Genes and gene expression products that are differentially regulated in prostate cancer

FIELD OF THE INVENTION

[0001] This invention relates to the area of diagnosis, prognosis, and treatment of cancer, tumor progression, hyperproliferative cell growth, and accompanying physical and biological manifestations. More specifically, the invention includes polynucleotides that are differentially regulated in prostatic disorders, such as metastatic prostate cancer, localized prostate cancer, and benign prostate hyperplasia (BPH).

BACKGROUND OF THE INVENTION

[0002] Genes that are up- or down-regulated in cancer or tumor progression are useful for therapeutic and diagnostic purposes. For example, detection of genes or gene expression products up-regulated in hyperproliferative cells can be a predictive or diagnostic marker of the onset or the progression of cancer. Early diagnosis can be useful if the cancer, tumors, or hyperproliferating cells can be inhibited, removed, or terminated to prevent metastasis or recurrence of cancerous growth. Such early warning is of particular use to prostate cancer patients, where removal of the growth, tumor, or cells is beneficial if the disease is confined to the prostate. There is a need in the art for genes related to cancer and tumor progression.

SUMMARY OF THE INVENTION

[0003] The present invention provides methods and reagents for diagnosing cancer, tumor progression, hyperproliferative cell growth, and accompanying biological and physical manifestations. Reagents for such diagnostic kits include:

[0004] (a) polynucleotides comprising a sequence capable of hybridizing to one or more of SEQ ID NO:1-339 or complement thereof,

[0005] (b) polypeptides comprising the amino acid sequence encoded by any one of SEQ ID NO:1-339; and

[0006] (c) antibodies capable of binding polypeptides comprising the amino acid sequence of (b).

[0007] The methods of diagnosis of the present invention include both nucleic acid assays and immunoassays.

[0008] In another embodiment, the present invention provides both compositions and methods for treating or ameliorating cancer, tumor progression, hyperproliferative cell growth, and accompanying biological and physical manifestations. The compositions for treatment or amelioration include:

[0009] (a) polynucleotides comprising the sequence capable of hybridizing to one or more of the sequences shown in SEQ ID NO:1-339 and complement thereof, including antisense, ribozyme and gene therapy nucleic acid constructs;

[0010] (b) polypeptides comprising the amino acid sequence encoded by any one of SEQ ID NO:1-339; and

[0011] (c) antibodies capable of binding polypeptides of polypeptides comprising the amino acid sequence (b).

[0012] Methods of treatment or amelioration include administering compositions of polynucleotides, polypeptides, antibodies, or combinations thereof and can be used

[0013] (a) to inhibit translation and/or transcription;

[0014] (b) to inhibit biological activity;

[0015] (c) as a vaccine antigen; and

[0016] (d) as an immune system inducer.

[0017] Such compositions can be administered systemically or locally to the desired site.

[0018] In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of

[0019] (a) any one of SEQ ID NOs:2, 5, 49, 50, 99, 100, 115, 116, 118, 130, 131, 140, 144, 145, 146, 157, 158, 159, 163, 164, 165, 166, 177, 178, 180, 211, 212, 213, 218, 219, 220, 221, 229, 232, 233, 242, 243, 248, 249, 254, 256, 257, 259, 272, 273, 277, 288, 289, 292, 293, 316, 317, and 330;

[0020] (b) a polynucleotide that encodes a variant of the polypeptide encoded by (a); and

[0021] (c) a polynucleotide encoding a protein expressed by a polynucleotide having the sequence of any one of the sequences of (a).

[0022] Preferably, the nucleic acid obtained from the biological material of part (b) above is genomic DNA or mRNA. The nucleic acid can also be cDNA complementary to the mRNA.

[0023] Another embodiment of the invention is the use of the isolated polynucleotides or parts thereof as diagnostic probes or as primers.

[0024] In another embodiment, the present invention provides a composition comprising a polypeptide, wherein said polypeptide is selected from the group consisting of

[0025] (a) a polypeptide encoded by any one of SEQ ID Nos:2, 5, 49, 50, 99, 100, 115, 116, 118, 130, 131, 140, 144, 145, 146, 157, 158, 159, 163, 164, 165, 166, 177, 178, 180, 211, 212, 213, 218, 219, 220, 221, 229, 232, 233, 242, 243, 248, 249, 254, 256, 257, 259, 272, 273, 277, 288, 289, 292, 293, 316, 317, and 330;

[0026] (b) a polypeptide encoded by full-length mRNA or cDNA corresponding to any one of SEQ ID NO:1-339, and

[0027] (c) a variant of the protein (a) or (b);

[0028] In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention further provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with the polynucleotide sequence. The invention also provides the full-length cDNA and the full length human gene corresponding to the polynucleotide.

[0029] Protein and polypeptide compositions of the invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody that specifically reacts with such protein or polypeptide are also provided by the present invention.

[0030] The invention further relates to a polypeptide or nucleic acid obtained by transforming a host cell with nucleic acid comprising at least one of SEQ ID NO:1-339, culturing the host cell, and recovering the replicated nucleic acid, the expressed RNA, and/or the expressed polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

[0031]
FIG. 1 provides the open reading frame for clone SL 195.

[0032]
FIG. 2 provides the open reading frame for clone SL 197.

[0033]
FIG. 3 provides the immunohistochemistry staining results for clone SL expression in a variety of normal and tumor tissues.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Genes that are up- or down-regulated in cancer or tumor progression are useful for therapeutic and diagnostic purposes. For example, a diagnostic assay to determine the stage of the disease also is useful in tailoring treatment of aggressive versus more mild cancer or tumor progression. The polynucleotide sequences and encoded polypeptides of the present invention are useful for these diagnostic or prognostic purposes.

[0035] Further, modulation of genes or gene expression products that are misregulated can be used to treat or ameliorate cancer, tumor progression, hyperproliferative cell growth, and the accompanying physical and biological manifestations. For example, the polynucleotide sequences provided herein as SEQ ID NO:1-339, can be used to construct the following polynucleotide and polypeptide compositions that are useful for treatment: antisense; ribozymes; antibodies; vaccine antigens; and immune system inducers, to induce dendritic cells, for example.

[0036] Identified herein are polynucleotide sequences that are upregulated in a cancer cell line, more specifically in a prostate cancer cell line. Thus, the present invention relates to methods and reagents for diagnosis, and to methods and compositions for treatment.

[0037] I. Use of Polynucleotides Having a Sequence of One or More of SEQ ID NO:1-339 to Obtain Full-Length cDNA and Full-Length Human Gene and Promoter Region

[0038] Full-length cDNA molecules comprising the disclosed sequences are obtained as follows. The polynucleotide or a portion thereof comprising at least 12, 15, 18, or 20 nucleotides is used as a hybridization probe to detect hybridizing members of a cDNA library using probe design methods, cloning methods, and clone selection techniques as described in U.S. Pat. No. 5,654,173, “Secreted Proteins and Polynucleotides Encoding Them,” incorporated herein by reference. Libraries of cDNA are made from selected tissues, such as normal or tumor tissue, or from tissues of a mammal treated with, for example, a pharmaceutical agent. Preferably, the tissue is the same as that used to generate the polynucleotides, as both the polynucleotides and the cDNA represent expressed genes. Most preferably, the cDNA library is made from the biological material described herein in the Examples. Alternatively, many cDNA libraries are available commercially. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989).

[0039] Members of the library that are larger than the polynucleotide, and preferably that contain the whole sequence of the native message, are obtained. In order to confirm that the entire cDNA has been obtained, RNA protection experiments are performed as follows. Hybridization of a full-length cDNA to an mRNA will protect the RNA from RNase degradation. If the cDNA is not full length, then the portions of the mRNA that are not hybridized will be subject to RNase degradation. This is assayed, as is known in the art, by changes in electrophoretic mobility on polyacrylamide gels, or by detection of released monoribonucleotides. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989). In order to obtain additional sequences 5′ to the end of a partial cDNA, 5′ RACE (PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc. 1990)) is performed.

[0040] Genomic DNA is isolated using polynucleotides in a manner similar to the isolation of full-length cDNAs. Briefly, the polynucldotides, or portions thereof, are used as probes to libraries of genomic DNA. Preferably, the library is obtained from the cell type that was used to generate the polynucleotides, but this is not essential. Most preferably, the genomic DNA is obtained from the biological material described herein in the Examples. Such libraries may be in vectors suitable for carrying large segments of a genome, such as P1 or YAC, as described in detail in Sambrook et al., 9.4-9.30. In addition, genomic sequences can be isolated from human BAC libraries, which are commercially available from Research Genetics, Inc., Huntville, Ala., USA, for example. In order to obtain additional 5′ or 3′ sequences, chromosome walking is performed, as described in Sambrook et al., such that adjacent and overlapping fragments of genomic DNA are isolated. These are mapped and pieced together, as is known in the art, using restriction digestion enzymes and DNA ligase.

[0041] Using the polynucleotides sequences of the invention, corresponding full length genes can be isolated using both classical and PCR methods to construct and probe cDNA libraries. Using either method, Northern blots, preferably, are performed on a number of cell types to determine which cell lines express the gene of interest at the highest rate.

[0042] Classical methods of constructing cDNA libraries are taught in Sambrook et al., supra. With these methods, cDNA can be produced from mRNA and inserted into viral or expression vectors. Typically, libraries of mRNA comprising poly(A) tails can be produced with poly(T) primers. Similarly, cDNA libraries can be produced using the instant sequences as primers.

[0043] PCR methods are used to amplify the members of a cDNA library that comprise the desired insert. In this case, the desired insert will contain sequence from the full length cDNA that corresponds to the instant ESTs. Such PCR methods include gene trapping and RACE methods. Gruber et al., PCT WO 95/04745 and Gruber et al., U.S. Pat. No. 5,500,356. Kits are commercially available to perform gene trapping experiments from, for example, Life Technologies, Gaithersburg, Md., USA. PCT Pub. No. WO 97/19110. (Apte and Siebert, Biotechniques 15:890-893, 1993; Edwards et al., Nuc. Acids Res. 19:5227-5232, 1991).

[0044] The promoter region of a gene generally is located 5′ to the initiation site for RNA polymerase II, and can be obtained by performing 5′ RACE using a primer from the coding region of the gene. Alternatively, the cDNA can be used as a probe for the genomic sequence, and the region 5′ to the coding region is identified by “walking up.” If the gene is highly expressed or differentially expressed, the promoter from the gene may be of use in a regulatory construct for a heterologous gene.

[0045] Once the full-length cDNA or gene is obtained, DNA encoding variants can be prepared by site-directed mutagenesis, described in detail in Sambrook et al., 15.3-15.63. The choice of codon or nucleotide to be replaced can be based on disclosure herein on optional changes in amino acids to achieve altered protein structure and/or function.

[0046] As an alternative method to obtaining DNA or RNA from a biological material, nucleic acid comprising nucleotides having the sequence of one or more polynucleotides of the invention can be synthesized. Thus, the invention encompasses nucleic acid molecules ranging in length from 15 nucleotides (corresponding to at least 15 contiguous nucleotides of one of SEQ ID NO:1-339) up to a maximum length suitable for one or more biological manipulations, including replication and expression, of the nucleic acid molecule. The invention includes but is not limited to (a) nucleic acid having the size of a full gene, and comprising at least one of SEQ ID NO:1-339; (b) the nucleic acid of (a) also comprising at least one additional gene, operably linked to permit expression of a fusion protein; (c) an expression vector comprising (a) or (b); (d) a plasmid comprising (a) or (b); and (e) a recombinant viral particle comprising (a) or (b).

[0047] The sequence of a nucleic acid comprising at least 15 contiguous nucleotides of at least any one of SEQ ID NO 1-339, preferably the entire sequence of at least any one of SEQ ID NO:1-339, is not limited and can be any sequence of A, T, G, and/or C (for DNA) and A, U, G, and/or C (for RNA) or modified bases thereof, including inosine and pseudouridine. The choice of sequence will depend on the desired function and can be dictated by coding regions desired, the intron-like regions desired, and the regulatory regions desired.

[0048] Where the entire sequence of any one of SEQ ID NO:1-339 is within the nucleic acid, the nucleic acid obtained is referred to herein as a polynucleotide comprising the sequence of any one of SEQ ID NO:1-339.

[0049] II. Expression of Polypeptide Encoded by Full-Length cDNA or Full-Length Gene

[0050] The polynucleotide, the corresponding cDNA, or the full-length gene is used to express the partial or complete gene product. Appropriate polynucleotide constructs are purified using standard recombinant DNA techniques as described in, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). The polypeptides encoded by the polynucleotides are expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Suitable vectors and host cells are described in U.S. Pat. No. 5,654,173.

[0051] Bacteria

[0052] Expression systems in bacteria include those described in Chang et al., Nature (1978) 275:615, Goeddel et al., Nature (1979) 281:544, Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776, U.S. Pat. No. 4,551,433, DeBoer et al., Proc. Natl. Acad. Sci. (USA) (1983) 80:21-25, and Siebenlist et al., Cell (1980) 20:269.

[0053] Yeast.

[0054] Expression systems in yeast include those described in Hinnen et al, Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze et al, J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen. Microbiol. (1986) 132:3459, Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302) Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt et al., J. Bacteriol. (1983) 154:737, Van den Berg et al., Bio/Technology (1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141, Cregg et al., Mol. Cell. Biol. (1985) 5:3376, U.S. Pat. Nos. 4,837,148 and 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr. Genet. (1985) 10:380, Gaillardin et al., Curr. Genet. (1985) 10:49, Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289; Tilbum et al., Gene (1983) 26:205-221, Yelton et al., Proc. Natl. Acad. Sci. (USA) (1984) 81:1470-1474, Kelly and Hynes, EMBO J. (1985) 4:475479; EP 0 244,234, and WO 91/00357.

[0055] Insect Cells.

[0056] Expression of heterologous genes in insects is accomplished as described in U.S. Pat. No. 4,745,051, Friesen et al. (1986) “The Regulation of Baculovirus Gene Expression” in: The Molecular Biology Of Baculoviruses (W. Doerfler, ed.), EP 0 127,839, EP 0 155,476, and Vlak et al., J. Gen. Virol. (1988) 69:765-776, Miller et al., Ann. Rev. Microbiol. (1988) 42:177, Carbonell et al., Gene (1988) 73:409, Maeda et al., Nature (1985) 315:592-594, Lebacq-Verheyden et al, Mol. Cell Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:8404, Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA (1988) 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al., Bio/Technology (1988) 6:47-55, Miller et al., Generic Engineering (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al., Nature, (1985) 315:592-594.

[0057] Mammalian Cells.

[0058] Mammalian expression is accomplished as described in Dijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S. Pat. No. 4,399,216. Other features of mammalian expression are facilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985.

[0059] Polynucleotide molecules comprising the polynucleotide sequence are propagated by placing the molecule in a vector. Viral and non-viral vectors are used, including plasmids. The choice of plasmid will depend on the type of cell in which propagation is desired and the purpose of propagation. Certain vectors are useful for amplifying and making large amounts of the desired DNA sequence. Other vectors are suitable for expression in cells in culture. Still other vectors are suitable for transfer and expression in cells in a whole animal or person. The choice of appropriate vector is well within the skill of the art. Many such vectors are available commercially. The polynucleotide is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence may be inserted by homologous recombination in vivo. Typically this is accomplished by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides, or by polymerase chain reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example.

[0060] Polynucleotides are linked to regulatory sequences as appropriate to obtain the desired expression properties. These may include promoters (attached either at the 5′ end of the sense strand or at the 3′ end of the antisense strand), enhancers, terminators, operators, repressors, and inducers. The promoters may be regulated or constitutive. In some situations it may be desirable to use conditionally active promoters, such as tissue-specific or developmental stage-specific promoters. These are linked to the desired nucleotide sequence using the techniques described above for linkage to vectors. Any techniques known in the art may be used.

[0061] When any of the above host cells, or other appropriate host cells or organisms, are used to replicate and/or express the polynucleotides or nucleic acids of the invention, the resulting replicated nucleic acid, RNA, expressed protein or polypeptide, is within the scope of the invention as a product of the host cell or organism. The product is recovered by any appropriate means known in the art.

[0062] Once the gene corresponding to the polypeptide is identified, its expression can be regulated in the cell to which the gene is native. For example, an endogenous gene of a cell can be regulated by an exogenous regulatory sequence as disclosed in U.S. Pat. No. 5,641,670, “Protein Production and Protein Delivery.”

[0063] Ribozymes

[0064] Trans-cleaving catalytic RNAs (ribozymes) are RNA molecules possessing endoribonuclease activity. Ribozymes are specifically designed for a particular target, and the target message must contain a specific nucleotide sequence. They are engineered to cleave any RNA species site-specifically in the background of cellular RNA. The cleavage event renders the mRNA unstable and prevents protein expression. Importantly, ribozymes can be used to inhibit expression of a gene of unknown function for the purpose of determining its function in an in vitro or in vivo context, by detecting the phenotypic effect.

[0065] One commonly used ribozyme motif is the hammerhead, for which the substrate sequence requirements are minimal. Design of the hammerhead ribozyme is disclosed in Usman et al., Current Opin. Struct. Biol. (1996) 6:527-533. Usman also discusses the therapeutic uses of ribozymes. Ribozymes can also be prepared and used as described in Long et al., FASEB J. (1993) 7:25; Symons, Ann. Rev. Biochem. (1992) 61:641; Perrotta et al., Biochem. (1992) 31:16-17; Ojwang et al., Proc. Natl. Acad. Sci. (USA) (1992) 89:10802-10806; and U.S. Pat. No. 5,254,678. Ribozyme cleavage of HIV-1 RNA is described in U.S. Pat. No. 5,144,019; methods of cleaving RNA using ribozymes is described in U.S. Pat. No. 5,116,742; and methods for increasing the specificity of ribozymes are described in U.S. Pat. No. 5,225,337 and Koizumi et al., Nucleic Acid Res. (1989) 17:7059-7071. Preparation and use of ribozyme fragments in a hammerhead structure are also described by Koizumi et al., Nucleic Acids Res. (1989) 17:7059-7071. Preparation and use of ribozyme fragments in a hairpin structure are described by Chowrira and Burke, Nucleic Acids Res. (1992) 20:2835. Ribozymes can also be made by rolling transcription as described in Daubendiek and Kool, Nat. Biotechnol. (1997) 15(3):273-277.

[0066] The hybridizing region of the ribozyme may be modified or may be prepared as a branched structure as described in Horn and Urdea, Nucleic Acids Res. (1989) 17:6959-67. The basic structure of the ribozymes may also be chemically altered in ways familiar to those skilled in the art, and chemically synthesized ribozymes can be administered as synthetic oligonucleotide derivatives modified by monomeric units. In a therapeutic context, liposome mediated delivery of ribozymes improves cellular uptake, as described in Birikh et al., Eur. J. Biochem. (1997) 245:1-16.

[0067] Therapeutic and functional genomic applications of ribozymes proceed beginning with knowledge of a portion of the coding sequence of the gene to be inhibited. Thus, for many genes, a polynucleotide sequence as disclosed herein provides adequate sequence for constructing an effective ribozyme. A target cleavage site is selected in the target sequence, and a ribozyme is constructed based on the 5′ and 3′ nucleotide sequences that flank the cleavage site. Retroviral vectors are engineered to express monomeric and multimeric hammerhead ribozymes targeting the mRNA of the target coding sequence. These monomeric and multimeric ribozymes are tested in vitro for an ability to cleave the target mRNA. A cell line is stably transduced with the retroviral vectors expressing the ribozymes, and the transduction is confirmed by Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR). The cells are screened for inactivation of the target mRNA by such indicators as reduction of expression of disease markers or reduction of the gene product of the target mRNA.

[0068] Antisense

[0069] Antisense nucleic acids are designed to specifically bind to RNA, resulting in the formation of RNA-DNA or RNA-RNA hybrids, with an arrest of DNA replication, reverse transcription or messenger RNA translation. Antisense polynucleotides based on a selected sequence can interfere with expression of the corresponding gene. Antisense polynucleotides are typically generated within the cell by expression from antisense constructs that contain the antisense EST strand as the transcribed strand. Antisense polynucleotides will bind and/or interfere with the translation of the corresponding mRNA. The expression products of control cells and cells treated with the antisense construct are compared to detect the protein product of the gene corresponding to the polynucleotide. The protein is isolated and identified using routine biochemical methods.

[0070] Antisense therapy for a variety of cancers is in clinical phase and has been discussed extensively in the literature. Reed reviewed antisense therapy directed at the Bc1-2 gene in tumors; gene transfer-mediated overexpression of Bc1-2 in tumor cell lines conferred resistance to many types of cancer drugs. (Reed, J. C., N. C. I. (1997) 89:988-990). The potential for clinical development of antisense inhibitors of ras is discussed by Cowsert, L. M., Anti-Cancer Drug Design (1997) 12:359-371. Additional important antisense targets include leukemia (Geurtz, A. M., Anti-Cancer Drug Design (1997) 12:341-358); human C-ref kinase (Monia, B. P., Anti-Cancer Drug Design (1997) 12:327-339); and protein kinase C (McGraw et al., Anti-Cancer Drug Design (1997) 12:315-326.

[0071] Given the extensive background literature and clinical experience in antisense therapy, one skilled in the art can use selected polynucleotides of the invention as additional potential therapeutics. The choice of polynucleotide can be narrowed by first testing them for binding to “hot spot” regions of the genome of cancerous cells. If a polynucleotide is identified as binding to a “hot spot”, testing the polynucleotide as an antisense compound in the corresponding cancer cells clearly is warranted.

[0072] Ogunbiyi et al., Gastroenterology (1997) 113(3):761-766 describe prognostic use of allelic loss in colon cancer; Barks et al., Genes, Chromosomes, and Cancer (1997) 19(4):278-285 describe increased chromosome copy number detected by FISH in malignant melanoma; Nishizake et al., Genes, Chromosomes, and Cancer (1997) 19(4):267-272 describe genetic alterations in primary breast cancer and their metastases and direct comparison using modified comparative genome hybridization; and Elo et al., Cancer Research (1997) 57(16):3356-3359 disclose that loss of heterozygosity at 16z24.1-q24.2 is significantly associated with metastatic and aggressive behavior of prostate cancer.

[0073] Dominant Negative Mutations

[0074] Dominant negative mutations are readily generated for corresponding proteins that are active as homomultimers. A mutant polypeptide will interact with wild-type polypeptides (made from the other allele) and form a non-functional multimer. Thus, a mutation is in a substrate-binding domain, a catalytic domain, or a cellular localization domain. Preferably, the mutant polypeptide will be overproduced. Point mutations are made that have such an effect. In addition, fusion of different polypeptides of various lengths to the terminus of a protein can yield dominant negative mutants. General strategies are available for making dominant negative mutants. See Herskowitz, Nature (1987) 329:219-222. Such a technique can be used for creating a loss of function mutation, which is useful for determining the function of a protein.

[0075] Identification of Secreted and Membrane-Bound Polypeptides

[0076] Both secreted and membrane-bound polypeptides of the present invention are of interest. For example, levels of secreted polypeptides can be assayed conveniently in body fluids, such as blood, urine, prostatic fluid and semen. Membrane-bound polypeptides are useful for constructing vaccine antigens or inducing an immune response. Such antigens would comprise all or part of the extracellular region of the membrane-bound polypeptides.

[0077] Because both secreted and membrane-bound polypeptides comprise a fragment of contiguous hydrophobic amino acids, hydrophobicity predicting algorithms can be used to identify such polypeptides.

[0078] A signal sequence is usually encoded by both secreted and membrane-bound polypeptide genes to direct a polypeptide to the surface of the cell. The signal sequence usually comprises a stretch of hydrophobic residues. Such signal sequences can fold into helical structures.

[0079] Membrane-bound polypeptides typically comprise at least one transmembrane region that possesses a stretch of hydrophobic amino acids that can transverse the membrane. Some transmembrane regions also exhibit a helical structure.

[0080] Hydrophobic fragments within a polypeptide can be identified by using computer algorithms. Such algorithms include Hopp & Woods, Proc. Natl. Acad. Sci. USA 78: 3824-3828 (1981); Kyte & Doolittle, J. Mol Biol. 157 105-132 (1982); and RAOAR algorithm, Degli Esposti et al., Eur. J. Biochem. 190: 207-219 (1990).

[0081] Another method of identifying secreted and membrane-bound polypeptides is to translate the present polynucleotides, SEQ ID NO:1-339, in all six frames and determine if at least 8 contiguous hydrophobic amino acids are present. Those translated polypeptides with at least 8; more typically, 10; even more typically, 12 contiguous hydrophobic amino acids are considered to be either a putative secreted or membrane bound polypeptide. Hydrophobic amino acids include alanine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine.

[0082] Putative secreted and/or membrane-bound polypeptides are encoded by the sequences of the following clones: SL-5, SL-6, SL-9, SL-11, SL-13, SL-90, SL-100, SL-107, SL-124, SL-135, SL-139, SL-143, SL-152, SL-153, SL-173, and SL-177.

[0083] Construction of Polypeptides of the Invention and Variants Thereof

[0084] The polypeptides of the invention include those encoded by the disclosed polynucleotides. These polypeptides can also be encoded by nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed polynucleotides. Thus, the invention includes within its scope nucleic acids comprising polynucleotides encoding a protein or polypeptide expressed by a polynucleotide having the sequence of any one of SEQ ID NO:1-339. Also within the scope of the invention are variants; variants of polypeptides include mutants, fragments, and fusions. Mutants can include amino acid substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, a phosphorylation site or an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino acid substituted. For example, substitutions between the following groups are conservative: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Ser/Cys,Thr, and Phe/Trp/Tyr.

[0085] Cysteine-depleted muteins are variants within the scope of the invention. These variants can be constructed according to methods disclosed in U.S. Pat. No. 4,959,314, “Cysteine-Depleted Muteins of Biologically Active Proteins.” The patent discloses how to substitute other amino acids for cysteines, and how to determine biological activity and effect of the substitution. Such methods are suitable for proteins according to this invention that have cysteine residues suitable for such substitutions, for example to eliminate disulfide bond formation.

[0086] The protein variants described herein are encoded by polynucleotides that are within the scope of the invention. The genetic code can be used to select the appropriate codons to construct the corresponding variants.

[0087] The invention encompasses polynucleotide sequences having at least 65% sequence identity to any one of SEQ ID NOs:1-339 as determined by the Smith-Waterman homology search algorithm as implemented in MSPRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty of 12, and gap extension penalty of 1.

[0088] Use of the Polynucleotides as Probes. in Mapping, and in Tissue Profiling

[0089] Probes

[0090] Polynucleotide probes comprising at least 12 contiguous nucleotides selected from the nucleotide sequence of a polynucleotide of SEQ ID NO:1-339 are used for a variety of purposes, including identification of human chromosomes and determining transcription levels.

[0091] The nucleotide probes are labeled, for example, with a radioactive, fluorescent, biotinylated, or chemiluminescent label, and detected by well known methods appropriate for the particular label selected. Protocols for hybridizing nucleotide probes to preparations of metaphase chromosomes are also well known in the art. A nucleotide probe will hybridize specifically to nucleotide sequences in the chromosome preparations which are complementary to the nucleotide sequence of the probe. A probe that hybridizes specifically to a polynucleotide should provide a detection signal at least 5-, 10-, or 20-fold higher than the background hybridization provided with other unrelated sequences.

[0092] In a non-limiting example, commercial programs are available for identifying regions of chromosomes commonly associated with disease, such as cancer. Polynucleotides of the invention can be used to probe these regions. For example, if through profile searching a polynucleotide is identified as corresponding to a gene encoding a kinase, its ability to bind to a cancer-related chromosomal region will suggest its role as a kinase in one or more stages of tumor cell development/growth. Although some experimentation would be required to elucidate the role, the polynucleotide constitutes a new material for isolating a specific protein that has potential for developing a cancer diagnostic or therapeutic.

[0093] Nucleotide probes are used to detect expression of a gene corresponding to the polynucleotide. For example, in Northern blots, mRNA is separated electrophoretically and contacted with a probe. A probe is detected as hybridizing to an mRNA species of a particular size. The amount of hybridization is quantitated to determine relative amounts of expression, for example under a particular condition. Probes are also used to detect products of amplification by polymerase chain reaction. The products of the reaction are hybridized to the probe and hybrids are detected. Probes are used for in situ hybridization to cells to detect expression. Probes can also be used in vivo for diagnostic detection of hybridizing sequences. Probes are typically labeled with a radioactive isotope. Other types of detectable labels may be used such as chromophores, fluors, and enzymes.

[0094] Expression of specific mRNA can vary in different cell types and can be tissue specific. This variation of mRNA levels in different cell types can be exploited with nucleic acid probe assays to determine tissue types. For example, PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes substantially identical or complementary to polynucleotides listed in the Sequence Listing can determine the presence or absence of cDNA or mRNA related to the polynucleotides of the invention.

[0095] Examples of a nucleotide hybridization assay are described in Urdea et al., PCT WO92/02526 and Urdea et al., U.S. Pat. No. 5,124,246, both incorporated herein by reference. The references describe an example of a sandwich nucleotide hybridization assay.

[0096] Alternatively, the Polymerase Chain Reaction (PCR) is another means for detecting small amounts of target nucleic acids, as described in Mullis et al., Meth. Enzymol. (1987) 155:335-350; U.S. Pat. No. 4,683,195; and U.S. Pat. No. 4,683,202, all incorporated herein by reference. Two primer polynucleotides nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers may be composed of sequence within or 3′ and 5′ to the polynucleotides of the Sequence Listing. Alternatively, if the primers are 3′ and 5′ to these polynucleotides, they need not hybridize to them or the complements. A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a large amount of target nucleic acids is generated by the polymerase, it is detected by methods such as Southern blots. When using the Southern blot method, the labeled probe will hybridize to a polynucleotide of the Sequence Listing or complement.

[0097] Furthermore, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al., “Molecular Cloning: A Laboratory Manual” (New York, Cold Spring Harbor Laboratory, 1989). mRNA or cDNA generated from mRNA using a polymerase enzyme can be purified and separated using gel electrophoresis The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labeled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labeled with radioactivity.

[0098] Mapping

[0099] Polynucleotides of the present invention are used to identify a chromosome on which the corresponding gene resides. Using fluorescence in situ hybridization (FISH) on normal metaphase spreads, comparative genomic hybridization allows total genome assessment of changes in relative copy number of DNA sequences. See Schwartz and Samad, Current Opinions in Biotechnology (1994) 8:70-74; Kallioniemi et al., Seminars in Cancer Biology (1993) 4 41-46; Valdes and Tagle, Methods in Molecular Biology (1997) 68:1, Boultwood, ed., Human Press, Totowa, N.J.

[0100] Preparations of human metaphase chromosomes are prepared using standard cytogenetic techniques from human primary tissues or cell lines. Nucleotide probes comprising at least 12 contiguous nucleotides selected from the nucleotide sequence shown in the Sequence Listing are used to identify the corresponding chromosome. The nucleotide probes are labeled, for example, with a radioactive, fluorescent, biotinylated, or chemiluminescent label, and detected by well known methods appropriate for the particular label selected. Protocols for hybridizing nucleotide probes to preparations of metaphase chromosomes are also well known in the art. A nucleotide probe will hybridize specifically to nucleotide sequences in the chromosome preparations that are complementary to the nucleotide sequence of the probe. A probe that hybridizes specifically to a polynucleotide-related gene provides a detection signal at least 5-, 10-, or 20-fold higher than the background hybridization provided with non-EST coding sequences.

[0101] Polynucleotides are mapped to particular chromosomes using, for example, radiation hybrids or chromosome-specific hybrid panels. See Leach et al., Advances in Genetics, (1995) 33:63-99; Walter et al., Nature Genetics (1994) 7:22-28; Walter and Goodfellow, Trends in Genetics (1992) 9:352. Such mapping can be useful in identifying the function of the polynucleotide-related gene by its proximity to other genes with known function. Function can also be assigned to the related gene when particular syndromes or diseases map to the same chromosome.

[0102] Tissue Profiling

[0103] The polynucleotides of the present invention can be used to determine the tissue type from which a given sample is derived. For example, a metastatic lesion is identified by its developmental organ or tissue source by identifying the expression of a particular marker of that organ or tissue. If a polynucleotide is expressed only in a specific tissue type, and a metastatic lesion is found to express that polynucleotide, then the developmental source of the lesion has been identified. Expression of a particular polynucleotide is assayed by detection of either the corresponding mRNA or the protein product. Immunological methods, such as antibody staining, are used to detect a particular protein product. Hybridization methods may be used to detect particular mRNA species, including but not limited to in situ hybridization and Northern blotting.

[0104] Use of Polymorphisms

[0105] A polynucleotide will be useful in forensics, genetic analysis, mapping, and diagnostic applications if the corresponding region of a gene is polymorphic in the human population. A particular polymorphic form of the polynucleotide may be used to either identify a sample as deriving from a suspect or rule out the possibility that the sample derives from the suspect. Any means for detecting a polymorphism in a gene are used, including but not limited to electrophoresis of protein polymorphic variants, differential sensitivity to restriction enzyme cleavage, and hybridization to an allele-specific probe.

[0106] Use of Polynucleotides to Raise Antibodies

[0107] Expression products of a polynucleotide, the corresponding mRNA or cDNA, or the corresponding complete gene are prepared and used for raising antibodies for experimental, diagnostic, and therapeutic purposes. The polynucleotide or related cDNA is expressed as described above, and antibodies are prepared. These antibodies are specific to an epitope on the polynucleotide-encoded polypeptide, and can precipitate or bind to the corresponding native protein in a cell or tissue preparation or in a cell-free extract of an in vitro expression system.

[0108] Immunogens for raising antibodies are prepared by mixing the polypeptides encoded by the polynucleotide of the present invention with adjuvants. Alternatively, polypeptides are made as fusion proteins to larger immunogenic proteins. Polypeptides are also covalently linked to other larger immunogenic proteins, such as keyhole limpet hemocyanin. Immunogens are typically administered intradermally, subcutaneously, or intramuscularly. Immunogens are administered to experimental animals such as rabbits, sheep, and mice, to generate antibodies. Optionally, the animal spleen cells are isolated and fused with myeloma cells to form hybridomas which secrete monoclonal antibodies. Such methods are well known in the art. According to another method known in the art, the polynucleotide is administered directly, such as by intramuscular injection, and expressed in vivo. The expressed protein generates a variety of protein-specific immune responses, including production of antibodies, comparable to administration of the protein.

[0109] Preparations of polyclonal and monoclonal antibodies specific for polynucleotide-encoded proteins and polypeptides are made using standard methods known in the art. The antibodies specifically bind to epitopes present in the polypeptides encoded by polynucleotides disclosed in the Sequence Listing. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, for example at least 15, 25, or 50 amino acids. A short sequence of a polynucleotide may then be unsuitable for use as an epitope to raise antibodies for identifying the corresponding novel protein, because of the potential for cross-reactivity with a known protein. However, the antibodies may be useful for other purposes, particularly if they identify common structural features of a known protein and a novel polypeptide encoded by a polynucleotide of the invention.

[0110] Antibodies that specifically bind to human polynucleotide-encoded polypeptides should provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in Western blots or other immunochemical assays. Preferably, antibodies that specifically bind polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate EST-encoded proteins from solution. For such immunoassays, any type of samples can be used, including tissue, organs, cells, urine, blood, prostatic fluid or semen.

[0111] Of interest are antibodies to the secreted polypeptides encoded by the present polynucleotide sequences, SEQ ID NO:1-339. Antibodies to secreted polypeptides can be used to test body fluids, such as blood, urine, prostatic fluid and semen.

[0112] To test for the presence of serum antibodies to the polypeptide in a human population, human antibodies are purified by methods well known in the art. Preferably, the antibodies are affinity purified by passing antiserum over a column to which a protein, polypeptide, or fusion protein is bound. The bound antibodies can then be eluted from the column, for example using a buffer with a high salt concentration.

[0113] In addition to the antibodies discussed above, genetically engineered antibody derivatives are made, such as single chain antibodies or humanized antibodies.

[0114] Antibodies to the polypeptides encoded by one or more of SEQ ID NO:1-339 also are contemplated for therapeutic compositions and uses. For example, antibodies directed to membrane-bound polypeptides that are up-regulated in cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations can be constructed. Antibodies can provide a useful therapeutic in inhibiting cell growth or inducing an immune reaction to cancer, tumor, or hyperproliferating cells. Typically, such antibodies are directed the extracellular regions of the membrane-bound polypeptide. The borders of such regions can be determined by identifying the location of the hydrophobic transmembrane fragment(s) in the encoded polypeptides of the present invention.

[0115] Exemplary antibodies were prepared using two sequences from clone SL-5: H2N-CGPRLPSFPCPTHEPSTGQLSK-CONH2 and H2N-CKDSQGLSDFKR-NSRTTRRSYKCCONH2. Using polyclonal antibodies raised against a mixture of these polypeptides, immunohistochemistry was performed on a variety of tumor tissues and corresponding normal tissue. The results are shown in FIG. 3, and discussed in the Examples. These polypeptides are useful for detecting a higher level of expression of clone SL-5 in tumor tissues.

[0116] Use of Polynucleotides to Construct Arrays for Diagnostics

[0117] The present polynucleotide sequences and gene products are useful for determining the occurrence of cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations. Specifically, the polynucleotides and encoded polypeptides of the instant invention can be utilized to determine the occurrence of prostatic disorders, such as BPH or localized prostate cancer.

[0118] A number of prostatic disorders exist, including adenocarcinoma, BPH, histologic prostate cancer, prostatic intraepithelial neoplasia, clinical prostate cancer, incidental prostate cancer, and localized prostate cancer. BPH is a common prostatic disorder in men which becomes clinically manifest usually after age fifty. In BPH, hyperplastic growth of prostatic cells in the periurethral glandular tissue in the central zone of the prostate gland cause an enlarged prostate which can compress or elongate the urethra and produce symptoms of urethral obstruction that may progress to urinary retention or to a constellation of symptoms known as prostatism. A host of physical manifestations can accompany prostatic disorders including: impotency, reduced urinary flow, hesitancy in initiating voiding, postvoid dribbling, a sensation of incomplete bladder emptying, and development of bladder or high urinary tract infections.

[0119] To determine the occurrence of cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations, the levels of polynucleotides and/or encoded polypeptides of the present invention in a sample are compared to the levels in a normal control of body tissues, cells, organs, or fluids. The normal control can include a pool of cells from a particular organ or tissue or tissues and/or cells from throughout the body. Either the immunoassays described above or the nucleic acid assays described below can be used for such measurements.

[0120] Any observed difference between the sample and normal control can indicate the occurrence of disease or disorder. Typically, if the levels of the polynucleotides and the encoded polypeptides of the present invention are higher than those found in the normal control, the results indicate the occurrence of cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations.

[0121] In addition, the present polynucleotides can be useful to diagnose the severity as well as the occurrence of cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations, including prostatic disorders. For example, the greater the difference observed in the sample versus the normal control of the present polynucleotides or encoded polypeptides, the greater the severity of the disorder, in particular, when higher levels as compared to a normal control are observed.

[0122] The present polynucleotides, as shown in SEQ ID NO:1-339, were expressed at higher levels in a prostate cancer cell line versus a normal prostate epithelial cell line.

[0123] Polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotide sequences in a sample. This technology can be used as a diagnostic and as a tool to test for differential expression to determine function of an encoded protein.

[0124] To create arrays, polynucleotide probes are spotted onto a substrate in a two-dimensional matrix or array. Samples of polynucleotides can be labeled and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away.

[0125] The probe polynucleotides can be spotted on substrates including glass, nitrocellulose, etc. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. The sample polynucleotides can be labeled using radioactive labels, fluorophors, etc.

[0126] Techniques for constructing arrays and methods of using these arrays are described in EP No. 0 799 897; PCT No. WO 97/29212; PCT No WO 97/27317; EP No. 0 785 280; PCT No. WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP No. 0 728 520; U.S. Pat. No. 5,599,695; EP No. 0 721 016; U.S. Pat, No. 5,556,752; PCT No. WO 95/22058; and U.S. Pat. No. 5,631,734.

[0127] Further, arrays can be used to examine differential expression of genes and can be used to determine gene function. For example, arrays of the instant polynucleotide sequences can be used to determine if any of the EST sequences are differentially expressed between normal cells and cancer cells, for example. High expression of a particular message in a cancer cell, which is not observed in a corresponding normal cell, can indicate a cancer specific protein.

[0128] Differential Expression

[0129] The present invention also provides a method to identify abnormal or diseased tissue in a human. For polynucleotides corresponding to profiles of protein families as described above, the choice of tissue may be dictated by the putative biological function. The expression of a gene corresponding to a specific polynucleotide is compared between a first tissue that is suspected of being diseased and a second, normal tissue of the human. The normal tissue is any tissue of the human, especially those that express the polynucleotide-related gene including, but not limited to, brain, thymus, testis, heart, prostate, placenta, spleen, small intestine, skeletal muscle, pancreas, and the mucosal lining of the colon.

[0130] The polynucleotide-related genes in the two tissues are compared by any means known in the art. For example, the two genes are sequenced, and the sequence of the gene in the tissue suspected of being diseased is compared with the gene sequence in the normal tissue. The polynucleotide-related genes, or portions thereof, in the two tissues are amplified, for example using nucleotide primers based on the nucleotide sequence shown in the Sequence Listing, using the polymerase chain reaction. The amplified genes or portions of genes are hybridized to nucleotide probes selected from the same nucleotide sequence shown in the Sequence Listing. A difference in the nucleotide sequence of the polynucleotide-related gene in the tissue suspected of being diseased compared with the normal nucleotide sequence suggests a role of the polynucleotide-encoded proteins in the disease, and provides a lead for preparing a therapeutic agent. The nucleotide probes are labeled by a variety of methods, such as radiolabeling, biotinylation, or labeling with fluorescent or chemiluminescent tags, and detected by standard methods known in the art.

[0131] Alternatively, polynucleotide-related mRNA in the two tissues is compared. PolyA+ RNA is isolated- from the two tissues as is known in the art. For example, one of skill in the art can readily determine differences in the size or amount of polynucleotide-related mRNA transcripts between the two tissues using Northern blots and nucleotide probes selected from the nucleotide sequence shown in the Sequence Listing. Increased or decreased expression of an polynucleotide-related mRNA in a tissue sample suspected of being diseased, compared with the expression of the same polynucleotide-related mRNA in a normal tissue, suggests that the expressed protein has a role in the disease, and also provides a lead for preparing a therapeutic agent.

[0132] Any method for analyzing proteins is used to compare two polynucleotide-encoded proteins from matched samples. The sizes of the proteins in the two tissues are compared, for example, using antibodies of the present invention to detect polynucleotide-encoded proteins in Western blots of protein extracts from the two tissues. Other changes, such as expression levels and subcellular localization, can also be detected immunologically, using antibodies to the corresponding protein. A higher or lower level of polynucleotide-encoded protein expression in a tissue suspected of being diseased, compared with the same polynucleotide-encoded protein expression level in a normal tissue, is indicative that the expressed protein has a role in the disease, and provides another lead for preparing a therapeutic agent.

[0133] Similarly, comparison of polynucleotide gene sequences or of polynucleotide gene expression products, e.g., mRNA and protein, between a human tissue that is suspected of being diseased and a normal tissue of a human, are used to follow disease progression or remission in the human Such comparisons of polynucleotide-related genes, mRNA, or protein are made as described above.

[0134] For example, increased or decreased expression of the polynucleotide-related gene in the tissue suspected of being neoplastic can indicate the presence of neoplastic cells in the tissue. The degree of increased expression of the polynucleotide gene in the neoplastic tissue relative to expression of the gene in normal tissue, or differences in the amount of increased expression of the polynucleotide gene in the neoplastic tissue over time, is used to assess the progression of the neoplasia in that tissue or to monitor the response of the neoplastic tissue to a therapeutic protocol over time. The expression pattern of any two cell types can be compared, such as low and high metastatic tumor cell lines, or cells from tissue which have and have not been exposed to a therapeutic agent.

[0135] Screening for Peptide Analogs and Antagonists

[0136] Polypeptides encoded by the instant polynucleotides and corresponding full length genes can be used to screen peptide libraries to identify binding partners, such as receptors, from among the encoded polypeptides.

[0137] Such binding partners can be useful in treating cancer, tumor progression, hyperproliferative cell growth, and/or accompanying biological or physical manifestations. For example, peptides or other compounds that are capable of binding or interacting with membrane-bound polypeptides encoded by one or more of SEQ ID NO:1-339, can be useful as a therapeutic. Also, peptides or other compounds capable of altering the conformation of any of the encoded polypeptides by one or more of SEQ ID NO:1-339 can inhibit biological activity and be useful as a therapeutic.

[0138] A library of peptides may be synthesized following the methods disclosed in U.S. Pat. No. 5,010,175, and in PCT WO91/17823.

[0139] Peptide agonists or antagonists are screened using any available method, such as signal transduction, antibody binding, receptor binding, mitogenic assays, chemotaxis assays, etc The methods described herein are presently preferred. The assay conditions ideally should resemble the conditions under which the native activity is exhibited in vivo, that is, under physiologic pH, temperature, and ionic strength. Suitable agonists or antagonists will exhibit strong inhibition or enhancement of the native activity at concentrations that do not cause toxic side effects in the subject. Agonists or antagonists that compete for binding to the native polypeptide may require concentrations equal to or greater than the native concentration, while inhibitors capable of binding irreversibly to the polypeptide may be added in concentrations on the order of the native concentration.

[0140] The end results of such screening and experimentation will be at least one novel polypeptide binding partner, such as a receptor, encoded by a cDNA polynucleotide or gene of the invention, and at least one peptide agonist or antagonist of the novel binding partner. Such agonists and antagonists can be used to modulate, enhance, or inhibit receptor function in cells to which the receptor is native, or in cells that possess the receptor as a result of genetic engineering. Further, if the novel receptor shares biologically important characteristics with a known receptor, information about agonist/antagonist binding may help in developing improved agonists/antagonists of the known receptor.

[0141] Therapeutics, whether polynucleotide or polypeptide or small molecule, can be tested, for example, in the mouse tumor assay described in Pei et al., Mol. Endo. 11: 433-441 (1997).

[0142] Other models for testing polynucleotides, polypeptides, antibodies, or small molecules useful for treatment include: animal models and cell lines disclosed in Bosland, Encyclopedia of Cancer, Volume II, pages 1283 to 1296 (1997) by Academic Press. Other useful cell lines are described in Brothman, Encyclopedia of Cancer, Volume II, pages 1303 to 1313 (1997) by Academic Press

[0143] Pharmaceutical Compositions and Therapeutic Uses

[0144] Pharmaceutical compositions can comprise polypeptides, antibodies, or polynucleotides of the claimed invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

[0145] The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician. Specifically, the compositions of the present invention can be used to treat, ameliorate, modulate, or prevent cancer, tumor progression, hyperproliferative cell growth and/or accompanying biological or physical manifestations, including prostatic disorders.

[0146] For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the polynucleotide, polypeptide or antibody compositions in the individual to which it is administered.

[0147] A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

[0148] Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington 's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0149] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

[0150] Delivery Methods

[0151] Once formulated, the polynucleotide compositions of the invention can be (1) administered directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) delivered in vitro for expression of recombinant proteins.

[0152] Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a tumor or lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

[0153] Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

[0154] Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

[0155] If a polynucleotide-related gene correlates with a proliferative disorder, such as neoplasia, dysplasia, and hyperplasia, the disorder may be amenable to treatment by administration of a therapeutic agent based on the polynucleotide or corresponding polypeptide.

[0156] Preparation of antisense polypeptides is discussed above. Neoplasias that are treated with the antisense composition include, but are not limited to, cervical cancers, melanomas, colorectal adenocarcinomas, Wilms' tumor, retinoblastoma, sarcomas, myosarcomas, lung carcinomas, leukemias, such as chronic myelogenous leukemia, promyelocytic leukemia, monocytic leukemia, and myeloid leukemia, and lymphomas, such as histiocytic lymphoma. Proliferative disorders that are treated with the therapeutic composition include disorders such as anhydric hereditary ectodermal dysplasia, congenital alveolar dysplasia, epithelial dysplasia of the cervix, fibrous dysplasia of bone, and mammary dysplasia. Hyperplasias, for example, endometrial, adrenal, breast, prostate, or thyroid hyperplasias or pseudoepitheliomatous hyperpiasia of the skin, are treated with antisense therapeutic compositions. Even in disorders in which mutations in the corresponding gene are not implicated, downregulation or inhibition of gene expression can have therapeutic application. For example, decreasing gene expression can help to suppress tumors in which enhanced expression of the gene is implicated.

[0157] Both the dose of the antisense composition and the means of administration are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. Administration of the therapeutic antisense agents of the invention includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. Preferably, the therapeutic antisense composition contains an expression construct comprising a promoter and a polynucleotide segment of at least 12, 22, 25, 30, or 35 contiguous nucleotides of the antisense strand. Within the expression construct, the polynucleotide segment is located downstream from the promoter, and transcription of the polynucleotide segment initiates at the promoter.

[0158] Various methods are used to administer the therapeutic composition directly to a specific site in the body. For example, a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of tumor. Alternatively, arteries which serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor. A tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor. The antisense composition is directly administered to the surface of the tumor, for example, by topical application of the composition. X-ray imaging is used to assist in certain of the above delivery methods.

[0159] Receptor-mediated targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues is also used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends in Biotechnol. (1993) 11:202-205; Chiou et al., (1994) Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.); Wu & Wu, J. Biol. Chem. (1988) 263-621-24; Wu et al., J. Biol. Chem. (1994) 269:542-46; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87:3655-59; Wu et al., J. Biol. Chem. (1991) 266:339-42. Preferably, receptor-mediated targeted delivery of therapeutic compositions containing antibodies of the invention is used to deliver the antibodies to specific tissue.

[0160] Therapeutic compositions containing antisense subgenomic polynucleotides are administered in a range of about 100 ng to about 200 mg of polynucleotides for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of polynucleotides can also be used during a gene therapy protocol. Factors such as method of action and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of EST antisense subgenomic polynucleotides or the same amounts readministered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. A more complete description of gene therapy vectors, especially retroviral vectors, is contained in U.S. Ser. No. 08/869,309, which is expressly incorporated herein, and in section G below.

[0161] For genes encoding polypeptides or proteins with anti-inflammatory activity, suitable use, doses, and administration are described in U.S. Pat. No. 5,654,173, incorporated herein by reference. Therapeutic agents also include antibodies to proteins and polypeptides, as described in U.S. Pat. No. 5,654,173.

[0162] Gene Therapy

[0163] The therapeutic polynucleotides and polypeptides of the present invention may be utilized in gene delivery vehicles. The gene delivery vehicle may be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51-64; Kimura, Human Gene Therpay (1994) 5:845-852; Connelly, Human Gene Therapy (1995) 1:185-193; and Kaplitt, Nature Genetics (1994) 6:148-153). Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention can be administered either locally or systemically These constructs can utilize viral or non-viral vector approaches. Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

[0164] The present invention can employ recombinant retroviruses which are constructed to carry or express a selected nucleic acid molecule of interest. Retrovirus vectors that can be employed include those described in EP 0 415 731; WO 90/07936; WO 94/03,622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; Vile and Hart, Cancer Res. (1993) 53:3860-3864; Vile and Hart, Cancer Res. (1993) 53:962-967; Ram et al., Cancer Res. (1993) 53:83-88; Takamiya et al., J. Neurosci. Res. (1992) 33:493-503; Baba et al., J. Neurosurg. (1993) 79:729-735; U.S. Pat. No. 4,777,127, GB Patent No. 2,200,651; and EP 0 345 242. Preferred recombinant retroviruses include those described in WO 91/02805.

[0165] Packaging cell lines suitable for use with the above-described retroviral vector constructs may be readily prepared (see PCT publications WO 95/30763 and WO 92/05266), and used to create producer cell lines (also termed vector cell lines) for the production of recombinant vector particles. Within particularly preferred embodiments of the invention, packaging cell lines are made from human (such as HT1080 cells) or mink parent cell lines, thereby allowing production of recombinant retroviruses that can survive inactivation in human serum.

[0166] The present invention also employs alphavirus-based vectors that can function as gene delivery vehicles. Such vectors can be constructed from a wide variety of alphaviruses, including, for example, Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532). Representative examples of such vector systems include those described in U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and PCT Publication Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO 95/07994.

[0167] Gene delivery vehicles of the present invention can also employ parvovirus such as adeno-associated virus (AAV) vectors. Representative examples include the AAV vectors disclosed by Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617.

[0168] Representative examples of adenoviral vectors include those described by Berkner, Biotechniques (1988) 6:616-627; Rosenfeld et al, Science (1991) 252:431-434; WO 93/19191; Kolls et al., PNAS (1994) 91:215-219; Kass-Eisler et al., PNAS (1993) 90:11498-11502; Guzman et al., Circulation (1993) 88:2838-2848; Guzman et al., Cir. Res. (1993) 73:1202-1207; Zabner et al., Cell (1993) 75:207-216; Li et al., Hum. Gene Ther. (1993) 4:403-409; Cailaud et al., Eur. J. Neurosci. (1993) 5:1287-1291; Vincent et al., Nat. Genet. (1993) 5:130-134; Jaffe et al., Nat. Genet. (1992) 1:372-378; and Levrero et al., Gene (1991) 101:195-202. Exemplary adenoviral gene therapy vectors employable in this invention also include those described in WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992)3:147-154 may be employed.

[0169] Other gene delivery vehicles and methods may be employed, including polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example Curiel, Hum. Gene Ther. (1992) 3:147-154; ligand linked DNA, for example see Wu, J. Biol. Chem. (1989) 264:16985-16987; eukaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796; deposition of photopolymerized hydrogel materials; hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033; nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411-2418, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581-1585.

[0170] Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859.

[0171] Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA (1994) 91(24):11581-11585.

[0172] Computer-Related Embodiments

[0173] In general, a library of polynucleotides is a collection of sequence information, which information is provided in either biochemical form (e.g., as a collection of polynucleotide molecules), or in electronic form (e.g., as a collection of polynucleotide sequences stored in a computer-readable form, as in a computer system and/or as part of a computer program). The sequence information of the polynucleotides can be used in a variety of ways, e.g., as a resource for gene discovery, as a representation of sequences expressed in a selected cell type (e.g., cell type markers), and/or as markers of a given disease or disease state. In general, a disease marker is a representation of a gene product that is present in all cells affected by disease either at an increased or decreased level relative to a normal cell (e.g., a cell of the same or similar type that is not substantially affected by disease).

[0174] The nucleotide sequence information of the library can be embodied in any suitable form, e.g., electronic or biochemical forms. For example, a library of sequence information embodied in electronic form comprises an accessible computer data file (or, in biochemical form, a collection of nucleic acid molecules) that contains the representative nucleotide sequences of genes that are differentially expressed (e.g., overexpressed or underexpressed) as between, for example, a cancerous cell and a normal cell. Biochemical embodiments of the library include a collection of nucleic acids that have the sequences of the genes in the library, where the nucleic acids can correspond to the entire gene in the library or to a fragment thereof, as described in greater detail below.

[0175] The polynucleotide libraries of the subject invention generally comprise sequence information of a plurality of polynucleotide sequences, where at least one of the polynucleotides has a sequence of any of SEQ ID NOs:1-339. By plurality is meant at least 2, usually at least 3 and can include up to all of SEQ ID NOs:1-339. The length and number of polynucleotides in the library will vary with the nature of the library, e.g., if the library is an oligonucleotide array, a cDNA array, a computer database of the sequence information, etc.

[0176] Where the library is an electronic library, the nucleic acid sequence information can be present in a variety of media. “Media” refers to a manufacture, other than an isolated nucleic acid molecule, that contains the sequence information of the present invention. Such a manufacture provides the genome sequence or a subset thereof in a form that can be examined by means not directly applicable to the sequence as it exists in a nucleic acid. For example, the nucleotide sequence of the present invention, e.g., the nucleic acid sequences of any of the polynucleotides of SEQ ID NOs:1-339, can be recorded on computer readable media, e.g., any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as a floppy disc, a hard disc storage medium, and a magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present sequence information. “Recorded” refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g., word processing text file, database format, etc. In addition to the sequence information, electronic versions of the libraries of the invention can be provided in conjunction or connection with other computer-readable information and/or other types of computer-readable files (e.g., searchable files, executable files, etc, including, but not limited to, for example, search program software, etc.).

[0177] By providing the nucleotide sequence in computer readable form, the information can be accessed for a variety of purposes. Computer software to access sequence information is publicly available. For example, the BLAST (Altschul et al., supra.) and BLAZE (Brutlag et al. Comp. Chem. (1993) 17:203) search algorithms on a Sybase system can be used to identify open reading frames (ORFs) within the genome that contain homology to ORFs from other organisms.

[0178] As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention. The data storage means can comprise any manufacture comprising a recording of the present sequence information as described above, or a memory access means that can access such a manufacture.

[0179] “Search means” refers to one or more programs implemented on the computer-based system, to compare a target sequence or target structural motif, or expression levels of a polynucleotide in a sample, with the stored sequence information. Search means can be used to identify fragments or regions of the genome that match a particular target sequence or target motif A variety of known algorithms are publicly known and commercially available, e.g., MacPattern (EMBL), BLASTN and BLASTX (NCBI). A “target sequence” can be any polynucleotide or amino acid sequence of six or more contiguous nucleotides or two or more amino acids, preferably from about 10 to 100 amino acids or from about 30 to 300 nt A variety of comparing means can be used to accomplish comparison of sequence information from a sample (e.g., to analyze target sequences, target motifs, or relative expression levels) with the data storage means. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer based systems of the present invention to accomplish comparison of target sequences and motifs. Computer programs to analyze expression levels in a sample and in controls are also known in the art.

[0180] A “target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration that is formed upon the folding of the target motif, or on consensus sequences of regulatory or active sites. There are a variety of target motifs known in the art. Protein target motifs include, but arc not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, hairpin structures, promoter sequences and other expression elements such as binding sites for transcription factors.

[0181] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. One format for an output means ranks the relative expression levels of different polynucleotides. Such presentation provides a skilled artisan with a ranking of relative expression levels to determine a gene expression profile.

[0182] As discussed above, the “library” of the invention also encompasses biochemical libraries of the polynucleotides of SEQ ID NOs:1-339, e.g., collections of nucleic acids representing the provided polynucleotides. The biochemical libraries can take a variety of forms, e.g., a solution of cDNAs, a pattern of probe nucleic acids stably associated with a surface of a solid support (i.e., an array) and the like. Of particular interest are nucleic acid arrays in which one or more of SEQ ID NOs:1-339 is represented on the array. By array is meant a an article of manufacture that has at least a substrate with at least two distinct nucleic acid targets on one of its surfaces, where the number of distinct nucleic acids can be considerably higher, typically being at least 10 nt, usually at least 20 nt and often at least 25 nt. A variety of different array formats have been developed and are known to those of skill in the art. The arrays of the subject invention find use in a variety of applications, including gene expression analysis, drug screening, mutation analysis and the like, as disclosed in the above-listed exemplary patent documents.

[0183] In addition to the above nucleic acid libraries, analogous libraries of polypeptides are also provided, where the where the polypeptides of the library will represent at least a portion of the polypeptides encoded by SEQ ID NOs:1-339.

[0184] The present invention will now be illustrated by reference to the following examples which set forth particularly advantageous embodiments. However, it should be noted that these embodiments are illustrative and are not to be construed as restricting the invention in any way.

EXAMPLES

Example 1

Isolation of the Polynucleotides

[0185] cDNA libraries were prepared from PrEC, normal human prostate epithelial cells, and LNCaP, a cell line derived from human lymph node metastasized prostate cancer. PrEC cells are available from Clonetics, San Diego, Calif., U.S.A. LNCaP cells are available from the ATCC, Manassas, Va., U.S.A.

[0186] Using a PCR technique and reagents available from Clontech, Palo Alto, Calif., USA (CLONTECH PCR-Select™), mRNA up-regulated in LNCaP was captured and amplified. The captured polynucleotide inserts were inserted in the pCR2.1 vector, available from Invitrogen, Carlsbad, Calif., U.S.A. The vectors with the inserts were transformed into E. coli cells.

Example 2

Confirmation of Differential Display

[0187] Ten clones were chosen at random, and up-regulation of the sequences of these clone inserts in LNCaP versus PrEC cells was confirmed by Northern blot. Dot blots were performed on 168 clones and up-regulation was confirmed.

[0188] Further, sequencing of the clones showed that prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) sequences were isolated by the process described in Example 1. A good correlation between increased serum PSA levels and prostate tumors has been observed. PSMA, a cell surface antigen, is another observed marker for prostate cancer. See Bosland, Encyclopedia of Cancer, Volume II, pages 1283-1296 (1997), Academic Press. Thus, the data confirm that up-regulated mRNA characteristic of gene expression in prostate cancer was cloned by the method of Example 1.

Example 3

Polynucleotide Sequences

[0189] The sequence results are shown in SEQ ID NO:1-339. For the sequencing experiments, each clone was named SL-1 to SL-209. Inserts from some of the clones were sequenced more than once. Each sequence was designated a unique combination of two names. This unique combination is shown in Table 1 in columns 2 and 3, denoted as “Sequence Name” and “Other Seq Name.”

[0190] Table 1 indicates all the sequences that correspond to each clone. Thus, all the sequences corresponding to clone SL-3, for example, are grouped together in Table 1.

[0191] Clones also were assigned cluster numbers. See column 4 of Table 1. Clones with the same cluster number generally comprise sequence derived from the same mRNA transcripts.

[0192] The last column of Table 1 indicates the nearest neighbor as determined by an alignment to sequences in a publicly available database.

[0193] A consensus for the sequence of each clone can be constructed by aligning the corresponding sequences or reverse complements thereof. Table 1 lists the names of all the sequences that correspond to each clone, and Table 2 shows the specific sequence that corresponds to each unique combination of Sequence Name and/or “Other Seq. Name.”

[0194] The entire insert of some clones may not be represented by the sequences presented in Table 2. For example, the 5′ and 3′ ends of a clone insert may have been sequenced, but the sequences do not overlap Additional sequence corresponding to the clone insert can be isolated and determined by constructing probes or primers from the sequences presented in Table 2 and a library of mRNA or cDNA from a prostate cell or prostate cancer cell line using the methods described above.

Example 4

Results of Public Database Search

[0195] Both the nucleotide sequence and translations of masked sequences shown in the Sequence Listing were aligned with individual sequences that were publicly available. Similarity with individual sequences is used to determine the activity of the polypeptides encoded by genes corresponding to the sequences referred to in Table 2.

[0196] The sequences in SEQ ID NO:1-333 first were masked to remove the pCR2.1 vector sequences. Masking was performed by aligning the pCR2.1 sequences with each of SEQ ID NO:1-333 using the BLASTN program. Any sequence that produced an alignment with a score of less that 0.1 was masked.

[0197] A BLASTN vs. Genbank search was performed using the masked sequences with search parameters of greater than 99% overlap, 99% identity, and a p value of less than 1×10−40 and this resulted in discard of sequences. Sequences from this search also were discarded if the inclusive parameters were met, but the sequence was ribosomal or vector-derived.

[0198] The resulting sequences from the previous search were classified into three groups (1, 2 and 3 below) and searched in a BLASTX vs. NRP (non-redundant proteins) database search: (1) unknown (no hits in the Genbank search), (2) weak similarity (greater than 45% identity and p value of less than 1×10−5), and (3) high similarity (greater than 60% overlap, greater than 80% identity, and p value less than 1×10−5). This search resulted in discard of sequences as having greater than 99% overlap, greater than 99% identity, and p value of less than 1×10−40.

[0199] The remaining sequences were classified as unknown (no hits), weak similarity, and high similarity (parameters as above). Two searches were performed on this set of sequences. First, a BLAST vs. EST database search resulted in discard of sequences with greater than 99% overlap, greater than 99% similarity and a p value of less than 1×10−40; sequences with a p value of less than 1×10−65 when compared to a database sequence of human origin were also excluded. Second, a BLASTN vs. Patent GeneSeq database resulted in discard of sequences with greater than 99% identity; p value less than 1×10−40; greater than 99% overlap.

[0200] The masked sequences were translated in all six reading frames to determine the best alignment with the individual sequences. These amino acid sequences and nucleotide sequences are referred, generally, as query sequences, which are aligned with the individual sequences.

[0201] Query and individual sequences were aligned using the BLAST programs, available over the world wide web.

[0202] Table 2 shows the results of the alignments. Table 2 refers to each sequence by its Sequence Name and/or “Other Seq. Name” and includes the accession numbers and descriptions of nearest neighbors from the Genbank and Non-Redundant Protein searches.

[0203] The activity of the polypeptide encoded by the sequences referred to in Table 2 is expected to be the same or similar to the nearest neighbor reported in Table 2. The accession number of the nearest neighbor is reported, providing a reference to the activities exhibited by the nearest neighbor. The search program and database used for the alignment also are indicated as well as a calculation of the p value.

[0204] Full length sequences or fragments of the polynucleotide sequences of the nearest neighbors can be used as probes and primers to identify and isolate the full length sequence corresponding to sequence referred to in Table 2. Although full length sequences can be obtained from the cell lines described above, the nearest neighbors can indicate a tissue or cell type to be used to construct a library for the full-length sequences of those referred to in Table 2.

[0205] The sequences referred to in Table 2 and the translations thereof may be human homologs of known genes of other species or novel allelic variants of known human genes. In such cases, these new human sequences may be suitable as diagnostics, prognostics, or therapeutics. As diagnostics, the human sequences exhibit greater specificity in detecting and differentiating human cell lines and types than homologs of other species. The human polypeptides are less likely to be immunogenic when administered to humans than homologs from other species. Further, on administration to humans, the encoded polypeptides can show greater specificity or can be better regulated by other human proteins than are homologs from other species.

[0206] In the preferred embodiments of the invention, the sequences shown in SEQ ID NO 1-339 consisting of the unmasked regions should be considered as the source of probes and primers, as these sequences are most representative of the distinguishing portions of these polynucleotides.

[0207] Generally, the masking itself does not influence the search results as shown in Table 2, except to eliminate multiple “hits” based on similarity to repetitive regions common to more than one polypeptide.

Example 5

Analysis of Clones SL-5, SL-9, SL-68, AND SL-173

[0208] Clone SL-5 (SEQ ID NO:14 and 334)

[0209] By Northern Blot, a 4.1 kb band was observed in expressed in normal prostate, testis, and lymphoblasic leukemia. It was also expressed in the cell lines LNCaP, and MDA PCa 2A and 2B (metastatic prostate cells into bone, androgen sensitive). Additional sequence corresponding to SEQ ID NO:14 is disclosed in SEQ ID NO:334.

[0210] Expression of SL-5 was investigated in normal and tumor tissues using immunohistochemistry. Antibody was prepared using two sequences from clone SL-5: H2N-CGPRLPSFPCPTHEPSTGQLSK-CONH2 and H2N-CKDSQGLSDFKRNSRTTR-RSYKCCONH2. Using polyclonal antibodies raised against a mixture of these polypeptides, immunohistochemistry (IHC) was performed on a variety of tumor tissues and corresponding normal tissue. The methods used were those described for the Manual IHC Protocol using BioGenex Reagents and Zymed AEC Solution, as known in the art. As shown in FIG. 3, SL-5 was detected in the following tumor tissue: adrenal, ovary, breast, colon, prostate, uterus, cervix, kidney, pancreas, liver, stomach, lymphoma, seminonia, thyroid, melanoma, basal cell carcinoma, and other tumor tissues. Where comparative normal tissue was available, expression in the corresponding normal tissue was lower than in the tumor tissue. Thus, SL-5 is a useful marker for cancer tissue including prostate.

[0211] Clone SL-9 (SEQ ID NO:18)

[0212] By Northern Blot, sequences from SL-9 were specifically expressed in normal spleen and normal peripheral blood leukocyte. Expression of the SL-9 sequences was observed also in promyelocytic leukemia HL-60, chronic mylogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma, and Raji cancer cell lines by Northern Blot.

[0213] Clone SL-173 (SEQ ID NO:153 and 154)

[0214] By Northern Blot, SL173 was found in every cancer cell line tested. Sequence from SL-173 has similarity to and may be a human homologue of the rat tumor transforming gene, which was found in the pituitary and described in Pei et al., Mol. Endo. 11: 433-441 (1997) and Pei, J. Biol. Chem. 273(9): 5219-5225 (1998). When the rat tumor transforming gene was injected in NIH3T3cells, the cells became transformed and were able to form a tumor when injected into mice. (Pei et al., Mol. Endo. supra).

[0215] Clone SL-68 (SEQ ID NO:218 and 219)

[0216] Two transcripts, 2.6 kb and 4.3 kb, were observed in normal spleen, thymus and peripheral blood leukocytes, as well as in promyelocytic leukemia, chronic myelogenous leukemia and lymphoblastic leukemia. The 4.3 kb transcript was seen in normal testis, colon, Hela cell S3, colorectal adenocarcinoma and melanoma. The 2.6 kb band was found in the following prostate cell lines: PC-3 (metastatic to bone, androgen insensitive); DU-145 (metastatic to brain, androgen insensitive); FFpz (primary cells derived from normal prostate epithelium); Ffca (primary cells derived from Gleason Grade 3 prostate cancer epithelium); and WO-CA (primary cells derived from Gleason Grade 4 prostate cancer epithelium). However, higher expression was observed in LNCaP, MDA PCa 2A, HPV-7 and HPV-10. A 9.5 kb transcript was also observed in MDA PCa 2A and 2B. Additional sequence corresponding to this clone is disclosed in SEQ ID NO:335.

[0217] Clone SL69 (SEQ ID NO:220 and 221)

[0218] A weak 2.6 kb band was observed in normal testis as well as in chronic myelogenous leukemia and lymphoblastic leukemia. Additional sequence corresponding to this clone is disclosed in SEQ ID NO:336.

[0219] Clone SL86 (SEQ ID NO:242 and 243)

[0220] The sequence was expressed in normal prostate (2.7 kb and 1.1 kb) and testis (1.1 kb). Low expression was observed in a cancer cell line blot using the cell lines described above. 1.1 kb and 2.7 kb transcripts were observed in the cell lines LNCaP, and MDA PCa 2a and 2b (metastatic prostate cells into bone, androgen sensitive), and weak 1.1 kb transcript was seen in HPV-7 (immortalized normal prostate cells) and HPV-10 (immortalized prostate cancer cells). Additional sequence corresponding to this clone is disclosed in SEQ ID NO:337.

[0221] Clone SL195 (SEQ ID NO:288 and 289)

[0222] The sequence was expressed in normal prostate as a 1.9 kb transcript, and the same transcript also observed in all cell lines in the cancer cell line blot described above. It was more heavily expressed in HeLa cell S3 and chronic myelogenous leukemia, and was expressed in all prostate cell lines. Additional sequence corresponding to this clone is disclosed in SEQ ID NO:338.

[0223] Clone SL97 (SEQ ID NO:292 and 293)

[0224] Two transcripts, 2.4 kb and 4 kb, were observed in normal prostate and testis. Two very weak 2.4 kb signals were observed in Hela cell S3 and chronic myelogenous leukemia. The 2.4 kb transcript was expressed in all prostate cell lines. A 4 kb transcript was found in LNCaP, MDA PCa 2A and 2B. Additional sequence corresponding to this clone is disclosed in SEQ ID NO:339.

[0225] Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such specific embodiments and equivalents are intended to be encompassed by the following claims.

[0226] All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein.

	Number	Date	Country
Parent	09328475	Jun 1999	US
Child	10202193	Jul 2002	US

Genes and gene expression products that are differentially regulated in prostate cancer

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