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 in many cases with the overproduction of a small family of membrane glycoproteins called p-glycoproteins (pgp's), which are thought to act as ATP-activated efflux pumps. Of the three family members in hamster, it is the homolog of human mdr1 (known to be overexpressed in human tumors), pgp1, that is most frequently involved with the establishment and maintenance of multidrug-resistance in tissue culture cell lines, and is the primary subject of this application. Little is known concerning the efflux mechanism supported by pgp1, the manner in which cross-resistance patterns are established, or how reversal of mdr by agents such as Verapamil and Cyclosporin A is achieved. Nor is it understood what function if any is played by the small pgp1 related RNA transcripts present in all mammalian cells. While each of these issues is of importance to pgp1 mediated mdr, it is clear that non-pgp related forms of mdr, supported by multidrug-resistance associated protein (MRP), and topoisomerase, also exist and must be accounted for if we are to extend our understanding of tumor cell drug resistance. Using a variety of recombinant DNA techniques, gene transfection assays, site directed mutagenesis, and short-term drug selection protocols we propose to 1) continue the molecular genetic study of transmembrane domain 6 in pgp1, a region that is known to mediate cross-resistance patterns and to be closely, if not directly, involved with the mechanism of Cyclosproin A reversal, 2) analyze additional naturally occurring mutants of pgp1 to further delineate its drug binding site(s) 3) determine the order of emergence of mdr mechanisms during short term selections using different antineoplastic drugs and 4) attempt to clarify the function of the 2.3kb poly (A)+ RNA transcript thought to be a splicing product of pgp1.