EVIDENCE FOR REDUCED DRUG INFLUX IN MULTIDRUG-RESISTANT CEM CELLS BY A FLUORESCENT DYE

Multidrug resistance (MDR) in cancer cells is commonly ascribed to a r educed drug accumulation mediated by an ATP dependent efflux pump. We have developed a new, rapid and quantitative method for measuring infl ux of BCECF-AM in sensitive (CEM) and MDR cells (CEM/VLB100). The fluo rescence of intracellular accumulated BCECF after hydrolysis of BCECF- AM is rapidly visualized by spectrofluorometry. The rate of BCECF-AM e ntry into CEM/VLB100 cells is considerably lower than that found in CE M cells, similar to 10-fold after 10 min of incubation. This phenomeno n is not in relation with a difference of esterase activities, it is n ot energy or intracellular pH-dependent, and BCECF efflux is negligibl e. CEM cells exhibited diffuse fluorescence within cytoplasm in contra st with numerous spots of intense labelling, related to the presence o f the cytoplasmic vesicles in CEM/VLB100 cells demonstrated by Nomarsk i's microscopy. MDR modulators such as verapamil, sodium orthovanadate , chlorpromazine or trifluoperazine induce an enhanced influx in CEM/V LB100 cells (150+/-4%; 204+/-17%; 410+/-17% and 229+/-7% respectively) whereas no major differences were noted with the parental sensitive c ells. Vinblastine (under conditions close to IC50) increases the influ x only in MDR cells (481+/-6%) by a process that is not linked to comp etitive inhibition of the P170 efflux pump. These results suggest that reduced influx of drugs could be a major defect in MDR cells, a possi ble role for P170-membrane lipids interactions is discussed.