Expression of the human MDR1 (multidrug resistance) gene results in cross-resistance to a diverse group of lipophilic drugs that includes many commonly used anticancer agents. Tbe MDR1 gene encodes P-glycoprotein, a 1280 amino acid transmembrane protein and a member of the """"""""ABC superfamily"""""""" of membrane transporters. Structural and biochemical studies have indicated that P-glycoprotein functions as an efflux pump that hydrolyzes ATP and actively transports various lipophilic substrates out of the cell. The details of the molecular mechanism of P-glycoprotein-mediated efflux, including the relationship between ATP hydrolysis and substrate translocation, the critical sites of P-glycoprotein interactions with its substrates and inhibitors, and the determinants of substrate specificity are still poorly understood. MDR1 expression is frequently observed in different types of cancer, where it has been associated with both the intrinsic and the acquired forms of resistance to chemotherapy. Except for tumors that are derived from MDR1-expressing normal tissues, little is known about the factors responsible for MDR1 expression in clinical cancer. Under the present proposal, the relationship between the ATPase activity and transport of different substrates by P-glycoprotein will be analyzed, using mutant forms of P-glycoprotein in which the binding or hydrolysis of ATP have been inactivated by specific mutations at each of the two nucleotide-binding sites. A retroviral mutation-selection system will be used to isolate and characterize MDR1 mutations that confer preferential resistance to different drugs and altered sensitivity to inhibitors, in order to identify structural features that determine the specificity of P-glycoprotein interactions with different ligands. In addition, genetic suppressor elements that interfere with P-glycoprotein mediated efflux will be isolated by expression selection of random fragments from MDR1 cDNA. These elements will be used to identify P-glycoprotein domains capable of independent functional interactions and to develop genetic reagents that would efficiently and selectively inhibit P-glycoprotein function. Another part of this proposal is based on a recent finding that treatment with various cytotoxic drugs or protein kinase C agonists can induce MDR1 expression in different types of human cells, and that such induction can be prevented with protein kinase inhibitors. The regulatory mechanisms of this potentially clinically relevant phenomenon will be analyzed, and cells that activate the MDR1 gene after exposure to cytotoxic drugs will be tested for other phenotypic changes that may be co-induced with MDR1 expression in human tumors.
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