JP2001514507A - Methods and compositions for diagnosis and treatment of cancer - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel gene, CaSm, which is highly expressed in cancer tissues and cell lines, especially pancreatic cancer. The CaSm full-length cDNA encodes a protein consisting of 133 amino acids. CaSm contains two Sm motifs found in the normal snRNP protein and has the highest homology (60% similarity) to the Sm G protein. The invention further includes CaSm peptides, fusion proteins, host cell expression systems, antibodies to CaSm, antisense CaSm molecules, and compounds that modulate CaSm gene expression or CaSm activity. Antisense CaS mRNA can alter the phenotype of transformed pancreatic cancer cells by reducing the ability of cancer cells to form large colonies in soft agar compared to untransfected cancer cells . The invention also encompasses methods of diagnosing disease, drug screening and treating cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION                  Methods and compositions for diagnosis and treatment of cancer                                 1.Introduction   The present invention encodes CaSm, a novel protein that is overexpressed in various cancer tissues Discovery, identification and characterization of nucleic acid molecules. The present invention relates to cancer diseases (including cancer). (But not limited to) diagnosis, disease monitoring, drug screening CaSm nucleotides that can be used for therapeutic and / or therapeutic purposes, Current system, CaSm protein, fusion protein, polynucleotide, peptide, gene Antibodies to the product, antisense CaSm nucleic acids, Transgenic animals, or recombinant knockout animals that do not express CaSm, and It relates to other compounds that modulate CaSm gene expression or CaSm activity.                               2.background                                 2.1cancer   Cancer is an increase in the number of abnormal cells from certain normal tissues, Infiltration of adjacent tissues and malignant cells in regional lymph nodes or distant sites (metastasis) Is characterized by lymphocytic or bloody spread. Premalignant abnormal cell proliferation is hyperplasia, Exemplified by dysplasia, or more particularly dysplasia (such abnormal growth For a review of conditions, see Robbins & Angell, 1976, Basic Pathology, Second Edition, W.W. B. Saunders Co., Philadelphia, pp. 68-79). Neoplastic lesions Lonely, especially under conditions where neoplastic cells escape host immune surveillance , Growth, metastasis, and heterogeneous abilities (Roitt, I., Brostoff, J. Kale, D., 1993, Immunology, Third Edition, Mosby, St. Louis, pp. 17.1-17.12). Clinical data and molecular biological studies indicate that cancer is a multi-step process that begins with small preneoplastic changes. Process, which indicates progression to a neoplasm under certain conditions.   Screening is the search for disease in asymptomatic humans. One individual Once the cleaning result is positive or symptoms or symptoms are confirmed, In addition, a diagnostic test is performed to determine the optimal course of treatment. The advantage of early detection is disease To treat the disease before the disease has spread (ie, when treatment or control is easiest) The meeting is obtained. The American Cancer Society is asymptomatic and at risk Routine cancer-related screening for a person (this includes breast, colon, skin, prostate, Tests (including glands) are recommended.   As the understanding of the pathophysiological role of cancer increases, cancer markers and genetic information Both roles of the report will become more important in the management and treatment of cancer patients. Tumor marker Is a histopathologic step to detect the cancer and to determine the extent of tumor burden. To help classify or identify the page, predict the outcome of drug therapy, and Quantification by biochemical or immunological means to monitor Substances in tissues or body fluids that are typically measured. Measurement of tumor markers is performed in the total population And screening of high-risk groups.   Abnormal control of mechanisms that regulate cell growth and differentiation leads to cell transformation. Minute Child analysis shows multiple mutations in oncogenes and tumor suppressor genes to indicate a malignant phenotype. Proved necessary. This multi-step process typically takes several decades Colonic colon that may require at least seven genetic events to evolve and complete It is better exemplified by cancer (Kinzler et al., 1996, Cell, 87: 159-170). Genetics of cancer Fundamental findings have important clinical implications, the most recent being diagnosed by genetic testing It is an improvement of.   For example, recent reports indicate that individuals with BRCA1 and BRCA2 mutations are more susceptible to cancer. The findings facilitate early detection of hereditary breast cancer (Easton et al., 1993, Cancer Sur v, 18: 95-1131; Miki et al., 1994, Science, 266: 66-71; Tavtigian et al., 1994, Natu. re Gen, 12: 333-337). However, the incidence of these cases is limited to all known breast cancers. 5-10% (Easton et al., 1993, Cancer Surv, 18: 95-1131; Miki et al., 199 4, Science, 266: 66-71 Tavtigian et al., 1994, Nature Gen, 12: 333-337). Ie sporadic malignant milk Early and late stage specific tumor markers still exist for more than 90% of cancers Is needed.   Colorectal and breast cancer have been extensively studied at the molecular and genetic level. It is an example of a malignant disease of the part. However, there are still many cancers that need molecular biological analysis I do. If tumor markers and oncogenes associated with these cancers are identified, New treatments that greatly help screen and identify individuals at risk for sexually transmitted diseases Will help you explore.                               2.2Pancreatic cancer   Pancreatic cancer is a disease in industrialized nations, for example, the incidence in Japan was 100,000 in 1960 The number increased from 1.8 to 5.2 out of 100,000 in 1985. Smoking and high-fat diets are It is said that there is a clerk. (Beazley et al., 1995, Clinical Oncology (Second Edition) Chapter 15, Ed. Murphey et al., American Cancer Society). Ductal adenoma of the exocrine pancreas of the pancreas It is the most common type of pancreatic cancer and ranks fourth in cancer deaths in the United States (Parker et al.) , 1996, CA-A Cancer Journal for Clinicians, 46: 5-27). Pancreatic cancer is very Malignant. Many patients are diagnosed at an advanced stage beyond their therapeutic potential ( Pancreatic cancer has a very poor prognosis with a 5-year survival rate of less than 3%; Warshaw et al., 1992, N . Engl. J. Med., 326: 455-465). Metastasis to distant sites (especially the liver) Occurs early in the course of the disease. The average survival rate after diagnosis is 6 months. Patients with chronic pancreatitis The incidence of pancreatic cancer is high among individuals. Clinical diagnosis of pancreatic cancer is made late in the disease Often. The first choice for a diagnostic test is a computed tomography (CT) scan And then the ultrasonic method. Fine needle biopsy with CT guidance to confirm diagnosis can get. Diagnostic tests give stage information. Generally, the tumor marker is the pancreas It is not effective in diagnosing or staging cancer.   Early identification and detection of pancreatic cancer is expected to improve survival. Recent surgical literature is mainly for patients with small (<2 cm) tumors Reported high 5-year survival rates (up to 20%) (Cameron et al., 1995, Surgical Clinics of North America, 75: 939-951). Pancreatic cancer staging is metastatic Patients with early stage disease have a much better prognosis than patients with late stage disease based on It is. Most survivors have small lesions and negative lymph nodes (T1, N0, M0) It is.   Surgery with Combination Therapy (5-Fluorouracil and Radiation) Successfully Treats Pancreatic Cancer Although the rate is highest, unfortunately the majority of patients at the time of decision are ineligible for this therapy. Drug treatment of unresectable cancer is relatively unsuccessful even with multiple drugs . Brennan et al., Chapter 27, "Cancer: Principles and Practice of Oncology", Chapter 4. Edition, edited by DeVita et al., L. B. See Lippincott Co., Philadelphia, 1993.   Thus, successful treatment depends on very early diagnosis and thus facilitates this early detection It is important to find additional pancreatic cancer markers.   The molecular cause of pancreatic cancer is undefined, but some genetic changes have been detected ing. For example, the most common change not yet recognized is the sudden onset of K-ras tumor development. Mutation (Almoguera et al., 1988, Cell, 53: 549-554) and TP53 (Redston et al., 19 94, Cancer Res., 54: 3025-3033), p16 / MTS-1 (Caldas et al., 1994, Nature Genet). ., 8: 27-32; Huang et al., 1996, Cancer Res., 56: 1137-1141) and DPC4 (Hahn et al.). , 1996, Science, 271: 350-353). Is a homozygous deletion. Gene amplification also plays a key role in some pancreatic cancers (Cheng et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 3636-3641. ). However, these multiple parameters may not be relevant to the multistep progression of pancreatic malignancy. Not well correlated with child events. In other words, the molecular biology of pancreatic cancer Information to promote understanding and develop new screening and early diagnostic tests There is a great need for additional genetic markers to provide.                              3.Summary of the Invention   The present invention provides a novel unregulated expression pattern in cancer tissues and cell lines. Gene identification and its potential as a target for diagnosis, drug screening and therapy The use of such genes and gene products.   In particular, the compositions of the present invention may comprise a recombinant DNA molecule, a cloned gene or a degenerate variant thereof. Novel cancers, including species and naturally occurring variants that encode novel CaSm gene products Related nucleic acid molecules encoding related Sm-like (CaSm) proteins. The composition of the present invention Further, a cloning vector containing an expression vector containing the nucleic acid molecule of the present invention. And a host comprising such a nucleic acid molecule. The compositions of the present invention also include CaSm Co-products, their variants and fragments, fusion proteins, and such CaSm gene products. Or antibodies to conserved variants or fragments thereof.   The nucleic acid sequence of the human CaSm gene (SEQ ID NO: 1) has been deposited with Genbank and No. AF000177 has been given. The CaSm gene produces a transcript of about 1.2 kb, and about 15,17 Encodes a 133 amino acid protein with a molecular weight of 9 daltons. The transcript is Several cancer cell lines, as well as various normal tissues (thymus, breast, colon, kidney, pancreas And heart). Predicted amino acids of the full-length CaSm gene product The sequence contains no recognizable signal sequence or transmembrane domain, This indicates that the Sm gene product is an intracellular protein. This amino acid sequence Has high homology to small nuclear ribonucleoprotein (snRNP) SmG protein.   The invention further relates to a diagnostic evaluation and prognosis of cancer, especially pancreatic cancer. For example, the present invention The nucleic acid molecule can be used as a diagnostic hybridization probe or as a CaSm gene. Can be used as a primer in diagnostic PCR analysis for detection of abnormal expression of You.   The antibody against the CaSm gene product of the present invention detects the presence of the CaSm gene product in body fluids. Can be used in diagnostic tests to issue. In a specific embodiment, CaSm Measurement of gene product levels can detect or detect cancer, especially pancreatic cancer. Can be performed to classify the stages.   The invention also relates to a method of identifying a subject having a predisposition to cancer. For example The nucleic acid molecule of the present invention may be used for mutation of CaSm gene, allele change and CaSm gene. A diagnostic hybridization procedure for diagnostic PCR analysis for the identification of regulatory defects Can be used as probes or primers.   Further, methods and compositions are provided for the treatment of cancer, especially pancreatic cancer. like this The methods and compositions provide for the level of CaSm gene expression and / or CaSm gene product activity. Can be modulated. Inhibition of CaSm expression by antisense RNA , Transformed phenotype of pancreatic cancer cell line, and cancer cells when injected into SCID mice Decreased carcinogenicity.   Further, the present invention provides a method for monitoring CaSm gene expression and / or CaSm gene product activity. CaSm gene and / or CaSm gene product for identification of a compound to be regulated About the usage of. Such compounds may be used to prevent and / or treat cancer. Can be used as a substance. Such compounds may also reduce the symptoms of the disease. It can be used for reduction and control of metastatic nature of cancer.                           4.BRIEF DESCRIPTION OF THE FIGURES   FIG. Expression of CaSm mRNA in pancreatic tissue. Total RNA from surgically obtained pancreatic samples ( 5 μg / lane) was electrophoresed on 1.2% agarose containing formaldehyde, Transfer to nylon membrane,³²Hybridized with a P-labeled CaSm probe. T, tumor ( Or suspect mass); N, normal: P, pancreatitis. The combined sample is This is a sample isolated from a human. These pairs form a laneset. You. Otherwise, a single sample from a separate individual is shown in the unpaired lane. Lane set 1, benign mass; lane set 2, adenocarcinoma; lane set 3, adenocarcinoma; Lane 4, insulinoma; lane 5, adenocarcinoma metastasized to large intestine; lane 6, adenocarcinoma; 7, pancreatitis: lane 8, low to moderately potentially malignant neoplasm; Lane 9, normal pancreas; lane 10, adenocarcinoma; lane 11 adenocarcinoma; lane 12, adenocarcinoma; Set 13, adenocarcinoma; lane set 14, adenocarcinoma; lane set 15, pancreatitis; Set 16, adenocarcinoma; lane set 17, adenocarcinoma; lane set 18, adenocarcinoma; lane 19, adenocarcinoma . The lower panel shows ethidium bromide stained RNA.   Figures 2A-2B. CaSm mRNA expression in human tissues and cancer cell lines. Surgically obtained pancreas Total RNA (10 μg / lane) from the sample was run on 1.2% agarose containing formaldehyde. Electrophoresis, transfer to nylon membrane,³²Hybridize with P-labeled CaSm probe Was. FIG. 2A shows Northern blot analysis using RNA from the indicated human tissues. Figure 2B Is Northern blot analysis using RNA from the described cancer cell lines. The cell line is Pancreas (CAPAN-1, HPAC), prostate (PC-3, LNCAP), breast (BT20, MCF-7), liver Viscera (HepG2, SKHEP-1), cervix (HeLa), ovary (OVCAR-3), lung (A-427), bladder Bladder (T24), rectum (SW1463), non-erythroid hematopoietic cells (MOLT-4, NC-37, Raji, H9, KG -1, HL-60), and from kidney (Caki-1) tumors. The lower panel is 3 shows ethidium bromide stained RNA.   Figures 3A-3C. Homology between CaSm protein and Sm G protein and putative protein . A diamond (◇) indicates that the sequence is not completely described. FIG. 3A. CaSm and human Sm G protein. The parenthesized area is as described Shows Sm motifs 1 and 2. The core consensus for the Sm motif was suggested by Hermann et al. (1995, EMBO J, 14: 2076-2088). U is uncharged hydrophobic Means amino acids (L, I, V, A, F, W, Y, C, M); Z is uncharged Means hydrophobic amino acids + T or S. FIG. 3B. Cosmid F40F8 (GenBank No. Z69302), estimated from reading frame J0714 (PIR S55137) CaSm tan with the gene product of Buditis elegans (Caenorhabditis elegans) Alignment of parkin. FIG. 3C. The code encoded by DNA clone accession number Z49399 ORF YJL124 gene product of Saccharomyces cerevisiae Alignment of CaSm protein with c.   FIG. Decreased endogenous CaSm expression in stable antisense transformants. CaSm Anti RNA (5 μg / lane) from each stable transformant containing the sense construct Electrophoresed in 1.5% agarose containing formaldehyde Transfer,³²Hybridized with a P-labeled CaSm probe. Endogenous CaSm mRNA size Size (1.2 kb) and the size of transfected antisense RNA (0.8 kb) Describe. The lower panel shows ethidium bromide stained RNA.   Figures 5A-5B. Reduction of fixed independent growth in antisense transformants. Figure 5A. Three weeks after parental pancreatic cancer cell line PANC-1 and four antisense transfections Clones (clone K, clone L, clone 1, clone 2) Soft agar colony. FIG. 5B. Soft agar colonies according to size from PNAC-1 and clone K Quantification.   FIG. Amino acid sequence predicted as the nucleotide sequence of CaSm.                           5.Detailed description of the invention   The present invention relates to CaSm proteins whose expression is increased in cancer tissues and cell lines. Related to discovery and characterization of encoding nucleic acid molecules.   Subsequences of genes that are specifically expressed at various stages during neoplastic growth And some of these are associated with metastatic spread of malignant tumors, especially diseases Very important for progression. Its expression is associated with pancreatic cancer at various stages of neoplastic growth. To identify and isolate related genes, we used subtractive hive Redidation cloning was performed (Schweinfest et al., 1990, Gene Anal. Techn., 7: 64-70; Schweinfest et al., 1993, Proc. Natl. Acad. Sci. 90: 4166-417 0). RNA preparation from pancreatic cancer cell line CAPAN-1 and more normal pancreatic epithelial cell line HS680.PAN did. Complementary cDNA cDNA obtained by subtractive hybridization Loans are differentially hybridized with total cDNA for CAPAN-1 and HS680.PAN mRNA Selected by the user. Much stronger hybridization than HS680.PAN cDNA One of the clones having the Called Sm. Label the CaSm cDNA insert for northern tumor and normal pancreatic tissue RNA To confirm increased expression levels in tumor cells by binding to blots and probes Used for The discovery of CaSm and other differentially expressed genes has diagnostic and disease progression. Facilitates row monitoring and molecular definition of specific stages of tumor growth Would be useful for This information also helps patients with prognosis and treatment options Will. In addition, the molecular definition of new genes involved in cancer is It will provide a new target for therapeutic intervention.   The compositions of the present invention described in the following sections provide recombinant mammalian CaS mDNA Offspring, cloned genes, or degenerate variants thereof. Further, in the present specification, Ca Nucleic acid probes useful for identifying Sm gene mutations and such Use of nucleic acid probes and modulation of CaSm gene expression in cancer cells Will be revealed. The composition of the present invention further comprises a CaSm gene encoded by the CaSm gene. Products (eg, peptides, proteins). The present invention also relates to a CaSm gene product. Or a conserved variant or fragment thereof. like that Antibodies measure levels of CaSm gene products in biological fluids and patient tissues Can be used for That is, the present invention also relates to the diagnosis and prognosis of cancer, particularly pancreatic cancer. Methods and kits for monitoring and staging, and monitoring the effectiveness of treatment. Includes   CaSm gene in identifying compounds that further modulate CaSm gene expression And / or uses of the CaSm gene product are provided. CaSm gene is used in various cancers It is a novel gene whose expression in cell lines and tissues is abnormal. Therefore Ca The Sm gene product is a mechanism underlying cancer onset and progression and cancer invasion and metastatic spread. May be involved in the introduction. That is, the present invention also provides for the prevention and / or treatment of cancer, and Providing a method for controlling metastatic spread of cancer based on modulation of CaSm expression You.                             5.1CaSm gene   The nucleic acid sequence of the identified CaSm gene is described herein. Full-length CaSm cDNA Partial cDNA clones identified by subtractive hybridization (CA3-30) (Schweinfest et al., 1990, Gene Anal. Techn., 7: 64-70; Scheweinfest et al., 1993, Proc. Natl. Acad. Sci. 90: 4166-4170).   The full-length cDNA clone was sequenced to nucleotides 878-883 as shown in FIG. It is a novel gene consisting of 894 nucleotides containing a polyadenylation signal. I understood. The translation initiation signal is a sequence containing the purines required at positions -3 and +4. TCAAAATGA(Nucleotides 160-168) (Kozak et al., 1991, J. Cell B) iol., 115: 887-903). The largest reading frame encodes a 133 amino acid polypeptide. (Nucleotides 165-163).   The CaSm cDNA clone was used as a plasmid in the E. coli DH5α strain in 1997. American Type Culture Collection (ATCC) with accession number 98497 ) (12301 Parklawn Drive Rockville, Maryland).   As used herein, the term “CaSm gene” refers to (a) described in FIG. E. coli D deposited with the American Type Culture Collection (ATCC) on the 11th DNA sequence contained in cDNA clone with ATCC accession number 98497 in H5α strain (B) described in FIG. 6 or on July 11, 1997, American Type C ATCC in E. coli DH5α strain deposited with the Culture Collection (ATCC) Encoding the amino acid sequence encoded by the cDNA clone having the number 98497. (C) as described in FIG. 6 or as published on July 11, 1997 by American Type Cu ATCC in E. coli DH5α strain deposited at lture Collection (ATCC) Phase of DNA sequence encoding amino acid sequence contained in cDNA clone No. 98497 Complement and high stringency conditions (eg, 0.5 M NaHPO_Four, 7% dodecyl sulfate Sodium (SDS), 1 mM EDTA at 65 ° C Hybridization and washing with 0.1 × SSC / 0.1% SDS at 68 ° C.) Any soybean DNA sequence (Ausubel F.M. et al. Eds., 1989, Current Protocols in Mo lecular Biology, Volume 1, Green Publishing Associates, Inc. And John Wi ley & Sons, Inc., New York, page 2.10.3); or (d) Is the large intestine deposited with the American Type Culture Collection (ATCC) on July 11, 1997 Contained in a cDNA clone of ATCC Accession No. 98497 in the E. coli strain DH5α. Of the DNA sequence encoding the amino acid sequence and moderate stringency Hybridization (Ausubel) under conditions (eg, washing at 42 ° C. with 0.2 × SSC / 0.1% SDS) Ed., 1989, supra) and described in FIG. 6 or on July 11, 1997, American Type ATCC in E. coli DH5α strain deposited with Culture Collection (ATCC) CaSm gene encoded by the sequence contained in the cDNA clone of No. 98497 Any DNA sequence that encodes a gene product that is functionally equivalent to the product.   In one embodiment of the present invention, the CaSm gene also has the fragment of the DNA sequence of (a) to (d). Pieces and degenerate variants, including naturally occurring variants thereof, may be included. CaSm remains A gene fragment comprises one or more intervening sequences or introns and a coding region. Contains control regions located beyond the 5 'or 3' end of the region or within the intron Genomic DNA molecules. One non-limiting example of a variant CaSm gene is Sm Mochi Amino acid residues corresponding to positions 22 to 32 of step 1 and all amino acid residues of Sm motif 2 Encodes a deleted CaSm gene product.   The CaSm gene sequence is preferably at the nucleotide level with the nucleic acid sequence described in FIG. It exhibits an overall similarity of at least about 80%, and more preferably the nucleic acid sequence of FIG. Showing at least about 85-90% overall similarity with the columns, most preferably as described in FIG. Shows at least about 95% overall similarity to the nucleic acid sequence.   The CaSm gene sequence of the present invention is preferably of mammalian origin, most preferably human. It is. Mammals include mice, rats, cats, dogs, cattle , Pigs, sheep, guinea pigs and rabbits.   The nucleic acid sequence of the human CaSm gene (SEQ ID NO: 1) has been deposited with GenBank and It is given the number AF000177.   The invention also relates to nucleic acid molecules encoding mutant CaSm, peptide fragments of CaSm, Truncated CaSm and CaSm fusion proteins are also included. With these nucleic acid molecules The encoded gene products are Sm motif 1 of CaSm, Sm motif 2 of CaSm, Sm motifs 1 and 2, or peptides corresponding to these portions; Is truncated CaSm lacking Sm motif 2 or both; one or more of CaSm Indicates that there are more amino acid residues, especially those in Sm motif 1 or Sm motif 2. Includes but is not limited to mutant CaSm that has been replaced or deleted. Like that The mutations in the CaSm mutants were Sm motif 1 and Sm mochi as shown in FIG. 3A. May also occur within the core consensus of the second party. Examples of such mutations are Glycine residue at position 13 in Sm motif 1 (amino acid residue 30 of CaSm) or Sm motif May occur at asparagine residue at position 23 (amino acid residue 40 of CaSm) .   The CaSm gene sequences of the present invention may further comprise high stringency or moderate stringency. At least about six of the CaSm gene sequences (a) to (d) Preferably about 12 and more preferably about 18 contiguous nucleotides. Isolated nucleic acid molecules.   The present invention also hybridizes to the DNA sequences (a)-(d) of the preceding paragraph, and thus Nucleic acid molecules, preferably DNA molecules, which are the complements of Such a hybrida As described above, the conditions for medium Agency. The nucleic acid molecule is a deoxyoligonucleotide ("oligo") In the case of high stringency conditions, for example, 6xSSC / 0.05% 37 ° C in thorium (for 14 base oligo), 48 ° C (for 17 base oligo), Washing at 55 ° C (for 20 base oligos) and 60 ° C (for 23 base oligos) Means You. These nucleic acid molecules are, for example, CaSm gene antisense useful for CaSm gene regulation. May encode or act as such. CaSm gene regulation Such methods modulate, for example, the phenotype and metastatic potential of cancer cells. Can be used to In addition, such sequences are also useful for CaSm gene regulation. May be used as part of a ribozyme and / or triple helix sequence.   Further, such molecules are used as components of diagnostic methods, where, for example, cancer is induced. Certain CaSm alleles or selections associated with causing or predisposing to cancer Alternatively, the presence of the spliced CaSm transcript may be deleted.   Furthermore, yeast systems (including but not limited to yeast two-hybrid systems) Inventions that include the CaSm gene as the screen in ().   The invention also relates to (a) any of the above CaSm coding sequences and / or their complements (eg, (B) CaSm coding sequence or antisense DNA vector Functionally related to control elements that direct the transcription and / or expression of a sense sequence A DNA expression vector containing the CaSm coding sequence of interest; and (c) Ca in the host cell. Controls that direct transcription and / or expression of Sm coding sequences or antisense sequences Created by genetic manipulation containing any of the above CaSm sequences functionally related to the element Host cells. The control elements herein are inducible and non-inducible To direct and control motors, enhancers, operators, and expression. Include, but are not limited to, other elements known to those of skill in the art. like that The control elements are cytomegalovirus (hCMV) immediate early promoter, SV40 adenovirus Ls early, late promoter, lac, TAC, TRC, phage A Operator and promoter regions required, fd coat protein control regions, Sphoglycerate kinase promoter, acid phosphatase promoter , And the promoter of yeast α-mating factor.   In addition to the above-mentioned CaSm gene sequence, the CaSm gene products present in other species Homologues of such sequences that exhibit extensive homology to those without undue experimentation. Can be easily identified and isolated by molecular biology methods known in the art. You. Genes encoding CaSm homologs include microorganisms such as yeast, nematodes (eg, Animals including Caenorhabditis elegans , Vertebrates, and mammals. Therefore, the present invention The reotide sequence is in reading frame J0 in cosmid F40F8 (GenBank accession number Z69302). Caenorhabditis elegance estimated from 714 (PIR S55137) (C.el. egans) and ORF YJL1124c in DNA clone accession number Z49399 Of the Saccharomyces cerevisiae gene product from Includes nucleotide sequences encoding CaSm homologs that do not load. Further CaSm Others in the genome that encode proteins with extensive homology to the gene product May have a homologous gene at the locus. These genes are also similar Can be identified by the method. Further alternatively spliced CaSm gene Variants may be present.   Using isolated human CaSm gene sequences, e.g., as disclosed herein, To clone animal CaSm gene homologues or variants, such human CaSm The gene sequence is labeled and appropriate cells or tissues obtained from the organism of interest (eg, , A cDNA library made from mRNA obtained from pancreatic epithelial cells) Used for For cloning such mammalian CaSm homologs Thus, for example, a mammalian cancer cell cDNA library is used for screening. Is also good.   The cDNA library is a different type of organism than the one from which the labeled sequence is obtained. The hybridization and washing conditions used when obtained from Agency. Low stringency conditions are known to those of skill in the art. Depending on the specific organism from which the library or labeled sequence is obtained. You. For guidance on such conditions, see, for example, Sambrook et al., 1989, Molecula. r Cloning, A Laboratoy Manual, Cold Springs Harbor Press, New York; And Ausubel 1989, Current Protocols in Molecular Biology, Green Publishing Assoc See iates and Wiley Interscience, New York   For cloning mammalian CaSm homologs using human CaSm sequences, for example, Use different stringency conditions to promote DNA hybridization Can be For example, hybridization at about 45 ° C in 6xSSC, then 2xSSC Washing at 50 ° C. in SC is used. Alternatively, the salt concentration during the washing step is about 5x at 50 ° C. Moderate stringency of about 2xSSC at 50 ° C from low stringency of SSC To a high stringency of about 0.2 × SSC at 50 ° C. Wash more The temperature of the purification process should be between room temperature and low stringency conditions to a medium stringency of about 42 ° C. From high stringency conditions to high stringency conditions of about 65 ° C. it can. Other conditions include 0.5M NaHPO_Four(PH 7.2) / 1 mM EDTA / 7% SDS at 68 ° C Or 50% formamide / 0.25M NaHPO_Four(PH 7.2) /. 25M NaCl / 1mM EDTA / Hybridize in 7% SDS, then 40 mM NaHPO_Four(PH 7.2) / 1mM EDTA / 5% SD Washing continues at 50 ° C. in S. Can change both temperature and salt, or One or other variables may be constant and others may vary.   Alternatively, the labeled fragments may again be obtained using appropriate stringency conditions known to those of skill in the art. Can be used to screen genomic DNA libraries of the organism of interest. You.   In addition, the CaSm gene homolog is an amino acid sequence within the CaSm gene disclosed herein. Using two degenerate oligonucleotide primer pools designed based on Perform polymerase chain reaction (PCR) to remove nucleic acids from the target organism May be isolated. The template for the reaction, for example, expresses the CaSm gene homolog or allele Prepared from mammalian cell lines or tissues known or suspected to be cDNA obtained by reverse transcription of mRNA may be used.   Confirm that the amplified sequence is the sequence of CaSm gene nucleotide sequence To do so, the PCR product may be subcloned and sequenced. Next, the PCR fragment Used to isolate full-length cDNA clones by various methods. For example, amplified breaks Label and use the pieces to create a cDNA library (eg, a bacteriophage cDNA library). Rally). Alternatively, labeled fragments can be obtained from the genomic library May be used to isolate genomic clones by screening.   PCR can be used to isolate full-length cDNA sequences. For example, RNA is a standard method A more suitable cell or tissue source (eg, known to express the CaSm gene). Or suspected, eg, pancreatic cancer cell line). The reverse transcription reaction Specific for the most 5 'end of the amplified fragment for priming first strand synthesis Performed on RNA using oligonucleotide primers. Obtained RNA The / DNA hybrid is then guanidized using a standard terminal transferase reaction. “Tail” with the enzyme, digest the hybrid with RNAase H, and then proceed to second strand synthesis. Prime with Re-C primer. That is, the cDNA sequence upstream of the amplified fragment is easily Separated. For a review of PCR and cloning methods, see, eg, PCR Primer, 1995. , Edited by Dieffenbach et al., Cold Spring Harbor Laboratory Press; Sambrook et al., 1989. See (supra).   The CaSm gene sequence further includes a CaSm gene allele and a mutant CaSm gene allele. May be used to isolate Such mutated alleles can cause cancer progression (metastasis) Is known or suspected to have a genotype that contributes to May be isolated from the individual. Next, the mutant allele and the mutant allele product Screening, therapeutic and diagnostic methods, and systems disclosed herein May be used. In addition, such CaSm gene sequences affect cancer progression and outcome Used to detect CaSm gene regulatory (eg, promoter) defects that can confer Can be   The cDNA of the mutated CaSm gene can be obtained using, for example, PCR which is a technique known to those skilled in the art. Isolated. In this case, the first cDNA strand is a mutant CaSm allele. Is known or suspected to be expressed in individuals presumed to have The oligo-dT oligonucleotide is hybridized to mRNA isolated from Alternatively, it may be synthesized by extending a new chain with a reverse transcriptase. Then cD Oligonucleotides that specifically hybridize the second strand of NA to the 5 'end of the normal gene Synthesized using nucleotides. Using these two primers, The product is amplified by PCR, cloned into a suitable vector and known to those skilled in the art. The DNA sequence is determined by the method described above. The DNA sequence of the mutant CaSm Loss of function or alteration of the mutant CaSm gene product compared to the The causative mutation can be identified.   Alternatively, an individual suspected or known to have a mutated CaSm allele Make a genomic library using DNA obtained from RNA from tissues known or suspected of expressing the mutant CaSm allele Can be used to create a cDNA library. Next, the normal CaSm gene or Will label any appropriate fragment thereof and the corresponding mutation in such a library. Used as a probe to identify different CaSm alleles. Next, mutation CaSm inheritance The clone containing the child sequence is purified and sequenced according to methods known to those skilled in the art. .   In addition, it is known or suspected to express, for example, a mutated CaSm allele. Expression libraries are created using cDNA synthesized from RNA isolated from Can be manufactured. In this way, the gene product created from the mutant allele is released. Reveals antibodies generated against the normal CaSm gene product (described below in Section 5.3). Can be screened in combination using standard antibody screening methods (For screening methods, see, for example, Harlow, E. and Lane, eds., 1988, "Antibo dies: A Laboratory Manual ", Cold Spring Harbor Press, Cold Spring Harbor Please refer to). CaSm mutation alters function (eg, missense or Or as a result of a frame shift), the CaSm gene The set of polyclonal antibodies to the product is a mutant CaSm May cross-react with gene products. Detection by reaction with such labeled antibody The resulting library clone is purified and sequenced using methods known to those skilled in the art. Can be determined.                     5.2CaSm Gene protein products   In another embodiment, the invention relates to the production of antibodies for diagnostic assays or to cancer (eg, pancreatic). Can be used to identify other cellular gene products involved in the development of An Sm gene product or a peptide fragment thereof is provided.   The amino acid sequence shown in FIG. 6 shows the CaSm gene product. CaSm gene product (this specification Is referred to interchangeably as "CaSm protein") is the mammalian CaSm gene Product, including the gene product encoded by the CaSm gene sequence described in Section 5.1 above. It further includes things.   In one embodiment, the CaSm gene product of the present invention shown in FIG. And has a predicted molecular weight of 15,179 daltons and an isoelectric point of 4.97. Predicted The amino acid sequence of the full-length CaSm gene product Contains no transmembrane domain, which indicates that the CaSm gene product is an intracellular protein It is shown that.   This 133 amino acid CaSm polypeptide is shared with the snRNP Sm G protein (Figure 3A). Have significant homology. Computerized BEST of CaSm and human Sm G protein FETs are 32% identical and 60% similar (allows conservative amino acid substitutions) . This similarity is almost completely restricted to the amino-terminal half of CaSm (amino acids 4-78). Is defined. Interestingly, this homology is characteristic of the Sm protein family. (Hermann et al., 1995, EMBO J., 14: 207). 6-2088). Sm motif 1 and Sm motif 2 (amino acid residues 18-49 and 61-74, respectively) ) Is involved in protein-protein interactions and is probably the assay of the snRNP complex. May be required for assembly (Hermann et al., 1995, EMBO J., 14: 2076-2088). Many important features that make up the Sm motif are retained in CaSm. In particular Consensus 13th and 23rd of Sm motif 1 are 100% conserved respectively The glycine and asparagine residues also correspond to amino acids 30 and 40, respectively, in CaSm. There are also places. Overall 15 defined positions in the consensus of Sm motif 1 Twelve of the locations are stored in CaSm. Furthermore, in the Sm motif 2 consensus Ten of the eleven defined locations are also conserved in CaSm (see FIG. 3A). . Gene production of Caenorhabditis elegans And the inheritance of Saccharomyces cerevisiae The offspring product has even greater similarity to CaSm (72.8% and 67.7%, respectively, FIG. 3B And 3C). These two gene products also contain the Sm motif, which In these regions, it is almost the same as CaSm. In addition to CaSm mammalian homologs, These two gene products, which have similar molecular weights, are the non-mammalian species of CaSm in each organism. Homolog. Therefore, in the present invention, the CaSm homolog is a cosmid F40F8 (GenBank acceptor). No. Z69302) Kaenorhub estimated from reading frame J0714 (PIR S55137) Not a C. elegans gene product, but a DNA clone Saccharomyces cerevisiae ORF YJL124c coded by accession number Z49399 All mammalian and non-saccharomyces cerevisiae gene products And non-mammalian CaSm homologs.   The predicted reading frame (ORF) of CaSm is a set of in vitro transcription and translation reactions. It was confirmed by the expression in the combination. Putative coding strand is 18 kDa polypep Translates the tide, while the putative non-coding strand produces a much smaller product. further Only the antisense probe to the putative coding strand, mR taken from pancreatic cancer cells Hybridizes with NA.   The CaSm gene product of the present invention controls cell growth by post-translational regulation of gene expression. Related to cellular mechanisms. In particular, CaSm promotes mRNA translation and / or Are involved in the inhibition of jar RNA degradation, both of which are on the 5 'end of eukaryotic mRNA. Includes a synergistic interaction between the denylated (poly (A)) mRNA tail and the cap structure Can be Such an interaction can result in (i) an mRNA cap, such as a ribosome, 'Start translation at end The complex eIF-4F, which contains the large subunit eIF4G and is (Containing eIF4A); and (ii) a protein that binds to the poly (A) tail. When combined, poly (A) binding facilitates assembly of the 40S ribosomal subunit into mRNA. Is known to be mediated by a protein bound to the synthetic protein Pablp ing. Tarun et al., 1996, EMBO J 15: 7168-7177; and Tarun et al., 1997, Proc. N atl. Acad. Sci. 94: 9046-9051. Homologous CaSm gene production in yeast (Encoded by ORF YJL11111111124c) within the Pablp gene (especially Pablp , In yeast cells containing mutations in the eIF-4E and eIF-4G genes) It is a bypass suppressor. Without being bound by theory, this observation is due to the fact that CaSm Gene products perform part of Pablp's function or promote translation, Pablp and eIF- Active in a pathway parallel to the interaction between 4G. Therefore, it promotes mRNA translation and suddenly The ability of CaSm homologs to rescue the death of mutant yeast cells is The emergence of a transformed phenotype due to overexpression of the mammalian CaSm gene product This is consistent with the observations described above.   In addition, CaSm gene products are proteins that are functionally equivalent gene products (all Mammalian and non-mammalian CaSm gene products). That's it Such an equivalent CaSm gene product is encoded by the CaSm gene sequence described in Section 5.1 above. Changes in the amino acid sequence (ie, functionally equivalent CaSm residues) Contains deletions, additions or substitutions of amino acid residues that cause May be. Amino acid substitutions are dependent on the polarity, charge, solubility, hydrophobicity, It is based on aqueous and / or amphoteric similarities. For example, non-polar (hydrophobic ) Amino acids are alanine, leucine, isoleucine, valine, proline, phenyl Including lualanine, tryptophan, and methionine; polar neutral amino acids Glycine, serine, threonine, cysteine, tyrosine, asparagine, and And glutamine; positively charged (basic) amino acids are arginine, lysine And histidine; and negative Charged (acidic) amino acids include aspartic acid and glutamic acid. Sm Conservative and non-conservative amino acids in the core consensus of chief 1 and / or 2 Acid substitution (eg, a conserved glycine residue at position 13 of Sm motif 1 or Sm mochi Conserved asparagine residue at position 23 of GF1 (see FIG. 3A). No).   As used herein, "functionally equivalent" means that the protein is as described in section 5.1 above. Substantially similar to the endogenous CaSm gene product encoded by the CaSm gene sequence of  It means that it can show in vivo activity. CaSm inheritance as used herein The in vivo activity of the progeny product means that it is present in the appropriate type of cells (eg, pancreatic cells) Is associated with the appearance of a preneoplastic or neoplastic phenotype of the cell when .   The CaSm gene product preferably has the amino acid sequence shown in FIG. At least about 80% overall similarity, more preferably the amino acid sequence as set forth in FIG. And at least about 90% overall similarity, and most preferably the amino acid described in FIG. Shows at least about 95% overall similarity to the acid sequence.   The CaSm gene products include peptide fragments of CaSm, truncated CaSm, and Naturally, variants can be included. These include CaSm Sm motif 1 and CaSm Sm Chief 2 or peptides corresponding to these parts; Sm motif 1 or Sm motif Truncated CaSm with or without trough 2 or both. Absent. The mutant CaSm peptide fragment is within the core consensus of Sm motifs 1 and 2. May contain one or more conservative or non-conservative amino acid substitutions.   The CaSm gene product can also be a CaSm gene described in this section that is operably linked to a heterologous component. A fusion protein containing the product sequence can be included. The heterogeneous component is a fusion tan Isolation of proteins (eg, matrix binding domains) or detectable labels Includes, but is not limited to, sequences that facilitate purification. Such isolation and labeling The minutes are known to those skilled in the art. For example, a CaSm-green fluorescent protein fusion (CaSm -GFP) is a CaSm protein It is expressed in cells to facilitate the localization and study of cellular intracellular trafficking.   The CaSm gene product or its peptide fragment or fusion protein Any assay that detects or measures the product, or the calibration and calibration of such an assay Can be used for standardization.   The CaSm gene product or peptide fragment thereof is isolated from a cellular source, Alternatively, it is produced by recombinant DNA technology using methods known in the art. sand That is, expression of the nucleic acid containing the CaSm gene sequence allows the production of the CaSm gene of the present invention. Methods for preparing products and peptides are described herein. The minute The CaSm gene product coding sequence and appropriate transcription and translation are determined using methods known in the art. Expression vectors containing control signals can be constructed. These methods are Examples include in vitro recombinant DNA technology, synthetic methods, and in vivo genetic recombination. See, for example, Sambrook et al., 1989 (supra) and Ausubel et al., 1989 (supra). Alternatively, use a synthesizer to convert RNA that can encode the CaSm gene product sequence And can be chemically synthesized. For example, "Oligonucleotide Synthesis", 1984, G ait, M.J., ed., IRL Press (Oxford). (Herein incorporated by reference).   Various host-expression vector systems can be used to express the CaSm gene coding sequence of the present invention. Can be used. Such a host-expression system produces the coding sequence of interest. Vehicle to be subsequently recovered and / or purified, but with the appropriate nucleotide When transformed or transfected with a nucleic acid sequence, the CaSm It is also a cell that shows a gene product. These contain the CaSm gene product coding sequence Recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors Bacteria transformed with the bacterium (for example, E. coli, Bacillus su microorganisms such as Btilis); recombinant yeast expression containing the CaSm gene product coding sequence Yeast transformed with a vector (eg, Saccharomyces (Saccharomyces), Pichia); contains CaSm gene product coding sequence Insects infected with a recombinant viral expression vector (eg, baculovirus) Cell line; a recombinant viral expression vector containing the CaSm gene product coding sequence (eg, For example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) Or recombinant plasmid expression vector (eg Ti plasmid) Plant cell lines transformed with; mammalian cells (eg, metallothionein promoter). Genomic or mammalian virus (eg, adenovirus late promoter) Contains a promoter obtained from vaccinia virus 7.5K promoter) Mammalian cell lines (eg, COS, CHO, BHK, 293, 3T3), but is not limited thereto.   In bacterial systems, many expression vectors are used, depending on the intended use of the expressed CaSm gene product. Are advantageously selected. For example, let's produce such proteins in large quantities If, for the preparation of a pharmaceutical composition containing CaSm protein, or CaSm To produce antibodies to proteins, fusion proteins that can be easily purified Vectors that direct high level expression of the product will be preferred. Such a bee The E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2). : 1791) (lacking the CaSm gene product coding sequence to lac PIN vector (individually linked in frame with the Z coding region and the vector); ー (Inouye & Inouye, 1989, Nucleic Acids Res. 13: 3101-3109; Van Heeke & Schuster, 1989, J.M. Biol. Chem. 264: 5503-5509); Not determined. The pGEX vector is also a glutathione S-transferase (GST ) Can be used to express foreign polypeptides as fusion proteins. Wear. Generally, such fusion proteins are soluble and can be lysed from lysed cells. Rix glutathione-adsorbed or bound to agarose beads followed by free glutathione Elution in the presence of tathione allows easy purification. pGEX The cloner must release the cloned target gene product from the GST moiety. It is designed to include a thrombin or factor Xa protease cleavage site.   Autographa californica in insect systems Nucleosomal virus (AcNPV) is used as a vector to express foreign genes Is done. The virus is Spodoptera frugiperda ) Proliferate in cells. The CaSm gene coding sequence is a non-essential region of the virus (eg, Cloned separately in the polyhedrin gene) and (Eg, a polyhedrin promoter). CaSm gene co Successful insertion of the nucleic acid sequence inactivates the polyhedrin gene, and Recombinant virus (ie, proteinaceous encoded by the polyhedrin gene) Virus that lacks a coat) is produced. These recombinant viruses are then Used to infect Spodoptera frugiperda cells (Where the inserted gene is expressed) (see, eg, Smith et al., 1993, J. . Virol. 46: 584; Smith, U.S. Patent No. 4,215,051).   In mammalian host cells, a number of viral-based expression systems are available. Expression When adenovirus is used as a vector, the CaSm gene coding sequence Denovirus transcription / translation control complex (eg, late promoter and three-component Sequence). This chimeric gene is then used in vitro or in vivo. o Inserted into the adenovirus genome by recombination. Non-essential of the viral genome Insertion into a region (eg, region E1 or E3) is viable and infected A recombinant virus capable of expressing the CaSm gene product in the host is obtained (eg, For example, Logan & Shenk, 1984, Proc. Natl. Acad. Sci. See USA 81: 3655-3659. I want to.)   In certain embodiments, retroviral vectors containing the CaSm gene Is used. See, for example, Miller et al., 1993, Meth. Enzymol. 217: 581-599 I want to. These retroviral vectors provide viral genome packaging and And for integration into host cell DNA Modified to delete retroviral sequences that are not required for In gene therapy The CaSm gene used is cloned into a vector, which To facilitate the delivery of For more information on retroviral vectors, see Boesen Et al., 1994, Biotherapy 6: 291-302, which treat stem cells with chemotherapy. To deliver the mdrl gene to hematopoietic stem cells in order to make it more resistant to Describes the use of rovirus vectors. Retro virus vector Other references illustrating use include Clowes et al., 1994, J. Am. Clin. Invest. 93: 644-651 Kiem et al., 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene. Therapy 4: 129-141; and Grossman and Wilson, 1993, Curr. Opin. Genetics an d Devel. 3: 110-114, there is.   Specific initiation sequences are required for efficient translation of the inserted CaSm gene product coding sequence. A gun is also needed. These signals include the ATG start codon and adjacent sequences. The complete CaSm gene, including the initiation codon and adjacent sequences, is inserted into an appropriate expression vector. If so, no additional translation control signals are required. However, the CaSm gene coding sequence If only a portion is inserted, an exogenous translational control signal (perhaps an ATG initiation Including the don) must be provided. In addition, the start codon is In the same phase as the reading frame for the desired coding sequence to ensure translation of the I have to. These exogenous translational control signals and initiation codons are native and Obtained from various sources of synthesis. Appropriate transcription enhancer element, transcription terminator Can increase the efficiency of expression (Bittner et al.) 1987, Methods in Enzymol. 153: 516-544).   Further, it modulates the expression of the inserted sequence in the particular manner desired or A host cell strain that modifies and processes the gene product may be selected. Tampa Such modifications (eg, glycosylation) and processing (eg, Cleavage, for example, is important for the function of the protein. Different host cells Post-translational processing of proteins and gene products It has a characteristic and specific mechanism of processing and modification. Foreign protein expressed The appropriate cell line or host system to ensure correct modification and processing of You can choose. To this end, the appropriate processing of the primary transcript of the gene product Eukaryotic host cells with cellular mechanisms of singing, glycosylation, and phosphorylation May be used. Such mammalian host cells include CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and especially for example, CAPAN-1, CAPAN-2, ASPC-1, P Cancer cell lines such as ANC-1 and HPAC, and normal pancreas such as HS680.PAN But not limited thereto.   For long-term, high-yield production of recombinant proteins, stable expression is preferred. example For example, a cell line stably expressing the CaSm gene product may be prepared. Virus replication start Rather than using an expression vector containing points, appropriate expression control elements (eg, Motor, enhancer, sequence, transcription terminator, polyadenylation site Can transform host cells with DNA and selectable markers You. After introduction of the foreign DNA, the prepared cells are grown for 1-2 days in a nutrient-supplemented medium. And then change to selective medium. The selectable marker in the recombinant plasmid The cell stably integrates the plasmid into its chromosome and proliferates Allows the formation of foci, which are then cloned and proliferated to Become a stock. This method is useful for generating cell lines that express the CaSm gene product. Can be used. Such engineered cell lines harbor endogenous CaSm gene products. It may be particularly useful for screening and evaluating compounds that affect the activity of the.   Many selection systems are available, for example, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosi Transferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. . USA 48: 2026), and adenine phosphoribosyltransferase (Lowy et al. , 1980, Cell 22: 817) gene in tk-, hgprt- or aprt- cells, respectively. You can use It is not limited to these. Also uses antimetabolite resistance as a basis for selection of the following genes: Can be: dhfr, which confers resistance to methotrexate (Wigl er et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl. Ac ad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); Confers resistance to the aminoglycoside G-418 (Cloberre-Garapin et al., 1981, J. . Mol. Biol. 150: 1); and hygro, which increases resistance to hygromycin (Santerre et al., 1984, Gene 30: 147).   Alternatively, using an antibody specific for the fusion protein to be expressed, The fusion protein can be easily purified. For example, Janknecht et al. System facilitates the purification of non-denatured fusion proteins expressed in human cell lines. Enable (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). . In this system, the gene reading frame is an amino acid consisting of six histidine residues. The gene of interest is a vaccinia recombinant plasmid so that it is Subcloned into sumid. Cells infected with recombinant vaccinia virus Extract from the cells²⁺-Nitriloacetic acid-agarose column, histidine The proteins tagged with are selectively eluted with a buffer containing imidazole.   The CaSm gene product can also be expressed in transgenic animals. Animals of any species (mouse, rat, rabbit, guinea pig, sheep, pig, minib Including bats, goats, and non-human primates, such as baboons, monkeys, and chimpanzees (But not limited to) to create CaSm transgenic animals Can be Non-mammalian homologues of CaSm also transgenic organisms Enorhabditis elegans and Saccharo Including, but not limited to, Saccharomyces cerevisiae No) can be expressed.   The CaSm transgene is introduced into the animal using any method known in the art. To create a founder line of transgenic animals. This Such methods include the pronuclear microinjection method (Hoppe, PC and Wagner, TE, 1989, US Patent No. 4,873,191); Retrovirus-mediated gene transfer into the germ line (Van der  Putten et al., 1985, Proc. Natl. Acad. Sci. USA 82: 6148-6152); in embryonic stem cells Gene targeting (Tompson et al., 1989, Cell 56: 313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3: 1803-1814); and sperm-mediated gene transfer (La. vitrano et al., 1989, Cell 57: 717-723); and the like. For a review of such methods, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115: 171-229, this is by reference in its entirety. And part of this specification.   The present invention relates to transgenic plants having the CaSm transgene in all of their cells. Transgenes in some but not all cells An animal (ie, a mosaic animal) is provided. Transgene is a single trans As a gene or in concatamers, eg head-to-head tandem or head -To-tail incorporated in tandem. The transgene is also used, for example, by the teachings of Lasko et al. It is selectively introduced and activated into specific cell types as indicated (Lasko et al., 1992 Proc. Natl. Acad. Sci. USA 89: 6232-6236). Such cell type specific activity The control sequences required for activation are dependent on the particular cell type of interest and will be apparent to those skilled in the art. . CaSm gene transgene integrates into the chromosomal site of the endogenous CaSm gene Is preferred, gene targeting is preferred. This is a simple explanation When such a method is used, endogenous CaSm inheritance through homologous recombination with chromosomal sequences Homologous to endogenous CaSm gene to introduce and disrupt function of offspring nucleotide sequence Design a vector containing several nucleotide sequences. Transgene Can also be selectively introduced into specific cell types, thus, for example, as described by Gu et al. (Gu et al. 94, Science 265: 103-106), and remove the endogenous CaSm gene only in that cell type. Activity Become The regulatory sequences required for such cell type-specific inactivation may be of particular interest. It depends on the cell type and will be apparent to those skilled in the art.   Methods for generating single copy transgenic animals with selected integration sites are also It is known to those skilled in the art. See, for example, Bronson et al., 1996, Proc. Natl. Acad. Sci. USA 93: 9067-9072, which is incorporated herein by reference in its entirety. Part).   Once a transgenic animal or organism has been created Thus, expression of the recombinant CaSm gene is performed using standard methods. First screen The animal is analyzed by Southern blot analysis or PCR to analyze animal tissues. This is done by measuring whether the incorporation of the gene has occurred. Trance The level of transgene mRNA expression in the tissue of the transgenic animal or organism Northern blot analysis of tissue samples obtained from different animals or organisms, in situ Including, but not limited to, hybridization analysis, and RT-PCR ) Evaluated using the method. Samples of CaSm gene expression tissues also It is assessed immunologically using antibodies specific for the gene product.                      5.3CaSm Antibodies to gene products   In another embodiment, the invention relates to an epitope of a CaSm gene product, Conserved mutant epitopes of the product, mutant CaSm gene product epitopes, Is an antibody that can specifically recognize one or more peptide fragments of the CaSm gene product. Also encompasses the body or fragments thereof. Such antibodies include polyclones Null antibody, monoclonal antibody (mAbs), human or chimeric antibody, single chain antibody, Fa b fragment, F (ab ') 2 fragment, Fv fragment, Fab expression library Fragments, anti-idiotype (Anti-Id) antibodies, and There are, but are not limited to, some epitope binding fragments.   These antibodies can be used, for example, to detect CaSm gene products in biological samples. And therefore used as part of a diagnostic and prognostic technique The patient's CaSm gene product is at abnormal levels, And / or to check for abnormal forms of the gene product Can be. Such antibodies may be useful as reagents in kits for diagnostic or prognostic techniques. Can be used as one. Such antibodies are also described, for example, in Section 5.4.2 below. As described in the above, effects on the CaSm gene product level and / or activity of the test compound It can be used in compound screening to assess effects. is there Alternatively, such antibodies may be relevant to the gene therapy techniques described in Section 5.4.3 below. For example, prior to introducing normal and / or modified CaSm-expressing cells into a patient. Can be used to evaluate   Furthermore, antibodies against anti-CaSm gene product suppress abnormal CaSm gene product activity For the method. Therefore, such antibodies are one of the cancer treatment methods. It can be used as a part.   Described herein are methods for producing such antibodies and fragments thereof. is there. Any such antibody or fragment thereof may be used for standard immunization. Encoding the antibody or fragment in a suitable host organism Can be produced by expressing a nucleic acid molecule by genetic recombination technology .   In order to produce an antibody against the CaSm gene product, the CaSm gene product or its Various host animals can be immunized by injecting the fragments. Known Ca It is possible to synthesize fragments of CaSm as antigenic peptides based on the amino acid sequence of Sm it can. Such host animals include rabbits, mice, and rabbits, to name a few. But not limited to them. To increase the immune response, the host animal Depending on the type of inorganic material, such as Freund (complete and incomplete) and aluminum hydroxide , Lysolecithin and other surfactants, pluronic polyols, polyanions , Peptide, fat emulsion, keyhole limpet hemocyanin, dinitrofe Knoll, and BCG (Bacille Calmette-Guerin) and Corynebacterium parvum Including but not limited to potentially useful human adjuvants Various adjuvants can be used.   Polyclonal antibodies are derived from the sera of animals immunized with an antigen such as the CaSm gene product. The heterogeneity of antibody molecules derived from them, or derivatives of those molecules with antigenic function It is a set. For example, a polyclonal antibody is an amino acid sequence of the CaSm protein. For synthetic peptides having the sequence of amino acid residues at positions 79-89 and 115-133 Made. For production of polyclonal antibodies, the host animal as described above is Immunization was performed by injecting the CaSm gene product that captured the adjuvant.   A monoclonal antibody is a homogeneous collection of antibodies to a particular antigen, Any production of antibody molecules by a continuous cell line It can also be obtained by techniques. Such techniques include Kohler and Milstein (1975, Nature 256: 495-497; and US Patent No. 4,376,110) , Human B cell hybridoma method (Kosbor et al., 1983, Immunology Today 4:72; Cole et al. , 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030), and EBV-hybrid (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Lis. s, Inc. pp.77-96), but are not limited thereto. Such antibodies are immunoglobulin Any class of IgG, IgM, IgE, IgA, IgD of Brin May be a subclass of The hybridoma producing the mAb of the present invention is in vitro o or may be cultured in vivo. Producing high titer mAbs in vivo is currently However, it is the preferred method of its production.   Furthermore, a gene derived from a mouse antibody molecule having appropriate antigen specificity is By splicing together with a gene derived from an active human antibody molecule Techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. A cad. Sci. 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al. 1985, Nature, 314: 452-454). Chimeric antibody is a mouse mAb A molecule having a variable region derived from Different molecules derived from different animal species, such as antibodies A molecule that has a moiety.   Alternatively, the method described as a technique for the production of single chain antibodies (US Patent No. 4,946,778; Bird, 1988, Science, 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature, 334: 544-54. 6) can be applied to the production of single-chain antibodies against the CaSm gene product. Single chain The body binds the heavy and light chain fragments of the Fv region via one amino acid. To form a single-chain polypeptide. In E. coli , Technique for Assembly of Functional Fv Fragments (Skerra et al., 1988, Science  242: 1038-1041) can also be used.   Antibody fragments that recognize specific epitopes can be made using known techniques. it can. For example, such fragments include pepsin digestion of antibody molecules. F (ab ') 2 fragments that can be produced, and disulfides of F (ab') 2 fragments Fab fragments obtained by reducing linkages include but are not limited to Not. Alternatively, a monoclonal Fab fragment with the desired specificity Fab expression libraries can be constructed to quickly and easily identify (Huse et al., 1989, Science, 246: 1275-1281).               5.4CaSm Use of genes, gene products, and antibodies   In various embodiments, the present invention relates to a CaSm gene, a CaSm gene product (a peptide thereof). Peptide fragments), and anti-CaSm gene products and peptide fragments thereof. Provides various uses of the body. Examples of such uses are described below. There is a prognostic and diagnostic evaluation of such cancers, and the identification of subjects predisposed to cancer.   In one embodiment, the present invention provides a variety of diagnostic and prognostic evaluations for cancer. Provide a way. Such a method can be performed, for example, using the CaSm gene nucleic acid described in Section 5.1. Otide sequences and the CaSm gene products (including peptide fragments thereof) described in Section 5.2 above. Reagents such as antibodies to it can.   In particular, such reagents include, for example: (1) detecting the presence of a mutation in the CaSm gene, or Or over- or under-expression of CaSm gene mRNA before or during tumor development Detection compared to normal cells or susceptibility to cancer or neoplastic changes Or qualification of other alleles of CaSm transcripts that may be associated with Quantitative detection, and (2) detection of excess CaSm gene product compared to non-disease states, or Or repairs associated with the presence of the tumor or progression or metastasis to the tumor It can be used to detect the presence of a decorated CaSm gene product (e.g., shorter than full-length). Wear.   The methods described herein may be used for cell samples or cellular samples taken directly from a patient. Applicable to sample of material. As a method for collecting and isolating desired cells or substances Any method known in the art can be used. Especially for pancreatic cancer For example, endoscopic retrograde cholangiopancreatography (ERCP) to collect pure pancreatic juice, Or percutaneous fine needle aspiration biopsy performed with pancreatic ultrasound imaging Obtain test samples using techniques known in the art such as e aspiration biopsy) be able to.   The methods described herein include, for example, at least one specific CaSm gene nucleic acid. Or a pre-packaged anti-CaSm gene antibody reagent as described herein. Can be performed by using a diagnostic test kit Patients with abnormalities before or at the onset of tumor Diagnosis and screening of humans predisposed to such neoplastic changes And can be conveniently used for identification.   The present invention relates to a method wherein a CaSm gene or gene product is related or suspected to be related. Useful for diagnosis and prognosis of certain malignant diseases. Such malignancies include the pancreas Cancers of the liver, ovaries, lungs, bladder, kidneys, colon, rectum, prostate, and cervix, Rabbits include, but are not limited to, mesothelioma. Detection technology based on nucleic acids The law is described below in Section 5.4.1. Peptide detection techniques are described in Section 5.4.2 below. It has been described.                      5.4.1CaSm Gene nucleic acid molecule detection   Mutations or polymorphisms in the CaSm gene can be detected using a number of techniques. The squid Nucleic acids from different nucleated cells can also be used as starting material for such assays And can be isolated by standard nucleic acid preparation methods well known to those skilled in the art. it can. As a starting source of genomic nucleic acids for the detection of CaSm mutations, Nucleated cells can also be used. Detection of CaSm transcripts or CaSm gene products For example, the CaSm gene in pancreatic cancer cells (including those that have metastasized) Any cell type or tissue available can be used.   Genomic DNA contains CaSm fragments, including point mutations, insertions, deletions, and chromosomal rearrangements. Hybridization of biological samples or detection of abnormalities in gene structure Can be used in amplification assays. Such assays include direct sequencing Determination method (Wong, C. et al., 1987, Nature, 330: 384-386), single-chain conformational Single stranded conformational polymorphism analysis (SSCP; Or ita, M .; Et al., 1989, Proc. Natl. Acad. Sci. USA 86: 2766-2770), heteroduplex Analysis (heteroduplex analysis) (Keen et al., 1991, Genomics, 11: 199-205; Perry, D .; J. & Carrell, R.W., 1992), denaturing gradient gel electrophoresis  electrophoresis) (DGGE; Myers, R.M. et al., 1985, Nucl. Acids Res. 13: 3131-31. 45), chemical mismatch cleavage (Cotton et al., 1988, Pr. oc. Natl. Acad. Sci. USA 85: 4397-4401), and oligonucleotide hybrids. Dilation (Wallace et al., 1981, Nucl. Acids Res. 9: 879-894; Lipshutz et al., 1). 995, Biotechniques, 19: 442-447) and the like.   A sample obtained from the patient (such as pancreatic juice or serum) or other appropriate cell supply Diagnostic methods for the detection of CaSm gene-specific nucleic acid molecules in a source include specific genes Amplification of sequences, for example by polymerase chain reaction (PCR; Mullis, K.B., 198 7; see U.S. Patent No. 4,683,202), but Yes, after amplification, the amplified molecule can be combined with techniques well known in the art, such as those described above. Analysis using such techniques as described above. Using these analytical techniques In order to determine whether the amplified sequence has a mutation in the CaSm gene, Assuming that the amplified nucleic acid contains only a normal copy of the CaSm gene Can be compared to the expected sequence.   In addition, well-known genotyping techniques are closely linked to mutations in the CaSm gene itself. Can be done to separate the types of sexual polymorphisms. These polymorphisms It can be used to identify individuals in a family who are thought to have the mutation. Can be. If the polymorphism shows linkage disequilibrium with a mutation in the CaSm gene, The polymorphism is used to identify individuals in the population who may have the mutation. It can also be used for The polymorphisms that can be used in this way include Fragment length polymorphism (RFLP), which includes sequence variations in the target sequence of the restriction enzyme ), Single nucleotide polymorphism, and simple sequence repeat polymorphism (SSLP).   For example, Weber (U.S. Pat.No. 5,075,217, which is incorporated herein by reference in its entirety). Is incorporated in (dC-dA) n- (dG-dT) n short tandem repeat blocks. Describes DNA markers based on length polymorphisms. (dC-dA) n- (dG-dT) The average separation length of n blocks is estimated at 30,000-60,000 bp. Very mutually Markers in close proximity indicate a high frequency of co-inheritance, such as Identification of genetic mutations, such as mutations in the CaSm gene, and CaSm mutations It is very useful in diagnosing related diseases and disorders.   Also, Caskey et al. (U.S. Pat.No. 5,364,759, which is hereby incorporated by reference in its entirety). (Incorporated in the text) is for the detection of short repetitive sequences of tri- or tetranucleotides. For DNA profiling assays. The process is an example For example, the target DNA such as CaSm gene is extracted, the extracted DNA is amplified, and the repetitive sequence Is used to generate a genotype map of each DNA.   In addition, CaSm probes can be used to directly identify RFLP. Furthermore, A CaSm probe or primer derived from the CaSm sequence is used for YAC, BAC, PAC, Used to isolate genomic clones such as mids, phages, or plasmids sell. The DNA contained in these clones is subjected to standard hybridization or Or single nucleotide polymorphisms or simple sequence length polymorphisms (SSLPs) using sequencing techniques? Can be screened.   Another diagnostic method for detecting CaSm gene-specific mutations or polymorphisms includes: For example, a nucleic acid and one or more labeled nucleic acid reagents may be labeled with a C Contact and contact under favorable conditions to specifically anneal to sequences within the aSm gene. And hybridization techniques. The nucleic acid may be a recombinant DNA molecule, a sample (eg, a patient sample or its Cloned genes from samples from other suitable cellular sources) , Or degenerate variants thereof, and as a labeled nucleic acid reagent, the combination described in Section 5.1. Includes recombinant DNA molecules, cloned genes or degenerate variants thereof. Preferably The length of these nucleic acid reagents is at least 15 to 30 nucleotides. Incube After the hybridization, remove all unannealed nucleic acids from the nucleic acid: CaSm molecular hybrid. Leave. The presence of the hybridized nucleic acid, if such a molecule is present, Is detected. Using such a detection scheme, the cell type or tissue Nucleic acids can be deposited, for example, on membranes or on microtiter plates or polystyrene vials. It can be immobilized on a solid support, such as a plastic surface, such as on a substrate. In this case, do not anneal after incubation, of the type described in section 5.1. The target nucleic acid reagent is easily removed. Remaining Annealing Label Ca Detection of the Sm nucleic acid reagent is performed using standard techniques well known in the art. Nucleic acid Is the CaSm gene sequence in which the reagent is annealed a CaSm gene mutation? To confirm, the annealing pattern expected from the normal CaSm gene sequence Be compared.   Quantitative and qualitative aspects of CaSm gene expression can also be assayed. example If the CaSm gene is expressed in pancreatic cancer cells (including those that have metastasized), RNA from a cell type or tissue that is or is suspected to be It can be tested using the hybridization or PCR techniques described above. So Can be from cell culture or from a patient. culture The analysis of cells harvested from material is part of a cell-based gene therapy technique. To investigate the effect of compounds on use or alternatively on CaSm gene expression This would be a necessary step in the evaluation of cells for Such a minute Analysis activates or inactivates CaSm gene expression and otherwise splices Transcripts [for example, amino acid residues 39-75 (this is the last 11 of Sm motif 1) Amino acids and all of Sm motif 2) were deleted. Quantitative and qualitative aspects of CaSm gene expression pattern Will.   In one embodiment of such a detection scheme, the reverse is performed from the RNA molecule of interest. CDNA molecules are synthesized by transcription. The resulting cDNA is either full-length or Is used as a template for a nucleic acid amplification reaction such as PCR. Reverse transcription and Used as a synthesis initiation reagent (e.g., primer) in the nucleic acid amplification step The nucleic acid reagent is selected from the CaSm gene nucleic acid reagents described in Section 5.1. Like that Preferably, the length of the nucleic acid reagent is at least 9-30 nucleotides.   In order to detect amplified products, radiolabeled or non-radioactive A nucleic acid amplification method can be performed using labeled nucleotides. Alternatively, Use either standard ethidium bromide staining or other appropriate nucleic acid staining method Enough amplification products can be made so that they can be visualized.   Such RT-PCR techniques can be used to replace normal splicing or Used to detect differences in size of CaSm transcripts likely due to icing be able to. In addition, such techniques are not Or full-length and higher levels detected in healthy individuals compared to individuals predisposed to neoplastic changes. And / or to detect quantitative differences in alternatively spliced CaSm transcripts , Using standard techniques.   If it is desired to detect a particular species that has been spliced into another form, such a arrangement may be used. Appropriate primers and / or Can use a hybridization probe. Alternatively, use Depends on the presence or absence of specific exons in the CaSm transcripts Using the sequence data shown in FIG. 6 to select primers that yield fragments of To select a pair of primers.   As an alternative to amplification techniques, the standard no Zan analysis can be performed. Quantitative analysis between CaSm transcripts using such techniques Differences in size and size can be detected.   Furthermore, such CaSm gene expression assays can be performed "in situ", i.e., A section of patient tissue obtained by examination or resection (fixed and / or frozen Can be performed directly on the substrate, in which case there is no need to purify the nucleic acid. Becomes Nucleic acid reagents such as those described in Section 5.1 can be used for such in situ procedures. Probes and / or primers (eg, Nuovo, G.J., 1 992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press , NY).   The results obtained by the methods described herein may be relevant to the pathology of cancer. Can be combined with diagnostic test results based on other genes. example For example, in pancreatic cancer, a K-ras mutation could be diagnosed clinically in 18 months (1995, Berthelemy et al., Ann. Intern. Med., 123: 188-191). Similarly, K-ras bumps account for 24% of the proliferative foci tested. Mutations were observed (1996, Tada et al., Gastroent., 110: 227-231).                       5.4.2CaSm Gene product detection   The wild-type or mutant CaSm gene product or Antibodies against conserved variants or peptide fragments thereof are As described in the detailed description, it can also be used for diagnosis and prognosis. Such a diagnostic method is Abnormality in CaSm gene expression level, or structure and / or time of CaSm gene product Can be used to detect abnormalities in typical tissue, cell, or subcellular locations Wear. For example, antibodies or antibody fragments as described below may be used for therapy. Compounds can be used for in vitro screening of CaSm gene expression and Ca It can be used to measure their effect on Sm peptide production. Beneficial for cancer Effective compounds can be identified and therapeutically effective dosages determined. Can be.   The tissue or cell type to be analyzed is CaSm Includes those generally known or suspected of expressing a child. Used herein For example, Harlow and Lane [Harlow, E .; And Lane, D ., 1988, "Antibodies: A Laboratory Manual", C old Spring Harbor Laboratory Press, Cold Spring Harbor, New York] Methods, etc., which are incorporated herein by reference in their entirety. Can be The isolated cells may be from cell culture or from a patient. . Analysis of cells removed from cultures tested the effect of compounds on CaSm gene expression. It may be a necessary stage to test.   For detection of CaSm gene products or conserved variants or peptide fragments thereof Preferred diagnostic methods are, for example, the CaSm gene product or a conserved mutant (selection). (Including gene products that are the result of alternatively spliced transcripts) or peptide cleavage Strip is detected by its interaction with an anti-CaSm gene product-specific antibody. An assay may be included.   For example, antibodies or antibodies useful in the invention as described in Section 5.3 above. Antibody fragments may be quantitatively or qualitatively identified as CaSm gene products or May be used to detect the presence of a variant or peptide fragment thereof. Departure Antibodies (or fragments thereof) useful in the context of the present invention additionally include, for example, immunofluorescence or In immunoelectron microscopy, CaSm gene products or conserved variants or For in situ detection of peptide fragments, it may be used histologically. in situ detection Removes a patient-derived histological specimen, such as a section of paraffin-embedded lung tissue Can be achieved by adapting to the labeled antibodies of the present invention. Antibodies (or antibodies Fragment) is adapted to allow the labeled antibody (or fragment) to overlap the biological sample. Is preferred. In addition, for example, antibodies can be introduced into cells by making the cell membrane permeable. It is desirable to introduce. Through the use of such procedures, CaSm remains The presence of the gene product or conserved variant or peptide fragment Its distribution within the weave can be detected. Using the present invention, a wide variety of Any of the histological methods (eg, staining procedures) can be used to achieve in situ detection, etc. It will be easily understood by those skilled in the art that it can be modified.   Immunoassay of CaSm gene products or conserved variants or peptide fragments thereof Say is typically a biological fluid, tissue extract, freshly harvested ) Cells or cell lysates (lysates) incubated in cell culture Identify CaSm gene products or conserved variants or peptide fragments thereof Incubate in the presence of detectable labeled antibodies Including detecting antibodies bound by any of a number of known techniques .   The biological sample can be a solid support or carrier, such as nitrocellulose, or Contacting cells, cell particles or other solid supports capable of immobilizing water-soluble proteins, It may be fixed thereon. The support is then washed with a suitable buffer and detectable Treated with a labeled CaSm gene-specific antibody. The solid support is then buffered Washing twice may remove unbound antibody. And on the solid support The amount of bound label can be detected by conventional means.   "Solid support or carrier" is any support capable of binding an antigen or antibody. You may. Well-known supports or carriers include glass, polystyrene and polypropylene. Ren, polyethylene, dextran, nylon, amylase, natural and modified cells Loin, polyacrylamide, gabbro and magnetite. The nature of the carrier May be, for the purposes of the present invention, partially water-soluble or water-insoluble . The support material can be used as long as the bound molecule can bind to the antigen or antibody. It may have qualitatively any possible structural shape. Thus, the shape of the support The shape can be spherical, such as a bead, or circular, such as the inner surface of a test tube or the outer surface of a rod. It may be cylindrical. Alternatively, the surface may be a sheet, test strip, etc. It may be flat. Preferred supports are polystyrene beads. Skilled person If so, do you know many other suitable carriers for binding antibodies or antigens? Or the use of routine experimentation could confirm the same. .   The binding activity of a given number of anti-CaSm gene product antibodies was determined according to well-known methods. Can be determined. Those of ordinary skill in the art will be able to perform each measurement by employing routine experimentation. Determining operative and optimal assay conditions for assay Will be able to.   One method of labeling a CaSm gene peptide-specific antibody in a detectable manner is Is the binding of a specific antibody to an enzyme and is used in enzyme immunoassays (EIA). [Voller, A., “Enzyme-linked immunosorbent assay (ELISA) (T he Enzyme Linked Immunosorbent Assay), 1978, Diagnostic Horizons 2: 1- 7, Microbiological Associates Quarterly Publication, Walkersville, MD] Voller et al., 1978, J .; Clin. Pathol. 31: 507-520; Butler 1981, Meth. Enzym ol. 73: 482-523; Maggio, E .; (Edit), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL ,; Et al., (Edited), 1981, Enzyme Immunoassay, Kag. aku Shoin, Tokyo｝. The enzyme that binds to the antibody can be, for example, spectroscopy, fluorescence, or visible. To create chemical residues that can be detected by means Similarly, reacts with a suitable substrate, preferably a chromogenic substrate. Anti The enzymes that can be used to detectably label the body are not limited to these. But not maleate dehydrogenase, micrococcus endonuclease, δ-5 -Steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase Hydrogenase, triose phosphate isomerase, horseradish peroxidase, Potassium phosphatase, asparaginase, glucose oxidase, β-gal Cuctosidase, ribonuclease, urease, catalase, glucose-6- Including phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection is by calorimetric method using a chromogenic substrate for the enzyme Can be achieved. Detection also measures the extent of enzymatic reaction of the substrate with a similarly prepared standard. This can be achieved by visual comparison.   Detection is also achieved by using any of a variety of other immunoassays it can. For example, radiolabeling an antibody or antibody fragment CaSm gene peptide can be detected by immunoassay (RIA) [eg For example, Weintraub, B., Principles of Radioimmunoassay assays), 7th training course on radioligand assay technology (Seventh Tr aining Course on Radioligand Assay Techniques), The Endocrine Society, March, 1986, which is incorporated herein by reference]. Radioactive Isotopes are measured by means such as the use of a gamma counter or a scintillation counter. Or by autoradiography.   The antibody can also be labeled with a fluorescent compound. Fluorescently labeled antibody at appropriate wavelength When exposed to light, its presence can be detected by fluorescence. Of the fluorescent labeling substances, Also commonly used are fluorescein isothiocyanate, rhodamine , Phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde Hyd and fluorescamine.   Antibodies are¹⁵²Fluorescent emitting metals such as Eu or other metals of the lanthanoid series Labeling can also be carried out so that it can be detected using These metals are diethylene Metals such as triaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) The activating group can be attached to the antibody.   Antibodies are labeled so that they can be detected by binding to a chemiluminescent compound You can also. The presence of a chemiluminescent-tagged antibody appears during the course of a chemical reaction. It is determined by detecting the presence of luminescence. Particularly useful chemiluminescent labeled compounds Examples of luminol, isoluminol, theromatic Lidinium ester, imidazole, acridinium salt and oxalate ester Le.   Similarly, bioluminescent compounds can be used to label the antibodies of the invention. Bioluminescence is a type of chemiluminescence found in biological systems. The proteins increase the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is Is measured by detecting the presence of Bioluminescent materials important for labeling purposes Quality is luciferin, luciferase and aequorin.   In various embodiments, the present invention provides methods for measuring CaSm gene products, and The use of the measurement method in applications is provided.   The method for measuring the CaSm gene product of the present invention can be used to detect cancer in a subject and / or Is staging, cancer screening in populations. In learning, in the differential diagnosis of the subject's physiological condition, and to the subject It is valuable in monitoring the effects of therapeutic treatments.   The present invention also relates to the detection, diagnosis, or staging of cancer, or CaSm gene products. In addition to at least one other such as a receptor or a differentiation antigen Methods for Monitoring Cancer Treatment by Measuring Markers of Cancer You. For example, but not limited to, cancer embryo (carcinoembryonic) antigens ( CEA), selected from CA19-9, CA195, DUPAN-2, SPAN-1 and CA50, eg serum Marker is for CaSm gene production Can be used in combination with a substance to detect, diagnose, stage, or treat pancreatic cancer. I can do it. In another embodiment, the prognostic indicator ) Are related to each other rather than the absolute concentration of the marker present at any given time Changes observed in the concentration of different markers. These measurements are It can help predict outcomes and assess and monitor the overall condition of the subject. Wear.   In certain embodiments of the invention, the CaSm gene product alone or with another marker Combinations include, but are not limited to, blood, serum, milk, urine, saliva, breast Membrane exudates, synovial fluid, cerebrospinal fluid, tissue infiltrations and It can be measured in any body fluid of a subject that contains a tumor infiltrate. In the blood or Measurement of the CaSm gene product in serum is based on test kits to be used in hospitals and homes. Good for development.   As described in section 5.4.2, any immunoassay may be used to implement the present invention. Can be used in Antibodies that can be used, or antibody fragments that contain a binding domain, are Without limitation, suitable antibodies from among the antibodies described in Section 5.3 Body and other antibodies known in the art or antibodies such as those described in Section 5.3. And antibodies obtained by standard procedures.                 5.4.3Detection and staging of cancer in subjects   In one embodiment of the present invention, the CaSm gene product or a fragment or Measurement of the circulating CaSm gene product can be used to detect cancer in the subject or to detect cancer in the subject. Can be used for staging.   Staging refers to classifying patients according to their degree of disease. Stay Zing is used to select treatments for individual patients, assess prognosis, and evaluate different treatment programs. Useful in comparison with ram results. Cancer staging involves physical examination and Performed early on a clinical basis according to laboratory radiologic evaluation.   Pancreatic cancer that can be detected and / or staged in a subject according to the present invention The disease or condition includes, but is not limited to, those listed in Table I (Beazley & Cohen, ch. 15, p. 255, “Clinical Oncology”, Second Edition, edited by Murphy et al., American Cancer Society, 1995).Table I                           Pancreatic cancer staging Early tumor (T) TX Early tumor cannot be evaluated T0 No evidence of early tumor Tumor limited to T1 pancreas     T1a Maximum tumor diameter is 2cm or less     T1b Tumor maximum diameter exceeds 2 cm T2 tumors spread directly to any of the following:                                       Tissue around duodenum, bile duct or pancreas T3 tumors spread directly to any of the following:                                       Stomach, spleen, colon or nearby large tube Lymph glands (N) NX Local lymph glands could not be evaluated N0 No local lymph gland metastasis N1 Local lymph gland metastasis Distant metastasis (M) MX Cannot assess the existence of distant metastases M0 No distant transition M1 distant metastasis Stage groupingStage I T1 N0 M0 T2 N0 M0 Phase II T3 N0 M0 Phase III Any T N1 M0 Phase IV Any T Any N M1   Any immunoassay as described in Section 5.4.2 will Used to measure the amount of CaSm gene product compared to baseline level it can. This baseline concentration may be different for subjects with varying degrees of disease or disorder. It is the amount confirmed to be normally present in tissue or body fluid. Specific stages of cancer A standard amount that is identified in the floor as normally present in the patient's tissue or body fluids Is present in the tissue or fluid of a subject, Indicates that it is on the floor. Baseline concentrations should be present in subjects before disease onset. It is also the concentration present or the amount present during the lull of the disease.   In certain embodiments of this aspect of the invention, measuring the concentration of the CaSm gene product Can be used in the detection of the presence of pancreatic cancer or metastasis or both You.   In another embodiment of the present invention, a CaSm gene product, a fragment thereof Measurement of biologically relevant molecules can be measured in two or more phenotypes or physiological states. Differential in subjects with a distinct disease phenotype or physiological condition as clear as It can be used for various diagnoses. At the end, for example, the CaSm gene product is measured Amount is a molecule normally present in body fluids of patients with one of the suspected physiological conditions. Compared to the amount of Not the amount normally present in patients with one or more physiological conditions The amount of molecules close to the amount normally present in patients with one of the physiological conditions is measured Indicates that the subject is in the physiological state. To baseline concentration In contrast, elevated levels of CaSm gene product in subjects indicate that the subject has cancer. And                   5.4.4Monitoring the effectiveness of therapeutic treatment   The present invention monitors the effect of a therapeutic treatment on a subject who has been administered the therapeutic treatment. Provide a way to:   Clinicians can use procedures (procedures) that can be used to monitor the effectiveness of these procedures. ure). Sensitive assays for therapy monitoring If so, identify CaSm gene products in cancer patients with different manifestations of the disease And can be detected. Therapeutic treatments that can be evaluated according to the present invention are limited to these Although not in translation, radiation therapy, surgery, chemotherapy, vaccination, endocrine therapy, It includes immunotherapy, gene therapy, and the like. Chemotherapy prescriptions are limited to these Including but not limited to the administration of medicaments such as fluorouracil and taxol .   The method of the present invention can be used to produce the CaSm gene at appropriate time intervals before, during, or after treatment. Measuring the quantity of an object. Some change or change in the amount of CaSm gene product Confirm that there are no subjects, e.g., a decrease in the transformed phenotype of the cancer cells. Can be shown to correlate with the effect of the treatment.   In a preferred embodiment, attempts that can be taken are to concentrate the CaSm gene product at different time points. Degree is measured and these values are compared to the baseline concentration. Bass line Concentration is the concentration of the marker present in a normal, disease-free solid; and / or treatment Either during pregnancy, during the lull of the disease, or during a stable period. It may be. These levels may be associated with disease progression or treatment outcome. it can. Elevated CaSm gene product concentrations relative to baseline concentrations indicate that Shows that there is almost no reaction.   5.5CaSm Screening assays for compounds that modulate activity   The present invention further provides, through its interaction with the CaSm gene or CaSm gene product, Identification of compounds that can affect the pathogenesis, progression and spread of metastases of cancer, especially pancreatic cancer Provide a way for   The following assays were performed for (i) the production of CaSm genes containing homologs of mammalian and non-mammalian CaSm. (Ii) CaSm gene containing homologs of mammalian and non-mammalian CaSm Compounds that bind to other intracellular proteins that interact with the product; (iii) mammals and Interaction of CaSm gene products, including non-mammalian homologues of CaSm, with other intracellular proteins Compounds that interfere with action; and (iv) modulate the activity of the CaSm gene (ie, Modulating the level of CaSm gene expression and / or CaSm gene product activity Designed to identify compounds that modulate the level of   The assay further comprises a CaSm gene capable of modulating the level of CaSm gene expression. Used to identify compounds that bind to control sequences (eg, promoter sequences). You may. See, for example, Platt, 1994, J.A. Biol. Chem. See 269: 28558-28562. This is hereby incorporated by reference in its entirety. Also provided CaSm A method for identifying a compound that modulates gene expression comprises: (a) converting a test compound to CaS m cells containing a reporter gene functionally related to the gene regulatory element or Contacting with a cell lysate; and (b) detecting expression of a reporter gene. including. Also, to identify compounds that modulate the provided CaSm gene expression. Another method comprises (a) converting a test compound to cells containing CaSm transcripts or Contacting with a cell lysate; and (b) detecting the translation of the CaSm transcript . Without limitation, green fluorescent protein (green fluorescent protein) protein), β-galactosidase, alkaline phosphatase, chloramphe Some reporters known in the art, such as Nicole acetyltransferase Genes can be used.   As described in Sections 5.2 and 6.2.2, the CaSm gene product and homologs of CaSm Is a protein that is a component of small nuclear ribonucleic acid proteins. It contains two sequence motifs that characterize the family. Sm motif 1 and These motifs, termed Sm motifs 2, belong to the spliceosome protein family. Required for interaction between members of the The CaSm gene product is Although it does not appear to be a member of the tribe, the CaSm gene product does not contain Sm proteins. Interacts with intracellular proteins that have one or both of these Sm motifs. it can. In addition, yeast CaSm homologs are found in yeast cells carrying the mutant Pablp gene. Can work as a bypass suppressor in Considering that, the CaSm gene product also contains eukaryotic mRNAs including Pablp, translation initiation complex, etc. Interacts with proteins related to poly (A) tail and 5 'cap structure sell.   Such intracellular proteins are uncontrolled cell growth as well as cancer pathogenesis, It can be involved in progression and transition diffusion.   Compounds identified by assays such as those described herein are examples For example, to investigate the biological function of the CaSm gene product in detail and to improve cancer symptoms Useful to Technologies such as those described in Section 5.5.1 Assays to test the effects of the compounds thus identified are described in the section below. Consider in 5.5.3. Fragments of the CaSm protein useful in these assays include: Although not limited to these, it can be used for CaSmSm motif 1 and CaSmSm motif 2. The corresponding peptides or parts thereof; and Sm motif 1 or Sm motif 2 or Both motifs have been deleted, including truncated CaSm. The composition of the present invention comprises Including a pharmaceutical composition comprising one or more compounds identified by such a method. You should be careful. Such pharmaceutical compositions are discussed, for example, in Section 5.7 below. Can be prepared. 5.5.1CaSm In vitro screening assays for compounds that bind to gene products   The in vitro system is used to prepare the CaSm gene product of the present invention and a homologue of CaSm (for example, Interaction with the yeast homologue encoded by QRF YJL124c) May be designed to identify compounds that can The identified compound is For example, to modulate the activity of a wild-type and / or mutant CaSm gene product. Useful for examining in detail the biological function of the CaSm gene product, To identify compounds that disrupt the interaction of the normal CaSm gene product Can be used for screening for Disintegrate. Such an interaction may be due to Sm motif 1, Sm motif 2, or both. May be mediated by a party.   Assay source used to identify compounds that interact with the CaSm gene product The reaction is performed by mixing the reaction mixture of the CaSm gene product or a fragment thereof and the test compound with two components. Are prepared under conditions sufficient for interaction, e.g., binding, and Form a complex, which is removed from the reaction mixture and / or detected Including. These assays can be performed in a variety of ways. An example For example, one way to perform such an assay is to use a CaSm gene product or a test substance. CaSm gene product attached to the solid phase at the end of the reaction Detecting the complex. In one embodiment of such a method, C The aSm gene product or a fragment thereof is immobilized on a solid surface The compound may be directly or indirectly labeled.   Actually, a microtitre plate is used as a solid phase. It can be used conveniently. Attached components may be non-covalent or covalent. Can be fixed by Non-covalent binding is simply a solid solution This can be achieved by coating the surface and drying. Or tongue to be fixed Using an immobilized antibody specific to the protein, preferably a monoclonal antibody, Proteins can be anchored to a solid surface. Prepare and store the surface in advance be able to.   To perform the assay, the non-immobilized component is removed from the immobilized component. Add to the coated surface containing the components. After the reaction is completed, formed Components that have not reacted under conditions such that the Is removed (eg, by washing). The detection of complexes anchored on solid surfaces Can be achieved in a number of ways. Components not previously immobilized are labeled (pr e-labeled), if a label immobilized on the surface is detected and detected , Indicating that a complex was formed. Non-pre-immobilized components If no labeling is used, the intermediate Indirect labeling, such as a labeled antibody specific for a component that has not been previously immobilized (anti- Can also be labeled directly or indirectly with a labeled anti-Ig antibody). Can be used.   Alternatively, the reaction can be carried out in the liquid phase, where the unreacted components The product is separated and the complex is detected. For example, specific immobilization specific to the CaSm gene product Test compound that adheres to the antibody or any complex formed in solution, and possible Immobilized complex using labeled antibodies specific for other components of the complex Can be detected.   5.5.2CaSm Assay for intracellular proteins that interact with gene products   Some suitable methods for detecting protein-protein interactions include CaSm Protein-intracellular protein interaction, especially Sm motif 1 or Sm motif 2 Or can be used to identify interactions mediated by both. You.   Among the traditional methods, only co-immunoprecipitati on), crosslinking and gradients or chromatography This is a co-purification method using a Raffy column. Using procedures like these, Ca Sm gene heritage, CaSm gene product fragments and CaSm homologs (eg, ORF YJL124 isolation of intracellular proteins that interact with the yeast homolog encoded by c) it can. Once isolated, such intracellular proteins can be identified. And similarly, in combination with standard techniques, Can be used to identify proteins. For example, interact with the CaSm gene product At least a portion of the amino acid sequence of the intracellular protein can be obtained by techniques known to those skilled in the art, for example, For example, Edman degradation [Creighton, 1983, “Proteins: Principles of Structure and Molecules (Pro teins: Structures and Molecular Principles) ", W. H. See Freeman & Co., N.Y., pp. 34-49 " . The amino acid sequence obtained is a gene sequence encoding such an intracellular protein. Guidance for the production of oligonucleotide mixtures that can be used to screen rows Can be used as a password. Screening can be performed, for example, by standard hybridization. Can be achieved by standard hybridization or PCR techniques. You. Techniques for producing and screening oligonucleotide mixtures are well known. (For example, Ausubel, supra., And PCR Protocols: A Guideto Method and Applic ations, 1990, edited by Innis, M. et al., Academic Press, Inc, New York)   Furthermore, a gene encoding an intracellular protein that interacts with the CaSm protein May be used that result in the simultaneous identification of These methods are, for example, labeled Has a modified CaSm protein or a fragment thereof (eg, Sm motif 1, Sm motif 2) Expression libraries are used for antibody purification of the λgt11 library. The CaSm protein or a fragment thereof is converted in the same manner as in the well-known technique of roving. And probing.   One method for detecting protein interactions in vivo, 2-hybrid A two-hybrid system can be used. One of this system Is described in Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 9578-95 82 and is commercially available from Clontech (Palo Alto, CA) .   5.5.3CaSm Compounds that interfere with gene product / intracellular macromolecular interactions Assay   The CaSm gene product of the present invention, a fragment thereof, and a homologue of CaSm (for example, CRF YJL124 c) is a protein and nucleic acid molecule in vivo. Interacts with any one or more intracellular macromolecules. Such macromolecules are [Polyadenylated (poly (A)) RNA and 5 'caps] RNA with a tipped structure] and the method described in Section 5.5.2 above. Thus, those proteins identified. For this discussion , Such intracellular macromolecules are referred to herein as "interacting partners. ing partners). " Compounds that disrupt the CaSm interaction in this way Are useful in controlling the activity of CaSm gene products, including mutant CaSm gene products. It is for. Such compounds include, but are not limited to, Including molecules such as the peptides described in Section 5.5.1, and It would be possible to gain access to the intracellular CaSm gene product. like this Compounds also include the amino acid sequence of Sm motif 1, Sm motif 2, or both. Also includes peptides or modified peptides.   Between the CaSm gene product and its intracellular interaction partner or partners Basic principles of assay systems used to identify compounds that interfere with interactions Under conditions long enough to allow the two to interact and bind A reaction mixture containing the gene product or a fragment thereof and an interaction partner is prepared, and the reaction mixture is prepared. And forming a complex. To test the inhibitory activity of a compound, mix Compounds are prepared in the presence and absence of the test compound. Test compound reacts first It may be included in the mixture, or it may contain the CaSm gene product and its intracellular interaction protein. -It may be added subsequent to the addition of the toner. The control reaction mixture contains the test compound Incubate without or with placebo. And the CaSm gene product Or the formation of some complex between a fragment thereof and an intracellular interaction partner It is. Control reactions that are not reaction mixtures containing the test compound The formation of a complex in the reaction indicates that the compound interacts with the CaSm gene protein Indicates that it interferes with the interaction with the toner. In addition, test compounds and normal CaSm Complex formation in the reaction mixture containing the gene protein can also This is compared to complex formation in a reaction mixture containing the type CaSm gene protein. This ratio Comparison of mutant CaSm gene protein with normal CaSm gene protein This is important if one wants to identify compounds that disrupt the interaction.   Assay of compounds that interfere with the interaction of the CaSm gene product with the interaction partner B) can be performed in a heterogeneous format or a homogeneous (homogeneous) format. Wear. Heterogeneous assays involve either the CaSm gene product or a binding partner on a solid phase. At the end of the reaction and at the end of the reaction. Including. In a homogeneous assay, the entire reaction is performed in the liquid phase. Any approach Again, the order of addition of reactants may give different information about the compound being tested. Can be varied to obtain. For example, the interaction part with the CaSm gene product Interaction with the donor, for example, by interfering with competition. The test compound is administered by performing the reaction in the presence of the test Quality prior to or concurrent with CaSm gene protein and intracellular interaction partners Can be identified by adding to the reaction mixture. Or pre-formed A test compound that disrupts the preformed complex, for example, A compound with a high binding constant that displaces one of its constituents will Can be tested by adding to the reaction mixture after coalescence has formed . The various types are briefly described below.   In heterogeneous assay systems, either the CaSm gene product or the interaction partner Non-sticky species are directly or indirectly labeled while attached to a solid surface. It is. In practice, microtiter plates are conveniently available. Anti stuck The species can be immobilized by non-covalent or covalent bonds. Non-covalent bonds are Simply the CaSm gene product or interaction Can be achieved by coating the solid surface with a solution of the toner and drying. There Is specific to the reactive species to be fixed in order to fix the reactive species on the solid surface. Immobilized antibodies can be used. Surface should be prepared and stored in advance be able to.   To perform the assay, the partner of the reactive species to be immobilized is the test compound With or without the test compound. After the reaction is completed, The unreacted components are removed (eg, by washing) and some complex formed. The body remains immobilized on the solid surface. Detection of complexes anchored on solid surfaces This can be achieved in a number of ways. Non-immobilized reactive species are labeled in advance. If the label immobilized on the surface is detected, the complex And If the non-immobilized reactive species is not pre-labeled, An indirect label to detect the anchored complex, e.g., first immobilized A labeled antibody specific for the non-reactive species (similarly, the antibody may be a labeled anti-Ig antibody (Directly or indirectly labeled by). Reaction Minutes, the complex formation is inhibited or the formed complex is disrupted. The test compound to be tested can be detected.   Alternatively, the reaction should be performed in the liquid phase in the presence or absence of the test compound. The reaction product is separated from unreacted components and the complex is detected. An example For example, one of the interacting components that anchors several complexes formed in solution. Use different immobilized antibodies and labeled antibodies specific for other partners Then, the fixed complex is detected. Furthermore, the order in which the reactants are added to the liquid phase Of compounds that inhibit the complex or disrupt the preformed complex Can be.   In another embodiment of the invention, a homogeneous assay can be used. This approach In H, a preformed complex of CaSm gene protein with an interaction partner Indicates that either the CaSm gene product or its interaction partner is labeled, Signals generated by intellect are lost by complex formation (eg, Was used for immunoassay See U.S. Pat. No. 4,109,496 to Rubenstein). From the preformed composite Addition of a test substance that competes with and displaces one of the counterparts will signal in the background above. Will produce the result. In this method, the CaSm gene protein / Test substances that disrupt intracellular interaction partner interactions can be identified You.   In a specific embodiment, the CaSm gene product or fragment thereof is in the section above. It can be prepared for immobilization using the recombinant DNA technology described in 5.1. For example, Ca The interaction activity of the Sm coding region is determined using a fusion vector such as pGEX-5X-1. Glutathione-S-transfer to be retained in the resulting fusion protein Can be fused to the Rase (GST) gene. Intracellular interaction partners can be purified, Use the methods described in section 5.2 above, which are routinely used in the industry. Can be used to make monoclonal antibodies. This antibody is, for example, Radioisotope by routine methods¹²⁵Can be labeled with I. Uneven For example, GST-CaSm fusion proteins can be converted to glutathione-agar beads Can be fixed. Next, in the presence or absence of the test compound, the interaction, For example, an intracellular interaction partner is added so that binding occurs. Reaction period At the end of the procedure, wash off any unbound material and label the monoclonal antibody Is added to the system and allowed to bind to the complexed components. CaSm gene protein and intracellular phase The interaction with the interaction partner remains bound to the glutathione-agar beads. It can be detected by measuring the amount of radioactivity present. Test compound interaction Successful inhibition of the effect will result in a decrease in the radioactivity measured.   Alternatively, the GST-CaSm gene fusion protein and the intracellular interaction partner are It can be mixed together in a liquid in the absence of solid glutathione-agar beads. Wear. The test compound can be added either during or after interacting with the reactive species. Wear. This mixture is then added to the glutathione-agar beads and allowed to bind. Missing substances are washed away. Again, the CaSm gene product / interaction partner interaction The degree of inhibition of action is Can be detected by adding the antibody and measuring the radioactivity bound to the beads. .   5.5.4CaSm Cell-based assays for the identification of compounds that modulate activity Surname   As used herein, compounds that can treat cancer by modulating CaSm activity A cell-based method for identifying In particular, such assays are limited Cell viability, cell morphology, cell division, differentiation, attachment, motility Or CaSm-dependent, such as phosphorylation and dephosphorylation of cellular proteins Identify compounds that act on the process. Limited as other CaSm-dependent processes Stimulation of translation, ribosome binding to mRNA, mRNA decapping Or protection from degradation. Compounds identified by such a method are: For example, it can be used in a method of treating cancer and metastasis.   In one embodiment, the cell-based assay is a CaSm gene product or C Using recombinant yeast cells comprising an expression construct that produces a yeast homolog of aSm, po Li (A) binding protein, Pablp, and the large subunit of the translation initiation complex, eIF-4G Have mutations in the genes encoding them. Code Pablp and eIF-4G Mutant yeast cells with a non-functional mutation in the gene act as a second suppressor It cannot survive unless CaSm or a CaSm homolog is present. In this assay Is a mutant yeast cell producing CaSm or a CaSm homolog, the compound is CaSm or CaSm The test compound is exposed at intervals sufficient to modulate the activity of the Sm homolog. Test compound The activity of CaSm when present is dependent on the viability or growth of the mutant yeast cells. evaluate. For example, compounds that inhibit CaSm activity have poor or no growth. Will not exist. Functional conservation of highly conserved poly (A) binding proteins Genes encoding isomeric Pablp and the large subunit eIF-4G of the translation initiation complex In mammalian cells with mutations in, a similar assay was performed using mammalian CaSm. Think you can do It is.   In another embodiment, the cell-based assay is in mammalian cells. Based on the expression of CaSm gene products and measurements of CaSm-dependent processes. Especially CA In pancreatic cancer cells such as PAN-1, CAPAN-2, ASPC-1, PANC-1 and HPAC Use any mammalian cell that expresses the CaSm gene and makes the CaSm gene product function Can be. If a detectable CaSm gene product is produced, Gland, liver, ovary, lung, rectum, kidney and non-erythroid hematopoietic cells Other cancer cell lines may be used. CaSm in these and other normal cells Recombinant expression of a gene can be performed by the method described in Section 5.2. These In assays, cells that produce a functional CaSm gene product are used to The test compound is exposed at intervals sufficient to modulate the activity of the test compound. CaSm gene product Activity is a CaSm-dependent intracellular process such as the appearance of a transformed phenotype Can be measured directly or indirectly by detecting or measuring. Contrast For example, cells that do not produce the CaSm gene product may be used for comparison. Intracellular processing Detect or measure it by applying any technique known in the art. You may.                             5.6 How to treat cancer   Method of treating cancer using CaSm gene or gene product as a therapeutic target and The composition is described below. The outcome of treatment should be at least Should benefit and, in the case of cancer, without limitation, cancer mitigation, Alleviation of cancer symptoms and control of metastatic spread of cancer.   All of these methods, in turn, involve the CaSm gene Modulating activity and / or expression.   As discussed above, the success of cancer treatment depends on techniques that work to reduce CaSm activity. It will be accomplished by art. For example, directly reducing the activity of the CaSm gene product And / or decrease the expression level of CaSm gene By doing so, the activity can be reduced.   In accordance with the present invention, for example, those identified by the assays described in Section 5.5 above A compound that reduces CaSm activity, such as, can be used to treat cancer. Said As described in Section 5.5, such molecules include, but are not limited to, peptides (Including soluble peptides) and small organic or inorganic molecules, It can be called a Sm antagonist. Prevents interaction between CaSm and intracellular macromolecules Amino acid sequence of Sm motif 1, Sm motif 2 or both, or a part thereof May be used. Effective doses and modes of administration for such compounds The techniques to be determined are described in section 5.7 below.   Further, in accordance with the present invention, antisense and ribosomes that inhibit CaSm gene expression Using the Zyme molecule to reduce the expression level of the CaSm gene, Effectively lowers the level of the Sm gene product, thereby reducing the level of CaSm activity It can also be done. Furthermore, to reduce the activity level of the CaSm gene Triple helix molecules can also be used. Such molecules may be wild-type or, if preferred, Designed to reduce or inhibit any of the mutant target gene activities be able to. Techniques for producing and using such molecules are well known to those of skill in the art. You.   Any technique that can be used to selectively administer a nucleic acid molecule to a cell population of interest For example, it can be used by using a delivery complex. Such de The liver complex may comprise a suitable nucleic acid molecule and a targeting means. Such a mark Specific means include, for example, sterols, lipids, viruses or target cell-specific binding An agent may be included. Viral vectors that can be used with recombinant viruses Include, but are not limited to, adenovirus, adeno-associated virus, Pure herpesvirus, vaccinia virus, and retroviral vectors These include other particles that introduce DNA into cells, such as liposomes.                          5.6.1 Antisense molecule   Use of antisense molecules as inhibitors of gene expression is based on genetics A special treatment approach (for review, see Chapter 69, Section 5 And Practice ”, 4th edition, edited by DeVita et al. B. Lippincott, Philadelphia 1993 See Stein). The present invention relates to a gene or cDN encoding CaSm or a part thereof. Treatment of nucleic acids consisting of at least 6 nucleotides that are antisense to A Provides top and prophylactic use. "Antisense" CaSm as used herein Nucleic acid refers to a portion of CaS mRNA (preferably mRNA) due to some sequence complementarity. A nucleic acid capable of hybridizing. The present invention is further described below. Thus, a pharmaceutically acceptable carrier comprises an effective amount of a CaSm antisense nucleic acid of the invention. A pharmaceutical composition comprising:   In another embodiment, the invention relates to the generation of a CaSm nucleic acid sequence in a mammalian cell. A method for inhibiting the current in vitro or in vivo, comprising the step of Providing a cell with an effective amount of a composition comprising a chisense nucleic acid. Turned to   The antisense nucleic acid of the present invention may comprise a coding region of CaSm mRNA and / or a non-coding region. It can be complementary to a region. Antisense molecule is complementary to mRNA transcript of CaSm gene Will bind and reduce or prevent translation. Although perfect complementarity is preferred Is not necessary. As described herein, a portion of the RNA is "complementary" Sequence is sufficient to hybridize to RNA and form a stable double helix. Sequence with sufficient complementarity, and thus in the case of double-stranded antisense nucleic acids May test a single strand of double-stranded DNA, or form a triple helix. May be assayed. Hybridization ability depends on degree of complementarity and antisense It depends on both the length of the nucleic acid. In general, the longer the hybridizing nucleic acid, It increases the number of base mismatches with the RNA that it may contain, and still provides a stable double helix (and May optionally be a triple helix). If you are skilled in the art, By determining the melting point of the hybridized complex using standard methods, You can check the allowable degree of pairing You.   A nucleic acid molecule complementary to the 5 'end of the message (eg, up to the AUG start codon, and And the 5 'untranslated sequence containing it) should work most effectively at inhibiting translation. is there. However, recently, sequences complementary to the 3 'untranslated sequence of mRNA Has been shown to be effective in inhibiting the translation of For an overview, see Wagner, R., 1994. , Nature 372: 333-335. Thus, for example, as shown in FIG. A nucleic acid molecule complementary to the untranslated non-coding region of either the 5'- or 3'- Antisense approach to inhibit the translation of endogenous CaSm gene mRNA. Noh.   Nucleic acid molecules complementary to the 5 'untranslated region of the mRNA are considered to contain the complement of the AUG start codon. available. Antisense nucleic acid molecules complementary to the mRNA coding region are less effective translation inhibitors Although a poison, it can be used in accordance with the present invention. Target or pathway gene mRNA 5'-, 3'- or those designed to hybridize to the coding region, Antisense nucleic acids are at least 6 nucleotides in length, preferably from 6 to Must be oligonucleotides ranging in length about 50 nucleotides. Special In a particular embodiment, the oligonucleotide is at least 10 nucleotides, Both 17 nucleotides, at least 25 nucleotides, at least 50 nucleotides, Or at least 200 nucleotides.   Regardless of the choice of target sequence, first perform an in vitro study, for example, Section 7. Quantify the ability of antisense molecules to inhibit gene expression, as described in Section 1. Preferably. These studies are based on oligonucleotide antisense gene inhibition. It is preferable to use controls that discriminate between harm and non-specific biological effects. these In research, target RNA or protein levels were compared to internal control RNA or protein levels. It is also preferred to compare with the level. Further antisense oligonucleotides Compare the results obtained with the control oligonucleotide to those obtained with the control oligonucleotide. Can be considered. The control oligonucleotide is almost identical to the test oligonucleotide. Have the same length and the nucleotide sequence of the oligonucleotide is Preferably, it differs from the antisense sequence, which is specific to the target sequence. Preferably not more than necessary to prevent hybridization .   Antisense molecules can be DNA or RNA or chimeric mixtures or derivatives thereof. Alternatively, it may be a modified variant thereof, single-stranded or double-stranded. For example, molecule, c In order to improve the stability such as hybridization, It may be modified at the base moiety, sugar moiety, or phosphate backbone. As an antisense molecule Other, such as peptides (eg, for target host cell receptors in vivo) Or cell membranes (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. . U.S.A. 86: 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-65 2; see PCT Publication No. WO 88/09810 published December 15, 1988) or the blood-brain barrier ( See, for example, PCT Publication No. WO 89/10134 published April 25, 1988). Enhancers, hybridization-triggered cleavage agents (eg, Krol et al., 1988, Bi). oTechniques 6: 958-976) or intercalating agents (see, for example, Zon, 1988, Pharm. Res. . 5: 539-549). Antisense molecules for this purpose are Molecules such as peptides, hybridization-triggered crosslinkers, transport agents, hybrids It may be conjugated to a ligation-induced cleavage agent or the like.   Antisense molecules include, but are not limited to, 5-fluorouracil , 5-bromouracil, 5-chlorouracil, 5-iodouracil, hydroxanthine , Xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) ura Sil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl Aminomethyluracil, dihydrouracil, β-D-galactosyl eosin, Nosin, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2 , 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyl Ruuracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl Queosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2- Methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wipe Xoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thio Uracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- Oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thioura Syl, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6 -Comprising at least one modified base moiety selected from the group comprising diaminopurines May be.   Antisense molecules also include, but are not limited to, arabinose, 2-flu Oroarabinose, xylulose, and hexoses. It may comprise at least one modified sugar moiety.   In yet another embodiment, the antisense molecule is a phosphorothioe , Phosphorodithioate, phosphoramidothioate, phosphoramidate , Phosphordiamidate, methyl phosphonate, alkyl phosphotriester, And at least one selected from the group consisting of formacetal or an analog thereof It may comprise one modified phosphate backbone.   In yet another embodiment, the antisense molecule is an α-anomeric oligomer. Gonucleotide. α-anomeric oligonucleotides are paired with normal β units. In contrast, the strand forms complementary double-stranded hybrids with complementary RNA that are parallel to each other. (Gautier et al., 1987, Nucl. Acids Res. 15: 6625-6641). Oligonucleo Tide is a 2'-0-methyl ribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15: 6131-6148), or chimeric RNA-DNA analogs (Inoue et al., 1987, FEBS Lett. 215: 32). 7-330).   The antisense molecules of the present invention can be prepared by standard methods known in the art, for example, Dynamic DNA synthesizers (eg, commercially available from Biosearch, Applied Biosystem, etc.) Can be synthesized. As an example, phosphorothioate oligonucleotides Leotide is synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16: 3209), methylphosphonate oligo The nucleotides are controlled porous glass polymer supports (Sarin et al., 1988, Proc. Natl. . Acad. Sci. U.S.A. 85: 7448-7451).   Antisense nucleotides complementary to the CaSm coding region, such as those described in Chapter 7.1 Otides could be used, but those complementary to the transcribed untranslated region are also preferred. Good. For example, the following sequence:Antisense oligonucleotides having the following can be used according to the present invention.   Express or preferably overexpress CaSm using CaSm antisense nucleic acids Can be treated or prevented from forming a cancer that includes such cell types. Express CaS mRNA Alternatively, a cell type that is overexpressed can be identified by various methods known in the art. Wear. Such methods include, but are not limited to, hybridization with CaSm-specific nucleic acids. Redidation (eg Northern hybridization, dot blot Hybridization, CaSm gene product by immunoassay (by in situ hybridization) And the like. In a preferred embodiment, immunocytochemistry or in situ high Prior to treatment by hybridization, etc., primary tissues from patients were expressed in CaSm expression Can be assayed for.   A pharmaceutically acceptable carrier comprising an effective amount of a CaSm antisense nucleic acid according to the present invention. The pharmaceutical composition is a type of disease that expresses or overexpresses CaS mRNA or protein. It can be administered to a patient with a disease or disorder.   CaSm antisense nucleic acids that may be effective in treating certain diseases or conditions The amount of can be determined by standard clinical techniques, depending on the nature of the cancer or condition. Possible Whenever possible, treat the antisense cytotoxicity of the tumor type being treated in vitro and then It is desirable to determine an effective animal model system prior to treatment and use it in humans.   Antisense molecules should be delivered to cells expressing the CaSm gene in vivo is there. Several methods developed to deliver antisense DNA or RNA to cells Eg, injecting the antisense molecule directly into the tissue site, or (Eg, an antisense molecule attached to a peptide or expressed on the surface of a target cell) Modifications designed to target an antibody that specifically binds to a receptor or antigen) Antisense molecules can be administered systemically. Delivery of antisense molecules -Can be delivered to the desired cell population via the complex. In special embodiments A pharmaceutical composition comprising a CaSm antisense nucleic acid, Li-β-1-> 4-N-acetylglucosamine polysaccharide), liposome, microparticle or microphone It is administered by capsule. In various embodiments of the invention, CaSm antisense It may be useful to use such compositions to achieve sustained release of the nucleic acid. In specific embodiments, targeted by antibodies to specific tumor antigens that can be identified. It may be desirable to use liposomes (Leonetti et al., 1990, Proc. tl. Acad. Sci. U.S.A 87: 2448-2451; Renneisen et al., 1990, J. Am. Biol. Chem. 265: 16337-16342).   However, intracellular concentrations of antisense are sufficient to suppress endogenous mRNA translation. Achieving degrees is often difficult. Therefore, the preferred approach is Pol III or pol with strong antisense oligonucleotide or polynucleotide  A recombinant DNA construct placed under the control of the II promoter is used. Patient target Using such a construct to transfect cells, the endogenous CaSm gene Produces sufficient transcription of single-stranded RNA that will form complementary base pairs with the transcript And it will prevent translation of the CaSm gene mRNA. For example, 7.1 As described in the chapter, once a vector is introduced in vivo, it is taken up by cells. And directs transcription of the antisense RNA. Such a vector is transcribed and As long as it can produce antisense RNA, it remains episomal Or can be integrated chromosomally. Such vectors are standard in the art. It can be constructed by standard recombinant DNA techniques. Vectors are in mammalian cells Plasmids, viruses, or publicly known in the art used for replication and expression in It may be something else of knowledge. Expression of the sequence encoding the antisense RNA Any known in the art to act on mammalian, preferably human cells These promoters may be used. Such a promoter may be inducible or , And may also be constructive. Such promoters are limited and None: SV40 early promoter region (Bernoist and Chambon, 1981, Nature 2 90: 304-310), a sequence contained in the 3 'long terminal repeat of Rous sarcoma virus. Lomotor (Yamamoto et al., 1980, Cell 22: 787-797), herpes thymidine kinase Promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445). And regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42). And so on. Any plasmid, cosmid, YAC or viral vector Either directly at the tissue site or via the delivery complex A recombinant DNA construct that can be introduced with this can be prepared. Alternatively, if desired Using a viral vector that selectively infects tissues You can also. Genetic variants available in the art, such as those described in Chapter 5.6.4 Any method for treating a child can be used. Representative methods are described below I do.                             5.6.2 Ribozyme   Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. (For an overview, see Rossi, J., 1994, Current Biology 4: 469-471, for example). The mechanism of ribozyme action includes the sequence of the ribozyme molecule to complementary target RNA. Specific hybridization is followed by endonucleolytic cleavage. Re The composition of the bozyme molecule comprises one or more sequences complementary to the target gene mRNA, and further comprises It must contain the well-known catalytic sequence involved in RNA cleavage. This array No. 5,093,246, which is incorporated herein by reference in its entirety. Teru. As such, within the scope of the present invention, the RNA sequence encoding the target gene protein Hammerhead motif specifically and efficiently catalyzes endonucleolytic cleavage of yeast The bozyme molecule is manipulated.   In addition, ribozyme components designed to catalytically cleave the CaSm gene mRNA transcript Disrupts CaSm gene mRNA translation and expression of target or pathway genes using offspring You can also. (For example, PCT International Publication No. WO90 / 11364 published October 4, 1990; Sa rver et al., 1990, Science 247: 1222-1225). MRNA by site-specific sequence recognition CaSm gene mRNA can be destroyed using a ribozyme that cleaves The use of marhead ribozymes is preferred. Hammerhead ribozyme is the target mRNA MR at the position indicated by flanking regions that form complementary base pairs with NA Cut NA. The only requirement is that the target mRNA has the following two base sequences: 5'-UG-3 'Is to have. Construction and manufacture of hammerhead ribozymes is described in the art. It is well known in the art and further described in Haseloff and Gerlach, 1988, Nature, 334: 585-591. Is well documented. A cleavage recognition site is located near the 5 'end of CaSm gene mRNA. To be efficient, ie increase efficiency, It is preferable to engineer ribozymes to minimize intracellular accumulation of New   For example, the following array: A hammerhead ribozyme having the following can be used according to the present invention.   The ribozymes of the present invention also include Tetrahymena Thermophila (IVS, or L-19 IVS (Known as RNA) and by Thomas Cech and co-workers. (Zaug et al., 1984, Science, 224: 574-578; Zaug and Cech, 1 986, Science, 231: 470-475; Zaug et al., 1986, Nature, 324: 429-433; University P International Patent Application No. WO 88/04300 published by atents Inc .; Been and Cech, 19 86, Cell, 47: 207-216) and other RNA endoribonucleases (hereinafter "Cech-type Bozyme "). Cech-type ribozymes are targeted at the target RNA after cleavage of the target RNA occurs. It has an eight base pair active site that hybridizes to the sequence. The present invention resides in the CaSm gene. Includes those Cech-type ribozymes that target an existing 8 base pair active site sequence.   As in the antisense approach, ribozymes are modified oligonucleotides. (Eg, related to improvements in stability, targeting, etc.) It should be delivered to cells that express the gene. The preferred method of delivery is a strong structure. D "encodes" a ribozyme under the control of the adult pol III or pol II promoter Transcription to include the use of NA constructs Transfected cells are sufficient to disrupt endogenous CaSm gene messages and inhibit translation Will produce large amounts of ribozymes. A ribozyme different from the antisense molecule Since it is a medium, it requires only a low intracellular concentration for efficiency.   The antisense RNA and DNA, ribozymes, and triple helix molecules of the invention are: The synthesis of DNA and RNA molecules can be controlled by any method known in the art. Can be manufactured. These include, for example, solid phase phosphoramidite chemical synthesis and the like. Oligodeoxyribonucleotides and oligoribonucleic acids well known in the art, such as Includes techniques for chemically synthesizing nucleotides. Alternatively, the RNA molecule is Can be produced by in vitro and in vivo transcription of a DNA sequence encoding an RNA molecule. You. Such a DNA sequence may be a suitable RNA such as a T7 or SP6 polymerase promoter. It can be incorporated into a variety of vectors that incorporate a polymerase promoter. Alternatively, depending on the promoter used, the antisense RNA may be constitutive or Stable introduction of inducibly synthesized antisense cDNA constructs into cell lines it can. These nucleic acid constructs can be selectively delivered to desired cells via a delivery complex. It can be administered to a population.   As a means to increase intracellular stability and half-life, Various modifications can be introduced. Possible modifications are not limited Is the 5 'and the flanking sequence of the ribo- or deoxynucleotide And / or addition to the 3 'end, or oligos in the oligodeoxyribonucleotide backbone. Phosphorothioate instead of phosphodiesterase linkage Is the use of 2'0-methyl.                             5.6.3 Therapeutic antibodies   Cancer using antibodies that show the ability to down-regulate the activity of the CaSm gene product Can be treated. Such antibodies are prepared using the standard techniques described in Section 5.3 above. For full-length wild-type or mutant CaSm protein, Or, for example, a portion of a protein such as Sm motif 1 or Sm motif 2. Can be prepared for any peptide. Some of these antibodies are limited But not polyclonal, monoclonal, Fab fragment, single chain antibody, chimera Antibodies.   Since CaSm is an intracellular protein, use antibodies that are internalized. Is preferred. However, using ribofectin or ribosomes, CaSm Delivery of antibodies or fragments of the Fab region that bind to a gene product epitope to cells. Can be. When using antibody fragments, they bind to the binding domain of the CaSm protein. The smallest inhibitory fragment is preferred. For example, various antibodies that bind to CaSm protein A peptide having an amino acid sequence corresponding to the domain of the region can be used . Such peptides may be chemically synthesized or recombinant using methods well known in the art. DNA techniques (see, for example, Creighton, 1983, supra; and Sambrook et al., 1989, supra). )). Alternatively, intracellular epitopes such as neutralizing antibodies Single-chain antibodies that bind to the loop can also be administered. Such single chain antibodies are available from Marsac o et al. (1993, Proc. Natl. Acad. Sci. USA 90: 7889-7893) and the like. For example, nucleotide sequences encoding single chain antibodies within a target cell population It can be administered by expressing the array.                             5.6.4 Gene therapy   Gene therapy refers to subjects with cancer or who want to prevent or control cancer. Refers to the treatment or prevention of cancer performed by administering an acid. This implementation of the invention In a form, the therapeutic nucleic acid is an antitherapeutic that interferes with the therapeutic effect by inhibiting CaSm expression. Produce nucleic acid molecules inside the cell. In another embodiment of the present invention, Copy the negative mutant CaSm protein or non-functional fragment or derivative thereof Administering nucleic acids comprising the sequence to be loaded, the interaction of CaSm and other intracellular Inhibits CaSm action by interfering with molecules.   For a general description of methods for gene therapy, see Goldspiel et al., 1993, Cli. nical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3: 87-95; Tolstoshe v, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan, 1993, Scienc e 260: 926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:19 1-217; May 1993, TIBTECH 11 (5): 155-215). Recombinant DNA technology available A method generally known in the art is described in Ausubel et al., (Eds.), 1993, C. urrent Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 19 90, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, N Y; and Chapters 12 and 13, Dracopoli et al., (Eds.), 1994, Current Protocols i. n Human Genetics, John Wiley & Sons, NY.   In one embodiment, the therapeutic nucleic acid is a dominant non-functional CaSm protein in cancer cells. Protein or a fragment thereof or a part of an expression vector for expressing a chimeric protein. A certain CaSm nucleic acid. The function of CaSm is that protein-protein interactions It is thought that it intervenes. Therefore, it is effective in binding to its interacting partner Competitive but functional CaSm mutant competes with wild-type CaSm It can be used as a dominant negative mutation. Dominant non-functional CaSm Can be manipulated to express CaSm in cancer cells that inappropriately overexpress CaSm.   In a preferred embodiment, the therapeutic nucleic acid produces an antisense molecule in a suitable host. An antisense CaSm nucleic acid that is part of an expression vector. In particular, take The nucleic acid has a promoter operably linked to the antisense CaSm sequence, The promoter may be inducible or constitutive and, if desired, tissue specific. You.   In another specific embodiment, the antisense CaSm sequence and any other desired These sequences are flanked by regions that promote homologous recombination at the desired genomic site. Thus, using a nucleic acid molecule that results in intrachromosomal expression of the antisense CaSm nucleic acid ( Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935; Zijlstra et al. , 1989, Nature 342: 435-438. ).   Delivery of the nucleic acid to the patient can be direct (the patient can be administered the nucleic acid or nucleic acid delivery vector or Direct exposure to the livery complex) or indirectly (first in vitro nucleation of cells in vitro). (Transformed with an acid and then transplanted into a patient). these The two approaches are known as in vivo or ex vivo gene therapy, respectively. I have.   In a specific embodiment, the nucleic acid is administered directly in vivo, where it is expressed. Produce antisense nucleic acid molecules or encoded non-functional CaSm gene products . This can be accomplished by any of a number of methods known in the art, including, for example, a suitable nucleic acid. Build it as part of an expression vector, e.g., defective or attenuated retrovirus Virus or other viral vectors (US Pat. No. 4,980,286) ), Or by direct injection of naked DNA, or by microparticle bombardment (eg, Genetic cancer; Biolistic, Dupont), or coating with lipids or cell surface receptors Or drug transfections, biopolymers (eg, poly-β-1-> 4-N- Acetylglucosamine polysaccharide; encapsulation in US Pat. No. 5,635,493), liposomes, By use of encapsulation in microparticles or microcapsules or into the nucleus By binding and administering a peptide that is known to be To bind to a ligand susceptible to mediated endocytosis and administer it (See, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432). It can be achieved by administering such that it is present in the cell. Another implementation In one embodiment, the nucleic acid-ligand complex can be formed, wherein the ligand Comprises a fusogenic viral peptide that disrupts endosomes, More nucleic acids can avoid lysosomal degradation. Yet another implementation In the form, the nucleic acid is targeted to a specific receptor, resulting in cell-specific uptake. And can be targeted in vivo for expression and expression (see, eg, PCT Publication No. WO 92/06180). Issue April 16, 1992 (Wu et al.); WO92 / 22635 No. WO92 / 20316, Dec. 23, 1992 (Wilson et al.); No. No. 3/14188 July 22, 1993 (Clarke et al.); No.WO93 / 20221 October 14, 1993 (Young) See). Alternatively, a host for expression by introducing the nucleic acid into cells and homologous recombination. It can be integrated into the DNA of cells (Koller and Smithies, 1989, Proc. Natl. A. cad. Sci. USA 86: 8932-8935; Zijlstra et al., 1989, Nature 342: 435-438).     In a specific embodiment, a viral vector containing an antisense CaSm nucleic acid is used. You. For example, retroviral vectors can be used (Miller et al., 1993, Meth. Enzymol. 217: 581-599). These retroviral vectors are Not required for packaging and integration of the ils genome into host cell DNA It has been modified to delete the Torovirus sequence. A used for gene therapy Antisense CaSm nucleic acids are cloned into a vector and gene delivery to the patient is achieved. It will be easier. For more information on retroviral vectors, see Boesen et al., 1994, Biot. herapy 6: 291-302, which may be more resistant to chemotherapy. Virus that delivers the mdrl gene to hematopoietic stem cells The use of vectors is described. Use of retroviral vectors in gene therapy Other references that exemplify applications include: Clowes et al., 1994, J. Am. Clin. Invest. 93:64 4-651; Kiem et al., 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Huma n Gene Therapy 4: 129-141; and Grossman and Wilson, 1993, Curr. Opin. i n Genetics and Devel. 3: There is 110-114.   Adenoviruses are other viral vectors that can be used for gene therapy. A Denovirus is a particularly attractive vehicle for delivering genes to airway epithelium . Adenoviruses naturally infect respiratory epithelia and cause mild disease. A Other targets for denovirus-based delivery systems include liver, central nervous system, and endothelial cells. There are vesicles and muscle. Adenovirus can infect nondividing cells It has advantages. Kozarsky and Wilson, 1993, Current Opinion in Genetic s and Development 3: 499-503 outlines adenovirus-based gene therapy. Provides a theory. Bout et al., 1994, Human Gene Therapy 5: 3-10 Demonstration of use of adenovirus vectors for transfer to rhesus epithelium in rhesus monkeys Was done. Other examples of the use of adenovirus in gene therapy include Rosenfel d et al., 1991, Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155; and Mastrangeli et al., 1993, J. Mol. Clin. Invest. 91: 225-234.   The use of adeno-associated virus (AAV) in gene therapy has also been proposed (W alsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300).   The form and amount of the therapeutic nucleic acid intended for use will depend on the cancer, the desired effect, the condition of the patient, and the like. And can be determined by one skilled in the art.   Unfavorable approaches to gene therapy include electroporation, Lipofection, calcium phosphate mediated transfection, or virus Antisense CaSm gene or dominant nonfunctional CaSm gene To the cancer cells in tissue culture. Introduction methods usually include selectable markers. Of the cells into cells. The cells are then placed under selection to incorporate the transgene. Cells that are expressed in E. coli are isolated. Then replace with cells overexpressing CaSm To deliver these cells to the patient. In this embodiment, the resulting recombination Prior to in vivo administration of the cells, the nucleic acid is introduced into the cancer cells. Such introductions are limited Although not required, transfection, electroporation, Cloinjection, viral vector or bacterio containing nucleic acid sequence Phage vector infection, cell fusion, chromosome-mediated gene transfer, microkaryotes (micr ocell) known in the art, including mediated gene transfer, spheroplast fusion, etc. Can be performed by any of the methods described above. Introduction of foreign genes into cells Many techniques are known in the art (eg, Loeffler and Behr, 19 93, Meth. Enzymol. 217: 599-618; Cohen et al., 1993, Meth. Enzymol. 217: 618-644 Cline, 1985, Pharmac. Ther. 29: 69-92). The technology is cheap for cells Results in the introduction of a defined nucleic acid, so that the nucleic acid can be expressed by the cells and Preferably, it can be inherited and expressed by the progeny of the cell.   The resulting recombinant cells can be delivered to a patient by various methods known in the art. Good In a preferred embodiment, the recombinant blood cells (eg, hematopoietic stem cells or progenitor cells) Preferably, it is administered intravenously.   Using targeted homologous recombination, inactivate the gene or its promoter or Knock-out can also reduce endogenous CaSm gene expression. (Eg, Smithies et al., Each of which is incorporated herein by reference in its entirety). 1985: Nature 317: 230-234; Thomas & Capecchl, 1987, Cell 51: 503-512; Thom. pson et al., 1989 Cell 5: 313-321). For example, the endogenous CaSm gene (CaSm gene Flanked by DNA homologous to either the coding or regulatory region of ) Mutant, non-functional CaSm gene (or completely unrelated DNA sequence) can be selected Together with or with a functional marker and / or a negative selectable marker Can be used to transfect cells that express the CaSm gene in vivo. it can. Insertion of the DNA construct allows inactivation of the CaSm gene by targeted homologous recombination. Causes sexualization. Such an approach is particularly useful for animals with inactive CaSm genes. Suitable for the use of modifications to ES (embryonic stem) cells to produce offspring of See, eg, Thomas & Capecchi 1987 and Thompson 1989, supra). Such techniques Surgery can also be used to create animal models of cancer. Direct injection of recombinant DNA construct Provided or a suitable viral vector, such as a herpes virus vector This approach will be useful in humans as long as It should be noted that it can be applied to different uses.   Alternatively, the regulatory regions of the CaSm gene (ie, the CaSm gene promoter and And / or enhancer) Forms a triple helix structure that blocks CaSm gene transcription in target cells in the body This can reduce the expression of the endogenous CaSm gene (as an overview, lene, C.E. 1991, Anticancer Drug Des., 6 (6): 569-84; Helene, C.E. Et al., 1992, Ann, N.Y. Acad. Sci., 660: 27-36; And Maher, L.J., 1992, Bioassays 14 (12): 807-15).                       5.7Pharmaceutical formulations and administration methods   The compounds and nucleic acid sequences described herein are administered to a patient at a therapeutically effective dose. To treat or prevent cancer. A therapeutically effective dose is the amount of the treated subject. Refers to an amount of the compound that is sufficient to provide a health benefit to the test subject. The therapeutic composition is a nucleic acid Formulations and methods of administration that can be used if they comprise are described in Chapter 5.6.4. You.                             5.7.1Effective dose   The toxicity and therapeutic efficacy of the compound may be, for example, LD₅₀(50% lethal dose of the population) and E D₅₀Cell culture or experiment to determine (the dose that is therapeutically effective in 50% of the population) It can be determined by standard pharmaceutical methods in animals. Toxicity and treatment The dose ratio of the effect is the therapeutic index, LD₅₀/ ED₅₀It can be expressed as a ratio. big Compounds that exhibit a high therapeutic index are preferred. Can also use compounds that have toxic side effects Yes, but minimizes the potential for damage to uninfected cells, thereby reducing side effects To design a delivery system that targets such compounds to diseased tissue sites Care should be taken.   The data obtained from cell culture assays and animal studies is based on the dosage used in humans. It can be used in formulating a range. The dose of such compounds is almost Is non-toxic ED₅₀Is preferably within the range of the circulating concentration including: The dose is It may vary within this range depending on the dosage form used and the route of administration used. Wear. For any of the compounds used in the methods of the invention, the therapeutically effective dose will vary. Can be estimated from cell culture assays. Dose is determined in cell culture I c₅₀(Ie, the concentration of test compound that achieves half-maximal inhibition of symptoms). Ring plasma concentration It can be formulated in animal models to reach a range of degrees. Using such information Thus, an effective dose in humans can be more accurately determined. Plasma levels can be measured, for example, by high performance liquid chromatography.                          5.7.2.Formulations and uses   A pharmaceutical composition for use according to the present invention may comprise one or more physiologically acceptable carriers. Alternatively, it can be formulated by an ordinary method using an excipient.   Thus, the compounds of the present invention and their physiologically acceptable salts and solvents are: Administration by inhalation or infusion (either through the mouth or nose), orally, orally It can be formulated for buccal, parenteral or rectal administration.   For oral administration, the pharmaceutical composition can be, for example, a binder (e.g., pregelled Corn starch, polyvinylpyrrolidone, or hydroxypropylmethyl Fillers (eg, lactose, microcrystalline cellulose, or phosphoric acid water) Lubricants (eg, magnesium stearate, talc or silica) Disintegrants (eg potato starch or starch glycolate nato) Or a pharmaceutically acceptable agent such as a wetting agent (eg, sodium lauryl sulfate) Tablets or capsules prepared by conventional methods using the excipients Can get. Tablets can be coated by methods well known in the art. Oral throw Liquid preparations for administration may take the form, for example, of solutions, syrups or suspensions. Or they may be composed with water or other suitable vehicle before use. As a dry preparation. Such liquid preparations may be suspended (e.g., Sorbitol syrup, cellulose derivatives or hydrogenated edible lipids); emulsifiers (eg Non-aqueous solvents (eg, almond oil, oily oils). Steal, ethyl alcohol or rectified vegetable oil); and preservatives (eg, methyl Or propyl-p-hydroxybenzoate or sorbic acid) Is It can be prepared in a customary manner using various additives. In addition, the formulation is Flavoring, coloring and sweetening agents can be included as appropriate.   Preparations for oral administration can be suitably formulated to give sustained release of the active compound. You.   For buccal administration, the composition may be formulated in conventional tablets or lozenges (lo lozenges). zenges).   For administration by inhalation, the compounds for use according to the invention are, for example, Lorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroe Pressurized packs using a suitable propellant such as tin, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from nebulizer or nebulizer. Pressurization In the case of aerosols, by providing a valve to deliver a metered amount, The unit of measurement can be determined. Caps made of, for example, gelatin for use in inhalers or insufflators The cell or package is prepared by combining the present compound with a suitable powder base, such as lactose or starch. Can be formulated to contain a powder mixture of   The compound can be administered parenterally, by injection, for example, by bolus injection or continuous infusion. It can be formulated for administration (ie, intravenous or intramuscular). Injectable preparations, for example Provided as unit dosage form in ampoules or multi-dose containers with added preservatives it can. The composition may be in the form of a suspension, solution or emulsion in an oily or aqueous solvent. Can be in the form of a mixture, and can be combined with suspending, stabilizing and / or dispersing agents, etc. It can contain reagents. Alternatively, the active substance may be added to a suitable vehicle, e.g., It may be in the form of a powder for constitution with sterile water containing no pyrogen.   The compounds may also be used in conventional suppositories, for example cocoa butter or other glycerides. Can also be formulated as rectal compositions such as suppositories or enemas, containing You.   In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long-acting preparations can be implanted (e.g., subcutaneously or intramuscularly) or It can be administered by internal injection. Thus, as an example, The compound can be a suitable polymeric or hydrophobic material, such as milk in an acceptable oil. ), Or with ion exchange resins, or with low solubility derivatives, such as low solubility It can be formulated as a degradable salt.                 6. Example:Identification of novel genes involved in cancer   This example describes the isolation and identification of the CaSm gene. Its expression is in the pancreas Subtractive hybridization clones to isolate genes associated with cancer Was performed. Overexpressed in pancreatic cancer cells for detailed characterization The CaSm gene was selected.                           6.1.Materials and methods Cell line   The cell lines HS680.PAN, CAPAN-1 and PANC-1 were converted to American type culture. -Obtained from the collection (Rockville, MD). DMEM / 10% FBS respectively , RPMI 1640/15% FBS and DMEM / 10% FBS. In a 35 mm well, 2 μg of DNA and 10 μl of LipofectAmine (Fibco-BRL, Bethesda, MD) (60-5 (0% confluent) to transfect PANC-1 cells. 500μ Stable transfected cells were selected in g / ml G418. Soft agar growth assay Was performed in a 6 well plate (35 mm well). At 1000, 5000, and 25,000 cells Duplicate assays started were counted after 3 weeks. Two independent runs of the soft agar assay Was. Tissue and serum samples   The Cooperative Human Tissue Network, Mt. Sinai Medical Center in Miam i Beach, Florida (from Dr. Saul Suster) and The Medical University of Sou Human tissue was obtained from th Carolina. RNA isolation and analysis   RNA from cultured cells and human tissues was purified according to the manufacturer's protocol using RNAzol B ( Purified using Tel-Test, Inc., Friendswood, TX). Lehrach et al. (1977, Bioche m, 16: 4743-4751) containing 0.66M formaldehyde (2.2M in the sample). Total RNA was fractionated on a 1.2 or 1.5% agarose gel. Separated in gel Transfer RNA to Duralon filters (Stratagene) in 0.1 M sodium phosphate, pH 6.8. After transfer and UV crosslinking, follow the manufacturer's instructions in Quik-Hyb (Stratagene). Hybridized with the labeled nucleic acid molecule. The quantity and quality of RNA is 28S and 18S-ribo Monitoring was performed by visualization of the some band with ethidium bromide.                                6.2.result                    6.2.1Cloning of cancer-related genes   In pancreatic cancer, mRNAs that are separately expressed are firstly the pancreatic cancer cell line CAPAN-1 and Hybridization subtracted with the more normal and more normal pancreatic epithelial cell line HS680.PAN It was identified by performing the zation. Subtractive hybridization first Performed as reported (1990, Schweinfest et al., Genet. Anal. Tech. Appl., 7: 64-70; 1993, Schweinfest et al., Proc. Natl. Acad. Sci., USA, 90: 4166-4170). Complementary DNA (cDNA) clones obtained by subtraction hybridization The expression was confirmed by two methods.   First, the DNA from 600 subtracted cDNA clones was dot-blocked onto a nylon membrane. Hybridization between CAPAN-1 and labeled total cDNA derived from HS680.PAN mRNA Analyzed according to the option. Clone CA3-30 showing different hybridization, Isolated from subtracted library clones. Full length partially included in CA3-30 The sequence is called the CaSm gene. Using the first CA3-30 cDNA clone, A full-length clone of the CaSm gene was isolated by surgery. CaSm is Among them, stronger hybridization for CAPAN-1 cDNA compared to HS680.PAN cDNA Had a solution signal.   Second, the CaSm cDNA insert was expressed differently (other, identified in trials). Northern blots of RNA from tumor and normal pancreatic tissue Used as a probe in the scanning. FIG. 1 shows a sample pair (from the same patient). Tumors and normal tissues) and CaS, including both individual tumors and normal subjects A representative Northern blot for m is shown. 8 out of 9 pairs are tumor / pancreas In flames, it shows significantly higher levels of 1.2 kb CaSm mRNA than normal. Because absolute levels of CaSm mRNA vary somewhat between samples, some tumors Samples had lower mRNA levels than unpaired normal samples (e.g., 17T and 18N). However, in paired samples, tumor tissue Shows a consistent pattern of overexpression in 9 individual tumor / pancreatitis subjects 9 show high levels of CaSm mRNA, comparable to paired tumor subjects.   In addition to pancreatic cancer, CaSm mRNA is found in normal thymus, breast, colon, spleen and esophageal tissues Is expressed in normal pancreas, lung, brain, placenta, kidney, ovary, It is also found in testis and heart (FIG. 2A). Some pancreatic cancer cell lines have high levels Expresses Bell CaSm mRNA. These include CAPAN-1, CAPAN-2, AsPC-1, PANC-1 and others. And HPAC (Figure 2B). Other cancer-derived cell lines that express high levels of CaSm mRNA Prostate (PC-3), liver (SK-HEP-1), ovary (OVCAR-3), lung (A-427), rectum (SW1 463), kidney (Caki-1) and non-erythropoietic hematopoietic cells (MOLT-4, NC-37, Raji, H9, KG-1) Origin (Figure 2B) as well as mesothelioma. As a result, CaSm gene expression is It was shown to be up-regulated in cells, especially pancreatic cancer cells. The results also show that the liver (SK-HEP-1), ovary (OVCAR-3), lung (A427) and kidney (Caki-1 ) -Derived cancer cell lines have increased CaSm expression compared to their cognate normal tissues (FIGS. 2A and 2B are compared).   In addition, a lower molecular weight CaSm variant is identified by polymerase chain reaction Have been. This mutant is clearly the amino acid 22- of Sm motif 1. All 32 and Sm motif 2 are deleted.                            6.2.2.CaSm cDNA   A full-length clone comprising the CaSm cDNA was isolated and sequenced, and nucleotides 878 -883 can be composed of 894 nucleotides containing a polyadenylation signal all right. The translation initiation signal is the sequence TCAAA containing the essential purines at positions -3 and +4.A TG A (nucleotides 160-168) (1991, Kozak et al., J. Cell Biol., 115 : 887-903). The largest open reading frame has an estimated molecular weight of 15,179 da 133 amino acid polypeptide (nucleotides 165-163) with a Luton and an isoelectric point of 4.97 Can be coded. The estimated open reading frame (ORF) of CaSm is And its expression in the coupled transcription and translation reactions. The putative code strand is 18 kg, somewhat larger than the estimated molecular weight of 15.2 kd from its deduced amino acid sequence Translate the Dalton polypeptide. Putative non-coding strands produce much smaller products Generate. In addition, only antisense probes for the putative coding strand Hybridized with mRNA from the cell, thus confirming expression of the putative ORF. It is.   Significant similarity to any motif listed in the PROSITE database No gender was observed. However, the 133 amino acid polypeptide of CaSm has the snRNP Sm G It has significant homology to the protein (FIG. 3A). Between CaSm and human Sm G protein Computer fit (BESTFIT) is 32% identity, 60% similarity (preserved) Consider amino acid substitution). This similarity is almost entirely limited to the amino-terminal half of CaSm. Is localized (amino acids 4-78). Interestingly, this homology indicates that the Sm protein It has been localized to two Sm motifs that characterize Amilly (Hermann et al., 1995, E MBO J., 14: 2076-2088). Sm motif 1 which is 32 and 14 amino acids respectively and Sm motif 2 is probably the protein-protein phase required for the construction of the snRNP complex (Hermann et al., 1995, EMBO J., 14: 2076-2088). CaSm and Sm  The level of identity between G proteins (32%) For example, Sm G protein identity (> 50% identity) between very marginal species such as plants and yeast Lower than that of one sex). Sm proteins derived from other snRNPs are better than Sm G proteins. Not similar to CaSm. In addition, at 133 amino acids, the CaSm gene product It is almost twice the size of the Sm G protein (76 amino acids). Finally, Sm F Tampa With the exception of proteins (p1 = 4.6), all Sm proteins have a basic isoelectric point (Wop pmann et al., 1990, Nuc. Acids Res., 18: 4427-4438). For this reason, CaSm It does not appear to be a true member of the quality family. Nevertheless, Most important properties that make up the Sm motif are retained in CaSm. Explicitly Are 100% conserved glycine and Aspa at positions 13 and 23 of Sm motif 1, respectively. Lagin residues are also found in CaSm. Overall, matches with Sm motif 1, Twelve of the fifteen identified positions are conserved in CaSm. In addition, Sm Mochi Of the 11 positions that were found to be identical in Roof 2, 10 were also conserved in CaSm (See FIG. 3A).   Among known proteins, the predicted CaSm protein is human Sm G protein. Ie, most similar to the “common protein” component of snRNP (1995, Hermann et al.) EMBO J., 14: 2076-2088). Interestingly, the largest region of homology is the Sm protein. It is found in the so-called Sm motifs 1 and 2, which characterize the protein. These mochi Is the protein-protein phase among members of the Sm protein family. Required for interaction, but they are also key Sm protein families (1995, Hermann et al., EMBO J., 14:20). 76-2088). All eight snRNP common core proteins have been cloned and sequenced. However, CaSm has very limited homology to this group. Therefore, CaSm is not considered a member of this common core group. Protein distribution Caenorhabditise legans and Saccharomyces ce Two gene products of DNA sequence from revisiae have higher levels than Sm G protein Similarity was shown (see FIGS. 3B and 3C). The homologues of these two CaSm gene products are also Including Sm motifs It is most similar to CaSm in the region. Each of these gene products is cosmid F40F 8 (GenBank accession number Z69302). elegans open reading frame J071 4 (PIR S55137), and the S. cerevisiae encoded by the DNA clone under accession number Z49399. cer Inferred from the evisiae gene product ORF YJL124c. C. elegans sequence is amino acids 3-12 1 has 54.4% identity, 72.8% similarity, while cerevisiae clone is amino The acid 4-130 has 37.8% identity and 67.7% similarity. Both of these Sm motifs Area. In addition, important amino acids that form a consensus It is also stored at this location. Thus, it has a molecular weight similar to CaSm. These two proteins are examples of non-mammalian homologues of CaSm in each microorganism .   Comprising genetically engineered peptides containing CaSm and FLAG epitopes Transfect the DNA construct encoding the fusion protein into COS-1 cells temporarily. I did it. Expression of the fusion protein and its subcellular distribution is specific to the FLAG epitope (Kodak scientific imaging system) ). Typically, but not in the same transfected cell, but in the cytoplasm and nucleus Both stainings were observed. The results indicate that CaSm is an intracellular protein that shuttles between cytoplasm and nucleus. It was suggested to be protein. Comprising CaSm and green fluorescent protein Similar results were obtained in expression experiments performed using the fusion protein (CaSm-GFP). Was.                    7.Example: Functional analysis of CaSm gene   This example illustrates the association of CaSm gene expression in pancreatic cancer cells with the transformed phenotype Things. Soft agar growth assay to assess anchorage-independent growth of cancer cells Was used to test for tumorigenicity of the cancer cells in mice.                           7.1.Materials and methods   Neomycin / G418-resistance cassette and multiple clones from pBluescript II SK Eukaryote, a modified version of pSG5 (Stratagene, La Jolla, CA) Insert the CaSm-derived insert into the product expression vector pSGneoSK in the antisense direction. Cloned. This construct is used to transfect PANC-1 cells. Used. Similar antisense CaSm expression constructs were transformed into another pancreatic cell line, ASPC-1 cells Was used for transfection.   The antisense CaSm expression construct was constructed in E. coli using an enhancer, Contains the combination of the denovirus major late promoter and the Adenovirus transport vector pAdBM5 (Quantum Biotechnologi es, Inc., Quebec, Canada). Adenovirus antisense CaS m construct is a human 2 that complements the essential viral EIA and EIB gene deletions. Replication is abnormal, except for cells such as 93 cells. Recombination product between two DNA molecules Has been engineered to produce recombinant infection of the adenovirus. The nome portion and this construct were co-transfected. These recombinant viruses To infect many different cell lines or tissues of human or non-human origin But they do not replicate once inside the cell.                                7.2.result   Up-regulation of CaSm in pancreatic cancer cells is responsible for the transformation of these cells In order to assess whether it is associated with a The experiment was performed. Expression constructs that constitutively express the 0.8 kb antisense RNA of CaSm , PANC-1 cells were stably transfected. For stable uptake of constructs After selection in G418, individual clones were Northern blot hybridized. Screening results in reduced endogenous 1.2 kb CaSm mRNA expression. I did. Ann Screening for antisense RNA is primarily expected to interfere with mRNA translation. Endogenous 1.2 kb CaSm mRNA levels in most clones cloned show no decrease Was. However, in order to confirm the decrease in CaSm expression, the knockdown of endogenous mRNA was confirmed. The clones that showed "p" were preferentially selected for further experiments. Fig. 4 Significant decrease in endogenous CaSm mRNA transcript in presence of chisense transcript (0.8 kb) This indicates that several clones were obtained.   Four clones were selected along with parental cells for analysis of anchorage-independent growth . Significant decrease in growth ability of antisense transfectants in soft agar (Figure 5). After 3 weeks in soft agar, only the parental cell line, PANC-1, Retained the ability to form large colonies in agar (FIG. 5A). Still many 4 antisense transfections, including clone 1 expressing low endogenous CaSm transcripts It is unlikely that all of the target bodies (clones K, L, 1 and 2) form large colonies. could not. The specific measurement of anchorage-independent colony formation of clone K is large (> 280 μm) and medium (140-280 μm) colonies are smaller than small (<140 μm) colonies Is also significantly greater (FIG. 5B). When growing on plastic, Cell K and parental cell line PANC-1 show very similar growth rates, The decrease in colony formation in the plant does not appear to be due to the growth rate.   ASPC-1 cells transfected with the antisense CaSm expression construct Similar results were obtained when tested in Say. When compared to the parent ASPC-1 cells, Growth of transfectants in soft agar was severely restricted. Evaluation of tumor formation ability To evaluate, transfected ASPC-1 cells were also tested in vivo. Fine Severe combined immunodeficiency (SCID) mice without vesicular and humoral immunity Infected ASPC-1 cells were injected. As a result, when compared to parent ASPC-1 cells, Infected ASPC-1 cells fail to form tumors in SCID mice, or The rate of tumor formation was shown to be low.   Recombinant infectious adenovirus carrying antisense CaSm expression construct PANC-1 cells stably transfected with the construct, as well as naive ASPC-1 cells Cells stably transfected with vesicles and antisense CaSm constructs Was used for infection. Unsensitized PANC-1 and ASPC-1 cells and tigers Infect both transfected PANC-1 and ASPC-1 cells with the recombinant virus. However, infected unsensitized PANC-1 cells and infected unsensitized ASPC-1 cells were I could not survive. Pre-transfected infected PANC-1 and ASPC-1 cells It survived and continued to show low growth in soft agar.                                7.3.Consideration   These results indicate that CaSm gene is up-regulated in pancreatic cancer tissues and cell lines. And induced by antisense RNA in pancreatic cancer cell lines. Inhibition of CaSm expression results in those cells forming anchorage-independent colonies in soft agar. Ability has been dramatically reduced. These findings indicate that CaSm is expressed in pancreatic epithelium Support the notion that they contribute to the transformed state in pancreatic cancer. PA In NC-1 cells or NIH3T3 cells, CaSm expression is not induced by serum stimulation, Stable transfectants expressing CaSm antisense RNA are nontransfected. Proliferates at the same rate as target cells and has no direct role in regulating growth. I think it is.   Increased CaSm mRNA expression was observed in the majority of pancreatic cancer samples tested Was. However, CaSm upregulation compared to the corresponding normal tissue Two of the samples that showed a failure were not neoplastic tissues, but rather they were Sample (see FIG. 1). A possible explanation for this finding is that CaSm is a disease of pancreatic cancer. It may also be elevated in the predisposing state of pancreatitis (199). 5, Bansal et al., Gastroent., 109: 247-251). In addition, the sample tested was Contains visceral cancer Maybe. Another possibility is high levels of CaSm in activated lymphocytes. Bell expression (see FIG.2B) was observed, indicating that Spike up-regulation is caused by a large number of activated lymphocytes that are part of the inflammatory response It may be due to.   However, implementation using other cell lines, such as prostate and lung cancer cell lines, Preliminary results indicate that CaSm plays an important role in the transformation of these cancer cell lines. And support for findings in pancreatic cancer. ing.   The possibility of using gene therapy for cancer treatment was also tested. Strategy is in vivo Cancer in patients causing down-regulation of endogenous CaSm gene expression Based on the delivery of antisense CaSm nucleic acid molecules to the Tumors regressed. The above results indicate that the antisense CaSm nucleic acid molecule Cancer cells that can be delivered to pancreatic cancer cells by using a vector system based on Phenotype could be changed. In addition, in the SCID mouse model The results were correlated with the findings in the in vitro soft agar growth assay, indicating that CaSm expression was Pancreatic cancer cells inhibited by antisense RNA have tumorigenic potential in vivo Indicates lower.                             8.Microbial deposit   An E. coli strain DH5α containing a cDNA clone encoding CaSm July 11, 1997, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms American Type Culture Collection (ATCC), 12301 Parklawn D Deposited with rive, Rockville, Maryland 20852 and obtained ATCC accession number 98497.   It is intended that the scope of the invention be limited to the specific embodiments described, which are intended to be merely illustrative of individual aspects of the invention. The invention is not limited to the embodiments, and functionally equivalent methods and components are It is within the scope of the invention. In fact, what is shown and described herein In addition, various modifications of the present invention are described above. And the accompanying drawings will make apparent to those skilled in the art. Such modifications are not Within the scope of the term.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61P 35/00 48/00 C07K 14/47 A61P 35/00 16/18 C07K 14/47 C12N 1/21 16/18 G01N 33/15 Z C12N 1/21 33/50 Z 5/10 33/53 D G01N 33/15 M 33/50 33/566 33/53 33/574 A C12N 15/00 ZNAA 33 / 566 5/00 B 33/574 A61K 37/02 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL , PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AU, AZ, BA, BB, BG, BR, BY, CA, CN, CU, CZ, EE, GE, GH, GW , HU, ID, IL, IS, JP, KG, KP, KR, KZ, LC, LK, LR, LT, LV, MD, MG, MK, MN, MX, NO, NZ, PL, RO, RU, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UZ, VN, YU (72) Inventor Papas, Takis, S. United States 29401 Charleston, South Carolina, Bull Street 84 Sea (72) Inventor Baron, Paul, El. United States 29407 Southport Drive, Charleston, South Carolina 1669 (72) Inventor Watson, Dennis, K. United States 29464 Mau, South Carolina Door Pleasant, Hobukau Bluff drive 617

Claims (1)

[Claims] 1. (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of FIG. 6, (b) a complement of the nucleotide sequence of (a), (c) an American Type Culture Collection contract Is capable of hybridizing with the CaSm gene or the nucleic acid molecule of (d) (a), (b) or (c) contained in the deposited microorganism having the number 98497, and is at least 80% identical to said nucleic acid molecule An isolated nucleic acid molecule comprising a nucleic acid molecule. 2. An isolated nucleic acid molecule that hybridizes to the nucleic acid of claim 1 and comprises a nucleotide sequence that encodes a naturally occurring CaSm polypeptide. 3. An isolated nucleic acid molecule comprising the nucleotide sequence of FIG. 4. The isolated nucleic acid molecule of claim 1, which is genomic DNA. 5. The isolated nucleic acid molecule of claim 1, which is a cDNA. 6. The isolated nucleic acid molecule of claim 1, which is an RNA. 7. The isolated nucleic acid molecule of claim 1, which hybridizes to at least six consecutive nucleotides of the CaSm gene shown in FIG. 8. a nucleotide sequence that hybridizes with the nucleic acid of claim 1 and encodes a polypeptide having the activity of a CaSm protein, linked to the nucleotide sequence encoding the heterologous protein or peptide without interruption by a stop codon. An isolated nucleic acid molecule comprising: 9. A nucleic acid molecule comprising a nucleotide sequence encoding (a) a CaSm deletion mutant or (b) the complement of the nucleotide sequence of (a). Ten. 10. The nucleic acid molecule according to claim 9, wherein Sm motif 1, Sm motif 2, or both Sm motif 1 and Sm motif 2 are deleted. 11． 10. The nucleic acid molecule according to claim 9, wherein one or more amino acid residues of Sm motif 1, Sm motif 2, or both Sm motif 1 and Sm motif 2 are deleted. 12． The glycine residue at position 13 in Sm motif 1, or the asparagine residue at position 23 in Sm motif 1, or both the glycine residue at position 13 and the asparagine residue at position 23 in Sm motif 1 have been deleted 10. The nucleic acid molecule of claim 9, wherein 13. 10. The nucleic acid molecule of claim 9, wherein amino acid residues 39-75 of CaSm have been deleted. 14. A nucleic acid molecule comprising a nucleotide sequence encoding CaSm Sm motif 1 or CaSm Sm motif 2. 15． 8. The isolated nucleic acid molecule of claim 7, further comprising a label. 16． A recombinant vector containing the nucleic acid molecule according to claim 1, 2, 8, 9, 10 or 14. 17． A regulation comprising a nucleic acid molecule according to claim 1, 2, 8, 9, 10 or 14, wherein the nucleotide sequence of said nucleic acid molecule comprises transcriptional and translational regulatory signals controlling the expression of said nucleotide sequence in a host cell. An expression vector operably linked to a nucleotide sequence. 18． A genetically engineered host cell containing the nucleic acid molecule of claim 1, 2, 8, 9, 10, or 14. 19. A regulation comprising a nucleic acid molecule according to claim 1, 2, 8, 9, 10 or 14, wherein the nucleotide sequence of said nucleic acid molecule comprises transcriptional and translational regulatory information controlling the expression of said nucleotide sequence in a host cell. A genetically engineered host cell operably linked to a nucleotide sequence. 20. 20. The genetically engineered host cell of claim 19, which is a prokaryote. twenty one. 20. The genetically engineered host cell of claim 19, which is a eukaryote. twenty two. A deposited microorganism having an American Type Culture Collection accession number 98497. twenty three. A transgenic animal, wherein the transgene comprises the nucleic acid molecule of claim 1. twenty four. A transgenic animal in which expression of a genomic sequence encoding a functional CaSm polypeptide is prevented or suppressed. twenty five. A delivery complex comprising the nucleic acid molecule according to claim 1 and targeting means. 26. 26. The delivery complex of claim 25, wherein the targeting means is selected from the group consisting of a sterol, a lipid, a virus, or a target cell-specific binding agent. 27. A delivery complex comprising the expression vector according to claim 17 and targeting means. 28. 28. The delivery complex of claim 27, wherein the targeting means is selected from the group consisting of sterols, lipids, viruses, or target cell specific binding agents. 29. 28. The delivery complex of claim 27, wherein the targeting means is selected from the group consisting of a recombinant adenovirus, a recombinant adeno-associated virus, a recombinant retrovirus, a recombinant herpes simplex virus, and a recombinant vaccinia virus. 30. A method for detecting the presence of a nucleic acid molecule according to claim 1 in a sample, comprising: (a) a nucleic acid capable of specifically hybridizing to a sample with at least a part of the nucleic acid molecule according to claim 1. Contacting the probe under hybridization conditions, and (b) measuring the hybridization of the probe to a sample nucleic acid, thereby detecting the presence of the nucleic acid sequence. 31. A method for detecting the presence of a nucleic acid molecule according to claim 1 in a sample, comprising: (a) a sample, a nucleic acid primer capable of specifically binding to at least a part of the nucleic acid molecule according to claim 1. Is contacted under hybridization conditions, (b) selectively amplifying at least a part of the nucleic acid molecule according to claim 1, and (c) detecting the amplified nucleic acid of the cell, whereby the nucleic acid Detecting the presence of the molecule. 32. A method for producing a CaSm polypeptide, comprising: (a) culturing the cells of claim 20 under conditions suitable for producing a CaSm polypeptide; and (b) isolating the CaSm polypeptide, A method comprising each step. 33. A method for producing a CaSm polypeptide, comprising: (a) culturing the cell of claim 21 under conditions suitable for producing a CaSm polypeptide; and (b) isolating the CaSm polypeptide, A method comprising each step. 34. An isolated mammalian polypeptide encoded by the nucleic acid molecule of claim 1. 35. An isolated CaSm polypeptide having the amino acid sequence of FIG. 36. 35. The polypeptide of claim 34, which is at least 90% identical to a CaSm polypeptide having the amino acid sequence of FIG. 37. 35. A fusion protein comprising the polypeptide of claim 34 and a polypeptide that is a detectable label or matrix binding domain. 38. A polypeptide encoded by the nucleic acid molecule according to claim 10. 39. A pharmaceutical formulation comprising a therapeutically effective amount of the polypeptide of claim 35 and a pharmaceutically acceptable carrier. 40. An antibody against the polypeptide according to claim 34 or 35. 41. 41. The antibody of claim 40, which is monoclonal. 42. An antibody preparation comprising an antibody fragment selected from the group consisting of F (ab) ' ₂ , Fab and Fv fragments, which specifically reacts with an epitope of a CaSm polypeptide. 43. 41. A pharmaceutical formulation comprising a therapeutically effective amount of the antibody preparation of claim 40 and a pharmaceutically acceptable carrier. 44. A method for detecting or measuring the polypeptide according to claim 34 or 36 in a sample, comprising: (a) contacting a sample with an antibody against a CaSm gene product under conditions that allow immunospecific binding; And (b) detecting or measuring the immunospecific binding of the antibody to the sample, thereby confirming the presence of the polypeptide in the sample, or measuring the amount of the polypeptide. . 45. A method for diagnosing cancer in a human subject, comprising detecting or measuring a CaSm gene product in a human subject, wherein the elevated level of the CaSm gene product is indicative of the presence of the cancer. 46. 46. The method of claim 45, wherein said cancer is pancreatic cancer. 47. 46. The method of claim 45, wherein the cancer is lung, mesothelioma, prostate, liver, ovarian, cervical, or breast cancer. 48. 48. The method of claim 46 or 47, wherein the cancer is associated with metastasis. 49. A method of tracking the effect of a therapeutic treatment in a human cancer patient, comprising measuring the CaSm gene product in a human cancer patient, wherein elevated levels of the CaSm gene product indicate an inadequate response to said treatment. There is a tracking method. 50. A method of identifying a compound that modulates CaSm gene expression, comprising: (a) contacting a test compound with a cell or cell lysate containing a reporter gene operably linked to a CaSm gene regulatory element; and (b) Detecting the expression of the reporter gene product. 51. A method for identifying a compound that modulates CaSm gene expression, comprising: (a) contacting a test compound with a cell or cell lysate containing a CaSm transcript; and (b) detecting translation of the CaSm transcript. A method comprising: 52. A method for identifying a compound that modulates the activity of a CaSm gene product or a homolog of a CaSm gene product, comprising: (a) bringing a test compound into contact with an organism or a cell containing the CaSm gene product or a homolog of CaSm; and b) comparing the phenotype of the organism or cell with the phenotype of the organism or cell that has not been contacted with the test compound, wherein the phenotypic change is such that the test compound is a CaSm gene product or a homolog of a CaSm gene product. Demonstrating that it can modulate the activity of a. 53. A method for identifying a compound that modulates the activity of a CaSm gene product or a homolog of a CaSm gene product, comprising: (a) contacting a test compound with a cell containing the CaSm gene product or a homolog of CaSm; The gene encoding the poly (A) binding protein and the large subunit of the translation initiation complex in it has been mutated so that the cell is not viable in the absence of the CaSm gene product or a homolog of CaSm. (B) comparing the viability or proliferation of the cells to the viability or proliferation of cells that have not been contacted with the test compound, wherein the decrease in proliferation or viability is due to the Ca Sm gene product or CaSm Demonstrating that it can modulate the activity of a homologue of the gene product. 54. 54. The method of claim 53, wherein the cell is a yeast cell, the poly (A) binding protein is Pablp, and the large subunit of the translation initiation complex is eIF-4G. 55. A method for treating cancer in a cancer patient, comprising: (a) administering to a cancer patient an effective amount of a delivery complex, wherein the delivery complex has an antisense nucleotide sequence of a CaSm gene sequence in a target cell. (B) an expression vector comprising an antisense nucleotide sequence of the CaSm gene sequence or a part thereof operably linked to a regulatory nucleotide sequence containing a transcription control signal for controlling expression, and targeting means; and (b) Expressing the antisense molecule in a target cell, thereby suppressing CaSm expression in the target cell. 56. A method of treating cancer in a cancer patient, comprising administering to the cancer patient an effective amount of a nucleic acid molecule encoding an antisense or ribozyme molecule that targets the CaSm transcript and prevents translation of the CaSm transcript. How to be. 57. A method for treating cancer in a cancer patient, comprising administering to the cancer patient an effective amount of a nucleic acid molecule that forms a triple helix with a CaSm gene promoter and inhibits transcription of the CaSm gene. 58. A method for treating cancer, comprising administering an effective amount of a compound that reduces CaSm expression in cells. 59. A method for treating cancer comprising administering an effective amount of a compound that antagonizes the activity of a CaSm gene product. 60. 60. The method of claim 55, 58 or 59, wherein said cancer is pancreatic cancer. 61. 60. The method of claim 55, 58 or 59, wherein said cancer is lung, mesothelioma, prostate, liver, ovarian, cervical or breast cancer. 62. 61. The method of claim 60, wherein said pancreatic cancer is associated with metastasis. 63. 62. The method of claim 61, wherein said cancer is associated with metastasis.