in SEQ ID NO: 1 or SEQ ID NO: 3. Suitable antisense S-oligonucleotides include, but are not limited to, the oligonucleotides set forth in SEQ ID NOs: 16, 17, and 20. The therapeutic agent may suitably be used to treat colorectal cells and tissues. The proposed course of action for therapeutic agents is to induce apoptosis in colorectal cancer cells. The therapeutic agent may be applied to mammals including human and domestic mammals.

  Antibodies that recognize the HELAD1 protein are also provided by this application. In part, antisense DNA, ribozymes, and RNAi (RNA interference) of the HELAD1 gene are provided as well.

  The present invention defines the step of determining the expression level of HELAD1 gene or HELAD1 protein level in a biological sample of a specimen, comparing the level determined for a normal sample, and the high expression level in the sample as having cancer A method for diagnosing cancer comprising the steps of: The cancer is suitably colorectal cancer.

  Furthermore, a method for screening candidate compounds for anticancer agents is provided. The method includes contacting the HELAD1 polypeptide with a candidate compound and selecting a compound that inhibits one or more of its activities.

  The present invention also provides a method of producing a protein by transfecting or transforming a host cell with a polynucleotide sequence encoding a HELAD1 protein and expressing the polynucleotide sequence.

  It is to be understood that the foregoing summary of the invention and the following detailed description are of the preferred embodiments and are not meant to limit the invention or other alternative embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION The terms “one”, “an”, and “the” as used herein refer to “at least one” unless otherwise specified. means.

  The human gene HELAD1, whose expression is markedly elevated in colorectal cancer compared to the corresponding non-cancerous colorectal tissue, was isolated and identified from a cDNA microarray. In one embodiment, the HELAD1 cDNA consists of 10,646 nucleotides comprising an open reading frame of 7,095 nucleotides (residues 230 to 7324) set forth in SEQ ID NO: 1. The open reading frame encodes a putative protein of 2365 amino acids set forth in SEQ ID NO: 2. In another embodiment, HELAD1 cDNA consists of 10,939 nucleotides comprising an open reading frame of 7,287 nucleotides (residues 331-7617) set forth in SEQ ID NO: 3. The open reading frame encodes a putative protein of 2429 amino acids set forth in SEQ ID NO: 4. The encoded protein is named HELAD1 (helicase, APC downregulation 1) due to the characteristics revealed in the following experiment.

  The HELAD1 gene encoded a protein containing a consensus motif of the CH domain at the amino terminus and AAA (ATPase associated with various cellular activities) at the carboxy terminus, and the product exhibited helicase and endonuclease activity in vitro.

  The CH domain is presumed to confer actin binding to various cytoskeletons and signaling molecules (Castresana and Saraste, 1995). Proteins containing CH domains are divided into three groups: (i) the fimbrin family of monomeric actin cross-linking molecules containing two actin binding domains, (ii) the opposite, each containing one actin binding domain And (iii) proteins containing a single amino-terminal CH domain (Stradal et al., 1998). According to the above classification, HELAD1 is classified into a third group including calponin, a molecule involved in smooth muscle contraction, IQGAP, a signaling molecule, and proto-oncogene Vav. HELAD1 is the first example that includes both CH and helicase domains, suggesting that proteins with CH domains may exert a wide range of biological functions.

The AAA family of proteins was first classified as a protein with one or two copies of a well-conserved cassette of 230-250 amino acids encompassing the Walker A and B motifs (Neuwald et al., 1999). The AAA family includes protease regulatory components, proteasome subunits, peroxisome biosynthetic proteins, Mg ²⁺ and Co ²⁺ chelators, vesicle synthesis proteins, dynein motor proteins, and DNA replication, recombination, restriction, and transcription, and Proteins involved in bacterial sporulation are included (Patel and Latterich, 1998; Neuwald et al., 1999). Among them, RuvBL1, a helicase homologous to E. coli RuvB, plays an important role in the late stages of homologous genetic recombination and in the repair of damaged DNA (Kanemaki et al., 1999). Similarly, MCM proteins containing AAA domains are components of the pre-replication complex and are essential for initiating DNA replication in eukaryotic cells (Takisawa et al., 2000; Tye and Sawyer, 2000). MCM protein is down-regulated in cells undergoing differentiation or quiescence and is more abundantly expressed in dysplastic or malignant tissue than normal tissue (Freeman et al., 1999). Thus, dysplastic cells can be characterized as functionally remaining in the cell cycle and / or accelerated DNA replication by deregulation of normal control over cell proliferation. Since HELAD1 expression is also enhanced in colorectal cancer, it may be involved in cell cycle progression and / or DNA replication. In addition, proteins containing AAA consensus motifs often have chaperone-like functions that assist in the construction, manipulation, or disassembly of protein complexes (Neuwald et al., 1999), and the hexameric structure of helicases is DNA Or it can provide a hole through which RNA can be passed and assembled into a DNA-protein complex (Tye and Sawyer, 2000; Patel and Picha, 2000). Therefore, HELAD1 may be able to interact with itself or other proteins. Future identification of its binding partner (s) will help to better understand the physiological role of this enzyme.

  In the experiments described herein, HELAD1 protein, like WRN, which is a protein that is more highly expressed in transformed and tumor cells than normal cells (Shiratori et al., 1999), has both helicase and exonuclease activity. showed that. Examination of WRN and BLM gene expression revealed that both were abundantly expressed in the corresponding normal tissues in 7 out of 12 colorectal cancers examined (data not shown). These results suggest that colon cancer cells may require high helicase expression in order to survive. However, the distribution of HELAD1 expression levels in normal human tissues was quite different from WRN; WRN is abundantly expressed in pancreas and placenta, but moderately expressed in heart and skeletal muscle (Yu et al., 1996) Although HELAD1 expression was abundant in fetal tissue, it could hardly be detected in any adult tissue examined. Interestingly, HELAD1 has 28% identity with C. elegans unc-53 (its overexpression causes growth cone elongation along the ap axis) (Hedgecock et al., 1987; Hekimi). And Kershaw, 1993). Furthermore, in the experiments reported herein, the expression of HELAD1 in cancer cells was down-regulated by transduction of APC (its product is essential for the control of axis formation, cell motility and differentiation) (Bienz And Clevers, 2000). This data strongly suggests that the expression of HELAD1 has a role in morphogenesis and / or differentiation. Although HELAD1 has not been proven to be a direct target of the Tcf / LEF transcription complex, activated HELAD1 may promote transcription of the target gene.

  Evidence herein demonstrates that the majority of colorectal cancers are abundantly expressed HELAD1, and that wild type APC can reduce the amount of HELAD1 in such tumors. This in turn strongly supports the notion that inappropriate expression of the HELAD1 gene is significantly involved in colorectal carcinogenesis. In particular, HELAD1 is not present in normal colon and other adult tissues, and when HELAD1 in cancer cells was suppressed by antisense S-oligonucleotide, it inhibited cancer cell proliferation and induced cell death. Although the exact molecular mechanism by which antisense S-oligonucleotides can suppress proliferation remains unclear, the data described herein show that HELAD1 is a molecular marker for the diagnosis of colorectal cancer Not only does it clearly show that it can serve as a molecular target for developing effective therapeutic reagents and strategies for patients with colorectal tumors.

Gene, protein, and partial peptide The present invention includes the human gene HELAD1 containing the polynucleotide sequence described in SEQ ID NO: 1 and SEQ ID NO: 3, and the degenerate and mutant forms thereof, the amino acid sequence described in SEQ ID NO: 2. HELAD1 proteins comprising the amino acid sequence set forth in SEQ ID NO: 4 and functional equivalents thereof are included to the extent they encode. Examples of proteins that are functionally equivalent to HELAD1 include, for example, variants of human HELAD1 protein, along with homologous proteins of other organisms corresponding to human HELAD1 protein.

  In the present invention, the term “functionally equivalent” means that the protein of the present invention has nuclease activity, helicase activity, or apoptosis inhibitory activity. If the protein of the invention has target activity, the activity can be routinely determined using conventional well-known assays. For example, nuclease activity may be determined by assaying for the degradation of a target nucleic acid such as DNA. Specifically, the target nucleic acid may be radiolabeled and incubated with the protein of the present invention. Next, nucleic acid degradation may be detected by electrophoresis. Alternatively, acid-soluble nucleotides released from nucleic acids may be quantified by scintillation counter (Sambrook, J. et al., Molecular Cloning, 2nd edition, 9.47-9.58, Cold Spring Harbor Laboratory Press, 1989). Similarly, helicase activity can be measured from double-stranded DNA substrates by assaying for unwinding of the target double-stranded DNA, as described, for example, by Suzuki et al. (1997) Nucleic Acids Res. 25: 2973-2978. May be determined by detecting substitution of the radiolabeled DNA fragment. Finally, apoptosis inhibitory activity may be determined by assaying for the induction of apoptosis or the presence or absence thereof. Specifically, apoptosis can be detected by TUNEL staining, detection of ladder DNA, microscopy, MTT assay, or other conventional methods (Methods in Enzymology: 322, pages 3-15).

  Methods for preparing proteins that are functionally equivalent to a given protein are well known to those of skill in the art and include known methods for introducing mutations into proteins. For example, those skilled in the art can prepare a protein functionally equivalent to the human HELAD1 protein by introducing an appropriate mutation into the amino acid sequence of the human HELAD1 protein by site-directed mutagenesis (Hashimoto-Gotoh, T (1995), Gene 152: 271-275; Zoller, MJ. And Smith, M. (1983), Methods Enzymol. 100: 468-500; Kramer, W. et al. (1984), Nucleic Acids Res. 12: 9441-9456; Kramer, W. and Fritz HJ. (1987), Methods Enzymol. 154: 350-367; Kunkel, TA. (1985), Proc. Natl. Acad. Sci. USA 82: 488-492; 1988), Methods Enzymol. 85: 2763-2766). Amino acid mutations can occur naturally. The protein of the present invention includes a protein having the amino acid sequence of human HELAD1 protein in which one or more amino acids are mutated under the condition that the obtained mutant protein is functionally equivalent to human HELAD1 protein. The number of amino acids mutated in such variants is generally 10 amino acids or less, preferably 6 amino acids or less, more preferably 3 amino acids or less.

  A mutated or modified protein, a protein having an amino acid sequence modified by deleting, adding, and / or substituting one or more amino acid residues of a specific amino acid sequence, may have an original biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81: 5562-5666; Zoller, MJ and Smith, M., Nucleic Acids Research (1982) 10: 6487). ~ 6500; Wang, A. et al., Science 224: 1431-1433; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79: 6409-6413).

  The amino acid residue to be mutated is preferably mutated to a different amino acid that preserves the properties of the amino acid side chain (a process known as conservative amino acid substitution). Examples of amino acid side chain properties are 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 with the following functional groups or common characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S, T Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); and aromatic-containing side chains (H, F, Y, W). Note that the letters in parentheses indicate the single letter code for amino acids.

  An example of a protein in which one or more amino acid residues are added to the amino acid sequence of human HELAD1 protein (SEQ ID NO: 2 or SEQ ID NO: 4) is a fusion protein comprising human HELAD1 protein. The fusion protein is a fusion of human HELAD1 protein and other peptides or proteins, and these are also included in the present invention. The fusion protein is a technique well known to those skilled in the art, for example, a DNA encoding the human HELAD1 protein of the present invention is ligated to a DNA encoding another peptide or protein so as to match the frame, and the fusion DNA is used as an expression vector. It can be made by inserting and expressing it in a host. There is no restriction on the peptide or protein fused to the protein of the invention.

  Known peptides that can be used as peptides fused to the protein of the present invention include, for example, FLAG (Hopp, TP et al., Biotechnology (1986) 6: 1204-1210), 6 × containing 6 His residues. His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin Fragments, B-tags, protein C fragments and the like are included. Examples of proteins that may be fused to the protein of the present invention include GST (glutathione-S-transferase), influenza agglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein), etc. It is.

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

  Another method known in the art for isolating functionally equivalent proteins is, for example, the hybridization technique (Sambrook, J. et al., Molecular Cloning, 2nd edition, 9.47-9.58, Cold Spring Harbor. This is the method using Institute Publishing, 1989). Those skilled in the art can easily isolate DNA having a high degree of homology with all or part of the HELAD1 DNA sequence encoding human HELAD1 protein (eg, SEQ ID NO: 1 or SEQ ID NO: 3), A protein functionally equivalent to the human HELAD1 protein can be isolated from the isolated DNA. The proteins of the present invention include proteins encoded by DNA that hybridizes with all or part of the DNA sequence encoding human HELAD1 protein, and proteins that are functionally equivalent to human HELAD1 protein. These proteins include mammalian homologues corresponding to proteins from humans or mice (eg, proteins encoded by monkey, rat, rabbit, and bovine genes). It is particularly preferred to use tissue from a fetal brain, fetal kidney, or colorectal tumor when isolating a cDNA very homologous to the DNA encoding the human HELAD1 protein from an animal.

  Hybridization conditions for isolating DNA encoding a protein that is functionally equivalent to the human HELAD1 protein can be routinely selected by those skilled in the art. For example, hybridization can be performed using “Rapid-hyb buffer” (Amersham LIFE SCIENCE) at 68 ° C. for 30 minutes or more, and a labeled probe is added at 68 ° C. You may carry out by heating for 1 hour or more. The subsequent washing step can be performed, for example, under low stringent conditions. Low stringency conditions are, for example, 42 ° C., 2 × SSC, 0.1% SDS, or preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, highly stringent conditions are used. High stringency conditions include, for example, 3 washes for 20 minutes at room temperature in 2 × SSC, 0.01% SDS, 3 washes for 20 minutes at 37 ° C. in 1 × SSC, 0.1% SDS, and 1 × SSC Wash twice for 20 minutes at 50 ° C in 0.1% SDS. However, several factors such as temperature and salt concentration can affect the stringency of hybridization, and those skilled in the art can appropriately select factors to obtain the required stringency.

  Instead of hybridization, it was synthesized based on gene amplification method, for example, based on sequence information of DNA (SEQ ID NO: 1 or SEQ ID NO: 3) encoding human HELAD1 protein (SEQ ID NO: 2 or SEQ ID NO: 4) Using primers, the polymerase chain reaction (PCR) method can be used to isolate DNA encoding a protein functionally equivalent to the human HELAD1 protein.

  A protein functionally equivalent to the human HELAD1 protein encoded by DNA isolated through the above hybridization technique or gene amplification technique usually has high homology with the amino acid sequence of the human HELAD1 protein. “High homology” typically refers to a homology of 40% or higher, preferably 60% or higher, more preferably 80% or higher, even more preferably 95% or higher. means. Protein homology can be determined according to the algorithm of “Wilbur, W.J. and Lipman, D.J., Proc. Natl. Acad. Sci. USA (1983) 80: 726-730”.

  Proteins useful in the context of the present invention vary in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains, or shape depending on the cell or host used to produce it or the purification method utilized. You may have. Nevertheless, so long as it has a function equivalent to that of the human HELAD1 protein of the present invention (SEQ ID NO: 2 or SEQ ID NO: 4), the protein is included in the scope of the present invention.

  Proteins useful in the context of the present invention can be prepared as recombinant or natural proteins by methods well known to those skilled in the art. The recombinant protein is obtained by inserting a DNA encoding the protein of the present invention (for example, a DNA containing the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3) into an appropriate expression vector, and inserting the vector into an appropriate host cell. Introducing, obtaining an extract, and subjecting the extract to chromatography, for example ion exchange chromatography, reverse phase chromatography, gel filtration, or affinity chromatography using a column immobilized with an antibody against the protein of the present invention. Or may be prepared by purifying the protein by combining a plurality of the above columns.

  Similarly, proteins useful in the context of the present invention are expressed in host cells (eg, animal cells and E. coli) as fusion proteins with glutathione-S-transferase proteins or as recombinant proteins with a number of histidines added. If so, the expressed recombinant protein can be purified using a glutathione column or a nickel column.

  After purifying the fusion protein, regions other than the target protein can be removed by cleavage with thrombin or factor Xa as necessary.

  The natural protein is isolated by methods known to those skilled in the art, for example, by contacting an affinity column coupled with an antibody that binds to the HELAD1 protein described below with an extract of a tissue or cell that expresses the protein of the present invention. be able to. The antibody can be a polyclonal antibody or a monoclonal antibody.

  The present invention also includes the use of partial peptides of the proteins of the present invention. The partial peptide has an amino acid sequence unique to the protein of the present invention, and consists of at least 7 amino acids, preferably 8 or more amino acids, more preferably 9 or more amino acids. Partial peptides may be used, for example, to prepare antibodies against the proteins of the invention, to screen for compounds that bind to the proteins of the invention, and to screen for promoters or inhibitors of the proteins of the invention. it can.

  The partial peptide of the present invention can be produced by genetic engineering, a known peptide synthesis method, or digesting the peptide of the present invention with an appropriate peptidase. In the case of peptide synthesis, for example, solid phase synthesis or liquid phase synthesis may be used.

  The isolated HELAD1 protein contains an AAA domain that exhibits 3 ′ to 5 ′ helicase activity and exonuclease activity in vitro. As noted above, helicases are known to have important roles in biological processes such as DNA replication, DNA repair, and transcription. Helicase inactivation is associated with many human disorders; similarly, helicase overexpression is found in certain cancers. Thus, the AAA domain is presumed to be important for the function of HELAD1. Therefore, the partial peptide of HELDA1 preferably contains an AAA domain.

  Furthermore, the present invention provides DNA encoding proteins useful in the context of the present invention. The DNA provided by the present invention can be used to produce the protein of the present invention in vivo or in vitro as described above, or of a disease attributable to a genetic abnormality in the gene encoding the protein of the present invention, It can be applied to gene therapy. Any form of DNA of the present invention can be used as long as it encodes the protein of the present invention. Specifically, cDNA synthesized from mRNA, genomic DNA, and chemically synthesized DNA can be used. The DNA of the present invention includes DNA containing a predetermined nucleotide sequence together with its degenerate sequence, as long as the resulting DNA encodes the protein of the present invention.

  The DNA of the present invention can be prepared by methods known to those skilled in the art. For example, for the DNA of the present invention, a cDNA library is prepared from a cell expressing the protein of the present invention, and a partial sequence of the DNA of the present invention (eg, SEQ ID NO: 1 or SEQ ID NO: 3) is used as a probe. It can be prepared by performing hybridization. The cDNA library can be prepared, for example, by the method described in Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Publishing (1989); or a commercially available cDNA library may be used. The cDNA library also extracts RNA from cells expressing the protein of the present invention, synthesizes oligo DNA based on the sequence of the DNA of the present invention (eg, SEQ ID NO: 1 or SEQ ID NO: 3), and uses the oligo as a primer. It can be prepared by performing PCR and amplifying the cDNA encoding the protein of the invention.

  Furthermore, by sequencing the nucleotides of the obtained cDNA, the translation region encoded by the cDNA can be routinely determined, and the amino acid sequence of the protein of the present invention can be easily obtained. Moreover, genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.

  More specifically, mRNA can first be prepared from a cell, tissue, or organ in which the protein of the invention is expressed (eg, fetal brain, fetal kidney, or colorectal tumor). MRNA can be isolated using known methods; for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry 18: 5294-5299 (1979)), or AGPC method (Chomczynski, P. and Sacchi, N. , Anal. Biochem. 162: 156-159 (1987)). Furthermore, mRNA may be purified from total RNA using an mRNA purification kit (Pharmacia) or the like, or mRNA may be purified directly with a quick prep mRNA purification kit (Pharmacia).

  Next, cDNA is synthesized using the obtained mRNA using reverse transcriptase. cDNA may be synthesized using a commercially available kit such as an AMV reverse transcriptase first strand cDNA synthesis kit (Seikagaku Corporation). Alternatively, the cDNA can be a primer such as those described herein, 5′-Ampli FINDER RACE kit (Clontech), and 5′-RACE method using polymerase chain reaction (PCR) (Frohman, MA et al., Proc Natl. Acad. Sci. USA 85: 8998-9002 (1988); Belyavsky A. et al., Nucleic Acids Res. 17: 2919-2932 (1989)).

  Desired DNA fragments are prepared from PCR products and ligated into vector DNA. E. coli and the like are transformed using the recombinant vector, and a desired recombinant vector is prepared from the selected colonies. The nucleotide sequence of the desired DNA can be confirmed by conventional methods such as the dideoxynucleotide chain termination method.

  The nucleotide sequence of the DNA of the present invention may be designed to be expressed more efficiently taking into account the codon usage in the host used for expression (Grantham R. et al., Nucleic Acids Res. 9: 43-74 (1981)). The DNA of the present invention may be changed by a commercially available kit or a usual method. For example, DNA can be altered by restriction enzyme digestion, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, or insertion of start codons (ATG) and / or stop codons (TAA, TGA, or TAG). Also good.

Specifically, the DNA of the present invention preferably includes DNA comprising a nucleotide sequence encoding the AAA domain of the protein of SEQ ID NO: 2 or the protein of SEQ ID NO: 4. The AAA ('A'TPases'A'ssociated with diverse cellular'A'ctivities) domain is the ATP-binding site of the large ATPase family, which has about 220 amino acids. Share the storage area. In general, the AAA domain has been proposed to act as an ATP-dependent protein clamp (Neuwald AF et al., Genome Res. 9 (1999): 27-43; Lenzen, CU. Et al., Cell 94 (1998): 525). Patel, S. et al., Trends Cell Biol. 8 (1998): 65-71; Confalonieri, F. and Duguet, M. (1995) Bioessays 17 (7): 639-50). The AAA domain of the HELAD1 protein of SEQ ID NO: 2 starts at position 2026 and ends at position 2180 of SEQ ID NO: 2. Similarly, the AAA domain of the HELAD1 protein of SEQ ID NO: 4 begins at position 2090 and ends at position 2244 of SEQ ID NO: 4. AAA domains include, for example, the Simple Modular Architecture Research Tool (SMART) (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95: 5857-5864; Letunic et al. (2002) Nucleic Acids Res. 30: 242-244; Schultz, J. et al. (2000) Nucleic Acids Res. 28: 231-234; Copley, RR et al. (1999) Curr. Opin. Struct. Biol. 9: 408-415; (1999) Nucleic Acids Res. 27: 229-232) or Protein Family Alignment & HMM Database (Pfam) (Bateman, A. et al. (2002) Nucleic Acids Res. 30 (1): 276-280; Bateman, A. et al. ( 2000) Nucleic Acids Res. 28: 263-266; Bateman, A. et al. (1999) Nucleic Acids Research 27: 260-262; Sonnhammer, ELL et al. (1998) Nucleic Acids Research 26: 320-322; Sonnhammer ELL et al. (1997) ) Proteins 28: 405-420). Preferably, SMART software, version 3.4, SMART HMMs number 654, and the sequence database of February 26, 2003 are used. When identified using SMART software, the E-value calculated using a hidden Markov model for the identified AAA domain may be 1 × 10 ⁻² or less, preferably 1 × 10 ⁻³ or less It may be smaller, even more preferably 1 × 10 ⁻⁴ or smaller.

  Furthermore, the present invention hybridizes under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and is functionally equivalent to a protein useful in the context of the present invention as described above. DNA encoding the various proteins is provided. Those skilled in the art may appropriately select stringent conditions. For example, low stringency conditions can be used. More preferably, highly stringent conditions can be used. These conditions are the same as the above conditions. The hybridizing DNA is preferably cDNA or chromosomal DNA.

Vectors and host cells The present invention also provides vectors into which the DNA of the present invention has been inserted. The vector of the present invention is useful for maintaining the DNA of the present invention in a host cell or for expressing the protein of the present invention. If E. coli is the host cell and the vector is amplified and produced in large quantities in E. coli (eg, JM109, DH5α, HB101, or XL1Blue), the vector is “ori” to be amplified in E. coli, and transformation Must have a marker gene (for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol, etc.) for selecting the selected E. coli. For example, M13-series vector, pUC series vector, pBR322, pBluescript, pCR-Script, etc. can be used. Furthermore, pGEM-T, pDIRECT, and pT7 can also be used for subcloning and extracting cDNA in the same manner as in the above vector. Expression vectors are particularly useful when vectors are used to produce proteins useful in the context of the present invention. For example, an expression vector expressed in E. coli must have the above characteristics in order to be amplified in E. coli. When E. coli such as JM109, DH5α, HB101, or XL1 Blue is used as a host cell, the vector can be a promoter that can efficiently express a desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341). : 544-546; FASEB J (1992) 6: 2422-2427), araB promoter (Better et al., Science (1988) 240: 1041-1043), T7 promoter or the like. In that aspect, for example, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP and pET (where the host is preferably BL21 expressing T7 RNA polymerase) instead of the above vector Can be used.

  In addition, the vector may also include a signal sequence for polypeptide secretion. An exemplary signal sequence that directs the protein to be secreted into the E. coli periplasm is the pelB signal sequence (Lei, S.P. et al., J. Bacteriol. (1987) 169: 4379). Means for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.

  In order to produce the protein of the present invention, in addition to E. coli, for example, expression vectors derived from mammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res. 1990, 18 (17): 5322), pEF, pCDM18), expression vectors derived from insect cells (eg, “Bac-to-BAC baculovirus expression system” (GIBCO BRL, pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2) Expression vectors derived from animal viruses (eg, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (eg, pZIpneo), expression vectors derived from yeast (eg, “Pichia expression kit” (Invitrogen), pNV11) , SP-Q01), and expression vectors derived from Bacillus subtilis (eg, pPL608, pKTH50) can be used.

  In order to express a vector in animal cells such as CHO, COS, or NIH3T3 cells, the vector may be a promoter required for expression in such cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277: 108. ), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18: 5322), CMV promoter and the like, and preferably a marker gene for selecting a transformant (eg selected by a drug) Must have a drug resistance gene (eg, neomycin, G418). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

  Furthermore, a method of stably expressing a gene and simultaneously amplifying the copy number of the gene in a cell may be used. For example, a vector containing a complementary DHFR gene (eg, pCHO I) may be introduced into CHO cells lacking the nucleic acid synthesis pathway and amplified with methotrexate (MTX). Furthermore, in the case of transient expression of a gene, a method of transforming a vector (such as pcD) containing an SV40 replication origin into COS cells containing an SV40 T antigen-expressing gene on the chromosome can be used.

  Proteins useful in the context of the present invention obtained as described above may be isolated from inside or outside the host cell (such as media) and purified as a substantially pure and homogeneous protein. The term “substantially pure” as used herein with reference to a given polypeptide means that the polypeptide is substantially free of other biopolymers. A substantially pure polypeptide is at least 75% (eg, at least 80%, 85%, 95%, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Protein isolation and purification methods are not limited to any particular method, and in practice any standard method may be used.

  For example, column chromatography, filtration, ultrafiltration, salting out precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization are appropriately selected. In combination, the proteins may be isolated and purified.

  Examples of chromatography include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R Included in Marshak et al., Cold Spring Harbor Laboratory Publishing (1996). These chromatographies may be performed by liquid chromatography such as HPLC and FPLC. Thus, the present invention provides a highly purified protein prepared by the above method.

  Proteins useful in the context of the present invention may be selectively altered or partially deleted by treatment with appropriate protein modifying enzymes before and after purification. Useful protein modifying enzymes include, but are not limited to, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like.

Antibodies, Vaccines, and Methods for Inducing Anti-Tumor Immunity The present invention further provides antibodies that bind to the proteins of the present invention. The antibody of the present invention can be used in any form such as a monoclonal antibody or a polyclonal antibody, and antiserum obtained by immunizing an animal such as a rabbit with the protein of the present invention, all classes of polyclonal antibodies and monoclonal antibodies. Antibodies, human antibodies, and humanized antibodies produced by genetic recombination are included.

  The protein of the present invention used as an antigen for obtaining 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 a human. Human derived proteins may be obtained from the nucleotide or amino acid sequences disclosed herein.

  In accordance with the present invention, a protein used as an immunizing antigen may be a complete protein or a partial peptide of the protein. The partial peptide may comprise, for example, an amino (N) -terminal or carboxy (C) -terminal fragment of the protein of the invention. As used herein, an antibody is defined as a protein that reacts with either the full length or a fragment of a protein of the invention.

  A gene encoding a protein of the invention or fragment thereof may be inserted into a known expression vector, which is then used to transform a host cell as described herein. The desired protein or fragment thereof may be recovered from the exterior or interior of the host cell by any standard method and then used as an antigen. Alternatively, whole cells expressing the protein or a lysate thereof, or a chemically synthesized protein may be used as the antigen.

  Any mammal can be immunized with the antigen, but preferably takes into account compatibility with the parental cell used for cell fusion. Generally, rodents, rabbits, or primate animals are used.

  Rodent animals include, for example, mice, rats, and hamsters. Rabbit eyes include, for example, rabbits. Primate animals include, for example, monkeys (Old World monkeys) such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys, baboons, and chimpanzees.

  Methods for immunizing animals with antigens are known in the art. Intraperitoneal or subcutaneous injection of antigen is a standard method for immunizing 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 may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, to form an emulsion, which may be administered to a mammal. Preferably, this is followed by several doses of antigen mixed with an appropriate amount of Freund's incomplete adjuvant every 4-21 days. A suitable carrier may be used for immunization as well. After immunization as described above, sera are examined for an increase in the amount of antibody desired by standard methods.

  Polyclonal antibodies against the proteins of the present invention may be prepared by collecting blood from immunized mammals that have been examined for an increase in the desired antibody in the serum, and separating the serum from the blood by any conventional method. Good. Polyclonal antibodies include serum containing polyclonal antibodies, and fractions containing polyclonal antibodies may be isolated from the serum. Immunoglobulin G or M can be obtained by further purifying this fraction from a fraction that recognizes only the protein of the present invention using, for example, an affinity column to which the protein of the present invention is bound and using a protein A or protein G column. Can be prepared.

  To prepare a monoclonal antibody, immune cells are collected from a mammal immunized with an antigen, and cell fusion is performed by confirming an increase in the level of the desired antibody in the serum as described above. The immune cells used for cell fusion are preferably obtained from the spleen. Other preferred parental cells that are fused with the above immune cells include, for example, mammalian myeloma cells, and more preferably myeloma cells that have acquired properties for selecting fused cells by drug.

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

  Hybridomas obtained by cell fusion can be selected by culturing them in a standard selection medium such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). Cell culture is typically continued in HAT medium for days to weeks for a period sufficient to kill all other cells (non-fused cells) except the desired hybridoma. Next, standard limiting dilution methods are performed to screen and clone hybridoma cells producing the desired antibody.

  In addition to the above methods of immunizing non-human animals with antigens to prepare hybridomas, human lymphocytes such as lymphocytes infected with EB virus are immunized in vitro with proteins, protein-expressing cells, or lysates thereof. May be. The immunized lymphocytes are then fused with human-derived myeloma cells that can divide indefinitely, such as U266, to form hybridomas that produce the desired human antibody that can bind to the protein (specifically Kaisho 63-17688).

  Next, the obtained hybridoma is transplanted into the abdominal cavity of a mouse to extract ascites. The resulting monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the protein of the present invention is bound. The antibody of the present invention can be used not only for purifying and detecting the protein of the present invention but also as a candidate for antagonist of the protein of the present invention. Furthermore, this antibody can be applied to antibody therapy for diseases associated with the protein of the present invention. When the obtained antibody is administered into a human body (antibody treatment), a human antibody or a humanized antibody is preferred in order to reduce immunogenicity.

  For example, a transgenic animal having a repertoire of human antibody genes may be immunized with an antigen selected from proteins, protein expressing cells, or lysates thereof. Next, antibody-producing cells can be recovered from the animal and fused with myeloma cells to obtain hybridomas, from which human antibodies against the protein can be prepared (WO 92-03918, WO No. 93-2227, International Publication No. 94-02602, International Publication No. 94-25585, International Publication No. 96-33735, and International Publication No. 96-34096).

  Alternatively, immune cells that produce antibodies, such as immunized lymphocytes, may be immortalized by oncogenes and used to prepare monoclonal antibodies.

  The monoclonal antibodies thus obtained can also be prepared recombinantly using genetic engineering techniques (eg Borrebaeck, CAK, published in the United Kingdom (1990) by MacMillan Publishers LTD). And Larrick, JW, Therapeutic Monoclonal Antibodies). For example, DNA encoding an antibody can be cloned from an immune cell such as an antibody-producing hybridoma or immune lymphocyte, inserted into an appropriate vector, and introduced into a host cell to prepare a recombinant antibody. Good. The present invention also provides a recombinant antibody prepared as described above.

Furthermore, the antibody of the present invention may be an antibody or a fragment of a modified antibody as long as it binds to one or more of the proteins of the present invention. For example, an antibody fragment may be Fab, F (ab ′) ₂ , Fv, or a single chain Fv (scFv) in which Fv fragments from H and L chains are linked by a suitable linker (Huston , JS et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). More specifically, antibody fragments may be generated by treating an antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding the antibody fragment may be constructed, inserted into an expression vector, and expressed in a suitable host cell (eg, Co, MS et al., J. Immunol. 152: 2968-2976 (1994); Better, M. and Horwitz, AH, Methods Enzymol. 178: 476-496 (1989); Pluckthun, A. and Skerra, A., Methods Enzymol. 178: 497-515 (1989); Lamoyi, E., Methods Enzymol 121: 652-663 (1986); Rousseaux, J. et al., Methods Enzymol. 121: 663-669 (1986); Bird, RE and Walker, BW, Trends Biotechnol. 9: 132-137 (1991). Wanna)

  The antibody may be modified by conjugation with a variety of molecules such as polyethylene glycol (PEG). The present invention provides such modified antibodies. A modified antibody can be obtained by chemically modifying an antibody. These modification methods are common 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 a complementarity determining region derived from a non-human antibody. (CDR), a humanized antibody comprising a framework region (FR) derived from a human antibody, and a constant region. Such antibodies can be prepared using known techniques.

  The antibody obtained as described above can be purified to homogeneity. For example, antibody separation and purification can be performed according to separation and purification methods used for general proteins. For example, an antibody is appropriately selected from column chromatography such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. (but not limited thereto). May be separated and isolated by use in combination (Antibodies: A Laboratory Manual, edited by Harlow and David Lane, Cold Spring Harbor Laboratory, 1988).

  Protein A columns and protein G columns can be used as affinity columns. Protein A columns used as examples include, for example, Hyper D, POROS, and Sepharose F.F. (Pharmacia).

  Examples of chromatography excluding affinity chromatography include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory (1996)). Chromatographic techniques can be performed by liquid phase chromatography such as HPLC, FPLC.

  For example, the antigen binding activity of the antibodies of the present invention can be measured using absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence. Also good. In ELISA, the antibody of the present invention is immobilized on a plate, the protein of the present invention is added to the plate, and then a sample containing a desired antibody such as a culture supernatant of antibody-producing cells or a purified antibody is added. Next, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme such as alkaline phosphatase is added 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. Protein fragments such as C-terminal fragments or N-terminal fragments may be used as proteins. BIAcore (Pharmacia) may be used to assess the activity of the antibody according to the invention.

  The above method comprises exposing the antibody of the present invention to a sample presumed to contain the protein of the present invention, and detecting or measuring an immune complex formed by the antibody and the protein, thereby Can be detected or measured.

  Since the method for detecting or measuring a protein according to the present invention can specifically detect or measure a protein, this method may be useful in various experiments in which the protein is used.

The invention also relates to a method of inducing anti-tumor immunity comprising administering a HELAD1 protein or an immunologically active fragment thereof, or a nucleic acid encoding any one of the protein and fragments thereof. The HELAD1 protein or immunologically active fragment thereof is useful as a vaccine against colon cancer. In the present invention, a vaccine against cancer means a substance having an effect of inducing antitumor immunity when it is inoculated into an animal. In general, anti-tumor immunity includes immune responses such as:
-Induction of cytotoxic lymphocytes against the tumor,
-Induction of antibodies recognizing tumors, and-induction of anti-tumor cytokine production.

  Thus, when inoculating an animal with a particular protein induces any one of these immune responses, the protein is said to have an anti-tumor immunity inducing effect. Induction of anti-tumor immunity by a protein can be detected by observing the response of the host immune system to the protein in vivo or in vitro.

  For example, methods for detecting the induction of cytotoxic T lymphocytes are well known. Foreign substances entering the living body are presented to T cells and B cells by the action of antigen presenting cells (APC). T cells that respond antigen-specifically to the antigen presented by APC differentiate into cytotoxic T cells (or cytotoxic T lymphocytes; CTLs) for stimulation by the antigen and then proliferate (this is T Called cell activation). Thus, CTL induction by a particular peptide can be assessed by presentation of the peptide to T cells by APC and detection of CTL induction. In addition, APC has the effect of activating CD4 + T cells such as macrophages, eosinophils, and NK cells. Since CD4 + T cells are also important in antitumor immunity, antitumor immunity that induces the action of peptides can be evaluated using the activation effect of these cells as an indicator.

For example, a method for evaluating the inducing action of CTL using dendritic cells (DC) as APC is well known. DC is a representative APC having the strongest CTL inducing action. In this method, the test polypeptide is first contacted with DC, which is then contacted with T cells. If a T cell having a cytotoxic effect on a cell of interest after contact with DC is detected, it indicates that the test polypeptide has activity to induce cytotoxic T cells. CTL activity against tumors can be detected, for example, using lysis of ⁵¹ Cr-labeled tumor cells as an indicator. Alternatively, a method for evaluating the degree of tumor cell damage using ³ H-thymidine uptake activity or LDH (lactate dehydrogenase) release as an index is also well known.

  APC is not limited to DC, and peripheral blood mononuclear cells (PBMC) may be used. In this case, it has been reported that CTL induction can be 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 confirmed to have CTL inducing activity by these methods is a polypeptide having a DC activation effect and subsequent CTL inducing activity. Therefore, polypeptides that induce CTL against tumor cells are useful as vaccines against tumors. Furthermore, APCs that have acquired the ability to induce CTLs against tumors by contacting with polypeptides are useful as vaccines against tumors. Furthermore, CTLs that have acquired cytotoxicity due to presentation of polypeptide antigens by APC can also be used as vaccines against tumors. Such a method for treating tumors using anti-tumor immunity with APC and CTL is called cellular immunotherapy.

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

  Alternatively, the induction of anti-tumor immunity by a polypeptide can be confirmed by observing the induction of antibody production against the tumor. For example, if an antibody against the polypeptide is induced in a laboratory animal immunized with the polypeptide and the growth of tumor cells is suppressed by the antibody, the polypeptide has the ability to induce anti-tumor immunity.

  Anti-tumor immunity is induced by administering the vaccine of the present invention, which enables the treatment and prevention of colon cancer. The effect of treating cancer or preventing the onset of cancer may be in any one of the following stages, for example, inhibitory activity on proliferation of cancerous cells, regression of cancer, and suppression of cancer development. Otherwise, it may be a decrease in mortality of individuals with cancer, a decrease in blood tumor markers, a reduction in detectable symptoms associated with cancer, and the like. Such an effect is preferably statistically significant, for example, a finding at a significance level of 5% or less of a therapeutic effect on colon cancer or a preventive effect on developing cancer compared to a non-vaccine administered control is preferred. . For example, Student's t-test, Mann-Whitney U-test, or ANOVA may be used for statistical analysis.

  The above-mentioned protein having immunological activity or a vector encoding the protein may be combined with an adjuvant. By adjuvant is meant a compound that enhances the immune response to the protein when administered together (or sequentially) with the protein having immune activity. Examples of adjuvants include, but are not limited to, cholera toxin, salmonella toxin, alum and the like. Furthermore, the vaccine of the present invention may be appropriately combined with a pharmaceutically acceptable carrier. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture solution and the like. Furthermore, you may contain a stabilizer, a suspension agent, a preservative, surfactant, etc. as needed. The vaccine is administered systemically or locally. The administration of the vaccine may be a single dose or may be enhanced by multiple doses.

  When APC or CTL is used as the vaccine of the present invention, the tumor can be treated or prevented, for example, by the ex vivo method. More specifically, after the PBMC of a subject to be treated or prevented is collected, the cells are contacted with the polypeptide ex vivo to induce APC or CTL, and then the cells can be administered to the subject. APC can also be induced by introducing a vector encoding the polypeptide into PBMCs ex vivo. In vitro induced APCs or CTLs can be cloned prior to administration. Cell immunotherapy can be performed more effectively by cloning and proliferating cells with high activity that damage target cells. In addition, APCs and CTLs isolated in this way may be used for cellular immunotherapy against similar types of tumors from other individuals as well as individuals from which the cells are derived.

Polynucleotides and Oligonucleotides The present invention also provides polynucleotides that hybridize to DNA encoding human HELAD1 protein (SEQ ID NO: 1 or SEQ ID NO: 3) or its complementary strand, and comprising at least 15 nucleotides. The polynucleotide of the present invention is preferably a polynucleotide that specifically hybridizes with DNA encoding the protein of the present invention. As used herein, the term “specifically hybridizes” refers to cross-hybridization with DNA encoding other proteins under normal hybridization conditions, preferably under stringent hybridization conditions. Means it doesn't happen significantly. Such polynucleotides include probes, primers, nucleotides, and nucleotide derivatives (eg, antisense oligonucleotides and ribozymes) that specifically hybridize to DNA encoding the protein of the invention or its complementary strand. . Moreover, such polynucleotides can be utilized to prepare DNA chips.

  The present invention includes antisense oligonucleotides that hybridize to any site within the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The antisense oligonucleotide is preferably against at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Even more preferred are the above antisense oligonucleotides comprising an initiation codon in at least 15 consecutive nucleotides.

  An example of an antisense molecule of the invention is a molecule having at least 15 contiguous nucleotides of SEQ ID NO: 20. Additional examples are described in the examples below and include, but are not limited to, the oligonucleotides set forth in SEQ ID NOs: 16 and 17.

  Antisense oligonucleotide derivatives or modified products can be used as antisense oligonucleotides. Examples of such modified products include lower alkyl phosphonate modifications such as methyl-phosphonate or ethyl-phosphonate types, phosphorothioate modifications, and phosphoramidate modifications.

  As used herein, the term “antisense oligonucleotide” means that DNA or mRNA and antisense oligonucleotides can specifically hybridize to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. It means not only an oligonucleotide in which nucleotides corresponding to nucleotides constituting a specific region of DNA or mRNA are completely complementary, but also an oligonucleotide having one or more nucleotide mismatches.

  Such polynucleotides are at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, in a “minimum 15 contiguous nucleotide sequence region”. Are included as polynucleotides having the homology of Using the algorithms described herein, homology can be determined. Such polynucleotides are useful as probes for isolating or detecting DNA encoding the proteins of the invention described in the Examples below, or as primers used to amplify.

  The antisense oligonucleotide derivative of the present invention binds to DNA or mRNA encoding a protein, inhibits its transcription or translation, promotes the degradation of mRNA, and inhibits the expression of the protein of the present invention. By acting on the cells producing the protein of the present invention.

  The antisense oligonucleotide derivatives of the present invention can be processed into topical preparations such as liniments or poultices by mixing with a suitable base material that is inert to the derivatives.

  Similarly, by adding excipients, isotonic agents, solubilizers, stabilizers, preservatives, analgesics, etc. as necessary, the derivatives are converted into tablets, powders, granules, capsules, liposome capsules, injections. It can be formulated into agents, solutions, nasal drops, and lyophilized drugs. These can be prepared according to the following conventional methods.

  The antisense oligonucleotide derivative is given to the patient by applying it directly on the affected area or by injecting it into the blood vessel so that it reaches the affected area. Antisense-mounting medium can also be used to increase durability and membrane permeability. Examples are liposomes, poly-L-lysine, lipids, cholesterol, lipofectin, or derivatives thereof.

  The dosage of the antisense oligonucleotide derivative of the present invention can be adjusted appropriately according to the patient's condition and used in desired amounts. For example, a dose range of 0.1-100 mg / kg, preferably 0.1-50 mg / kg can be administered.

  The antisense oligonucleotides of the invention are useful for inhibiting the expression of the protein of the invention and thus suppressing the biological activity of the protein of the invention. Similarly, an expression inhibitor comprising the antisense oligonucleotide of the present invention is useful in that it can inhibit the biological activity of the protein of the present invention.

Screening Method Furthermore, the present invention provides a method for screening a compound that binds to the protein of the present invention by using the protein of the present invention. This screening method includes the following steps: (a) contacting the test sample with the protein of the present invention or a partial peptide thereof; (b) detecting the binding activity between the test protein or the protein of the present invention or a partial peptide thereof; And (c) selecting a compound that binds to the protein of the present invention or a partial peptide thereof.

  The protein of the present invention used for screening may be a recombinant protein or a protein derived from nature, or a partial peptide thereof. Any test sample, such as cell extract, cell culture supernatant, fermented microbial product, extract from marine organisms, plant extract, purified or crude protein, peptide, non-peptide compound, synthetic micromolecular compound and natural compound Can be used. The protein of the present invention to be contacted with the test sample can be, for example, a purified protein, a soluble protein, a form bound to a carrier, or a fusion protein fused to another protein.

  For example, as a method for screening a protein that binds to the protein of the present invention using the protein of the present invention, many methods well known to those skilled in the art can be used. Such screening can be performed specifically by the immunoprecipitation method, for example, as follows. A gene encoding the protein of the present invention is expressed in an animal cell or the like by inserting the gene into an expression vector for a foreign gene such as pSV2neo, pcDNA I, and pCD8. The promoter used for expression may be any commonly used promoter, such as the SV40 early promoter (Rigby (Williamson), Genetic Engineering Vol. 3, Academic Publishing, London). 83-141 (1982)), EF-1α promoter (Kim et al., Gene 91 217-223 (1990)), CAG promoter (Niwa et al., Gene 108: 193-200 (1991)), RSV LTR promoter (Cullen Methods in Enzymology 152: 684-704 (1987)), SRα promoter (Takebe et al., Mol. Cell. Biol. 8: 466 (1988)), CMV immediate early promoter (Seed and Aruffo, Proc. Natl. Acad. Sci. USA) 84: 3365-3369 (1987)), SV40 late promoter (Gheysen and Fiers, J. Mol. Appl. Genet. 1: 385-394 (1982)), adenovirus late promoter (Kaufman et al., Mol. Cell. Biol. 9: 946 (1989)), including HSV TK promoter . Introduction of a gene into an animal cell for expressing a foreign gene can be performed by, for example, electroporation (Chu G. et al., Nucl. Acids. Res. 15: 1311-1326 (1987)), calcium phosphate method (Chen, C. And Okayama, H., Mol. Cell. Biol. 7: 2745-2852 (1987)), DEAE dextran method (Lopata, MA et al. Nucl. Acids. Res. 12: 5707-5717 (1984); Sussman, DJ and Milman, G., Mol. Cell. Biol. 4: 1642-1643 (1985)), lipofectin method (Derijard, B., Cell 7: 1025-1037 (1994); Lamb, BT et al., Nature Genetics 5: 22- 30 (1993); Rabindran, SK et al., Science 259: 230-234 (1993)) and the like. The protein of the present invention is a fusion protein containing a monoclonal antibody recognition site (epitope) by introducing an epitope of a monoclonal antibody whose specificity is known to the N-terminus or C-terminus of the protein of the present invention. Can be expressed. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). For example, vectors that can express a fusion protein with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP), etc. by using a multicloning site are commercially available.

  A fusion protein prepared by introducing a very small epitope consisting of several to 12 amino acids so that the properties of the protein of the present invention are not changed by fusion has also been reported. Polyhistidine (His tag), influenza agglutinin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV- Tags), E-tags (epitopes on monoclonal phages), etc., and monoclonal antibodies that recognize them can be used as epitope-antibody systems for screening proteins that bind to the proteins of the present invention. (Experimental Medicine 13: 85-90 (1995)).

  In immunoprecipitation, immune complexes are formed by adding these antibodies to cell lysates prepared with an appropriate detergent. The immune complex consists of the protein of the present invention, a protein capable of binding to the protein, and an antibody. Immunoprecipitation can be performed by using an antibody against the protein of the present invention in addition to using an antibody against the above-described epitope. The antibody against the protein of the present invention can be obtained by, for example, introducing a gene encoding the protein of the present invention into a suitable E. coli vector, expressing the gene in E. coli, purifying the expressed protein, and rabbit, mouse, rat, goat. It can be prepared by immunizing poultry against proteins. Antibodies can also be prepared by immunizing the above animals against a synthetic partial peptide of the protein of the invention.

  The immune complex can be precipitated by, for example, protein A sepharose or protein G sepharose when the antibody is a mouse IgG antibody. When the protein of the present invention is prepared as a fusion protein with an epitope such as GST, the immune complex can be prepared by using a substance that specifically binds to these epitopes such as glutathione sepharose 4B. It can be formed in the same manner as when an antibody against is used.

  Immunoprecipitation can be performed, for example, by or according to methods described in the literature (Harlow, E. and Lane, D .: Antibodies, 511-552, Cold Spring Harbor Laboratory Publishing, New York (1988)). .

SDS-PAGE is commonly used to analyze immunoprecipitated proteins, and bound proteins can be analyzed by protein molecular weight using an appropriate concentration of gel. Since the protein bound to the protein of the present invention is difficult to detect by a general staining method such as Coomassie staining or silver staining, the detection sensitivity of the protein is determined by radioisotope ³⁵ S-methionine or ³⁵ S-cysteine. It can be improved by culturing the cells in a culture medium containing, labeling the protein in the cells and detecting the protein. If the molecular weight of the protein is known, the target protein can be purified directly from an SDS-polyacrylamide gel and its sequence determined.

  As a method for isolating a protein that binds to the protein of the present invention using the protein of the present invention, for example, West-Western blot analysis (Skolnik, EY et al., Cell (1991) 65: 83-90) can be used. . Specifically, the protein that binds to the protein of the present invention is a cell, tissue, organ (for example, ovary) that is expected to express the protein that binds to the protein of the present invention by using a phage vector (for example, ZAP). , Tissue such as testis and placenta, or cultured cells), expressing the protein on LB-agarose, immobilizing the expressed protein on the filter, and purifying the purified labeled protein of the present invention It can be obtained by reacting with the filter described above and detecting plaques expressing the protein bound to the protein of the invention according to the label. The protein of the present invention utilizes an antibody that specifically binds to the protein of the present invention, or a peptide or polypeptide (eg, GST) fused to the protein of the present invention by utilizing the binding between biotin and avidin. Can be labeled. A method using a radioisotope or fluorescence may also be used.

  Alternatively, in another embodiment of the screening method of the present invention, a cell-based 2-hybrid system may be used (“MATCHMAKER 2-hybrid system”, “mammalian MATCHMAKER 2-hybrid assay kit”, “MATCHMAKER 1- Hybrid system "(Clontech);" HybriZAP 2-hybrid vector system (Stratagene) "; References" Dalton, S. and Treisman, R. (1992) Cell 68: 597-612 "," Fields, S " And Sternglanz R., Trends Genet. (1994) 10: 286-292 ").

  In the two-hybrid system, the protein of the present invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells that, when expressed, are expected to express a protein that binds to a protein of the invention such that the library is fused to a VP16 or GAL4 transcriptional activation region. Next, the cDNA library is introduced into the above yeast cells, and the cDNA derived from the library is isolated from the detected positive clones (when a protein that binds to the protein of the present invention is expressed in yeast cells, The reporter gene is activated by the binding of, and positive clones can be detected). The protein encoded by the cDNA can be prepared by introducing the cDNA isolated above into E. coli and expressing the protein.

  In addition to the HIS3 gene, for example, Ade2 gene, LacZ gene, CAT gene, luciferase gene and the like can be used as a reporter gene.

  Compounds that bind to the protein of the invention can be screened using affinity chromatography. For example, the protein of the present invention is immobilized on a carrier of an affinity column, and a test sample containing a protein capable of binding to the protein of the present invention that is expected to be expressed is applied to the column. The test sample described herein may be, for example, a cell extract, a cell lysate, or the like. After loading the test sample, the column can be washed to prepare the protein bound to the protein of the present invention.

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

  A biosensor using the surface plasmon resonance phenomenon may be used as a means for detecting or quantifying the binding compound in the present invention. When such a biosensor is used, the interaction between the protein of the present invention and the test compound can be observed in real time as a surface plasmon resonance signal using only a very small amount of protein and without labeling ( (For example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the protein of the present invention and the test compound using a biosensor such as a BIA core.

  Methods for screening for molecules that bind when the immobilized proteins of the present invention are exposed to synthetic chemical compounds, natural substance banks, or random phage peptide display libraries, or proteins of the present invention (agonists and antagonists) High-throughput screening methods based on combinatorial chemistry techniques (Wrighton Nc, Farrel FX, Chang R, Kashyap AK, Barbone FP, Mulcahy LS, Johnson DL, Barret RW) , Jolliffe LK, Dower WJ; Small peptides as potent mimetics of the protein hormone erythropoietin, Science (USA) July 26th 1996, 273: p458-64; Verdine GL., The combinatorial chemistry of nature, Nature (UK) 1996 November 7, 384: p11-13; Hogan JC Jr., Directed combinatorial chemistry, Nature (UK) November 7, 1996, 384: p17-9) It is well known.

  A compound isolated by screening is a candidate for a drug that promotes or inhibits the activity of the protein of the present invention in order to treat or prevent a disease caused by a disease such as cancer. A compound obtained by the screening method of the present invention, in which a part of the structure of the compound capable of binding to the protein of the present invention has been converted by addition, deletion, and / or substitution, can also be obtained by the screening method of the present invention. Included in the resulting compound.

  Moreover, the present invention provides a method for screening for compounds that promote or inhibit the activity of the protein of the present invention. Since the HELAD1 protein of the present invention has nuclease, helicase, and apoptosis inhibitory activity, a compound that promotes or inhibits any one of these activities of the HELAD1 protein of the present invention is screened using this activity as an index. be able to.

  One embodiment of the screening method includes the following steps: (a) contacting HELAD1 protein or a partial peptide thereof with DNA in the presence of a test sample; (b) detecting DNA degradation; And (c) selecting a compound that promotes or inhibits degradation as compared to degradation detected in the absence of the test sample.

  Another embodiment of the screening method includes the following steps: (a) contacting HELAD1 protein or a partial peptide thereof with double-stranded DNA in the presence of a test sample; (b) unwinding DNA And (c) selecting a compound that promotes or inhibits unwinding as compared to unwinding detected in the absence of the test sample.

  Further embodiments of the screening method include the following steps: (a) culturing cells expressing HELAD1 protein or a partial peptide thereof in the presence of a test sample, (b) detecting apoptosis of the cells, And (c) selecting a compound that promotes or inhibits apoptosis as compared to apoptosis detected in the absence of the test sample.

  Compounds that inhibit the expression and / or activity of HELAD1 are useful as anticancer agents.

  Any HELAD1 protein can be used for screening as long as they have at least one activity selected from the group consisting of helicase, nuclease, and apoptosis inhibitory activity. For example, human HELAD1 protein can be used, and proteins functionally equivalent to these proteins can also be used. HELAD1 protein may be expressed endogenously or exogenously by the cell.

  Use any test sample, such as cell extract, cell culture supernatant, fermented microbial product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptide compound, synthetic micromolecular compound, natural compound be able to. A compound obtained by the above screening for a compound that binds to the protein of the present invention can also be used as a test compound.

  The compound isolated by this screening is a candidate agonist or antagonist of the protein of the present invention. The term “agonist” refers to a molecule that activates its function by binding to a protein of the invention. Similarly, the term “antagonist” means a molecule that inhibits its function by binding to a protein of the invention. Moreover, the compounds isolated by this screening are candidates for compounds that inhibit in vivo interactions between the proteins of the invention and molecules (including DNA and proteins).

  Nuclease activity can be detected by assaying the degradation of nucleic acids such as DNA. Helicase activity can be detected, for example, by assaying for double-stranded DNA unwinding, as described in the Examples below. Apoptosis-inhibiting or inducing activity can be detected by assaying for cell death, for example, as described in the Examples below, and comparing the measured cell death to the cell death that occurred in the control group.

  Compounds isolated by screening are drug candidates that inhibit the activity of the protein of the present invention and can be applied in the treatment of diseases associated with the protein of the present invention, such as cancer, and more particularly colorectal cancer.

  In addition, compounds in which a part of the structure of the compound that inhibits the activity of HELAD1 protein is converted by addition, deletion, and / or substitution are also included in the compounds that can be obtained by the screening method of the present invention. It is.

Pharmaceutical Formulations Compounds isolated by the methods of the present invention for humans and other mammals such as mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows, monkeys, baboons, chimpanzees The isolated compounds can be administered directly or can be formulated into dosage forms using known pharmaceutical preparation methods. For example, if desired, the drug is administered orally as sugar-coated tablets, capsules, elixirs and microcapsules, or injection of a sterile solution or suspension in water or any other pharmaceutically acceptable liquid It can be administered parenterally in dosage form. For example, the compound may be a pharmacologically acceptable carrier or vehicle, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient. , Solvents, preservatives, binders, etc., can be mixed in unit doses generally required for acceptable drug administration. The amount of active ingredient in these preparations makes it possible to obtain a suitable dose within the indicated range.

  Examples of additives that can be mixed into tablets and capsules are binders such as gelatin, corn starch, gum tragacanth and gum arabic; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin, and alginic acid Lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; flavors such as peppermint, Gaultheria adenothrix oil and cherries. Where the unit dosage form is a capsule, a liquid carrier such as oil can also be included in the above ingredients as well. Sterile injectable composites can be formulated according to conventional drug implementation using a solvent such as distilled water for injection.

  Physiological saline, glucose, and other isotonic solutions containing adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as an aqueous solution for injection. These should be used with suitable solubilizers such as alcohols, specifically polyhydric alcohols such as ethanol, propylene glycol and polyethylene glycol, nonionic surfactants such as Polysorbate 80 ™ and HCO-50 Can do.

  Sesame oil or soybean oil can be used as an oily liquid, may be used with benzyl benzoate or benzyl alcohol as a solubilizing agent, and buffering agents such as phosphate buffer and sodium acetate buffer; such as procaine hydrochloride It may be formulated with analgesics; stabilizers such as benzyl alcohol, phenol; and antioxidants. The prepared injection may be filled into a suitable ampoule.

  Well known to those skilled in the art to administer the pharmaceutical compounds of the invention to patients, for example as intraarterial injection, intravenous injection, subcutaneous injection, as well as intranasal, transbronchial, intramuscular, or oral administration. The method may be used. The dose and method of administration will vary according to the weight and age of the patient and the method of administration. However, those skilled in the art can routinely select them. If the compound can be encoded by DNA, the DNA can be inserted into a gene therapy vector and administered to administer the vector. The dose and administration method vary depending on the patient's weight, age, and symptoms, and those skilled in the art can appropriately select them.

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

  When administered parenterally in the form of injections to normal adults (body weight 60 kg), there are some differences depending on the patient, target organ, symptoms, and method of administration, but about 0.01 mg to about 30 mg / day, preferably about It is convenient to administer a dose of 0.1 mg to about 20 mg / day, more preferably about 0.1 mg to about 10 mg / day intravenously. Similarly, in the case of other animals, it is possible to administer an amount converted to a body weight of 60 kg.

Diagnostic Method The present invention further provides a method of diagnosing cancer using HELAD1 protein as a diagnostic marker. This diagnostic method includes the following steps:
(A) measuring the expression level of a gene encoding the protein of the present invention in a biological sample of the specimen;
(B) comparing the expression level of the gene with the expression level in a normal sample, and (c) defining the cancer as having a higher expression level of the HELAD1 gene in the sample.

  The expression level of the HELAD1 gene in a specific specimen can be estimated by quantifying the mRNA corresponding to the HELAD1 gene or the protein encoded by the HELAD1 gene. Quantification methods for mRNA are known to those skilled in the art. For example, the level of mRNA corresponding to the HELAD1 gene can be estimated by Northern blotting or RT-PCR. Since the nucleotide sequences of the HELAD1 gene are all shown in SEQ ID NO: 1 and SEQ ID NO: 3, those skilled in the art can design a nucleotide sequence for a probe or primer for quantifying the HELAD1 gene.

  Similarly, the expression level of the HELAD1 gene can be analyzed based on the activity or amount of the protein encoded by the HELAD1 gene. A method for determining the amount of HELAD1 protein is shown below. For example, immunoassay methods are useful for quantifying proteins in biological materials. Any biological material can be used to quantify the protein or its activity. For example, colon tissue is analyzed to estimate the protein encoded by the HELAD1 gene. On the other hand, a method suitable for determining the activity of the protein encoded by the HELAD1 gene can be selected according to the activity of each protein to be analyzed.

  The expression level of the HELAD1 gene in the specimen (test sample) is estimated and compared with the level of a normal sample. If such a comparison indicates that the expression level of the HELAD1 gene is higher than that of the normal sample, the subject is determined to be suffering from cancer. The expression level of the HELAD1 gene in normal samples and specimens from subjects can be determined simultaneously. Alternatively, the normal range of expression levels can be determined by statistical methods based on the results obtained by analyzing the expression level of the HELAD1 gene in a sample collected so far from the control group. The result obtained by examining the subject's sample is compared with the normal range; if the result does not fall within the normal range, the subject is deemed to have cancer. In the present invention, the cancer to be diagnosed is preferably colorectal cancer.

  In the present invention, a diagnostic agent for diagnosing colorectal cancer is also provided. The diagnostic agent of the present invention contains a compound that binds to the DNA or protein of the present invention. Preferably, an oligonucleotide that hybridizes with the polynucleotide of the present invention or an antibody that binds to the protein of the present invention may be used as these compounds.

  The following examples are provided to illustrate the present invention and to assist one of ordinary skill in making and using the present invention. The examples are not to be construed as limiting the scope of the invention in any way.

  Any patents, patent applications, and publications cited herein are hereby incorporated by reference.

EXAMPLES BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail by the following examples, but the present invention is not limited to these examples.

Materials and methods
1． Cell lines and tissue samples Mouse fibroblasts (NIH3T3), monkey fibroblasts (COS7), and human colon cancer cell lines (SW480, SNUC2A, and LoVo), American Type Culture Collection (ATCC, Rockville, MD) ) Human fetal kidney cell line HEK293 was purchased from Takara (TaKaRa, Tokyo, Japan). All cell lines were grown as monolayers in appropriate media and maintained at 37 ° C. in a humid atmosphere with 5% CO ₂ (NHDF, NIH3T3, COS7, and HEK293) or without CO ₂ (SW480). . Informed consent was obtained from 20 patients who had undergone colectomy to obtain cancerous tissue and corresponding non-cancerous tissue.

2． Construction and Transfection of Recombinant Adenovirus Construction and preparation of adenovirus expressing the wild type AXIN1, LacZ, or APC 20 amino acid repeat domain was performed as previously described (Satoh et al., 2000). SW480 cells grown in a monolayer were infected with the virus solution at MOI 100 and incubated for 1 hour at 37 ° C. with gentle agitation every 15 minutes, after which fresh medium was added. Transfected cells were maintained in an incubator for 72 hours.

3． cDNA microarray
The preparation of cDNA microarray glass slides has been described in the literature (Ono et al., 2000). For each analysis of the expression profile, we prepared two sets of glass slides containing two sets of 23040 cDNA spots to reduce variability between experiments. Total RNA was extracted from SW480 cells 72 hours after infection with adenovirus constructs expressing either wild-type APC (Ad-APC) or LacZ (Ad-LacZ). RNA amplification based on T7 was performed using poly A RNA purified from the extract as described elsewhere (Ono et al., 2000). Aliquots (2.5 μg) of amplified RNA (aRNA) from SW480 cells containing Ad-APC or Ad-LacZ were labeled with Cy5-dCTP or Cy3-dCTP (Amersham Pharmacia Biotech), respectively. Hybridization, washing, and detection were performed as previously described (Ono et al., 2000). A gene was excluded from further testing if the intensity of both Cy3 and Cy5 was less than 100,000 fluorescence units. Of the rest, we selected genes with a Cy3 / Cy5 signal ratio> 2.0 for further testing.

Four. Isolation and sequencing of human HELAD1 cDNA
Among the genes that showed a Cy3 / Cy5 signal ratio> 2.0, we focused on the gene identified as “HELAD1” in-house because its expression is often elevated in colon cancer tissue. A database search at NCBI (National Center for Biotechnology Information) using the BLAST program identified a homology with the 6.2 kb assembled nucleotide sequence (Unigene accession number Hs.23467). Since this sequence was cleaved from the 5 ′ region of the cDNA, the present inventors transferred human fetal brain cDNA library (Stratagene) to HELAD1 (5′-AGAGACAGAAGAGACTGGAGCAA-3 ′ (SEQ ID NO: 5) and 5′- About 9.0 kb positive clone was isolated by screening with a PCR product amplified with a primer corresponding to CCTAAGGTTCCCTGTGTCGTAG-3 ′ (SEQ ID NO: 6). The present inventors determined the sequence of the 5 ′ end by performing 5 ′ rapid amplification (5′RACE) of the cDNA end using the Marathon cDNA amplification kit (Clontech). A cDNA template is synthesized from human fetal brain mRNA for amplification and PCR is performed using the gene-specific reverse primer (5'-GTATCGGGAGAGAGGCTGAAGAAGAGG-3 '(SEQ ID NO: 7)) and the AP1 primer provided in the kit went. The nucleotide sequence was determined with an ABI PRISM 3700 DNA sequencer (PE Applied Biosystems) according to the manufacturer's instructions.

Five. Northern Blot Analysis Human multi-tissue blots (Clontech) were hybridized with HELAD1 PCR products labeled by random oligonucleotide priming using the Mega Label kit (Amersham Pharmacia Biotech). Prehybridization, hybridization and washing were performed according to the supplier's recommendations. Blots were autoradiographed using intensifying screens at -80 ° C for 48 hours.

RNA extraction and RT-PCR
Total RNA was extracted from cell lines or tissues using TRIZOL reagent (Life Technologies) according to the manufacturer's instructions. Extracted RNA was treated with DNase I (Boehringer Mannheim) and reverse transcribed into single-stranded cDNA using oligo (dT) _12-18 primer with Superscript II reverse transcriptase (Gibco-BRL) . Each single-stranded cDNA was diluted for subsequent PCR amplification by monitoring the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a quantitative control. Primer sets for amplification include GAPDH, GAPDHF (5'-ACAACAGCCTCAAGATCATCAG-3 '; SEQ ID NO: 8) and GAPDHR (5'-GGTCCACCACTGACACGTTG-3'; SEQ ID NO: 9), and HELAD1F (5 ' -GAATTGCGCCAGTACCTGTCCAA-3 '; SEQ ID NO: 10) and HELAD1R (5'-GTCATCGTGATGGGTGTTGCTGTT-3'; SEQ ID NO: 11). Each PCR reaction was performed on a GeneAmp PCR system 9700 (PE Applied Biosystems) by first denaturing in 25 μl of 1 × PCR buffer (Takara) at 94 ° C. for 5 minutes, then at 94 ° C. for 30 seconds and at 58 ° C. 20 cycles (for GAPDH) or 32 cycles (for HELAD1) were performed for 30 seconds at 72 ° C.

6． Immunocytochemical staining
To obtain flag-tagged HELAD1, we cloned the complete coding sequence into the appropriate site of the pCMV-FLAG5a plasmid vector (Sigma) and transferred the plasmid clone pFLAG-HELAD1 into COS7 cells. I did it. Transiently transfected COS7 cells were fixed with PBS containing 4% paraformaldehyde and then permeabilized with PBS containing 0.1% Triton X-100 at 4 ° C. for 3 minutes. Cells were covered with blocking solution (2% BSA in PBS) for 30 minutes at room temperature to block nonspecific antibody binding sites. The cells were then incubated with mouse anti-flag antibody (Sigma). The antibody was stained with a goat anti-mouse secondary antibody conjugated with rhodamine and observed with an ECLIPSE E8000 microscope (Nikon). To confirm the expression of flag-tagged HELAD1 protein in transfected cells, we also extracted whole cells using anti-flag antibody (Sigma) as previously described (Ishiguro et al., 2000). Western blotting with either product or nuclear extract was also performed. Nuclei from the cells were isolated as previously described (Chauveau et al., 1956).

7． Preparation of recombinant protein representing AAA domain of HELAD1 Recombinant protein of AAA domain of HELAD1 fused to glutathione S-transferase was transferred to the appropriate site of plasmid vector (pGEX-5X; Amersham Pharmacia Biotech). The RT-PCR product corresponding to codon positions 1958-2234 was prepared by cloning. Bacteria harboring pGEX-HELAD1 were grown overnight in 1 × LB medium, then cultured in fresh medium for an additional 3 hours, then at 37 ° C. with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) Incubated for 4 hours. Bacteria were sedimented and sonicated in lysis buffer (1% Triton-X and 1 mM PMSF in PBS). The recombinant fusion protein was purified from the supernatant using glutathione sepharose beads (Amersham Pharmacia Biotech).

8． Helicase Assay Helicase activity was measured by removing radiolabeled DNA fragments from double stranded DNA substrates as described elsewhere (Suzuki et al., 1997). Each substrate DNA complex consisting of M13mp18 (Takara) and ³² P-labeled complementary oligonucleotide (5′-CAGGGTTTTCCCAGTCAC-3 ′; SEQ ID NO: 12) was added to 20 mM Tris-HCl (pH 7.5), 10 mM MgCl. _2. Prepared by annealing the two components in 100 mM NaCl, and 1 mM DTT. Annealing DR45 (5'-TCGCGGAAGCTTGGCTGCAGAATATTGCTAGCGGGAATTCGGCGCG-3 '; SEQ ID NO: 13) and DR1735 (5'-GCAGCCAAGCTTCCGCG-3'; SEQ ID NO: 14) as a substrate for determining helicase activity from 3 'to 5' The 5 ′ to 3 ′ substrate was prepared by annealing DR45 and DR1753 (5′-TCGCCCTTAAGCCGCGC-3 ′; SEQ ID NO: 15). Unhybridized oligonucleotides were removed using SUPREC02 according to the supplier's protocol (Takara). Each DNA substrate (˜2.5 pg) is added to 20 μg of GST in a reaction mixture containing 50 mM Tris-HCl buffer (pH 7.5), 1 mM MgCl ₂ , 0.5 mg / ml BSA, and 2 mM 2-mercaptoethanol. -Incubated with 1 μg of HELAD1 protein or recombinant WRN protein for 60 minutes at 37 ° C. Recombinant WRN protein was donated by Dr. Furuichi (Agene).

9． Colony formation assay
Plasmids designed to express HELAD1 sense and antisense transcripts were constructed by cloning the entire coding region of HELAD1 cDNA into the appropriate sites of the pcDNA3.1 vector (Invitrogen). COS7 cells (5 × 10 ⁵ cells each) were seeded in 10 cm culture dishes, FuGENE6 used as recommended (Roche (Roche)) the manufacturer, plasmids designed to express the sense strand of HELAD1 pcDNA HELAD1S, pcDNA-HELAD1AS designed to express the antisense strand, or pcDNA vector were transfected. Cells were cultured for 14 days in the presence of 400-800 μg / ml geneticin (G418) and stained with Giemsa solution (MERCK).

Ten. Inhibition of HELAD1 by antisense oligonucleotides
HPLC-purified antisense oligonucleotide phosphorothioate HELAD1-AS2 (5'-CAGAAACCGATTCCAT-3 '; SEQ ID NO: 16), HELAD1-AS3 (5'-GACTCAGAAACCGATT-3', each designed to suppress the expression of HELAD1 SEQ ID NO: 17), and FITC-labeled-AS2 (5′-CAGAAACCGATTCCAT-3 ′; SEQ ID NO: 16), and control oligonucleotide phosphorothioate HELAD1-SE2 (5′-ATGGAATCGGTTTCTG-3 ′; SEQ ID NO: 18) And HELAD1-SE3 (5′-AATCGGTTTCTGAGTC-3 ′; SEQ ID NO: 19) were prepared. 1 μM of each oligonucleotide phosphorothioate was transfected into SW480 cells using Lipofectin reagent (Gibco-BRL) according to the supplier's protocol. Transfection efficiency was the same for all oligonucleotides (data not shown). Cells were maintained at 37 ° C. for 5 days and stained with Giemsa solution (Merck). In order to confirm inhibition of HELAD1, semi-quantitative RT-PCR was performed using template RNA extracted from cells 12 hours after transfection.

11． MTT assay
10 cm plates of SW480 cells (1 × 10 ⁵ ) were transfected with antisense or control oligonucleotides using Lipofectin reagent (Gibco-BRL) according to the supplier's protocol. Cell viability was assessed by MTT assay 7 days after treatment. MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] (Sigma) was added to each culture dish at a concentration of 500 μg / ml, and the plate was further incubated at 37 ° C. Incubated for 4 hours. Next, acid-SDS (0.01 N hydrochloric acid / 10% SDS) was added to all wells and mixed well. The plate was incubated overnight at 37 ° C. to dissolve the dark blue crystals, and then the absorbance at 570 nm was measured with a microplate reader 550 (BIO-RAD).

12． Flow cytometry and TUNEL assay
SW480 cells (5 × 10 ⁵ cells / 10 cm culture dish) were transfected with 1 μM of each oligonucleotide phosphorothioate using Lipofectin reagent (Gibco-BRL). On day 7, cells were trypsinized and collected, and a cell suspension was prepared using Cycle TEST PLUS (Becton Dickinson) according to the manufacturer's protocol. Flow cytometry analysis was performed on a FACSCalibur (Becton Dickinson). The number of nuclei in the G0-G1, S, and G2-M phases of the cell cycle and the sub-G1 population were determined from at least 20000 ungated cells. Apoptotic cells were detected by TUNEL assay using the ApopTaq kit (Intergen) according to the manufacturer's protocol.

13. Statistical analysis Statistical significance was analyzed by ANOVA with Scheffe test using commercially available software (Statview, Berkeley).

Example 1: Detection of HELAD1 When SW480 cells expressing mutant APC and wild-type β-catenin were infected with Ad-Axin, or Ad-APC, a construct that expresses the 20-amino acid repeat domain of APC, A decrease in β-catenin expression and a decrease in Tcf / LEF-specific transcriptional activity have been previously reported (Kawasoe et al., 2000). When comparing the expression profile between SW480 cells infected with either Ad-APC or Ad-LacZ on a cDNA microarray containing 23040 independent target cDNAs, the gene corresponding to Unigene accession number Hs.23467 is It was found that there was a significant decrease in expression in response to APC. Subsequent RT-PCR experiments confirmed that Ad-APC decreased the expression of this gene in SW480 cells, but not Ad-Axin or Ad-LacZ (FIG. 1A). Similarly, Ad-APC infection of other colon cancer cell lines, including SNUC2A and LoVo, down-regulated gene expression (data not shown). In the present specification, this gene is referred to as HELAD1 in consideration of the characteristics revealed in the following experiment (helicase, adenomatous polyposis coli down-regulated 1). .

Example 2: Expression of HELAD1 in colon cancer tissue
Accumulation of β-catenin as a result of either APC or β-catenin mutations is a frequent feature of colorectal tumors, so semiquantitative expression of HELAD1 in human colon cancer tissues and the corresponding non-cancerous mucosa When examined by dynamic RT-PCR, it was found that expression increased in 16 (80%) of the 20 tumors examined (FIG. 1B). In the other four examples, the expression levels were similar to the corresponding normal tissue levels. This result was consistent with the notion that HELAD1 is upregulated in response to activation of the β-catenin / Tcf transcription complex.

Example 3: Expression of HELAD1 in human tissues Northern blot analysis using the RT-PCR product of HELAD1 as a probe detects an 11 kb transcript in human fetal brain and lung and a 7.5 kb transcript in fetal kidney and liver (Figure 1C). However, these transcripts were not detectable in any adult human tissue examined.

  Since the UniGene clone (Hs. 23467) is smaller than the transcript detected by Northern analysis, a human fetal brain cDNA library was screened and a 5′RACE experiment was performed. This effort identified two HELAD1 transcripts with different 5 ′ sequences (GenBank Accession Nos. AB063115 (SEQ ID NO: 1) and AB063116 (SEQ ID NO: 3)). A homology search with its deduced amino acid sequence using NCBI's BLAST program showed 28% identity with nematode unc-53. Comparison of the genome sequence in NCBI with the HELAD1 cDNA sequence using the BLAST program revealed that HELAD1 consists of 38 exons and belongs to chromosome band 11p15.1. Simple module construction search tool (SMART, http://smart.embl-heidelberg.de) (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95: 5857-5864; Letunic et al. (2002), Nucleic Acids Res 30: 242-244) predicts that the predicted protein is CH (calponin homology) domain (codons 23-126 in SEQ ID NO: 2; codons 87-190 in SEQ ID NO: 4) and AAA (various cells) ATPase associated with activity) domain (codons 2026-2180 in SEQ ID NO: 2; codons 2090-2244 in SEQ ID NO: 4) were found to contain. Furthermore, the A and B motifs of the Walker-type NTP binding domain were identified in codons 2034 to 2041 (SEQ ID NO: 2) (GPSGTGKT) and 2099 to 2104 (SEQ ID NO: 2) (LVIILD), respectively (sequence) In No. 4, codons 2098 to 2105 (GPSGTGKT) and 21163 to 2168 (LVIILD), respectively.

Example 4: Subcellular localization of HELAD1 protein To examine the position of HELAD1 protein in mammalian cells, a plasmid expressing flag-tagged HELAD1 protein (pFLAG-HELAD1) was transiently transfected into COS7 cells. The tagged proteins were observed by immunocytochemical staining. HELAD1 protein was detected by anti-Flag antibody on Western blot (FIG. 2A), and subsequent microscopic analysis showed that it was mainly localized in the cytoplasm and nucleus (FIG. 2B). Nuclei were separated from the cells and Western blot analysis was performed with anti-flag antibodies. HELAD1 expression was identified in the nucleus (FIG. 2C).

Example 5: DNA helicase activity
The AAA domain is found in many proteins encoding DEAD / DEAH box helicases along with the ABC transporter, and the helicase of the HELAD1 protein, since the A and B motifs of the Walker-type NTP binding domain are commonly found in helicases. The activity was examined. A recombinant GST-fusion protein (GST-HELAD1) containing the AAA domain of HELAD1 was prepared. Partially double-stranded DNA substrate was mixed with GST-HELAD1 or Werner helicase (WRN) recombinant protein in the presence of ATP (FIG. 3A). The substrate unwound by both GST-HELAD1 and WRN is smaller, and the oligonucleotide is also smaller compared to the original size (18-mer), which adds to the helicase activity of HELAD1 and WRN Nuclease activity. Further testing revealed that the helicase activity of the recombinant GST-HELAD1 protein was in the 3 ′ to 5 ′ direction and not the 5 ′ to 3 ′ direction (data not shown).

Example 6: Induction of apoptosis by antisense S-oligonucleotide of HELAD1 To investigate the effect of this gene on cell proliferation, antisense (AS2 and AS3) S-oligos corresponding to the DNA sequence near the first ATG Nucleotides and control (SE2 and SE3) S-oligonucleotides were synthesized. Almost all SW480 cells incorporated transfected FITC-labeled S-oligonucleotides into their nuclei (data not shown). Antisense S-oligonucleotide significantly suppressed HELAD1 expression within 24 hours after transfection, but not sense S-oligonucleotide (FIG. 4A). The number of cells transfected with antisense S-oligonucleotide (AS2 = 0.32 ± 0.02, AS3 = 0.31 ± 0.08) is the number of cells transfected with control S-oligonucleotide (SE2 = 0.69 ± 0.07, SE3 = 0.84 ± 0.05) As compared to the transfection significantly decreased (FIG. 4B). By FACS analysis, cells transfected with antisense S-oligonucleotide AS2 (32.3 ± 5.1%) or AS3 (46.4 ± 4.6%) are transfected with SE2 (6.5 ± 3.7%) or SE3 (14.0 ± 5.8%) A larger proportion of sub-G1 population was detected compared to the cells (Figure 4C for AS3, data not shown for AS2). Consistently, TUNEL assay confirmed the induction of apoptosis in SW480 cells transfected with AS2 or AS3 (FIG. 4D for AS3, data not shown for AS2).

Industrial Applicability The expression of the human gene HELAD1 is markedly elevated in colorectal cancer compared to non-cancerous colorectal tissue. Thus, this gene may serve as a diagnostic marker for colorectal cancer, and the protein encoded thereby may be used in diagnostic assays.

  The present invention also showed that suppression of HELAD1 expression induces apoptosis in colorectal cancer cells. Thus, an agent that blocks the expression of HELAD1 or suppresses its activity may find therapeutic utility as an anticancer agent, particularly as an anticancer agent for treating colorectal cancer. Examples of such agents include antisense oligonucleotides and antibodies that recognize HELAD1.

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  All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Furthermore, while the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. It is believed that there is.

2 shows HELAD1 expression in cancer cells and fetal tissue. FIG. 1A shows a decrease in the expression of HELAD1 in response to Ad-APC or Ad-Axin in SW480 cells. Semi-quantitative RT for RNA extracted from intact (pseudo) cells or cells infected with adenovirus containing either wild type AXIN1, the 20 amino acid repeat region of the wild type APC gene, or LacZ for 72 hours -PCR was performed. FIG. 1B shows the expression of HELAD1 in colon cancer tissue (T) and adjacent non-cancerous mucosa (N) as examined by semi-quantitative RT-PCR. GAPDH expression was used as an internal control. FIG. 1C shows a multi-tissue northern blot analysis of HELAD1 in human fetal tissue. The size of the molecule is indicated in kb on the left side. 2 shows sublocalization of tagged HELAD1 protein in COS7 cells. FIG. 2A shows the expression of flag-tagged HELAD1 protein in COS7 cells detected by anti-Flag antibody on Western blot. FIG. 2B shows immunocytochemical staining (600 times magnification) of the flag-tagged HELAD1 protein. Cells transfected with pFLAG-HELAD1 were stained with anti-Flag antibody and visualized with anti-mouse secondary antibody conjugated with rhodamine. FIG. 2C shows a Western blot analysis of nuclear extracts from cells that exogenously express the flag-tagged HELAD1 protein. 1 shows helicase activity of HELAD1. FIG. 3A shows 5 ′ to 3 ′ helicase activity analyzed by incubating recombinant GST-HELAD1 protein with a radiolabeled partially double stranded DNA substrate. Recombinant WRN protein and GST-axin protein served as controls. FIG. 3B shows an analysis of 3 ′ to 5 ′ helicase activity. FIG. 6 shows apoptosis induction by an antisense S-oligonucleotide that suppresses the expression of HELAD1. FIG. 4A shows the expression of HELAD1 in response to antisense S-oligonucleotides (AS2, AS3) or control S-oligonucleotides (SE2, SE3) in SW480 cells, analyzed by semi-quantitative RT-PCR. FIG. 4B shows the viability of SW480 cells in response to AS2, SE2, AS3, or SE3. Cells were treated with S-oligonucleotide and incubated for 7 days, after which their viability was assessed by MTT assay. Data are expressed as the number of viable cells treated with S-oligonucleotide relative to untreated cells (NC). Error bars indicate standard deviation. FIG. 4C shows FACS analysis of cells transfected with either AS3 or SE3. FIG. 4D shows the TUNEL staining result of the same cells, and yellow or green cells show apoptosis.

Claims (19)

A method of diagnosing colorectal cancer in a sample comprising the following steps:
(A) a step of measuring the expression level of a gene in a sample, wherein the gene encodes a protein selected from the group consisting of: (i) a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 (Ii) one or more amino acids have been modified such that the protein retains biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: A protein having the amino acid sequence described in 4; (iii) the nucleotide sequence described in SEQ ID NO: 1 or SEQ ID NO: 3 so that the protein has a biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4. A protein encoded by a DNA that hybridizes under stringent conditions with DNA having: and (b) the presence of colorectal cancer in the sample with increased expression levels Step of comparing the expression level associated correlating the measured expression level in step (a) the normal samples.   2. The method of claim 1, wherein the protein comprises the amino acid sequence set forth in SEQ ID NO: 2.   2. The method of claim 1, wherein the protein comprises the amino acid sequence set forth in SEQ ID NO: 4.   The amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 having 10 or fewer amino acid modifications such that the protein retains the biological activity of the protein of SEQ ID NO: 2 or SEQ ID NO: 4. The method of claim 1 comprising:   An amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4 having 5 or fewer amino acid modifications such that the protein retains the biological activity of the protein of SEQ ID NO: 2 or SEQ ID NO: 4 The method of claim 1 comprising:   5. The method of claim 4, wherein the modification comprises a conservative amino acid substitution.   Colorectal cancer comprising the step of suppressing expression in colorectal cells of a gene having the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 so that cell cycle arrest and apoptosis are induced in colorectal cells How to treat or prevent onset.   A method of treating or developing colorectal cancer, comprising inhibiting the activity in a colorectal cell of a protein selected from the group consisting of such that cell cycle arrest and apoptosis are induced in the colorectal cell. How to prevent: (i) a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (ii) the protein has a biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4 A protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, wherein one or more amino acids have been modified; (iii) the protein is equivalent to the protein of SEQ ID NO: 2 or SEQ ID NO: 4 Highly stringent under stringent conditions with DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 to have biological activity A protein encoded by DNA to be bridged.   An antisense molecule that hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and comprises a polynucleotide comprising at least 15 nucleotides, wherein SEQ ID NO: 1 or SEQ ID NO: in colorectal cells 3. An antisense molecule that induces apoptosis by suppressing the expression of a gene having the DNA sequence according to 3.   10. The antisense molecule of claim 9, having a nucleotide sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 16, and SEQ ID NO: 17. A vaccine that induces anti-tumor immunity comprising a material selected from the group consisting of:
(A) a protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
(B) one or more amino acids have been modified such that the protein retains biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4, SEQ ID NO: 2 or SEQ ID NO: 4 A protein having the described amino acid sequence;
(C) under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 so that the protein has biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4. A protein encoded by DNA that hybridizes with
(D) an antigenic fragment of a protein as defined in paragraph (a), (b), or (c);
(E) a vector comprising a nucleotide sequence encoding a protein as defined in paragraph (a), (b), or (c);
(F) a vector comprising a nucleotide sequence encoding an antigenic fragment as defined in paragraph (d);
(G) an antigen presenting cell contacted with the protein defined in paragraph (a), (b), or (c);
(H) an antigen presenting cell contacted with an antigenic fragment as defined in paragraph (d);
(I) an antigen-presenting cell into which a vector defined in (e) or (f) is introduced; and (j) activated by an antigen-presenting cell as defined in (g), (h), or (i) T lymphocytes.   12. A method for inducing anti-tumor immunity comprising the step of administering to a subject a vaccine according to claim 11. A method of screening a compound for treating colorectal cancer comprising the following steps:
(A) contacting a test compound with a protein selected from the group consisting of: (i) a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (ii) the protein is SEQ ID NO: 2 or A protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, wherein one or more amino acids have been modified to have a biological activity equivalent to the protein of SEQ ID NO: 4; (iii) Hybridize under stringent conditions with DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 so that the protein has biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4 A protein encoded by DNA
(B) assaying the binding of the test compound to the protein; and (c) selecting a test compound that binds to the protein. A method of screening a compound for treating colorectal cancer comprising the following steps:
(A) measuring the biological activity of a protein selected from the group consisting of: (i) a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 in the presence of the test compound; ii) One or more amino acids have been modified such that the protein retains biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4, described in SEQ ID NO: 2 or SEQ ID NO: 4 (Iii) having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 such that the protein has a biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4. A protein encoded by DNA that hybridizes with DNA under stringent conditions;
(B) comparing the activity measured in step (a) with the same biological activity of the protein observed in the absence of the test compound;
(C) selecting a test compound that inhibits the biological activity of the protein.   15. The method of claim 14, wherein the biological activity of the protein is selected from the group consisting of nuclease activity, helicase activity, and antiapoptotic activity in colorectal tissue. (I) a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (ii) one such that the protein retains biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4. A protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, wherein one or more amino acids are modified; (iii) a biological protein equivalent to the protein of SEQ ID NO: 2 or SEQ ID NO: 4 An activity of a protein selected from the group consisting of a protein encoded by a DNA that hybridizes under stringent conditions with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 so as to have activity A method of screening for a compound that inhibits, comprising the following steps:
(A) contacting a protein or a partial peptide thereof with DNA in the presence of a test sample containing at least one test compound;
(B) detecting degradation of DNA; and (c) selecting a test compound that inhibits degradation as compared to degradation detected in the absence of the test sample. (I) a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (ii) one such that the protein retains biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4. A protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, wherein one or more amino acids are modified; (iii) a biological protein equivalent to the protein of SEQ ID NO: 2 or SEQ ID NO: 4 An activity of a protein selected from the group consisting of a protein encoded by a DNA that hybridizes under stringent conditions with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 so as to have activity A method of screening for a compound that inhibits, comprising the following steps:
(A) contacting a protein or a partial peptide thereof with double-stranded DNA in the presence of a test sample containing at least one test compound;
(B) detecting DNA unwinding; and (c) selecting a test compound that inhibits unwinding as compared to unwinding detected in the absence of the test sample. (I) a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; (ii) one such that the protein retains biological activity equivalent to that of SEQ ID NO: 2 or SEQ ID NO: 4. A protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, wherein one or more amino acids are modified; (iii) a biological protein equivalent to the protein of SEQ ID NO: 2 or SEQ ID NO: 4 An activity of a protein selected from the group consisting of a protein encoded by a DNA that hybridizes under stringent conditions with a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 so as to have activity A method of screening for a compound that inhibits, comprising the following steps:
(A) culturing cells expressing the protein or a partial peptide thereof in the presence of a test sample containing at least one test compound;
(B) detecting apoptosis of the cells; and (c) selecting a test compound that inhibits apoptosis as compared to apoptosis detected in the absence of the test sample.   19. A compound identified by the method of claim 13, 14, 15, 16, 17, or 18.

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