STUDIES OF MULTIDRUG RESISTANCE IN MAMMALIAN CELLS

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The development of resistance to chemotherapeutic agents is observed in the clinic, and has been studied extensively in tissue culture cells. One unusual phenotype is that of multidrug resistance, in which cells that are challenged with any one of a variety of cytotoxic drugs develop resistance not only to the selective agent but also cross-resistance to other, seemingly unrelated, compounds. This multidrug-resistant (mdr) phenotype is associated with the overproduction of membrane glycoproteins, including p-glycoprotein, and in some cases with the overproduction of cytosolic proteins as well. The phenotype is complex, therefore, and the genetic basis for resistance and cross-resistance is obscured by the fact that the differential amplification of multiple genes (which we call MDRA genes) mediates the overproduction of the glycoproteins, whereas other coamplified genes mediate the expression of at least one cytosolic protein and probably other proteins as well. While the ultimate cause of multidrug resistance is the lowered intracellular concentration of drug, the role of any of the amplified gene products in the mechanism of resistance or cross-resistance in model tissue culture systems and in human tumors refractory to chemotherapy, remains obscure. With the use of recombinant DNA technology, DNA transfection techniques, and nuclear runoff transcription assays, we propose to define the role of the MDRA gene products in the establishment of the mdr phenotype, and to identify other genes whose products may also be involved in its expression. Additionally, when amplified, the expression of MDRA genes can far exceed their gene copy number, suggesting that overexpression is not mediated by amplification alone. We will, therefore, determine the molecular basis for this enhanced expression for it may indicate a mechanism for overexpression in the absence of amplification. Finally, we propose to measure the levels of expression of MDRA genes in a large number of human tumor cell lines and normal cell lines, and tumors and normal tissues in an effort to determine the extent to which these genes are expressed in human cells and whether their overexpression can be shown to be associated with the resistance that many patients display to a wide variety of chemotherapeutic agents.