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 reduced drug accumulation mediated by an ATP dependent efflux pump. We have developed a new, rapid and quantitative method for measuring influx of BCECF-AM in sensitive (CEM) and MDR cells (CEM/VLB100). The fluorescence of intracellular accumulated BCECF after hydrolysis of BCECF-AM is rapidly visualized by spectrofluorometry. The rate of BCECF-AM entry into CEM/VLB100 cells is considerably lower than that found in CEM cells, similar to 10-fold after 10 min of incubation. This phenomenon is not in relation with a difference of esterase activities, it is not energy or intracellular pH-dependent, and BCECF efflux is negligible. CEM cells exhibited diffuse fluorescence within cytoplasm in contrast with numerous spots of intense labelling, related to the presence of the cytoplasmic vesicles in CEM/VLB100 cells demonstrated by Nomarski's microscopy. MDR modulators such as verapamil, sodium orthovanadate, chlorpromazine or trifluoperazine induce an enhanced influx in CEM/VLB100 cells (150+/-4%; 204+/-17%; 410+/-17% and 229+/-7% respectively) whereas no major differences were noted with the parental sensitive cells. Vinblastine (under conditions close to IC50) increases the influx only in MDR cells (481+/-6%) by a process that is not linked to competitive inhibition of the P170 efflux pump. These results suggest that reduced influx of drugs could be a major defect in MDR cells, a possible role for P170-membrane lipids interactions is discussed.