| Abstract: |
Recently (Wei er al., Biophys J 69: 883-895, 1995), several 3T3/hu cystic fibrosis transmembrane conductance regulator (CFTR) transfectant clones were found to exhibit a low-level multidrug resistance (MDR) phenotype. This phenotype is similar, but not identical to that found for MDR transfectants not previously exposed to chemotherapeutic drugs. Both MDR and CFTR transfectants are depolarized (exhibit lower plasma membrane ΔΨ), but the former have alkaline pH(i) whereas the latter are acidic. It has been proposed (Roepe et al., Biochemistry 32: 11042-11056, 1993) that both decreased ΔΨ and increased pH(i) contribute to altered cellular retention of chemotherapeutic drugs in MDR tumor cells, but the relative contribution of each to altered cellular drug accumulation, drug retention, and drug efflux has not been studied in detail. We therefore examined doxorubicin transport for hu CFTR and mu MDR 1 transfectants using sensitive continous monitoring of fluorescence techniques. Both CFTR and MDR transfectants exhibited significantly reduced doxorubicin accumulation, relative to drug-sensitive control cells. Plots of the initial rate of accumulation versus doxorubicin concentration were linear for the control cells and the CFTR and MDR transfectants between 0.1 to 0.5 μM drug, but better fit by a quadratic between 0.1 to 1.5 μM drug. The slopes of these curves were proportional to measured ΔΨ. Low-level selection of either CFTR or MDR transfectants with chemotherapeutic drug did not decrease further the initial rate of drug accumulation or change ΔΨ. Accumulation experiments for control cells performed in the presence of various concentrations of K+ further suggests that the rate of accumulation is related to ΔΨ. By measuring the kinetics of doxorubicin release for CFTR and MDR transfectants preloaded with drug, we concluded that alkaline pH(i) perturbations are more important for determining relative intracellular binding efficiency. We also concluded, similar to the case previously made for MDR protein (Roepe, Biochemistry 31: 12555-12564, 1992) that CFTR overexpression does not enhance the rate of drug efflux. These data better define the role of lowered ΔΨ and elevated pH(i) in altering the cellular retention of doxorubicin in MDR tumor cells. |
