| Atp-binding cassette protein responsible for cytotoxin resistance -> Monitor Keywords |
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  Atp-binding cassette protein responsible for cytotoxin resistanceRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate, Animal Cell, Tumor Cell Or Cancer CellThe Patent Description & Claims data below is from USPTO Patent Application 20070048810. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] The present application is a division of U.S. Ser. No. 09/856,927, which is the national phase under 35 U.S.C. .sctn.371 of PCT/US99/28107, filed Nov. 24, 1999, which claims priority to U.S. Ser. No. 60/110,473, filed Nov. 30, 1998. The contents of each of the above-mentioned documents are incorporated herein in the entirety. FIELD OF THE INVENTION [0002] This invention provides for a novel ATP-binding cassette protein which is responsible for cytotoxin resistance. The invention also provides for methods of expressing the protein and assays for identification of inhibitors of the protein. BACKGROUND OF THE INVENTION [0003] The multidrug resistance/ATP-binding cassette (MDR/ABC) superfamily in humans includes genes whose products represent membrane proteins involved in energy-dependent transport of a wide variety of substrates across a membrane (see, e.g., Dean, M. and Allikmets, R. (1995) Curr. Opin. Genet. Dev. 5, 79-785). The overexpression of ABC transporters has been linked with drug resistance since the 1976 discovery of P-glycoprotein and the subsequent cloning of the encoding gene, MDR-1. Resistance ensues from reduced intracellular drug concentrations, a result of active drug efflux. The subsequent identification of the multidrug resistance associated protein (MRP), encoded by the MRP gene, heralded a new era that recognized the complexity of the problem and catalyzed the search for additional transporters. MDR-1 and MRP are members of the expanding superfamily of ATP-binding cassette proteins (ABC proteins). This superfamily is comprised of a large and diverse group of proteins that transport solutes across biological membranes. Transmembrane domains are thought to form a pathway through which substrates cross cell membranes, while two ATP-binding domains hydrolyze ATP to accomplish substrate transport. Mutations in ABC transporters have been identified as etiologic in diseases including hyperinsulinemic hypoglycemia of infancy, adrenoleukodystrophy, and cystic fibrosis. The transporters MDR-1 and MRP, and possibly the multispecific organic anion transporter, cMOAT, are thought to be involved in both normal excretion of xenobiotics and in drug resistance. The ABC superfamily also includes a number of transporters without known function and the potential exists to identify additional transporters which mediate drug resistance. [0004] Recent studies have described a number of cell lines with resistance to mitoxantrone that exhibit multidrug resistance without overexpression of MRP. In addition to mitoxantrone, these cell lines are particularly resistant to anthracyclines, and have an energy-dependent reduction in the accumulation of daunomycin and mitoxantrone. Cell lines possessing this phenotype include sublines derived by selection of leukemic cells, as well as breast, colon, and gastric carcinomas. SUMMARY OF THE INVENTION [0005] The present invention thus provides for the first time, nucleic acids encoding a new transporter protein that mediates drug resistance. These proteins are generically called ATP binding cassette proteins (ABC proteins). The ABC protein of the invention is referred to as MXR1. It is also known as ABCP, and is also known as ABCG2. [0006] In one aspect, the present invention provides an isolated ATP-binding cassette protein that confers mitoxantrone resistance to S1-M1-80 human colon carcinoma cells when expressed in the cells; and specifically binds to polyclonal antibodies which specifically bind to a member of the group of proteins depicted in SEQ ID NO. 2 or SEQ ID. NO. 4; and has a molecular weight between about 70 kDa and about 75 kDa. [0007] In one embodiment, the MXR1 protein has the sequence depicted in SEQ ID NO. 2 or SEQ ID NO. 4. In another embodiment, the protein has 95% identity to the amino acids depicted in SEQ ID NO. 2 or SEQ ID. NO. 4. [0008] In another aspect, the present invention provides a eukaryotic cell genetically altered to overexpress an ATP-binding cassette protein that confers mitoxantrone resistance on S1-M1-80 human colon carcinoma cells when expressed in the cells; and specifically binds to polyclonal antibodies which specifically bind to a member of the group of proteins depicted in SEQ ID NO. 2 or SEQ ID. NO. 4. [0009] In one embodiment, the cells of the invention are genetically altered by transformation of the cell with an exogenous DNA comprising an expression cassette encoding the ATP-binding cassette protein. In another embodiment, the expression cassette also employs a heterologous promoter operatively linked to the DNA encoding the ATP-binding cassette protein. In another embodiment, the cell may have an endogenous copy of the ATP-binding cassette protein with a genetic alteration comprising insertion of DNA that serves as an enhancing element or as a second promoter where the insertion is upstream of the endogenous promoter operatively linked to the ATP-binding cassette protein and where the inserted DNA increases the basal expression levels of ATP-binding cassette protein. [0010] In another aspect, the present invention provides for DNA encoding an ATP-binding cassette protein wherein the protein confers mitoxantrone resistance on S1-M1-80 human colon carcinoma cells when expressed in the cells and specifically binds to polyclonal antibodies which specifically bind to the proteins depicted in SEQ ID NO. 2 or SEQ ID NO. 4. [0011] In one embodiment, the DNA encodes for the protein of SEQ ID NO. 2 or SEQ ID NO. 4 and in other embodiments the DNA encodes a protein that has 95% identity to the amino acids depicted in SEQ ID NO. 2 or SEQ ID NO. 4. In another embodiment, the DNA has the sequence depicted in SEQ ID NO. 1 or SEQ ID NO. 3. [0012] In another aspect, the present invention provides a process for over expressing ATP-binding cassette protein in a cell comprising a first step of either (1) transforming the cell with an expression cassette which directs the expression of ATP-binding cassette protein; or, (2) selecting a cell having an endogenous copy of the ATP-binding cassette protein, and transforming the cell with DNA which can serve as an enhancing element or as a second promoter where the insertion is upstream of the endogenous promoter operatively linked to the ATP-binding cassette protein and where the inserted DNA increases the basal expression levels of ATP-binding cassette protein; and a second step of culturing the transformed cell under conditions where the levels of ATP-binding cassette protein are increased above the basal levels of the non-transformed cells. The ATP binding protein of this embodiment is one that confers mitoxantrone resistance on S1-M1-80 human colon carcinoma cells when expressed in the cells; and, specifically binds to polyclonal antibodies which specifically bind to a member of the group of proteins depicted in SEQ ID NO. 2 or SEQ ID No. 4. [0013] In one embodiment, the ATP binding cassette protein has 95% homology to the amino acids depicted in SEQ ID NO. 2 or SEQ ID No. 4. In yet another embodiment, the protein has the amino acids depicted in SEQ ID NO. 2 or SEQ ID No. 4. [0014] In another aspect, the present invention provides a method of screening for inhibitors of cytotoxin resistance in cells. The method comprises (1) culturing a cell genetically altered by the introduction of heterologous DNA which permits the overexpression an ATP-binding cassette protein that confers mitoxantrone resistance on S1-M1-80 human colon carcinoma cells when expressed in the cells; and specifically binds to polyclonal antibodies which specifically bind to a member of the group of proteins depicted in SEQ ID NO. 2 or SEQ ID NO. 4; and, (2) contacting the cell with a cytotoxin in an amount that permits cell survival due to the resistance conferred by the ATP-binding cassette protein; and, (3) contacting the cell with a compound that inhibits the biological activity of the ATP-binding cassette protein; and, (4) detecting the inhibition by measuring growth inhibition of the cells. [0015] In one embodiment, the cytotoxin is mitoxantrone. In another embodiment, the cytotoxin is daunomycin. In another embodiment, the cell is a carcinoma cell. In another embodiment, the ATP-binding cassette protein has 95% homology to the amino acids depicted in SEQ ID NO. 2 or SEQ ID NO. 4. In another embodiment, the ATP binding cassette protein has the amino acid sequence depicted in SEQ ID NO. 2 or SEQ ID NO. 4. [0016] In another aspect, the invention provides a binding protein that specifically binds to an ATP-binding cassette protein which has 95% homology to the amino acids depicted in SEQ ID NO. 2 or SEQ ID NO. 4. In one embodiment, the binding protein is an antibody, and in another embodiment, the binding protein is a monoclonal antibody. Non-Competitive Assay Formats. [0017] Immunoassays for detecting the ATP binding cassette protein may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case the protein) is directly measured. In one preferred "sandwich" assay, for example, the capture agent (anti-ABC antibodies) can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the ATP binding cassette protein present in the test sample. The ATP binding cassette protein thus immobilized is then bound by a labeling agent, such as a second ATP binding cassette antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin. DETAILED DESCRIPTION [0018] Introduction Continue reading... 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