The objective of this application is to study the regulatory effects of post-translational phosphorylation of the drug efflux pump, P-glycoprotein (PGP), in multidrug-resistant (MDR) human breast cancer cells and in vitro using recombinant baculovirus expression. PGP is an ATP-dependent plasma membrane transporter that is responsible for conferring resistance to structurally diverse natural product anticancer drugs. PGP is the product of the MDR1 gene, a highly conserved multigene family consisting of two genes in man, and of which only MDR1 confers the MDR phenotype after transfection. PGP serves as a substrate for protein-serine kinases of the protein kinase C (PKC) family. Recent studies from the applicant's laboratory have shown that MDR can be increased in MDR1- expressing human breast cancer cells following transfection with PKCa, and is associated with decreased drug retention and increased phorbol ester-stimulated PGP phosphorylation. This effect can be partially reversed by antisense expression of PKCa. Moreover, site directed mutagenesis of Ser671 in PGP attenuates drug binding and the ability of PKCa to activate drug-induced PGP ATPase in a baculovirus expression system. Therefore, the goals of this application are to determine the roles of different isoforms of PKC in modulating PGP activity, and in this context, to explore ways in which to down-regulate PGP activity by selectively inhibiting specific PKC isoforms. Based on the applicant's previous characterization of PKC isoforms in breast cancer cells, the first goal of this application will be to determine the effect of wild type or constitutively active forms of PKCa on MDR in human breast carcinoma cells stably expressing PGP. In instances where the MDR phenotype shows a reduction or absence of a particular PKC isoform, eg. PKC-beta-2, delta and epsilon, cells will be transfected with the low abundance form of PKC. The second goal will be to selectively inhibit the PKC isoform which is over-expressed in MDR cells, eg. PKC-alpha, by stably expressing the antisense cDNA. The third goal will be to determine the effect of mutating one or more PKC consensus phosphorylation sites in PGP on its ability to transport drugs. The function of mutated PGP (drug accumulation, drug binding and ATPase activity) will be assessed in MCF-7 cells by stable expression and in insect cells after baculovirus infection.
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