WO2001023426A2

WO2001023426A2 - Hypoxia-related human genes, proteins, and uses thereof

Info

Publication number: WO2001023426A2
Application number: PCT/US2000/027189
Authority: WO
Inventors: Nicholas C. Denko; Amato J. Giaccia; Christopher J. Green; Keith R. Laderoute; Cornelia Schindler; Albert Ching-Wei Koong
Original assignee: Varian Associates, Inc.
Priority date: 1999-09-30
Filing date: 2000-10-02
Publication date: 2001-04-05
Also published as: AU7748200A; WO2001023426A3

Abstract

The polynucleotide and polypeptide sequences of two novel hypoxia-inducible human genes, HIG1 and HIG2, are described. In addition, a number of known genes and ESTs have now been established as being hypoxia-inducible and hypoxia-repressible. Polynucleotide and polypeptide arrays comprising the hypoxia-inducible and hypoxia-repressible gene sequences, proteins, or antibodies which specifically bind the proteins are disclosed. Methods for using the hypoxia-inducible and hypoxia-repressible gene sequences and proteins, and arrays thereof, to diagnose and treat hypoxia-related conditions such as cancer and ischemia are also provided.

Description

HYPOXIA-RELATED HUMAN GENES, PROTEINS, AND USES

THEREOF

BACKGROUND OF THE INVENTION

a) Field of the Invention

The present invention relates to hypoxia-inducible and hypoxia-repressible genes, and fragments thereof, and to the use of these sequences in the diagnosis and treatment of disease conditions involving hypoxia, including stroke, heart attack, and cancer .

b) Description of Related Art

Hypoxia is responsible for regulating a number of cellular and systemic processes, including angiogenesis, erythropoiesis, and glycolysis. Hypoxic insult and hypoxia-induced gene expression also play a role in a variety of severe pathological conditions including ischemia, retinopathy, neonatal distress, and cancer.

Hypoxia-induced gene expression is associated with ischemia (and reperfusion) in many tissues including the liver, heart, eyes, brain, and vasculature. Many of the hypoxia-induced genes are believed to be involved in the protection or repair of the cells exposed to hypoxia. Enhancement of the body's protective expression of some stress-induced genes is therefore likely to be beneficial in many ischemia/reperfusion-related conditions such as liver transplantation, bypass operations, cardiac arrest, \ and stroke. For instance, in the brain, the response to brain ischemia includes the enhanced expression of growth factors and anti-apoptosis genes (Koistinaho et al . (1997) Neuroreport 20 :i- viii) .

However, the ischemic induction of gene expression is not always favorable. For example, brain ischemia can also result in the expression of apoptosis genes or other genes which promote degeneration of the neuronal cells. Ischemia can also induce an extreme inflammatory reaction in the injured brain via the upregulation of proinflammatory cytokines, chemokines, and endothelial-leukocyte adhesion molecules (Feuerstein et al . (1997) Ann . N. Y. Acad. Sci . 15:179-93). There is some evidence that this hypoxia-induced inflammatory response is a major cause of brain damage.

Eye diseases associated with neovascularization also involve hypoxia. These eye diseases include diabetic retinopathy, retinopathy of prematurity, and sickle cell retinopathy. All can be serious enough to lead to blindness. The feasibility of treatment of retinopathy of prematurity by antisense inhibition of a hypoxia-induced gene, vascular endothelial growth factor (VEGF) , has been demonstrated (Robinson, Patent No. 5,661,135).

A tissue associated with hypoxia-induced and hypoxia- repressed gene expression is the vasculature. There are four major cell types that comprise the vasculature, such as vascular endothelial cells, vascular smooth muscle cells, fibroblasts, and macrophages. The study of hypoxia-induced and hypoxia-repressed gene expression in these cell types in vi tro and in vivo as a result of normal or pathophysiological conditions promises new insight into vascular diseases.

Hypoxia affects several mechanisms in cellular physiology, such as the transcriptionally regulated expression of vasoactive substances and matrix proteins involved in modulating vascular tone or remodeling the vasculature and surrounding tissue. Hypoxia results in the transcriptional induction of genes encoding vasoconstrictors and smooth muscle, and genes encoding matrix or remodeling molecules. Hypoxia also results in transcriptional inhibition of vasodilators such as endothelial nitric oxide synthase (eNOS) (Faller, D.V. (1999; Clin . Exp . Pharmacol . Physiol . ^'26(1) :74-84) .

The process of wound healing also involves the induction of gene expression by hypoxia (Anderson et al . , Patent No. 5,681,706). TNF-α (tumor necrosis factor-α) expression and secretion by macrophages is one response involved in wound healing that is induced by low oxygen. Other hypoxia-induced effects include the formation of scar tissue. In addition to playing a manor regulatory role m the body's response to stress in postnatal life, tissue hypoxia is responsible for regulating expression of genes m the developing embryo, particularly with regard to angiogenesis and vasoformation (Iyer et al . (1998) Genes and Developmen t 12:149- 162; Maltepe et al . (1997) Na ture 386:403-407). Hypoxia also plays a role m neonatal stress and pregnancy-related diseases. For instance, oxygen tension appears to regulate cytotrophoblast proliferation and differentiation within the uterus (Genbacev et al. (1997) Science 277:1669-1672). Some disease conditions related to pregnancy, such as preeclampsia, are associated with abnormal cytotrophoblast differentiation and behavior. A number of studies have shown that an increased concentration of a hypoxia-induced gene product, insulin-like Growth Binding Protein (IGFBP-1), is associated with preeclampsia once manifest in the third trimester, even though US Patent No. 5,712,103 teaches that reduced levels of IGFBP-1 m maternal blood in the first and second trimester, especially during the middle of the second trimester, can be used as a predictive indicator of preeclampsia. Hypoxia has also been established to play a key role in neoplastic tissues. The progression of human tumors to malignancy is an evolutionary process involving the differential expression of multiple genes m response to unique microenvironments . Low oxygen conditions create a dominant tumor microenvironment which directly favors processes driving malignant progression, such as angiogenesis or elimination of p53 tumor suppressor activity.

In addition to promoting further tumor growth, the abnormally low oxygen levels that are found m nearly all solid tumors negatively impact therapeutic efforts. Hypoxic tumors often demonstrate resistance to radiation therapy and chemotherapy .

The connection between tumor hypoxia and the treatment of cancer is further exemplified by a study of cervical cancer that showed that the oxygen level of a tumor was an independent prognostic factor (Hoeckel et al . (19961 Semm . Radia t . Oncol . 6:1-8) . The prognostic value of the oxygen level of a tumor was found to be more significant than all other indicators such as the age of the patient, clinical stage, or tumor size.

A number of oxygen-regulated genes have been identified in the art. Expression of many of these genes is induced by the interaction of hypoxia inducible factor-1 (HIF-1) , a transcription factor complex, with the factor's DNA recognition site on the gene, the hypoxia-responsive element (HRE) . HIF-1 has been cloned and found to not be activated by stressors such as heat shock and ionizing radiation. Differential-display polymerase chain reaction (PCR) has been used to identify additional genes induced by hypoxia (O'Rourke et al. (1996) Eur . J. Biochem . 241:403-410). Six hypoxia-induced genes were identified, three of which were of known function. In addition to the known genes, two expressed sequence tags (ESTs) , and one full-length sequence were identified. The differential-display PCR method used by O'Rourke et al . to screen for hypoxically induced genes was found to be limited in its ability to identify hypoxically-induced genes. In addition to the identification of hypoxia-induced and hypoxia-repressed genes, the identification of the stress- responsive regulatory elements of those genes is also of interest. The identification of such regulatory elements may provide for an inherently tumor-specific form of gene therapy. The HRE from a previously identified hypoxically induced gene, mouse phosphoglycerate kinase-1, has been used to control expression of heterologous genes both in vi tro and in vivo (within a tumor) under hypoxic conditions (Dachs et al . (1997) Nature Medicine 3: 515-520) . Similarly, a method for utilizing an anoxia-responsive element to effect controlled expression of a heterologous protein has been reported (Anderson et al., Patent No. 5,681,706) . SUMMARY OF THE INVENTION

The present invention relates to genes whose expression is modified under hypoxic conditions. The genes may be induced or repressed.

One aspect of the present invention provides the isolated polynucleotide having the sequence shown as SEQ ID NO:l (Fig. 1A) , comprising the cDNA of the hypoxia-induced human gene HIGl , and encoding the polypeptide sequence of SEQ ID NO: 2 (HIGl; Fig. IB) . Polynucleotides with sequences complementary to SEQ ID NO:l, fragments of SEQ ID NO:l which are at least twelve nucleotides in length, and sequences which hybridize to SEQ ID NO:l are also contemplated by the present invention. In particular, one aspect of the invention concerns the fragment of the sequence set forth in SEQ ID NO:l comprising nucleotides 62- 343, the nucleotides representing the coding sequence of human HIGl . The complements to the coding sequence, at least twelve nucleotide-long fragments of the coding sequence, and sequences which hybridize to the coding sequence of HIGl are also provided by the invention.

Another aspect of the present invention provides the isolated polynucleotide having the sequence shown as SEQ ID NO: 3 (Fig. 2A) , comprising the cDNA of the hypoxia induced gene HIG2 , and encoding the polypeptide sequence of SEQ ID NO: 4 (HIG2; Fig. 2B) . The complements to SEQ ID NO: 3, as well as at least twelve nucleotide-long fragments thereof and sequences which hybridize thereto are also provided. The invention refers in particular to a polynucleotide having a sequence corresponding to nucleotides 274-465 of the sequence set forth in SEQ ID NO: 3, or complements thereof, or at least twelve nucleotide-long fragments thereof, or sequences which hybridize thereto. Nucleotides 274-465 represent the coding sequence of human HIG2 .

The present invention also encompasses expression vectors and delivery vehicles which contain polynucleotides of the present invention and host cells that are genetically engineered with polynucleotides of the present invention.

In another embodiment, the invention provides for an oligonucleotide probe comprising fragments, preferably at least about 15 nucleotides long, of the polynucleotides of SEQ ID NO:l or SEQ ID NO: 3, or the complement thereto.

Polypeptides of the sequences set forth in SEQ ID NO: 2 (HIGl) and SEQ ID NO:4 (HIG2), or biochemically equivalent fragments of the polypeptides of either sequence, are further contemplated by the present invention.

Antibodies that are specifically lmmunoreactive to the hypoxia-induced polypeptides HIGl or HIG2 of the present invention are also provided. In still another embodiment, the present invention provides for arrays of polynucleotides or polypeptides corresponding to at least two different hypoxia-inducible genes, hypoxia-induced polypeptides, or antibodies immunoreactive with hypoxia-induced polypeptides . Hypoxia-inducible genes suitable for use in the arrays, diagnostic methods, and treatment methods of the invention described herein are not limited to HIGl and HIG2 , or derivatives thereof, but also include a number of known genes now determined to be hypoxia-inducible. Additional hypoxia-induced genes useful in the methods and arrays of the present invention include, but are not limited to, the genes of annexin V, lipocortin 2 , heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen , phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal LI, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activa tor inhibi tor-1 (PAI-1) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase , lysyl hydroxylase-2 , endothelin-1 , endothelin-2 , B-cell transloca tion gene-1 (BTG-1 ) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1) , quiescin , growth arrest DNA damage- inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1 ) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophilin , cyclooxygenase-1 (COX-1) , fructose hisphospha tase , crea tine transporter , fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2-interacting killer (BIK) , 19 kDa - interacting protein 3, Nip3L/Nix, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferπtin , msulin-like growth factor binding protein 3 (IGFBP-3) , phosphofructokmase (PFK) , aldolase A, aldolase C, mtegπn alpha 5, integrin alpha 5 receptor, placental growth factor, mterleukm-1 (IL-1) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycerate kinase 1 (PGK-1) , monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor, trisephosphate isomerase, lg associated alpha, terferon regulatory factor 6 (IRF6) , putative ORF KIAA0113 , c- fos, glucose transporter-like protein 3/ glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta, brain HHCPA78, muc 1, RNAse L, Mxi 1, glucose-regulated protein 18 (GRP78) , quiescm , lysl oxidase, prostaglandm endoperoxide synthetase, msulin-mducible protein 1, MHC-cIllDQB, myocyte- specific factor 2 (MEF2) , bacteria permeating protein, hexok ase , Cap43 (nickel inducible) , cyclm G2 , carbonic anhydrase IX, TPI, angiogemn, and SDK3.

In one aspect, the present invention provides diagnostic and prognostic tools for assaying for the expression of hypoxia- inducible genes m a tissue of an animal, for determining the presence of hypoxia m a tissue in an animal, and for evaluating a hypoxia-related condition m an animal particularly in order to tailor therapy to a known hypoxic state. The detection of expression products, such as mRNA transcripts or proteins, of the hypoxia-inducible genes of HIGl, HIG2 , annexm V, lipocort 2, heterogeneous nuclear πbonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoπbosylpyrophosphate synthetase , acetoacetylCoA thiolase, πbosomal L¹ , fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activator mhιbιtor-1 (PAI-1), macrophage migration inhibitory factor (MIF) , fibronectin receptor , fibronectin 1, lysl hydroxylase , lysyl hydroxylase-2 , endothelιn-1 , endothelιn-2 , B-cell translocation gene-1 (BTG-1) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kmase-1 (clk-1), quiescm, growth arrest DNA damage- inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498, differentiation of embryo chondrocytes (DEC1), low density lipoprotein receptor related protein (LDLR) , namster hairy gene homologue , adipophil , cyclooxygenase-1 (COX-1) , fructose bisphosphatase , creatme transporter, fatty acid binding protein, lactate dehydrogenase (LDH) , Bcl-2- interacting killer (BIK) , 19 kDa- interacting protein 3, Nιp3L/Nιx, Pιm-1, vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferπtm, msulm-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, mtegπn alpha 5 receptor , placental growth factor, ιnterleukιn-1 (IL-1) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycerate kinase 1 (PGK-1), monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor , trisephosphate isomerase, lg associated alpha, mterferon regulatory factor 6 (IRF6), putative ORF KIAA0113, c- fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta, brain HHCPA78, mucm 1, RNAse L, Mxi 1, glucose-regulated protein 18 (GRP18) , quiescm, lysl oxidase , prostaglandm endoperoxide synthetase, insulin- inducible protein 1, MHC-cIllDQB , myocyte- specific factor 2 (MEF2) , bacteria permeating protein , hexokmase , Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, angiogenm, or SDK3 , or combinations tnereof, to determine the presence of hypoxia in a tissue or evaluate a hypoxia-related condition in an animal is encompassed by the present invention. Methods of diagnosing and treating hypoxia- related conditions via such methods are also encompassed by the present invention.

Other methods of assaying for expression of hypoxia- mducible genes, determining the presence of hypoxia in a tissue m an animal, or evaluating a hypoxia-related condition m an animal involves the use of the arrays of the invention. First, a polynucleotide array or antibody array of the invention may be contacted with polynucleotides or polypeptides, respectively, either from or derived from a sample of body fluid or tissue obtained from the animal. Next, the amount and position of polynucleotide or polypeptiαe from the animal's sample which binds to the sites of the array is determined. Optionally, the gene expression pattern observed may be correlated with an appropriate treatment.

In one aspect, the present invention provides for an expression array of polynucleotides to determine the presence of hypoxia in a tissue in an animal or a human, or to evaluate a hypoxia-related condition in an animal or a human. First, an expression array may be contacted with polynucleotides either purified or unpurified derived from a sample of body fluid or tissue obtained from the animal or human. Next, the amount and position of polynucleotide from the animal's sample which binds to the sites of the expression array is determined. Optionally, the gene expression pattern observed may be correlated with an appropriate treatment.

In a preferred embodiment of the invention, a gene chip ( vide infra ) may be contacted with polynucleotides either purified or unpurified derived from a sample of body fluid or tissue obtained from the animal or human. The amount and position of polynucleotide from the animal's sample which binds to the sites of the gene chip can then be determined. The gene expression pattern observed on the gene chip may be correlated with an appropriate treatment.

Another embodiment of the present invention provides for a diagnostic blood test for assaying for the expression of hypoxia- inducible genes in a tissue of an animal or human, and for detecting the presence of hypoxia-inducible gene products in a tissue in an animal or human. The detection of expression products, such as diagnostic marker proteins, of the hypoxia- inducible genes of PAI-1 , IGF-BP3, placental growth factor, adipophilin , mucin 1 , endothelin-1 , endothelin-2 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferri tin, EPH receptor ligand, angiogenin, TGF beta , HIGl , HIG2 , annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen , phosphoribosylpyrophospha te synthetase , acetoacetylCoA thiolase, ribosomal L I, fibroblast growth factor-3 (FGF-3) , macrophage migra tion inhibitory fa ctor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2 , B-cell transloca tion gene-1 (BTG-1 ) , reducing agent and tunicamycin - responsive protein (RTP) , CDC-like kinase-1 (clk-1) , quiescin, growth arrest DNA damage- inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498 , differentia tion of embryo chondrocytes (DEC1) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue , cyclooxygenase-1 (COX-1) , fructose bisphospha tase, crea tine transporter, fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2- interacting killer (BIK) , 19 kDa -interacting protein 3, Nip3L/Nix, Pim-1 , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor , interleukin-1 (IL-1 ) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1 ) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor , trisephospha te isomerase , lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113 , c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, brain HHCPA78, RNAse L, Mxi 1 , glucose- regula ted protein 18 (GRP18) , quiescin , lysl oxidase , prostaglandin endoperoxide synthetase, insulin- inducible protein 1 , MHC-cIl lDQB, myocyte-specific factor 2 (MEF2) , ba cteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, or SDK3, or combinations or derivatives thereof, to determine the presence of hypoxia in a tissue or evaluate a hypoxia-related condition in an animal or human is encompassed by the present invention.

Another aspect of the present invention provides for a diagnostic blood test for assaying for the expression of hypoxia- inducible genes in a tumor tissue of an animal or human, and for detecting the presence of hypoxia-inducible gene products in a tumor tissue in an animal or human. The detection of expression products, such as diagnostic marker proteins, of the hypoxia- inducible genes of PAI-1 , IGF-BP3, placental growth factor, adipophilin , mucin 1 , endothelin-1 , endothelin-2 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferritin , EPH receptor ligand, angiogenin , IGF beta , HIGl , HIG2 , annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal L I, fibroblast growth factor-3 (FGF-3) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2 , B-cell transloca tion gene-1 (BTG-1 ) , reducing agent and tunicamycin- responsive protein (RTF) , CDC-like kinase-1 (clk-1) , quiescin, growth arrest DNA damage- inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498, differentiation of embryo chondrocytes (DEC1 ) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue , cyclooxygenase-1 (COX-1 ) , fructose bisphospha tase, crea tine transporter, fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2- interacting killer (BIK) , 19 kDa -interacting protein 3, Nip3L/Nix, Pim-1 , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor , interleukin-1 (IL-1 ) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1 ) , monocarboxyla te transporter 3, DNA binding protein A20 , peroxisome prolifera tion receptor, trisephospha te isomerase, lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113, c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, brain HHCPA18, RNAse L, Mxi 1 , glucose- regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1 , MHC-cIl lDQB, myocyte- specif ic factor 2 (MEF2) , bacteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI , or SDK3, or combinations or derivatives thereof, to determine the presence of hypoxia in a tumor tissue or evaluate a hypoxia-related tumor condition in an animal or human is encompassed by the present invention.

In a preferred embodiment of the invention, the diagnostic marker proteins in the blood test are the hypoxia-inducible genes of PAI-1 , IGF-BP3, placental growth factor, adipophilin, mucin 1 , endothelin-1 , endothelin-2 , vascular endothelial growth fa ctor (VEGF) , erythropoietin (EPO) , trans ferritin , EPH receptor ligand, angiogenin , or TGF beta . Another embodiment of the invention is a nuclear medicine based assay designed to non-mvasively identify tumors of hypoxia in vivo by assaying for the expression of hypoxia-inducible genes in a tumor tissue of an animal or human, and by detecting the presence of hypoxia-inducible gene products in a tumor tissue in an animal or human. The detection of expression products, such as diagnostic cell surface ligands and receptors, of the hypoxia- mducible genes of integrin alpha 5 receptor , mterleukm-1 (IL- 1 ) receptor , fibronectin , EPH receptor ligand, APO-1 (Fas Receptor) , mucm-1 , crea tme transporter, monocarboxyla te transporter, or combinations or derivatives thereof, to determine the presence of hypoxia in a tumor tissue or evaluate a hypoxia- related tumor condition in an animal or human is encompassed by the present invention. Other aspects of the invention concern treating a tissue which is a tumor by first determining the presence of hypoxia m the tumor and, second, treating the tumor with an established form of therapy for cancers such as radiation therapy, chemotherapy, and surgery. In other aspects, the invention provides for methods of attenuating the hypoxic response of a tissue by blocking expression of a hypoxia-inducible gene HIGl , HIG2 , annexm V, lipocortm 2, heterogeneous nuclear πbonucleoprotem Al (hnRNP Al ) , Ku autoantigen , phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal LI, fibroblast growth fa ctor-3 (FGF-3) , EPH receptor ligand, plasmmogen activa tor mhιbιtor-1 (PAI-1 ) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor , fibronectin 1 , lysl hydroxylase , lysyl hydroxylase-2 , endothelm-1 , endothelm-2 , B-cell transloca tion gene-1 (BTG-1) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kιnase-1 (clk-1) , quiescm , growth arrest DNA damage- inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1 ) , low densi ty lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipopmlm, cyclooxygenase-1 (COX-1) , fructose bisphospha tase , crea tme transporter, fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2-mtera ctmg killer (BIK) , 19 kDa -interacting protein 3, Nip3L/Nix, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin , insulin-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor, trisephospha te isomerase, lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113, c- fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta , brain HHCPA18, mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1 , MHC-cIllDQB , myocyte- specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, angiogenin , or SDK3 in the cell or by neutralizing the polypeptide expression products of these genes in the tissue. The invention also provides for methods of treating hypoxia-related conditions by attenuating the hypoxic response of a tissue in an animal such as a human.

Methods for enhancing the response of tissue to hypoxia are provided in other embodiments of the present invention. These methods involve administering expression vectors comprising the hypoxia-inducible genes of the present invention or administering polypeptide expression products of hypoxia-inducible genes to the tissue .

Methods for identifying stress-inducibie and stress repressible genes are also provided.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the human HIGl cDNA and protein sequences. The nucleotide sequence for the human HIGl gene is shown in Figure 1A from 5' to 3' (SEQ ID NO : 1 ) . The coding sequence is underlined. The other regions are untranslated regions (5' and 3' UTR) of the gene. The protein sequence of human HIGl is shown m Figure IB (SEQ ID NO:2) .

Figure 2 shows the human HIG2 cDNA and protein sequences. The nucleotide sequence for the human HIG2 gene is shown m Figure 2A from 5' to 3' (SEQ ID NO: 3) . The coding sequence is underlined. The other regions are untranslated regions (5' and 3' UTR) of the gene. The protein sequence of human HIG2 is shown in Figure 2B (SEQ ID NO:4) .

Figure 3 shows the murine HIGl cDNA and protein sequences. The nucleotide sequence for the murine HIGl gene is shown m Figure 3A from 5' to 3' (SEQ ID NO: 5) . The coding sequence is underlined. The other regions are untranslated regions (5' and 3' UTR) of the gene. The protein sequence of murine HIGl is shown in Figure 3B (SEQ ID NO: 6).

Figure 4 shows the HIGl cDNA and protein sequences of seπola quinqueradia ta . The nucleotide sequence for this fish HIGl is shown in Figure 4A from 5' to 3' (SEQ ID NO:7). The coding sequence is underlined. The other regions are untranslated regions (5' and 3' UTR) of the gene. The protein sequence of fisn HIGl is shown m Figure 4B (SEQ ID NO : 8 ) .

Figure 5 shows the murine HIG2 cDNA and protein sequences. The nucleotide sequence for the murine HIG2 gene is shown in Figure 5A from 5' to 3' (SEQ ID NO: 9). The coding sequence is underlined. The other regions are untranslated regions of the gene (5' and 3' UTR) . The protein sequence of murine HIG2 is shown m Figure 5B (SEQ ID NO: 10).

Figure 6 shows the alignment of human HIGl and HIG2 protein sequences with the HIGl and HIG2 sequences of other species. The HIGl homologues from humans (hHIGl), mice (mHIGl), and fish

( seπola quinqueradia te) (fHIGl or GHL1) are aligned m Figure 6A; the HIG2 homologues from humans (hHIG2) and mice (mHIG2) are aligned m figure 6B . Figure 7 schematically illustrates the addition of linkers to cDNA library fragments. The linker addition is followed by PCR amplification .

Figure 8 illustrates how the subtraction protocol is used to enrich the tester cDNA library with sequences unique to the tester cDNAs .

DETAILED DESCRIPTION OF THE INVENTION

a) Definitions and General Parameters

The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

By the term "hypoxia" (or "hypoxic") is meant, for the purposes of the specification and claims, an environment of reduced oxygen tension such that the oxygen content is less than or equal to about 5%. In most cases, hypoxic tissue will have an oxygen content that is less than or equal to about 2%.

"Normoxic" or "oxic" conditions are conditions comprising a normal level of oxygen for that particular environment. Normoxic or oxic tissue typically has an oxygen content above about 5% . The terms "hypoxia-induced" or "hypoxia-mducible" when referring to a gene means that the gene is expressed at a higher level when the host cell is exposed to hypoxic conditions than when exposed to normoxic conditions. Typically, the number of mRNA transcripts of a hypoxia-induced gene would is at least about 20% higher n a hypoxic cell versus a normoxic cell. Preferably, expression of the hypoxia-induced gene is at least about 2-fold higher m hypoxic versus normoxic cells. Most preferably, expression of the hypoxia-mducible gene is at least about 5-fold higher in hypoxic cells versus normoxic cells.

A "hypoxia-related condition" in an animal is a condition where hypoxia or altered (typically, enhanced) levels of expression of hypoxia-mducible genes in a tissue of the animal is involved. The hypoxia or altered expression of hypoxia- mducible genes may either be a symptom or play a role in the cause, development, progression, amelioration, or cure of the condition. A hypoxia-related condition may optionally be a disease or pathological condition. Hypoxia-related conditions include, but are not limited to, cancer, ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, stroke, and wound healing.

The term "hypoxia-induced protein" or " hypoxia-mduced gene product" means a protein encoded by a gene whose expression is induced by hypoxia. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, naturally-occurring polynucleotides or polypeptides present a living animal are not isolated, but the same polynucleotides or polypeptides could be part of a vector or composition, and be isolated in that such vector or composition is not part of its natural environment.

A "sample obtained from a patient" or a "sample obtained from an animal" may be a sample of tissue or a sample of body fluid. The term "tissue" is used herein to refer to any biological matter made up of one cell, multiple cells, an agglomeration of cells, or an entire organ. The term tissue, as used herein, encompasses a cell or cells which can be either normal or abnormal (i.e. a tumor) . A "body fluid" may be any liquid substance extracted, excreted, or secreted from an organism or a tissue of an organism. The body fluid need not necessarily contain cells. Body fluids of relevance to the present invention include, but are not limited to, whole blood, serum, plasma, urine, cerebral spinal fluid, tears, and amniotic fluid. The term "biochemically equivalent variations" means protein or nucleic acid sequences which differ in some respect from the specific sequences disclosed herein, but nonetheless exhibit the same, or substantially the same, functionality. In the case of cDNA, for example, this means that modified sequences which contain other nucleic aciαs than those specifically disclosed are encompassed, provided that the alternate cDNA encodes mRNA which in turn encodes a protein of this invention. Such modifications may involve tne substitution of only a few bases, or many. The modifications may involve substitution of degenerate coding sequences or replacement of one coding sequence with another; introduction of non-natural nucleic acids is contemplated. It is not necessary for the alternate DNA to hybridize with that disclosed herein provided that the functional criterion is met. Preferably, the modified nucleic acid sequence hybridizes to and is at least 95% complementary to the sequence of interest.

Similarly, in the case of the proteins of this invention, alterations in the amino acid sequence which do not affect functionality may be made. Such variations may involve replacement of one amino acid with another, use of side chain modified or non-natural amino acids, and truncation. The skilled artisan will recognize which sites are most amenable to alteration without affecting the basic function.

A "polynucleotide", "oligonucleotide", or "nucleic acid" includes, but is not limited to, mRNA, cDNA, genomic DNA, and synthetic DNA and RNA sequences, comprising the natural nucleoside bases adenine, guanine, cytosine, thymine, and uracil. The term also encompasses sequences having one or more modified nucleosides. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. No limitation as to length or to synthetic origin are suggested by the use of either of these terms herein. The term "polypeptide" means a poly (amino acid) comprising at least two amino acids linked by peptide bonds. A "protein" is a polypeptide which is encoded by a gene.

"Neutralizing" a polypeptide or protein means inhibiting, partially or wholly, the bioactiviry of the polypeptide or protein. This inhibition of activity may mean inhibition of catalytic activity, prevention of binding to a receptor or ligand, blockage or dimer formation, or the like.

The term "sequences which hybridize thereto" means polynucleotide sequences which are capable of forming Watson- Crick hydrogen bonds with another polynucleotide sequence under normal hybridization conditions, such as in buffered (pH. 7.0- 7.5) aqueous, saline solutions (for instance, 1 to 500 mM NaCI) at room temperature. Although normal hybridization conditions will depend on the length of the polynucleotides involved, typically they include the presence of at least one cation such as Na⁺, K Mg , or Ca'⁺, a near neutral pH, and temperatures less than 55°C. Although the sequences which hybridize to a polynucleotide may be about 90%-100% complementary to the polynucleotide, if the sequences are of sufficient length, in solutions with high salt concentrations, and/or under low temperature conditions, polynucleotides with complementarity of 70% or above, or even just 50% or above, may hybridize to the polynucleotide. Sequences which hybridize thereto typically comprise at least 15 nucleotides, and preferably at least about 30 nucleotides, which are complementary to the target polynucleotide .

A "coding sequence" is a polynucleotide or nucleic acid sequence which is transcribed and translated (in the case of DNA) or translated (in the case of mRNA) into a polypeptide m vi tro or m vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' (ammo) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence will usually be located 3' to the coding sequence.

Nucleic acid "control sequences" refer to translational start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, as necessary and sufficient for the transcription and translation of a given coding sequence in a defined host cell. Examples of control sequences suitable for eucaryotic cells are promoters, polyadenylation signals, and enhancers. All of these control sequences need not be present m a recombinant vector so long as those necessary and sufficient for the transcription and translation of the desired gene are present.

"Operably or operatively linked" refers to tne configuration of the coding and control sequences so as to perform the desired function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. A coding sequence is operably linked to or under the control of transcriptional regulatory regions in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA that can be translated into the encoded protein. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

The expression products described herein may consist of proteinaceous material having a defined chemical structure. However, the precise structure depends on a number of factors, particularly chemical modifications common to proteins. For example, since all proteins contain ionizable amino and carboxyl groups, the protein may be obtained in acidic or basic salt form, or in neutral form. The primary amino acid sequence may be derivatized using sugar molecules (glycosylation) or by other chemical derivatizations involving covalent or ionic attachment with, for example, lipids, phosphate, acetyl groups and the like, often occurring through association with saccharides. These modifications may occur in vi tro, or in vivo, the latter being performed by a host cell through posttranslational processing systems. Such modifications may increase or decrease the biological activity of the molecule, and such chemically modified molecules are also intended to come within the scope of the invention .

"Vector" means a polynucleotide comprised of single strand, double strand, or circular DNA or RNA. An "expression vector" is comprised of the following elements operatively linked at appropriate distances for allowing functional gene expression: replication origin, promoter, enhancer, 5' mRNA leader sequence, ribosomal binding site, nucleic acid cassette, termination and polyadenylation sites, and selectable marker sequences. One or more of these elements may be omitted in specific applications. The nucleic acid cassette can include a restriction site for insertion of the nucleic acid sequence to be expressed. In a functional vector the nucleic acid cassette contains the nucleic acid sequence to be expressed including translation initiation and termination sites. An expression vector is constructed so that the particular coding sequence is located m the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences. Modification of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, m some cases it may be necessary to modify the sequence so that ±t may be attached to the control sequences with the appropriate orientation; or to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site which is m reading frame with and under regulatory control of the control sequences.

A "regulatory element" is a segment of DNA to which a transcription factor (s) binds ana alters the activity of a gene's promoter either positively (induction¹ or negatively (repression) ,

A "stress-responsive element" or "stress-responsive regulatory element" is a regulatory element which binds transcription factors activated by the cell in response to environmental stress. Environmental stressors may include one or more of the following: oxygen depletion; radiation; heat shock; pH change; hypothermia; or glucose starvation.

A "delivery vehicle", as used herein, refers to a means of delivering a polypeptide or a polynucleotide to a cell. The delivery vehicle is preferably used to deliver an expression vector to a cell or a cell m an organism. A delivery vehicle may be a virus, such as a retrovirus, an adenovirus, an adeno- associated virus, a herpes simplex virus, or a vaccinia virus. Other possible delivery vehicles are non-viral. For instance, one of the many liposome formulations known to those skilled in the art, such as Lipofectm, may serve as a delivery vehicle. Liposomes are hollow spnerical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. They have internal aqueous space useful for entrapping water soluble compounds such as polynucleotides. Recognition molecules can be attached to their surface for the targeting of the delivery vehicles to specific tissues. As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Antibodies may exist as intact immunoglobulins or as a number of fragments, including those well-characterized fragments produced by digestion with various peptidases . While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that antibody fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibody fragments encompassed by the use of the term "antibodies" include, but are not limited to, Fab, Fab', F(ab')₂, scFv, Fv, dsFv diabody, and Fd fragments. The phrase "specifically binds to a polypeptide" or "specifically immunoreactive with", when referring to an antibody refers to a binding reaction which is determinative of the presence of the polypeptide (or protein) in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein or polypeptide. A variety of immunoassay formats may be used to select anitbodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are rountinely used to select monoclonal antibodies specifically immunoreactive with a protein.

b) Hypoxia-inducible Genes and Expression Products We have discovered a novel human gene, herein referred to as HIGl , whose expression is induced by cellular response to hypoxia (see the specific examples, Examples 1-6 below) . We have isolated a cDNA of the human HIGl gene (SEQ ID NO:l; Fig. 1A) and identified the coding sequence to be nucleotides 62-343 of SEQ ID NO:l. The protein encoded by HIGl comprises the ammo acid sequence shown in Figure IB (SEQ ID NO:2). Polynucleotides with sequences complementary to SEQ ID NO:l, polynucleotides that are fragments of SEQ ID NO : 1 of at least twelve nucleotides length and polynucleotides which hybridize to SEQ ID NO:l are also within the scope of the present invention. The fragments of SEQ ID NO : 1 are preferably at least 15 nucleotides long.

In particular, polynucleotides comprising the nucleotides 62-343 of SEQ ID NO:l, or complements thereto, or at least twelve nucleotide long fragments thereof, or sequences which hybridize thereto are preferred. Fragments of the coding sequence of HIGl are preferably at least fifteen nucleotides in length.

We have also discovered a second, novel human gene, herein referred to as HIG2, whose expression is induced by cellular response to hypoxia. We have isolated a cDNA clone of this gene. The cDNA sequence of the HIG2 gene is shown in Fig. 2A (SEQ ID NO: 3). The coding sequence of HIG2 comprises nucleotides 274-465 of SEQ ID NO: 3. Fragments of the HIG2 sequence, and of the HIG2 coding sequence in particular, of at least twelve, and preferably fifteen, nucleotides in length are provided by the present invention as well. Polynucleotides of sequence which is complementary to SEQ ID NO: 3 (especially to nucleotides 274-465) or polynucleotides which hybridize to polynucleotides of the sequence set forth in SEQ ID NO: 3 (especially to nucleotides 274- 465), are also contemplated.

Polypeptides encoded by the polynucleotides of HIGl (SEQ ID NO:2; Fig. IB) and HIG2 (SEQ ID NO: 4; Fig. 2B) , or biochemically equivalent variations of either protein, are also provided by the present invention. Fragments of these polypeptides which consist of at least eight ammo acids are provided as well. Preferably, the fragments are at least 15 ammo acids in length. All biochemically equivalent variations of the aforementioned polynucleotides and polypeptides are considered to be fully within the scope of this invention. The mouse and fish HIGl polynucleotide and polypeptide sequences (Figs. 3, 4, and 6) can be considered biochemically equivalent variations of the human HIGl . The mouse HIG2 polynucleotide and polypeptide sequences (Figs. 5 and 6) are likewise understood to be biochemically equivalent variations of the human HIGl.

The polynucleotides of this invention may readily be incorporated within expression vectors by one of ordinary skill in the art. In a preferred embodiment, the polynucleotide sequence comprising nucleotides 62-343 of SEQ ID NO:l (the coding sequence of HIGl) or nucleotides 274-465 of SEQ ID NO: 2 (the coding sequence of HIG2) is operably linked with appropriate control sequences, such as a promoter.

Alternatively, larger fragments of the polynucleotides of SEQ ID NO:l or SEQ ID NO: 2 which comprise portions of the untranslated regions of the genes may be used in an expression vector instead. This may be particularly useful when hypoxia- mduciblity is desired, since the untranslated regions may contain critical regulatory regions such as hypoxia-responsive elements.

The polynucleotides of this invention may also be incorporated within a host cell. In one embodiment, transfection may be used to introduce an expression vector containing one of the polynucleotides of the invention into the cell. The polynucleotide of the transfected vector may also be operably linked with control sequences including regulatory elements to effect the expression within the cell of exogenous protein or polypeptide sequences encoded by the polynucleotides of the present invention. Methods of cloning, amplification, expression, and purification will be apparent to the skilled artisan. Representative methods are disclosed in Molecular Cloning: a Labora tory Manual , 2nd Ed . , Vol . 1 -3, eds. Sambrook et al . , Cold Spring Harbor Laboratory (1989).

A HIGl or HIG2 polynucleotide may be introduced into an animal either by first incorporating the vector into a cell and then transferring the cell to the animal (ex vivo) or by incorporating the vector into a cell within an animal directly The introduction of a HIGl or HIG2 polynucleotide into a cell may be achieved by directly injecting the nucleic acid into the cell or by first mixing the nucleic acid with polylys e or cationic lipids which will help facilitate passage across the cell membrane. However, introduction of the polynucleotide into the cell is preferably achieved through the use of a delivery vehicle such as a liposome or a virus. Viruses which may be used to introduce a HIGl or HIG2 polynucleotide or expression vector into a cell include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and vaccinia viruses .

Antisense oligonucleotides complementary to HIGl and HIG2, particularly those which are capable of blocking expression of HIGl or HIG2 are provided by the present invention. The antisense oligonucleotide is preferably an oligonucleotide having a sequence complementary to at least a portion (preferably at least about 12 nucleotides in length) of SEQ ID NO:l or SEQ ID NO: 3. The antisense oligonucleotide is preferably between about 15 and about 22 nucleotides in length. Modifications of the sequence or bases of the antisense oligonucleotide may be desirable to facilitate transfer into a cell, stability, or tight binding to the HIGl or HIG2 mRNA.

An oligonucleotide probe is provided by another embodiment of the invention. The probe consists of one of the polynucleotides of this invention, or an at least 12 nucleotide- long fragment thereof. The probe may be used to assay for, and if the probe is properly labeled, quantitate, the hypoxia-induced expression of HIGl or HIG2 in a cell. In a preferred embodiment, the probe is at least about 15 nucleotides in length. In a particularly preferred embodiment, the probe is between 15 and 22 nucleotides in length.

Antibodies specifically immunoreactive with the HIGl or HIG2 polypeptides represent still another embodiment of the mvention. These antibodies may be monoclonal or polyclonal. The antibodies may optionally be recombinant or purely synthetic. The antibody may be an intact antibody or fragment. The preparation of antibodies specific to the HIGl and HIG2 polypeptides would be routine for those skilled m the art. In addition to the identification of the new genes HIGl and HIG2 which were found to be hypoxia-inducible, we have also established for the first time that several previously known genes are hypoxia-inducible in humans (see the specific examples, Examples 2 and 9, below and Tables 6, 7, 8, and 9. These genes annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen , phosphoribosylpyrophospha te synthetase , acetoacetylCoA thiolase, ribosomal LI, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activa tor inhibitor-1 (PAI-1 ) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase , lysyl hydroxylase-2 , endothelin-1 , endothelin-2 , B- cell transloca tion gene-1 (BTG-1 ) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1) , quiescin , growth arrest DNA damage- inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1 ) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophilin , cyclooxygenase-1 (COX-1) , fructose bisphospha tase , crea tine transporter, fa tty acid binding protein , lacta te dehydrogenase

(LDH) , Bcl -2-interacting killer (BIK) , 19 kDa -inter acting protein 3, Nip3L/Nix, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferritin , insulin-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor, trisephospha te isomerase, lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113, c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta , brain HHCPA18, mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin- inducible protein 1 , MHC- cIHDQB, myocyte-specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI , angiogenin , and SDK3. Furthermore, expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , expression previously known to be hypoxia-inducible only in endothelial cells (Graven et al. (1998) Am. J. Physiol., 274(2 Pt 1) : C347-355) , is now shown by our work to be greatly induced in transformed cells. Additionally, a multitude of EST sequences from the databases have now been identified as being hypoxia- inducible (Table 3, Example 8, Table 5, Example 9, and Tables 7 and 8) .

c) Polynucleotide, Polypeptide, and Antibody Arrays.

Another aspect of the invention involves the presentation of multiple (at least two, and preferably more than four) hypoxia-inducible gene sequences, polynucleotide probes complementary to the hypoxia-inducible gene sequences, hypoxia- induced polypeptides, or antibodies (immunoreactive with hypoxia- induced polypeptides) on an array. In particularly preferred arrays, more than about 10 different polynucleotides, polypeptides, or antibodies are presented on the array. In an alternative preferred embodiment, the number of different polynucleotides, proteins, or antibodies on the array is greater than about 25, even greater than about 100, or even greater than about 1000.

One aspect of the invention provides an array of polynucleotides which comprises at least two different hypoxia- inducible genes, or complements thereto, or at least twelve nucleotide-long fragments thereof, or sequences which hybridize thereto. The hypoxia-inducible genes or their fragments may optionally be selected from HIGl, HIG2, any of the hypoxia- inducible genes listed in Table 1 (below), Table 3 (Example 8, below), Table 5 (Example 9, below), and Tables 6, 7, 8, and 9. However, it is understood that all of the hypoxia-inducible gene sequences on the array need not be derived only from those hypoxia-inducible listed herein. The polynucleotides on the array are typically single-stranded.

For instance, in one embodiment of the polynucleotide array, on of the multiple polynucleotides on the array is derived from either the HIGl or HIG2 gene sequences. The polynucleotides of the array may comprise the entire sequence of one strand of the gene, or may comprise at least 12 nucleotide long fragments thereof, or sequences which hybridized thereto. In an alternative embodiment, one of the polynucleotides of the array comprises a polynucleotide corresponding to nucleotides 62-343 of SEQ ID NO:l ( HIGl ) or nucleotides 274-465 of SEQ ID NO: 2 ( HIG2) , or complements to one of the coding sequences, or at least twelve nucleotide-long fragments of one of the coding sequences, or sequences which hybridize to one of the coding sequences. In another embodiment of the polynucleotide array, at least one of the polynucleotide sequences of HIGl , HIG2 , annexm V, lipocortm 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al ) , Ku autoantigen , phosphoribosylpyrophospha te synthetas , acetoacetylCoA thiolase, ribosomal L I, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasmmogen activa tor ιnhιbιtor-1 (PAI-1) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase , lysyl hydroxylase-2 , endothelιn-1 , endothelιn-2 , B-cell transloca tion gene-1 (BTG-1 ) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kmase-1 (clk-1 ) , quiescin , growth arrest DNA damage- inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1 ) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophil n , cyclooxygenase-1 (COX-1) , fructose bisphospha tase , crea tme transporter , fa tty acid binding protein, lacta te dehydrogenase (LDH) , Bcl -2-ιnteractιng killer (BIK) , 19 kDa - interacting protein 3, Nιp3L/Nιx, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferπtm , msulm-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor , placental growth factor, mterleukm-1 (IL-1) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1) , monocarboxyla te transporter 3, DNA binding protein A20 , peroxisome prolifera tion receptor , tπsephospha te isomerase , lg associa ted alpha , mterferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113 , c- fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT- 3) , glycogen branching enzyme, TGF beta , brain HHCPA18, mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin, lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1 , MHC-cIllDQB, myocyte- specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase , Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, angiogenin , and SDK3 is represented on the array in combination with a second, different polynucleotide sequence from a hypoxia-inducible gene. The second polynucleotide sequence may be selected from HIGl , HIG2, any of the hypoxia-inducible genes represented in Table 1 shown below, or Tables 6, 7, 8, or 9, any of the expressed sequence tags of hypoxia-inducible genes shown in Table 3 (see Example 8) or Tables 7 and 8, or any other hypoxia-inducible gene or expressed sequence tag from a hypoxia-inducible gene. Furthermore, the second polynucleotide sequence selected from any of the represented hypoxia-inducible genes can be derived from normal cells or tumor cells, exposed to hypoxic conditions. Table 7 ranks hypoxia-inducible genes by normal cell induction. Table 8 ranks hypoxia-inducible genes by tumor cell induction. Table 9 illustrates a hypoxic induction comparison of normal keratinocytes whereby genes are listed by increasing levels of hypoxic gene induction in normal dermal keratinocytes (NDK) and normal cervical keratinocytes (NCK) . These are the normal cell counterparts of the cervical cancer cell lines (Siha and C33a) , and the head and neck cancer cell lines (Fadu) .

It is understood that regardless of which genes are represented on the array, the gene sequences do not have to be represented in their entirety. The polynucleotide sequences that are immobilized on the array are most preferably, single-stranded and complementary to the mRNA transcripts of the relevant hypoxia-inducible genes. The immobilized polynucleotides may be fragments or complementary sequences of the gene or EST sequence that contain at least twelve nucleotides and preferably at least fifteen nucleotides. Alternatively, longer gene fragments including EST fragments of at least 50 or at least 100 nucleotides may be used. In a preferred embodiment of the array, the array is made up of many different gene sequences.

In another embodiment of the polynucleotide array, only polynucleotides correlating to hypoxia-mducible genes expressing gene products of a similar function are included on the array. At least two, but preferentially more than two, different hypoxia-mduced genes encoding proteins from a single functional category are represented on the array. Examples of eight functional categories of hypoxia-mducible proteins are as follows: (1) glycolytic enzymes/protems; (2) angiogenesis/tissue remodeling proteins; (3) erythropoiesis/vascular regulatory proteins; (4) metabolic/homeostatic proteins; (5) apoptosis proteins; (6) DNA repair proteins; (7) cell-cycle proteins; and (8) transcriptional regulatory proteins. These categories are shown in Table 1, below, along with some representative members of each of the categories. It is understood that the members of each of the eight functional categories of hypoxia-mducible proteins are not limited to the lists shown in Table 1. It is further understood, that the list of functional categories of hypoxia-mducible genes is not limited to the eight categories listed in Table 1. Again, a preferred embodiment of this array comprises polynucleotide sequences complementary to the mRNA transcripts of the relevant hypoxia inducible genes. A particularly preferred embodiment of an array displays multiple polynucleotide sequences, each of which is complementary to a different gene which encodes a protein involved in angiogenesis and/or tissue remodeling.

Table 1. Eight Functional Categories of Hypoxia-inducible Genes

GLYCOLYTIC ENZYMES/PROTEINS ractate αehyαrogenase (LDH) phosphoglycerate kinase (PGK) aldolase A

L-phosphofructokmase (PFKL) glucose transporter isoform 3 (Glut-3) mterleukιn-2 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adenylate kinase isoenzyme 3 (AK-3) ANGIOGENESIS/TISSUE REMODELING PROTEINS vascular endothelial growth factor (VEGF) placental growth factor platelet-derived growth factor β (PDGFβ) transforming growth factor β (TGFβ) tumor necrosis factor α (TNFα) ιnterleukιn-6 (IL-6) ιnterleukιn-2 (IL-2) tissue factor fibroblast growth factor (FGF-3)

EPH receptor ligand plasm nogen activator nhιbιtor-1 (PAI-1) macrophage migration inhibitory factor (MIF) fibronectin receptor lysyl hydroxylase-2 endothelιn-2 integrin alpha 5 mucin 1

ERYTHROPOIEISIS/VASCULAR REGULATORY PROTEINS eryt ropoietin (EPO) tyrosine hydroxylase heme oxygenase alpha-fetoprotem (AFP) endothelin

METABOLIC/HOMEOSTATIC PROTEINS insulin-like growth factor binding proteιn-1 (IGFBP-1) metalloth onein creatme kinase inducible nitric oxide synthase (ι-NOS-1) epidermal growth factor receptor (EGFR) huntingtin-associated protein 1 (HAP-1) glucose-regulated protein 78 (GRP78) glucose-regulated protein 90 (GRP90) thioredoxm annexin V glyceraldehyde-3-phosphate dehydrogenase (GAPDH) heterogeneous nuclear ribonucleoprotein Al (hnRNP Al ) gamma-glutamyl cysteme synthetase heavy subunit phosphoribosylpyrophosphate synthetase (PRPP synthetase) acetoacetylCoA thiolase fructose bisphosphatase creatme transporter fatty acid binding protein glucose transporter isoform 3 (Glut-3) adenylate kinase lsoenzyme 3 (AK-3) lactate dehyrogenase (LDH) phosphofructokinase (PFK) aldolase lactate dehydrogenase (LDH) glycogen branching enzyme phsophoglycerate kinase 1 (PGK-1)

APOPTOSIS PROTEINS sulm-l ke growth factor binding protem-3 (IGFBP-3j c-myc c-]un

Bcl-2-ιnteractιng killer (BIK)

19 kDa-mteractmg protein 3 long/Nιp3-lιke protein X

(NιpP3L/Nιx)

APO-1

DNA binding protein A20

DNA-REPAIR PROTEINS

Ku (70)

CELL-CYCLE PROTEINS

B-cell translocation gene-1 (BTG-1) reducing agent and tunicamycm responsive protein (RTP)

CDC-like kmase-1 (clk-1) quiescm (Q6) growth arrest DNA damage-mducible protein 45 (GADD45)

GENE EXPRESSION AND TRANSCRIPTIONAL REGULATORY PROTEINS

RNAse L differentiation of embryo chondrocytes (DEC1) c-fos

Mxι-1

In an alternative embodiment of the polynucleotide array, polynucleotides correlating to the gene sequences encoding proteins belonging to at least two different functional categories of hypoxia-mducible genes are displayed on a single array. Although at least two different polynucleotide sequences are required to form the array, m a preferred embodiment many more than two are used. Again, a preferred embodiment of this array comprises polynucleotide sequences complementary to the mRNA transcripts of the relevant hypoxia inducible genes of at least 12 nucleotides in length, and preferably fifteen.

The present invention also provides for polypeptide arrays analogous to the polynucleotide arrays discussed above, except that the polypeptide sequences of the hypoxia-inducible genes, or fragments thereof, are displayed in an array. The polypeptide array comprises the polypeptide expression products of at least two hypoxia-inducible genes, or biochemically equivalent fragments thereof. For instance, the polypeptide array my comprise the protein HIGl or HIG2 and at least one other protein which is a hypoxia induced gene product. Alternatively, the polypeptide array may instead comprise at least one protein selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF) , fibronectin receptor, fibronectin 1, lysl hydroxylase, lysyl hydroxylase-2, endothelin-1 , endothelin-2 , B-cell translocation gene-1 (BTG-1), reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1), quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentiation of embryo chondrocytes (DEC1), low density lipoprotein receptor related protein (LDLR) , hamster hairy gene homologue, adipophilin, cyclooxygenase-1 (COX-1), fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase (LDH) , Bcl-2-interacting killer (BIK), 19 kDa-interacting protein 3, Nip3L/Nix, Pim-1, vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin, insulin-like growth factor binding protein 3 (IGFBP-3), phosphofructokinase (PFK), aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor), LDHM, phosphoglycerate kinase 1 (PGK-1), monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor, trisephosphate isomerase, lg associated alpha, mterferon regulatory factor 6 (IRF6), putative ORF KIAA0113, c- fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta, brain HHCPA78, mucm 1, RNAse L, Mxi 1, glucose-regulated protein 78 (GRP78), quiescm, lysl oxidase, prostaglandm endoperoxide synthetase, msulm- ducible protein 1, MHC-cIllDQB, myocyte- specific factor 2 (MEF2), bacteria permeating protein, hexokmase, Cap43 (nickel inducible), cyclm G2 , carbonic anhydrase IX, TPI, angiogenm, and SDK3, or a biochemically equivalent fragment thereof; and at least one of a second polypeptide which is a second hypoxia-mduced gene product, or a biochemically equivalent fragment thereof.

Another aspect of the invention concerns a polypeptide array comprising at least two different hypoxia-mduced proteins, or biochemically equivalent fragments thereof, wherem each hypoxia- duced protein belongs to a different functional category. Alternatively, the polypeptide array comprises at least two different hypoxia-mduced proteins or biochemically equivalent fragments thereof, wherein said hypoxia-mduced proteins are all proteins belonging to a single functional category. Optionally, the functional category may be selected from the group consisting of glycolytic enzymes/protems, metabolic/homeostatic proteins, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, erythropoiesis/vascular regulatory proteins, and transcriptional regulatory proteins. (See Table 1, above.)

Yet another alternative embodiment of the invention, is an array analogous to a polypeptide array described above, except that antibodies immunoreactive with the hypoxia-mduced polypeptides are immobilized to form the array, rather than the polypeptide sequences themselves. Each array comprises at least two different antibodies, each of which is immunoreactive with a different hypoxia-mduced protein. Each of the two antibodies is specifically immunoreactive with the polypeptide expression products of hypoxia-mducible genes, such as, but not limited to, HIGl or HIG2. For instance, m one embodiment, the antibody array comprises at least one antibody immunoreactive with a protein selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF) , fibronectin receptor, fibronectin 1, lysl hydroxylase, lysyl hydroxylase-2, endothelin-1 , endothelin-2 , B- cell translocation gene-1 (BTG-1) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1) , quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentiation of embryo chondrocytes (DEC1), low density lipoprotein receptor related protein (LDLR) , hamster hairy gene homologue, adipophilin, cyclooxygenase-1 (COX-1), fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase

(LDH) , Bcl-2-interacting killer (BIK) , 19 kDa-interacting protein 3, Nip3L/Nix, Pim-1, vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin, insulin-like growth factor binding protein 3 (IGFBP-3), phosphofructokinase (PFK), aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor), LDHM, phosphoglycerate kinase 1 (PGK-1), monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor, trisephosphate isomerase, lg associated alpha, interferon regulatory factor 6 (IRF6) , putative ORF KIAA0113, c-fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta, brain HHCPA78, mucin 1, RNAse L, Mxi 1, glucose-regulated protein 78 (GRP78), quiescin, lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1, MHC- cIHDQB, myocyte-specific factor 2 (MEF2) , bacteria permeating protein, hexokinase, Cap43 (nickel inducible), cyclin G2 , carbonic anhydrase IX, TPI, angiogenin, and SDK3. The antibody array further comprises at least one of a second antibody, wherein said second antibody specifically binds a second hypoxia- induced gene product or a biochemically equivalent fragment thereof. The antibodies on the array may be monoclonal or polyclonal. They may be intact antibodies or fragments of antibodies that are capable of specifically binding the polypeptides of the present invention. As is the case with the polynucleotide and polypeptide arrays of the invention, the antibody array preferably comprises at least four different antibodies, and preferably more than about 10 different antibodies .

Methods of constructing arrays of biomolecules , especially polynucleotides, have been previously established m the art. For instance, some methods for preparing particularly high density polynucleotide arrays are disclosed in Pirrung et al., Patent No. 5,143,854, Pirrung et al., Patent No. 5,405,783, Fodor et al., Patent No. 5,445,934, Fodor et al., Patent No. 5,510,270, Fodor et al . , Patent No. 5,744,305, and Fodor et al., Patent No. 5,800,992, all of which are herein incorporated by reference. The polypeptides, antibodies, or polynucleotides may be immobilized on the array either covalently or noncovalently . Methods for immobilizing biomolecules are well known to those of ordinary skill in the art. The material to which the polynucleotides or polypeptides are immobilized m the array may vary. Possible substrates for construction of a biomolecule array include, but are not limited to, cellulose, glass, silicon, silicon oxide, silicon nitride, polystyrene, germanium, (poly) tetrafluorethylene, and gallium phosphide.

A gene expression array (gene chip) provides a quantitative method for monitoring and measuring hypoxia-related gene expression and may contain hundreds to thousands of genes and/or ESTs that are screened simultaneously. This allows for more rapid coverage of the genome. Once genes have been identified with the use of the gene expression array, their hypoxia mducibility and hypoxia repressability can be confirmed at the RNA level by Northern blotting or other techniques. There are many commercially available expression arrays such as the Atlas™ gene arrays (CLONETECH) , GDA™ arrays and GEM™ microarrays (Incyte Pharmaceuticals, Inc.), the Affymetπx GeneChιp®System (Affymetrix, Inc.), and others. However, expression arrays can also be produced directly with any number of genes and/or ESTs on any number of materials, such as cellulose, glass, silicon, silicon oxide, silicon nitride, polystyrene, germanium, (poly) tetrafluorethylene, and gallium phosphide. For example, an array comprising from about 100 to about 1000 hypoxia-inducible and/or -repressible genes or more can be used to assess hypoxia- related conditions in animals and humans. The arrays can be carefully engineered to minimize non-specific hybridization with DNA or RNA probes. When hybridization is performed, the background levels are sufficiently low to permit detection of genes present at only few copies per cell. The sensitivity of the array permits the identification of genes that are expressed as low as only once per cell, which makes the array highly suitable to detect rare transcripts. An array comprising from about 100 to about 1000 hypoxia-repressible genes including, but not limited to, thrombospondin 1, stathmin, survivin, beta- tubulin or any of the genes or ESTs from Table 4 can be used to assess hypoxia-repression in animals and humans. Additionally, the small format and high density of the arrays not only permits the detection of rare transcripts but also the screening of many genes in parallel. This makes the use of expression arrays a valuable tool in research, diagnostics, and other pharmaceutical applications .

In a preferred embodiment of the invention, a gene chip may be contacted with polynucleotides either purified or unpurified derived from a sample of body fluid or tissue obtained from the animal or human. The amount and position of polynucleotide from the animal's sample which binds to the sites of the gene chip can then be determined. The gene expression pattern observed on the gene chip may be correlated with an appropriate treatment.

d) Methods of Use In all of the methods of use described below, the animal is preferably a mammal. Most preferably, the mammal is a human.

We have demonstrated that the expression of HIGl or HIG2 and a number of other genes is indicative of a cell's response to hypoxia as shown in the specific examples shown below (Examples 1-9) . Accordingly, detection of abnormal levels of the transcripts of hypoxia-inducible genes such as HIGl or HIG2 , or combinations thereof, in the tissues or body fluids of an animal can be used in both a diagnostic and prognostic manner for hypoxia-related conditions. The abnormal levels may be characterized by either increased levels or decreased levels, depending upon the hypoxia-related condition being analyzed. In other cases, either the complete absence or any presence of a hypoxia-inducible gene transcript may be indicative of an abnormal condition. Similarly, detection of abnormal levels of the hypoxia-induced polypeptides, or combinations thereof, can be used in either a diagnostic or prognostic manner for hypoxia- related conditions. The presence of hypoxia in a tissue can be evaluated by testing for the presence or absence of the transcripts or polypeptides encoded by the polynucleotides of the invention in either the tissue or in the body fluids of the animal. Detection of the transcripts or polypeptides can be either qualitative or quantitative.

One aspect of the invention, therefore, provides a method of determining the presence of hypoxia in a tissue in an animal or evaluating a hypoxia-related condition in a tissue in an animal. These methods comprise assaying for either the messenger RNA (mRNA) transcripts or the polypeptide expression product of at least one gene selected from the group consisting of HIGl , HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al ) , Ku autoantigen, phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal LI, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activa tor inhibitor-1 (PAI-1) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2 , endothelin- 1 , endothelin-2 , B-cell transloca tion gene-1 (BTG-1) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1 ) , quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1 ) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophilin , cyclooxygenase-1 (COX-1 ) , fructose bisphospha tase, crea tine transporter, fa tty acid binding protein , lactate dehydrogenase (LDH) , Bcl -2-interacting killer (BIK) , 19 kDa - interacting protein 3, Nip3L/Nix, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferri tin , sulm-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1 ) receptor , APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1) , monocarboxyla te transporter 3, DNA binding protein A20 , peroxisome prolifera tion receptor, trisephospha te isomerase , lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113 , c-fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT- 3) , glycogen branching enzyme, TGF beta , brain HHCPA18, mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin- inducible protein 1 , MHC-cIllDQB, myocyte-specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, angiogenin , and SDK3 in a body fluid or the tissue of the animal. This method determining the presence of hypoxia in a tissue may be used to diagnose a hypoxia-related condition in a animal.

The presence of hypoxia in a tissue or the degree of expression of hypoxia-inducible genes determined by these methods may be used to select an appropriate treatment for the animal. For instance, the hypoxia-related condition being evaluated may be cancer and the tissue which is the target of the evaluation may optionally be a tumor. The degree to which the tumor is showing gene expression patterns characteristic of hypoxia or the activation of genes involved in angiogenesis, for instance, can be usefully correlated with appropriate treatment of tumors of that particular type. The hypoxia-related condition, however, need not necessarily be cancer. The hypoxia-related condition may instead be any condition in which hypoxic conditions play a role (favorable or detrimental to the animal). Such conditions include, but are not limited to, ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, stroke and wound healing.

The transcripts of hypoxia-inducible genes may be detected by any of several means known to those skilled in the art. One embodiment of diagnostic detection involves annealing to the transcript, in vivo or in vitro, a labeled nucleic acid probe complementary to the transcript sequence. The labeled probe can be fluorescent, radioactive, immunoreactive, colormetric or otherwise marked for detection. To detect very minute quantities of a transcript, amplification of the transcript in a tissue or fluid sample from the animal may first be performed to aid subsequent detection of the transcript. Amplification of the hypoxically-induced transcripts can be readily achieved using the polynucleotides of the present invention as primers, using reverse transcriptase to make a cDNA copy of the transcript, and then using polymerase chain reaction to achieve exponential amplification.

Detection of expression of the polypeptide products of the HIGl or HIG2 genes, or any of the other hypoxia-induced genes could be achieved, for instance, by the application of labeled antibodies specifically immunoreactive with the polypeptide products. The antibodies can be applied to the tissue in vivo, or to tissue or body fluid samples removed from the animal. Various forms of typical immunoassays known to those skilled in the art would be applicable here. These assays include both competitive and non-competitive assays. For instance, in one type of assay sometimes referred to as a "sandwich assay", immobilized antibodies that specifically react with HIG2 polypeptide are contacted with the biological tissue or fluid sample. Presence of the immobilized HIG2-antibody complex could then be achieved by application of a second, labeled antibody immunoreactive with either the HIG2 polypeptide or the HIG2- antibody complex. A Western blot type of assay could also be used in an alternative embodiment of the present invention.

If a removed tissue is to be analyzed in vi tro, typically, degradation of the tissue is preferred prior to testing for the presence of either an mRNA transcript or a gene product. For instance, if detection of polynucleotides is desired, proteolytic degradation is useful (Temsamani et al . , Patent No. 5,693,466) . Extraction or isolation of proteins or nucleic acids in the sample is also preferred prior to carrying out a diagnostic screen. Numerous methods for the isolation of proteins or nucleic acids from cells or biological fluids are well established in the art.

In a preferred embodiment, a diagnostic evaluation of hypoxia-induced gene expression involves assaying the expression levels of more than one hypoxia-inducible inducible genes at a time. The arrays of the invention are particularly useful for assaying the expression of multiple hypoxia-inducible genes in parallel. The diagnostic detection methods mentioned above in regard to in vi tro detection would also apply as methods for detecting the presence of polynucleotides and polypeptides in a tissue or a body fluid upon administration of a sample of the tissue or fluid to one of the arrays of the present invention.

Use of the polynucleotide or antibody arrays of the present invention for determining the presence of hypoxia in a tissue of an animal or for evaluating a hypoxia-related condition in a tissue of an animal allows for an unprecedented look at the exact nature and stage of the hypoxic response of a tissue, since the hypoxia-induced expression of a combination of genes is screened at one time. Patterns of expression of hypoxia-inducible (or hypoxia-repressible) genes are complex and highly indicative of hypoxia in a tissue, as demonstrated in the specific examples shown below, Examples 8 and 9. The pattern of expression of hypoxia-inducible genes can therefore be used in a diagnostic or prognostic manner to aid in the treatment of a hypoxia-related condition in an animal. Information on the pattern of expression of a combination of hypoxia- duced genes can readily be correlated with the aggressiveness of a tumor for instance, thereby providing knowledge critical for establishing the best line of treatment. The polypeptide arrays of the present invention also can be used to screen for drugs useful m the treatment of hypoxia- related conditions. These drugs may be drugs which are capable of inhibiting the hypoxic response of a tissue.

For instance, methods of assaying for expression of hypoxia- ducible genes m a tissue in an animal, determining the presence of hypoxia in a tissue in an animal, or evaluating a hypoxia-related condition in a tissue in an animal comprise first contacting the proteins or messenger RNA of a sample of body fluid or tissue obtained from the animal with an antibody array or polynucleotide array, respectively, of the invention. Tissue or fluid samples from an animal may be contacted directly with an array, and binding of the proteins or mRNA transcripts on the array detected. (The cells in a tissue to be assayed would preferably be lysed prior to application to the array.) Alternatively, the tissue or fluid sample may be purified to isolate the proteins or mRNA transcripts prior to application to the array. In an alternative embodiment of the method, cDNA is first prepared from the messenger RNA of the sample by reverse transcription and then the cDNA is applied to a polynucleotide array. Once the protein, mRNA or cDNA is delivered to the array, the method comprises detecting the amount and position of the protein, mRNA or cDNA which remains bound to the array after removal of excess or non-bound protein, mRNA, or cDNA.

Additionally, a method of diagnosing a hypoxia-related condition m an animal may optionally comprise the additional step of correlating the result of the evaluation of the hypoxia- related condition in the tissue in the animal with an appropriate treatment for the animal. The hypoxia-related condition which may be evaluated, diagnosed or treated by any of the above methods may a condition such as cancer, ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, or stroke . Another aspect of the invention provides for a method of treating a tumor. This method involves first determining the presence of hypoxia m a tumor by any of the methods described above (with or without arrays) . The method further comprises treating said tumor with any combination of an established form of therapy for cancer such as radiation therapy, chemotherapy, or surgery .

The HIGl or HIG2 polynucleotides or the polynucleotides corresponding to the gene sequences of other hypoxia-mducible gene sequences, such as those listed in Table 1, may be used to attenuate the response of a tissue to hypoxia. These hypoxia- mducible sequences can be targeted within a tissue by the introduction of antisense oligonucleotides, triple-helix probes, catalytic nucleic acids or the like in a manner which inhibits expression of the HIG genes or other hypoxia-mducible genes within the tissue.

Therefore, m one embodiment, the method of attenuating the hypoxic response of tissue comprises inhibiting the expression of a gene selected from the group consisting of HIGl , HIG2 , annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al ) , Ku autoantigen , phosphoribosylpyrophospha te synthetase , acetoacetylCoA thiolase, ribosomal L I, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasmmogen activator inhibitor -1 (PAI-1) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2 , endothelιn-1 , endothelιn-2 , B- cell transloca tion gene-1 (BTG-1 ) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kιnase-1 (clk-1) , quiescm, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage- inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1 ) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophilin , cyclooxygenase-1 (COX-1) , fructose bisphospha tase, crea tine transporter , fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2- interacting killer (BIK) , 19 kDa - interacting protein 3, Nιp3L/Nιx, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferπtin , insulin- l ke growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor , placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1 ) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor , trisephospha te isomerase, lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113, c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta , brain HHCPA18 , mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1 , MHC- cIHDQB, myocyte-specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase , Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, and SDK3 in said cell. This inhibition of expression of a hypoxia-inducible gene may optionally be achieved by introducing into the cells of said tissue a nucleic acid molecule such as an antisense oligonucleotide, a triple-helix probe, a deoxyribozyme, or a ribozyme which is specific to the hypoxia-inducible gene.

In an alternative embodiment of the invention, the HIGl or HIG2 proteins or other expression products of hypoxia-inducible genes may instead be targeted to attenuate the hypoxic response of a tissue. For this purpose, antibodies, antagonists, inhibitors, or proteases that are specific to the expression products of hypoxia-induced genes may be introduced to the tissue .

In one embodiment, a method of attenuating the hypoxic response of a tissue comprises neutralizing a protein selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF) , fibronectin receptor, fibronectin 1, lysl hydroxylase, lysyl hydroxylase-2, endothelin-1 , endothelin-2 , B-cell translocation gene-1 (BTG-1) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1), quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentiation of embryo chondrocytes (DEC1), low density lipoprotein receptor related protein (LDLR) , hamster hairy gene homologue, adipophilin, cyclooxygenase-1 (COX-1), fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase (LDH) , Bcl-2-interacting killer (BIK), 19 kDa-interacting protein 3, Nip3L/Nix, Pim-1, vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin, insulin-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor), LDHM, phosphoglycerate kinase 1 (PGK-1), monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor, trisephosphate isomerase, lg associated alpha, interferon regulatory factor 6 (IRF6) , putative ORF KIAA0113, c- fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3), glycogen branching enzyme, TGF beta, brain HHCPA78, mucin 1, RNAse L, Mxi 1, glucose-regulated protein 78 (GRP78), quiescin, lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1, MHC-cIllDQB, myocyte- specific factor 2 (MEF2) , bacteria permeating protein, hexokinase, Cap43 (nickel inducible), cyclin G2 , carbonic anhydrase IX, TPI, angiogenin, and SDK3. In this method an agent specifically targeting the protein is optionally introduced into the cells of the tissue and can be an antibody, an antagonist, an inhibitor, or a protease. The methods described above for attenuating the hypoxic response of a tissue may be used to treat a hypoxia-related condition in an animal. For instance, the treatment of a hypoxia-related condition in an animal may be effected by targeting the hypoxia-induced gene sequences of the hypoxic (or potentially hypoxic) tissue via one or more of the techniques known to those skilled in the art. These techniques include, but are not limited, to introduction of antisense oligonucleotides, triple-helix probes, deoxyribozymes, or ribozymes into the subject's tissue of concern. In a preferred embodiment, the animal to be treated is a human. The hypoxia-related condition towards which this treatment may be directed is ischemia, stroke, heart attack, neonatal distress, retinopathy, or any other disease condition in which hypoxia plays a significant role. In another embodiment, the hypoxia-related condition to be treated is cancer and the tissue is a tumor. The disclosed treatment of the tumor may be coupled with any combination of other cancer therapies such as radiation therapy, chemotherapy, or surgery. Similarly, treatment of the hypoxia-related conditions may also be achieved by neutralizing the protein expression products of hypoxia-inducible genes, as described above. In accordance with this method, antibodies, antagonists, inhibitors, proteases, or the like which target and neutralize HIGl and HIG2 polypeptides may be introduced into the animal, preferably human, containing the tissue to be treated.

The protein expression products of the genes which have been newly identified as being hypoxia-inducible may be used to identify or screen for drugs, such as inhibitors, useful in the treatment of hypoxia-related conditions. For instance, small molecule drug candidates or peptides may be tested against the any of the proteins of HIGl, HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al), Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activator inhibitor-1 (PAI-1), macrophage migration inhibitory factor (MIF) , fibronectin receptor, fibronectin 1, lysl hydroxylase, lysyl hydroxylase-2, endothelin-1 , endothelin-2, B-cell translocation gene-1 (BTG-1), reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1), quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentiation of embryo chondrocytes (DEC1), low density lipoprotein receptor related protein (LDLR) , hamster hairy gene homologue, adipophilin, cyclooxygenase-1 (COX-1) , fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase (LDH) , Bcl-2-interacting killer (BIK) , 19 kDa-interacting protein 3, Nip3L/Nix, Pim-1, vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin, insulin-like growth factor binding protein 3 (IGFBP-3), phosphofructokinase (PFK), aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor), LDHM, phosphoglycerate kinase 1 (PGK-1), monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor, trisephosphate isomerase, lg associated alpha, interferon regulatory factor 6 (IRF6), putative ORF KIAA0113, c- fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta, brain HHCPA78, mucin 1, RNAse L, Mxi 1, glucose-regulated protein 78 (GRP78), quiescin, lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1, MHC-cIllDQB, myocyte- specific factor 2 (MEF2), bacteria permeating protein, hexokinase, Cap43 (nickel inducible), cyclin G2 , carbonic anhydrase IX, TPI, angiogenin, or SDK3 to see if inactivation of the enzymatic activity or prevention of crucial binding activity of the hypoxia-induced protein occurs. Combinatorial libraries of small molecules or libraries of peptides such as those produced by phage display may alternatively be screen against one of the hypoxia-induced proteins described herein.

The expression of some gene products induced by hypoxia can be helpful in protecting cells from damage or death. Thus, this invention also provides for methods of enhancing the hypoxic response of a tissue and thereby and treating hypoxic tissue (or potentially hypoxic tissue) . The method comprises introducing an expression vector into the tissue and allowing for expression of the coding sequence on the vector to take place. The coding sequence of the expression vector comprises the sequence of at least one of the genes HIGl , HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen , phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal LI, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activa tor inhibitor-1 (PAI-1 ) , macrophage migra tion inhibi tory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase , lysyl hydroxylase-2 , endothelin-1 , endothelin-2 , B-cell transloca tion gene-1 (BTG-1 ) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1 ) , quiescin , growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1) , low densi ty lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophilin , cyclooxygenase-1 (COX-1 ) , fructose bisphospha tase, crea tine transporter, fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2- interacting killer (BIK) , 19 kDa - interacting protein 3, Nip3L/Nix, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferritin , insulin-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5 , integrin alpha 5 receptor , placental growth fa ctor, interleukin-1 (IL-1 ) receptor, APO-1 (Fas receptor) , LDHM, phosphogly cera te kinase 1 (PGK-1 ) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor, trisephospha te isomerase , lg associa ted alpha , interferon regula tory fa ctor 6 (IRF6) , puta tive ORF KIAA0113 , c- fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta , brain HHCPA18, mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1 , MHC-cIl lDQB, myocyte- specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, angiogenin, or SDK3. Expression of the vector' s hypoxia-inducible gene within the tissue should occur at a level which is higher than would occur in the absence of the expression vector. Depending on the particular use, the coding sequence of the expression vector may be operably linked to its native promoter, another hypoxia-inducible promoter, or a constitutive promoter.

Alternatively, the proteins of the hypoxia-inducible genes may be introduced into the tissue directly to enhance the hypoxic response of the tissue and for treatment of hypoxia. Delivery of the proteins may be achieved through the use of liposomes, hydrogels, controlled-release polymers, or any of the other vehicles known m the art to be useful for the delivery of polypeptides as drugs .

e) Methods for Identifying Stress-Inducible Genes To facilitate efforts to identify hypoxia-mducible genes, we modified and improved a PCR subtraction method known as Representational Difference Analysis (RDA) (see specific example, Example 1, below, and Figures 7 and 8) . The RDA method has been used to distinguish differences between genomic DNA from two related, but different sources (Wigler et al., Patent No.

5,436,142) . The RDA technique involves selectively amplifying via polymerase chain reaction only fragments of those sequences contained withm one DNA sample, but not the other. The selectivity of the amplification step used in this method is not precise, but is sufficient to detect differences in the genomes of two human individuals.

The present invention provides for methods of identifying both stress-mducible and stress-repressible genes. The methods identify differences between mRNA from cell populations exposed to different stress conditions. A representative protocol for the identification of stress-mducible genes is outlined in detail in a specific example below (Example 1) .

The method for identifying stress-mducible or stress- repressible genes and fragments of genes involves first subjecting one of two populations of cells to stress prior to preparation of two cDNA libraries from the mRNA libraries of the two populations. Protocols for the generation of cDNA libraries through reverse transcription of mRNA sequences are well known in the art and kits for doing so are commercially available (from Gibco BRL, for instance) . In a preferred embodiment of the method, the cDNAs are synthesized by using a mixture of oligo-dT primers containing equal proportions of oligomers having a G, A, or C residue at the 3' -end ("indexed" or "registered" primers) . This approach ensures that a given primer will hybridize at the start of a polyA tail sequence of an mRNA rather than randomly within the sequence. These oligo-dT primers also have a defined DNA sequence (20 to 24 base pairs m length) that is incorporated into each cDNA fragment. This tag permits the use of two PCR primers to specifically amplify the 3' -end of each cDNA. The two cDNA libraries are digested separately with restriction enzymes and then linker sequences are ligated to the ends of the digested cDNA fragments, as shown in Fig. 7. Restriction digests and ligation of linkers may be performed in any manner known to those skilled in the art. Some examples of such methods may be found in Sambrook et al. (1989) Molecular Cloning: A Labora tory Manual , 2nd. ed, Cold Spring Harbor Laboratory Press, herein incorporated by reference. The cDNA library from one of the two cell populations is amplified with tagged oligonucleotide primers by means of the polymerase chain reaction (PCR) . In a preferred embodiment, the "tag" on the oligonucleotide primers is biotin. However, any chemical or biological moiety which provides a means of selection or isolation of the tagged entity (by affinity chromatography, for instance) is suitable as a tag. In the preferred embodiment, use of biotin as a tag allows for removal of the tagged sequences on a streptavidin resin. In an alternative embodiment, however, oligonucleotides bearing a thiol group, for example, may instead be used as the tagged primer, since oligonucleotides with attached thiol groups can be retained on a variety of affinity resins, such as thiopropyl sepharose columns or mercurial resins. The cDNA library PCR-amplified with tagged primers is referred to herein as "driver" cDNA. The cDNA library from the stressed cells is amplified with normal, non-tagged, oligonucleotide primers in a separate polymerase chain reaction. The cDNA PCR-amplified in this manner is referred to herein as "tester" cDNA.

The non-tagged, amplified, tester cDNA is heated and then reannealed in the presence of a large excess (typically about 5- to about 100-fold) of the tagged, amplified, driver cDNA. See Fig. 8. Next, those DNA strands which either are themselves tagged or are duplexed with tagged DNA are removed from the mixture. This removal is typically done via exposure of the mixture of DNA strands to a resin or matrix which has affinity for the tag used on the primers earlier. In a preferred embodiment, magnetic beads coated with streptavidin are used. Other resins, such as streptavidin agarose could be used in conjunction with a biotin tag. Tagged single-stranded or duplex cDNA will be retained on the affinity resin, and the non-tagged species, which are not retained, can be found in the flowthrough or supernatant. In this technique, the cDNA from the non- stressed cell population is "subtracted" from the cDNA of the stressed cell population. The remaining, non-tagged cDNA library is said to be "enriched". The remaining, non-tagged cDNA sequences are then again amplified by means of the polymerase chain reaction with non-tagged primers. After amplification of the remaining non-tagged cDNA sequences, the non-tagged cDNA library is again heated and reannealed in the presence of a large excess (typically about 5- to about 100-fold) of the original tagged cDNA library. Removal of all tagged DNA molecules and reamplification of remaining tagged sequences again follows. The combination of steps involving heating and reannealing, removed tagged molecules, and reamplifying remaining, non-tagged molecules constitutes one round. The methods of the present invention involve repeating the rounds from zero to many times. In a preferred embodiment, the method involves a total of approximately 3 to 5 rounds.

In a particularly preferred embodiment, the method involves performing the steps as described above in parallel with a second set of steps in which the cDNA library from the stressed population of cells is instead subtracted from the cDNA library from the non-stressed population. This means that in the second set of steps, the cDNA library from the stressed cell population is amplified with tagged primers and the cDNA library from the non-stressed cell population is amplified with non-tagged primers. The original cDNA of the stressed cell population is repeatedly subtracted from the cDNA of the non-stressed cell population, and separately, the original cDNA of the non-stressed cell population is repeatedly subtracted from the stressed cell population .

In the final round of the preferred embodiment of the method, one of the two enriched cDNA libraries obtained from the two sets of steps is subtracted from the other enriched cDNA library. Which enriched library is subtracted from which is entirely dependent upon whether stress-inducible or stress- repressible sequences are sought. If stress-inducible sequences are sought, the enriched, non-stressed cDNA library is subtracted from the enriched, stressed, cDNA library. If stress-repressible sequences are sought, the enriched, stressed-cell cDNA library is subtracted from the enriched non-stressed-cell cDNA library.

The final subtraction step of one enriched library against another is beneficial since the initial subtraction rounds of the procedure tend to remove only the cDNAs that are in common and present at high frequency in the two populations, because cDNA fragments derived from rare messages will initially be present at such low concentrations that they might not find a complementary strand during the hybridization step. After the major sequences in common are removed by subtraction, the rare sequences will begin to increase in concentration so that they can then be effectively subtracted. After multiple cycles of subtraction are performed, the rarest sequences from both conditions are enriched in the libraries, and subtraction of one enriched library from another yields an effective isolation of either stress-inducible or stress-repressible genes. After the desired number of rounds of subtraction have been completed, the enriched cDNA library may be cloned and sequenced using any one of the multitude of techniques known to those skilled in the art. A particularly convenient method of inserting PCR-amplified DNA strands into vectors suitable for cloning and sequencing, known as "T-A cloning", is commercially available from companies such as Invitrogen and Novagen. Other alternative methods can be found in Molecular Cloning: A Labora tory Manual , 2nd. ed, Vol . 1 -3, eds. Sambrook et al., Cold Spring Harbor Laboratory Press (1989) . In one embodiment, the stress to which one of the two cell populations is exposed is hypoxia. The method may also be applied to the investigation of responses to other stresses, such as ionizing radiation, heat, glucose starvation, hypothermia, or pH change. Alternatively, the response to a stress such as a toxin or a drug may be investigated by employment of the disclosed method. f) Diagnostic Blood Test Using Hypoxia-inducible Marker Gene Products

A new diagnostic blood test, disclosed herein, allows for the detection of hypoxia-related conditions. Hypoxia-responsive genes produce marker gene products that can be measured in the blood stream of humans and animals. A blood test has been devised to test for these diagnostic marker gene products whereby secreted proteins are the basis for measuring tumor hypoxia (see Example 10 below) . Secreted proteins can be measured in the bloodstream of humans or animals with solid tumors. The oxygen status of each tumor sample is determined through independent measurement techniques including, but not limited to, a nitroimidazole-binding technique (EF5) or the Eppendorf oxygen electrode. These applications can determine if there is a relationship between the oxygen status and blood levels of the hypoxia marker gene. Serum levels of secreted marker proteins are assayed through commercially available ELISA kits that are well known in the art. Serum levels can also be assayed through proteomic techniques, immunohistochemistry, immune blotting, and other techniques that are well known in the art.

One aspect of the present invention provides for a diagnostic blood test for assaying for the expression of hypoxia- inducible genes in a tissue of an animal or human, and for detecting the presence of hypoxia-inducible gene products in a tissue in an animal or human. The detection of expression products, such as diagnostic marker proteins, of the hypoxia- inducible genes of PAI-1 , IGF-BP3, placental growth factor, adipophilin , mucin 1 , endothelin-1 , endothelin-2 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin, EPH receptor ligand, angiogenin, TGF beta , HIGl , HIG2 , annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al ) , Ku autoantigen, phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal LI, fibroblast growth factor-3 (FGF-3) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2, B-cell transloca tion gene-1 (BTG-1) , reducing agent and tunicamycin- responsive protein (RTP) , CDC-like kinase-1 (clk-1) , quiescin , growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, cyclooxygenase-1 (COX-1) , fructose bisphospha tase , crea tine transporter, fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2- interacting killer (BIK) , 19 kDa - interact ing protein 3, Nip3L/Nix, Pim-1 , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, interleukin-1 (IL-1) receptor , APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1 ) , monocarboxyla te transporter 3, DNA binding protein A20 , peroxisome prolifera tion receptor , trisephospha te isomerase, lg associa ted alpha , interferon regula tory fa ctor 6 (IRF6) , puta tive ORF KIAA0113 , c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, brain HHCPA18, RNAse L, Mxi 1 , glucose- regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1 , MHC-cIllDQB, myocyte-specific factor 2 (MEF2) , bacteria permea ting protein, hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI , or SDK3 , or combinations or derivatives thereof, to determine the presence of hypoxia in a tissue or evaluate a hypoxia-related condition in an animal or human is encompassed by the present invention. A further aspect of the present invention provides for a diagnostic blood test for assaying for the expression of hypoxia- inducible genes in a tumor tissue of an animal or human, and for detecting the presence of hypoxia-inducible gene products in a tumor tissue in an animal or human. The detection of expression products, such as diagnostic marker proteins, of the hypoxia- inducible genes of PAI-1 , IGF-BP3, placental growth factor, adipophilin , mucin 1 , endothelin-1 , endothelin-2 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferri tin , EPH receptor ligand, angiogenin , TGF beta , HIGl , HIG2 , annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal L I, fibroblast growth factor-3 (FGF-3) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2 , B-cell transloca tion gene-1 (BTG-1) , reducing agent and tunicamycin- responsive protein (RTP) , CDC-like kinase-1 (clk-1 ) , quiescin , growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1) , low density lipoprotein receptor related protein (LDLR) , hamster hairy gene homologue , cyclooxygenase-1 (COX-1 ) , fructose bisphospha tase, crea tine transporter, fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2- interacting killer (BIK) , 19 kDa -interacting protein 3, Nip3L/Nix, Pim-1 , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor , interleukin-1 (IL-1 ) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor , trisephospha te isomerase, lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113 , c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, brain HHCPA18, RNAse L, Mxi 1 , glucose- regula ted protein 18 (GRP18) , quiescin , lysl oxidase , prostaglandin endoperoxide synthetase, insulin-inducible protein 1 , MHC-cIllDQB, myocyte- specif ic factor 2 (MEF2) , bacteria permea ting protein , hexokinase , Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, or SDK3, or combinations or derivatives thereof, to determine the presence of hypoxia in a tumor tissue or evaluate a hypoxia-related tumor condition in an animal or human is encompassed by the present invention. In a preferred embodiment of the invention, the diagnostic marker proteins used in the blood test are the hypoxia-inducible genes of PAI-1 , IGF-BP3 , placental growth factor, adipophilin , mucin 1 , endothelin-1 , endothelin-2 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin , EPH receptor ligand, angiogenin , or TGF beta . g) Nuclear Medicine Used To Identify Hypoxia-Related Conditions Through Surface Markers

One possible avenue to assess conditions related to tumors with hypoxia in vivo, is the use of nuclear medicine based assays designed to non-invasively identify tumors. Tumor hypoxic regions can be detected through the non-invasive imaging of the cell surface using nuclear medicine approaches such as Magnetic Resonance Imaging (MRI), Nuclear Magnetic Resonance (NMR), or others well known in the art. Imaging reagents that assist in the detection of tumor regions may be adminstered intravenously or orally. Cell surface ligands and receptors such as integrin alpha 5 and the interleukin-1 (IL-1) receptor are good targets for this type of nuclear medicine based imaging of hypoxia. These gene products are good candidates because the are localized to the tumor areas where they are expressed on the cell surface, and they are accessible to systemically administered imaging reagents .

One embodiment of the invention is a nuclear medicine based assay designed to non-invasively identify tumors of hypoxia in vivo by assaying for the expression of hypoxia-inducible genes in a tumor tissue of an animal or human, and by detecting the presence of hypoxia-inducible gene products in a tumor tissue in an animal or human. The detection of expression products, such as diagnostic cell surface ligands and receptors, of the hypoxia- inducible genes of integrin alpha5 receptor , interleukin-1 (IL-1 ) receptor , fibronectin , EPH receptor ligand, APO-1 (Fas Receptor) , mucin-1 , crea tine transporter , monocarboxyla te transporter, or combinations or derivatives thereof, to determine the presence of hypoxia in a tumor tissue or evaluate a hypoxia-related tumor condition in an animal or human is encompassed by the present invention.

h) Examples

The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims. Example 1. Generation of Enrichment cDNA Libraries

Normal human cervical epithelial cells stably immortalized with the human papillomavirus E6 and E7 oncoproteins (HCE.E6.E7) served as the starting material for the construction of a cDNA library enriched by representational difference analysis (RDA) . HCE.E6E7 were cultured in synthetic medium PFMR-4A (Kim et al . (1997) Cancer Res . 57:4200-4).

A total of 5 μg of poly A⁺ mRNA from both HCE.E6E7 cells cultured under hypoxic (5% C0₂/5% H₂/90% N₂ for 16 hours at 37°C) conditions and HCE.E6E7 cells cultured under aerobic (5% C0₂ /

20% 0₂ / 75% N at 37°C) conditions were used to generate double- stranded cDNA preparations by using the Gibco BRL cDNA Synthesis System.

Hypoxic conditions were generated by the use of an anaerobic chamber (Sheldon Laboratories, Cornelius OR) that is flushed with a gas mixture of 90% N₂, 5% C0₂ and 5% H₂. Any oxygen that was introduced into the chamber was consumed over a catalyst with hydrogen. A monitoring oxygen electrode was used to confirm an environment of 0.05% oxygen or less during experimentation.

One-fifth of the cDNA product (approximately 1-1.5 μg) from the hypoxic or oxic cells was digested with 20 units of the Nla III restriction enzyme, 50 mM potassium acetate, 1 mM DTT, and 100 μg/ml bovine serum albumin for 60 min at 37°C. The reaction mixture was extracted with phenol and chloroform, precipitated with ethanol, redissolved in lOuL of water and lyophilized. Ethidium agarose gel electrophoresis was used to verify that the cleavage was successful.

For each pair of cDNAs used for the RDA procedure (i.e. the "test" and the "driver"), two different DNA linkers were attached by ligation to the Nla III cleaved ends. The 3' -end of the linker sequence opposite the ligation site was terminated with an amine so that it cannot be used as an acceptor or donor for a ligase. The linker oligonucleotides used were as follows (where "X" denotes the animo-terminated residue at the 3' -end of the shorter of the two strands) : 5'-TTTTACCAGCTTATTCAATTCGGTCCTCTCGCACAGGATGCATG-3' (SEQ ID NO: 11) XATGGTCGAATAAGTTAAGCCAGGAGAGCGTGTCCTAC-5' (SEQ ID NO: 12)

5'-TTTTTGTAGACATTCTAGTATCTCGTCAAGTCGGAAGGATGCATG-3' (SEQ ID NO: 13) XAACATCTGTAAGATCATAGAGCAGTTCAGCCTTCCTAC-5' (SEQ ID NO: 14)

(The linker pair of SEQ ID NO: 13 and SEQ ID NO: 14 was used for the hypoxically incubated cell cDNAs . ) The two separate linker strands were dissolved in 10 mM Tris-HCl (pH 7.6), 10 mM MgCl₂ buffer (10 μM of each oligomer) , then heat-denatured and slowly cooled to room temperature before use in a ligation reaction.

Next, 1 μg of the Nla III cleaved cDNA was ligated in a 100 μL volume of 1 μM double-stranded linker, 5% polyethylene glycol, 50 mM Tris-HCl (pH 7.6), 10 mM MgCl₂, 1 mM ATP, and 1 mM DTT at 16°C for 3 h. Since the linkers used to ligate to the cDNA fragments do not have a phosphate at the 3' -end of the Nla III overhang, the resulting ligation products have a single-stranded nick. Performing the reaction in this way had the advantage of preventing self-ligation of the linkers. The excess linkers were removed by gel filtration through a spin-column containing Sephacryl S-300HR. The linker-ligated cDNA fragments were collected in the microfuge tube while the excess unligated linkers were trapped in the Sephacryl with other low molecular- weight components. The gel-filtered, linker-ligated cDNA fragments were then lyophilized to dryness.

The linker-ligated cDNA fragments were amplified by a single-primer PCR technique. Again, if the preparation was to be used as the driver cDNA, it was amplified by using PCR primers with a biotin residue at the 5' -end. If the preparation was to be used as the test cDNA from which the driver is used to subtract sequences, then it was amplified by using untagged primers .

The ligated cDNA (0.1 μg aliquot) was amplified in a standard PCR buffer containing 1 μM primer, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl₂, and 0.01% gelatin. Before PCR amplification, the nicked PCR template had to be repaired by TAQ polymerase during a 5-min extension reaction at 72°C. After this initial incubation, a standard PCR reaction of 35 cycles (94°C, 30s; 56°C, 30s; 72°C, 60s) was performed in a Perkin Elmer DNA Thermal Cycler. The oligonucleotide primers used in the amplification step were as follows:

5' -CCAGCTTATTCAATTCGGTCC-3' (SEQ ID NO: 15) 5'-GTAGACATTCTAGTATCTCGT-3' (SEQ ID NO: 16)

(SEQ ID NO: 16 was the primer used to amplify cDNA from the hypoxically incubated cells.) The entire PCR reaction was passed through a 1 ml Sephacryl spin column as described above to remove salts, dNTPs, and excess primers. The yield of the amplification was determined by ethidium agarose gel electrophoresis. The product appeared as expected as a smear of DNA fragments ranging from 100 to 2,000 base pairs (bp) in size.

The first round of subtraction was performed by mixing 3 μg of the biotinylated driver cDNA with 0.1 μg of the test cDNA. The mixture was lyophilized in a 0.5 mL microfuge tube and carefully redissolved in 2 μL of 50 mM HEPES (pH 7.5), 10 mM EDTA, 1.5 mM NaCI, and 2% sodium dodecyl sulfate (SDS). This very small amount of solution was overlaid with 50 μL of mineral oil to prevent evaporation, and the tube was place in the thermal cycler and heated at 95°C for 10 min. It was then slowly cooled to 68°C over a period of 1 h, after which the incubation at 68°C was continued for a further 4 hours. At the end of the incubation, 100 μl of the same HEPES buffer at 68°C was added to the tube. The diluted solution was then cooled to room temperature and the mineral oil removed.

The biotinlyated cDNAs and any hybridized sequences were removed by mixing the diluted solution with a 100 μL slurry containing 1 mg of M-280 Streptavidin Dynabeads (Dynal) in the same incubation buffer. The incubation was continued at room temperature for 30 min with slow tumbling. The beads were then pelleted to the bottom of the tube by using a magnet and the supernatant was removed and desalted by passing through a 1 mL

Sephacryl spin column as described above. The cDNA solution was then lyophilized and redissolved in 10 μL of water. The small amount of cDNA remaining after subtraction was reamplified by PCR using the same primers. A single-stranded binding protein was added to the PCR reaction mixture used to reamplify the subtracted cDNA fragments: 1 μL (one-tenth volume) of the subtracted cDNA preparation was placed in 100 μL of PCR buffer containing 1 μg of Escherchia Coli single-stranded binding protein (Perfect Match™, Stratagene) . The cDNA was amplified during 25 PCR cycles (94°C, 30 s; 54°C, 30 s; 72°C, 60 s), and the product was analyzed by ethidium agarose gel electrophoresis. The appearance of this reamplified cDNA was similar to that of the initial material described above.

Multiple rounds of subtraction were performed. The subtraction libraries were prepared in parallel, so that the library enriched for sequences expressed under hypoxic conditions was prepared at the same time as the library enriched for sequences expressed under normoxic conditions. In each case, the driver used for the initial rounds of subtraction was the original set of cDNA fragments . After three rounds of subtraction, the enriched library prepared in parallel was used as the driver for the fourth round. In this way, the rarest sequences from both conditions were enriched in the final library. For instance, to obtain hypoxically induced sequences in this final round, the cDNA library enriched for sequences expressed under normoxic conditions served as the driver library and the cDNA library enriched for sequences expressed under hypoxic conditions served as the test library.

Example 2. Sequence Identification and Northern Blot Analysis of Significant Isolated Expressed Sequence Tags (ESTs) .

Several hundred cDNA fragments were sequenced from each of the two enrichment libraries produced by the subtraction protocol of Example 1 from HCE.E6E7 cells cultured under hypoxic and aerobic conditions. Four rounds of RDA subtraction of the oxic cDNAs from the hypoxic cDNAs generated a population of fragments in one of the enrichment libraries representing genes that theoretically are induced by hypoxic treatment. Five hundred randomly chosen clones from the cDNA library were partially sequenced. The obtained sequences were analyzed by NCBI-blast to determine the frequency of each of the genes/ESTs in the enriched population and to identify whether the isolated, hypoxia-mduced ESTs corresponded to previously identified genes or ESTs.

The frequencies of EST sequences among the 500 randomly chosen cDNA fragments obtained from the cDNA library enriched for sequences expressed under hypoxic conditions (after all four rounds of subtraction) is shown in Table 2, below. The two most frequently occurring ESTs, the HIGl EST and the HIG2 EST, corresponded to no known genes . Because these most frequently repeated clones were unknown, the full-length cDNAs representing HIGl and HIG2 were isolated (see Example 3, below) .

All the ESTs present m the clones of each library that were represented more than one time and that did not contain a highly repetitive element were tested by Northern blot for induction by hypoxia m Siha cervical carcinoma cells (and/or HCE.E6E7 cells). Selected probes representing ESTs found more than once were applied to Northern blots of total RNA from cell cultures harvested following different aerobic and hypoxic exposures to verify hypoxia mducibility or repressibility . For instance, the northern blot assays were used to confirm that, α- tubulin mRNA, detected in the HCE.E6E7 aerobic enrichment library, decreased m response to hypoxia in HCE.E6E7 cells, whereas mRNA corresponding to the HIG2 EST, found in the hypoxic enrichment library, strongly increased under the same hypoxic conditions.

Table 2. Tags isolated from the cDNA library following four rounds of RDA subtraction of oxic cDNAs from hypoxic cDNAs .

* as determined by Northern blot

** minor 4.2kB acetoacylCoA thiolase message only s induced

The procedure for the Northern blot assay was essentially as follows. Total RNA was isolated with Trizol (Gibco BRL) following the directions of the manufacturer. 5-10 μg of total RNA was denatured with glyoxal and size fractionated on a 1% agarose phosphate gel. The gel was capillary transferred to Hybond nylon (Schleicher and Shuell) and UV cross-linked. Probes were radiolabeled by random priming of gel-purified full length HIGl, or a fragment of HIG2 containing only the coding sequence a Stul fragment (Redipπme, Amersham) . Hybridization was carried out 0.5 M Na₂HP0₄, 7% SDS, 1 mM EDTA at 56°C for HIGl and 65°C for HIG2, washed to 0.2-0.5 x SSC at 56°C or 65°C, exposed to a phosphorimager plate, and visualized on a Storm 860 phosphoimager (Molecular Dynamics) .

The hypoxia-inducibility of ESTs as determined by Northern blot is summarized m Table 2, above. The HIGl and HIG2 sequences both demonstrated hypoxia-inducibility in the Northern blot assay.

Northern blots of total RNA from various aerobic and hypoxic human cells [HCE.E6E7s; SiHa cervical squamous carcinoma, MCF-7 breast carcinoma, H1299 lung carcinoma, Hctllβ colonic carcinoma cells; human cervical fibroblasts (HCFs) and HCF.E6E7s] probed for HIG2 expression demonstrated the following: (1) the gene is expressed as a single 1.5 kb transcript (the original EST cross-hybridizes with unknown 1.6- and 4-kb transcripts in HCE.E6E7s); (2) HIG2 mRNA increases from undetectable in 21% 0₂ (air) to abundant in 0.02% 0₂ in HCE.E6E7, SiHa, and MCF-7 cells after 6 h of hypoxia; (3) HIG2 is moderately expressed in H1299 and Hctllβ cells after 6 h of hypoxia; (4) there is no detectable HIG2 mRNA in HCFs and HCF.E6E7s; (5) in SiHa cells, HIG2 remains elevated for 48 h of hypoxia but decreases moderately by 72 h of exposure; and (6) no HIG2 induction is found in SiHa cells 6 h and 24 h after treatment with UV-C (20 J/m²) , γ-irradiation (6 Gy) , MMS (100 μg/mL for 1 h) , serum deprivation (0.1%), or glucose starvation (4%, <1 mM) ; (7) HIG2 expression is extinguished after exposure of hypoxic cells to 2 hours of reoxygenation .

The hypoxia inducibility of HIGl has been found to range between about 2-fold and about 5-fold across a variety of different human cell lines studied. The hypoxia-inducibility of HIG2 ranges between about 10- and about 20-fold across the various human cell lines studied. (See also Example 4, below).

In addition to the novel genes HIGl and HIG2, several known genes identified by the subtraction method in Example 1 were confirmed by Northern blots to be hypoxia inducible. These genes are also listed in Tables 2, 6, 7, 8, and 9. ESTs corresponding to the genes of annexin V, lipocortin 2, hnRNP Al, Ku (70) autoantigen, glyceraldehyde-3-phosphate dehydrogenase, ribosomal L7, acetoacetylCoA thiolase, and PRPP synthetase were identified by multiple hits in the hypoxia screen. All of these previously known genes were confirmed to be hypoxia-inducible by Northern blot.

It should be noted that although acetoacetyl CoA thiolase sequence tag is listed as induced, the reported, major RNA (1.8 kb) for the gene does not change. However, there is a larger, hybridizing, RNA species (4.2 kb) that is induced after 24-48 h hypoxia (data not shown) .

ESTs corresponding to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were especially prevalent amongst the cDNA clones. The hypoxia-induced expression of glyceraldehyde-3- phosphate dehydrogenase had been previously identified only in normal, non-transformed cells.

Example 3. Isolation and Analysis of Full-Length HIGl and HIG2 cDNA Sequence

The HIG2 EST (142 bp) was used to probe a conventional cDNA library constructed from mRNA isolated from SiHa cells exposed to 16 h hypoxia to obtain the full-length cDNA clone HIG2. This library was probed with radiolabelled HIG2 tag using conventional methods. Full length HIGl was isolated by first identifying overlapping ESTs from the NCBI human EST database, until a full length sequence was generated (1.35 kb) . PCR primers were then synthesized corresponding 5' and 3' UTRs in order to amplify the complete sequence using RT-PCR of SiHa RNA isolated after a 16 h hypoxia treatment. The full-length HIGl cDNA was then cloned and sequenced to confirm the predicted sequence.

The full-length cDNA sequence of HIGl is shown in Figure 1A. The full-length cDNA sequence of HIG2 is shown in Figure 2A. The translations of the putative open reading frames from HIGl and HIG2 are listed in Figure IB and 2B, respectively, and both encode small peptides (95 and 64 aa residues respectively) without obvious functional motifs.

Example 4. Hypoxic induction of HIGl and HIG2 in cervical cancer cell lines .

Because HIGl and HIG2 represent two novel genes whose functions are unknown, these genes were investigated in more detail. The expression of HIGl and HIG2 was examined in a series of human cervical cancer cell lines (SiHa, CaSki and C33a) under oxic and hypoxic conditions in vi tro . (The cell lines SiHa, CaSki and C33a were obtained from the ATCC and were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI1640 supplemented with 10% fetal bovine serum.) Although HIGl is induced moderately within 2 hours of hypoxia in all the cell lines tested, it remains elevated only in the Siha cells. HIG2 is more consistently induced from low basal levels in all the cervical cancer cells tested. The major HIG2 mRNA species is 1.4 kb in length, but there are two other mRNA species of minor abundance (8.0 and 9.0 kb) that are induced with identical kinetics to the major species.

Example 5. Hypoxic induction of HIGl and HIG2 in tumor xenografts .

The hypoxic induction of HIGl and HIG2 in vivo was also tested in tumor xenografts generated from the C33a cell line by Northern blot analysis of total tumor RNA. Gene expression in untreated xenografts was compared to that in xenografts that were made hypoxic by treatment of the host animal with flavone acetic acid (FAA) 24 hours prior to explantation and RNA isolation. To generate tumor xenografts, 2.5-5 x 10⁶ cells were injected subcutaneously into the flank of scid mice and allowed to grow into tumors that reached 1-2 cm in diameter before harvest. FAA (Lipha Chemical, NY) was injected IP into the animals at 200 mg/kg in 5% sodium bicarbonate 24 hours prior to tumor harvest. FAA treatment resulted in increased tumor hypoxia as measured by ependorff electrode and increased HIGl and HIG2 expression by 1.2 and 2.4 fold respectively. The moderate level of HIGl induction in vivo is not unexpected, due to the in vi tro data. The portion of the human gene used for a probe in these experiments has low homology with mouse RNA and under the conditions used, did not cross-hybridize .

Example 6. Specificity of the induction of HIGl and HIG2.

We next investigated whether HIGl and HIG2 induction is unique to hypoxic stress, or if it is elicited by other tumor microenvironment stresses such as glucose deprivation, serum starvation, or by genotoxic stresses such as UV or ionizing radiation. We also tested the hypoxia-mimetic, iron-chelating compound desferoxamine that has been shown to induce expression from HIF-1 responsive genes. For stress treatments, cells were plated overnight and then treated the next day with either 256 nm UV at 1.2 J/m²/sec, or gamma irradiation from ¹³⁷Cs source at 3.8 Gy/min. Glucose and serum deprivation experiments were performed by washing the cells three times m phosphate-buffered saline (PBS) and replacing the indicated media (glucose free RPMI with dialyzed serum, or 0.1% FBS RPMI).

Northern blot analyses was performed on RNA isolated from C33a cells exposed to these stresses. HIGl was poorly responsive to hypoxic stress over this time course, but strongly induced by glucose deprivation. HIG2 was induced strongly by hypoxia, the hypoxia-mimetic stress desferoxamine (DFO) , and glucose deprivation. UV light seemed to have little effect upon either HIGl or HIG2 expression. In contrast, while ionizing radiation did not change HIGl expression levels, it did result in a moderate 2.5 fold induction of HIG2 by 24 hours. There were similarities in the pattern of stress responsiveness of HIG2 and that of the HIF-responsive VEGF gene, suggesting that HIF-1 may be important in HIG2 expression.

Example 7. Identification of HIGl and HIG2 sequences from non- human species.

A search of the NCBI-dbEST database for fragments of genes from other species that might represent evolutionaπly conserved orthologues identified overlapping mouse EST fragments that encode for similar peptides to the human version of HIGl and

HIG2. The murine HIGl and HIG2 orthologues are shown in Figures 3A and 5A, respectively. These mouse genes code for predicted peptides (Figures 3B amd 5B, respectively) with 84% and 76% identity to the human peptides respectively. There also existed a cDNA cloned from fish ( seriola qumqueradia ta ) in the database that coded for a HIGl orthologue (Figure 4A and 4B) . A sequence comparison of the HIGl homologues is shown in Figure 6A. A sequence comparison of the HIG2 homologues is shown in Figure 6B. We confirmed the existence of murine HIGl and HIG2 by cloning the presumed genes and assaying for their expression. We designed oligonucleotide primers corresponding to sequences in the 5' and 3' untranslated regions that would amplify these genes. We were able to make primers that amplified the entire murine HIG2 cDNA, but were only able to make primers that would amplify the coding sequence for murine HIGl :

mHIGl forward primer (SEQ ID NO:17): 5' -CCGATCTAGAGGAAGGGACCCCGCGTCTCGGA-3' mHIGl reverse primer (SEQ ID NO: 18):

5' -GGCGCTCGAGTCTAAACCCACATGTTATTTATTG-3' mHIG2 forward primer (SEQ ID NO: 19):

5' -CCTTACTCCTGCACGACCTGG-3' mHIG2 reverse primer (SEQ ID NO:20):

5' -GGCGCTCGAGCACATGTGCATTACACTGGAGA-3 '

These primers were then used to amplify the coding sequences of HIGl OR HIG2 from reverse-transcribed RNA isolated from the murine squamous cell tumor cell line SCCVII (cultured in DMEM supplemented with 10% FBS) . The amplified fragments were cloned and sequenced, confirming the predicted sequence.

The cloned genes were then used as probes for Northern blot analysis of RNA isolated from SCCVII cells. Both mHIGl (murine HIGl ) and HIG2 (murine HIG2) have hypoxia-inducible species of

RNA by this analysis. Murine HIGl has two major RNA species that strongly hybridize to the probe, at approximately 1.2-1.4 kb in length. The larger message is modestly induced, while the smaller message is strongly induced to approximately 5 fold by a 12h exposure to hypoxia. Murine HIG2 also has two RNA species at approximately 1.4 and 2.2 kb . Both the murine HIG2 mRNAs seem to be mildly hypoxia-inducible with 2-3 fold induction by 6-12 hours. For comparison, the same blot was probed with vascular endothelial growth factor (VEGF) and this message shows an approximately 5-fold induction by 6h .

Example 8. Analysis of Gene Expression under Hypoxia using Gene Discovery Arrays (GDA) .

Nylon filters containing GDA arrays were purchased from Genome Systems (St Louis, MO) that have affixed to them nucleic acids that were originally characterized by the I.M.A.G.E. consortium (LLNL) . This array represents 18,394 cDNA clones that have been categorized as either known genes or ESTs (expressed sequence tags) isolated by the consortium. This filter was used to quantitatively determine the mRNA expression levels of all these arrayed cDNAs in SIHA tumor cells both under oxic conditions and hypoxic conditions (18 hrs, <0.2 %). Messenger RNA was isolated from control and hypoxic SIHA cells and cDNA probe was generated using MoML reverse transcriptase. 2 μg mRNA was incubated with 500 ng of oligonucleotide primer (T)ι₈ NM (N=A/C/G, M=A/C/G/T) in the presence of reaction buffer, 4mM dATP, 4mM dGTP, 4 mM dTTP and 4mM alpha ^l331P dCTP and 200U reverse transcriptase. The radioactively labeled first strand cDNA that was produced from this reaction was then used to probe the respective filter. The filters were then exposed to a phosphoimager plate, the image collected and digitized for analysis, and the relative counts on each cDNA were quantitated and compared using GDA analysis software. The results are shown in Table 3 for the 500 genes or ESTs with the greatest level of hypoxic induction and in Table 4 for the 500 genes or ESTs with the greatest level of hypoxic repression.

Table 3. Genes (identified by Genbank Accession Number) whose expression was induced in hypoxic cells, shown with the ratio of their expression in hypoxic cells over their expression in oxic cells . ratio Ace . No . ratio Ace . No . ratio Ace . No . ratio Ace . No .

5.18 AA069408 7.013 AA134027 4.027 AA155910 3.372 AA037436

7.528 T48883 5.236 N35559 5.343 T87461 2.025 H22698

9.999 N58711 5.753 R63553 3.478 24548 5.454 07146

5.678 H04904 6.525 N27733 5.699 T48772 5.209 AA101069

6.453 H82707 3.146 N94304 4.095 N94916 4.599 24109

7.825 56465 4.218 H70730 6.52 K14897 4.996 R17409

9.999 N31409 1.659 AA053856 1.159 R34659 4.932 17090

9.999 H19264 8.836 05763 2.492 H93923 5.391 R24601

3.626 R73213 3.394 R54524 5.054 N28535 5.189 R26954

9.999 R08251 8.246 W15599 2.08 AA069499 2.12 AA187216

9.999 H91612 4.216 R09918 5.021 38635 3.852 H86677

9.999 AA196038 9.238 AA005185 8.59 T54127 4.653 K67329

9.999 W55913 4.654 T95404 4.289 R26319 4.993 W19173

5.143 R94248 9.262 R19946 6.048 07082 5.641 06851

9.999 R85589 4.308 AA068998 3.12 AA194330 6.186 00378

2.9 H50204 5.203 N47831 4.594 N31417 4.458 AA204792

4.333 H52973 4.175 AA151009 2.424 AA069173 4.64 W48584

9.999 H60510 5.33 W46682 3.645 52472 3.742 R84764

9.915 R13129 5.39 AA176700 6.109 H09049 4.049 R21449

9.999 AA040826 3.612 H47207 3.843 H81775 5.074 AA160325

2.186 N76562 7.666 87527 5.664 H71710 2.356 R97269

8.468 R13125 5.965 47502 2.494 17237 3.68 H86214

9.999 52400 6.338 N44758 3.516 H06318 4.654 N40829

3.376 AA054303 2.313 AA126937 4.006 T63499 5.114 W19928

4.263 AA042800 6.232 R31353 5.829 R22383 5.526 H18298

2.681 46165 4.846 R18798 3.457 T87920 2.867 N39173

8.287 24084 4.648 T78246 5.766 47525 3.251 R76943

9.999 T77247 6.726 H29713 5.493 W32575 4.139 07720

8.506 R13073 4.524 AA026304 5.622 R84635 3.604 AA085920

9.999 T95699 6.113 AA053162 5.284 R18138 3.814 H18258

8.7 N30952 1.702 H65775 3.414 R34648 3.192 AA035131

8.372 N29065 3.685 32710 4.46 H80175 2.275 AA033736

7.37 H13744 6.264 T51305 4.479 R76163 3.896 R71065

9.999 H46657 7.182 R14403 4.381 17182 3.534 AA074177

6.115 H83517 3.9 38235 5.81 AA126956 4.358 R92264

6.587 AA129780 5 W47083 3.781 AA088390 3.561 N34634

6.587 N42542 5.209 W35243 4.048 R52046 3.695 AA114861

9.999 H67546 3.288 W24343 5.474 N98916 4.673 R31970

6.678 AA181350 5.209 H93373 3.226 R51929 3.989 H70974

7.187 T67190 6.255 R26331 3.033 H69334 4.646 AA156193 ratio Ace . No . ratio Ace . No . ratio Ace . No . ratio Aec . No .

6.498 AA034932 2.549 R70082 5.458 20511 3.258 7AA085385

9.999 H41372 4.698 W32969 3.742 T40473 4.453 49770

9.999 H08885 4.038 R66920 5.132 R82723 4.056 37672

8.221 H90627 6.586 25455 5.886 T50788 2.503 38097

4.812 T91423 7.516 38478 3.266 AA036758 5.118 N48838

5.535 N49031 1.961 AA039447 3.564 46219 5.105 H41937

7.673 AA079020 3.163 H89835 3.535 N64406 1.544 AA159807

5.718 H44892 3.311 AA156148 4.839 W17076 3.436 AA112478

2.058 H38180 5.592 38424 5.314 H18129 4.396 R07212

3.101 T48613 6.954 H75477 4.118 H93835 4.917 AA128281

3.236 H12952 3.507 31707 5.306 AA167017 4.303 N39630

2.553 T87585 3.968 N40606 5.012 W24939 4.165 39234

2.961 R76842 3.417 67757 3.887 38826 1.983 H68587

4.505 N54105 5.57 AA074760 3.791 H62991 3.358 H78277

3.424 72986 2.311 H18350 4.853 AA132801 2.968 T87597

3.738 H53662 2.707 31757 2.065 R54128 3.142 H64978

1.682 W05551 3.252 T89571 3.35 AA214334 4.376 16557

2.942 H62026 3.202 07148 3.553 W16484 3.316 H45068

3.16 AA146611 2.404 H66389 3.902 AA122157 3.559 H08997

3.702 H70850 3.621 R68331 3.451 H44677 3.277 H08983

4.118 49687 4.089 AA099075 3.277 W20192 2.455 H66256

4.359 AA100113 4.46 H91361 3.534 T47067 2.632 T70457

3.338 AA133312 2.057 N42428 3.581 T82048 2.569 H13942

4.367 T49117 3.423 48763 3.694 AA134135 2.59 T77584

3.79 H58461 3.844 R60387 2.79 R25979 2.346 R71543

4.806 N69323 3.421 W19744 1.586 R21064 3.168 T98705

2.664 05089 2.473 R59435 1.972 H29698 3.43 T78542

3.176 AA070823 3.189 R07238 3.507 R89708 2.887 T74951

1.966 W24455 2.496 AA074340 3.189 R06568 3.768 W16974

3.258 R63252 3.287 N78038 1.957 AA009869 3.48 R88098

4.543 H80571 3.935 07144 1.627 31940 2.021 48791

3.593 AA131550 4.133 R06175 3.545 38638 3.686 R80458

4.34 N42413 3.912 N46036 1.634 R60752 1.812 H29706

2.313 R62688 2.535 78057 2.917 R28459 3.501 AA130339

2.952 H73881 3.614 R16609 3.614 H63610 2.642 H10811

5.245 AA007484 3.208 H01260 3.568 R66182 2.323 AA074067

6.009 N57562 1.818 04913 2.77 H95908 2.419 T56791

3.62 H25971 3.591 R61631 2.83 H24644 2.432 R48720

3.583 H19106 3.528 H71668 2.458 AA044130 2.881 R20222

3.085 W46660 4.66 R18905 2.039 R23341 3.274 H03764

3.694 R36401 2.311 H64449 3.785 H01679 2.71 R55247

3.945 R09905 1.619 W07452 2.82 T91461 3.324 AA005419

4.416 H40081 3.462 AA203284 3.202 T74959 2.494 T97640

4.498 AA156956 4.843 N66473 1.924 T39976 4.157 R23556

3.22 AA112421 3.777 H78279 3.135 H68817 3.956 N53743

2.991 AA147722 3.256 AA156298 3.739 H83559 1.947 01963 ratio Ace . No . ratio Ace . No . ratio Ace . No . ratio Aee . No .

4.341 W20171 3.25 N75101 1.841 T79362 3.858 N94762

1.778 W21312 3.961 H75277 2.551 N36269 3.655 AA007521

3.42 N28517 4.366 R80450 3.285 T79536 1.506 R00903

3.436 07026 3.532 AA057729 3.193 T87507 2.775 H83982

4.393 R21898 2.373 T92805 3.87 T71354 2.61 H12686

3.543 R36586 3.167 AA005286 3.572 W19860 2.577 H78353

2.784 T48691 3.33 H12796 2.896 AA039258 3.227 N45476

4.06 W16946 1.679 R60831 4.245 T79534 2.799 R55692

3.071 T85481 3.64 T85390 2.096 R59467 3.196 31182

4.116 T83199 2.606 N73091 3.168 H91401 2.371 T91436

2.479 H19169 2.648 T56084 2.074 24718 2.951 T54610

3.768 37172 3.4 37084 2.318 T54602 2.735 AA001324

2.146 H70732 3.531 07043 2.636 H65057 4.007 H02412

4.475 N75228 2.86 N44537 3.254 R52482 3.547 N92284

3 428 W00630 3.546 T74332 2.377 H95979 2.257 H78485

2.951 N57398 2.2 R35560 2.4 R80523 3.216 R20594

3.62 15521 3.945 R69162 2.953 H79197 3.416 H12419

3.262 R61036 2.337 T48694 3.711 R85335 1.522 R91771

2.572 T68568 3.228 N31889 2.546 T56622 3.225 H09280

1.5 N40660 2.72 R60420 3.107 AA205009 2.957 H63806

2.193 N98743 3.234 AA004897 2.83 00950 2.588 R70441

3.464 16685 2.893 R24648 2.271 N73209 3.093 R19326

2.372 T82120 2.187 AA063234 2.596 N31231

2.765 T61346 3.766 R96692 2.953 T85879

4.054 AA214079 3.217 AA004891 2.655 R32750

1.542 AA164677 3.915 W00391 1.922 N32733

2.937 AA211776 3.291 R82770 3.495 17002

3.067 T87472 4.545 H18766 2.984 W20484

3.058 H61812 3.167 R87818 1.377 AA136789

2.937 N45602 2.214 88806 2.364 H45241

2.927 R14907 3.318 T54086 1.678 17311

3.476 H12508 2.992 H61280 3.688 R87413

3.25 19104 3.13 AA167039 3.432 R14301

2.441 R87193 3.287 T87358 2.927 R80475

2.521 H92713 2.711 03009 3.258 T79546

2.617 R32216 2.674 N98348 3.18 T78497

2.262 AA126184 3.311 N47460 1.595 R52015

3.281 H09869 2.187 H73438

2.02 AA054041 2.751 78830

2.639 H66558 2.953 T49575

3.709 R07731 3.122 N47660

3.032 T80874 3.435 H03226

3.673 H51373 3.371 H42536

3.114 T51726 3.049 T85153

3.137 H09884 2.421 N53255

3.202 T98755 2.379 R70814 ratio Ace . No . ratio Ace . No . ratio Ace. No. ratio Ace. No.

3.201 AA194172 2.33 47650 1.981 H13009 3.072 T78498

Table 4. Genes (identified by Genbank Accession Number) whose expression was repressed in hypoxic cells , shown with the ratio of their expression in oxic cells over their expression in hypoxic cells . ratio Ace . No . ratio Aee . No . ratio Ace . No . ratio Ace . No .

9.999 H38055 7.873 N33752 4.55 R59009 9.999 R31317

6.948 AA057425 6.351 T54424 3.78 AA121166 6.082 AA057398

9.999 AA116099 8.097 T87470 8.936 R01823 6.504 R89643

9.999 AA057428 7.137 H06343 7.628 AA085375 7.172 H70359

9.999 R73197 4.887 AA058878 3.185 AA054115 5.273 N48132

9.999 R48415 9.999 H46055 8.052 H96006 4.543 AA054096

9.999 H14999 6.651 T40066 5.324 T67226 5.207 AA054071

9.999 T96535 9.672 R89521 5.895 R62231 4.911 AA078915

9.999 H27140 9.999 AA065190 4.767 AA112466 4.439 H52742

9.999 T99054 7.455 H43837 4.271 N34169 3.092 T67415

9.999 H46382 4.601 81199 5.635 H51983 3.457 R49895

9.999 R90757 9.999 H27344 6.176 N52679 4.953 H16042

9.999 AA121402 9.999 H49310 6.465 H50403 6.039 H45590

9.999 H38676 9.999 R69813 4.812 H26200 6.767 AA029349

7.52 AA057511 9.999 N32666 6.495 N30528 6.456 H51981

9.999 N43944 2.046 AA053873 7.015 H47146 5.186 R61165

9.999 AA134018 9.999 AA125970 5.891 R11999 2.541 R85183

5.741 H14566 4.712 H83338 5.442 N24303 4.764 H85692

9.999 AA035019 9.999 R94457 3.989 H14332 5.288 DROS-A8

9.285 N29018 4.545 N31674 6.206 R64420 4.603 R24904

7.813 AA069149 5.6 T50828 6.203 R37898 3.307 72875

9.999 R76214 7.861 R74161 6.251 R76298 4.033 H71729

9.999 R23999 7.437 T99984 5.33 N98261 6.431 H14193

9.999 R23880 5.5 R48041 6.206 R24405 8.731 N48042

9.999 AA078826 7.565 H82390 4.865 H39089 4.063 H70778

7.739 T92655 4.998 T54422 9.999 W72342 6.486 W24476

6.577 W01565 6.317 H98046 4.976 T65484 9.999 01642

9.59 R90884 7.321 N30514 3.745 H96724 7.103 T98068

5.535 R56663 5.137 H38881 9.999 H49809 3.784 AA100388

8.157 00931 8.737 T99046 3.286 N57334 4.15 H60927

9.999 H50385 5.465 H47440 8.214 R51931 3.897 R28248

8.922 H28503 6.734 N42979 3.912 T79680 7.319 H56754

5.182 H84008 6.802 01319 3.824 H21568 4.617 R92111

7.947 W01051 7.572 R95136 5.746 T80382 6.422 H94177

9.05 R66879 9.999 AA112231 9.999 R87923 4.375 R87352

9.999 H45773 6.035 H53489 5.902 R23778 6.6 R31364

9.999 AA126109 5.53 N94798 8.584 AA101044 3.713 AA059302

9.999 R54784 6.009 N30964 3.638 AA112340 5.179 H51160

9.999 N98857 9.999 N44142 8.09 R90895 5.423 R25798

9.999 AA056159 7.759 03129 6.3 N90458 4.448 R63455 ratio Aee . No . ratio Ace . No . ratio Ace . No . ratio Ace . No .

9.999 R77028 4.156 03125 4.874 R87886 6.596 T77139

9.999 N36070 7.184 R79618 4.621 N32679 4.909 R63498

5.356 R89245 4.281 H38147 7.755 W02372 4.523 R22272

9.999 H51782 4.445 R38004 4.624 H30637 6.954 H45355

9.999 N48735 9.999 R88190 8.387 R01888 5.624 R75964

5.379 H86672 9.999 N41573 8.644 AA070426 4.742 N32681

9.999 T95210 9.723 H52741 9.999 31524 4.494 R06552

7.355 R81942 4.401 T54426 6.574 H81786 9.097 R87320

9.999 N53883 4.914 AA054102 3.719 N40437 5.369 R55451

7.685 N24364 6.874 R31243 7.48 N42402 7.075 R84765

7.473 H91761 7.618 AA113044 6.86 R32757 4.058 H84844

4.537 W16945 5.394 R96571 8.926 H21214 6.525 W21173

4.091 DROS-A8 2.424 R71723 4.111 H18154 1.955 AA054211

9.999 AA115819 4.366 H86277 2.981 R53678 3.214 N50075

4.996 N36347 2.214 T53945 3.552 R06539 3.359 T93912

5.587 N30932 5.37 R09668 4.474 H43816 4.156 T77415

2.24 R80470 4.809 H40716 6.663 AA146629 2.779 AA040227

2.746 H16193 2.192 R70132 4.402 R01530 3.468 N26148

6.109 AA004785 2.555 R81899 1.884 H86896 2.744 R26215

3.155 N28396 4.968 N42806 4.013 R68198 3.757 H58331

3.525 AA047581 2.243 H66535 3.632 H16160 2.892 N34217

4.396 H84204 4.049 R46282 1.867 R85333 2.987 AA059324

6.795 R54918 5.522 R49786 1.993 16980 3.314 R23635

5.972 R78728 8.225 T67978 2.042 T60294 1.29 R77994

4.684 R80601 3.362 R81838 3.009 W32352 3.188 R84692

7.734 R73050 5.364 H19572 1.589 W15194 3.837 N75231

4.832 H12682 4.739 R33409 2.503 H52012 4.043 AA043598

6.87 N56601 3.959 AA069498 2.192 R22397 2.645 R22392

4.153 R80286 4.115 H44733 4.219 R55158 3.933 R11541

9.999 N73428 5.33 AA029010 3.069 AA100975 3.244 H73503

2.754 AA002135 9.999 AA113853 8.61 AA113299 2.909 AA053288

2.948 H39058 4.264 N31362 1.999 R87373 3.059 H38003

6.235 R01799 2.793 R20924 4.285 N45686 4.04 7ΛA088387

6.179 R09942 3.664 R98517 3.87 02494 4.006 H89896

1.849 R72766 4.656 N24477 3.84 R73063 4.744 AA126881

2.963 R82691 4.443 AA032034 2.614 H14441 6.651 AA088231

2.716 R83247 3.799 AA054203 4.806 AA058898 3.346 N59684

9.375 AA.070943 2.852 W95433 1.683 H89823 4.122 T92415

4.051 N43796 5.221 N72527 4.392 AA037418 3.81 AA088190

4.754 N46182 5.408 02353 4.142 T83106 2.846 N32669

5.892 R32571 4.626 01717 4.241 T81175 4.096 T58881

3.03 W72046 7.031 W79028 3.335 R31471 2.097 T99924

5.486 H85829 4.702 R12648 3.253 R86304 3.441 AA046822

5.718 R75796 3.427 N32672 2.914 H86156 3.813 H85193

2.697 N45640 3.989 N90836 4.004 H87311 3.134 R91315

2.888 H49806 3.692 N92684 2.595 T94530 3.132 T71293 ratio Ace . No . ratio Ace . No . ratio Aee . No . ratio Aee . No .

5.419 H52350 2.552 06829 2.108 H27617 3.45 H79957

4.515 AA071099 2.537 H53375 1.565 H14143 3.198 AA058615

3.535 H51993 4.752 H67462 4.018 H49818 3.309 N34153

3.287 AA069031 4.076 AA029883 1.677 R97857 3.734 H61493

5.108 R79519 4.394 H12075 3.544 N90841 1.687 R56760

5.336 N29042 3.253 T86612 3.901 AA076660 1.796 AA132756

4.075 R48060 9.23 T85558 4.49 N53295 2.934 H20613

4.343 N77703 5.32 N29155 1.67 AA190622 6.358 AA129486

5.491 W56898 3.579 H44664 4.118 AA099647 3.07 H44888

2.202 H28534 5.57 16814 4.007 AA069114 3.583 04937

4.449 H62445 2.133 R85191 2.606 W92014 2.521 AA047388

4.196 R90798 2.161 H50471 4.047 87655 4.349 R52735

7.226 R35606 3.275 H30629 3.891 01911 2.165 AA065193

3.224 R25899 4.284 R80273 4.32 R54194 4.009 N42453

9.999 AA113119 4.063 H27411 2.085 N57249 3.688 72149

4.2 N30471 4.231 H44693 3.472 R74281 4.603 AA044942

5.466 T98994 2.471 W47021 4.044 AA054209 3.445 AA187560

2.577 AA075652 4.544 H69415 2.621 H62639 2.273 R74076

2.448 R06926 2.425 AA181061 1.461 T78546 2.268 H65149

2.535 45582 3.336 T97872 3.677 40228 5.781 T48877

4.173 T82054 3.553 H51540 6.08 AA101477 3.152 AA033945

3.359 T98110 2.63 N44493 3.907 00916 3.032 H49111

2.703 R86735 2.587 AA088784 1.846 N90969 3.741 H22503

2.77 AA045397 3.082 R25304 3.006 H83363 2.252 N78086

2.893 R53671 3.06 AA058600 4.484 17108 2.755 H71635

2.077 AA088407 2.741 H81782 1.482 N64281 2.673 N77126

2.213 T95166 3.466 H84604 2.899 AA045672 4.796 AA083226

1.963 H27400 7.264 AA113169 2.994 H43746

5.333 T62191 3.859 N63192 2.65 R91004

2.671 N32657 2.887 N29162 2.439 AA047858

3.374 T83919 1.926 R28329 2.867 W21593

3.108 AA191189 3.17 AA172313 2.374 N34202

2.292 R74157 2.057 R34929 3.144 N80131

1.787 R21095 4.695 W55902 2.458 AA043774

4.011 AA180772 2.179 R13273 3.306 T56558

2.901 H82540 2.354 AA121148 3.896 AA205980

3.707 R73398 5.805 AA088299 2.565 AA179841

2.658 H55982 2.233 H84617 2.973 H68783

1.826 H45128 2.011 AA059207 3.142 30866

4.006 25183 2.742 H39778 2.992 AA028961

4.299 T56400 3.034 H83022 2.489 R26026

4.086 T95526 3.245 AA113900 5.751 AA085171

1.691 AA053901 2.716 R19726 1.822 AA150817 Example 9. Analysis of Gene Expression under Hypoxia using GEM™ microarrays

The hypoxic induction of genes in FaDu cells was analyzed by comparing the expression of genes in FaDu cells exposed to hypoxic conditions (5% C0₂/5% H₂/90% N₂ for 16 hours at 37°C) to those exposed to normal, oxic conditions. This differential expression was analyzed using GEM™ technology provided by Genome Systems Inc. Messenger RNA (mRNA) was extracted from hypoxic FaDu cells, and separately from oxic FaDu cells. The total RNA was isolated from the cells essentially according to the standard Genome Systems Inc. protocol, as follows. 500 μl Trizol was added 50-100 mg of fresh frozen cells. The cells were then immediately homogenized. 500 μl Trizol was then added, and the sample was mixed well. The sample was homogenized for five minutes at room temperature. Next, 0.2 ml chloroform was added per 1 ml Trizol. The mixture was shaken vigorously for 15 seconds and then allowed to incubate three minutes at room temperature. The sample was then centrifuged at 12,000X g for 15 minutes at 4°C. The aqueous phase was transferred to a fresh centrifuge tube without disturbing the interphase. 0.5 ml of isopropanol was added and the samples were incubated for 10 minutes at room temperature. The RNA was pelleted by centrifuging at 12,000X g for 10 minutes at 4°C. The supernatant was then removed. 1 ml of 75% ethanol was added to the pellet, which was then vortexed. This was followed by centrifugation at 7,500X g for 5 minutes at 4°C. The ethanol was removed. The pellet was dried for 10 minutes at room temperature and then dissolved in 10 μl nuclease-free water and stored at - 80°C. Next, the poly A+ RNA was isolated from total RNA essentially according to the standard Genome Systems Inc. protocol, as follows. To purify polyA RNA, the total RNA sample was passed twice over OligoTex mRNA isolation columns from Qiagen. After the elution of the polyA RNA, the polyA RNA was ethanol precipitated, and the final product was brought up in DEPC H₂0 or TE . For 50 μl of elution from the OligoTex column, 40 μl of IX TE and 1 μl of glycogen (5 mg/ml) was added. Then 120 μl of 100% EtOH was added and the sample was frozen at -80°C for 10 minutes. The sample was then spun at 12,000 x g for 10 minutes at 4°C. The supernatant was removed and 250 μl of 75% EtOH was added. The pellet was spun at 12,000 x g for 5 minutes at 4°C. The supernatant was again removed and the pellet dried for 10 minutes at room temperature. The pellet was then dissolved in DEPC H20 to a concentration of 50 ng/μl.

The purified RNA samples were sent to Genome Systems Inc. to perform a GEM microarray analysis. From the mRNA samples, fluorescent labeled cDNA probes were prepared by Genome Systems Inc. using standard methodologies familiar to those skilled in the art. The cDNA probes corresponding to the mRNA sample from the oxic FaDu cells were labeled with a different, distinguishable fluorescent label than the cDNA probes corresponding to the mRNA sample from the hypoxic FaDu cells. The two fluorescent probe samples (one from hypoxic FaDu cells, the other from oxic FaDu cells) were then simultaneously applied by Genome Systems Inc. to their Human UniGEM V microarray for hybridization to the arrayed cDNA molecules. The Human UniGEM V microarray contains sequence verified Genome Systems

Inc. proprietary cDNA clones representing more than 4,000 known human genes and up to 3,000 ESTs mapped to the UniGene database. (All of the genes on the microarray were selected for criteria such as known functions, homologies, and presence on the human transcript map.) The genes or gene fragments of the GEM microrarray (each 500-5000 base pairs in length) are arrayed on glass surface to which they have been chemically bonded.

Once the two fluorescent cDNA samples were sufficiently incubated with the arrayed cDNA molecules to allow for hybridization to occur, the microarray was washed free of probe molecules which had not hybridized. The different gene/EST sites of the GEM microarray are then scanned for the each of the two fluorescent labels. Presence of the fluorescent label at a particular gene site indicates the expression of that gene in the cell corresponding to that fluorescent label.

The 30 genes or ESTs which were determined on the microarray to have the greatest level of induction in hypoxic FaDu cells (versus oxic FaDu cells) are listed below m Table 5, along with their levels of induction, functional category if known, and GenBank accession number.

GEM™ technology was also used to analyze the differential gene expression of Siha cells, C33a cells, and normal keratinocytes as a result of hypoxic induction. The genes or ESTs which were determined on the microarray to have the greatest level of induction in the assayed hypoxic cells (versus oxic cells) including FaDu cells, Siha cells, C33a cells, and normal keratinocytes are shown below in Tables 7, 8, and 9 along with their levels of induction and GenBank accession number. Table 6 illustrates hypoxic gene induction by functional category.

Table 5. Genes Induced by Hypoxia in FaDu cells .

Example 10. Analysis of Hypoxia-Associated Tumors In Vivo by Analyzing Potential Marker Gene Products Present in Blood

Blood samples were collected from patients with solid tumors (head, neck, and cervical malignancies) and the oxygen status of each sample was determined with the use of EF5, a nitroimidazole-binding technique well known in the art (Lord et al., Cancer Res . (1993) 53 (23) : 5721-6; and Evans et al . , Bri tish Journal of Cancer (1995) 72 (4 ) : 875-82 ) .

The blood samples were tested for the presence of plasminogen activator inhibitor-1 (PAI-1). Human serum levels of PAI-1 protein were determined by using a commercially available ELISA assay called TintElize® kit (Biopool International, Inc.). The assay utilizes a monoclonal antibody that recognizes all forms of human PAI-1 including active, inactive (latent) , and complexed to tPA/uPA. The secondary antibody is conjugated to horseradish peroxidase (HRP) and visualization is achieved by conversion of HRP substrate to a yellow-colored product.

According to the TintElize® kit protocol, blood samples are prepared as follows: 9 volumes of blood are collected in 1 volume of 0.1 M trisodium citrate. Alternatively, 99 volumes of blood are collected in 1 volume of 0.5 M EDTA. The samples are then centrifuged at 2500 x g for 15 minutes. During collection, 1/3 of the plasma supernatant is harvested with a plastic pipette. Plasma samples are stored at 2-5^cC degrees and assayed within 2 hours. Plasma can be stored for longer periods of time at -20°C and thawed at 37°C for 30 mintues before use. The assay is performed at room temperature according to the TintElize® kit protocol (Biopool International, Inc., Catalog #210221).

PAI-1 levels that were assayed from patient sera were determined to be between 2-20 ng/ml in normal individuals and between 40-110 ng/ml in patients with tumor hypoxia. The increased levels of PAI-1 in the blood stream of patients with tumor hypoxia in comparison to normal individuals establish its use as a diagnostic marker protein.

Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the claims.

Claims

What is claimed is:

1. An array of polynucleotides, comprising at least two different hypoxia-inducible genes, or complements thereto, or at least thirty nucleotide-long fragments thereof, or sequences which hybridize thereto.

2. An array of Claim 1, comprising at least two different polynucleotides, each comprising a hypoxia-inducible gene, or an at least thirty nucleotide-long fragments thereof, or the complement thereto, wherein said hypoxia-inducible genes encode proteins belonging to different functional categories selected from the group consisting of glycolytic enzymes/proteins, metabolic/homeostatic proteins, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, erythropoiesis/vascular regulatory proteins, and transcriptional regulatory proteins.

3. An array of Claim 1, comprising at least two different polynucleotides, each comprising a hypoxia-inducible gene, or an at least thirty nucleotide-long fragment thereof, or the complement thereto, wherein said hypoxia-inducible genes all encode proteins belonging to a single functional category selected from the group consisting of glycolytic enzymes/proteins, metabolic/homeostatic proteins, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, erythropoiesis/vascular regulatory proteins, and transcriptional regulatory proteins.

4. An array of Claim 1, comprising:

(a) at least one gene selected from the group consisting of HIGl , HIG2 , annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal LI, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activa tor inhibitor-1 (PAI-1) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2, endothelin- 1 , endothelin-2 , B-cell transloca tion gene-1 (BTG-1) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1 ) , quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophilin , cyclooxygenase-1 (COX-1 ) , fructose bisphospha tase , crea tine transporter , fa t y acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2-intera cting killer (BIK) , 19 kDa - interacting protein 3, Nip3L/Nix, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferritin , insulin-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1 ) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor, trisephospha te isomerase, lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113, c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT- 3) , glycogen branching enzyme, TGF beta , brain HHCPA18 , mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin , lysl oxidase, prostaglandin endoperoxide synthetase, insulin- inducible protein 1 , MHC-cIl lDQB, myocyte-specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, angiogenin, and SDK3, or an at least thirty nucleotide-long fragment thereof; and

(b) a second polynucleotide, wherein said second polynucleotide comprises a second hypoxia-inducible gene or an at least thirty nucleotide-long fragment thereof.

5. An array of polypeptides, comprising at least two different hypoxia-induced proteins, or biochemically equivalent fragments thereof, wherein each hypoxia-induced protein belongs to a different functional category selected from the group consisting of glycolytic proteins, metabolic enzymes/protems, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, erythropoiesis/vascular regulatory proteins, and transcriptional regulatory proteins.

6. The array of Claim 5, comprising at least two different hypoxia- duced proteins or biochemically equivalent fragments thereof, wherem said hypoxia-mduced proteins are all proteins belonging to a single functional category selected from the group consisting of glycolytic enzymes/protems, metabolic/homeostatic proteins, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, erythropoiesis/vascular regulatory proteins, and transcriptional regulatory proteins.

7. The array of Claim 5, comprising:

(a) at least one protein selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al), Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3 (FGF-3), EPH receptor ligand, plasminogen activator ιnhιbιtor-1 (PAI-1), macrophage migration inhibitory factor (MIF) , fibronectin receptor, fibronectin 1, lysl hydroxylase, lysyl hydroxylase-2, endothel - 1, endothelm-2, B-cell translocation gene-1 (BTG-1), reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kmase-1 (clk-1), quiesc , growth arrest DNA damage-mducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentiation of embryo chondrocytes (DEC1) , low density lipoprotein receptor related protein (LDLR) , hamster hairy gene homologue, adipophilm, cyclooxygenase-1 (COX-1) , fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase (LDH) , Bcl-2-mteractmg killer (BIK) , 19 kDa- interacting protein 3, Nιp3L/Nιx, Pim-1, vascular endothelial growth factor (VEGF) , erythropoietm (EPO) , transfemtm, msulm-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, mtegrm alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor), LDHM, phosphoglycerate kinase 1 (PGK-1) , monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor, trisephosphate isomerase, lg associated alpha, interferon regulatory factor 6 (IRF6) , putative ORF KIAA0113, c-fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT- 3), glycogen branching enzyme, TGF beta, brain HHCPA78, mucin 1, RNAse L, Mxi 1, glucose-regulated protein 78 (GRP78), quiescin, lysl oxidase, prostaglandin endoperoxide synthetase, insulin- inducible protein 1, MHC-cIllDQB, myocyte-specific factor 2 (MEF2), bacteria permeating protein, hexokinase, Cap43 (nickel inducible), cyclin G2, carbonic anhydrase IX, TPI, angiogenin, and SDK3, or a biochemically equivalent fragment thereof; and (b) at least one of a second polypeptide, wherein said second polypeptide is a second hypoxia-induced gene product, or a biochemically equivalent fragment thereof.

8. An array of antibodies, comprising: (a) at least one antibody immunoreactive with a protein selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al) , Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activator inhibitor-1 (PAI-1) , macrophage migration inhibitory factor (MIF) , fibronectin receptor, fibronectin 1, lysl hydroxylase, lysyl hydroxylase-2, endothelin-1, endothelin-2, B-cell translocation gene-1 (BTG-1), reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1), quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentiation of embryo chondrocytes (DEC1), low density lipoprotein receptor related protein (LDLR) , hamster hairy gene homologue, adipophilin, cyclooxygenase-1 (COX-1) , fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase (LDH) , Bcl-2-interacting killer (BIK), 19 kDa-interacting protein 3, Nip3L/Nix, Pim-1, vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin, insulin-like growth factor binding protein 3 (IGFBP-3), phosphofructokinase (PFK), aldolase A, aldolase C, integrin alpha 5, integrin alpha 5 receptor, placental growth factor, interleukin-1 (IL-1) receptor, APO-1 (Fas receptor), LDHM, phosphoglycerate kinase 1 (PGK-1), monocarboxylate transporter 3, DNA binding protein A20, peroxisome proliferation receptor, trisephosphate isomerase, lg associated alpha, interferon regulatory factor 6 (IRF6) , putative ORF KIAA0113, c- fos, glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT-3) , glycogen branching enzyme, TGF beta, brain HHCPA78, mucin 1, RNAse L, Mxi 1, glucose-regulated protein 78 (GRP78), quiescin, lysl oxidase, prostaglandin endoperoxide synthetase, insulin-inducible protein 1, MHC-cIllDQB, myocyte- specific factor 2 (MEF2), bacteria permeating protein, hexokinase, Cap43 (nickel inducible), cyclin G2 , carbonic anhydrase IX, TPI, angiogenin, and SDK3; and

(b) at least one of a second antibody, wherein said second antibody specifically binds a second hypoxia-induced gene product or a biochemically equivalent fragment thereof.

9. A method of assaying for expression of hypoxia-inducible genes in a tissue of an animal, comprising:

(a) contacting the proteins of a sample of body fluid or tissue obtained from said animal with the array of Claim 7; and (b) detecting the amount and position of protein from said sample that binds to the array.

10. A method of evaluating a hypoxia-related condition in a tissue of an animal, comprising: (a) contacting the proteins of a sample of body fluid or tissue obtained from said animal with the array of Claim 7; and

(b) detecting the amount and position of protein from said sample that binds to the array.

11. A method of determining the presence of hypoxia in a tissue in an animal, comprising:

12. A method of assaying for expression of hypoxia-mducible genes in a tissue of an animal, comprising:

(a) contacting messenger RNA from a sample of body fluid or tissue obtained from said animal, or cDNA derived therefrom, with the array of Claim 1; and

(b) detecting the amount and position of messenger RNA or cDNA from said sample that binds to the array.

13. A method of evaluating a hypoxia-related condition m a tissue of an animal, comprising:

(b) detecting the amount and position of the messenger RNA or the cDNA that binds to the array.

14. A method of determining the presence of hypoxia in a tissue in an animal, comprising:

(a) contacting messenger RNA from a sample of body fluid or tissue obtained from said animal, or cDNA derived therefrom, with the array of Claim 1; and (b) detecting the amount and position of the messenger RNA or the cDNA that binds to the array.

15. A method of determining the presence of hypoxia in a tissue in an animal, comprising: assaying for either the mRNA transcript or the polypeptide expression product of a gene selected from the group consisting of HIGl , HIG2, annexin V, lipocortin 2, heterogeneous nuclear ribonucleoprotein Al (hnRNP Al ) , Ku autoantigen, phosphoribosylpyrophospha te synthetase, acetoacetylCoA thiolase, ribosomal L , fibroblast growth factor-3 (FGF-3) , EPH receptor ligand, plasminogen activa tor mhιbι tor-1 (PAI-1) , macrophage migra tion inhibitory factor (MIF) , fibronectin receptor, fibronectin 1 , lysl hydroxylase, lysyl hydroxylase-2 , endothelm- 1 , endothelin-2 , B-cell transloca tion gene-1 (BTG-1) , reducing agent and tunicamycin-responsive protein (RTP) , CDC-like kinase-1 (clk-1 ) , quiescin, growth arrest DNA damage-inducible protein 45 (GADD45) , DNA damage-inducible transcript 124498, differentia tion of embryo chondrocytes (DEC1) , low density lipoprotein receptor rela ted protein (LDLR) , hamster hairy gene homologue, adipophilin, cyclooxygenase-1 (COX-1 ) , fructose bisphospha tase, crea tine transporter, fa tty acid binding protein , lacta te dehydrogenase (LDH) , Bcl -2- intera cting killer (BIK) , 19 kDa - interacting protein 3, Nip3L/Nix, Pim-1 , vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , trans ferritin , insulin-like growth factor binding protein 3 (IGFBP-3) , phosphofructokinase (PFK) , aldolase A, aldolase C, integrin alpha 5 , integrin alpha 5 receptor , placental growth factor, interleukin-1 (IL-1 ) receptor, APO-1 (Fas receptor) , LDHM, phosphoglycera te kinase 1 (PGK-1 ) , monocarboxyla te transporter 3, DNA binding protein A20, peroxisome prolifera tion receptor, trisephospha te isomerase, lg associa ted alpha , interferon regula tory factor 6 (IRF6) , puta tive ORF KIAA0113 , c-fos , glucose transporter-like protein 3/glucose transporter isoform 3 (GLUT- 3) , glycogen branching enzyme, TGF beta , brain HHCPA18 , mucin 1 , RNAse L, Mxi 1 , glucose-regula ted protein 18 (GRP18) , quiescin, lysl oxidase, prostaglandin endoperoxide synthetase , insulin- inducible protein 1 , MHC-cIl lDQB, myocyte-specific factor 2 (MEF2) , bacteria permea ting protein , hexokinase, Cap43 (nickel inducible) , cyclin G2 , carbonic anhydrase IX, TPI, angiogenin, and SDK3 in a body fluid or the tissue of said animal.

16. The method of Claim 15, wherein said tissue is a tumor.

17. An array of polynucleotides, comprising:

(a) at least one hypoxia-mducible gene, or the complement thereto, or an at least thirty nucleotide-long fragment thereof, or a sequence which hybridizes thereto, wherein said hypoxia- inducible gene is bound to a solid surface; and

(b) at least one hypoxia-repressible gene, or the complement thereto, or an at least thirty nucleotide-long fragment thereof, or a sequence which hybridizes thereto, wherein said hypoxia-repressible gene is bound to said solid surface.

18. A method of assaying for differential expression of hypoxia-related genes in a tissue of an animal, comprising:

(a) contacting messenger RNA from a sample of body fluid or tissue obtained from said animal, or cDNA derived therefrom, with the array of Claim 17; and

19. An array of polynucleotides, comprising at least one hypoxia-repressible gene, or the complement thereto, or an at least thirty nucleotide-long fragment thereof, or a sequence which hybridizes thereto, wherein said hypoxia-repressible gene is bound to a solid surface.

20. A method of assaying for differential expression of hypoxia-repressible genes in a tissue of an animal, comprising: (a) contacting messenger RNA from a sample of body fluid or tissue obtained from said animal, or cDNA derived therefrom, with the array of Claim 19; and

21. A method for detecting the presence of tumor hypoxia in body fluid, comprising:

(a) measuring the amount of a marker protein in said body fluid; and (b) quantifying the amount of said marker protein, wherein said marker protein is selected from the group consisting of PAI- 1, IGF-BP3, placental growth factor, adipophilin, mucin 1, endothelin-1, endothelin-2, vascular endothelial growth factor (VEGF) , erythropoietin (EPO) , transferritin, EPH receptor ligand, angiogenin, and TGF beta.

22. A method for detecting the presence of a hypoxic condition in a tissue or body fluid of an animal, which method comprises assaying a sample of said tissue or body fluid for the presence of the expression products of one or more genes of claim 4.

23. A method for detecting the presence of a hypoxic condition in a tissue or body fluid of an animal, which method comprises assaying a sample of said tissue or body fluid for the presence of one or more polypeptides of claim 7.

24. A method for detecting the presence of a hypoxic condition in a tissue or body fluid of an animal, which method comprises assaying a sample of said tissue or body fluid for the presence of one or more antibodies of claim 8.