L1210 cells cultivated under the selection pressure of doxorubicin or vincristine express common mechanisms of multidrug resistance based on the overexpression of P-glycoprotein

Abstract

Multidrug resistance of neoplastic tissue is often associated with the overexpression and increased drug transport activity of plasma membrane transporters like P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) or breast cancer resistance protein, as well as with the elevation of the glutathione detoxification pathway. We have already described the overexpression of P-gp under the selection pressure of vincristine in L1210 mouse leukemia cells. In the present study, mechanisms of multidrug resistance induced in L1210 cells cultivated in the presence of doxorubicin were analyzed. The selection pressure of both vincristine (yielding a resistant subline of L1210 cells, R V) and doxorubicin (yielding a resistant subline of L1210 cells, R D) induced a dramatic depression of cell sensitivity to both drugs. Both R V and R D cells demonstrated a lack of ability to accumulate calcein/AM and fluo-3/AM as fluorescent substrates of P-gp and MRP. The retention of dyes could be reached in both cell sublines by the application of inhibitors of P-gp (like verapamil) but not by probenecid – an inhibitor of anion transporters, including MRPs. Massive protein bands, at a M r range of 130–180 kDa that interact with c219 antibody against P-gp, were detected in the crude membrane fraction isolated from both R V and R D (but not from L1210) cells by Western blot. The cytosolic activity of glutathione S-transferase was found to be similar in R V and R D cells and did not differ significantly from the activity ascertained in parental L1210 cells. Neither the R V nor R D cell sublines differed considerably, as measured by cell ultrastructure. In conclusion, based on P-gp overexpression, both doxorubicin and vincristine induce a common multidrug resistance phenotype in L1210 cells.