Multiple drug resistance of tumor cells is a major obstacle in the successful treatment of cancer by chemotherapy. Multiple drug resistance frequently results from amplification and overexpression of a family of proteins termed multiple drug resistance (MDR) P-glycoproteins. The MDR proteins have multiple sites for phosphorylation by protein kinase C (PKC), and a number of recent observations suggest that MDR may be directly activated by PKC phosphorylation. First, expression of specific PKC isoenzymes is increased in multiple drug resistant cell lines in comparison with the drug-sensitive parent cell lines. Second, treatment of MDR cell lines with PKC activators enhances drug resistance, while treatment with PKC inhibitors reverses the MDR phenotype. Finally, transfection of some PKC isoenzymes enhances the MDR phenotype. In addition, the proteins coded by all of the MDR cDNA's isolated to date have multiple potential PKC phosphorylation sites, indicating that PKC may play a direct role in the phosphorylation of MDR. Evidence from our lab demonstrates that MDR P-glycoproteins play an important role in cholesterol biosynthesis and esterification, possibly by transporting cholesterol and cholesterol precursors between membranes within the cell. This finding suggests that PKC regulation of MDR activity may play a normal physiologic role in cholesterol metabolism. The current proposal describes experiments designed to test the hypothesis that specific PKC isoenzymes directly regulate MDR activity by phosphorylation. and that this regulation has consequential effects on cellular cholesterol metabolism. To test this hypothesis, I will assess the ability of each of ten different PKC isoenzymes (alpha, BetaI, BetaII, gamma, delta, epsilon, eta, theta, mu, and zeta) to alter MDR activity when overexpressed in CHO cells; I will assay changes in drug accumulation, drug-induced ATPase activity, and cell growth in the presence of cytotoxic MDR substrate drugs.