GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

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We have continued to analyze the mechanism of action of the multidrug transporter and have worked on the development of new strategies to circumvent multidrug resistance in cancer and to exploit molecular knowledge of the multidrug transporter to design new cancer treatments. The multidrug transporter (P-glycoprotein) has been purified to near homogeneity and shown to be an active drug-dependent ATPase of high specific activity after reconstitution into proteoliposomes. Vesicles containing P-glycoprotein capable of transport have very active P- glycoprotein kinases, and this activity is stimulated by GTP. At least one novel plasma membrane associated P-glycoprotein-kinase has been partially purified, but its role in regulating activity of the multidrug transporter has not yet been determined. Kinetic studies demonstrate that the transporter interacts with drugs within the lipid bilayer, and indirect evidence suggests that drug may be removed from both the inner and outer leaflets of the bilayer. Molecular manipulation of P-glycoprotein by analysis of point mutations and chimeras with other members of the ATP- binding cassette (ABC) superfamily of transporters has revealed multiple regions of the molecule near or within the transmembrane domains which affect substrate specificity, and has indicated the interchangeability of ABCs between MDR1 and MDR2, a related transporter of unknown specificity. Function of P-glycoprotein has been explored by insertional inactivation of the mdr1b gene in mouse adrenal Y-1 cells, with loss of ability of these cells to secrete steroids above basal levels. We have continued to develop the MDR1 gene as a dominant selectable marker for gene therapy. Retroviral vectors expressing the human MDR1 cDNA are able to confer resistance to taxol on transduced and transplanted mouse bone marrow cells, and this strategy is under consideration for gene therapy in humans to protect bone marrow during high dose chemotherapy for cancer. Two other multidrug resistant genetic systems are under development to aid in the analysis of other mechanisms of multidrug resistance: (1) A human melanoma line cross-resistant to epipodophyllotoxins (VP-16 and VM-26) and anthracyclines which has a deletion of Ala 428 in topoisomerase II; and (2) High level cis-platinum resistant human hepatoma and KB adenocarcinoma cells with multiple protein alterations.