Antibodies to mitogenic oxygenases Number:7,048,923 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to new genes encoding for the production of novel proteins involved in generation of reactive oxygen intermediates that affect cell division. The present invention also provides vectors containing these genes, cells transfected with these vectors, antibodies raised against these novel proteins, kits for detection, localization and measurement of these genes and proteins, and methods to determine the activity of drugs to affect the activity of the proteins of the present invention.<H oxygenases, Antibodies, Antibodies, to, m, 7048923.html, Patent, pto, 7048923

Title: Antibodies to mitogenic oxygenases

Abstract: The present invention relates to new genes encoding for the production of novel proteins involved in generation of reactive oxygen intermediates that affect cell division. The present invention also provides vectors containing these genes, cells transfected with these vectors, antibodies raised against these novel proteins, kits for detection, localization and measurement of these genes and proteins, and methods to determine the activity of drugs to affect the activity of the proteins of the present invention.

Patent Number: 7,048,923 Issued on 05/23/2006 to Lambeth, et al.

Inventors: Lambeth; J. David (Decatur, GA); Griendling; Kathy K. (Stone Mountain, GA); Lassegue; Bernard P. (Decatur, GA); Arnold; Rebecca S. (Tucker, GA); Cheng; Guangjie (Atlanta, GA)
Assignee: Emory University (Atlanta, GA)

Appl. No.: 319236

Filed: December 13, 2002

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09437568	Nov., 1999	6620603
60151242	Aug., 1999
60149332	Aug., 1999
60107911	Nov., 1998

Current U.S. Class: 424/133.1 ; 424/130.1; 530/388.1; 530/388.8

Current International Class: A61K 39/395 (20060101); A61K 39/40 (20060101); C07K 16/00 (20060101)

Field of Search: 424/130.1,133.1,141.1,178.1,183.1 530/387.1,388.1,388.8,388.85,530.85,89.1,389.7,389.1,391.1,350

References Cited [Referenced By]

U.S. Patent Documents


4340535	July 1982	Voisin et al.
4690914	September 1987	Callut et al.
5191066	March 1993	Bieniarz et al.
5585103	December 1996	Raychaudhuri et al.
5593966	January 1997	Malech et al.
5733748	March 1998	Yu et al.
5929033	July 1999	Tang et al.
5985270	November 1999	Srivastava
6620603	September 2003	Lambeth et al.
6858386	February 2005	Macina et al.
2003/0044783	March 2003	Williams et al.
2003/0109690	June 2003	Ruben et al.

Foreign Patent Documents


WO 96/39419	Dec., 1996	WO
WO 97/42506	Nov., 1997	WO
WO 98/44159	Aug., 1998	WO
WO 98/44133	Oct., 1998	WO
WO 99/01020	Jan., 1999	WO
WO 99/24463	May., 1999	WO
WO 99/25828	May., 1999	WO
WO 99/47658	Sep., 1999	WO
WO 99/53051	Oct., 1999	WO
WO 99/55858	Nov., 1999	WO
WO 99/60161	Nov., 1999	WO
WO 99/63088	Dec., 1999	WO
WO 00/07632	Feb., 2000	WO
WO 00/12708	Mar., 2000	WO
WO 00/28031	May., 2000	WO
WO 00/32221	Jun., 2000	WO
WO 00/37643	Jun., 2000	WO
WO 01/96388	Dec., 2001	WO
WO 01/096388	Dec., 2001	WO

Other References

Batot et al. Eur. J. Biochem. 1995, 234, 208-215. cited by examiner .
Candia et al. (Int. J. Dev. Biol. 1996; 40 (6): 1179-84). cited by examine- r .
Harlow et al. teach (Antibodies, A Laboratory Manual, Chapter 5, p. 76, 1988). cited by examiner .
Abdelrahim, M., et al., "Liquid chromatographic assay of dityrosin in human cerebrospinal fluid," J. Chromatogr. B. Biomed. Sci. Appl., vol. 696, pp. 175-182 (1997). cited by other .
Anderson, S.O., "Covalent cross-links in a structural protein, resilin," Acta Physiol. Scand., vol. 66 Suppl. 263, pp. 1-81 (1966). cited by other .
Burdon, R.H., "Superoxide and hydrogen peroxide in relation to mammalian cell proliferation," Free Radical Biol. Med., vol. 18, No. 4, pp. 775-794 (1995). cited by other .
Church, S.L., et al., "Increased managanese superoxide dismutase expression suppresses the malignant phenotype of human melanoma cells," Proc. Natl. Acad. Sci. USA, vol. 90, pp. 3113-3117 (1993). cited by other .
Emmendorffer, A., et al., "Production of oxygen radicals by fibroblasts and neutrophils from a patient with x-linked chronic granulomatous disease," Eur. J. Haematol, vol. 51, pp. 223-227 (1993). cited by other .
Fernandez-Pol, J.A., et al., "Correlation between the loss of the transformed phenotype and an increase in superoxide dismutase activity in a revertant subclone of sarcoma virus-infected mammalian cells," Can. Res., vol. 42, pp. 609-617 (1982). cited by other .
Fire, A., et al., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature, vol. 391, No. 6669, pp. 806-811 (1998). cited by other .
Frohman, M.A., et al., "Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer, " Proc. Natl. Acad. Sci. USA, vol. 85, pp. 8998-9002 (1988). cite- d by other .
Fukui, T., et al., "p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats, " Cir. Res., vol. 80, No. 1, pp. 45-51 (1997). cited by other .
Gardner, P.R., et al., "Superoxide radical and iron modulate aconitase activity in mammalian cells," J. Biol. Chem., vol. 270, No. 22, pp. 13399-13405 (1995). cited by other .
Griendling, K. K., et al., "Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells, " Cir. Res., vol. 74, No. 6, pp. 1141-1148 (1994). cited by other .
Irani, K., et al., "Mitogenic signaling mediated by oxidants in ras-transformed fibroblasts," Science, vol. 275, No. 5306, pp. 1649-1652 (1997). cited by other .
Li, Y., et al., "Validation of lucigenin (Bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems, " J. Biol. Chem., vol. 273, No. 4, pp. 2015-2023 (1998). cited by other .
Mastsubara, T., et al. "Increased superoxide anion release from human endothelial cells in response to cytokines," J. Immun., vol. 137, No. 10, pp. 3295-3298 (1986). cited by other .
Meier, B., et al., "Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumor necrosis factor-.alpha., " Biochem. J., vol. 263, No. 2, pp. 539-545 (1989). cited by other .
Pagano, P. J., et al., "Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II," Proc. Natl. Acad. Sci. USA, vol. 94, No. 26, pp. 14483-14488 (1997). cited by other .
Schmidt, K. N., et al. "The roles of hyrdogen peroxide and superoxide as messengers in the activation of transcription factor NF-.kappa.B," Chem. & Bio., vol. 2, No. 1, pp. 13-22 (1995). cited by other .
Schreck, R., et al., "Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-.kappa.B transcription factor and HIV-1, " EMBO J., vol. 10, No. 8, pp. 2247-2258 (1991). cited by othe- r .
Suh, Y., et al., "Cell transformation by the superoxide-generating oxidase Mox1, " Nature, vol. 401, No. 6748, pp. 79-82 (1999). cited by other .
Sundaresan, M., et al., "Requirement for generation of H.sub.2O.sub.2 for platelet-derived growth factor signal transduction," Science, vol. 270, pp. 296-299 (1995). cited by other .
Szatroski, T.P., et al., "Production of large amounts of hydrogen peroxide by human tumor cells," Canc. Res., vol. 51, No. 3, pp. 794-798 (1991). cited by other .
Uhlinger, D.J., "Nucleoside triphosphate requirements for superoxide generation and phosphorylation in a cell-free system from human neutrophils," vol. 266, No. 31, pp. 20990-20997 (1991). cited by other .
Ushio-Fukai M., et al., "p22.sup.phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells," J. Biol. Chem., vol. 271, No. 38, pp. 23317-23321 (1996). cited by other .
Yan, T., et al., "Manganese-containing superoxide dismutase overexpression causes phenotypic reversion in SV40-transformed human lung fibroblasts," Canc. Res., vol. 56, pp. 2864-2871 (1996). cited by other .
Yu, L., et al., Biosynthesis of the phagocyte NADPH oxidase cytochrome b.sub.558, J. Biol. Chem., vol. 272, No. 43, pp. 27288-27294 (1997). cite- d by other .
U.S. Appl. No. 60/086,266, filed May 21, 1998, Macina et al. cited by othe- r .
Adams, M.D., et al., EMBL Accession No. AA305700, Mar. 8, 2000. cited by other .
Adams, M.D., et al., EMBL Accession No. AA305700, Apr. 18, 1997. cited by other .
Arbiser, Jack L., et al., "Reactive Oxygen Generated by Nox1 Triggers the Angiogenic Switch, " PNAS, vol. 99, No. 2, pp 715-720, Jan. 22, 2002. cit- ed by other .
Banfi, Botond, et al., "A Mammalian H.sup.+Channel Generated Through Alternative Splicing of the NADPH Oxidase Homolog NOH-1," Science, vol. 287, pp 138-142, Jan. 7, 2000. cited by other .
Bayraktutan, Ulvi, et al., "Molecular Characterization and Localization of the NAD(P)H Oxidase Components gp91-phox and p22-phox in Endothelial Cells," Arterioscler Thromb Vasc Biol., vol. 20, pp 1903-1911, Aug. 2000. cited by other .
Davis, A. R., et al., Genbank Accession No. 046522, May 30, 2000. cited by other .
Dupuy, C., et al., EMBL Accession No. AF181972, Dec. 29, 1999. cited by other .
Dupy, Corinne et al., "Purifucation of a Novel Flavoprotein Involved in the Thyroid NADPH Oxidase --Cloning of the Porcine and Human cDNAs, " The Journal of Biological Chemistry, vol. 274, No. 52, pp 37265-37269, 1999. cited by other .
Griffin, Thomas W., et al., "Initial Clinical Study of Indium-111-Labeled Clone 110 Anticarcinoembryonic Antigen Antibody in Patients with Colorectal Cancer," Journal of Clinical Oncology, vol. 9, No. 4, pp 631-640, April 1991 cited by other .
Hillier L., et al., EMBL Accession No. W52750, Mar. 4, 2000. cited by othe- r .
Kikuchi, Hideaki, et al., "NADPH Oxidase Subunit, gp91.sup.phox Homologue, Preferentially Expressed in Human Colon Epithelial Cells, " GENE, vol. 254, pp 237-243, 2000. cited by other .
Lambeth, J. David, et al., "Novel Homologs of gp91phox," TIBS, vol. 25 pp 459-461, Oct. 2000. cited by other .
Lauffer, Randall B "Targeted Relaxation Enhancement Agents for MRI," Magnetic Resonance in Medicine, vol. 22, pp 339-342, 1991. cited by other .
Li, Fei et al., "CD34.sup.+ Peripheral Blood Progenitors as a Target for Genetic Correction of the Two Flavocytochrome b .sub.558 Defective Forms of Chronic Granulomatous Disease," Blood, vol. 84, No. 1, pp 53-58, Jul. 1, 1994. cited by other .
Lloyd, D., Genbank Accession No. CAB06073, Nov. 23, 1999. cited by other .
Lloyd, D., EMBL Accession No. Z83819, Nov. 23, 1999. cited by other .
Suh, Y.-A., et al., Genbank Accession No. AF127763, Jun. 10, 1999. cited by other .
Banfi, B., et al., Genbank, Accession No. AF166326, Jan. 6, 2000. cited by other .
Banfi, B., et al., Genbank, Accession No. AF166327, Jan 5, 2000. cited by other .
Banfi, B., et al., Genbank, Accession No. AF166328, Jan. 6, 2000. cited by other .
Haenze, J., Genbank, Accession No. AJ438989, Mar. 15, 2002. cited by other .
Suh, Y. A., et al., Genbank, Accession No. NM.sub.--013954, Apr. 28, 2000. cited by other .
Banfi, B., et al., Genbank, Accession No. NM.sub.--013955, Apr. 28, 2000. cited by other .
Suh, Y.A. et al., Genbank, Accession No. NM.sub.--007052, Dec. 22, 1999. cited by other .
Teahan, C. et al., Genbank, Accession No. NP.sub.--000388. cited by other .
Suh, Y.A. et al., Genbank, Accession No. Q9Y5S8, Feb. 15, 2000. cited by other .
NCBI Annotation Project, Genbank, Accession No. XP.sub.--010073, August 27, 2001. cited by other .
Oka, Hiroshi et al., Expression of E-Cadherin Cell Adhesion Molecules in Human Breast Cancer Tissues and Its Relationship to Metastasis, Cancer Reaserch, vol. 53, pp 1696-1701, Apr. 1, 1993. cited by other .
Palmer, S., EMBL Accession No. AL031773, Nov. 23, 1999. cited by other .
Rosenberg, Steven et al., "Use of Tumor-Infiltrating Lymphocytes and Interleukin-2 in the Immunotherapy of Patients with Metastatic Melanoma", The New England Journal of Medicine, vol. 319, No. 25, pp. 1676-1680. cit- ed by other .
Royer-Pokora, Brigitte, et al., "Cloning the Gene for an Inherited Human Disorder --Chronic Granulomatous Disease --on the Basis of its Chromosomal Location, " Nature, vol. 322, pp 32-38, Jul. 3, 1986. cited by other .
Shinozaki, S., et al., "Upregulation of Reg 1a and GW112 in the Epithelium of Inflamed Colonic Mucosa," Gut, vol. 48, pp 623-629, 2001. cited by other .
NCI-CGAP, EMBL Accession No. AA493362, Mar. 3, 2000. cited by other .
NCI-CGAP, EMBL Accession No. AA641653, Mar. 3, 2000. cited by other .
Suh, YA, Genbank, Accession No. Q9WV87, May 30, 2000. cited by other .
Suh, Y.A., Genbank, Accession No. NP.sub.--00893, Feb. 3, 2001. cited by other .
Suh, Y.A., Genbank, Accession No. NP.sub.--039249, Feb. 2, 2001. cited by other .
Sumerdon, Gail A., et al., "An Optimized Antibody-Chelator conjugate for Imaging of Carcinoembryonic Antigen with Indium-111," Nucl. Med. Biol., vol. 17, No. 2, pp 247-254, 1990. cited by other .
Tanigami, A., et al., Genbank, Accession No, AK000683, Feb. 22, 2000. cite- d by other .
Tockman, Melvyn S., et al., "Considerations in Bringing a Cancer Biomarker to Clinical Application," Cancer Research (Suppl.), vol. 52, pp 2711s-2718s, May 1, 1992. cited by other .
Weeraratna, Ashani T., et al., "Loss of Uteroglobin Expression in Prostate Cancer: Relationship to Advancing Grade," Clinical Cancer Research, vol. 3, pp 2295-2300, Dec. 1997. cited by other .
Wilson, R. et al., EMBL Accession No. AF003139, Mar. 3, 2000. cited by oth- er .
Zhou, Yaling, et al., "Interleukin- 4 Suppresses the Expression of Macrophage NADPH Oxidase Heavy Chain Subunit (gp91-phox)," Biochimica et Biophysica Acta, vol. 1265, pp 40-48, 1995. cited by other .
Leusen, Jeanette H. W., et al., "A Point Mutation in gp91-phox of Cytochrome b.sub.558 of the Human NADPH Oxidase Leading to Defective Translocation of the Cytosolic Proteins p47-phox and p67-phox," J. Clin. Invest., vol. 93, pp. 2120-2126, May 1994. cited by other.

Primary Examiner: Siew; Jeffrey
Assistant Examiner: Fetterolf; Brandon
Attorney, Agent or Firm: Klarquist Sparkman LLP

Government Interests

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of National Institutes of Health grants HL38206 and HL58000.

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent application Ser. No. 09/437,568, filed Nov. 10, 1999 now U.S. pat. No. 6,620,603 which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/107,911, filed Nov. 10, 1998, U.S. Provisional Patent Application Ser. No. 60/149,332, filed Aug. 17, 1999, and U.S. Provisional Patent Application Ser. No. 60/151,242, filed Aug. 27, 1999. Each of the aforementioned patent applications is hereby incorporated by reference in its entirety.

Claims

The invention claimed is:

1. An isolated antibody, wherein the isolated antibody specifically binds a polypeptide having the amino acid sequence set forth as SEQ ID NO: 2, wherein the isolated antibody is a monoclonal antibody.

2. An isolated antibody, wherein the isolated antibody is a monoclonal antibody that specifically binds to amino acids 243 256 of SEQ ID NO: 2.

3. An isolated antibody, wherein the antibody is a monoclonal antibody that specifically binds to amino acids 538 551 of SEQ ID NO: 2.

4. The isolated antibody of claim 2, wherein the isolated antibody is labeled.

5. The isolated antibody of claim 4, wherein the label is a radiolabel, dye, magnetic particle, biotin-avidin, fluorescent molecule, ferritin, chemiluminescent molecule, or colloidal gold.

6. The isolated antibody of claim 2, wherein the isolated antibody is linked to a cytotoxin.

7. The isolated antibody of claim 6, wherein the cytotoxin is ricin.

8. The isolated antibody of claim 3, wherein the isolated antibody is labeled.

9. The isolated antibody of claim 8, wherein the label is a radiolabel, dye, magnetic particle, biotin-avidin, fluorescent molecule, ferritin, chemiluminescent molecule, or colloidal gold.

10. The isolated antibody of claim 3, wherein the isolated antibody is linked to a cytotoxin.

11. The isolated antibody of claim 10, wherein the cytotoxin is ricin.

12. The isolated antibody of claim 1, wherein the isolated antibody is labeled.

13. The isolated antibody of claim 12, wherein the label is a radiolabel, dye, magnetic particle, biotin-avidin, fluorescent molecule, ferritin, chemiluminescent molecule, or colloidal gold.

14. The isolated antibody of claim 12, wherein the isolated antibody is linked to a cytotoxin.

15. The isolated antibody of claim 14, wherein the cytotoxin is ricin.

Description

TECHNICAL FIELD

The present invention relates to the field of normal and abnormal cell growth, in particular mitogenic regulation. The present invention provides the following: nucleotide sequences encoding for the production of enzymes that are mitogenic regulators; amino acid sequences of these enzymes; vectors containing these nucleotide sequences; methods for transfecting cells with vectors that produce these enzymes; transfected cells; methods for administering these transfected cells to animals to induce tumor formation; and antibodies to these enzymes that are useful for detecting and measuring levels of these enzymes, and for binding to cells possessing extracellular epitopes of these enzymes.

BACKGROUND OF THE INVENTION

Reactive oxygen intermediates (ROI) are partial reduction products of oxygen: 1 electron reduces O.sub.2 to form superoxide (O.sub.2.sup.-), and 2 electrons reduce O.sub.2 to form hydrogen peroxide (H.sub.2O.sub.2). ROI are generated as a byproduct of aerobic metabolism and by toxicological mechanisms. There is growing evidence for regulated enzymatic generation of O.sub.2.sup.- and its conversion to H.sub.2O.sub.2 in a variety of cells. The conversion of O.sub.2.sup.- to H.sub.2O.sub.2 occurs spontaneously, but is markedly accelerated by superoxide dismutase (SOD). High levels of ROI are associated with damage to biomolecules such as DNA, biomembranes and proteins. Recent evidence indicates generation of ROI under normal cellular conditions and points to signaling roles for O.sub.2.sup.- and H.sub.2O.sub.2.

Several biological systems generate reactive oxygen. Phagocytic cells such as neutrophils generate large quantities of ROI as part of their battery of bactericidal mechanisms. Exposure of neutrophils to bacteria or to various soluble mediators such as formyl-Met-Leu-Phe or phorbol esters activates a massive consumption of oxygen, termed the respiratory burst, to initially generate superoxide, with secondary generation of H.sub.2O.sub.2, HOCl and hydroxyl radical. The enzyme responsible for this oxygen consumption is the respiratory burst oxidase (nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) oxidase).

There is growing evidence for the generation of ROI by non-phagocytic cells, particularly in situations related to cell proliferation. Significant generation of H.sub.2O.sub.2, O.sub.2.sup.-, or both have been noted in some cell types. Fibroblasts and human endothelial cells show increased release of superoxide in response to cytokines such as interleukin-1 or tumor necrosis factor (TNF) (Meier et al. (1989) Biochem J. 263, 539 545.; Matsubara et al. (1986) J. Immun. 137, 3295 3298). Ras-transformed fibroblasts show increased superoxide release compared with control fibroblasts (Irani, et al. (1997) Science 275, 1649 1652). Rat vascular smooth muscle cells show increased H.sub.2O.sub.2 release in response to PDGF (Sundaresan et al. (1995) Science 270, 296 299) and angiotensin II (Griendling et al. (1994) Circ. Res. 74, 1141 1148; Fukui et al. (1997) Circ. Res. 80, 45 51; Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317 23321), and H.sub.2O.sub.2 in these cells is associated with increased proliferation rate. The occurrence of ROI in a variety of cell types is summarized in Table 1 (adapted from Burdon, R. (1995) Free Radical Biol. Med. 18, 775 794).

TABLE-US-00001 TABLE 1 Superoxide Hydrogen Peroxide human fibroblasts Balb/3T3 cells human endothelial cells rat pancreatic islet cells human/rat smooth muscle cells murine keratinocytes human fat cells rabbit chondrocytes human osteocytes human tumor cells BHK-21 cells fat cells, 3T3 L1 cells human colonic epithelial cells

ROI generated by the neutrophil have a cytotoxic function. While ROI are normally directed at the invading microbe, ROI can also induce tissue damage (e.g., in inflammatory conditions such as arthritis, shock, lung disease, and inflammatory bowel disease) or may be involved in tumor initiation or promotion, due to damaging effects on DNA. Nathan (Szatrowski et al. (1991) Canc. Res. 51, 794 798) proposed that the generation of ROI in tumor cells may contribute to the hypermutability seen in tumors, and may therefore contribute to tumor heterogeneity, invasion and metastasis.

In addition to cytotoxic and mutagenic roles, ROI have ideal properties as signal molecules: 1) they are generated in a controlled manner in response to upstream signals; 2) the signal can be terminated by rapid metabolism of O.sub.2.sup.- and H.sub.2O.sub.2 by SOD and catalase/peroxidases; 3) they elicit downstream effects on target molecules, e.g., redox-sensitive regulatory proteins such as NF kappa B and AP-1 (Schreck et al. (1991) EMBO J. 10, 2247 2258; Schmidt et al. (1995) Chemistry & Biology 2, 13 22). Oxidants such as O.sub.2.sup.- and H.sub.2O.sub.2 have a relatively well defined signaling role in bacteria, operating via the SoxI/II regulon to regulate transcription.

ROI appear to have a direct role in regulating cell division, and may function as mitogenic signals in pathological conditions related to growth. These conditions include cancer and cardiovascular disease. O.sub.2.sup.- is generated in endothelial cells in response to cytokines, and might play a role in angiogenesis (Matsubara et al. (1986) J. Immun. 137, 3295 3298). O.sub.2.sup.- and H.sub.2O.sub.2 are also proposed to function as "life-signals", preventing cells from undergoing apoptosis (Matsubara et al. (1986) J. Immun. 137, 3295 3298). As discussed above, many cells respond to growth factors (e.g., platelet derived growth factor (PDGF), epidermal derived growth factor (EGF), angiotensin II, and various cytokines) with both increased production of O.sub.2.sup.-/H.sub.2O.sub.2 and increased proliferation. Inhibition of ROI generation prevents the mitogenic response. Exposure to exogenously generated. O.sub.2.sup.- and H.sub.2O.sub.2 results in an increase in cell proliferation. A partial list of responsive cell types is shown below in Table 2 (adapted from Burdon, R. (1995) Free Radical Biol. Med. 18, 775 794).

TABLE-US-00002 TABLE 2 Superoxide Hydrogen peroxide human, hamster fibroblasts mouse osteoblastic cells Balb/3T3 cells Balb/3T3 cells human histiocytic leukemia rat, hamster fibroblasts mouse epidermal cells human smooth muscle cells rat colonic epithelial cells rat vascular smooth muscle cells rat vascular smooth muscle cells

While non-transformed cells can respond to growth factors and cytokines with the production of ROI, tumor cells appear to produce ROI in an uncontrolled manner. A series of human tumor cells produced large amounts of hydrogen peroxide compared with non-tumor cells (Szatrowski et al. (1991) Canc. Res. 51, 794 798). Ras-transformed NIH 3T3 cells generated elevated amounts of superoxide, and inhibition of superoxide generation by several mechanisms resulted in a reversion to a "normal" growth phenotype.

O.sub.2.sup.- has been implicated in maintenance of the transformed phenotype in cancer cells including melanoma, breast carcinoma, fibrosarcoma, and virally transformed tumor cells. Decreased levels of the manganese form of SOD (MnSOD) have been measured in cancer cells and in vitro-transformed cell lines, predicting increased O.sub.2.sup.- levels (Burdon, R. (1995) Free Radical Biol. Med. 18, 775 794). MnSOD is encoded on chromosome 6q25 which is very often lost in melanoma. Overexpression of MnSOD in melanoma and other cancer cells (Church et al. (1993) Proc. of Natl. Acad. Sci. 90, 3113 3117; Fernandez-Pol et al. (1982) Canc. Res. 42, 609 617; Yan et al. (1996) Canc. Res. 56, 2864 2871) resulted in suppression of the transformed phenotype.

ROI are implicated in growth of vascular smooth muscle associated with hypertension, atherosclerosis, and restenosis after angioplasty. O.sub.2.sup.- generation is seen in rabbit aortic adventitia (Pagano et al. (1997) Proc. Natl. Acad. Sci. 94, 14483 14488). Vascular endothelial cells release O.sub.2.sup.- in response to cytokines (Matsubara et al. (1986) J. Immun. 137, 3295 3298). O.sub.2.sup.- is generated by aortic smooth muscle cells in culture, and increased O.sub.2.sup.- generation is stimulated by angiotensin II which also induces cell hypertrophy. In a rat model system, infusion of angiotensin II leads to hypertension as well as increased O.sub.2.sup.- generation in subsequently isolated aortic tissue (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317 23321.; Yu et al. (1997) J. Biol. Chem. 272, 27288 27294). Intravenous infusion of a form of SOD that localizes to the vasculature or an infusion of an O.sub.2.sup.- scavenger prevented angiotensin II induced hypertension and inhibited ROI generation (Fukui et al. (1997) Circ. Res. 80, 45 51).

The neutrophil NADPH oxidase, also known as phagocyte respiratory burst oxidase, provides a paradigm for the study of the specialized enzymatic ROI-generating system. This extensively studied enzyme oxidizes NADPH and reduces oxygen to form O.sub.2.sup.-. NADPH oxidase consists of multiple proteins and is regulated by assembly of cytosolic and membrane components. The catalytic moiety consists of flavocytochrome b.sub.558, an integral plasma membrane enzyme comprised of two components: gp91phox (gp refers to glycoprotein; phox is an abbreviation of the words phagocyte and oxidase) and p22phox (p refers to protein). gp91phox contains 1 flavin adenine dinucleotide (FAD) and 2 hemes as well as the NADPH binding site. p22phox has a C-terminal proline-rich sequence which serves as a binding site for cytosolic regulatory proteins. The two cytochrome subunits, gp91phox and p22phox appear to stabilize one another, since the genetic absence of either subunit, as in the inherited disorder chronic granulomatous disease (CGD), results in the absence of the partner subunit (Yu et al. (1997) J. Biol. Chem. 272, 27288 27294). Essential cytosolic proteins include p47phox, p67phox and the small GTPase Rac, of which there are two isoforms. p47phox and p67phox both contain SH.sub.3 regions and proline-rich regions which participate in protein interactions governing assembly of the oxidase components during activation. The neutrophil enzyme is regulated in response to bacterial phagocytosis or chemotactic signals by phosphorylation of p47phox, and perhaps other components, as well as by guanine nucleotide exchange to activate the GTP-binding protein Rac.

The origin of ROI in non-phagocytic tissues is unproven, but the occurrence of phagocyte oxidase components has been evaluated in several systems by immunochemical methods, Northern blots and reverse transcriptase-polymerase chain reaction (RT-PCR). The message for p22phox is expressed widely, as is that for Rac1. Several cell types that are capable of O.sub.2.sup.- generation have been demonstrated to contain all of the phox components including gp91phox, as summarized below in Table 3. These cell types include endothelial cells, aortic adventitia and lymphocytes.

TABLE-US-00003 TABLE 3 Tissue gp91phox p22phox p47phox p67phox neutrophil +.sup.1,2 +.sup.1,2 +.sup.1,2 +.sup.1,2 aortic adventitia +.sup.1 +.sup.1 +.sup.1 +.sup.1 lymphocytes +.sup.2 +.sup.2 +.sup.1,2 +.sup.1,2 endothelial cells +.sup.2 +.sup.2 +.sup.1,2 +.sup.1,2 glomerular mesangial -- +.sup.1,2 +.sup.1,2 +.sup.1,2 cells fibroblasts -- +.sup.2 +.sup.1,2 +.sup.2 aortic sm. muscle -- +.sup.1,2 ? ? .sup.1= protein expression shown. .sup.2= mRNA expression shown.

However, a distinctly different pattern is seen in several other cell types shown in Table 3 including glomerular mesangial cells, rat aortic smooth muscle and fibroblasts. In these cells, expression of gp91phox is absent while p22phox and in some cases cytosolic phox components have been demonstrated to be present. Since gp91phox and p22phox stabilize one another in the neutrophil, there has been much speculation that some molecule, possibly related to gp91phox, accounts for ROI generation in glomerular mesangial cells, rat aortic smooth muscle and fibroblasts (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317 23321). Investigation of fibroblasts from a patient with a genetic absence of gp91phox provides proof that the gp91phox subunit is not involved in ROI generation in these cells (Emmendorffer et al. (1993) Eur. J. Haematol. 51, 223 227). Depletion of p22phox from vascular smooth muscle using an antisense approach indicated that this subunit participates in ROI generation in these cells, despite the absence of detectable gp91phox (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317 23321). At this time the molecular candidates possibly related to gp91phox and involved in ROI generation in these cells are unknown.

Accordingly, what is needed is the identity of the proteins involved in ROI generation, especially in non-phagocytic tissues and cells. What is also needed are the nucleotide sequences encoding for these proteins, and the primary sequences of the proteins themselves. Also needed are vectors designed to include nucleotides encoding for these proteins. Probes and PCR primers derived from the nucleotide sequence are needed to detect, localize and measure nucleotide sequences, including mRNA, involved in the synthesis of these proteins. In addition, what is needed is a means to transfect cells with these vectors. What is also needed are expression systems for production of these molecules. Also needed are antibodies directed against these molecules for a variety of uses including localization, detection, measurement and passive immunization.

SUMMARY OF THE INVENTION

The present invention solves the problems described above by providing a novel family of nucleotide sequences and proteins encoded by these nucleotide sequences termed mox proteins and duox proteins. In particular the present invention provides compositions comprising the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, and fragments thereof, which encode for the expression of proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, respectively, and fragments thereof. While not wanting to be bound by the following statement, it is believed that these proteins are involved in ROI production. The present invention also provides vectors containing these nucleotide sequences, cells transfected with these vectors which produce the proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, and fragments thereof, and antibodies to these proteins and fragments thereof. The present invention also provides methods for stimulating cellular proliferation by administering vectors encoded for production of the proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof. The present invention also provides methods for stimulating cellular proliferation by administering the proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof. The nucleotides and antibodies of the present invention are useful for the detection, localization and measurement of the nucleic acids encoding for the production of the proteins of the present invention, and also for the detection, localization and measurement of the proteins of the present invention. These nucleotides and antibodies can be combined with other reagents in kits for the purposes of detection, localization and measurement.

Most particularly, the present invention involves a method for regulation of cell division or cell proliferation by modifying the activity or expression of the proteins described as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 or fragments thereof. These proteins, in their naturally occurring or expressed forms, are expected to be useful in drug development, for example for screening of chemical and drug libraries by observing inhibition of the activity of these enzymes. Such chemicals and drugs would likely be useful as treatments for cancer, prostatic hypertrophy, benign prostatic hypertrophy, hypertension, atherosclerosis and many other disorders involving abnormal cell growth or proliferation as described below. The entire expressed protein may be useful in these assays. Portions of the molecule which may be targets for inhibition or modification include but are not limited to the binding site for pyridine nucleotides (NADPH or NADH), the flavoprotein domain (approximately the C-terminal 265 amino acids), and/or the binding or catalytic site for flavin adenine dinucleotide (FAD).

The method of the present invention may be used for the development of drugs or other therapies for the treatment of conditions associated with abnormal growth including, but not limited to the following: cancer, psoriasis, prostatic hypertrophy, benign prostatic hypertrophy, cardiovascular disease, proliferation of vessels, including but not limited to blood vessels and lymphatic vessels, arteriovenous malformation, vascular problems associated with the eye, atherosclerosis, hypertension, and restenosis following angioplasty. The enzymes of the present invention are excellent targets for the development of drugs and other agents which may modulate the activity of these enzymes. It is to be understood that modulation of activity may result in enhanced, diminished or absence of enzymatic activity. Modulation of the activity of these enzymes may be useful in treatment of conditions associated with abnormal growth.

Drugs which affect the activity of the enzymes represented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, or fragments thereof, may also be combined with other therapeutics in the treatment of specific conditions. For example, these drugs may be combined with angiogenesis inhibitors in the treatment of cancer, with antihypertensives for the treatment of hypertension, and with cholesterol lowering drugs for the treatment of atherosclerosis.

Accordingly, an object of the present invention is to provide nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to provide vectors containing these nucleotide sequences, or fragments thereof.

Yet another object of the present invention is to provide cells transfected with these vectors.

Still another object of the present invention is to administer cells transfected with these vectors to animals and humans.

Another object of the present invention is to provide proteins, or fragments thereof, that are involved in ROI production.

Still another object of the present invention is to provide antibodies, including monoclonal and polyclonal antibodies, or fragments thereof, raised against proteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to administer genes containing nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans and also to cells obtained from animals and humans.

Another object of the present invention is to administer antisense complimentary sequences of genes containing nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans and also to cells obtained from animals and humans.

Yet another object of the present invention is to provide a method for stimulating or inhibiting cellular proliferation by administering vectors containing nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans. It is also an object of the present invention to provide a method for stimulating or inhibiting cellular proliferation by administering vectors containing antisense complimentary sequences of nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans. These methods of stimulating cellular proliferation are useful for a variety of purposes, including but not limited to, developing animal models of tumor formation, stimulating cellular proliferation of blood marrow cells following chemotherapy or radiation, or in cases of anemia.

Still another object of the present invention is to provide antibodies useful in immunotherapy against cancers expressing the proteins represented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 or fragments thereof.

Yet another object of the present invention is to provide nucleotide probes useful for the detection, localization and measurement of nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to provide antibodies useful for the detection, localization and measurement of nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production.

Another object of the present invention is to provide kits useful for detection of nucleic acids including the nucleic acids represented in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, or fragments thereof, that encode for proteins, or fragments thereof, that are involved in ROI production.

Yet another object of the present invention is to provide kits useful for detection and measurement of nucleic acids including the nucleic acids represented in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, or fragments thereof, that encode for proteins, or fragments thereof, that are involved in ROI production.

Still another object of the present invention is to provide kits useful for the localization of nucleic acids including the nucleic acids represented in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, or fragments thereof, that encode for proteins, or fragments thereof that are involved in ROI production.

Another object of the present invention is to provide kits useful for detection of proteins, including the proteins represented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, or fragments thereof, that are involved in ROI production.

Yet another object of the present invention is to provide kits useful for detection and measurement of proteins, including the proteins represented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, or fragments thereof, that are involved in ROI production.

Still another object of the present invention is to provide kits useful for localization of proteins, including the proteins represented in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48, or fragments thereof, that are involved in ROI production.

Yet another object of the present invention is to provides kits useful for the detection, measurement or localization of nucleic acids, or fragments thereof, encoding for proteins, or fragments thereof, that are involved in ROI production, for use in diagnosis and prognosis of abnormal cellular proliferation related to ROI production.

Another object of the present invention is to provides kits useful for the detection, measurement or localization of proteins, or fragments thereof, that are involved in ROI production, for use in diagnosis and prognosis of abnormal cellular proliferation related to ROI production.

These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1(a d). Comparison of amino acid sequences of the human mox1 protein (labeled mox1.human, SEQ ID NO:2), rat mox1 protein (labeled mox1.rat, SEQ ID NO:21), human mox2 protein (labeled mox2.human., SEQ ID NO:4) of the present invention to human (gp 91phox/human.pep, SEQ ID NO:12) bovine (gp 91 phox/bovine.pep, SEQ ID NO:37), and murine (gp 91 phox/mouse.pep, SEQ ID NO:38) proteins. Also included are related plant enzyme proteins cytb 558.arabidopsis.pep (SEQ ID NO:39) and cytb558.rice.pep, (SEQ ID NO:40). Enclosed in boxes are similar amino acid residues.

FIG. 2. Sequence similarities among proteins related to gp91phox including human mox1 (SEQ ID NO:2), human mox2 (SEQ ID NO:4), and rat mox1 (SEQ ID NO:21). The dendrogram indicates the degree of similarity among this family of proteins, and also includes the related plant enzymes.

FIG. 3. Cell free assay for mox-1 activity. Superoxide generation was measured using the chemiluminescent reaction between lucigenin and superoxide in cell lysates from vector control NEF2 and mox1 transfected NIH3T3 cells.

FIG. 4. Superoxide generation by human mox1. Reduction of NBT in mox1 transfected and control fibroblasts was measured in the absence (filled bars) or presence (open bars) or superoxide dismutase.

FIG. 5. Aconitase (filled bars), lactate dehydrogenase (narrow hatching) and fumarase (broad hatching) were determined in lysates of cells transfected with vector alone (NEF2) or with mox1 (YA26, YA28 and YA212).

DETAILED DESCRIPTION OF THE INVENTION

The present invention solves the problems described above by providing a novel family of nucleotide sequences and proteins, encoded by these nucleotide sequences, termed mox proteins and duox proteins. The term "mox" refers to "mitogenic oxidase" while the term "duox" refers to "dual oxidase". In particular, the present invention provides novel compositions comprising the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, and fragments thereof, which encode, respectively, for the expression of proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof.

Both the mox and duox proteins described herein have homology to the gp91phox protein involved in ROI generation, however, the mox and duox proteins comprise a novel and distinct family of proteins. The mox proteins included in the present invention have a molecular weight of approximately 65 kDa as determined by reducing gel electrophoresis and are capable of inducing ROI generation in cells. As described in more detail below, the mox proteins of the present invention also function in the regulation of cell growth, and are therefore implicated in diseases involving abnormal cell growth such as cancer. The present invention describes mox proteins found in human and rat, however, it is likely that the mox family of genes/proteins is widely distributed among multicellular organisms.

The duox proteins described herein are larger than the mox proteins and have three distinct regions: the amino terminal region having homology to peroxidase proteins, the internal region having homology to calmodulin (CAM) proteins and the carboxy-terminal region having homology to mox proteins. Human duox1 is shown in SEQ ID NO:46 and a portion of human duox2 is shown in SEQ ID NO:48. Nucleotides encoding duox1 and duox2 proteins are also shown in SEQ ID NO: 45 and SEQ ID NO:47, respectively. In addition to the human duox proteins, comparison of the sequence of human duox1 and human duox2 with genomic databases using BLAST searching resulted in the identification of two homologs of duox in C. elegans (Ce-duox1 and Ce-duox2). Drosophila also appears to have at least one duox homolog. Thus, the duox family of genes/proteins is widely distributed among multicellular organisms.

Although not wanting to be bound by the following statement, it is believed that duox1 and duox2 have dual enzymatic functions, catalyzing both the generation of superoxide and peroxidative type reactions. The latter class of reactions utilize hydrogen peroxide as a substrate (and in some cases have been proposed to utilize superoxide as a substrate). Since hydrogen peroxide is generated spontaneously from the dismutation of superoxide, it is believed that the NAD(P)H oxidase domain generates the superoxide and/or hydrogen peroxide which can then be used as a substrate for the peroxidase domain. In support of this hypothesis, a model for the duox1 protein in C. elegans has been developed that has an extracellular N-terminal peroxidase domain, a transmembrane region and a NADPH binding site located on the cytosolic face of the plasma membrane. By analogy with the neutrophil NADPH-oxidase which generates extracellular superoxide, human duox1 is predicted to generate superoxide and its byproduct hydrogen peroxide extracellularly where it can be utilized by the peroxidase domain.

While the ROI generated by duox1 and duox2 may function as does mox1 in regulation of cell growth, the presence of the peroxidase domain is likely to confer additional biological functions. Depending upon the co-substrate, peroxidases can participate in a variety of reactions including halogenation such as the generation of hypochlorous acid (HOCl) by myeloperoxidase and the iodination of tyrosine to form thyroxin by thyroid peroxidase. Peroxidases have also been documented to participate in the metabolism of polyunsaturated fatty acids, and in the chemical modification of tyrosine in collagen (by sea urchin ovoperoxidase). Although not wanting to be bound by this statement, it is believed that the predicted transmembrane nature of duox1 facilitates its function in the formation or modification of extracellular matrix or basement membrane. Since the extracellular matrix plays an important role in tumor cell growth, invasion and metastasis, it is believed that the duox type enzymes play a pathogenic role in such conditions.

In addition to the nucleotide sequences described above, the present invention also provides vectors containing these nucleotide sequences and fragments thereof, cells transfected with these vectors which produce the proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof, and antibodies to these proteins and fragments thereof. The present invention also provides methods for stimulating cellular proliferation by administering vectors, or cells containing vectors, encoded for production of the proteins comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 and fragments thereof. The nucleotides and antibodies of the present invention are useful for the detection, localization and measurement of the nucleic acids encoding for the production of the proteins of the present invention, and also for the detection, localization and measurement of the proteins of the present invention. These nucleotides and antibodies can be combined with other reagents in kits for the purposes of detection, localization and measurement. These kits are useful for diagnosis and prognosis of conditions involving cellular proliferation associated with production of reactive oxygen intermediates.

The present invention solves the problems described above by providing a composition comprising the nucleotide sequence SEQ ID NO:1 and fragments thereof. The present invention also provides a composition comprising the nucleotide sequence SEQ ID NO:3 and fragments thereof. The present invention also provides a composition comprising the nucleotide sequence SEQ ID NO:22 and fragments thereof. The present invention also provides a composition comprising the nucleotide sequence SEQ ID NO:41 and fragments thereof. The present invention also provides a composition comprising the nucleotide sequence SEQ ID NO:45 and fragments thereof. The present invention also provides a composition comprising the nucleotide sequence SEQ ID NO:47 and fragments thereof.

The present invention provides a composition comprising the protein SEQ ID NO:2 encoded by the nucleotide sequence SEQ ID NO:1. The present invention provides a composition comprising the protein SEQ ID NO:4 encoded by the nucleotide sequence SEQ ID NO:3. The present invention provides a composition comprising the protein SEQ ID NO:21 encoded by the nucleotide sequence SEQ ID NO:22. The present invention provides a composition comprising the protein SEQ ID NO:42 encoded by the nucleotide sequence SEQ ID NO:41. The present invention provides a composition comprising the protein SEQ ID NO:46 encoded by the nucleotide sequence SEQ ID NO:45. The present invention provides a composition comprising the protein SEQ ID NO:48 encoded by the nucleotide sequence SEQ ID NO:47.

The present invention provides a composition comprising the protein SEQ ID NO:2 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:1 or fragments thereof. The present invention also provides a composition comprising the protein SEQ ID NO:4 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:3 or fragments thereof. The present invention also provides a composition comprising the protein SEQ ID NO:21 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:22 or fragments thereof. The present invention also provides a composition comprising the protein SEQ ID NO:42 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:41 or fragments thereof. The present invention also provides a composition comprising the protein SEQ ID NO:46 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:45 or fragments thereof. The present invention also provides a composition comprising the protein SEQ ID NO:48 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:47 or fragments thereof.

The present invention also provides vectors containing the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47 or fragments thereof. The present invention also provides cells transfected with these vectors. In addition, the present invention provides cells stably transfected with the nucleotide sequence SEQ ID NO:1 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:22 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:41 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:45 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:47 or fragments thereof.

The present invention provides cells stably transfected with the nucleotide sequence SEQ ID NO:1 or fragments thereof, which produce the protein SEQ ID NO:2 or fragments thereof. In addition, the present invention provides cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof which produce the protein SEQ ID NO:4 or fragments thereof. In addition, the present invention provides cells stably transfected with the nucleotide sequence SEQ ID NO:22 or fragments thereof which produce the protein SEQ ID NO:21 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:41 or fragments thereof which produce the protein SEQ ID NO:42 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:45 or fragments thereof which produce the protein SEQ ID NO:46 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:47 or fragments thereof which produce the protein SEQ ID NO:48 or fragments thereof.

The present invention provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:1 which produce the protein SEQ ID NO:2 or fragments thereof. The present invention also provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof, which produce the protein SEQ ID NO:4 or fragments thereof. The present invention also provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:22 or fragments thereof, which produce the protein SEQ ID NO:21 or fragments thereof. The present invention also provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:41 or fragments thereof, which produce the protein SEQ ID NO:42 or fragments thereof. The present invention also provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:45 or fragments thereof, which produce the protein SEQ ID NO:46 or fragments thereof. The present invention also provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:47 or fragments thereof, which produce the protein SEQ ID NO:48 or fragments thereof.

Specifically, the present invention provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:1 or fragments thereof, which produce the protein SEQ ID NO:2 or fragments thereof. The present invention also provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof, which produce the protein SEQ ID NO:4 or fragments thereof. The present invention also provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:22 or fragments thereof, which produce the protein SEQ ID NO:21 or fragments thereof. The present invention also provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:41 or fragments thereof, which produce the protein SEQ ID NO:42 or fragments thereof. The present invention also provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:45 or fragments thereof, which produce the protein SEQ ID NO:46 or fragments thereof. The present invention also provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:47 or fragments thereof, which produce the protein SEQ ID NO:48 or fragments thereof.

The present invention may also be used to develop anti-sense nucleotide sequences to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47 or fragments thereof. These anti-sense molecules may be used to interfere with translation of nucleotide sequences, such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:22, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:47, or fragments thereof, that encode for proteins such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:48 or fragments thereof. Administration of these anti-sense molecules, or vectors encoding for ant