Description

LOW-GLUCOSE-INDUCIBLE or LOW-GLUCOSE-AND-HYPOXIA-INDUCIBLE GENES, PROTEINS, AND USES THEREOF

BACKGROUND OF THE INVENTION a) Field of the Invention

The present invention relates to use of low-glucose-inducible genes or fragments thereof, or use of low-glucose-and-hypoxia-inducible genes and fragments thereof in the diagnosis and treatment of disease conditions involving low-glucose or hypoxia-and-low-glucose, including stroke, heart attack, cancer, proliferative disorders, cardiac disorders, occlusive circulative disorders, arteriosclerosis, myocardial infarction, ischemic-reperfusion-related-disorders, aged macular degeneration, diabetic retinopathy, neurodegenerative disorders, autoimmune disorders, rheumatoid arthritis, conditions associated with organ transplant, inflammatory disorders, immunologically mediated disorders, viral diseases, bone disorders and neonatal distress.

b)Background Art

(Low glucose)

Deficiencies in nutrients, especially of glucose are involved for regulating a number of cellular and systemic processes, including angiogenesis, hematopoiesis, and glycolysis.

Aging and local chronic inflammation are established risk factors for epithelial tumorigenesis. These risk factors can act individually and/or synergistically to increase the incidence of age-related carcinomas. The pro-cancer microenvironment (PCM) facilitates the selective survival and growth of transformed cells. The cancer-induced environment has certain features in common with chronic inflammatory-induced PCM. Namely, there are: enhanced oxidative cell resistance against apoptosis, increased production of matrix-degrading enzymes, switching to glycolytic metabolism, angiogenesis and vasorelaxation, thus providing nutrient delivery, but restriction of the immune cell mobilization and/or its activation. PCM genesis takes place between the growth-inhibiting (cytotoxic) and growth promoting (regenerative) stages of inflammatory response. According to this model, age-related metabolic changes create opportunities for chronic (not acute) inflammatory response, which supports the PCM condition with the non-healing wound state that often occurs around carcinomas.

Cell replication is tightly controlled in normal tissues and aberrant during disease progression, such as in tumorigenesis. The replication of cells can be divided into four distinct phases: Gap 1 (Gl), synthesis (S), gap 2 (G2), and mitosis (M). The progression from one phase to the next is intricately regulated and involves many "checkpoints" that take into account cellular status and environmental cues. Among the modulators of cell cycle progression are specific nutrients, that function as energy sources or regulate the production and/or function of proteins needed to advance cells through a replicative cycle (30).

(Hypoxia)

Oxygen is essential for the life of most aerobic organisms. Oxygen homeostasis represents an indispensable organizing principle for human physiology(l). The requirement for oxidative phosphorylation to generate ATP is balanced at the cost of oxidative damage to cellular compositions (2,3).

Hypoxia has been also known to play a key role in neoplastic tissues. The progression of human tumors to malignancy involves differential expression of multiple genes in response to unique microenvironments. Low-oxygen conditions create a dominant tumor microenvironment that directly favors processes driving malignant progression, such as angiogenesis or elimination of p53, mTOR, and PTEN tumor suppressor activity.

Cumulative acquisition of genetic alterations affecting oncogenes or tumor suppressor genes may select for tumor cell clones with enhanced proliferation and survival potential. As a result oxygen and nutrient consumption increases, leading to a tumor microenvironment characterized by low oxygen tension, low glucose levels, and an acidic pH (4).

Most solid tumors develop regions of low oxygen tension because of an imbalance in oxygen supply and consumption. As a tumor expands, the vigorous growth of cancer cells creates a hypoxic microenvironment, which, if not alleviated, may restrict tumor growth or even cause cell death (6). Deficiencies in oxygenation are widespread in solid tumors and the growth thereof is critically dependent on the regulators of blood vessel growth, shown to play a coordinated role in the progression of tumors (7, 8). Angiogenesis and increased glycolysis, two universal characteristics of solid tumors, represent adjustments to a hypoxic microenvironment (9). Clinical and experimental evidence suggests that tumor hypoxia is associated with a more aggressive phenotype (10).

Solid tumors consist of malignant cells and stromal tissues, which includes new blood vessels, matrix components and cells responsible for their production, a fibrin-gel matrix, and inflammatory leukocytes (11, 12). Tumor hypoxia can lead to chemotherapy resistance by depriving tumor cells of the oxygen essential for the cytotoxic activities of these agents. Hypoxia may also reduce tumor sensitivity to chemotherapy through complicated mechanisms that include proteomic and genomic changes. These effects, in turn, can lead to increased invasiveness and metastatic potential, loss of apoptosis, and chaotic angiogenesis, thereby further increasing drug resistance (13). In addition to proteomic changes, hypoxia may promote genomic instability, thereby increasing genetic diversity (14-17).

Recent reports have revealed that, in some cancers, constitutively activated NF- K B regulates the expression of multiple genes, including those encoding (18-20), matrix metalloproteinase, and vascular endothelial growth factor (VEGF) (21, 22). It has been also implicated that hypoxia can induce resistance of pancreatic cancer cells to gemcitabine mainly through the PI3K/Akt pathways (23).

Hypoxia in tumor tissue regulates a variety of transcription factors including hypoxia-inducible factor- 1 (HIF-I) (24). There is a growing body of evidence demonstrating a dual role for HIF-I in tumor growth by enabling both cell survival and angiogenesis through its trans-activation of hypoxia-inducible genes (25). The HIF system induces adaptive responses including angiogenesis, glycolysis, and pH regulation, which confer increased resistance towards the hostile tumor microenvironment. Apart from protumorigenic, the wide-ranging HIF pathway may also have antitumorigenic components, which might, however, be counteracted by specific genetic mechanisms. Thus, mounting evidence suggests that the HIF system plays a decisive role in tumor physiology and progression.

However, it is unlikely that the cellular response to hypoxia is mediated solely through HIF-I (26-29). Recent reports have suggested that phosphatidylinositol 3-kinase/Akt signaling can induce angiogenesis and oncogenic transition in both HIF-I dependent and HIF-I -independent manners (26).

Hypoxia-induced gene expression is associated with ischemia (and reperfusion) in many tissues including those of the liver, heart, eyes, artery and brain. Enhancement of the body's protective expression of some stress-induced genes is therefore likely to be beneficial in many ischemia/reperfusion-related conditions such as in the case of liver transplantation, bypass operations, cardiac arrest, and stroke. The adaptive response as illustrated by gene expression, either systemically or locally, of host or parasites is similarly associated with nutrient

deficiencies, including glucose deprivation, frequently observed under pathological conditions.

REFERENCES

1. Semenza, G.L. Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology. Trends MoI. Med. 7, 345-350, (2001).

2. Semenza, GX. Perspectives on oxygen sensing. Cell. 98, 281-284, (1999)

3. Lin S., et al. Berberine Inhibits HIF-I Expression via Enhanced Proteolysis. MoI Pharmacol. 66, 612-619, (2004).

4. Acker T., Plate K.H. A role for hypoxia and hypoxia-inducible transcription factors in tumor physiology. J MoI Med. 2002 ;80:562-575

5. Hoeckel M, Schlenger K, Hockel S, Vaupel P. Hypoxia and Radiation Response in Human Tumors. (1996) Semin. Radiat. Oncol. 6: 1-8.

6. Bergers G & Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3, 401-410, (2003).

7. Jiang C.Q., et al. Expression levels and significance of hypoxia inducible factor-1 alpha and vascular endothelial growth factor in human colorectal adenocarcinoma. Chin Med J. 117, 1541-6, (2004).

8. Hockel M., Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J. Natl. Cancer Inst., 93, 266-276, (2001).

9. Mantovani, A., et al. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23, 549-555, (2002).

10. Harrison L, Blackwell K. Hypoxia and anemia: factors in decreased sensitivity to radiation therapy and chemotherapy? The Oncologist. 9 (Suppl 5), 31-40, (2004).

11. Vaupel P. The role of hypoxia-induced factors in tumor progression. The Oncologist 9 (suppl 5), 10-17, (2004).

12. Vaupel P, Harrison L. Tumor hypoxia: causative factors, compensatory mechanisms, and cellular response. The Oncologist 9(suppl 5), 4-9, (2004).

13. Graeber T. G., et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379, 88-91, (1996).

14. Bardeesy N, Depinho RA. Pancreatic cancer biology and genetics. Nature Rev, 2, 897-909, (2002).

15. Akakaura N., et al. Constitutive expression of hypoxia-inducible factor-I D (HIF-Ia) renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and nutrient

deprivation. Cancer Res. 61, 6548-6554, (2001).

16. Chen J., et al. Dominant negative HIF-Ia reduces tumorigenicity of pancreatic cancer cells through the suppression of hypoxia-inducible anaerobic metabolism. Am J Pathol., 162, 1282-1291, (2003).

17. Yokoi K and Fidler IJ. Hypoxia Increases Resistance of Human Pancreatic Cancer Cells to Apoptosis Induced by Gemcitabine. Clin Cancer Res. 102, 299-2306, (2004).

18. Arsham A.M., Plas D.R., Thompson CB. & Simon M.C. Akt and Hypoxia-Inducible Factor-1 Independently Enhance Tumor Growth and Angiogenesis Cancer Res., 64, 3500-3507, (2004)

19. Mizukami Y ₅ et al. Hypoxia-Inducible Factor-1 -Independent Regulation of Vascular Endothelial Growth Factor by Hypoxia in Colon Cancer. Cancer Research 64, 1765-1772, (2004).

20. Levy A. P., Levy N. S., Goldberg M. A. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J. Biol. Chem., 271, 2746-2753, (1996).

21. Selten, G, et al. The primary structure of the putative oncogene pim-1 shows extensive homology with protein kinases. Cell 46, 603-611, (1986).

22. Zippo A., et al. Identification of FIk-I target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro. Blood 103, 4536-4544, (2004)

23. Yan B., et al. The PIM-2 Kinase Phosphorylates BAD on Serine 112 and Reverses BAD-induced Cell Death. J. Biol. Chem., 278, 45358-45367, (2003).

24. Winn LM, Lei W, Ness SA. Pim-1 phosphorylates the DNA binding domain of c-Myb. Cell Cycle 2, 258-262, (2003).

25. Valdman A., et al. Pim-1 expression in prostatic intraepithelial neoplasia and human prostate cancer. Prostate 60, 367-371, (2004)

26. Cuypers H.T., et al. Murine leukemia virus-induced T-cell lymphomagenesis: integration of proviruses in a distinct chromosomal region. Cell 37, 141-150, (1984).

27. Lilly M & Kraft A. Enforced expression of the Mr 33,000 Pim-1 kinase enhances factor-independent survival and inhibits apoptosis in murine myeloid cells. Cancer Res. 57, 5348-5355, (1997)

28. Lilly M., et al. The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway. Oncogene 18, 4022-4031, (1999)

29. van Lohuizen M., et al. Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors. Cell 56,

673-682, (1989)

302. Bohnsack BL ₅ Hirschi KK. Nutrient regulation of cell cycle progression. Annu RevNutr.

2004; 24: 433-53

SUMMARY OF THE INVENTION

A. Problem to be solved

The present inventors have long studied solid tumors. Solid tumors are often resistant to chemotherapy by depriving tumor cells of the oxygen essential for the cytotoxic activities of these agents.

B. Solution to the problem

The present inventors have long been striving to treat solid tumors such as pancreatic carcinoma. Pancreatic carcinoma is often resistant to chemotherapy. The hypoxia condition surrounding solid tumors is associated with this resistance to chemotherapy. It is now considered that HIF-I alpha induced in hypoxia condition are involved in protecting the tumor cells from hypoxia and hypoxia induced apoptosis or inhibition of cell growth.

However, HIF-I is not considered to be a single gene involved in protecting solid tumors against hypoxia-and- low- glucose conditions.

The present inventors discovered that the low-glucose environment surrounding a tumor also contributes to its resistance to chemotherapy, and inhibition of low-glucose-induced gene leads to the apoptosis of tumor cells.

The present inventors also discovered that inhibition of genes induced in the low-glucose micoroenviroment or low-glucose-and-hypoxia micoroenviroment may cause apoptosis to tumor cells. With this discovery, the inventors provide microarrays for detection of a low -glucose micoroenviroment or a low-glucose-and-hypoxia micoroenviroment, a method of treatment of solid tumors and a method of detections of solid tumors.

The inventors have established for the first time that several previously known genes are low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes in humans. Genes (l)-(4) may be referred as the target genes hereinafter.

Specifically, the present inventors have found that following four groups of genes are involved in the low-glucose or low-glucose-and-hypoxia condition.

1) Type 1 genes are induced only under condition in which both low glucose and hypoxia conditions exist.

2) Type 2 genes are induced under hypoxic conditions and further enhanced under additional low glucose conditions.

3) Type 3 genes are induced under low glucose conditions and further enhanced under additional hypoxic conditions.

4) Type 4 genes are low-glucose-inducible genes that are not affected by additional hypoxic condition.

Above mentioned Type 1-4 genes are suitable for use in the microarrays, diagnostic methods, and treatment methods.

More, specifically, the type 1 genes includes, but are not limited to, the following genes:

(a) ANGPTL4, RGS4, DKKl, SLC2A1, JUN, ABL2, TM4SF1, BCL6, NOG, PHLDAl, CLECSF2, MDM4, VEGF, VIL2, P4HA2, LGALS8, ZFP36L1, ADFP, SPAG9, PTPN14, PPARA, ARHGAP5, SAP30, TP53BP2, BHLHB2, DUSP5, SLC21A12, SLC2A3, P2RX1, MIG-6, SIAH2, PDElOA, GJA7, MYC, LIF, PDKl, SIRT7, RLF, PTP4A1, DDEFl, SLC16A6, RAP2B, LPP, PORIMIN, TAZ_2, SEC14L1, RYBP, STXlA, PROL2, KCNA3, EGLNl, MAPK7, SPRY2, ASCL2, DKFZp434K1210, GROl, SLC16A3, CEBPE, SYN47, SGK, ARL7, GMEBl, POLS, RRN3, VEGFB, ZNF84, SAA4, MS4A6A, NT5C, MLLT4, ADAM30, INGlL, EGRl, FOSLl, SERPINB8, RTVPl, MAP2, MTlH, RABEX5, CD44, ARHGEF7, AF5Q31, CGGBPl, RAP2A, N0X4, FHL2, RASAL2, NR2C2, RERE, FOXO3A, NFAT5, and USP3.

Furthermore, type 2 genes includes, but are not limited to, the following genes:

(b) PFKFB3, HK2, ADM, HKl, and BNIP3L.

Furthermore, type 3 genes includes, but are not limited to, the genes:

(c) SLC38A2, EROl-L (BETA), FOXDl, HSPA6, SAT, SCD, RAB27B, RTP801, COPEB, IL8, NFIL3, EREG ₅ and SERPINEl.

Furthermore, type 4 genes includes, but are not limited to, the following genes:

(d) HERPUDl, SDF2L1, PLAB, ATF3, ARMET, HMOXl ₅ DSCRl ₅ GADD45A, DDIT3, SES2, CTH, ARG2, DNAJB9, GPR56, BTGl, AREG, FOS, RNASE4, CYR61, GABARAPL3, GARS, GRBlO, UAPl ₅ GCLC ₅ PTPRH, XBPl, TNFRSFlOB ₅ STX3A, BIRC3, CRYAB ₅ GR03, CPR8 ₅ I_966269, EIF5, PPIB ₅ G8 ₅ ASNS ₅ FKBP2, OPTN ₅ GFPTl ₅ PCK2, MTHFD2, SEC23B, TGIF, CANX, AARS, PYCRl ₅ ARNTL, GR02, MGEA6, RELB ₅ ILlO ₅ KITLG ₅ SLC3A2, SARS ₅ PREB ₅ DFNA5, SGKL, PRSS 15, CEP3,

B4GALT6, UGTRELl, TFRC, KDELR2, UNG2 ₅ ATP2B1, SSR3, SSRl ₅ SHMT2, PIGA ₅

TXNRDl, TAXlBPl, DYRK3, CNK, and D3R3.

A detailed explanation of these genes is provided in Tables 1-4.

Table 1 List of Type 1 Genes

Table 2 List of Type 2 Genes

Gene Description Function name

(1) signal transducing proteins

f(3) metabolic/homeostatic enzymes/proteins,

(4) apoptosis proteins,

BNIP3L Adenovirus E1B 19kD interacting protein 3 like, a member of the Apoptosis BCL2 family of proapoptotic proteins, promotes apoptosis and may function as a tumor suppressor

[(5) DNA repair proteins

(6) angiogenesis/tissue remodeling proteins,

ADM Precursor of adrenomedullin and proadrenomedullin-N20, Angiogenesis/ hypotensive peptides involved in cardiovascular, renal and Remodelling endocrine function, increased expression may contribute to type II diabetes in some individuals

(7) cell-cycle proteins,

(8) proteins belonging to the category of transcription factors

(9) proteins regulating multi-drug resistance and

(10) growty factors/cytokines/chemokines and related protein

(11) others

Table 3 List of T e 3 Genes

Table 4 List f T e 4 Genes

C. Present invention

Present invention includes the following.

1.Microarray of polynucleotides or oligonucleotide, or polypeptides

The present invention includes microarrays of polynucleotides or oligonucleotide, or polypeptides corresponding to at least two different low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes such as those selected from type 1-4 genes.

Furthremore, present invention includes microarrays of low-glucose-induced polypeptide or low-glucose-and-hypoxia-induced polypeptides, or antibodies that are immunoreactive with low-glucose-induced polypeptides or low-glucose-and-hypoxia-induced polypeptides.

A microarray to determine the presence of low glucose or low glucose and hypoxia in a tissue, or to evaluate a low-glucose or low-glucose-and-hypoxia-related conditions in an animal is encompassed by the present invention.

The present invention also entails microarrays of polynucleotides or polypeptides corresponding to at least two different low-glucose-and-hypoxia-inducible genes, low-glucose-and-hypoxia-induced polypeptides, or antibodies that are immunoreactive with low-glucose-and-hypoxia-induced polypeptides .

2. Use of genes whose expression is induced under low-glucose conditions or low-glucose-and-hypoxic conditions to (1) treat a pathological condition or (2) detect a pathological condition

The present invention relates to use of genes or fragments there of whose expression is induced under low-glucose conditions or low-glucose-and-hypoxic conditions to detect or treat a pathological condition.

In other words, present invention relates to the use of genes selected from type 1-4 genes above or fragments thereof.

3. Method of treatment and agents therefore

The present invention relates to a method of treating a patient who has elevated expression of genes that are induced under low-glucose conditions or low-glucose-and-hypoxic conditions, which comprises administrating antisense nucleotides, siRNAs or dominant negative polynucleotides or other inhibitory agents or factors directed to genes whose

expressions are induced under low-glucose conditions or low-glucose-and-hypoxic conditions, or genes selected from type 1-4 genes above.

Present invention relates to antisense nucleotides, siRNAs, dominant negative polynucleotides, or other inhibitory agents or factors directed to genes whose expressions are induced under low-glucose conditions or and low-glucose-and-hypoxic conditions, or genes selected from Type 1-4 genes above.

The present invention also encompasses expression vectors and delivery vehicles that contain antisense nucleotides, siRNAs, the dominant negative polynucleotides and host cells that are genetically engineered with the use of such vectors or delivery vehicles.

4. Method of measuring or evaluating a low-glucose-inducible gene, low-glucose-induced polypeptide, or antibody immunoreactive with low glucose-induced polypeptide

The present invention includes method for measuring or evaluating a low-glucose-inducible gene, low-glucose-induced polypeptide, or antibody that is immunoreactive with a low-glucose-induced polypeptide.

The present invention includes method for measuring or evaluating a low-glucose-and -hypoxia-inducible gene, low-glucose-and-hypoxia-induced polypeptide, or antibody that is immunoreactive with a low-glucose-and-hypoxia-induced polypeptide.

In other words, present invention includes method for measuring or evaluating a gene selected from type 1-4 genes above, a polypeptide expressed by the gene selected from such type 1-4 genes, or antibody that is immunoreactive with a polypeptide expressed by the gene selected from such Type 1-4 genes.

More specifically, the present invention includes method for measuring or evaluating a low-glucose-inducible gene, low-glucose-induced polypeptide, or antibody that is immunoreactive with a low-glucose-induced polypeptide, or a low-glucose-and-hypoxia-inducible gene, low-glucose-and-hypoxia-induced polypeptide, or antibody that is immunoreactive with a low-glucose-and-hypoxia induced polypeptide, in a test sample obtained from test subject so as to determine or detect the presence of pathological conditions in test subject.

The present invention entails microarrays of polynucleotides or polypeptides corresponding to at least two different low-glucose-inducible genes, low-glucose-induced polypeptides, or antibodies that are immunoreactive with low-glucose-induced polypeptides.

Other methods of determining the existence and degree of expression of hypoxia/low-glucose-inducible genes, determining the presence of hypoxia and low glucose in a tissue in an animal, or evaluating a low-glucose-and-hypoxia related condition in an animal involves the use of the microarrays of the invention. First, a polynucleotide microarray or antibody microarray of the invention may be caused to come into contact 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 polynucleotides or polypeptides from the animal's sample which bind to the sites of the microarray is determined. Optionally, the gene expression pattern observed may be correlated with an appropriate treatment.

5. Methods of detection or diagnosis of a pathological condition and a probe, a primer or an antibody therefor

Methods of detecting or diagnosing low-glucose- or low-glucose-and-hypoxia related conditions via above methods are also encompassed by the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the mRNA expression of leukemia inhibitory factor (LIF) under different conditions.

Figure 2 shows the mRNA expression of serum glucocorticoid regulated kinase (SGK) under different conditions.

Figure 3 shows the inhibitory inhibitory effects of siRNA for LIF on the growth of HeIa cells.

The cells were cultured under hypoxic (1% O ₂ ) and glucose-deprived conditions (13 mg/dl).

Figure 4 shows the inhibitory inhibitory effects of siRNA for SGK on the growth of HeIa cells.

The cells were cultured under hypoxic (1% O ₂ ) and glucose-deprived conditions (13 mg/dl).

DETAILED DESCRIPTION OF THE INVENTION .1. 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. 1-1. Low glucose or low glucose and hypoxia

By the term "low glucose" is meant, for the purposes of the specification and claims, an environment of reduced glucose supply to tissues or cells such that the glucose content is less than or equal to about 50 mg/dl. In most cases, low-glucose tissue has a glucose supply that is less than or equal to about 50 mg/dl.

The terms " low-glucose-induced" or " low-glucose-inducible" when referring to a gene means that the gene is expressed at a higher level when the host cell is exposed to low-glucose conditions than when exposed to normal glucose supply conditions.

The term "hypoxia" (or "hypoxic") means, 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" conditions are conditions comprising a normal level of oxygen for a particular environment. Normoxic tissue typically has an oxygen content above about 5%.

The terms "low-glucose-and-hypoxia-induced" or "low-glucose-and-hypoxia-inducible" when referring to a gene mean that the gene is expressed at a higher level when the host cell is exposed to both hypoxic-and-low-glucose conditions than when exposed to normoxic or to normal glucose supply conditions.

Typically, the number of mRNA transcripts of a low-glucose-and-hypoxia-induced gene would be at least about 50% higher in a hypoxic and low-glucose cell versus a normoxic and normal-glucose-supplied cell. Preferably, expression of the hypoxia/low-glucose-induced gene is at least about 2-times higher in hypoxic-and-low-glucose cells versus a normoxic and normal-glucose-supplied cells.

A "low-glucose-inducible genes or low-glucose-and-hypoxia inducible genes" means the following 1-4

1) Type 1 genes are induced only under conditions in which both low glucose and hypoxic conditions exist.

2) Type 2 genes are induced under hypoxic conditions and further enhanced under additional low glucose conditions.

3) Type 3 genes are induced under low glucose conditions and further enhanced under additional hypoxic conditions.

4) Type 4 genes are low-glucose-inducible genes that are not affected by additional hypoxic condition.

A "low-glucose-related condition" or a "low-glucose-and-hypoxia-related condition" in an animal is a condition in which altered (typically, enhanced) levels of expression of low-glucose-inducible genes, or low-glucose-and-hypoxia-inducible genes in a tissue of the animal is involved. In this specification, a "low-glucose-related condition" or a "low-glucose-and-hypoxia-related condition" is sometimes referred as hypoxia/low-glucose-related conditions.

In this specification, low-glucose-and-hypoxia induced genes" or "low-glucose-and-hypoxia inducible genes" include genes listed in tables 1-4 above.

1-2. Low-glucose-inducible genes, hypoxia/low-glucose-induced protein

The low-glucose-inducible genes may play a role in the cause, development, progression, aggravation, amelioration, or cure of a given condition. A low-glucose-related condition may optionally be a disease or pathological condition. Low-glucose-related conditions include, but are not limited to, stroke, heart attack, cancer, proliferative disorders, cardiac disorders, occlusive circulative disorders, arteriosclerosis, myocardial infarction, ischemic-reperfusion-related disorders, aged macular degeneration, diabetic retinopathy, neurodegenerative disorders, autoimmune disorders, rheumatoid arthritis, conditions associated with organ transplant, inflammatory disorders, immunologically mediated disorders, viral diseases, bone disorders, neonatal distress, and wound healing.

The term "hypoxia/low-glucose-induced protein" or "hypoxia/low-glucose-induced gene product" means a protein encoded by a gene whose expression is induced by hypoxia/low glucose.

1-3. Polynucleotide, Oligonucleotide, or Nucleic acid

A"polynucleotide", an "oligonucleotide", or a "nucleic acid" includes, but is not limited to,

mRNA, cDNA, genomic DNA, synthetic DNA, and synthetic RNA, comprising the natural nucleoside bases adenine, guanine, cytosine, thymidine, and uracil, and optionally, one or more modified nucleosides or artificial nucleosides.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. No limitation as to length or to synthetic origin is suggested by the use of either of these terms herein.

A probe or primer includes a polynucleotide or oligonucleotide which hybridize the target polynucletoide and typically comprises at least 12 nucleotides, and preferably at least about 15 nucleotides, which are completely or partially complementary to the target polynucleotide.

1-4. Protein polypeptide

The term "polypeptide" means a polypeptide comprising at least two amino acids linked by peptide bonds. A "protein" is a polypeptide which is encoded by a gene.

"Neutralizing" a polypeptide or protein means inhibiting, partially or wholly, the 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.

Similarly, in the case of the proteins of this invention, alterations in the amino acid sequence that do not affect functionality may be made. Such variations may involve substitution, insertion, addition and/or deletion of one or several amino acids in the amino acid sequences,, 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.

1-5. Genetic engineering

"Operably" or "operatively linked" refers to the configuration of the coding and control sequences allowing performance of a 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 binds the promoter sequence and transcribes the coding sequence into mRNA that can be translated into the encoded protein. The control

sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered" operably linked" to the coding sequence.

A "delivery vehicle" as used herein, refers to a means of delivering a polypeptide, siRNA, 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 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 manner similar to as those lipids that make up the cell membrane. They have internal aqueous space useful for entrapping water soluble compounds such as polynucleotides or siRNA. Recognition molecules can be attached to their surface for the targeting of the delivery vehicles to specific tissues.

As used herein, an "antibody"refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Antibodies may exist as intact immunoglobulins or as a number of fragments, including those well-characterized fragments produced by digestion with various peptidases. While various antibody fragments are defined in terms of the digestion of an intact antibody, persons skilled in the art will appreciate that antibody fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Antibody fragments encompassed by the use of the term "antibodies" include, but are not limited to, Fab, Fab ¹ , F (ab ¹ ) 2, scFv, Fv, dsFv diabody, and Fd fragments.

2. Polynucleotide, oligonucleotide, polypeptide, and antibody microarrays.

Another aspect of the invention involves the presentation of multiple (at least two, and preferably more than four) hypoxia/low-glucose-inducible gene sequences, polynucleotide probes complementary to the hypoxia/low-glucose-inducible gene sequences,

hypoxia/low-glucose-induced polypeptides, or antibodies (immunoreactive with hypoxia/low-glucose induced polypeptides) on a microarray. In particularly preferred microarrays, more than about 10 different polynucleotides, polypeptides, or antibodies are presented on the microarray. In an alternative preferred embodiment, the number of different polynucleotides, proteins, or antibodies on the microarray is greater than about 25, or even greater than about 100.

2-1. Polynucleotide Micro Array or oligonucleotide microarray

One aspect of the invention provides a microarray of polynucleotides that comprises at least two different low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes, complements thereof, or fragments thereof at least twelve nucleotides in length, or sequences which hybridize to any thereof. The low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes or their fragments may optionally be selected from any of the group consisting of genes of Type 1, genes of Type 2, genes of Type 3 and genes of Type 4. However, it is understood that all of the low-glucose-inducible-gene sequences or low-glucose-and-hypoxia-inducible genes on the microarray need not be derived only from those of Type 1, Type 2, Type 3 or Type 4 listed herein. The gene sequences on the microarray may preferably include low glucose-inducible gene sequences together with those induced by hypoxia alone, low glucose alone, or hypoxia and low glucose., preferably of which expression is enhanced by more than three times under low-glucose conditions or under hypoxia-and-low-glucose-condition. The polynucleotides on the microarray are typically single-stranded.

The polynucleotides of the microarray may comprise the entire sequence of one strand of the gene, or may comprise at least 12 nucleotide long fragments thereof, or may compose sequences that hybridize thereto. In an alternative embodiment, one of the polynucleotides of the microarray comprises a polynucleotide that complements one of the coding sequences, or at least fifteen nucleotide-long fragments of one of the coding sequences, or sequences that hybridize to one of the coding sequences.

hi another embodiment of the polynucleotide microarray, at least one of the polynucleotide sequences of Type 1 genes listed above is immobilized on the microarray in combination with a different polynucleotide sequence from a low-glucose-inducible gene. The polynucleotide sequence may be selected from any of the low-glucose-inducible genes or

low-glucose-and-hypoxia-inducible genes of Type 2, Type 3 or Type 4 listed in tables above, shown below, any of the expressed sequence tags of low-glucose-inducible genes of Type2, Type 3 or Type 4 listed in tables above, or any other low-glucose-and-hypoxia-inducible-gene or expressed sequence tag from a low-glucose-and-hypoxia-inducible gene. It is understood that regardless of which genes are immobilized on the microarray, the gene sequences do not have to be represented in their entirety.

The polynucleotide sequences that are immobilized on the microarray are most preferably single-stranded and complementary to the mRNA transcripts of the relevant low-glucose-inducible genes or low-glucose- and hypoxia inducible genes. The immobilized polynucleotides may be fragments or complementary sequences of the gene or EST sequence that contain at least twelve nucleotides and preferably at least fifteen nucleotides.

Alternatively, longer gene fragments including EST fragments of at least 50 or at least 100 nucleotides may be used. In a preferred embodiment of the microarray, the microarray is made up of many different gene sequences.

In another embodiment of the polynucleotide microarray, only polynucleotides correlating to low-glucose-inducible genes or low-glucose-and-hypoxia inducible genes expressing gene products of a similar function are included on the microarray. At least two, but preferentially more than two, different low-glucose-induced genes or low-glucose-and-hypoxia-inducible genes encoding proteins from a single functional category are represented on the microarray.

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

2-2. Polypeptide microarray The present invention also provides polypeptide microarrays analogous to the

polynucleotide microarrays discussed above, except that the polypeptide sequences of the low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes, or fragments thereof, are displayed in a microarray. The polypeptide microarray comprises the polypeptide expression products of at least two low-glucose-inducible genes and/or low-glucose-and-hypoxia-inducible genes, or biochemically equivalent fragments thereof. For instance, the polypeptide microarray may comprise the protein that is a product of a low glucose-induced gene and /or protein that is a product of a low-glucose-and-hypoxia-inducible gene. Alternatively, the polypeptide microarray may instead comprise at least one protein coded by a gene selected from the group consisting of genes of Type 1, genes of Type 2, genes of Type 3, and genes of Type 4.

Another aspect of the invention concerns a polypeptide microarray comprising at least two different low-glucose-induced proteins or low-glucose-and-hypoxia-induced protein, or biochemically equivalent fragments thereof, wherein each low-glucose-induced protein or low-glucose-and-hypoxia-induced protein belongs to a different functional category. Alternatively, the polypeptide microarray comprises at least two different low-glucose-induced proteins or low-glucose-and-hypoxia-induced protein, or biochemically equivalent fragments thereof, wherein said low-glucose-induced proteins or low-glucose-and-hypoxia-induced proteins are all proteins belonging to a single functional category.

2-3. Antibody Microarray

Yet another alternative embodiment of the invention is a microarray analogous to a polypeptide microarray described above, except that antibodies immunoreactive with the low-glucose-induced polypeptides or low-glucose-and-hypoxia-induced polypeptides are immobilized to form the microarray, rather than the polypeptide sequences themselves. Each microarray comprises at least two different antibodies, each of which is immunoreactive with a different low-glucose-induced protein or low-glucose-and-hypoxia-induced protein. Each of the two antibodies is specifically immunoreactive with the polypeptide expression products of low-glucose-inducible genes low-glucose-and-hypoxia-inducible genes, such as, but not limited to, a gene selected form a group consisting of genes of Type 1, genes of Type 2, genes of Type 3, and genes of Type 4.

The antibody microarray further comprises at least one second antibody, wherein said

second antibody specifically binds to a second low-glucose-induced gene product or low-glucose-and-hypoxia-inducible gene product, or a biochemically equivalent fragment thereof. The antibody microarray antibody may further comprise at least one third antibody, wherein said third antibody specifically bind to a gene products induced under low-glucose alone condition or a biochemically equivalent fragment thereof.

The antibodies on the microarray 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 microarrays of the invention, the antibody microarray preferably comprises at least four different antibodies, and preferably more than about 10 different antibodies.

2-4. Preparation of microarray

Methods of constructing microarrays of biomolecules, such as polynucleotides, have been previously established in the art.

Polynucleotides and oligonucleotides, for, example, a DNA microarray may be prepared through immobilization of prepared DNAs on a base such as that disclosed in Science Vol. 270, pp. 467-470. Oligo DNA microarray may be prepared through direct synthesis of oligonuclotides on a base such as that disclosed in Science VoI 251, pp. 767-773. Furtheremore, for instance, some methods for preparation disclosed in Pirrung et al., US Patent No. 5,143,854, Pirrung et al., US Patent No. 5,405,783, and Fodor et al., US Patent No. 5,510,270, all of which are incorporated herein by reference. The polypeptides, antibodies, or polynucleotides may be immobilized on the microarray either covalently or noncovalently. Methods for immobilizing biomolecules are well known to those of ordinary skill in the art. For example, Collections of Standards Technologies, "Amplification and dection of nucleic acids" published by The Japan Patent Office discloses such basic technologies.

The material upon which the polynucleotides or polypeptides are immobilized in the microarray may vary. Possible substrates for construction of a biomolecule microarray include, but are not limited to, cellulose, glass, silicon, silicon oxide, silicon nitride, polystyrene, germanium, (poly) tetrafluorethylene, and gallium phosphide.

3. Method of treatment and composition therefore The present inventors discovered that inhibition of expression or function of genes induced

by low glucose or of genes induced by low glucose and hypoxia may cause apoptosis or cell growth inhibition.

3-1 Diseases to be treated by present methods

The hypoxia/low-glucose-related condition towards which this treatment may be directed is preferably age-related macular degeneration (AMD), further preferably occlusive circulative disorder, and most preferably cancer.

Examples of the hypoxia/low-glucose-related condition towards which this treatment may be directed includes ischemia, stroke, heart attack, cancer, proliferative disorders, cardiac disorders, occlusive circulative disorders, arteriosclerosis, myocardial infarction, ischemic reperfusion-related-disorders, age-related macular degeneration, diabetic retinopathy, neurodegenerative disorders, autoimmune disorders, rheumatoid arthritis, conditions associated with organ transplant, inflammatory disorders, immunologically mediated disorders, viral diseases, bone disorders, neonatal distress, stroke, wound healing and any other disease condition in which hypoxia/low-glucose plays a significant role. In another embodiment, the hypoxia/low-glucose-related condition to be treated is cancer and the tissue is a tumor to improve the sensitivity to cancer therapy (relief from multi-drug resistance induced by a gene or a gene product of the present invention disclosed above). The disclosed treatment of the tumor may be coupled with any combination of other cancer therapies such as radiation therapy, chemotherapy, or surgery.

Another aspect of the invention provides for a method of treating the patient of age-related macular degeneration or diabetic retinopathy.

Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. AMD can be categorized into an atrophic (dry) form and a neovascular (wet, exudative) form. The neovascular form of AMD, the most devastating, occurs because of the formation of an abnormal choroidal neovascular network (choroidal neovascularization, or CNV) beneath the neural retina. The newly formed blood vessels are excessively leaky. This leads to accumulation of subretinal fluid and blood leading to loss of visual acuity. Eventually, there is a total loss of the functional retina in the involved region, as a large disciform scar involving choroids and retina forms. The forms of AMD do not necessarily comprise a continuum. It is important to note that CNV occurs not only in wet AMD but also in other ocular pathologies such as ocular histoplasmosis syndrome, angiod streaks, ruptures in

Bruch's membrane, myopic degeneration, ocular tumors, and some retinal degenerative diseases.

AMD affects about 15 million Americans over the age of 50, and another 1.7 million new patients are being diagnosed annually. Dry AMD causes vision loss which is occasionally severe. Worldwide numbers are about double or more. Wet AMD, the more severe form of the disease, accounts for about 200,000 new cases every year and a total patient population of about 1.6 million in the U.S. alone. Approximately 100,000 are blind from the disease. Aging populations in the industrialized countries are expected to dramatically increase these numbers. Until the recent approval of Macugen, there were two methods available for treating wet AMD-laser photocoagulation and photodynamic therapy-but each is effective in only a small subset of affected individuals and recurrences after photocoagulation are common. No treatment is available for dry AMD.

Diabetic retinopathy, a progressive microvascular complication of diabetes, is a major cause of blindness. In the diabetic state, hyperglycemia leads to decreased retinal blood flow, retinal hyperpermeability, delays in photoreceptor nerve conduction, and retinal neuronal cell death. The combination of these events renders the retina hypoxic and ultimately leads to the development of diabetic retinopathy. Retinal ischemia leads to abnormal proliferation of new vessels through release of growth factors, which also increase retinal vascular permeability, a hallmark of the disease. This abnormal permeability results in a thickening of the retina called macular edema. Slowing, preventing, or reversing the progressive changes caused by the disease would be of significant therapeutic value for avoiding subsequent, more severe complications.

The National Society to Prevent Blindness has estimated that 4 to 6 million diabetics in the U.S. have diabetic retinopathy. The estimated annual incidence of new cases of proliferative diabetic retinopathy and diabetic macular edema are 65,000 and 75,000, respectively, with a prevalence of 700,000 and 500,000 cases respectively. There are no drug treatments available.

Another aspect of the invention provides also for a method of treating disorders of circulative/cardial systems, such as an occlusive circulative disorder, arteriosclerosis, myocardial infarction, an ischemic-reperfusion-related disorder or cardiac stroke.

3-2. Inhibition of the expression of genes induced by low glucose or low glucose and hypoxia Expression of genes induced by low glucose or low glucose and hypoxia may be inhibited by any known means or methods including but not limited to using: (1) inhibitory

polynucleotides such as an antisense oligonucleotide, a triple-helix probe, a deoxyribozyme, or a ribozyme, a dominant negative polynucleotides, or a siRNA, directed to a low-glucose-inducible gene or a low-glucose-and-hypoxia-inducible gene, or (2) a low molecular weight compound or the like which is specific to the low-glucose-inducible gene or low-glucose-and-hypoxia-inducible genes. Any molecule identified by the method below may also be used.

3-2-1. The polynucleotides capable of inhibiting expression of a gene induced by low glucose or low glucose and hypoxiafreferred as inhibitory polynucleotides hereafter)

The polynucleotides capable of inhibiting expression of a gene induced by low glucose or low glucose and hypoxia include an antisense oligonucleotide, a triple-helix probe, a deoxyribozyme, or a ribozyme, a dominant negative polynucleotides which may be referred to as dominant negative mutant, or siRNA.

(1) Antisense oligonucleotides

Antisense oligonucleotides complementary to genes of the present invention such as those liste in tables 1-4 , particularly those that are capable of blocking the expression of genes of the present invention are provided by the present invention. The antisense oligonucleotide is preferably an oligonucleotide having a sequence complementary to at least a portion that is preferably at least about 12 nucleotides in length. The antisense oligonucleotide is preferably between about 15 and about 22 nucleotides in length. Modifications of the sequence or bases of the antisense oligonucleotide may be desirable to facilitate transfer into a cell, stability, or tight binding to the mRNA.

An antisense DNA may ususally be designed so as to contain the start site of the coding sequences of the target gene. (This is because the inhibition of ribosome translocation has a greater effect on the inhibition of translation by antisense DNA than the decomposition by RNase H.)

(2) SiRNA: siRNA against a gene may be designed by known method. For example, siRNA may be designed against genes listed in tables 1-4 above by using known design software such as siSearch available at http://sonhammer.cgb.ki.se/siSearch/siSearch_l .7.html or in accordance with Biochem Biophys Res Commun 2004 Jun 18, Vol. 319(1) pp. 264-274,.

(3) A triple-helix oligonucleotide against regulatory region of the genes of type 1-4 may be designed by a known method, for example by the method of Prog. Nucleic Acid Res. MoI. biol., 67, 163-192, or Biochim. Biophys. Acta, 1489, 181-206.

(4) Deoxyribozyme and ribozyme may be used as disclosed in Pharmacological reviews Vol. 52, Issue 3, 325-348, September 2000 or Curr MoI Med. 2004 Aug 4 (5):489-506.

(5) Morpholino oligo may be designed by a publicly know method such as that disclosed in Antisense Nucleic Acid Drug Dev. 1997 Jun 7 (3): 187-95 or Biochim Biophys Acta. 1999 Dec l0 1489 (l):141-58.

(6) Dominant-negative mutants may be designed by well known methods, such as mutating well conserved regions within a gene as disclosed in BMC Cell Biology 2003, 4:16.

3-2-2. A low molecular weight compound or the like identified by the present screening method or neutralising protein expression products

Similarly, treatment of the hypoxia/low-glucose-related conditions may also be achieved by neutralizing the protein expression products of hypoxia/low-glucose-inducible genes, as described above. In accordance with this method, antibodies, antagonists, inhibitors, proteases, or the like that target and neutralize the function of polypeptides produced by newly identified genes as being hypoxia/ low-glucose-inducible may be introduced into an animal, preferably a human, either alone or together with the tissue to be treated.

3-3. Delivery or administration of an inhibitory polynuceotide or other agent

These techniques include, but are not limited to, introduction of antisense oligonucleotides, triple-helix probes, deoxyribozymes, ribozymes, dominant negative polynucleotides, siRNAs, low molecular weight compounds, or the like into the subject tissue of concern. In a preferred embodiment, the animal to be treated is a human.

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

An inhibitory polynucleotide, such as an antisense nucleotide or siRNA contained in a

expression vector may be introduced into an animal either by first incorporating the vector into a cell and then transferring the cell to the animal (ex vivo) or by incorporating the vector into a cell within an animal directly (in vivo).

The introduction of an inhibitory polynucleotide into a cell may also be achieved by directly injecting the inhibitory polynucleotide or expression vector carrying the inhibitory polynucleotide into the cell or by first mixing the nucleic acid with polylysine or cationic lipids that will help facilitate passage across the cell membrane. However, introduction of the polynucleotide into the cell is preferably achieved through the use of a delivery vehicle such as a liposome or a virus. Viruses that may be used to introduce a polynucleotide or expression vector into a cell include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and vaccinia viruses.

3-4. The polynucleotides corresponding to the gene sequences of hypoxia/low-glucose-inducible gene sequences, such as Type 1 genes, Type 2 genes, Type 3 genes, or Type 4 genes in Tables 1-4 above, may be used to attenuate the response of a tissue to hypoxia.

3-5. Embodiments

These low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes can be targeted within a tissue by the introduction of inhibitory polynucleotides such as antisense oligonucleotides, triple-helix probes, catalytic nucleic acids, dominant negative polynucleotides, siRNA, or low molecular weight compounds, or the like in a manner that inhibits expression of the low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes within the tissue.

Therefore, in one embodiment, the method of attenuating the low-glucose response or hypoxia-and-low-glucose response of tissue comprises inhibiting the expression in said cell of a gene selected from the group consisting of genes listed in table 1-4 above, including ANGPTL4, RGS4, DKKl, SLC2A1, JUN, ABL2, TM4SF1, BCL6, NOQ PHLDAl, CLECSF2, MDM4, VEGF, VIL2, P4HA2, LGALS8, ZFP36L1, ADFP, SPAG9, PTPN14, PPARA, ARHGAP5, SAP30, TP53BP2, BHLHB2, DUSP5, SLC21A12, SLC2A3, P2RX1, MIG-6, SIAH2, PDElOA, GJA7, MYC, LIF, PDKl, SIRT7, RLF, PTP4A1, DDEFl,

SLC16A6, RAP2B, LPP, POREVIIN, TAZ_2, SEC14L1, RYBP, STXlA, PROL2, KCNA3,

EGLNl, MAPK7, SPRY2, ASCL2, DKFZp434K1210, GROl, SLC16A3, CEBPE ₅ SYN47, SGK ₅ ARL7, GMEBl ₅ POLS ₅ RRN3, VEGFB ₅ ZNF84, SAA4, MS4A6A, NT5C, MLLT4, ADAM30, INGlL ₅ EGRl ₅ FOSLl, SERPINB8, RTVPl, MAP2, MTlH ₅ RABEX5, CD44, ARHGEF7, AF5Q31, CGGBPl, RAP2A, N0X4, FHL2, RASAL2, NR2C2, RERE, F0X03A, NFAT5, USP3 in said cell.

This inhibition of expression of a low-glucose-inducible gene or low-glucose-and-hypoxia-inducible gene may optionally be achieved by introducing into the cells of said tissue a nucleic acid molecule such as an antisense oligonucleotide, a triple-helix probe-, a deoxyribozyme, a ribozyme, a dominant negative polynucleotide, or siRNA, a low molecular weight compound, or the like, which is specific to the hypoxia/low-glucose-inducible gene.

hi an alternative embodiment of the invention, the expression products of low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes may instead be targeted to attenuate the low-glucose response or hypoxia-and-low-glucose response of a tissue. For this purpose, antibodies, antagonists, inhibitors, or proteases that are specific to the expression products of low-glucose-induced genes or low-glucose-and-hypoxia-inducible genes may be introduced into the tissue.

In one embodiment, a method of attenuating the low-glucose response or hypoxia-and-low-glucose-response of a tissue comprises neutralizing a protein selected from the group consisting of the genes listed in Tables 1-4 above, including: ANGPTL4, RGS4, DKKl, SLC2A1, JUN, ABL2, TM4SF1, BCL6, NOG ₅ PHLDAl, CLECSF2, MDM4, VEGF, VIL2, P4HA2, LGALS8, ZFP36L1, ADFP ₅ SPAG9, PTPN14, PPARA, ARHGAP5, SAP30, TP53BP2, BHLHB2, DUSP5, SLC21A12, SLC2A3, P2RX1, MIG-6, SIAH2, PDElOA ₅ GJA7, MYC ₅ LIF ₅ PDKl ₅ SIRT7, RLF ₅ PTP4A1, DDEFl ₅ SLC16A6, RAP2B, LPP, PORIMIN, TAZ_2, SEC14L1, RYBP ₅ STXlA ₅ PR0L2, KCNA3, EGLNl, MAPK7, SPRY2, ASCL2, DKFZp434K1210, GROl ₅ SLC16A3, CEBPE ₅ SYN47, SGK, ARL7, GMEBl, POLS, RRN3, VEGFB, ZNF84, SAA4, MS4A6A, NT5C, MLLT4, ADAM30, INGlL, EGRl ₅ FOSLl ₅ SERPINB8, RTVPl, MAP2 ₅ MTlH, RABEX5, CD44, ARHGEF7, AF5Q31, CGGBPl ₅ RAP2A, N0X4, FHL2, RAS AL2, NR2C2, RERE ₅ F0X03A, NFAT5, and USP3.

In this method, an agent specifically targeting the protein is optionally introduced into the cells of the tissue and can be an antibody, an antagonist, an inhibitor, or a protease.

The methods described above for attenuating the low-glucose response or hypoxia-and-low-glucose-response of a tissue may be used to treat a low-glucose-related condition or low-glucose-and-hypoxia-related condition in an animal. For instance, the treatment of a low-glucose-related condition or low-glucose-and-hypoxia-related condition in an animal may be effected by targeting the low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes in the low-glucose tissue or hypoxia-and-low-glucose tissue via one or more of the techniques known to those skilled in the art.

Methods for inhibiting the response of tissue to low-glucose or hypoxia-and-low-glucose-are provided in other embodiments of the present invention. These methods involve administering expression vectors comprising an inhibitory polynucleotide such an anti-sense nucleotide, a dominant negative polynucleotide or siRNA against low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes or administering a polypeptide or a antagonistic compound inhibiting the function of expression products of low-glucose-inducible-genes or low-glucose-and-hypoxia-inducible genes to the tissue.

Methods for inhibiting the response of the host to low glucose or hypoxia and low glucose are provided in other embodiments of the present invention. These methods involve administering expression vectors comprising an inhibitory polynucleotide such as an anti-sense nucleotide, a dominant negative polynucleotide, or siRNA directed to low glucose-inducible genes or low glucose-and-hypoxia-inducible genes or administering a polypeptide or an antagonistic compound inhibiting the function of expression products of low-glucose-inducible genes or low-glucose-and-hypoxia- inducible genes to the host.

The protein expression products of the genes that have been newly identified as being hypoxia/ low-glucose-inducible may be used to identify or screen for drugs, such as inhibitors, useful in the treatment of hypoxia/low-glucose-related conditions. For instance, small molecule drug candidates or peptides may be tested against the any of the proteins which are coded by any of genes listed in Tables 1-4 above, including: ANGPTL4, RGS4, DKKl ₅ SLC2A1, JUN, ABL2, TM4SF1, BCL6, NOG, PHLDAl, CLECSF2, MDM4, VEGF, VIL2, P4HA2, LGALS8, ZFP36L1, ADFP, SPAG9, PTPN14, PPARA, ARHGAP5, SAP30, TP53BP2, BHLHB2, DUSP5, SLC21A12, SLC2A3, P2RX1, MIG-6, SIAH2, PDElOA,

GJA7, MYC, LIF, PDKl ₅ SIRT7, RLF, PTP4A1, DDEFl, SLC16A6, RAP2B, LPP, PORIMIN, TAZ_2, SEC14L1, RYBP, STXlA, PROL2, KCNA3, EGLNl, MAPK7, SPRY2, ASCL2, DKFZp434K1210, GROl, SLC16A3, CEBPE, SYN47, SGK, ARL7, GMEBl, POLS, RRN3, VEGFB, ZNF84, SAA4, MS4A6A, NT5C, MLLT4, ADAM30, INGlL, EGRl, FOSLl, SERPINB8, RTVPl, MAP2, MTlH, RABEX5, CD44, ARHGEF7, AF5Q31, CGGBPl, RAP2A, N0X4, FHL2, RASAL2, NR2C2, RERE ₅ F0X03A, NFAT5, and USP3, to see if inactivation of the enzymatic activity or prevention of crucial binding activity of the hypoxia/low-glucose-, low-glucose- or hypoxia-induced protein occurs. Combinatorial libraries of small molecules or libraries of peptides such as those produced by phage display may alternatively be screened for those which are capable of inactivation of the enzymatic activity or prevention of crucial binding activity of the one of the hypoxia/low-glucose-induced proteins described herein.

hi other aspects, the invention provides for methods of attenuating the low-glucose response or hypoxia-and-low-glucose response of a tissue by blocking expression of a low-glucose-inducible gene, or low-glucose-and hypoxia inducible genes such as genes listed in Tables 1-4 above, including ANGPTL4, RGS4, DKKl, SLC2A1, JUN ₅ ABL2, TM4SF1, BCL6, NOG, PHLDAl, CLECSF2, MDM4, VEGF ₅ VIL2, P4HA2, LGALS8, ZFP36L1, ADFP ₅ SPAG9, PTPN14, PPARA ₅ ARHGAP5, SAP30, TP53BP2, BHLHB2, DUSP5, SLC21A12, SLC2A3, P2RX1, MIG-6, SIAH2, PDElOA ₅ GJA7, MYC, LIF ₅ PDKl ₅ SIRT7, RLF ₅ PTP4A1, DDEFl ₅ SLC16A6, RAP2B, LPP, PORIMIN ₅ TAZ_2 ₅ SEC14L1, RYBP ₅ STXlA, PROL2, KCNA3, EGLNl ₅ MAPK7, SPRY2, ASCL2, DKFZp434K1210, GROl ₅ SLC16A3, CEBPE, SYN47, SGK ₅ ARL7, GMEBl ₅ POLS ₅ RRN3, VEGFB, ZNF84, SAA4, MS4A6A, NT5C, MLLT4, ADAM30, INGlL, EGRl ₅ FOSLl ₅ SERPINB8, RTVPl, MAP2, MTlH ₅ RABEX5, CD44, ARHGEF7, AF5Q31, CGGBPl ₅ RAP2A, N0X4, FHL2, RASAL2, NR2C2, RERE ₅ F0X03A, NFAT5, and USP3 in the cell or by neutralizing the polypeptide expression products of these genes in the tissue. The invention also provides for methods of treating hypoxia/low-glucose-related conditions by attenuating the low-glucose response or hypoxia-and-low-glucose response of a tissue in an animal such as a human.

4. Detection of low-glucose-related condition and low-glucose-and-hvpoxia-related condition We have demonstrated that the expression of a number of other genes is indicative of a cell's response to hypoxia/low-glucose as described in the specific examples below. Accordingly, detection of abnormal levels of the transcripts of hypoxia-inducible genes such

as ANGPTL4, RGS4, DKKl, SLC2A1, JUN, ABL2, TM4SF1, BCL6, NOG, PHLDAl, CLECSF2, MDM4, VEGF, VIL2, P4HA2, LGALS8, ZFP36L1, ADFP, SPAG9, PTPN14, PPARA, ARHGAP5, SAP30, TP53BP2, BHLHB2, DUSP5, SLC21A12, SLC2A3, P2RX1, MIG-6, SIAH2, PDElOA, GJA7, MYC, LIF, PDKl, SIRT7, RLF, PTP4A1, DDEFl, SLCl 6A6, RAP2B, LPP, PORIMIN, TAZ_2, SEC14L1, RYBP, STXlA, PR0L2, KCNA3, EGLNl, MAPK7, SPRY2, ASCL2, DKFZp434K1210, GROl, SLC16A3, CEBPE, SYN47, SGK, ARL7, GMEBl, POLS, RRN3, VEGFB, ZNF84, SAA4, MS4A6A, NT5C, MLLT4, ADAM30, INGlL, EGRl, FOSLl, SERPINB8, RTVPl, MAP2, MTlH, RABEX5, CD44, ARHGEF7, AF5Q31, CGGBPl, RAP2A, N0X4, FHL2, RASAL2, NR2C2, RERE, FOXO3A, NFAT5, USP3, or combinations thereof, in the tissues or body fluids of an animal can be used in both a diagnostic and prognostic manner for hypoxia/low-glucose-, hypoxia-, or low-glucose-related conditions.

4-1 Diseases to be detected or diagnosed by present methods

The hypoxia/low-glucose-related condition towards which this detection or diagnosis may be directed is preferably age-related macular degeneration (AMD), further preferably occlusive circulative disorder, and most preferably cancer.

Examples of hypoxia/low-glucose-related conditions towards which this detection or diagnosis may be directed include ischemia, stroke, heart attack, cancer, proliferative disorders, cardiac disorders, occlusive circulative disorders, arteriosclerosis, myocardial infarction, ischemic-reperfusion-related disorders, age-related macular degeneration, diabetic retinopathy, neurodegenerative disorders, autoimmune disorders, rheumatoid arthritis, conditions associated with organ transplant, inflammatory disorders, immunologically mediated disorders, viral diseases, bone disorders, neonatal distress, stroke, wound healing and any other disease conditions in which hypoxia/low-glucose plays a significant role. hi another embodiment, the hypoxia/low-glucose-related condition to be detected or diagnosed is cancer and the tissue is a tumor to improve sensitivity to cancer therapy (relief from multi-drug resistance induced by a gene or a gene product disclosed above).

Another aspect of the invention provides a method for detection or diagnosis with reference to the patient suffering from age-related macular degeneration or diabetic retinopathy mentioned in 3.1 above.

Abnormal levels may be characterized by either increased levels or decreased levels, depending upon the low-glucose-related condition or low-glucose-and-hypoxia-related

condition being analyzed. In other cases, either the complete absence or any presence of a hypoxia/ low-glucose-inducible gene transcript may be indicative of an abnormal condition. Similarly, detection of abnormal levels of the hypoxia/ low- glucose-induced polypeptides, or combinations thereof, can be used in either a diagnostic or prognostic manner for hypoxia/ low-glucose-related conditions.

The presence of hypoxia/low-glucose, hypoxia or low glucose in a tissue can be evaluated by testing for the presence or absence of the transcripts or polypeptides encoded by the Typel genes, Type 2 genes, Type 3 genes, or Type 4genes shown above in either the tissue or in the body fluids of an animal. Detection of the transcripts or polypeptides can be either qualitative or quantitative.

4-2. Determination or evaluation of the presence of low-glucose-related conditions or low-glucose-and-hypoxia-related conditions

One aspect of the invention, therefore, provides a method of determining the presence of low-glucose-related conditions or low-glucose-and-hypoxia-related conditions in tissue in an animal or evaluating low-glucose-related conditions or low-glucose-and-hypoxia-related conditions in tissue in an animal. These methods comprise conducting assay regarding either the messenger RNA (mRNA) transcripts or the polypeptide expression product of at least one gene selected from the group consisting of genes of Type 1, genes of Type 2, genes of Type 3 and genes of Type 4 in a body fluid or the tissue of the animal. This method for determining the presence of low-glucose-related conditions or low-glucose-and-hypoxia-related conditions in a tissue may be used to diagnose low-glucose-related conditions or low-glucose-and-hypoxia-related conditions in an animal.

The presence of hypoxia/low-glucose in a tissue or the degree of expression of low-glucose- inducible genes or low-glucose-and-hypoxia-inducible genes determined by these methods may be used to select an appropriate treatment for the animal. For instance, the low-glucose-related conditions or low-glucose-and-hypoxia-related conditions being evaluated may indicate cancer and the tissue that is evaluated may optionally be a tumor. The degree to which the tumor exhibits gene expression patterns characteristic of low glucose or low glucose and hypoxia, or the activation of genes involved in angiogenesis, multi-drug resistance or antiapoptosis, for instance, can be usefully correlated with appropriate treatment of tumors of a particular type.

4-3. Means for detection of the transcripts of low-glucose-inducible genes or low-glucose-and-hvpoxia-inducible genes.

The transcripts of low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes may be detected by any means known to those skilled in the art. One embodiment of diagnostic detection involves annealing to the transcript, in vivo or in vitro, a labeled nucleic acid probe complementary to the transcript sequence. The labeled probe can be fluorescent, radioactive, immunoreactive, colormetric, or otherwise marked for detection. To detect very minute quantities of a transcript, amplification of the transcript in a tissue or fluid sample from an animal, may first be performed to aid subsequent detection of the transcript. Amplification of the transcripts under low-glucose-related conditions or low-glucose-and-hypoxia-related conditions can be readily achieved using the polynucleotides designed for genes selected from the group consisting of genes of Typel, genes of Type 2, genes of Type 3, and genes of Type 4 as primers, using reverse transcriptase to make a cDNA copy of the transcript, and then using a polymerase chain reaction to achieve exponential amplification.

4-4. Means for detection of polypeptides

Detection of expression of the polypeptide products of any of the low-glucose-induced genes or low-glucose-and-hypoxia-inducible genes could be achieved, for instance, by the application of labeled antibodies specifically immunoreactive with the polypeptide products. The antibodies can be applied to the tissue in vivo, or to tissue or body fluid samples removed from the animal. Various forms of typical immunoassays known to those skilled in the art would be applicable here. These assays include both competitive and non-competitive assays. For instance, in one type of assay sometimes referred to as a "sandwich assay," immobilized antibodies that specifically react with a polypeptide are contacted with a biological tissue or fluid sample. Presence of the immobilized polypeptide-antibody complex could then be achieved by application of a second, labeled antibody immunoreactive with either the polypeptide or the polypeptide-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 tissue is preferred prior to testing for the presence of either an mRNA transcript or a gene product.

4-5 Treatment based on the determination bv the present method

One aspect of the invention concerns treating a tissue that is a tumor by first determining the presence of low glucose or low glucose and hypoxia in the tumor and, second, treating the tumor with an established form of therapy for cancers such as radiation therapy, chemotherapy, or surgery.

This method involves first determining the presence of hypoxia/low-glucose in a tumor by any of the methods described above. The method further comprises treating said tumor with any combination of established forms of therapy for cancer such as radiation therapy, chemotherapy, and surgery.

4-6. Embodiment

Li a preferred embodiment, a diagnostic evaluation of low-glucose-induced genes or low-glucose-and-hypoxia-inducible gene expression involves assaying the expression levels of more than one low-glucose-inducible gene or low-glucose-and-hypoxia-inducible genes at a time. The microarrays of the invention are particularly useful for assaying the expression of multiple hypoxia/low-glucose-inducible genes 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 microarrays of the present invention.

Use of the polynucleotide or antibody microarrays of the present invention for determining the presence of hypoxia/low-glucose, hypoxia, or low glucose in a tissue of an animal or for evaluating a hypoxia/low-glucose-related condition in a tissue of an animal allows for an unprecedented look at the exact nature and stages of the low-glucose response or hypoxia-and-low-glucose response of a tissue, since the hypoxia/low-glucose-induced expression of a combination of genes is screened for at one time.

Patterns of expression of low-glucose-inducible genes or low-glucose-and-hypoxia- inducible genes are complex and highly indicative of low glucose, or low glucose and hypoxia, in a tissue, as demonstrated in the specific examples shown in Examples below. The pattern of expression of low-glucose-inducible genes or hypoxia-and-low-glucose-inducible genes can therefore be used in a diagnostic or prognostic manner to aid in the treatment of a low-glucose-related condition or low-glucose-and-hypoxia-related condition in an animal.

Information on the pattern of expression of a combination of low-glucose-induced genes or low-glucose-and-hypoxia-inducible genes can readily be correlated with the aggressiveness of a tumor, retinopathy, or cardiac disorder, for instance, thereby providing knowledge critical for establishing the best line of treatment.

The polypeptide microarrays of the present invention also can be used to screen for drugs useful in the treatment of hypoxia/low-glucose-related conditions. These drugs may be drugs that are capable of inhibiting the low-glucose response or hypoxia-and-low-glucose response of a tissue.

For instance, methods of assaying for expression of low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes in a tissue in an animal, determining the presence of low glucose or hypoxia and low glucose in a tissue in an animal, or evaluating a low-glucose-related condition or a low-glucose-and-hypoxia-related condition in a tissue in an animal comprise first contacting the proteins or messenger RNA of a sample of body fluid or tissue obtained from the animal with an antibody microarray or polynucleotide microarray, respectively, of the invention. Fluid samples from an animal may come into direct contact with an microarray, and binding of the proteins or mRNA transcripts on the microarray may be detected. The cells in a tissue to be subjected to assay would preferably be lysed prior to application to the microarray. Alternatively, the tissue or fluid sample may be purified to isolate the proteins or mRNA transcripts prior to application to the microarray. 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 microarray. Once the protein, mRNA, or cDNA is delivered to the microarray, the method comprises detecting the amount and position of the protein, mRNA, or cDNA that remains bound to the microarray after removal of excess or non-bound protein, mRNA, or cDNA.

Additionally, a method of diagnosing a low-glucose-related condition or a low-glucose-and-hypoxia-related condition in an animal may optionally comprise the additional step of correlating the result of the evaluation of the low-glucose-related condition or the low-glucose-and-hypoxia-related condition in the tissue in the animal with an appropriate treatment for the animal. The low-glucose-related condition or low-glucose-and-hypoxia-related condition that may be evaluated, diagnosed or treated by any of the above methods may be a condition such as cancer, a proliferative disorder, a

cardiac disorder, an occlusive circulative disorder, arteriosclerosis, myocardial infarction, an ischemic reperfusion related disorder, aged macular degeneration, diabetic retinopathy, a neurodegenerative disorder, an autoimmune disorder, rheumatoid arthritis, a condition associated with organ transplant, an inflammatory disorder, an immunologically mediated disorder, a viral disease, a bone disorder, neonatal distress, stroke, or wound healing.

5. Others

The expression of some gene products induced by low-glucose or low-glucose-and-hypoxia can be helpful in protecting cells from damage or death. Thus, this invention also provides methods of enhancing the low-glucose response or hypoxia-and-low-glucose response of a tissue and thereby and treating low-glucose or low-glucose-and-hypoxic tissue. The method comprises introducing an expression vector into the tissue and allowing for expression of the coding sequence on the vector to take place. The coding sequence of the expression vector comprises one of the genes selected from group consisting of genes of Type 1, genes of Type 2, genes of Type 3 or genes of Type 4. Expression of the vector's low glucose-inducible gene or low-glucose-and-hypoxia-inducible gene within the tissue should occur at a level that is higher than that which would occur in the absence of the expression vector. Depending on use, the coding sequence of the expression vector may be operably linked to its native promoter, another low-glucose-inducible promoter or low-glucose-and-hypoxia-inducible promoter, or a constitutive promoter.

Alternatively, the proteins of the low-glucose-inducible genes or low-glucose-and-hypoxia-inducible genes may be introduced into the tissue directly to enhance the low-glucose response or hypoxia-and-low-glucose response of the tissue and for treatment of low-glucose conditions or hypoxia-and-low-glucose conditions. Delivery of the proteins may be achieved through 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.

EXAMPLES

The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims.

[Example 1]

DNA microarray

Pancreatic cancer cell line, PCI43 cells were cultured under hypoxic (1% O ₂ ) and/or glucose-deprived (13 mg/dl) conditions for 16 h. Three conditions were employed.

1) Condition 1 : under hypoxic (1% O ₂ ) and glucose-deprived (13 mg/dl) conditions for 16 h.

2) Condition 2 : under hypoxic (1% O ₂ ) for 16 hours followed by glucose-deprived (13 mg/dl) conditions for 16 h.

3) Condition 3 : under glucose-deprived (13 mg/dl) conditions for 16 h followed by hypoxic (1% O ₂ ) condition for 16 hours.

The incubation under hypoxic condition (1 % O ₂ ) took place in a hypoxic chamber charged with 95 % N ₂ and 5 % CO ₂ (Wakenyaku Co. Ltd., Tokyo). Total RNA was extracted from the cells with the use of TRIZOL Reagent (LIFE TECHNOLOGIES, Tokyo, Japan). mRNA was purified from the total RNA with the use of a Quickprep mRNA purification kit (Amersham Pharmacia Biotech, Tokyo, Japan). Extracted RNAs were examined for human genome- wide gene expression with a Human Genome Ul 33 A GeneChip probe array (Affymetrix) and with the use of oligonucleotide probe sets interrogating approximately 14,000 transcripts. Quality control, GeneChip hybridization, and raw data analysis were performed according to the manufacturer's instructions. Double-stranded cDNAs were synthesized by means of the Superscript Choice system (Invitrogen) and a GeneChipT7-Oligo(dT) promoter primer kit. The cDNAs were subjected to in vitro transcription in the presence of biotin-labeled ribonucleotides by means of a BioArray High- Yield RNA transcript labeling kit. The biotin-labeled cRNAs were chemically fragmented and hybridized to the GeneChip probe array. Using the EukGE-WS2 fluidics protocol, we stained the probe array with a streptavidin R-phycoerythrin conjugate (Molecular Probes, Eugene, Oreg.). To detect the hybridized cyanine 3- and cyanine 5-labeled cRNA or cDNA, a microarray slide scanner (Agilent's dual-laser scanner (G2565AA) capable of exciting and detecting the fluorescence from the cyanine 3 and cyanine 5 fluorescent molecules (532 and 633 nm laser lines) was used. The data generated were analyzed with Microarray Suite Expression Analysis software (Agilent Technologies).

We defined the genes expressed at levels more than 1.5 times higher under hypoxic conditions than under non-hypoxic conditions as possible hypoxia-inducible genes in this study. We defined the genes expressed at levels more than 2 times higher under glucose-deprived conditions than under normal-glucose conditions as possible

glucose-deprivation-inducible genes in this study.

Obtained glucose-deprivation-inducible genes and obtained low-glucose-and-hypoxia-inducible genes are listed in Tables 5-8 below in accordance with types of genes as follows:

1) Type 1 genes are induced only when both low-glucose and hypoxia conditions exist.

2) Type 2 genes are induced under hypoxic conditions and further enhanced under additional low-glucose conditions.

3) Type 3 genes are induced under low-glucose conditions and further enhanced under additional hypoxic conditions.

4) Type 4 genes are low-glucose-inducible genes that are not affected by additional hypoxic conditions.

Table 5. type 1 genese inducible only where low gkucose + hypoxia 1/8

Table 5. type 1 genese inducible only where low gkucose + hypoxia 2/8

Table 5. type 1 genese inducible only where low gkucose + hypoxia 3/8

Table 5. type 1 genese inducible only where low gkucose + hypoxia 4/8

Table 5. type 1 genese inducible only where low gkucose + hypoxia 5/8

Table 5. type 1 genese inducible only where low gkucose + hypoxia 6/8

Table 5. type 1 genese inducible only where low gkucose + hypoxia 7/8

Table 5. type 1 gehese inducible only where low gkucose + hypoxia 8/8

Table 6. type 2 genese induced under hypoxia with further enhancement under additional 1/1 low glucose conditions

Table 7. type 3 genes inducible under low-glucose condition with further enhancement under 1/2 additional hypoxic conditions

Table 7. type 3 genes inducible under low-glucose condition with further enhancement under 2/2 additional hypoxic conditions

Table 8. type 4 low^glucose inducible genes without any affect by additional hypoxic 1/8 conditions

Table 8. type 4 low-glucose inducible genes without any affect by additional hypoxic 2/8 conditions

Table 8. type 4 loW-glucose inducible genes without any affect by additional hypoxic 3/8 conditions

Table 8. type 4 low-glucose inducible genes without any affect by additional hypoxic 4/8 conditions

Table 8. type 4 low-glucose inducible genes without any affect by additional hypoxic 5/8 conditions

Table 8. type 4 low-glucose inducible genes without any affect by additional hypoxic 6/8 conditions

Table 8. type 4 low-glucose inducible genes without any affect by additional hypoxic 7/8 conditions

Table 8. type 4 low-glucose inducible genes without any effect by additional hypoxic 8/8 conditions

[Example 2] Expression of LIF mRNA in different conditions

HeIa cells were cultured for 16 hours under hypoxic (1% O ₂ ) and/or glucose-deprived (13 mg/dl) conditions. Total RNA was extracted from the cells with the use of TRIZOL Reagent (LIFE TECHNOLOGIES, Tokyo, Japan). Briefly, each RNA sample (5 μg) was subjected to cDNA synthesis in 50 μl of reaction mixture containing 75 mM KCl, 50 niM Tris-HCl (pH 8.3), 3 mM MgC12, 10 mM dithiothreiotol, 0.5 mM each dNTP, 2 μM random primer, and 1000 U AMLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) by incubation at 37°C for 1 h. Each cDNA (10 ng) was amplified in triplicates with the use of the SYBR-Green PCR assay kit and then detected on an ABI PRISM® 7900HT Sequence Detection System. The β-actin RNA was used to standardize the total amount of cDNA. The primers used were as follows: LIF,

Forward: 5'-AGGTTTCCTCCAAGGCCCTCTG-S', Reverse: 5'-GTATGGCACAGGTGGCGTTGAC-S ' .

Relative mRNA levels were determined by comparing the PCR cycle thresholds between cDNA of the gene of interest and that of β-actin. Results are shown in Fig.1.

[Example 3] Expression of SGK mRNA in different conditions

HeIa cells were cultured for 16 hours under hypoxic (1% O ₂ ) and/or glucose-deprived (13 mg/dl) conditions. Total Rna was extracted from the cells with the use of TRIZOL Reagent (LIFE TECHNOLOGIES, Tokyo, Japan). Briefly, each RNA sample (5 μg) was subjected to cDNA synthesis in 50 μl of reaction mixture containing 75 mM KCl, 50 mM Tris-HCl (pH 8.3), 3 mM MgC12, 10 mM dithiothreiotol, 0.5 mM each dNTP, 2 μM random primer, and 1000 U AMLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) by incubation at 37°C for 1 h. Each cDNA (10 ng) was amplified in triplicates with the use of the SyBR-Green PCR assay kit and then detected on an ABI PRISM® 7900HT Sequence Detection System. The β-actin RNA was used to standardize the total amount of cDNA.

The primers used were as follows: SGK

Forward: 5'- TCCTGGTGGGCCTTCACTTCTC-3', Reverse: 5'- GTGGTTCCAGGAAGCAGCGTTC-3'. Relative mRNA levels were determined by comparing the PCR cycle thresholds between

cDNA of the gene of interest and that of β-actin. Results are shown in Fig.2.

[Example 4]. Inhibitory effects of siRNAfor LIF on the growth of cells .

SiRNAs for LIF were synthesized by Silencer™ siRNA Construction kit (Ambion, Cat 1620). Sequences were as follows: No. 1

Sense: 5'-AACAACCTGGACAAGCTATGTCCTGTCTC-S ' Antisense: 5 '-AAACATAGCTTGTCCAGGTTGCCTGTCTC-S ' No. 2

Sense: 5'-AAGAAGAAGCTGGGCTGTCAACCTGTCTC-S ' Antisense: 5'-AATTGACAGCCCAGCTTCTTCCCTGCTTC-S' No.3

Sense: 5'-AATGCCCTCTTTATTCTCTATCCTGTCTC-S ' Antisense: 5'-AAATAGAGAATAAAGAGGGCACCTGTCTC-S ', GFP-control-siRNA: 5 ' -ggctacgtccaggagcgcacc-3 ' . siRNA transfection was done with Lipofectamine™ 2000 (Invitrogen) at the 30 % to 40 % cell confluence in antibiotic-free medium. After 48hours transfection, the cells were cultured under hypoxic (1% O ₂ ) and glucose-deprived (13 mg/dl) conditions. Viable cells were counted by Trypan Blue analysis. Results are shown in Fig.3.

[Example 5] Inhibitory effects of siRNA for SGK on the growth of HeIa cells

SiRNAs for SGK were synthesized via a Silencer™ siRNA Construction kit (Ambion, Cat 1620). Sequences were as follows: No. 1

Sense: 5'-AAGGACTTTATTGAGAAGATTCCTGTCTC-S ' Antisense: 5'-AAAATCTTCTGAATAAAGTCGCCTGTCTC-S ' No. 2

Sense: 5'-AAGGATGACTTCATGGAGATTCCTGTCTC-S' Antisense:5'-AAAATCTCCATGAAGTCATCCCCTGTCTC-3' No. 3 Sense: 5 '-AACTGGGATGATCTCATTAATCCTGTCTC-3 '

Antisense: 5'-AAATTAATGAGATCATCCCAGCCTGTCTC -3',

GFP-control-siRNA: 5 ' -ggctacgtccaggagcgcacc-3 ' .

SiRNA transfection was performed with Lipofectamine™ 2000 (Invitrogen) at 30% to 40% cell confluence in antibiotic-free medium. After 48 hours transfection, the cells were cultured under hypoxic (1% O ₂ ) and glucose-deprived (13 mg/dl) conditions. Viable cells were counted by Trypan Blue analysis.Results are shown in Fig.4.

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

All publications, patents, and patent applications cited herein including US Patent Application No.60/786,356, which is a priority document of the present application, are incorporated herein by reference in their entirety.