AU3455599A - Hypoxia-inducible human genes, proteins, and uses thereof - Google Patents

Description

WO 99/48916 PCT/US99/06860 HYPOXIA-INDUCIBLE HUMAN GENES, PROTEINS, AND USES THEREOF BACKGROUND OF THE INVENTION 5 a) Field of the Invention The present invention relates to hypoxia-inducible 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. 10 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 15 cancer. Hypoxia-induced gene expression is associated with ischemia (and reperfusion) in many tissues including the liver, heart, eyes, and brain. 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 20 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). 25 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- WO 99/48916 PCT/US99/06860 2 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. s 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). 10 The process of wound healing also involves the induction of gene expression by hypoxia (Anderson et al., Patent No. 5,681,706). TNF-a (tumor necrosis factor-a) 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. is In addition to playing a major regulatory role in the body's response to stress in postnatal life, tissue hypoxia is responsible for regulating expression of genes in the developing embryo, particularly with regard to angiogenesis and vasoformation (Iyer et al. (1998) Genes and Development 12:149-162; Maltepe et al. (1997) Nature 386:403-407). Hypoxia also plays a role in neonatal stress and 20 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 25 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 in maternal WO 99/48916 PCT/US99/06860 3 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 5 involving the differential expression of multiple genes in 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 1o levels that are found in 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 15 of a tumor was an independent prognostic factor (Hoeckel et al. (1996) Semin. Radiat. 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. 20 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. 25 Differeitial-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 WO 99/48916 PCT/US99/06860 4 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. 5 In addition to the identification of hypoxia-induced 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 1o expression of heterologous genes both in vitro 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). 15 SUMMARY OF THE INVENTION The present invention relates to genes whose expression is induced under hypoxic conditions. One aspect of the present invention provides the isolated polynucleotide 2o having the sequence shown as SEQ ID NO: 1 (Fig. 1A), comprising the cDNA of the hypoxia-induced human gene HIG1, and encoding the polypeptide sequence of SEQ ID NO:2 (HIG1; Fig. IB). Polynucleotides with sequences complementary to SEQ ID NO:1, fragments of SEQ ID NO:1 which are at least twelve nucleotides in length, and sequences which hybridize to SEQ ID NO: 1 are also contemplated 25 by the present invention. In particular, one aspect of the invention concerns the fragment of the sequence set forth in SEQ ID NO: 1 comprising nucleotides 62 343, the nucleotides representing the coding sequence of human HIG]. The complements to the coding sequence, at least twelve nucleotide-long fragments of WO 99/48916 PCT/US99/06860 5 the coding sequence, and sequences which hybridize to the coding sequence of HIG1 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 5 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 10 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 15 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: 1 or SEQ ID NO:3, or the complement thereto. Polypeptides of the sequences set forth in SEQ ID NO:2 (HIGI) and SEQ 20 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 immunoreactive to the hypoxia-induced polypeptides HIGI or HIG2 of the present invention are also provided. In still another embodiment, the present invention provides for arrays of 25 polynucleotides or polypeptides corresponding to at least two different hypoxia inducible genes, hypoxia-induced polypeptides, or antibodies immunoreactive with hypoxia-induced polypeptides.

WO 99/48916 PCT/US99/06860 6 Hypoxia-inducible genes suitable for use in the arrays, diagnostic methods, and treatment methods of the invention described herein are not limited to HIG1 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 5 the methods and arrays of the present invention include, but are not limited to, the genes of annexin V, lipocortin 2, hnRNP A ], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl 10 hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate 15 dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1. In one aspect, the present invention provides diagnostic and prognostic tools for assaying for the expression of hypoxia-inducible genes in a tissue of an animal, for determining the presence of hypoxia in a tissue in an animal, and for evaluating a hypoxia-related condition in an animal particularly in order to tailor 20 therapy to a known hypoxic state. The detection of expression products, such as mRNA transcripts or proteins, of the hypoxia-inducible genes of HIG], HIG2, annexin V, lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 25 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related WO 99/48916 PCT/US99/06860 7 protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, or Pim-1, or combinations. thereof, to determine the presence of hypoxia in a tissue or evaluate a hypoxia 5 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-inducible genes, determining the presence of hypoxia in a tissue in an animal, or evaluating a lo hypoxia-related condition in 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 polypeptide from the animal's sample 15 which binds to the sites of the array is determined. Optionally, the gene expression pattern observed may be correlated with an appropriate treatment. Other aspects of the invention concern treating a tissue which is a tumor by first determining the presence of hypoxia in the tumor and, second, treating the tumor with an established form of therapy for cancers such as radiation therapy, 20 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 HIG], HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, 25 fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA WO 99/48916 PCT/US99/06860 8 damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, or Pim-1 in the cell or by 5 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 io 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-inducible and stress repressible genes are also provided. 15 BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the human HIGI cDNA and protein sequences. The nucleotide sequence for the human HIG] gene is shown in Figure 1A from 5' to 3'(SEQ ID 20 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 HIGI is shown in Figure 1B (SEQ ID NO:2). Figure 2 shows the human HIG2 cDNA and protein sequences. The nucleotide 25 sequence for the human HIG2 gene is shown in 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).

WO 99/48916 PCT/US99/06860 9 Figure 3 shows the murine HIGI cDNA and protein sequences. The nucleotide sequence for the murine HIG] gene is shown in Figure 3A from 5' to 3' (SEQ ID NO:5). The coding sequence is underlined. The other regions are untranslated 5 regions (5' and 3' UTR) of the gene. The protein sequence of murine HIGI is shown in Figure 3B (SEQ ID NO:6). Figure 4 shows the HIGI cDNA and protein sequences of seriola quinqueradiata. The nucleotide sequence for this fish HIG] is shown in Figure 4A from 5' to 3' 1o (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 fish HIGI is shown in Figure 4B (SEQ ID NO:8). Figure 5 shows the murine HIG2 cDNA and protein sequences. The nucleotide 15 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 in Figure 5B (SEQ ID NO:10). 20 Figure 6 shows the alignment of human HIGI and HIG2 protein sequences with the HIGI and HIG2 sequences of other species. The HIGI homologues from humans (hHIGI), mice (mHIGI), and fish (seriola quinqueradiate) (flIG1 or GHL1) are aligned in Figure 6A; the HIG2 homologues from humans (hHIG2) and mice (mHIG2) are aligned in figure 6B. 25 Figure 7 schematically illustrates the addition of linkers to cDNA library fragments. The linker addition is followed by PCR amplification.

WO 99/48916 PCT/US99/06860 10 Figure 8 illustrates how the subtraction protocol is used to enrich the tester cDNA library with sequences unique to the tester cDNAs. 5 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. 10 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 15 of oxygen for that particular environment. Normoxic or oxic tissue typically has an oxygen content above about 5%. The terms "hypoxia-induced" or "hypoxia-inducible" 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. 2o Typically, the number of mRNA transcripts of a hypoxia-induced gene would is at least about 20% higher in a hypoxic cell versus a normoxic cell. Preferably, expression of the hypoxia-induced gene is at least about 2-fold higher in hypoxic versus normoxic cells. Most preferably, expression of the hypoxia-inducible gene is at least about 5-fold higher in hypoxic cells versus normoxic cells. 25 A "hypoxia-related condition" in an animal is a condition where hypoxia or altered (typically, enhanced) levels of expression of hypoxia-inducible genes in a tissue of the animal is involved. The hypoxia or altered expression of hypoxia inducible genes may either be a symptom or play a role in the cause, development, WO 99/48916 PCTIUS99/06860 11 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 5 healing. The term "hypoxia-induced protein" or "hypoxia-induced 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 10 example, naturally-occurring polynucleotides or polypeptides present in 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" is 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 20 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 25 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 acids than those specifically disclosed are encompassed, WO 99/48916 PCTIUS99/06860 12 provided that the alternate cDNA encodes mRNA which in turn encodes a protein of this invention. Such modifications may involve the 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 5 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 10 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. 15 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 20 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. 25 "Neutralizing" a polypeptide or protein means inhibiting, partially or wholly, the bioactivity 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.

WO 99/48916 PCT/US99/06860 13 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 NaCl) 5 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 2 +, or Ca 2 +, 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 1o 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 12 nucleotides, and preferably at least about 15 nucleotides, which are complementary to the target 15 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 in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are 20 determined by a translation start codon at the 5' (amino) 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, 25 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 WO 99/48916 PCTIUS99/06860 14 these control sequences need not be present in 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 the configuration of the coding 5 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 10 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 15 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 20 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 25 vitro, 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.

WO 99/48916 PCT/US99/06860 15 "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, s 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 io including translation initiation and termination sites. An expression vector is constructed so that the particular coding sequence is located in 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 15 of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it 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 20 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 in reading frame with and under regulatory control of the control sequences. A "regulatory element" is a segment of DNA to which a transcription 25 factor(s) binds and alters the activity of a gene's promoter either positively (induction) 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 WO 99/48916 PCT/US99/06860 16 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 5 polypeptide or a polynucleotide to a cell. The delivery vehicle is preferably used to deliver an expression vector to a cell or a cell in 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 1o many liposome formulations known to those skilled in the art, such as Lipofectin, may serve as a delivery vehicle. Liposomes are hollow spherical 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 15 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 20 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 25 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.

WO 99/48916 PCT/US99/06860 17 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 biologics. Thus, 5 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 10 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 is We have discovered a novel human gene, herein referred to as HIG1, 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 HIG1 gene (SEQ ID NO: 1; Fig. 1A) and identified the coding sequence to be nucleotides 62-343 of SEQ ID NO: 1. The protein encoded by HIG] comprises the 2o amino acid sequence shown in Figure 1B (SEQ ID NO:2). Polynucleotides with sequences complementary to SEQ ID NO: 1, polynucleotides that are fragments of SEQ ID NO: 1 of at least twelve nucleotides in length and polynucleotides which hybridize to SEQ ID NO: 1 are also within the scope of the present invention. The fragments of SEQ ID NO: 1 are preferably at least 15 nucleotides long. 25 In particular, polynucleotides comprising the nucleotides 62-343 of SEQ ID NO: 1, or complements thereto, or at least twelve nucleotide long fragments thereof, or sequences which hybridize thereto are preferred. Fragments of the coding sequence of HIG] are preferably at least fifteen nucleotides in length.

WO 99/48916 PCT/US99/06860 18 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 5 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 1o the sequence set forth in SEQ ID NO:3 (especially to nucleotides 274-465), are also contemplated. Polypeptides encoded by the polynucleotides of HIG1 (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 15 polypeptides which consist of at least eight amino acids are provided as well. Preferably, the fragments are at least 15 amino 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 HIG1 polynucleotide and polypeptide 20 sequences (Figs. 3, 4, and 6) can be considered biochemically equivalent variations of the human HIG1. The mouse HIG2 polynucleotide and polypeptide sequences (Figs. 5 and 6) are likewise understood to be biochemically equivalent variations of the human HIG1. The polynucleotides of this invention may readily be incorporated within 25 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: 1 (the coding sequence of HIG1) or nucleotides 274-465 of SEQ ID NO:2 (the coding WO 99/48916 PCT/US99/06860 19 sequence of HIG2) is operably linked with appropriate control sequences, such as a promoter. Alternatively, larger fragments of the polynucleotides of SEQ ID NO: 1 or SEQ ID NO:2 which comprise portions of the untranslated regions of the genes s may be used in an expression vector instead. This may be particularly useful when hypoxia-induciblity 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 1o 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 i5 will be apparent to the skilled artisan. Representative methods are disclosed in Molecular Cloning: a Laboratory Manual, 2nd Ed., Vol. 1-3, eds. Sambrook et al., Cold Spring Harbor Laboratory (1989). A HIG] 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 2o animal (ex vivo) or by incorporating the vector into a cell within an animal directly (in vivo). The introduction of a HIG1 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 polylysine or cationic lipids which will help facilitate passage 25 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 HIG1 or HIG2 polynucleotide or expression vector into a cell include, but are not limited to, WO 99/48916 PCT/US99/06860 20 retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and vaccinia viruses. Antisense oligonucleotides complementary to HIG1 and HIG2, particularly those which are capable of blocking expression of HIG1 or HIG2 are provided by 5 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: 1 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 1o may be desirable to facilitate transfer into a cell, stability, or tight binding to the HIG] 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 15 for, and if the probe is properly labeled, quantitate, the hypoxia-induced expression of HIG] 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 HIG 1 or HIG2 20 polypeptides represent still another embodiment of the invention. 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 HIG1 and HIG2 polypeptides would be routine for those skilled in the art. 25 In addition to the identification of the new genes HIGI 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). These genes include annexin V, lipocortin WO 99/48916 PCT/US99/06860 21 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell 5 translocation gene-], reducing agent and tunicamycin-responsive protein, CDC like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density Hpoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, 1o Nip3L/Nix, and Pim-1. 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 15 identified as being hypoxia-inducible (Table 3, Example 8 and Table 5, Example 9). c) Polynucleotide, Polypeptide, and Antibody Arrays. Another aspect of the invention involves the presentation of multiple (at 20 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 25 the array. In an alternative preferred embodiment, the number of different polynucleotides, proteins, or antibodies on the array is greater than about 25, or even greater than about 100.

WO 99/48916 PCTIUS99/06860 22 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 5 selected from HIGI, HIG2, any of the hypoxia-inducible genes listed in Table 1 (below), Table 3 (Example 8, below), and Table 5 (Example 9, below). 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. 10 For instance, in one embodiment of the polynucleotide array, on of the multiple polynucleotides on the array is derived from either the HIG 1 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 15 embodiment, one of the polynucleotides of the array comprises a polynucleotide corresponding to nucleotides 62-343 of SEQ ID NO: 1 (HIG1) 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 2o embodiment of the polynucleotide array, at least one of the polynucleotide sequences of HIG], HIG2, annexin V, lipocortin 2, hnRNP A1, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl 25 hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose WO 99/48916 PCT/US99/06860 23 bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1 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 s from HIG], HIG2, any of the hypoxia-inducible genes represented in Table 1, shown below, any of the expressed sequence tags of hypoxia-inducible genes shown in Table 3 (see Example 8), or any other hypoxia-inducible gene or expressed sequence tag from a hypoxia-inducible gene. It is understood that regardless of which genes are represented on the array, the gene sequences do not 10 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 15 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 20 correlating to hypoxia-inducible genes expressing gene products of a similar function are included on the array. At least two, but preferentially more than two, different hypoxia-induced genes encoding proteins from a single functional category are represented on the array. Examples of seven functional categories of hypoxia-inducible proteins are as follows: (1) glycolytic enzymes/proteins; (2) 25 angiogenesis/tissue remodeling proteins; (3) erythropoiesis/vascular regulatory proteins; (4) metabolic/homeostatic proteins; (5) apoptosis proteins; (6) DNA repair proteins; and (7) cell-cycle proteins. These categories are shown in Table 1, below, along with some representative members of each of the categories. It is WO 99/48916 PCTIUS99/06860 24 understood that the members of each of the seven functional categories of hypoxia-inducible proteins are not limited to the lists shown in Table 1. It is further understood, that the list of functional categories of hypoxia-inducible genes is not.limited to the seven categories listed in Table 1. Again, a preferred 5 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. 10 Table 1. Seven Functional Categories of Hypoxia-Inducible Genes GLYCOLYTIC ENZYMES/PROTEINS lactate dehydrogenase (LDH) phosphoglycerate kinase (PGK) aldolase A L-phosphofructokinase (PFKL) glucose transporter isoform 3 (Glut-3) interleukin-2 glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) adenylate kinase isoenzyme 3 (AK-3) ANGIOGENESIS/TISSUE REMODELING PROTEINS vascular endothelial growth factor (VEGF) platelet-derived growth factor P (PDGFp) transforming growth factor P (TGFp) WO 99/48916 PCT/US99/06860 25 tumor necrosis factor a (TNFa) interleukin-6 (IL-6) interleukin-2 (IL-2) tissue factor fibroblast growth factor (FGF-3) EPH receptor ligand plasminogen activator inhibitor-I (PAl- 1) macrophage migration inhibitory factor (MIF) fibronectin receptor lysyl hydroxylase-2 endothelin-2 ERYTHROPOIEISIS/VASCULAR REGULATORY PROTEINS erythropoietin (EPO) tyrosine hydroxylase heme oxygenase alpha-fetoprotein (AFP) endothelin METABOLIC/HOMEOSTATIC PROTEINS insulin-like growth factor binding protein-I (IGFBP- 1) metallothionein creatine kinase inducible nitric oxide synthase (i-NOS-1) WO 99/48916 PCT/US99/06860 26 epidermal growth factor receptor (EGFR) huntingtin-associated protein 1 (HAP- 1) glucose-regulated protein 78 (GRP78) glucose-regulated protein 90 (GRP90) thioredoxin annexin V glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) heterogeneous nuclear ribonucleoprotein Al (hnRNP A1) gamma-glutamyl cysteine synthetase heavy subunit phosphoribosylpyrophosphate synthetase (PRPP synthetase) acetoacetylCoA thiolase fructose bisphosphatase creatine transporter fatty acid binding protein glucose transporter isoform 3 (Glut-3) adenylate kinase isoenzyme 3 (AK-3) lactate dehyrogenase (LDH) APOPTOSIS PROTEINS insulin-like growth factor binding protein-3 (IGFBP-3) c-myc c-jun Bcl-2-interacting killer (BIK) WO 99/48916 PCTIUS99/06860 27 19 kDa-interacting protein 3 long/Nip3-like protein X (NipP3L/Nix) Pim-1 DNA-REPAIR PROTEINS Ku (70) CELL-CYCLE PROTEINS B-cell translocation gene-I (BTG- 1) reducing agent and tunicamycin responsive protein (RTP) CDC-like kinase- 1 (clk- 1) quiescin (Q6) growth arrest DNA damage-inducible protein 45 (GADD45) 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-inducible genes are displayed on a single array. Although at least two different polynucleotide sequences are 5 required to form the array, in 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 1o 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 HIG 1 or HIG2 and at least 15 one other protein which is a hypoxia induced gene product. Alternatively, the WO 99/48916 PCTIUS99/06860 28 polypeptide array may instead comprise at least one protein selected from the group consisting of HIGI, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen s activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase- 1, quiescin, growth arrest DNA damage-inducible protein 45, DEC 1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase- 1, fructose 10 bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim- 1, or a biochemically equivalent fragment thereof; and at least one of a second polypeptide which is a second hypoxia-induced gene product, or a biochemically equivalent fragment thereof. 15 Another aspect of the invention concerns a polypeptide array comprising at least two different hypoxia-induced proteins, or biochemically equivalent fragments thereof, wherein each hypoxia-induced protein belongs to a different functional category. Alternatively, the polypeptide array comprises at least two different hypoxia-induced proteins or biochemically equivalent fragments thereof, 20 wherein said hypoxia-induced proteins are all proteins belonging to a single functional category. Optionally, the functional category may be selected from the group consisting of glycolytic enzymes/proteins, metabolic/homeostatic proteins, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, and erythropoiesis/vascular regulatory proteins. (See Table 1, 25 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-induced polypeptides are immobilized to form the array, rather WO 99/48916 PCTIUS99/06860 29 than the polypeptide sequences themselves. Each array comprises at least two different antibodies, each of which is immunoreactive with a different hypoxia induced protein. Each of the two antibodies is specifically immunoreactive with the polypeptide expression products of hypoxia-inducible genes, such as, but not 5 limited to, HIGI or HIG2. For instance, in one embodiment, the antibody array comprises at least one antibody immunoreactive with a protein selected from the group consisting of HIGI, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen 10 activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC 1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase- 1, fructose 15 bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim- 1. 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. 20 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 25 antibodies. Methods of constructing arrays of biomolecules, especially polynucleotides, have been previously established in the art. For instance, some methods for preparing particularly high density polynucleotide arrays are WO 99/48916 PCT/US99/06860 30 disclosed in Pirrung et al., Patent No. 5,143,854, Pirrung et al., Patent No. 5,405,783, and Fodor et al., Patent No. 5,510,270, 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 5 immobilizing biomolecules are well known to those of ordinary skill in the art. The material to which the polynucleotides or polypeptides are immobilized in 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. 10 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 HIG1 or HIG2 and a number 15 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 HIG1 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 20 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, 25 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 WO 99/48916 PCT/US99/06860 31 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 5 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 HIG], HIG2, annexin V, lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, fibroblast growth factor-3, 10 EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, 15 adipophilin, cyclooxygenase-], fructose bisphosphatase, creatine transporter, fatty acid binding protein, glucose transporter-like protein III, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1 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. 20 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 25 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.

WO 99/48916 PCT/US99/06860 32 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 5 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 1o 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 15 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 HIG 1 or HIG2 genes, or any of the other hypoxia-induced genes could be achieved, for instance, 2o 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 25 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 WO 99/48916 PCT/US99/06860 33 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 vitro, typically, degradation of the 5 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 10 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 15 in parallel. The diagnostic detection methods mentioned above in regard to in vitro 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 20 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 25 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.

WO 99/48916 PCT/US99/06860 34 Information on the pattern of expression of a combination of hypoxia-induced 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 5 for drugs useful in 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-inducible genes in 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 10 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 is 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 20 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 in an animal may optionally comprise the additional step of correlating the result of the 25 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 WO 99/48916 PCT/US99/06860 35 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 in a tumor by any 5 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 HIG] or HIG2 polynucleotides or the polynucleotides corresponding to the gene sequences of other hypoxia-inducible gene sequences, such as those io listed in Table 1, may be used to attenuate the response of a tissue to hypoxia. These hypoxia-inducible 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-inducible genes within the tissue. is Therefore, in one embodiment, the method of attenuating the hypoxic response of tissue comprises inhibiting the expression of a gene selected from the group consisting of HIG], HIG2, annexin V, lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen 20 activator inhibitor-], macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, 25 fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1 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 WO 99/48916 PCT/US99/06860 36 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 HIG 1 or HIG2 proteins or other expression products of hypoxia-inducible genes may instead be targeted 5 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 lo HIGI, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin 15 responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage inducible protein 45, DEC 1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase- 1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim- 1. In this method an 20 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 25 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 WO 99/48916 PCT/US99/06860 37 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 5 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 10 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 HIGI and HIG2 polypeptides may be introduced into the animal, preferably human, containing the tissue to be treated. 15 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 HIGI, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku 20 autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase- 1, quiescin, growth arrest DNA 25 damage-inducible protein 45, DEC 1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase- 1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, or Pim- 1 to see if inactivation WO 99/48916 PCT/US99/06860 38 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. 5 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 10 sequence on the vector to take place. The coding sequence of the expression vector comprises the sequence of at least one of the genes HIG], HIG2, annexin V, lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7,fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-], macrophage migration 15 inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, 20 fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, or Pim-1. 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 25 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 WO 99/48916 PCTIUS99/06860 39 the use of liposomes, hydrogels, controlled-release polymers, or any of the other vehicles known in 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-inducible genes, we modified and 5 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 io only fragments of those sequences contained within 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 inducible and stress-repressible genes. The methods identify differences between 15 mRNA from cell populations exposed to different stress conditions. A representative protocol for the identification of stress-inducible genes is outlined in detail in a specific example below (Example 1). The method for identifying stress-inducible or stress-repressible genes and fragments of genes involves first subjecting one of two populations of cells to 20 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 25 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 WO 99/48916 PCTIUS99/06860 40 have a defined DNA sequence (20 to 24 base pairs in 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 5 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 Laboratory Manual, 2nd. ed, Cold Spring Harbor Laboratory Press, herein incorporated by reference. 10 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 15 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 20 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. 25 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 WO 99/48916 PCTIUS99/06860 41 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 5 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". 1o 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. 15 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 20 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 25 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 WO 99/48916 PCT/US99/06860 42 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 5 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 1o 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 15 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 20 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 25 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 WO 99/48916 PCT/US99/06860 43 be found in Molecular Cloning: A Laboratory 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 5 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) Examples 10 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 15 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 pg of poly A+ mRNA from both HCE.E6E7 cells cultured under hypoxic (5% C0 2 /5% H 2 /90% N 2 for 16 hours at 37 0 C) conditions and 2o HCE.E6E7 cells cultured under aerobic (5% CO 2 / 20% 02 / 75% N 2 at 37 0 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% 25 N 2 , 5% C0 2 and 5% H 2 . 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.

WO 99/48916 PCT/US99/06860 44 One-fifth of the cDNA product (approximately 1-1.5 ptg) 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 ig/ml bovine serum albumin for 60 min at 37 0 C. The reaction mixture was extracted with phenol and chloroform, 5 precipitated with ethanol, redissolved in 1 OuL 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 1o 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' 15 (SEQ ID NO: 11) XATGGTCGAATAAGTTAAGCCAGGAGAGCGTGTCCTAC-5' (SEQ ID NO:12) 5'-TTTTTGTAGACATTCTAGTATCTCGTCAAGTCGGAAGGATGCATG 3'(SEQ ID NO:13) 20 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 25 dissolved in 10 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 buffer (10 pLM of each oligomer), then heat-denatured and slowly cooled to room temperature before use in a ligation reaction.

WO 99/48916 PCT/US99/06860 45 Next, 1 ptg of the Nla III cleaved cDNA was ligated in a 100 ptL volume of 1 piM double-stranded linker, 5% polyethylene glycol, 50 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 1 mM ATP, and 1 mM DTT at 16 0 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 5 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 1o 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 15 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 ptg aliquot) was amplified in a standard PCR buffer containing 1 jM primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , and 0.01% gelatin. Before PCR amplification, the nicked PCR template had to be 2o repaired by TAQ polymerase during a 5-min extension reaction at 720 C. After this initial incubation, a standard PCR reaction of 35 cycles (940 C, 30s; 560 C, 30s; 720 C, 60s) was performed in a Perkin Elmer DNA Thermal Cycler. The oligonucleotide primers used in the amplification step were as follows: 25 5'-CCAGCTTATTCAATTCGGTCC-3' (SEQ ID NO:15) 5'-GTAGACATTCTAGTATCTCGT-3' (SEQ ID NO:16) WO 99/48916 PCTIUS99/06860 46 (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. 5 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 pg of the biotinylated driver cDNA with 0.1 pg of the test cDNA. The mixture was lyophilized in a 0.5 mL microfuge tube and carefully redissolved in 2 p1L of 50 10 mM HEPES (pH 7.5), 10 mM EDTA, 1.5 mM NaCl, and 2% sodium dodecyl sulfate (SDS). This very small amount of solution was overlaid with 50 pL of mineral oil to prevent evaporation, and the tube was place in the thermal cycler and heated at 95 0 C for 10 min. It was then slowly cooled to 68 C over a period of 1 h, after which the incubation at 680 C was continued for a further 4 hours. At 1 the end of the incubation, 100 pl of the same HEPES buffer at 68 0 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 p.L slurry containing 1 mg of M-280 20 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 25 pL 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 pL WO 99/48916 PCT/US99/06860 47 (one-tenth volume) of the subtracted cDNA preparation was placed in 100 pL of PCR buffer containing 1 pig of Escherchia Coli single-stranded binding protein (Perfect Match T M , Stratagene). The cDNA was amplified during 25 PCR cycles (94 0 C, 30 s; 54 0 C, 30 s; 72 0 C, 60 s), and the product was analyzed by ethidium 5 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 10 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 15 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 20 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 25 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 WO 99/48916 PCT/US99/06860 48 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-induced ESTs corresponded to previously identified genes or ESTs. The frequencies of EST sequences among the 500 randomly chosen cDNA 5 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 HIGI EST and the HIG2 EST, corresponded to no known genes. Because these most frequently repeated clones were unknown, the full-length cDNAs representing HIG1 and HIG2 were 1o isolated (see Example 3, below). All the ESTs present in 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 in Siha cervical carcinoma cells (and/or in HCE.E6E7 cells). Selected probes representing ESTs found more than once 15 were applied to Northern blots of total RNA from cell cultures harvested following different aerobic and hypoxic exposures to verify hypoxia inducibility or repressibility. For instance, the northern blot assays were used to confirm that, x-tubulin mRNA, detected in the HCE.E6E7 aerobic enrichment library, decreased in response to hypoxia in HCE.E6E7 cells, whereas mRNA 20 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. # of hits tag gene to which EST accession # response comment name corresponds of gene to hypoxia* 106 HIG 1 HIG 1 induced novel gene 98 HIG2 HIG2 induced novel gene WO 99/48916 PCT/US99/06860 49 48 HIG3 GAPDH J04038 induced inducibility prev. known 11 HIG4 HNRNP X12671 induced 11 HIG5 Annexin V U01691 induced 8 HIG6 AcetoacetylCoA S70154 induced** thiolase 7 HIG7 Tissue Factor X67698 induced inducibility prev. known 7 5-2A bp X76388 not induced 6 unknown gene clone 68 no signal 5 Alu-like not determined 5 HIG8 Lipocortin 2 M14043 induced 5 HIG9 Ribosomal L7 X57959 induced 4 unknown gene clone 24 not induced 3 Vacuolar ATPase X71490 no signal 3 HIG1O PRPP synthase D00860 induced 3 Alu-like not determined 2 RNA poll 40Kd AF047441 not induced subunit 2 HIG 11 thioredoxin X77584 induced inducibility prev. known 2 hSRP 1 (nuc loc) U28386 not induced 2 HIG12 Ku(70) J04611 induced 2 Sm protein F X85382 not induced 1 168 different clones * as determined by Northern blot ** minor 4.2kB acetoacylCoA thiolase message only is induced WO 99/48916 PCT/US99/06860 50 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 [tg of total RNA was denatured with glyoxal and size fractionated on a 1% agarose phosphate gel. The gel was capillary transferred to 5 Hybond nylon (Schleicher and Shuell) and UV cross-linked. Probes were radiolabeled by random priming of gel-purified full length HIG1, or a fragment of HIG2 containing only the coding sequence in a Stul fragment (Rediprime, Amersham). Hybridization was carried out in 0.5 M Na 2

HPO

4 , 7% SDS, 1 mM EDTA at 56 0 C for HIGI and 65 0 C for HIG2, washed to 0.2-0.5 x SSC at 56 0 C or 1o 650C, 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 in Table 2, above. The HIGI and HIG2 sequences both demonstrated hypoxia-inducibility in the Northern blot assay. 15 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, Hct1 16 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 20 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 Hct1 16 cells after 6 h of hypoxia; (4) there is no detectable HIG2 mRNA in HCFs and HCF.E6E7s; (5) in SiHa cells, HIG2 25 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/m 2 ), y-irradiation (6 Gy), MMS (100 pg/mL for 1 h), serum deprivation (0.1%), or glucose starvation (4%, <1 mM); (7) HIG2 WO 99/48916 PCTIUS99/06860 51 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 5 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 HIG1 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 Table 2. ESTs 1o 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. 15 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 20 (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 25 Sequence The HIG2 EST (142 bp) was used to probe a conventional cDNA library constructed froin mRNA isolated from SiHa cells exposed to 16 h hypoxia to obtain the full-length cDNA clone HIG2. This library was probed with WO 99/48916 PCT/US99/06860 52 radiolabelled HIG2 tag using conventional methods. Full length HIG1 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 5 sequence using RT-PCR of SiHa RNA isolated after a 16 h hypoxia treatment. The full-length HIG1 cDNA was then cloned and sequenced to confirm the predicted sequence. The full-length cDNA sequence of HIG1 is shown in Figure 1A. The full length cDNA sequence of HIG2 is shown in Figure 2A. The translations of the 10 putative open reading frames from HIG] and HIG2 are listed in Figure lB and 2B, respectively, and both encode small peptides (95 and 64 aa residues respectively) without obvious functional motifs.

WO 99/48916 PCT/US99/06860 53 Example 4. Hypoxic induction of HIG1 and HIG2 in cervical cancer cell lines. Because HIG1 and HIG2 represent two novel genes whose functions are unknown, these genes were investigated in more detail. The expression of HIG1 and HIG2 was examined in a series of human cervical cancer cell lines (SiHa, 5 CaSki and C33a) under oxic and hypoxic conditions in vitro. (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 HIG] 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 io 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. is Example 5. Hypoxic induction of HIG] and HIG2 in tumor xenografts. The hypoxic induction of HIG] 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 20 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 25 resulted in increased tumor hypoxia as measured by ependorff electrode and increased HIG] and HIG2 expression by 1.2 and 2.4 fold respectively. The moderate level of HIG] induction in vivo is not unexpected, due to the in vitro data. The portion of the human gene used for a probe in these experiments has WO 99/48916 PCT/US99/06860 54 low homology with mouse RNA and under the conditions used, did not cross hybridize. Example 6. Specificity of the induction of HIG1 and HIG2. 5 We next investigated whether HIG 1 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 1o genes. For stress treatments, cells were plated overnight and then treated the next day with either 256 nm UV at 1.2 J/m 2 /sec, or gamma irradiation from 1 37 Cs source at 3.8 Gy/min. Glucose and serum deprivation experiments were performed by washing the cells three times in phosphate-buffered saline (PBS) and replacing the indicated media (glucose free RPMI with dialyzed serum, or 15 O.1%FBSRPMI). Northern blot analyses was performed on RNA isolated from C33a cells exposed to these stresses. HIG] was poorly responsive to hypoxic stress over this timecourse, but strongly induced by glucose deprivation. HIG2 was induced strongly by hypoxia, the hypoxia-mimetic stress desferoxamine (DFO), and 20 glucose deprivation. UV light seemed to have little effect upon either HIG] or HIG2 expression. In contrast, while ionizing radiation did not change HIG] 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 25 HIG2 expression. Example 7. Identification of HIG] and HIG2 sequences from non-human species.

WO 99/48916 PCT/US99/06860 55 A search of the NCBI-dbEST database for fragments of genes from other species that might represent evolutionarily conserved orthologues identified overlapping mouse EST fragments that encode for similar peptides to the human version of HIG1 and HIG2. The murine HIG] and HIG2 orthologues are shown 5 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 quinqueradiata) in the database that coded for a HIG 1 orthologue (Figure 4A and 4B). A sequence comparison of the HIGI homologues is shown in Figure 1o 6A. A sequence comparison of the HIG2 homologues is shown in Figure 6B. We confirmed the existence of murine HIGI 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 15 entire murine HIG2 cDNA, but were only able to make primers that would amplify the coding sequence for murine HIG]: mHIG1 forward primer (SEQ ID NO: 17): 5'-CCGATCTAGAGGAAGGGACCCCGCGTCTCGGA-3' 20 miHIGI 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): 25 5'-GGCGCTCGAGCACATGTGCATTACACTGGAGA-3' These primers were then used to amplify the coding sequences of HIG] OR HIG2 from reverse-transcribed RNA isolated from the murine squamous cell tumor cell WO 99/48916 PCT/US99/06860 56 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 mHIG1 (murine HIG1) and mHIG2 5 (murine HIG2) have hypoxia-inducible species of RNA by this analysis. Murine HIG] 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 10 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. 15 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 20 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 25 cells and cDNA probe was generated using MoML reverse transcriptase. 2 pg mRNA was incubated with 500 ng of oligonucleotide primer (T) 18 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 [ 33 ]P dCTP and 200U reverse transcriptase.

WO 99/48916 PCT/US99/06860 57 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 5 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 10 was induced in hypoxic cells, shown with the ratio of their expression in hypoxic cells over their expression in oxic cells. ratio Acc. No. ratio Acc. No. ratio Acc. No. ratio Acc. 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 W24548 5.454 W07146 5.678 H04904 6.525 N27733 5.699 T48772 5.209 AA101069 6.453 H82707 3.146 N94304 4.095 N94916 4.599 W24109 7.825 W56465 4.218 H70730 6.52 H14897 4.996 R17409 9.999 N31409 1.659 AA053856 1.159 R34659 4.932 W17090 9.999 H19264 8.836 W05763 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 W38635 3.852 H86677 9.999 AA196038 9.238 AA005185 8.59 T54127 4.653 H67329 9.999 W55913 4.654 T95404 4.289 R26319 4.993 W19173 5.143 R94248 9.262 R19946 6.048 W07082 5.641 W06851 9.999 R85589 4.308 AA068998 3.12 AA194330 6.186 W00378 2.9 H50204 5.203 N47831 4.594 N31417 4.458 AA204792 Substitute Sheet (Rule 26) WO 99/48916 PCT/US99/06860 58 4.333 H52973 4.175 AA151009 2.424 AA069173 4.64 W48584 9.999 H60510 5.33 W46682 3.645 W52472 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 W87527 5.664 H71710 2.356 R97269 8.468 R13125 5.965 W47502 2.494 W17237 3.68 H86214 9.999 W52400 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 W46165 4.846 R18798 3.457 T87920 2.867 N39173 8.287 W24084 4.648 T78246 5.766 W47525 3.251 R76943 9.999 T77247 6.726 H29713 5.493 W32575 4.139 W07720 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 W32710 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 W17182 3.534 AA074177 6.115 H83517 3.9 W38235 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 6.498 AA034932 2.549 R70082 5.458 W20511 3.258 AA085385 9.999 H41372 4.698 W32969 3.742 T40473 4.453 W49770 9.999 H08885 4.038 R66920 5.132 R82723 4.056 W37672 8.221 H90627 6.586 W25455 5.886 T50788 2.503 W38097 4.812 T91423 7.516 W38478 3.266 AA036758 5.118 N48838 Substitute Sheet (Rule 26) WO 99/48916 PCT/US99/06860 59 5.535 N49031 1.961 AA039447 3.564 W46219 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 W38424 5.314 H18129 4.396 R07212 3.101 T48613 6.954 H75477 4.118 H93835 4.917 AA128281 3.236 H12952 3.507 W31707 5.306 AA167017 4.303 N39630 2.553 T87585 3.968 N40606 5.012 W24939 4.165 W39234 2.961 R76842 3.417 W67757 3.887 W38826 1.983 H68587 4.505 N54105 5.57 AA074760 3.791 H62991 3.358 H78277 3.424 W72986 2.311 H18350 4.853 AA132801 2.968 T87597 3.738 H53662 2.707 W31757 2.065 R54128 3.142 H64978 1.682 W05551 3.252 T89571 3.35 AA214334 4.376 W16557 2.942 H62026 3.202 W07148 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 W49687 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 W48763 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 W05089 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 W07144 1.627 W31940 2.021 W48791 3.593 AA131550 4.133 R06175 3.545 W38638 3.686 R80458 4.34 N42413 3.912 N46036 1.634 R60752 1.812 H29706 2.313 R62688 2.535 W78057 2.917 R28459 3.501 AA130339 Substitute Sheet (Rule 26) WO 99/48916 PCT/US99/06860 60 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 W04913 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 W01963 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 W07026 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 W31182 4.116 T83199 2.606 N73091 3.168 H91401 2.371 T91436 2.479 H19169 2.648 T56084 2.074 W24718 2.951 T54610 3.768 W37172 3.4 W37084 2.318 T54602 2.735 AA001324 2.146 H70732 3.531 W07043 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 W15521 3.945 R69162 2.953 H79197 3.416 H12419 Substitute Sheet (Rule 26) WO 99/48916 PCT/US99/06860 61 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 W00950 2.588 R70441 3.464 W16685 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 W17002 3.067 T87472 4.545 H18766 2.984 W20484 3.058 H61812 3.167 R87818 1.377 AA136789 2.937 N45602 2.214 W88806 2.364 H45241 2.927 R14907 3.318 T54086 1.678 W17311 3.476 H12508 2.992 H61280 3.688 R87413 3.25 W19104 3.13 AA167039 3.432 R14301 2.441 R87193 3.287 T87358 2.927 R80475 2.521 H92713 2.711 W03009 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 W78830 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 Substitute Sheet (Rule 26) WO 99/48916 PCT/US99/06860 62 3.673 H51373 3.371 H42536 3.114 T51726 3.049 T85153 3.137 H09884 2.421 N53255 3.202 T98755 2.379 R70814 3.201 AA194172 2.33 W47650 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 5 cells over their expression in hypoxic cells. ratio Acc. No. ratio Acc. No. ratio Acc. No. ratio Acc. 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 W81199 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 Substitute Sheet (Rule 26) WO 99/48916 PCTIUS99/06860 63 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 W72875 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 W01642 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 W00931 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 W01319 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 W03129 6.3 N90458 4.448 R63455 9.999 R77028 4.156 W03125 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 Substitute Sheet (Rule 26) WO 99/48916 PCTIUS99/06860 64 5.379 H86672 9.999 N41573 8.644 AA070426 4.742 N32681 9.999 T95210 9.723 H52741 9.999 W31524 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 W16980 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 AA088387 Substitute Sheet (Rule 26) WO 99/48916 PCTIUS99/06860 65 6.179 R09942 3.664 R98517 3.87 W02494 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 AA070943 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 W02353 4.142 T83106 2.846 N32669 5.892 R32571 4.626 W01717 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 5.419 H52350 2.552 W06829 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 W16814 4.007 AA069114 3.583 W04937 4.449 H62445 2.133 R85191 2.606 W92014 2.521 AA047388 4.196 R90798 2.161 H50471 4.047 W87655 4.349 R52735 7.226 R35606 3.275 H30629 3.891 W01911 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 W72149 Substitute Sheet (Rule 26) WO 99/48916 PCT/US99/06860 66 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 W45582 3.336 T97872 3.677 W40228 5.781 T48877 4.173 T82054 3.553 H51540 6.08 AA101477 3.152 AA033945 3.359 T98110 2.63 N44493 3.907 W00916 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 W17108 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 W30866 4.006 W25183 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 Substitute Sheet (Rule 26) WO 99/48916 PCT/US99/06860 67 Example 9. Analysis of Gene Expression under Hypoxia using GEMTM microarrays 5 The hypoxic induction of genes in FaDu cells was analyzed by comparing the expression of genes in FaDu cells exposed to hypoxic conditions (5% C0 2 /5%

H

2 /90% N 2 for 16 hours at 37 C) to those exposed to normal, oxic conditions. This differential expression was analyzed using GEMTM technology provided by Genome Systems Inc. Messenger RNA (mRNA) was extracted from hypoxic 1o 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 pl Trizol was added 50 100 mg of fresh frozen cells. The cells were then immediately homogenized. 500 pl Trizol was then added, and the sample was mixed well. The sample was 15 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,OOOX 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 20 isopropanol was added and the samples were incubated for 10 minutes at room temperature. The RNA was pelleted by centrifuging at 12,OOOX 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 25 minutes at room temperature and then dissolved in 10 pl nuclease-free water and stored at -80 0 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, WO 99/48916 PCTIUS99/06860 68 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 H 2 0 or TE. For 50 tl of elution from the OligoTex column, 40 tl of IX TE and 1 p.l of glycogen (5 5 mg/ml) was added. Then 120 pl 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 pl 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 io pellet was then dissolved in DEPC H20 to a concentration of 50 ng/ptl. 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 15 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. 20 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, 25 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.

WO 99/48916 PCT/US99/06860 69 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 5 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 cells (versus oxic cells) are listed below 1o in Table 5, along with their levels of induction, functional category if known, and GenBank accession number. Table 5. Genes Induced by Hypoxia in FaDu cells. Functional Gene GenBan Induction Category k fibroblast growth factor (FGF-3) X14445 5.6 Angiogenesis vascular endothelial growth factor X54936 4.0 (VEGF) EPH receptor ligand M57730 3.9 plasminogen activator inhibitor -1 (PAl- M14083 13.5 1) Tissue macrophage migration inhibitory factor M25639 9.3 Remodeling (MIF) fibronectin receptor X06256 6.0 lysyl hydroxylase-2 U84573 4.8 endothelin-2 M65199 4.4 B-cell translocation gene-I (BTG-1) X61123 4.9 WO 99/48916 PCT/US99/06860 70 reducing agent and tunicamycin- D87953 3.8 responsive protein (RTP) Cell Cycle CDC-like kinase (clk-1) L29219 3.0 quiescin (Q6) U97276 2.9 growth arrest DNA damage-inducible L24498 2.9 protein 45 (GADD45) Differentiation of Embryo AB0040 8.2 Chondrocytes- 1 (DEC 1) 66 low density lipoprotein receptor (LDLR) X13916 6.0 related protein Miscellaneous human hairy gene homologue (HRY) L19314 4.7 adipophilin X97324 3.9 cyclooxygenase-1 (COX-1) U63846 3.0 fructose bisphosphatase AF0549 4.5 87 creatine transporter U36341, 4.1 U41163 Metabolism fatty acid binding protein M94856 3.9 glucose transporter-like protein III M20681 3.4 (GLUT-3) lactate dehyrogenase (LDH) X02152 2.9 insulin-like growth factor (IGFBP-3) M35878 11.1 Apoptosis Bcl-2-interacting killer (BIK) X89986 7.6 19 kDa-interacting protein 3 long/Nip3- AB0047 5.3 like protein X (NipP3L/Nix) 88 Pim-1 M54915 4.4 EST AA0447 5.0 68 WO 99/48916 PCT/US99/06860 71 EST AA0545 4.7 43 KIAA0242 D87684 3.9 Unknown EST AA1562 3.5 40 EST AA1868 3.4 03 EST AA0630 3.0 84 EST H15429 2.9 EST U60873 2.9 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 5 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 (57)

1. An isolated polynucleotide comprising: the sequence set forth in SEQ ID NO:1, the complement thereof, an 5 at least twelve nucleotide-long fragment thereof, or a sequence which hybridizes thereto; or the sequence set forth in SEQ ID NO:3, the complement thereof, an at least twelve nucleotide-long fragment thereof, or a sequence which hybridizes thereto. 10

2. The isolated polynucleotide of Claim 1, which comprises nucleotides 62 343 of SEQ ID NO: 1, the complement thereof, an at least twelve nucleotide-long fragment thereof, or a sequence which hybridizes thereto. 15

3. The isolated polynucleotide of Claim 1, which comprises nucleotides 274 465 of SEQ ID NO:3, the complement thereof, an at least twelve nucleotide-long fragment thereof, or a sequence which hybridizes thereto.

4. An expression vector comprising: 20 (i) the polynucleotide of Claim 1; and (ii) a promoter, wherein said promoter is operably linked to said polynucleotide.

5. A delivery vehicle comprising the polynucleotide of Claim 1. 25

6. An isolated cell comprising the polynucleotide of Claim 1. WO 99/48916 PCTIUS99/06860 73

7. An antisense oligonucleotide capable of blocking expression of the polynucleotide of Claim 1.

8. A probe comprising a polynucleotide of Claim 1, or a fragment thereof, that 5 is at least 12 nucleotides in length.

9. An array of polynucleotides, comprising: (a) at least one polynucleotide of Claim 1; and (b) a second polynucleotide, wherein said second: polynucleotide io comprises the sequence of a second hypoxia-inducible gene, or an at least twelve nucleotide-long fragment thereof.

10. An isolated polypeptide encoded by the polynucleotide sequence of Claim 1, or a biochemically equivalent fragment thereof. 15

11. An isolated polypeptide comprising SEQ ID NO:2, a biochemically equivalent fragment of SEQ ID NO:2, SEQ ID NO:4, or a biochemically equivalent fragment of SEQ ID NO:4. 20

12. An array of polypeptides, comprising: (a) at least one polypeptide of Claim 11; and (b) at least one of a second polypeptide, wherein said second polypeptide is a hypoxia-induced gene product or a biochemically equivalent fragment thereof. 25

13. An antibody specifically immunoreactive with a polypeptide of Claim 11. WO 99/48916 PCT/US99/06860 74

14. An array of antibodies, comprising: (a) at least one antibody of Claim 13; and (b) at least one of a second antibody, wherein said second antibody specifically binds a second hypoxia-induced gene product or a fragment thereof. 5

15. An array of polynucleotides, comprising at least two different hypoxia inducible genes, or complements thereto, or at least twelve nucleotide-long fragments thereof, or sequences which hybridize thereto. 10

16. The array of Claim 15, comprising at least two different polynucleotides, each comprising a hypoxia-inducible gene, or an at least twelve 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 15 proteins, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, and erythropoiesis/vascular regulatory proteins.

17. The array of Claim 15, comprising at least two different polynucleotides, each comprising a hypoxia-inducible gene, or an at least twelve nucleotide-long 20 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, and erythropoiesis/vascular regulatory proteins. 25

18. The array of Claim 17, comprising at least two different polynucleotides, each comprising a hypoxia-inducible gene, or an at least twelve nucleotide-long WO 99/48916 PCT/US99/06860 75 fragment thereof, or the complement thereto, wherein all of the hypoxia-inducible genes encode angiogenesis or tissue remodeling proteins.

19. The array of Claim 15, comprising: 5 (a) at least one gene selected from the group consisting of HIG], HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl 10 hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate 15 dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1, or an at least twelve nucleotide-long fragment thereof; and (b) a second polynucleotide, wherein said second polynucleotide comprises a second hypoxia-inducible gene or an at least twelve nucleotide-long fragment thereof. 20

20. An array of polypeptides, comprising the polypeptide expression products of at least two hypoxia-inducible genes, or biochemically equivalent fragments thereof. 25

21. The array of Claim 20, 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/proteins, apoptosis proteins, WO 99/48916 PCT/US99/06860 76 DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, and erythropoiesis/vascular regulatory proteins.

22. The array of Claim 20, comprising at least two different hypoxia-induced s proteins or biochemically equivalent fragments thereof, wherein said hypoxia induced proteins are all 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, and erythropoiesis/vascular regulatory proteins. 10

23. The array of Claim 20, comprising: (a) at least one protein selected from the group consisting of HIGI, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, is fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin responsive protein, CDC-like kinase- 1, quiescin, growth arrest DNA damage inducible protein 45, DEC 1, low density lipoprotein receptor related protein, 20 hamster hairy gene homologue, adipophilin, cyclooxygenase- 1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim- 1, or a biochemically equivalent fragment thereof; and (b) at least one of a second polypeptide, wherein said second 25 polypeptide is a second hypoxia-induced gene product, or a biochemically equivalent fragment thereof. WO 99/48916 PCT/US99/06860 77

24. An array of antibodies, comprising at least two different antibodies specifically immunoreactive with the polypeptide expression products of hypoxia inducible genes. s

25. An array of antibodies of Claim 24, comprising: (a) at least one antibody immunoreactive with a protein selected from the group consisting of HIG1, HIG2, annexin V, lipocortin 2, hnRNP A1, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen 1o activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase- 1, quiescin, growth arrest DNA damage-inducible protein 45, DEC 1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose 15 bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim- 1; 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. 20

26. 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 24; and 25 (b) detecting the amount and position of protein from said sample that binds to the array. WO 99/48916 PCT/US99/06860 78

27. 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 24; and 5 (b) detecting the amount and position of protein from said sample that binds to the array.

28. The method of Claim 27, wherein said hypoxia-related condition is cancer, ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, 10 or stroke.

29. A method of diagnosing a hypoxia-related condition in an animal, said method comprising: (a) evaluating the hypoxia-related condition in a tissue of the animal by is the method of Claim 27; and (b) correlating the result of the determination of step(a) with an appropriate treatment for the animal.

30. A method of treating a hypoxia-related condition in a tissue of an animal, 20 said method comprising: (a) diagnosing the hypoxia-related condition in the tissue of the animal by the method of Claim 29; and (b) treating said animal with said appropriate treatment. 25

31. A method of determining the presence of hypoxia in a tissue in an animal, comprising: (a) contacting the proteins of a sample of body fluid or tissue obtained from said animal with the array of Claim 24; and WO 99/48916 PCT/US99/06860 79 (b) detecting the amount and position of protein from said sample that binds to the array.

32. A method of assaying for expression of hypoxia-inducible genes in a tissue 5 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 15; and (b) detecting the amount and position of messenger RNA or cDNA from 10 said sample that binds to the array.

33. A method of evaluating a hypoxia-related condition in a tissue of an animal, comprising: (a) contacting messenger RNA from a sample of body fluid or tissue 15 obtained from said animal, or cDNA derived therefrom, with the array of Claim 15; and (b) detecting the amount and position of the messenger RNA or the cDNA that binds to the array.

34. The method of Claim 33, wherein said hypoxia-related condition is cancer, 20 ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, or stroke.

35. A method of diagnosing a hypoxia-related condition in an animal, said method comprising: 25 (a) evaluating the hypoxia-related condition in a tissue of the animal by the method of Claim 33; and (b) correlating the result of the determination of step (b) with an appropriate treatment for the animal. WO 99/48916 PCT/US99/06860 80

36. A method of treating a hypoxia-related condition in an animal, said method comprising: (a) diagnosing the hypoxia-related condition in the tissue of the animal by the method of Claim 35; and 5 (c) treating said animal with said appropriate treatment.

37. 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 10 obtained from said animal, or cDNA derived therefrom, with the array of Claim 15; and (b) detecting the amount and position of the messenger RNA or the cDNA that binds to the array. is

38. A method of treating a hypoxia-related condition in a tissue in an animal, comprising: (a) assaying for either the mRNA transcript or the polypeptide expression product of at least one gene selected from the group consisting of HIG], HIG2, annexin V, lipocortin 2, hnRNP A ], Ku autoantigen, 20 phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA 25 damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, glucose WO 99/48916 PCT/US99/06860 81 transporter-like protein III, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1 in a body fluid or the tissue of said animal; (b) correlating the result of the determination of step (a) with an appropriate treatment for the hypoxia-related condition; and 5 (c) treating said tissue with said appropriate treatment.

39. The method of Claim 38 wherein said hypoxia-related condition is cancer, ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, or stroke. 10

40. The method of Claim 39, wherein (a) said hypoxia-related condition is cancer; and (b) said appropriate treatment is selected from the group consisting of radiation therapy, chemotherapy, and surgery. 15

41. 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 HIG], HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate 20 synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-], macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], 25 low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, glucose transporter-like protein III, lactate WO 99/48916 PCT/US99/06860 82 dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1 in a body fluid or the tissue of said animal.

42. The method of Claim 41, wherein said tissue is a tumor. 5

43. A method of treating a tumor, comprising: (a) determining the presence of hypoxia in a tumor by the method of Claim 42; and (b) treating said tumor with an established form of therapy for cancer. 10

44. The method of Claim 43, wherein said established form of therapy for cancer is selected from the group consisting of radiation therapy, chemotherapy, and surgery. 15

45. The method of diagnosing a hypoxia-related condition in an animal, comprising: determining the presence of hypoxia in a tissue in the animal by the method of Claim 41. 2o

46. A method of attenuating the hypoxic response of tissue in an animal, comprising: inhibiting the expression of a gene selected from the group consisting of HIG], HIG2, annexin V lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, 25 fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA WO 99/48916 PCT/US99/06860 83 damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1. 5

47. A method of treating a hypoxia-related condition in an animal, comprising: attenuating the hypoxic response of a tissue in said animal by the method of Claim 46. 10

48. A method of attenuating the hypoxic response of a tissue, comprising: neutralizing a protein selected from the group consisting of HIG1, HIG2, annexin V, lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 15 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose 20 bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1.

49. A method of treating a hypoxia-related condition in an animal, comprising: attenuating the hypoxic response of a tissue in said animal by the method of 25 Claim 48. WO 99/48916 PCT/US99/06860 84

50. A method of treating a hypoxia-related condition in a tissue, comprising: (a) introducing an expression vector into said tissue; and (b) expressing the coding sequence of said expression vector within said tissue, wherein said coding sequence is a gene selected from the group consisting of 5 HIG], HIG2, annexin V, lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L 7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-i, reducing agent and 10 tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1. 15

51. A method of treating a hypoxia-related condition in a tissue, comprising: administering to the tissue a polypeptide expressed by a gene selected from the group consisting of HIG], HIG2, annexin V, lipocortin 2, hnRNP A], Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, 20 ribosomal L7,fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-], reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC], low density lipoprotein receptor 25 related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1. WO 99/48916 PCT/US99/06860 85

52. A method for identifying stress-inducible genes and fragments of genes, comprising the steps of: (a) subjecting one of two populations of cells to stress; (b) preparing cDNA libraries from the two populations of cells; 5 (c) digesting the cDNA libraries with restriction enzymes and then ligating linker sequences to the ends of said digested cDNA; (d) amplifying the cDNA library from the non-stressed cells with tagged primers by means of the polymerase chain reaction and amplifying the other cDNA library from the stressed cells with non-tagged primers by means of the io polymerase chain reaction; (e) heating and reannealing the non-tagged, amplified cDNA in the presence of an excess of the tagged, amplified cDNA; (f) removing from the mixture those DNA strands which either are themselves tagged or are duplexed with tagged DNA; is (g) amplifying remaining non-tagged cDNA sequences by means of the polymerase chain reaction; (h) repeating, from 0 to 5 times, steps (c) through (g) using as the two cDNA libraries the remaining non-tagged cDNA sequences and the original tagged cDNA library; 20 (i) in a separate, second part of the method, performing steps (c) through (h), except that in step (d) the cDNA library from the non-stressed cells are amplified with non-tagged primers and the cDNA library from the stressed cells are amplified with tagged primers; (j) in a third part of the method, repeating steps (c) through (g), wherein 25 in step (c) the two cDNA libraries are the enriched cDNA libraries obtained from the first and second part of this method, and wherein in step (d) the enriched cDNA library from the non-stressed cells is tagged during amplification and the WO 99/48916 PCT/US99/06860 86 enriched cDNA library from the stressed cells is amplified with non-tagged primers.

53. The method of Claim 52, wherein said stress is selected from the group 5 consisting of hypoxia, ionizing radiation, hypothermia, and heat shock.

54. The method of Claim 52, wherein said cDNA libraries of step (b) are prepared with indexed primers. 10

55. The method for identifying stress-repressible genes and fragments of genes, comprising the steps of: (a) subjecting one of two populations of cells to stress; (b) preparing cDNA libraries from the two populations of cells; (c) digesting the cDNA libraries with restriction enzymes and then 15 ligating linker sequences to the ends of said digested cDNA; (d) amplifying the cDNA library from the non-stressed cells with tagged primers by means of the polymerase chain reaction and amplifying the other cDNA library from the stressed cells with non-tagged primers by means of the polymerase chain reaction; 20 (e) heating and reannealing the non-tagged, amplified cDNA in the presence of an excess of the tagged, amplified cDNA; (f) removing from the mixture those DNA strands which either are themselves tagged or are duplexed with tagged DNA; (g) amplifying remaining non-tagged cDNA sequences by means of the 25 polymerase chain reaction; (h) repeating, from 0 to 5 times, steps (c) through (g) using as the two cDNA libraries the remaining non-tagged cDNA sequences and the original tagged cDNA library; WO 99/48916 PCT/US99/06860 87 (i) in a separate, second part of the method, performing steps (c) through (h), except that in step (d) the cDNA library from the non-stressed cells are amplified with non-tagged primers and the cDNA library from the stressed cells are amplified with tagged primers; 5 (j) in a third part of the method, repeating steps (c) through (g), wherein in step (c) the two cDNA libraries are the enriched cDNA libraries obtained from the first and second part of this method, and wherein in step (d) the enriched cDNA library from the stressed cells is tagged during amplification and the enriched cDNA library from the non-stressed cells is amplified with non-tagged 1o primers.

56. The method of Claim 55, wherein said stress is selected from the group consisting of hypoxia, ionizing radiation, hypothermia, and heat shock. 15

57. The method of Claim 55, wherein said cDNA libraries of step (b) are prepared with indexed primers.