GENETIC ANALYSIS OF THE MULTIDRUG RESISTANCE PHENOTYPE IN TUMOR CELLS

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We have been interested in defining the major mechanisms of simultaneous resistance of cancer cells to multiple chemotherapeutic agents. One major mechanism is expression of an energy-dependent efflux pump, termed P-glycoprotein (P-gp), or the multidrug transporter, encoded in humans by the MDR1 gene. The sequence of the MDR1 cDNA led to a model of the transporter as a pump with 12 transmembrane domains and 2 adenosine 5'- triphosphate (ATP) sites; determination of the domains of P-gp responsible for substrate binding and coupling of ATPase activity to substrate transport are the major goals of our work. Model systems based on stable expression or transient expression of mutated P-gps by a vaccinia virus expression system or a baculovirus system have been developed to assay functional effects of these mutations on drug binding, drug-dependent ATPase, drug resistance and drug transport. Mutations in either or both ATP sites eliminate the ability of P-gp to pump fluorescent substrates or confer drug resistance. These ATP sites are not fully functionally interchangeable as demonstrated by creation of P-gp chimeras and by labeling experiments with 32P-azido-ATP, supporting a model of alternating use of ATP sites in which the N- terminal site is utilized first. Evidence for the interaction of the C- terminal ATP sites with an N-terminal substrate binding site has been obtained by analysis of a mutation in the TM6 which affects substrate binding, but also allows a deletion in the "C" region of the C-terminal ATP site to be expressed on the cell surface. We have constructed bicistronic retroviral expression vectors carrying MDR1 and several other genes for treatment of immunodeficiency, X-linked severe combined, adenosine deaminase deficiency, Fabry disease, Gaucher disease, and chronoic granulomatous disease, as well as a ribozyme directed against the long terminal repeat (LTR) in human immunodeficiency virus (HIV), luciferase and beta-galactosidase as marker genes, and other drug- resistance genes, dihydrofolate reductase (DHFR), and methylguanine methyltransferase (MGMT). These vectors may be delivered to bone marrow stem cells grown ex vivo or complexed to liposomes in vivo. We have analyzed the mechanism of multidrug resistance resulting from selection in cisplatin of hepatoma cells and KB adenocarcinoma cells. Cisplatin- resistant hepatoma and KB cells are cross-resistant to methotrexate, arsenite and antimonite and show reduced accumulation of these toxic agents due to the pleiotropic absence of specific uptake systems for these toxic agents.