COMPOSITIONS AND METHODS FOR THE MODIFYING HYPOXIA INDUCED GENE REGULATION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U. S. C. § 119 (e) to provisional application Serial No. 60/461,712, filed April 9,2003, the contents of which are hereby incorporated by reference into the present disclosure.

TECHNICAL FIELD This invention relates to isolated polynucleotides shown to be up or down regulated in a pathological cell as compared to a counterpart normal, healthy cell.

BACKGROUND OF THE INVENTION It is known that many, but not all genes present in a cell are expressed at any given time. Fundamental questions of biology require knowledge of which genes are transcribed and the relative abundance of transcripts in different cells. Typically, when and to what degree a given gene is expressed has been analyzed one gene at a time.

Thus, information regarding the identity of all expressed genes in a cell and the level of expression of these genes would facilitate the study of many cellular processes such as activation, differentiation, aging, viral transformation, morphogenesis, and mitosis. A comparison of the expressed genes of a particular cell or the same cell from various individuals or species, under the same or different environmental stimuli, provides valuable insight into the molecular biology of the cell. SAGE (Serial Analysis of Gene Expression or"SAGE") disclosed in Velculescu, et al. (1995) Science 270: 484-487 and U. S. Patent No. 5,695, 937) is a technique that provides such information. Using SAGE, the expressed genes in normal renal proximal tubule cells were compared to renal carcinoma cells that do,

and do not express the tumor suppressor gene known as the von Hippel-Lindau tumor suppressor ("VHL").

It is widely accepted that most solid tumors larger than several mm3 have undergone an angiogenic switch, a crucial step for tumor growth and metastasis.

Thus, blocking angiogenesis could be a strategy to arrest tumor growth (Folkman (1971) New Eng. J. Med. 285 (21): 1182-1186). Various signals from both genetic mutations and environmental factors are involved in this switch (Carmeliet and Jam (2000) Nature 407: 249-257), with one of the most potent environmental signals being hypoxia (low oxygen tension). Although new blood vessel formation is one of the consequences of hypoxia and results in the alleviation of the hypoxic environment, some regions of large solid tumors continue to be under hypoxia. Adaptation to hypoxia is of fundamental importance in developmental, physiological, and pathophysiological processes (Bunn and Poyton (1996) Physiol. Rev. 76 (3): 839-885; Guillemin and Krasnow (1997) Cell 89 (1) : 9-12). At the molecular level, this adaptation to hypoxia depends in part on appropriate expression of many physiologically relevant genes that are regulated transcriptionally, posttranscriptionally or posttranslationally. The gene expression changes in response to hypoxia are important to many fundamental biological processes such as apoptosis, cell cycle control, stress adaptation, anaerobic metabolism, tissue remodeling and angiogenesis. Genes whose expression change in response to hypoxia include: erythropoietin (Epo) which plays a crucial role in regulating the oxygen-carrying capacity of the blood; vascular endothelial growth factor (VEGF) which is a potent angiogenic growth factor that induces new blood vessel formation in both physiological and pathophysiological processes; and genes involved in the long-term adaptation of energy metabolism in an environment of decreased oxygen tension such as glucose transporter 1 (GLUT 1) and phosphoglycerate kinase 1 (PGKI) (Bunn and Poyton (1996) supra ; Semenza (1999) Ann. Rev. Cell Dev. Biol. 15: 551-578).

Enhanced glucose metabolism and angiogenesis are hallmarks of tumor growth and involve up-regulation of genes that are normally induced by hypoxia.

Hypoxia-inducible factor (HIF) is a critical transcriptional regulator which modulates many hypoxia-associated genes, including GLUT I and VEGF (Semenza (2000) J. Appl. Physiol. 88 (4): 1474-1480; and Semenza (2000) Genes Dev.

14 (16): 1983-1991). HIF is a heterodimer comprised of HIF-a and ARNT, two basic

helix-loop-helix proteins in the PAS family (Semenza (1999) supra). ARNT mRNA and protein levels are not significantly effected by ambient oxygen tension. There are at least two mammalian HIF-a isoforms, HIF-la and HIF-2a, that appear to be predominantly regulated by protein stability, although there is some evidence that HIF mRNA levels may also be effected by oxygen (Wenger (2000) J. Exp. Biol.

203 (Pt. 8): 1253-1263). HIF-a proteins are only minimally present under normoxia due to their rapid degradation through the ubiquitin-proteasome pathway (Wenger (2000) supra). In contrast, under low oxygen tension, HIF-a proteins are stabilized (Semenza (2000) Genes Dev. 14 (16): 1983-1991).

Inactivation of the VHL tumor suppressor gene results in the development of von Hippel-Lindau disease, a hereditary cancer syndrome distinguished by highly angiogenic tumors of the kidney, retina, pancreas and central nervous system (Clifford and Maher (2001) Adv. Cancer Res. (82): 85-105; Kondo and Kaelin (2001) Exp. Cell Res. 264 (1) : 117-125). Several lines of evidence have emerged to suggest that pVHL, the VHL gene product, is a multifunctional protein. The most well characterized function of pVHL is its role as a component of the ubiquitin E3 ligase complex that targets HIF-a for ubiquitin-dependent proteasome degradation (Cockman et al. (2000) J. Biol. Chem. 275 (33): 25733-25741). Recent studies demonstrated that the VHL-dependent proteolytic degradation of both HIF-la and HIF-2a occurs through enzymatic hydroxylation of specific prolyl residues within the HIF-a ODDD (oxygen dependent degradation domain) (Ivan et al. (2001) Science 292 (5516): 464-468).

In hypoxic cells, HIF-a degradation is suppressed leading to enhanced transcription of target genes, including pro-angiogenic genes. In renal cell carcinoma (RCC), a common manifestation of VHL disease, HIF-a overexpression is observed throughout the tumor. Two of the known transcriptional targets of HIF-a, GLUT 1 and VEGF (Wiesener et al. (2001) Cancer Res. 61 (13): 5215-5222), are also overexpressed in RCC tumors and likely contribute to the activation of the angiogenic switch found in tumorigenesis. The renal cell carcinoma cell line 786-O does not express detectable HIF-la, although expression of HIF-2a is constitutively high in these cells (Maxwell et al. (1999) Nature 399 (6733): 271-275).

Thus, there is a need in the art to elucidate the molecular mechanisms of tumor cell behavior, including hypoxia signaling pathways, that are now believed to have a

profound impact on the diagnosis, prognosis, and treatment of tumors (Livingston and Shivdasani (2001) JAMA 285 (5): 588-593). This invention satisfies this need and provides related advantages as well.

DESCRIPTION OF THE INVENTION The von Hippel-Lindau tumor suppressor, pVHL, is a key player in one of the best-characterized hypoxia signaling pathways, the VHL-HIF pathway. Serial Analysis of Gene Expression (SAGE) supra, was used to investigate hypoxia-regulated gene expression in renal carcinoma cells (786-0) with and without VHL. The gene expression profiles of the cancer cells were compared to SAGE profiles from normal renal proximal tubule cells grown under both normoxia and hypoxia.

The genes identified in Table 2 infra, were shown to be differentially expressed in cancer cells as compared to normal renal cells when grown under normoxia and hypoxia. Most of these have not been previously connected to differential expression under these conditions.

Based on these findings, this invention provides screening methods to identify candidate agents capable of altering the biological activity of a polypeptide encoded by a polynucleotide involved in hypoxia-related tumorigenesis by contacting a test agent with a target cell expressing the polynucleotide, and monitoring activity of the expressed polypeptide product, wherein the test agent which modifies the activity of the polypeptide is a candidate agent. In one aspect, the biological activity is the induction of hypoxia-related gene enolase 2 (GenBank No. Y00691M27) or a biological equivalent thereof.

In another aspect, the biological activity is the induction of a hypoxia-related gene, inducible in the absence of VHL, wherein the gene is selected from the group comprising at least one of integrin alpha E (GenBank No. L25851), endothelin 2 (GenBank No. X55177), stress-induced endoplastic reticulum protein 1 (GenBank No. AB022427), phosphoglutamase 1 (GenBank No. M83088), cell-division cycle 25B (AL10980) and biological equivalents thereof.

In a further aspect, the biological activity is differential expression in a neoplastic cell under hypoxia, as compared to a normal counterpart cell and in a

neoplastic cell that has lost VHL and the gene is selected from the group comprising at least one of enolase 2 (GenBank No. Y00691 M27) glia maturation factor B (GenBank No. AB001106) and biological equivalents thereof.

In yet a further aspect, the biological activity is induction of a gene, induced in the absence of VHL but not hypoxia, wherein the gene is selected from the group comprising at least one of metalloprotease 1 (GenBank No. AK001183), insulin-like growth factor binding protein 3 (GenBank No. Hs. 77326), and biological equivalents thereof. In one aspect, the target cell of the assay is a renal carcinoma cell.

In another aspect, the screen further requires contacting a normal, healthy counterpart cell (e. g., renal cell such as a renal proximal tubule cell) to the test cell and monitoring the activity of the polypeptide.

The screen also is useful to determine if a drug or therapy is effective for an individual in need of such treatment by obtaining the target cell from the individual, wherein the target cell is suspected of being involved in the pathology or tissue to be treated. It also is useful to monitor the efficacy of a drug or therapy by performing the screen prior to treatment and/or at different time points during treatment, and comparing the results obtained at different time points.

In one aspect the target cell is present in a subject and therefore in vivo.

Subjects, for example, rats, mice, simians or the like, containing a target cell can be used as animal models for the discovery of new potential therapeutics and/or to personalize treatment to the individual subject (as noted above) or to monitor treatment.

The screen can be practiced in silico by comparing the transciptome of a test or target cell to a database containing one or more sequences identified in Table 2. A computer-readable medium having stored one or more gene sequences or biological equivalents thereto is further provided by this invention. Several therapeutic benefits are achievable by the modulation, e. g., enhancement or suppression of expression, of the genes of this invention. For example, by inhibiting the biological activity of enolase 2 (GenBank No. Y00691M27) or a biological equivalent thereof, one can negatively affect cell metabolism and inhibit cell growth and/or promote apoptosis (cell death). Alternatively, by enhancing the expression of this gene or its equivalent,

one can enhance growth in adverse oxygen environments such as ischemic heart disease.

Also provided are methods and compositions to inhibit a hypoxia-related gene, inducible in the absence of VHL, wherein the gene is selected from the group comprising at least one of integrin alpha E (GenBank No. L25851), endothelin 2 (GenBank No. X55177), stress-induced endoplastic reticulum protein 1 (GenBank No. AB022427), phosphoglutamase 1 (GenBank No. M83088), cell-division cycle 25B (AL10980), and biological equivalents thereof. By use of these compositions and methods, one can specifically target mutant neoplastic cells and prevent cell growth and/or promote apoptosis (cell death). Detection of such genes can be diagnostic for neoplastic cells that have lost VHL expression and thus can dictate treatment options for such cancers. Alternatively, by enhancing the expression of one of these genes or its equivalent, one can enhance cell growth or prevent apoptosis.

In a further aspect, inhibition of a gene selected from the group comprising at least one of enolase 2 (GenBank No. Y00691 M27), glia maturation factor B (GenBank No. AB001106) and biological equivalents thereof, provides a method to negatively affect cell metabolism to inhibit cell growth and/or promote apoptosis (cell death). Alternatively, by enhancing the expression of at least one of these genes or its equivalent, one can enhance growth in adverse oxygen environments such as ischemic heart disease.

In yet a further aspect, inhibition of a gene, induced in the absence of VHL but not hypoxia, wherein the gene is selected from the group comprising at least one of metalloprotease 1 (GenBank No. AK001183), insulin-like growth factor binding protein 3 (GenBank No. Hs. 77326), and biological equivalents thereof, provides a method to distinguish mutant neoplastic cells from cells grown in low oxygen.

Inhibition of such a gene can negatively affect cell growth specifically in neoplastic cells that have lost VHL but not in cells growing under hypoxia or in normal cells.

Alternatively, by enhancing the expression of one of these genes or its equivalent, one can enhance cell growth in populations of cells that have lost VHL gene function.

Agents and compositions known to inhibit gene function include but are not limited to agents and compositions that degrade mRNA (ribozymes), anti-sense

nucleotides, small interfering RNAs, antibodies that block action of polynucleotides and small molecule drug inhibitors. For the purpose of illustration only, specific examples include, but are not limited to, HerceptinTM (Genentech, Inc.), GleevacTM (Novartis) and thalidomide.

Agents and compositions known to enhance gene function include but are not <BR> <BR> limited to agents and compositions that enhance gene expression (e. g. , enhancers) or that block or inhibit negative gene regulators such as activator proteins and proteins or nucleotides that bind to negative regulatory elements on DNA. For the purpose of illustration only, specific examples include, but are not limited to, AP-2, and the trichothecene mycotoxin vomitoxin (VT).

Compositions useful in the screens and methods described herein are further provided by this invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows genes that are negatively regulated by VHL in 786-0 cells.

The black circle on the left denotes genes that are up-regulated 24-fold in 786-0 VHL-Nor. cells compared to 786-0 VHL+Nor. cells; the white circle on the right indicates the genes that are up-regulated 2 4-fold in 786-0 VHL+Hyp. cells compared to 786-0 VHL+Nor. cells. The gray intersection of the two circles denotes the genes that are up-regulated in both comparisons. The number of genes included in each set is indicated. Letters in each section refer to representative genes from each set which can be found in the corresponding section of Tables 2 and 3.

Figure 2 shows the effects of VHL on gene expression in 786-0 cells. The black circle on the left denotes the genes that are up-regulated 24-fold in 786-0 VHL-Nor. cells compared to Renal Proximal Tubule (RPTEC) Nor. cells; the white circle on the right indicates the genes that are up-regulated 24-fold in 786-0 VHL+Nor. cells. Because two independent SAGE libraries were made from the RPTECs under each growth condition, the tag values were averaged before comparing. The gray intersection of the two circles denotes the genes that are up- regulated in both comparisons. The number of genes induced in each set is indicated.

Letters in each section refer to representative genes from each set which can be found in the corresponding sections of Table 2 and 4.

Figure 3 shows an alternative hypoxia-responsive pathway in 786-0 cells expressing VHL. The black circle on the left denotes the genes that are up-regulated 24-fold in 786-0 VHL+Hyp. cells compared to 786-0 VHL+Nor. cells; the white circle on the right indicates the genes that are up-regulated 24-fold in RPTEC Hyp. cells compared to RPTEC Nor. cells. SAGE tag values for the two independent libraries of RPTECs grown under each condition were averaged before comparing. The gray intersection of the two circles denotes the genes that are up-regulated in both comparisons. The number of genes induced in each set is indicated. Letters in each section refer to representative genes from each set which can be found in the corresponding sections of Table 2 and 5.

Figure 4 schematically shows hypoxia-responsive genes in 786-0 cells lacking VHL. The black circle on the left denotes the genes that are up-regulated 24-fold in 786-0 VHL-Hyp. cells compared to 24-fold in 786-0 VHL-Nor. cells; the white circle on the right indicates the genes that are up-regulated 2 4-fold in 786-0 VHL+Hyp. compared to 786-0 VHL+Nor. cells. The gray intersection of the two circles denotes the genes that are up-regulated in both comparisons. The number of genes included in each set is indicated. Letters in each section refer to representative genes from each set which can be found in the corresponding sections of Table 2 and 6.

Figure 5 graphically shows that real-time quantitative RT-PCR expression confirms SAGE expression. Quantitative RT-PCR was performed on three genes observed to be induced by SAGE. RNA from an independently generated set of 786- 0-VHL+ cells (WT8) and 786-0 VHL cells (pRC3) was used to confirm induced expression of CCNßl, TGFI, and GLUT1 in the RCC cells compared to RPTECs.

BRIEF DESCRIPTION OF THE TABLES Table 1 shows SAGE libraries of cancer cells (TRCC 768-0) and normal cells.

Table 2, subsections (a) through (i), identifies several SAGE tags that were found to be differentially expressed in renal cells grown under deficient conditions.

Table 3A identifies 116 genes represented in Figure 1 (A) (109 genes; 7 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold in 786-0 VHL-Nor. (V2) when compared to 786-0 VHL+Nor. (V1). Also shown, for

comparison, is the SAGE data for each gene in the 786-0 VHL+Hyp. (V3) with respect to 786-0 VHL+Nor. (V1). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending order of V2/V1.

Table 3B identifies 38 genes represented in Figure 1 (B) (36 genes; 2 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold in 786-0 VHL-Nor. (V2) compared to 786-0 VHL+Nor. (V1), and induced by hypoxia in 786-0 VHL+Hyp. (V3) compared to 786-0 VHL+Nor. (V1). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending order of V2/V1.

Table 3C identifies 122 genes represented in Figure 1 (105 genes; 17 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold by hypoxia in 786-0 VHL+Hype. (V3) when compared to 786-0 VHL+Nor. (V1). Also shown, for comparison, is the SAGE data for each gene in the 786-0 VHL-Nor. (V2) with respect to 786-0 VHL+Nor. (V1). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending order of V3/V1.

Table 4A identifies 416 genes represented in Figure 2 (C) (369 genes; 47 tags with no matches are not shown). SAGE data for genes up-regulated &gt;4-fold in 786-0 VHL-Nor. (V2) when compared to the average of two independent cultures of Renal Proximal Tubule cells (RPTECs) grown under nomoxia (V5, V6). Also shown, for comparison, is the SAGE data for each gene in the 786-0 VHL+Nor. (V1) with respect to its average expression in the two independent cultures of Renal Proximal Tubule cells (RPTECs) grown under nomoxia (V5, V6). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending order of V2/V5, V6.

Table 4B also identifies 468 genes represented in Figure 2 (D) (421 genes; 47 tags with no matches are not shown). SAGE data for genes up-regulated &gt;4-fold in 786-0 VHL+Nor. (V1) when compared to the average of two independent cultures of Renal Proximal Tubule cells (RPTECs) grown under nomoxia (V5, V6). Also shown, for comparison, is the SAGE data for each gene in the 786-0 VHL-Nor. (V2) with respect to its average expression in the two independent cultures of Renal Proximal Tubule cells (RPTECs) grown under nomoxia (V5, V6). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending

order of V1/V5, V6.

Table 4C further identifies 371 genes represented in Figure 2 (E) (351 genes; 20 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold in 786-0 cells, regardless of VHL status (VHL+, V1 and VHL-, V2) with respect to the average of two independent cultures of Renal Proximal Tubule cells (RPTECs) grown under nomoxia (V5, V6). The table is presented in descending order of V21V5, V6.

Table 5A identifies 153 genes represented in Figure 3 (F) (135 genes; 18 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold by hypoxia in 786-0 VHL+Hyp. (V3) when compared to 786-0 VHL+Nor. (V 1). Also shown, for comparison, is the SAGE data for each gene as a ratio of its average expression in the two independent culture of RPTECs grown under hypoxia (V7, V8) with respect to their average expression in the two independent cultures of RPTECs grown under nomoxia (V5, V6). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending order of V3/V 1.

Table 5B also identifies 7 genes represented in Figure 3 (G) (6 genes; 1 novel tag are not shown). SAGE data for genes up-regulated 2 4-fold by hypoxia in both 786-0 VHL+ cells and RPTECs. Normalized SAGE data from 786-0 VHL+Hyp. (V3) was compared to normalized data from 786-0 VHL+Nor. (V1). The average tag abundance for the two independent cultures of RPTECs grown under hypoxia (V7, V8) and nomoxia (V5, V6) was used. The table is presented in descending order of V3/V1.

Table 5C further identifies 407 genes represented in Figure 3 (347 genes; 60 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold by hypoxia in RPTECs. The average tag abundance for the two independent cultures of RPTECs grown under hypoxia (V7, V8) and nomoxia (V5, V6) was used. Also shown, for comparison, is the SAGE data for each gene in 786-0 VHL+Hyp. (3) compared to 786-0 VHL-Nor. (V1). SAGE tag abundance were normalized before calculating ratios, as shown. The table is presented in descending order of V7, V8/V5, V6.

Table 6A identifies 87 genes represented in Figure 4 (H) (83 genes; 4 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold by hypoxia in

786-0 VHL-Hyp. (V4) when compared to those in 786-0 VHL-Nor. (V2). Also shown, for comparison, is the SAGE data for each gene in 786-0 VHL+Hyp. (V3) compared to 786-0 VHL+Nor. (V1). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending order of V4/V2.

Table 6B also identifies 4 genes represented in Figure 4 (I). SAGE data for genes up-regulated 24-fold by hypoxia in 786-0 cells, regardless of VHL status (VHL+, V3 and VHL-, V4) with respect to 786-0 cells grown under nomoxia (VHL+, V1 and VHL-, V2) SAGE tag abundances were normalized before calculating ratio, as shown. The table is presented in descending order of V4/V2.

Table 6C further identifies 156 genes represented in Figure 4 (137 genes; 19 tags with no matches are not shown). SAGE data for genes up-regulated 24-fold by hypoxia in 786-0 VHL+Hyp. (V3) when compared to 786-0 VHL+Nor. (V1). Also shown, for comparison, is the SAGE data for each gene 786-0 VHL-Hyp. (V4) when compared to 786-0 VHL-Nor. (V2). SAGE tag abundances were normalized before calculating ratios, as shown. The table is presented in descending order of V3/V1.

MODE (S) FOR CARRYING OUT THE INVENTION Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Definitions The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, organic chemistry, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e. g., Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds, (1987) ) ; the series METHODS<BR> IN ENZYMOLOGY (Academic Press, Inc. ) ; "PCR : A PRACTICAL APPROACH<BR> " (M. MacPherson, et al. , IRL Press at Oxford University Press (1991) ) ; PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.

(1988) ); and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

As used in the specification and claims, the singular form"a,""an"and"the" include plural references unless the context clearly dictates otherwise. For example, the term"a cell"includes a plurality of cells, including mixtures thereof.

The term"comprising"is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.

"Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention.

Embodiments defined by each of these transition terms are within the scope of this invention.

The terms"polynucleotide"and"nucleic acid molecule"are used interchangeable to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term"polynucleotide"includes, for example, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules.

A"gene"refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

A biologically equivalent polynucleotide is a polynucleotide that hybridizes under moderate or stringent conditions to a sequence identified in Table 2 or its complement. "Hybridization"refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding

between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

Hybridization reactions can be performed using traditional hybridization techniques under different stringency. In general, a low stringency hybridization reaction is carried out at about 40°C in 10 X SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at 10 about 50°C in 6 X SSC, and a high stringency hybridization reaction is generally performed at about 60°C in 1 X SSC. Alternatively, TMAC hybridization technology can be used for hybridization reactions probed with pooled oligonucleotides such as the SAGE tags. The advantage of using TMAC hybridization is that the reaction condition is not dependent on the G+C content of the oligonucleotide, and the melting temperature is determined only by the length of the oligomers to be used.

When hybridization occurs in an anti-parallel configuration between two single-stranded polynucleotides, the reaction is called"annealing"and those <BR> <BR> polynucleotides are described as"complementary. "A double-stranded polynucleotide can be"complementary"or"homologous"to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.

"Complementarity"or"homology" (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules. A polynucleotide that is 100% complementary to a second polynucleotide are understood to be"complements"of each other.

Equivalent polynucleotides also include polynucleotides that are greater than 75%, or 80%, or more than 90%, or more than 95% homologous to the sequence shown in Table 2 and as further isolated and identified using sequence homology searches. Sequence homology is determined using a sequence alignment program run under default parameters and correcting for ambiguities in the sequence data, changes

in nucleotide sequence that do not alter the amino acid sequence because of degeneracy of the genetic code, conservative amino acid substitutions and corresponding changes in nucleotide sequence, and variations in the lengths of the aligned sequences due to splicing variants or small deletions or insertions between sequences that do not affect function.

A variety of sequence alignment software programs are available in the art.

Non-limiting examples of these programs are BLAST family programs including BLASTN, BLASTP, BLASTX, TBLASTN, and TBLASTX (BLAST is available from the worldwide web at ncbi. nlm. nih. gov/BLAST/), FastA, Compare, DotPlot, BestFit, GAP, FrameAlign, ClustalW, and Pileup. These programs are obtained commercially available in a comprehensive package of sequence analysis software such as GCG Inc. 's Wisconsin Package. Other similar analysis and alignment programs can be purchased from various providers such as DNA Star's MegAlign, or the alignment programs in GeneJockey. Alternatively, sequence analysis and alignment programs can be accessed through the world wide web at sites such as the CMS Molecular Biology Resource at sdsc. edu/ResTools/cmshp. html. Any sequence database that contains DNA or protein sequences corresponding to a gene or a segment thereof can be used for sequence analysis. Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS.

Parameters for determining the extent of homology set forth by one or more of the aforementioned alignment programs are known. They include but are not limited to p value, percent sequence identity and the percent sequence similarity. P value is the probability that the alignment is produced by chance. For a single alignment, the p value can be calculated according to Karlin et al. (1990) PNAS 87: 2246. For multiple alignments, the p value can be calculated using a heuristic approach such as the one programmed in BLAST. Percent sequence identify is defined by the ratio of the number of nucleotide or amino acid matches between the query sequence and the known sequence when the two are optimally aligned. The percent sequence similarity is calculated in the same way as percent identity except one scores amino acids that are different but similar as positive when calculating the percent similarity. Thus, conservative changes that occur frequently without altering function, such as a change from one basic amino acid to another or a change from one hydrophobic amino acid to

another are scored as if they were identical.

A"gene product"refers to the amino acid (e. g., peptide or polypeptide) generated when a gene is transcribed and translated.

As used herein, the term"modulate"means to alter or modify a process or biological function.

The term"peptide"is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e. g. ester, ether, etc. As used herein the term"amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.

The term"cDNAs"refers to complementary DNA, that is mRNA molecules present in a cell or organism made into cDNA with an enzyme such as reverse transcriptase. A"cDNA library"is a collection of all of the mRNA molecules present in a cell or organism, all turned into cDNA molecules with the enzyme reverse transcriptase, then inserted into"vectors." A"probe"when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.

A"primer"is a short polynucleotide, generally with a free 3'-OH group that binds to a target or"template"potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. A"polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers"or a"set of primers"consisting of an"upstream"and a "downstream"primer, and a catalyst of polymerization, such as a DNA polymerase,

and typically a thermally-stable polymerase enzyme. Methods for PCR are known in the art, and taught, for example in"PCR: A PRACTICAL APPROACH" <BR> <BR> (M. MacPherson et al. , IRL Press at Oxford University Press (1991) ). All processes of producing replicate copies of a polynucleotide, such as PCR or gene cloning, are <BR> <BR> collectively referred to herein as"replication. "A primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses.

Sambrook et al. , supra.

The term"genetically modified"means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. "Foreign nucleic acid"includes, but is not limited to promoters, enhancers and gene activators. For example, a genetically modified cell includes a cell that contains a polynucleotide that may be in its native environment, but not expressed and expression has been turned on or alternatively, the level of expression has been enhanced or lowered by the upstream insertion of a gene activator.

"Differentially expressed"as applied to a gene, refers to the differential production of the mRNA transcribed from the gene or the protein product encoded by the gene. A differentially expressed gene may be overexpressed or underexpressed as compared to the expression level of a normal or control cell. The term"differentially expressed"also refers to nucleotide sequences in a cell or tissue which are expressed where silent in a control cell or not expressed where expressed in a control cell.

A"composition"is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label or a pharmaceutically acceptable carrier) or active, such as an adjuvant.

A"pharmaceutical composition"is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term"pharmaceutically acceptable carrier"encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin,

REMINGTON'S PHARM. SCI., 15th Ed. , Mack Publ. Co. , Easton, PA (1975).

An"effective amount"is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.

A"gene delivery vehicle"is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, cationic liposomes, viruses, such as baculovirus, adenovirus, adeno-associated virus, and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

A"viral vector"is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors and the like. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and the inserted polynucleotide. As <BR> <BR> used herein, "retroviral mediated gene transfer"or"retroviral transduction"carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Retroviruses carry their genetic information in the form of RNA ; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form that integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a polynucleotide to be inserted. Adenoviruses (Ads) are a relatively well characterized, homogenous group

of viruses, including over 50 serotypes. (see, e. g. , WO 95/27071). Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad-derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, WO 95/00655; WO 95/11984. Wild-type AAV has high infectivity and specificity integrating into the host cells genome (Hermonat and Muzyczka (1984) PNAS 81: 6466-6470; Lebkowski et al. (1988) Mol. Cell. Biol. 8: 3988-3996).

Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5'and/or 3'untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5'of the start codon to enhance expression.

Gene delivery vehicles also include several non-viral vectors, including DNA/liposome complexes, and targeted viral protein DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention. To enhance delivery to a cell, the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e. g. , TCR, CD3 or CD4.

Polynucleotides are inserted into vector genomes using methods known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of restricted polynucleotide. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector DNA. Additionally, an oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in

mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; stabilizing elements 3'to the inserted polynucleotide, and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Other means are known and available in the art.

"Host cell"is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous polynucleotides, polypeptides and/or proteins. It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, plant cells, insect cells, animal cells, and mammalian cells, e. g., murine, rat, simian or human.

An"antibody"is an immunoglobulin molecule capable of binding an antigen.

As used herein, the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity.

As used herein, "expression"refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al.

(1989) supra). Similarly, an eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors can be obtained commercially or assembled by the sequences described in methods known in the art.

A"subject"is a vertebrate, preferably a mammal, more preferably a human.

Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

A"control"is an alternative subject or sample used in an experiment for comparison purpose. A control can be"positive"or"negative".

The term"culturing"refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i. e. , morphologically, genetically, or phenotypically) to the parent cell. By"expanded"is meant any proliferation or division of cells.

The terms"cancer, ""neoplasm,"and"tumor,"are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a"clinically detectable"tumor is one that is detectable on the basis of tumor mass; e. g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition. Tumor cells often express antigens that are tumor specific. The term"tumor associated antigen"or"TAA"refers to an antigen that is associated with or specific to a tumor.

As used herein, "solid phase support"is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include, but are not limited to, silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, arrays and chips. A suitable solid phase support may be selected on the basis of desired end use and suitability for various synthetic protocols. For example, for peptide synthesis, solid phase support may refer to resins such as polystyrene (e. g.,

PAM-resin obtained from Bachem Inc. , Peninsula Laboratories, etc.), POLYHIPER resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGelTM, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California). In one embodiment for peptide synthesis, solid phase support can refer to polydimethylacrylamide resin.

A"transgenic animal"refers to a genetically engineered animal or offspring of genetically engineered animals. The transgenic animal can contain genetic material from at least one unrelated organism (such as from a bacteria, virus, plant, or other animal) or can contain a mutation which interferes with expression of a gene product.

Polynucleotides This invention provides methods for modulating cellular function by delivering to a cell or tissue a gene identified in Table 2, or its biological equivalent.

In a further aspect, agents that modulate the expression of a gene or its equivalent of Table 2 can be identified by insertion of the one or more genes into a cell that will express the gene. In this way, target cells are generated for use in the in vitro screens, noted above. Alternatively, when the gene is administered to a subject under conditions that allow for expression of the gene, such as an animal, the animal also is useful in the screens identified above. Alternatively, deficient expression of a gene can be restored by administration of a gene or its equivalent of Table 2 under conditions that allow expression of the gene. This gene therapy is useful to treat or ameliorate the symptoms associated with various disorders, e. g., enzyme deficiencies or replacement of tumor suppressor genes in cancers.

A number of alternative approaches can be employed to administer or deliver the polynucleotide. The gene can be delivered to cells deficient in the gene's specific activity using a gene delivery vehicle, wherein the polynucleotide is operatively linked to an appropriate promoter element. Methods for administering an effective amount of a gene delivery vehicle to a cell have been developed and are known to those skilled in the art and described herein. Alternatively, the polypeptide encoded by the polynucleotide of interest can be delivered in an effective amount to a cell that is deficient in the polypeptide's function.

SAGE analysis revealed that certain genes are up-regulated in tumor cells.

Thus, these genes are useful as markers for neoplastic cells and targets for visualizing and diagnosing these cells. Methods for detecting gene expression in a cell are known in the art and include techniques such as in hybridization to DNA microarrays, in situ hybridization, PCR, RNase protection assays and Northern blot analysis. Such methods are useful to detect and quantitate expression of the gene in a cell.

Alternatively expression of the encoded polypeptide can be detected by various methods. In particular it is useful to prepare polyclonal or monoclonal antibodies that are specifically reactive with the target polypeptide. Such antibodies are useful for visualizing cells that express the polypeptide using techniques such as immunohistology, ELISA, and Western blotting. These techniques can be used to determine expression level and diagnose tumors in a subject.

As noted above, the genes identified on Table 2 and their equivalents were selected for further study as they were found to be differentially expressed in cancer cells. Genes that are up-regulated or down-regulated encode proteins involved in several distinct biochemical pathways. These include, but are not limited to, cell growth, attachment to extracellular matrix components, inter-cellular signaling, intra-cellular signaling and metabolism.

The genes identified in Table 2 and their equivalents are useful candidates for developing therapeutic agents for a variety of disease conditions related to tumorigenesis and angiogenesis. These include, but are not limited to, well-vascularized tumors and hyperplasia.

The agents and compositions of the present invention can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.

The pharmaceutical compositions can be administered orally, intranasally, parenterally, transdermally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of gene therapy, suspensions, solutions and emulsions of the ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to an agent of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of

the invention.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavoring agents. It also is intended that the agents, compositions and methods of this invention be combined with other suitable compositions and therapies.

The polynucleotides of the invention can comprise additional sequences, such as coding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, and polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.

The polynucleotides of the invention can be introduced by any suitable gene delivery method or vector. They also can be expressed in a suitable host cell for generating a cell-based therapy. These methods are known in the art.

This invention also provides genetically modified cells that produce enhanced expression of the genes of Table 2 or their equivalents. The genetically modified cells can be produced by insertion of upstream regulatory sequences such as promoters or gene activators (see, U. S. Patent No. 5,733, 761).

The polynucleotides and polypeptides can be conjugated to a detectable marker, e. g., an enzymatic label or a radioisotope for detection of nucleic acid and/or expression of the gene in a cell. A wide variety of appropriate detectable markers are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In one aspect, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples. Thus, this invention further provides a method for detecting a

single-stranded polynucleotide identified encoding are of more gene of Table 2 or its equivalent, or its complement, by contacting target single-stranded polynucleotides with a labeled, single-stranded polynucleotide (a probe) which is a portion of the polynucleotides identified in Table 2 (or the corresponding complement) under conditions permitting hybridization (preferably moderately stringent hybridization conditions) of complementary single-stranded polynucleotides, or more preferably, under highly stringent hybridization conditions. Hybridized polynucleotide pairs are separated from un-hybridized, single-stranded polynucleotides. The hybridized polynucleotide pairs are detected using methods known to those of skill in the art and set forth, for example, in Sambrook et al. (1989) supra.

The polynucleotides and sequences embodied in this invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service.

Suitable cell or tissue samples used for this invention encompass body fluid, solid tissue samples, tissue cultures or cells derived therefrom and the progeny thereof, and sections or smears prepared from any of these sources, or any other samples that may contain neoplastic tumor tissue.

The polynucleotides of this invention can be isolated or replicated using PCR.

The PCR technology is the subject matter of U. S. Patent Nos. 4,683, 195; 4,800, 159; 4,754, 065; and 4,683, 202 and described in PCR: THE POLYMERASE CHAIN REACTION (Mullis et al. eds, Birkhauser Press, Boston (1994) ) or MacPherson, et al. (1991) and (1995) supra, and references cited therein. Alternatively, one of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to replicate the DNA. Accordingly, this invention also provides a process for obtaining the polynucleotides of this invention by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides. In a separate embodiment, these polynucleotides are further isolated. Still further, one of skill in the art can insert the polynucleotide into a suitable replication vector and insert the

vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA so amplified can be isolated from the cell by methods known to those of skill in the art. A process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained.

RNA can be obtained by first inserting a DNA polynucleotide into a suitable host cell. The DNA can be delivered by any appropriate method, e. g., by the use of an appropriate gene delivery vehicle (e. g., liposome, plasmid or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA; the RNA can then be isolated using methods known to those of skill in the art, for example, as set forth in Sambrook et al. (1989) supra. For instance, m-RNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. (1989) supra, or extracted by nucleic-acid-binding resins following the accompanying instructions provided by manufactures.

Polynucleotides exhibiting sequence complementarity or homology to a polynucleotide identified in Table 2 find utility as hybridization probes. Since the full coding sequence of the transcript is known, any portion of this sequence or homologous sequences, can be used in the methods of this invention.

It is known in the art that a"perfectly matched"probe is not needed for a specific hybridization. Minor changes in probe sequence achieved by substitution, deletion or insertion of a small number of bases do not affect the hybridization specificity. In general, as much as 20% base-pair mismatch (when optimally aligned) can be tolerated. Preferably, a probe useful for detecting the aforementioned mRNA is at least about 80% identical to the homologous region. More preferably, the probe is 85% identical to the corresponding gene sequence after alignment of the homologous region; even more preferably, it exhibits 90% identity.

These probes can be used in radioassays (e. g. Southern and Northern blot analysis) to detect, prognose, diagnose or monitor various cells or tissues containing these cells. The probes also can be attached to a solid support or an array such as a chip for use in high throughput screening assays for the detection of expression of the gene corresponding a polynucleotide of this invention. Accordingly, this invention also provides a probe comprising or corresponding to a polynucleotide identified in

Table 2 or its equivalent, or its complement, or a fragment thereof, attached to a solid support for use in high throughput screens.

The total size of fragment, as well as the size of the complementary stretches, will depend on the intended use or application of the particular nucleic acid segment.

Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between at least 5 to 10 to about 100 nucleotides, or even full length according to the complementary sequences one wishes to detect.

Nucleotide probes having complementary sequences over stretches greater than 5 to 10 nucleotides in length are generally preferred, so as to increase stability and selectivity of the hybrid, and thereby improving the specificity of particular hybrid molecules obtained. More preferably, one can design polynucleotides having gene-complementary stretches of 10 or more or more than 50 nucleotides in length, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology with two priming oligonucleotides as described in U. S. Patent No. 4,603, 102 or by introducing selected sequences into recombinant vectors for recombinant production. In one aspect, a probe is about 50-75 or more alternatively, 50-100, nucleotides in length.

The polynucleotides of the present invention can serve as primers for the detection of genes or gene transcripts that are expressed in neoplastic cells described herein. In this context, amplification means any method employing a primer-dependent polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. For illustraton purposes only, a primer is the same length as that identified for probes, above.

One method to amplify polynucleotides is PCR. However, PCR conditions used for each reaction are empirically determined. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg2+ concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides. After amplification, the resulting DNA fragments can be

detected by any appropriate method known in the art, e. g., by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.

The invention further provides the isolated polynucleotide operatively linked to a promoter of RNA transcription, as well as other regulatory sequences for replication and/or transient or stable expression of the DNA or RNA. As used herein, the term"operatively linked"means positioned in such a manner that the promoter will direct transcription of RNA off the DNA molecule. Examples of such promoters are SP6, T4 and T7. In certain embodiments, cell-specific promoters are used for cell-specific expression of the inserted polynucleotide. Vectors which contain a promoter or a promoter/enhancer, with termination codons and selectable marker sequences, as well as a cloning site into which an inserted piece of DNA can be operatively linked to that promoter are known in the art and commercially available.

For general methodology and cloning strategies, see GENE EXPRESSION <BR> <BR> TECHNOLOGY (Goeddel ed. , Academic Press, Inc. (1991) ) and references cited<BR> therein and VECTORS: ESSENTIAL DATA SERIES (Gacesa and Ramji, eds. , John<BR> Wiley &amp; Sons, N. Y. (1994) ) which contains maps, functional properties, commercial suppliers and a reference to GenEMBL accession numbers for various suitable vectors.

In one embodiment, polynucleotides derived from the polynucleotides of the invention encode polypeptides or proteins having diagnostic and therapeutic utilities as described herein as well as probes to identify transcripts of the protein that may or may not be present. These nucleic acid fragments can by prepared, for example, by restriction enzyme digestion of larger polynucleotides and then labeled with a detectable marker. Alternatively, random fragments can be generated using nick translation of the molecule. For methodology for the preparation and labeling of such fragments, see Sambrook, et al. (1989) supra.

Expression vectors containing these nucleic acids are useful to obtain host vector systems to produce proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include plasmids, viral vectors and liposomes. Adenoviral vectors are particularly useful for introducing genes into tissues in vivo because of their high levels of expression and

efficient transformation of cells both in vitro and in vivo. When a nucleic acid is inserted into a suitable host cell, e. g., a prokaryotic or a eukaryotic cell and the host cell replicates, the protein can be recombinantly produced. Suitable host cells will depend on the vector and can include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells constructed using known methods. See Sambrook, et al. (1989) supra. In addition to the use of viral vector for insertion of exogenous nucleic acid into cells, the nucleic acid can be inserted into the host cell by methods known in the art such as transformation for bacterial cells; transfection using calcium phosphate precipitation for mammalian cells; or DEAE-dextran ; electroporation ; or microinjection. See, Sambrook et al. (1989) supra, for methodology. Thus, this invention also provides a host cell, e. g. a mammalian cell, an animal cell (rat or mouse), a human cell, or a prokaryotic cell such as a bacterial cell, containing a polynucleotide encoding a protein or polypeptide or antibody.

When the vectors are used for gene therapy in vivo or ex vivo, a pharmaceutically acceptable vector is preferred, such as a replication-incompetent retroviral or adenoviral vector. Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression of the inserted polynucleotide. As used herein, the term"pharmaceutically acceptable vector"includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into dividing cells. An example of such a vector is a"replication-incompetent"vector defined by its inability to produce viral proteins, precluding spread of the vector in the infected host cell. An example of a replication-incompetent retroviral vector is LNL6 (Miller A. D. et al.

(1989) BioTechniques 7 : 980-990). The methodology of using replication-incompetent retroviruses for retroviral-mediated gene transfer of gene markers has been established. (Bordignon (1989) PNAS USA 86: 8912-8952; Culver, K. (1991) PNAS USA 88: 3155; and Rill, D. R. (1991) Blood 79 (10): 2694-2700).

Clinical investigations have shown that there are few or no adverse effects associated with the viral vectors. (Anderson (1992) Science 256: 808-813).

This invention also provides"knock-out"animals, in which the gene of interest selected from Table 2, is deleted or mutated sufficiently to disrupt its function. The resulting transgenic animals can be used to further analyze the function

of the gene or as an assay system for therapeutic agents and methods. These "knock-out"animals are made by taking advantage of the phenomena of homologous recombination using methods known in the art. Briefly, targeting DNA vectors contain (1) two blocks of DNA sequences that are homologous to separate regions of the target site; (2) a DNA sequence that codes for resistance to the compound G418 (Neo') between the two blocks of homologous DNA (i. e. positive selection marker) and (3) DNA sequences coding for herpes simplex virus thymidine kinases (HSV-tkl and HSV-tk2) outside of the homologous blocks (i. e. negative selection marker).

When this vector is introduced into the embryonic stem cell, homologous recombination inserts the Neor gene into the target genome, disrupting function of that gene.

Proteins This invention provides proteins or polypeptides expressed from the polynucleotides or their equivalents, which is intended to include wild-type and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells, as well as muteins, analogs and fragments thereof. In some embodiments, the term also includes antibodies and anti-idiotypic antibodies. Such polypeptides can be isolated or produced using the methods identified below.

It is understood that functional equivalents or variants of the wild-type polypeptide or protein also are within the scope of this invention, for example, those having conservative amino acid substitutions of the sequences identified in Table 2.

Other analogs include fusion proteins comprising a protein or polypeptide, identified in Table 2, or a fragment of that protein or polypeptide.

The proteins and polypeptides of this invention are obtainable by a number of processes known to those of skill in the art, which include purification, chemical synthesis and recombinant methods. Full length proteins can be purified from a neoplastic cell or a tumor biopsy as identified above. Sources for purifying the protein can also be from an individual, such as a patient with ischemic heart disease.

Proteins can be purified by methods such as immunoprecipitation with antibody, and standard techniques such as gel filtration, ion-exchange, reversed-phase, and affinity chromatography using a fusion protein as shown herein. For such methodology, see for example Deutscher et al. (1999) GUIDE TO protein PURIFICATION:

METHODS IN ENZYMOLOGY (Vol. 182, Academic Press). Accordingly, this invention also provides the processes for obtaining these proteins and polypeptides as well as the products obtainable and obtained by these processes.

The proteins and polypeptides also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by Perkin/Elmer/Applied Biosystems, Inc. , Model 430A or 431A, Foster City, CA, USA. The synthesized protein or polypeptide can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Accordingly, this invention also provides a process for chemically synthesizing the proteins of this invention by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.

Alternatively, the proteins and polypeptides can be obtained by well-known recombinant methods as described, for example, in Sambrook et al. (1989) supra, using the host cell and vector systems described above.

Also provided by this application are the polypeptides and proteins described herein conjugated to a detectable agent for use in the diagnostic methods. For example, detectably labeled proteins and polypeptides can be bound to a column and used for the detection and purification of antibodies. They also are useful as immunogens for the production of antibodies as described below. The proteins and fragments of this invention are useful in an in vitro assay system to screen for agents or drugs, which modulate cellular processes.

The proteins of this invention also can be combined with various liquid phase carriers, such as sterile or aqueous solutions, pharmaceutically acceptable carriers, suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. When used to prepare antibodies, the carriers also can include an adjuvant that is useful to non-specifically augment a specific immune response. A skilled artisan can easily determine whether an adjuvant is required and select one. However, for the purpose of illustration only, suitable adjuvants include, but are not limited to Freund's Complete and Incomplete, mineral salts and polynucleotides.

This invention also provides a pharmaceutical composition comprising any of

a protein, analog, mutein, or polypeptide fragment of this invention, alone or in combination with each other or other agents, and an acceptable carrier. These compositions are useful for various diagnostic and therapeutic methods as described herein.

Antibodies Also provided by this invention is an antibody capable of specifically forming a complex with the proteins or polypeptides as described above. The term"antibody" includes polyclonal antibodies and monoclonal antibodies. The antibodies include, but are not limited to mouse, rat, and rabbit or human antibodies, and varients thereof.

Laboratory methods for producing polyclonal antibodies and monoclonal antibodies, as well as deducing their corresponding nucleic acid sequences, are known in the art, see Harlow and Lane (1988) supra, and Sambrook, et al. (1989) supra. The monoclonal antibodies of this invention can be biologically produced by introducing protein or a fragment thereof into an animal, e. g., a mouse or a rabbit. The antibody producing cells in the animal are isolated and fused with myeloma cells or heteromyeloma cells to produce hybrid cells or hybridomas. Accordingly, the hybridoma cells producing the monoclonal antibodies of this invention also are provided.

Thus, using the protein or fragment thereof, and known methods, one of skill in the art can produce and screen the hybridoma cells and antibodies of this invention for antibodies having the ability to bind the proteins or polypeptides.

If a monoclonal antibody being tested binds with the protein or polypeptide, then the antibody being tested and the antibodies provided by the hybridomas of this invention are equivalent. It also is possible to determine without undue experimentation, whether an antibody has the same specificity as the monoclonal antibody of this invention by determining whether the antibody being tested prevents a monoclonal antibody of this invention from binding the protein or polypeptide with which the monoclonal antibody is normally reactive. If the antibody being tested competes with the monoclonal antibody of the invention as shown by a decrease in binding by the monoclonal antibody of this invention, then it is likely that the two antibodies bind to the same or a closely related epitope. Alternatively, one can pre-incubate the monoclonal antibody of this invention with a protein with which it is

normally reactive, and determine if the monoclonal antibody being tested is inhibited in its ability to bind the antigen. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or a closely related, epitopic specificity as the monoclonal antibody of this invention.

The term"antibody"also is intended to include antibodies of all isotypes.

Particular isotypes of a monoclonal antibody can be prepared either directly by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class switch variants using the procedure described in Steplewski et al. (1985) PNAS 82: 8653 or Spira et al. (1984) J. Immunol. Meth. 74 : 307.

This invention also provides biological active fragments of the polyclonal and monoclonal antibodies described above. These"antibody fragments"retain some ability to selectively bind with its antigen or immunogen. Such antibody fragments can include, but are not limited to: Fab; Fab' ; F (ab') 2, Fv; and SCA. A specific example of"a biologically active antibody fragment"is a CDR region of the antibody.

Methods of making these fragments are known in the art, see for example, Harlow and Lane (1988) supra.

The antibodies of this invention also can be modified to create chimeric antibodies and humanized antibodies (Oi et al. (1986) BioTechniques 4 (3): 214).

Chimeric antibodies are those in which the various domains of the antibodies'heavy and light chains are coded for by DNA from more than one species.

The isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibodies of the invention can be accomplished by one of ordinary skill in the art by producing anti-idiotypic antibodies (Herlyn et al. (1986) Science 232: 100). An anti-idiotypic antibody is an antibody that recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest.

Idiotypic identity between monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using antibodies to the epitopic determinants on a monoclonal antibody it is possible to identify other hybridomas expressing monoclonal antibodies of the same epitopic specificity.

It is also possible to use the anti-idiotype technology to produce monoclonal antibodies that mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the mirror image of the epitope bound by the first monoclonal antibody. Thus, in this instance, the anti-idiotypic monoclonal antibody could be used for immunization for production of these antibodies.

As used in this invention, the term"epitope"is meant to include any determinant having specific affinity for the monoclonal antibodies of the invention.

Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

The antibodies of this invention can be linked to a detectable agent or label.

There are many different labels and methods of labeling known to those of ordinary skill in the art.

The antibody-label complex is useful to detect the protein or fragments in a sample, using standard immunochemical techniques such as immunohistochemistry as described by Harlow and Lane (1988) supra. Competitive and non-competitive immunoassays in either a direct or indirect format are examples of such assays, e. g., enzyme linked immunoassay (ELISA) radioimmunoassay (RIA) and the sandwich (immunometric) assay. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

The coupling of antibodies to low molecular weight haptens can increase the sensitivity of the assay. The haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts avidin, or dinitropherryl, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See Harlow and Lane (1988) supra.

The monoclonal antibodies of the invention also can be bound to many different carriers. Thus, this invention also provides compositions containing the antibodies and another substance, active or inert. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those

skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.

Compositions containing the antibodies, fragments thereof or cell lines which produce the antibodies, are encompassed by this invention. When these compositions are to be used pharmaceutically, they are combined with a pharmaceutically acceptable carrier.

Functional Analysis with Antibodies Antibodies of this invention can be used to purify the polypeptides of this invention and to identify biological equivalent polynucleotides. They also can be used to identify agents that modify the function of the polynucleotides of this invention. These antibodies include polyclonal antisera, monoclonal antibodies, and various reagents derived from these preparations that are familiar to those practiced in the art and described above. Antibodies can be used in immuno-histochemistry to determine the physical location of the proteins encoded by the identified genes in subjects.

Antibodies that neutralize the activities of proteins encoded by identified genes can also be used in vivo and in vitro to demonstrate function by adding such neutralizing antibodies into in vivo and in vitro test systems. They also are useful as pharmaceutical agents to modulate the activity of polypeptides of the invention.

Various antibody preparations can also be used in analytical methods such as ELISA assays or Western blots to demonstrate the expression of proteins encoded by the identified genes by test cells in vitro or in vivo. Fragments of such proteins generated by protease degradation during metabolism can also be identified by using appropriate polyclonal antisera with samples derived from experimental samples.

Screening Assays The present invention provides methods for screening various agents that modulate the expression of a polynucleotide of the invention or the function of a protein product encoded by the polynucleotide of interest in a neoplastic cell. For the purposes of this invention, an"agent"is intended to include, but not be limited to a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein (e. g. antibody), a polynucleotide (e. g. anti-sense) or a

ribozyme. A vast array of compounds can be synthesized, for example polymers, such as polypeptides and polynucleotides, and synthetic organic compounds based on <BR> <BR> various core structures, and these are also included in the term"agent. "In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. It should be understood, although not always explicitly stated that the agent is used alone or in combination with another agent, having the same or different biological activity as the agents identified by the inventive screen.

One preferred embodiment is a method for screening small molecules capable of interacting with the protein or polynucleotide of the invention. For the purpose of <BR> <BR> this invention, "small molecules"are molecules having low molecular weights (MW) that are, in one embodiment, capable of binding to a protein of interest thereby altering the function of the protein. Preferably, the MW of a small molecule is no more than 1,000. Methods for screening small molecules capable of altering protein function are known in the art. For example, a miniaturized arrayed assay for detecting small molecule-protein interactions in cells is discussed by You et al. (1997) Chem. Biol. 4: 961-968.

To practice the screening method in vitro, suitable cell cultures or tissue cultures containing this type of neoplastic cell are first provided. The cell can be a cultured cell or a genetically modified cell in which a transcript from a gene of Table 2 or its biological equivalent or its complement is expressed. Alternatively, the cells can be from a tissue biopsy. The cells are cultured under conditions (temperature, growth or culture medium and gas (COZ)) and for an appropriate amount of time to attain exponential proliferation without density dependent constraints. It also is desirable to maintain an additional separate cell culture; one which does not receive the agent being tested as a control.

As is apparent to one of skill in the art, suitable cells can be cultured in microtiter plates and several agents can be assayed at the same time by noting genotypic changes, phenotypic changes or cell death.

When the agent is a composition other than a DNA or RNA, such as a small molecule as described above, the agent can be directly added to the cell culture or added to culture medium for addition. As is apparent to those skilled in the art, an "effective"a mount must be added which can be empirically determined. When agent

is a polynucleotide, it can be directly added by use of a gene gun or electroporation.

Alternatively, it can be inserted into the cell using a gene delivery vehicle or other method as described above.

Kits containing the agents and instructions necessary to perform the screen in vitro method as described herein also are claimed.

The assays also can be performed in a subject. When the subject is an animal such as a rat, mouse or simian, the method provides a convenient animal model system that can be used prior to clinical testing of an agent. In this system, a <BR> <BR> candidate agent is a potential drug if transcript expression is altered, i. e. , upregulated et al. (such as restoring tumor suppressor function), downregulated or eliminated as with drug resistant genes or oncogenes, or if symptoms associated or correlated to the presence of cells containing transcript expression are ameliorated, each as compared to untreated, animal having the pathological cells. It also can be useful to have a separate negative control group of cells or animals that are healthy and not treated, which provides a basis for comparison. After administration of the agent to subject, suitable cells or tissue samples are collected and assayed for altered gene expression or protein function.

Identification, Analysis, and Manipulation of Genetic Polymorphisms with SNP Technology The polynucleotides of Table 2 are useful to search for and identify single nucleotide polymorphism (SNP's), which are mutant variants of the gene in the human population. Identification of such polymorphisms is useful to define human diseases to which mutations in the gene contribute and to perfect therapies for disease processes in which the protein encoded by these genes. Mutant variants of the gene identified in this manner can then be employed in the development, screening, and analysis of pharmaceutical agents to treat these diseases. Methods to detect such SNP's can be formatted to create diagnostic tests. Furthermore, various mutations in the gene which effect the response of different individuals to therapeutic agents can be identified and then diagnosed through analysis of SNP's, to guide the prescription of appropriate treatments. Also, SNP's identified in the gene can provide useful sequence markers for genetic tests to analyze other genes and mutations in the region of the genome where the gene is located. Thus it is useful to incorporate these SNP's

into polymorphism databases.

Skilled practitioners of the art are familiar with an array of methods for identifying and analyzing SNP's. High throughput DNA sequencing procedures such as sequencing by hybridization (Drmanac et al. (1993) Science 260: 1649-1652), mini-sequencing primer extension (Syvanen (1999) Hum. Mutut. 13 (1) : 1-10), or other sequencing methods can be used to detect SNP's in defined regions of the gene.

Alternatively, other methods such as hybridization to oligonucleotides on DNA microarrays (Lipshutz et al. (1999) Nat. Genet. 21 (1 Suppl. ) : 20-24); analysis of single strand conformational polymorphisms in DNA or RNA molecules by various analytical methods (Nataraj (1999) Electrophoresis 20 (6): 1177-85); PCR-based mutational analyses such as PCR with primers spanning the polymorphic sequence; or protection of SNP-containing oligonucleotides from nuclease protection such as by use of the bacterial mutS protein, can be employed. Many sophisticated high-throughput technologies based on methods such as automated capillary electrophoresis (Larsen et al. (1999) Hum. Mut. 13 (4): 318-327), time-of-flight mass spectroscopy (Li et al. (1999) supra), high density micro-arrays (Sapolsky et al.

(1999) Genet. Anal. 14 (5-6) : 187-92), semiconductor microchips (Gilles et al. (1999) Nat. Biotechnol. 17 (4): 365-70), and others have been demonstrated that can be employed with the oncogenic osteomalacia-related gene to perform the uses described above.

Genomics Applications This invention also provides a process for preparing a database for the analysis of a cell's expressed genes by storing in a digital storage medium information related to the sequences of the invention. Using this method, a data processing system for standardized representation of the expressed genes of a cell is compiled. The database contains at least one polynucleotide, its biological equivalent or complement, of this invention. In an alternative embodiment, the database contains any combination of polynucleotides of this invention, alone or in combination with other polynucleotides.

The data processing system is useful to characterize the genotype or phenotype of a cell and to analyze gene expression between two cells by first selecting a cell and then identifying and sequencing the expressed sequences (transcriptome) of the cell. This information also is stored in a computer-readable storage medium as the

transcriptome. The transcriptome is then compared with at least one sequence (s) of transcription fragments from a polynucleotide of this invention. The compared sequences are then analyzed. Uniquely expressed sequences and sequences differentially expressed between the polynucleotides of this invention and the selected cell can be identified by this method.

In other words, this invention provides a computer based method for screening the homology of an unknown DNA or mRNA sequence against one or more of the polynucleotides of this invention by first providing the complete set of expressed genes, i. e. , the transcriptome, in computer readable form and homology screening the DNA or mRNA of the unknown sequence against the polynucleotides of this invention or database containing same and determining whether the DNA sequence of the unknown contains similarities to any portion of the transcriptome listed in the computer readable form. In one aspect, the transcriptome of the test cell is compared to a gene identified in Table 2.

Thus, the information provided herein also provides a means to compare the relative abundance of gene transcripts in different biological specimens by use of high-throughput sequence-specific analysis of individual RNAs or their corresponding cDNAs using a modification of the systems described in WO 95/2068, WO 96/23078 and U. S. Patent No. 5,618, 672.

Non-Human Transgenic Animals In another aspect, the novel polynucleotide sequences associated with a pathological state of a cell can be used to generate transgenic animal models. In recent years, geneticists have succeeded in creating transgenic animals, for example mice, by manipulating the genes of developing embryos and introducing foreign genes into these embryos. Once these genes have integrated into the genome of the recipient embryo, the resulting embryos or adult animals can be analyzed to determine the function of the gene. The mutant animals are produced to understand the function of known genes in vivo and to create animal models of human diseases. (Joyner et al.

(1992) in POSTIMPLANTATION DEVELOPMENT IN THE MOUSE (Chadwick and Marsh, eds. , John Wiley &amp; Sons, United Kingdom) pp: 277-297; Dorin et al.

(1992) Nature 359: 211-215).

The following examples are intended to illustrate, but not limit the invention.

EXPERIMENTAL A total of eight independent SAGE libraries were generated from eight different normal and tumor samples, as shown in Table 1. The samples varied in wild type VHL gene expression and exposure to hypoxia. Two samples were derived from the RCC 786-0 cell line, which is defective in the VHL gene, engineered to express wild type VHL through infection with a retroviral vector carrying the gene. These cells were grown under normoxic (786-0 VHL+ Nor. ) and hypoxic (786-0 VHL+<BR> Hyp. ) conditions. Two samples were generated from the RCC 786-0 cells infected with an empty retroviral vector, also grown under normoxic (786-0 VHL-Nor.) and hypoxic (786-0 VHL-Hyp.) conditions. Two samples were from independent primary cultures of normal renal proximal tubule cells (RPTECs), which are believed to give rise to renal cell carcinoma, grown under normoxic conditions (RPTECs VHL+ Nor. ). Finally, two samples were from the same independent RPTECs grown<BR> under hypoxic conditions (RPTECs VHL+ hyp. ). In total, over 380,000 SAGE tags were sequenced and more than 61,000 unique tags were identified. Gene expression profiles of the cells grown under different conditions were compared to find potential regulators or targets of VHL and/or hypoxia-mediated signaling. For this analysis, normalized SAGE tag abundance was used to compare data from two or more libraries, and genes were considered induced or repressed if their expression levels changed by at least 4-fold.

Effects of VHL on gene expression in 786-0 cells. As a multifunctional tumor suppressor, pVHL plays an important role in many aspects of cell biology.

Genes exclusively up-regulated in 786-0 cells lacking VHL include genes that encode growth factors and their receptors, proteins involved in cell-cell and cell-extracellular matrix interactions, and proteins that control cellular metabolism (Table 2) were assayed. Many of the genes in this group were shown to be hypoxia-inducible genes (Table 2c), including GLUT 1, insulin-like growth factor binding protein 3 (IGFBP3) and VEGF, all of which are regulated by HIF-la (Semenza (1999) Am. Rev. Cell Dev.

Biol. 15: 551-578).

It is worth noting that a SAGE tag corresponding to VEGF was induced 4-fold in the 786-0 VHL-cells compared to RPTEC, and was also induced in the RPTECs

grown under hypoxia (Table 2c). This SAGE tag is located in the 3'untranslated region of multiple isoforms of VEGF (Claffey et al. (1998) Mol. Biol. Cell 9: 469-481) and therefore does not identify which isoform (s) is predominantly expressed in these cells. Interestingly, VEGFI89 was previously observed by SAGE at induced levels in glioblastoma cells grown under hypoxia (Lal et al. (2001) J. Natl. Cancer Inst.

93 (17): 1337-1343) but was not induced at least 4-fold in any of our samples exposed to hypoxia.

Hypoxia-overexpressed gene 3 (HOG3) was also expressed more highly in the 786-0 VHL-Nor. cells than in the RPTEC Nor. cells (Table 2c). HOG3 is an HIF-1-dependent hypoxia inducible gene originally identified in human glioblastoma cells (Lal et al. (2001) supra.). HIG2 (Hypoxia inducible gene 2), which was previously identified as a hypoxia inducible gene in cultured human cervical epithelial cells and in cervical tumor xenografts deprived of oxygen (Denko, N. et al. (2000) Clin. Cancer Res. 6 (2): 480-487), was also induced in the 786-0 VHL-Nor. cells.

Overexpression of these genes is consistent with the role of VHL in negatively regulating HIF-responsive genes.

468 genes are exclusively up-regulated in the 786-0 cells that have been engineered to re-express VHL, with respect to their expression in RPTECs. Some genes were previously implicated in playing a functional role in angiogenesis and tumorigenesis (Table 2d). These include syndecan 2 (SDC2) (Iozzo and San Antonio (2001) J. Clin. Invest. 108 (3): 349-355), neuropilin 1 (NRP1) (Soker, S. et al. (1990) Cell 92 (6): 735-745), plasminogen activator urokinase receptor (PLAUR) (Roland, et al. (1990) EMBO J. 9 (2): 467-474; Graham, et al. (1998) Blood 91 (9): 3300-3307; Graham, et al. (1999) Int. J. Cancer 80 (4): 617-623) and integrin beta 3 binding protein (ITGB3BP) (Shattil, et al. (1995) J. Cell Biol. 131 (3): 807-816 and Li, et al.

(1999) Mol. Cell Biol. 19 (10): 7191-7202). The role of VHL in negatively regulating genes involved in angiogenesis has now been extensively studied. This data shows that VHL also positively regulates genes that promote angiogenesis, at least in the context of a cancer cell.

This analysis also identified 371 genes that are constitutively over-expressed in the 786-0 cells with respect to RPTECs, regardless of VHL expression (Figure 2e).

This set reflects genetic differences between a malignant cell line and normal cells.

Some of the genes represent potential biomarkers or tumor specific antigens for renal

cell carcinoma since their overexpression is independent of the genetic status of VHL.

Indeed, cyclin BI (CCNBI), a protein that regulates cell growth and proliferation, is induced about 7 to 10-fold, by SAGE, in the 786-0 cells. CCNB1 was recently identified as an epithelial tumor-specific antigen and is a candidate for cancer vaccine therapy (Kao, et al. (2001) J. Exp. Med. 194 (9): 1313-1324).

Although 371 genes are induced at least 4-fold in both the presence and absence of VHL, many of these genes are more highly expressed in the VHL-cells.

This shows that VHL plays a role in their transcriptional regulation. Many of these genes are involved in regulating tumor proliferation, differentiation, and angiogenesis.

HIF-a is regulated through prolyl hydroxylation by PHD (PH domain-containing) proteins 1,2 and 3 (Bruick and McKnight (2001) Science 294: 1337-1340 and Epsteen et al. (2001) Cell 107 (1) : 43-54). In vitro studies using recombinant PHD proteins suggest that PHDs may be oxygen sensors (Epstein et al.

(2001) supra, and Semenza (2001) Cell 107 (1) : 1-3). Importantly, PHD-1 is up-regulated in the cancer cells grown under normoxia compared to RPTECs (Table 2d). Conceivably, alterations in PHD-1 expression impact the response of the cancer cells to oxygen changes.

Overall, more than 1000 genes showed induced expression in the 786-0 cells (relative to their expression in RPTECS), with and without VHL, suggesting that the cancer cells have undergone significant changes compared to non-transformed renal epithelial cells. These changes have resulted in the induction of genes that promote and sustain tumor growth.

Genes that are hypoxia-inducible in the presence of VHL. Hypoxia within a tumor is primarily a pathophysiologic consequence of structurally and functionally disturbed microcirculation, accompanied by the deterioration of diffusion conditions.

Tumor hypoxia is a central issue in tumor physiology and cancer treatment, since it is associated with adverse outcomes. The identification of genes specifically induced under hypoxic conditions may be of functional importance in a variety of critical cellular pathways, including cell cycle arrest, apoptosis, metabolism, tissue remodeling and angiogenesis. The role of VHL in regulating gene expression in tumors and in response to hypoxic conditions is under intense study. Because the renal cell carcinoma cell line 786-0 carries a defective VHL gene, it is an ideal model

system to delineate the role of VHL in both hypoxia signaling and tumorigenesis. By comparing the genes that are hypoxia-inducible in the 786-0 VHL+ cells with the hypoxia-inducible genes in RPTECs, genes were identified that are uniquely hypoxia-regulated in a tumor cell environment. Many of these genes have not previously been shown to be hypoxia-inducible in other tumor or normal cells. 160 genes are up-regulated by hypoxia in 786-0 VHL+ cells, whereas 414 genes are induced by hypoxia in the RPTECs. It is noteworthy that only seven genes are hypoxia-inducible at least 4-fold in both RPTECs and in the 786-0 VHL+ cells. The vast majority of genes (153 out of 160) that are hypoxia-inducible in the 786-0 VHLt cells are not induced at least 4-fold in RPTECs grown under hypoxia. As discussed above, VEGF is induced more than 5-fold in RPTECs grown under hypoxia, but because basal expression of VEGF is elevated in 786-0 VHL+ cells, the hypoxic induction is not as dramatic in these cells. The observation that greater than 95% of the hypoxia-inducible genes differ between the malignant cells and the normal renal cells suggests the establishment of an alternative hypoxia sensitive pathway (s) in these tumor cells. The TGFß-induced gene (TGFBI; Table 2f) is among the 153 hypoxia-inducible genes in 786-0 VHL+ cells but not in the RPTECs. TGFBI was also induced in the 786-0 cells regardless of VHL status (Table 2e), although the SAGE tags identified under these different conditions were distinct. Both tags are located in the 3'UTR of the gene and therefore correspond to the same protein, although the alternative mRNAs may be regulated differently. TGFBI was previously reported to be elevated in adenomas and colorectal cancer (Zhang et al. (1997) Science 276 (5316): 1268-1272 and Buckhaults et al. (2001) Cancer Res.

61 (19): 6996-7001). TGFBI is known to bind to collagens, and is believed to play an important role in cell-collagen interactions (Munier (1997) Nature Genetics 15 (3): 247-251). Overexpression of this gene may therefore be a critical component in the tissue remodeling that accompanies tumorigenesis.

Genes in this group also include zinc finger protein 36 (ZFP36L1) which is thought to be involved in regulating the response to growth factors (Barnard et al.

(1993) Nucleic Acids Res 21: 3580; (Bustin et al. (1994) DNA Cell Biol. 13: 449-459) and p53-induced protein PIGPCI, also called the THW tumor suppressor (Hildebrandt et al. (2000) Anticancer Research 20: 2801-2809). THW is a putative transmembrane receptor that was initially identified as a gene that was down-regulated in a melanoma

cell line capable of metastasis when compared to a non-metastasizing line. It was also down-regulated in mammary carcinoma cell lines compared with cell lines derived from non-malignant mammary epithelium, and in pancreas cell lines derived from metastases compared with a cell line derived from a primary pancreatic tumor (Hildebrandt et al. (2000) supra). Indeed, it was genetic data that led to THW's assignment as a tumor suppressor gene; the THW gene is located on chromosome 6q, a frequent site of loss of heterozygosity (LOH) associated with many malignancies (Hildebrandt et al. (2000) supra). Consistent with previous data, THW was found to be down-regulated in the cancer cell line, 786-0 VHL+ Nor., compared with normal RPTECs grown under normoxic conditions (8. 22-fold). Interestingly, the down-regulation of THW is less dramatic in 786-0 VHL-Nor. compared with normal RPTECs (3. 79-fold,). Thus, VHL plays a role in THW expression, and establishes a pathway of regulation for tumor suppressor genes.

Recently, a set of hypoxia-overexpressed genes (HOGs) was identified in human glioblastoma cells using SAGE (Lal et al. (2001) J. Natl. Cancer Inst.

93 (17): 1337-1343), and six of the HOGs were found to be controlled by HIF-a (Lal et al. (2001) supra). Interestingly, none of the 6 HOGs was induced in the 786-0 VHL+ cells grown under in this study. As discussed above, the 786-0 cells lack HIF-1 a expression so the failure to see HOG induction here may be explained by exclusive HIF-1 a regulation of the HOGs.

The seven genes that are hypoxia-inducible in both RPTECs and in the 786-0 VHL+ cells may be regulated by HIF-a proteins. Since HIF-2a is constitutively expressed in the 786-0 VHL-cells, regardless of oxygen pressure, higher expression of these seven genes is expected in the 786-0 VHL-Nor. cells with respect to RPTECs grown under normoxia. Interestingly, this is only true for two of the seven genes, PAI-1 and a novel gene (no match for the SAGE tag in the public databases). Thus the other 5 genes are controlled through proteins other than HIF, and again separates the VHL and HIF-a gene-induction pathways. Glycolytic enzyme enolase 2 (EN02), which was previously found in nervous tissue (Oliva et al. (1991) Genomics 10: 157- 165), is among the seven hypoxic genes. Interestingly, the a-, or non-neuronal enolase (ENO1), is ubiquitously expressed and is known to be induced by hypoxia in an HIF-la dependent manner (Iyer et al. (1998) Genes Dev 12: 149-162).

414 genes were induced at least 4-fold in the RPTECs grown under hypoxic conditions. As expected, this group includes genes previously identified as hypoxic genes, such as GLUT 1, insulin-like growth factor binding protein 3 (IGFBP3), adenylate kinase 3 (ADK3), and VEGF (Table 2c, 2cl) (Semenza (1999) supra).

HIF-la mRNA was also detected at induced levels in the hypoxia treated RPTECs, which confirms that HIF-la can be regulated at a transcriptional level as well (Semenza (2000) supra ; Wenger (2000) supra). In addition to the genes known to be hypoxia-inducible, many of the detected genes are not well characterized and/or were not previously known to be regulated by hypoxia. Examples include retinoic acid induced gene 3 (RAI3) which encodes a putative G protein-coupled receptor (Cheng and Lotan (1998) J. Biol. Chem. 273: 35008-35015), lysyl oxidaselike gene 2 (LOXL2) which encodes an extracellular protein that may be specifically involved in cell adhesion and senescence (Csiszar (2001) Prog Nucleic Acid Res Mol Biol. 70: 1- 32); Jourdan-Le Saux et al. (1998) Genomics 51: 305-307); Saito et al. (1997) J Biol Chem. 272: 8157-8160) and a presumably novel gene since the SAGE tag does not match any known genes or ESTs in the public databases.

VHL-independent Gene Expression in 786-0 Cells under Hypoxic Conditions. VHL is a known regulator of the hypoxia pathway through HIF. A defect in VHL will result in the constitutive activation of the HIF-a pathway and lead to up-regulation of hypoxia-inducible genes, even under normoxic condition.

Interestingly, there are still 91 genes that are inducible by hypoxia in 786-0 VHL- cells. 87 genes are exclusively hypoxia-inducible in 786-0 in the absence of VHL, whereas 156 are only induced by hypoxia in the presence of VHL. Thus, tumor cells evolve VHL-dependent and VHL-independent mechanisms to respond to hypoxic stress so that cells can survive in this adverse environment. Among the 87 genes which are uniquely hypoxia-inducible in 786-0 VHL-cells are genes known to be involved in cell-cell interactions and vascular remodeling (Table 2h), including integrin a E (ITGAE) (Cerf-Bensussan et al. (1987) Eur. J. Immunol.

17 (9): 1279-1285) and endothelin 2 (EDN2) (Ohkubo et al. (1990) FEBS Lett. 274 (1- 2): 136-140); Saida et al. (2000) Genomics 64 (1) : 51-61). Interestingly, EDN2 was previously identified as a hypoxia-inducible gene in hypopharyngeal tumor cells (Koong et al. (2000) Cancer Res. 60 (4): 883-887). This list also includes genes involved in the stress response (stress-associated endoplasmic reticulum protein 1;

SERPI) (Yamaguchi et al. (1999) J. Cell Biol. 147 (6) 1195-1204), cellular metabolism (phosphoglucomutase 1 ; PGMI) (Whitehouse et al. (1992) PNAS 89 (1) : 411-415), and cell growth control (cell division cycle 25B; CDC25B) (Bulavin et al. (2001) Nature 411 (6833): 102-107). In addition, many of the genes encode proteins of unknown function that are candidate targets for regulators of hypoxia-responsive pathways.

Interestingly, 4 genes are induced by hypoxia in 786-0 cells regardless of VHL expression (Table 2i). These 4 genes are not among the 414 genes that are hypoxia inducible in normal kidney cells (RPTECs). The specificity of induction in renal cancer cells, regardless of VHL status, suggests that these four genes may be useful biomarkers for RCC and/or effective therapeutic targets. Among the 4 genes induced is HAX-1. The HAX-I gene product is known to interact with various proteins including the polycystic kidney disease protein (PKD2) (Gallagher et al. (2000) PNAS 97 (8): 4017-4022) and the F-actin binding protein, cortactin (Gallagher et al.

(2000) supra). It has been proposed that HAX-1 connects PK D2 to the actin cytoskeleton. This study shows that expression of HAX-1 is increased in a hypoxic tumor environment suggests that this protein may contribute to the cell-matrix contact changes associated with tumor progression. Two of the remaining 3 genes induced in 786-0 cells, regardless of VHL, encode proteins with uncharacterized functions and the final SAGE tag in this group corresponds to multiple genes.

Genes which are negativelv regulated by VHL in 786-0 cells. It has been shown that VHL negatively regulates many hypoxia-inducible genes through the VHL-HIF pathway. In addition, VHL has been shown to regulate the mRNA stability of some hypoxia-inducible genes (Iliopoulos et al. (1996) Nature Med. 1 (8): 822-826).

Thus, a defect in VHL is expected to result in the overexpression of VHL target genes. This is consistent with the observation that many genes which are induced by hypoxia in the VHL 786-0 VHL+ cells are also induced by the absence of VHL, under normoxia in the 786-0 VHL-cells. 38 genes that are hypoxia-inducible in the 786-0 VHL+ cells are also up-regulated in 786-0 VHL-cells under normoxic conditions, when compared to 786-VHL+ cells grown under normoxia. Again, the neuronal enolase, EN02, described above, is among these genes (Table 2b). Another interesting gene in this category is glia maturation factor beta (GMFB) (Table 2b) which is involved in the differentiation of brain cells and in the stimulation of neural regeneration (Kaplan et al. (1991) J Neurochem 57: 483-490). Overexpression of

genes involved in neuronal metabolism, growth and differentiation is intriguing since other manifestations of VHL disease are the development of hemangioblastomas of the retina and central nervous system and pheochromocytomas (Kondo and Kaelin (2001) Exp Cell Res 264: 117-125; Maher and Kaelin (1997) Medicine (Baltimore) 76: 381-391).

In the tumor cells, there are 154 genes that appear to be negatively regulated by VHL. These genes are up-regulated in 786-0 cells that lack VHL compared to those expressing VHL (Table 2a). Among those genes are matrix metalloprotease 1 (MMPI) which cleaves collagens of types I, II, III, VII and X (Templeton et al.

(1990). Numerous studies on MMPs have demonstrated that these proteases play an important role in angiogenesis, morphogenesis, and tissue remodeling, processes which are all associated with the timely breakdown of extracellular matrix during tumorigenesis (Nagase and Woessner (1999) J Biol Chem 274: 21491-21494; Stetler-Stevenson (1999) J Clinic Invest 103: 1237-1241; Vu and Werb (2000) Genes Dev 14: 2123-2133). This group also contains genes that are involved in cell growth and differentiation, including insulin-like growth factor binding protein 3 (IGFBP3) (Cubbage et al. (1990) J Biol Chem 265: 12642-12649). This shows that the VHL tumor suppressor functions by directly regulating genes that control cell growth and differentiation, as well as angiogenesis.

In summary, the gene expression profile analyses of the renal cell carcinoma line 786-0 and the normal kidney cells, grown under different environmental conditions, demonstrated that 786-0 cells have evolved genetic alterations that affect cell growth and differentiation as well as an altered response to hypoxia. The molecular profiles presented here identified genes in the alternative pathways that are either VHL-dependent or-independent. The lists include genes with potential importance in the physiological and pathologic regulation of tumor growth, metabolism, and angiogenesis. Since renal cell tumors are highly vascularized, this data shows that the tumor cells induce angiogenic factors, such as VEGF and TGF-P.

Moreover, this analysis identified genes that are inducible by hypoxia specifically in the cancer cells that have lost VHL expression, such as SERP1 and HAX1, which identifies them as valuable targets specific for mutant tumor cells. Importantly, genes such as MMP-1, that are known to play a critical role in tissue remodeling and angiogenesis, are VHL-regulated in these cells.

It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and the following examples are intended to illustrate and not limit the scope of the invention.

Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.