| Non-steroidal anti-inflammatory drug activated gene with anti-tumorigenic properties -> Monitor Keywords |
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Non-steroidal anti-inflammatory drug activated gene with anti-tumorigenic propertiesRelated Patent Categories: Multicellular Living Organisms And Unmodified Parts Thereof And Related Processes, Nonhuman Animal, Transgenic Nonhuman Animal (e.g., Mollusks, Etc.), MammalNon-steroidal anti-inflammatory drug activated gene with anti-tumorigenic properties description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070180543, Non-steroidal anti-inflammatory drug activated gene with anti-tumorigenic properties. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] This invention generally relates to drug screens for agents that are agonistic or antagonistic to activation of the promoter region of the NAG-1 gene. BACKGROUND OF THE INVENTION [0002] Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of inflammatory disease. Their anti-inflammatory effects are believed to result from their ability to inhibit the formation of prostaglandins by prostaglandin H synthase (COX). Two isoforms of prostaglandin H synthase, COX-1 and COX-2 have been identified. COX-1 is constitutively expressed in many tissues, while the expression of COX-2 is regulated by mitogens, tumor promoters, and growth factors. High expression of COX-2 has been reported in human colorectal tumors and tumors from other tissues, suggesting a role for this enzyme in regulating tumor growth. NSAIDs are effective in reducing human and rodent colorectal and possibly, breast and lung cancer. In addition, retrospective and prospective studies link NSAID usage to a reduced risk for colorectal cancer death. NSAIDs are effective in reducing the number and size of polyps in animal models, and epidemiological studies indicate that use of NSAIDs provide a 40-50% reduction in mortality from colorectal cancer. [0003] Our understanding of the mechanisms by which NSAIDs exert their anti-tumor effect is not clear. One possible mechanism is altered arachidonic acid (AA) metabolism, since NSAIDs inhibit the formation of prostaglandins by COX-1 and COX-2. Recent data suggest that inhibition of COX by NSAIDs may increase the cellular pool of AA, resulting in the hydrolysis of sphingomyelin to ceramide, which promotes apoptosis. Some data ink NSAID chemoprevention in colorectal cancer cells to prevention of angiogenesis and induction of apoptosis. COX appears to regulate angiogenesis induced by colon cancer cells and NSAIDs appear to limit tumor growth. In epithelial cells of the intestinal crypt, elevated COX-2 expression appears to attenuate apoptosis. Some evidence links NSAID-induced apoptosis to inhibition of COX, but other data suggest a prostaglandin-independent mechanism for the induction of apoptosis. PPAR.gamma. agonists also have anti-tumorigenic activity and stimulate apoptosis. For example, ligand activation of PPAR.gamma. inhibits proliferation of breast cancer cells, and the addition of PPAR.gamma. ligands to cancer cells induces apoptosis. PPAR.gamma. agonists also inhibit the growth of transplantable tumors in a nude mouse model. [0004] As is evident from the foregoing, NSAIDs, and the pathways they activate, have great potential for use in the war against cancer. However, as is also evident from the above, insufficient information has been available regarding the NSAID-induced or associated anti-cancer pathways, to help identify agents that are potentially advantageous as cancer treatments. What is needed are novel reagents and methods that can be used to screen for compounds that show promise as anti-cancer agents. SUMMARY OF THE INVENTION [0005] The present invention generally relates to compositions and methods of identifying and testing NAG-1 gene-activating agonists and antagonists. Activation of the NAG-1 gene by NSAIDs has been correlated with the apoptotic elimination of certain types of cancer cells. Although not limited to any particular mechanism, one embodiment of the present invention involves the use of the promoter region of the NAG-1 gene to test for agents that are agonistic or antagonistic to promoter activation. In this regard, test kits also constitute an embodiment of the present invention. In addition, the invention provides methods to identify other members of the NAG-1 gene family, methods to identity homologs of NAG-1 which are native to other tissue or cell types and methods to generate reagents derived from the invention. [0006] The present invention is not limited by the method of the employed screen. In one embodiment the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one preferred embodiment, the cells are transfected transiently, while in another preferred embodiment the cells are stably transfected. In yet another preferred embodiment, transgenic animals are generated. In yet another embodiment, high throughput screens are contemplated. For example, the present invention provides means to screen compound libraries, peptide libraries and the like, in order to identify suitable antagonists or agonists. [0007] The present invention also contemplates the use of a reporter gene construct operationally liked to the NAG-1 promoter. The present invention is not limited to any particular reporter gene construct, as many reporter gene constructs will find use with the present invention. For example, in a preferred embodiment, a luciferase reporter gene is used. In other embodiments, genes encoding fluorescent proteins (e.g., green fluorescent protein), .beta.-galactosidase, or proteins that may be precipitated (e.g., by immunoprecipitation) are used. [0008] It is further contemplated that the present invention will find use in the identification of native activators or inhibitors of the NAG-1 gene. For example, it is contemplated that peptide libraries produced from native genes are screened by the present invention. Non-natively occurring peptides are also be screened by the present invention. In another embodiment combinatorial libraries are screened. In yet another embodiment, mixtures of compounds are screened. In some preferred embodiments, if a compound mixture gives a positive test result, the mixture is then subdivided into constitutory components and retested. In still further embodiments, this procedure is repeated until the active component or components are identified. [0009] The present invention also be finds use in the identification of new homologs of NAG-1, as well as genes that share similarities in the promoter region or natural mutations thereof. The present invention contemplates screening for homologs using standard molecular procedures. In one preferred embodiment, screens are conducted using Northern and Southern blotting. [0010] Furthermore, as shown in Example 8 below, the NAG-1 peptide has anti-tumor and pro-apoptotic effects. It is contemplated that this peptide, or a portion thereof, will be effective in the treatment of certain cancers when administered directly to tumors or to cancer patients. In this regard, the present invention also provides compositions and methods for the use of the NAG-1 peptide for the treatment of cancers. The present invention further provides the compositions and methods for use of modified or derived variations of the NAG-1 peptide for the treatment of cancers. [0011] The present invention is not limited to the treatment of any particular cancer as it is contemplated that all cancers may be treated with the NAG-1 peptide. The present invention is also not limited to any particular portion of the NAG-1 peptide for the treatment of cancers, as it is contemplated that any suitable portion of the NAG-1 peptide will find use in cancer treatment. Furthermore, the present invention is not limited to any particular modification of the NAG-l peptide for the treatment of cancer. Indeed, any modification that changes the ability of the NAG-1 peptide to treat cancer is embodied in the present invention. Such changes include, but are not limited to, increased stability, increased solubility, increased tumor killing ability, decreased toxicity, increased half-life before and after administration to the patient, etc. Also, the present invention is not limited to any particular method of administration, as the NAG-1 peptide may be administered by any suitable means including, but not limited to, oral, nasal, intravenous, intramuscular, intrathecal and subcutaneous modes of administration. Additionally, the NAG-1 peptide may be administered as an ointment, lotion or gel (i.e., for the treatment of skin and mucosal tumors). [0012] The present invention further provides purified DNA having an oligonucleotide sequence comprising SEQ ID NO:1, as well as compositions comprising this sequence or portion(s) thereof. In addition, the present invention provides expression vectors comprising at least a portion of the oligonucleotide sequence of SEQ ID NO:1. Further still, the present invention provides compositions comprising the translation product of at least a portion of SEQ ID NO:1. The present invention further provides compositions comprising antibodies reactive with at least a portion of the translation product of SEQ ID NO:1. The present invention also provides transgenic animals generated from an expression vector comprising at least a portion of the oligonucleotide sequence of SEQ ID NO:1. [0013] The present invention provides methods for screening compounds, comprising the steps of: a) providing in any order, cells comprising a recombinant expression vector, wherein the vector comprises at least a portion of the oligonucleotide sequence of SEQ ID NO:1 and at least one reporter construct; and, at least one compound; b) contacting the cells with the compound(s) to produce treated cells; and, c) detecting the readout of the reporter construct or constructs in the treated cells. [0014] The present invention also provides methods for screening compounds, comprising the steps of: a) providing in any order, at least one transgenic animal generated from a recombinant expression vector wherein, the vector comprises at least a portion of the oligonucleotide sequence of SEQ ID NO:1 and at least one reporter construct; and, at least one compound; b) exposing the transgenic animal(s) to at least one compound to produce at least one treated animal; and, c) detecting the readout of the reporter construct(s) in the treated animal(s). The present invention further provides methods wherein, at least one transgenic animal is repeatedly (i.e., more than once) exposed to at least one compound. The present invention also further provides methods wherein the treated animal further comprises cancer cells and a decrease or increase in the number of cancer cells present in the treated animal is measured. [0015] The present invention also provides methods comprising: providing in any order, a patient with symptoms of cancer and, a composition comprising at least a portion of the NAG-1 peptide; and b) administering the composition to the patient. The present invention is not limited to any particular type of cancer as it is contemplated that the present invention will find use with all types of cancer. Additionally, the present invention is not limited to any particular means of administration of NAG-1 to the patient, as the NAG-1 peptide may be administered by any suitable means including, but not limited to, oral, nasal, intravenous, intramuscular, intrathecal and subcutaneous modes of administration. Additionally, the NAG-1 peptide may be administered as an ointment, lotion or gel (i.e., for the treatment of skin and mucosal tumors). DESCRIPTION OF THE FIGURES [0016] FIG. 1 shows the identification and expression of NAG-1 by INDO. Panel A provides a schematic diagram for reported genes PLAB, PTGFB, PDDF, MIC-1, and HP00269. The bar indicates the coding region of cDNA with amino acids reported previously. Panel B provides Northern and Western analysis results that indicate NAG-1 induction is time-dependent. Panel C is a graph showing the apoptosis and cell cycle kinetics of INDO-treated HCT-116 cells at different time points. Panel D provides Northern and Western data showing the concentration-dependent expression of NAG-1. Panel B is a graph showing the apoptosis and cell-cycle kinetics of INDO-treated HCT-116 cells at different concentrations. [0017] FIG. 2 shows NAG-1 induction and NSAID-induced apoptosis by several NSAIDs. Panel A shows results for HCT-116 cells treated with various NSAIDs.1 Panel B is a graph showing the correlation between induction of apoptosis and increased expression of NAG-1. [0018] FIG. 3 shows NAG-1 induction by INDO and aspirin in different cell lines. Panel A provides Northern blot results for A549 lung carcinoma cells, while Panel B provides Northern blot results for MCF-7 breast carcinoma cells, Panel C provides Northern blot results for U937 leukemia cells, and Panel D provides Northern blot results for PC-3 prostate cells. [0019] FIG. 4 shows PPAR.gamma. ligands also increase NAG-1 expression. Panel A provides results for 15dPGJ.sub.2 and TGZ. Panel B provides Northern and Western results indicating the time-dependent expression of NAG-1 mRNA and protein in HCT-116 cells. Panel C is a graph showing the apoptosis and cell cycle kinetics of TGZ-treated HCT-116 cells at different times. [0020] FIG. 5 shows the genomic structure and promoter analysis of NAG-1 in HCT-116 cells. Panel A provides a restriction map of the NAG-1 gene. Panel B is a deletion analysis of the NAG-1 promoter. Panel C is a graph showing the luciferase activity of 2021 bp NAG-1 promoter in the presence of TGZ and various NSAIDs. 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