P-GLYCOPROTEIN EXPRESSION AND FUNCTION IN RAT HEPATOCYTES IN CULTURE

Expression of P-glycoprotein, which confers multidrug resistance to a broad range of anticancer drugs, was studied in rat hepatocytes in culture. P-glycoprotein was localized in the plasma membrane by immunohistochemical staining and was evaluated by western blotting with C219 as primary antibody and quantification of the coloured spots. The conditions of culture (time in culture, cell density at seeding) had a strong effect on the expression of P-glycoprotein. Expression increases with time in culture. At 10 x 10(6) cells/75 cm(2) flasks, which is the normal density seeding for hepatocytes in culture, the increase of P-glycoprotein was 17% between 4 and 24 hr in culture, 52% between 24 and 48 hr and 37% between 48 and 96 hr. At low density cell seeding (2 x 10(6) cells/75 cm(2)), the expression of P-glycoprotein was higher than at normal density from the first day in culture (+20%). This difference of expression was maintained until 96 hr of culture and was maximum at 48 hr (+44%). This P-glycoprotein was functional and this overexpression was correlated with a decrease of doxorubicin retention in hepatocytes.