Effects of membrane potential versus pHi on the cellular retention of doxorubicin analyzed via a comparison between cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) transfectants.
Recently (Wei et 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 delta psi ), but the former have alkaline pHi whereas the latter are acidic. It has been proposed (Roepe et al., Biochemistry 32: 11042-11056, 1993) that both decreased delta psi and increased pHi 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 continuous 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 microM drug, but better fit by a quadratic between 0.1 to 1.5 microM drug. The slopes of these curves were proportional to measured delta psi. Low-level selection of either CFTR or MDR transfectants with chemotherapeutic drug did not decrease further the initial rate of drug accumulation or change delta psi. Accumulation experiments for control cells performed in the presence of various concentrations of K+ further suggests that the rate of accumulation is related to delta psi. By measuring the kinetics of doxorubicin release for CFTR and MDR transfectants preloaded with drug, we concluded that alkaline pHi 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 delta psi and elevated pHi in altering the cellular retention of doxorubicin in MDR tumor cells.
Robinson
L J
LJ
Molecular Pharmacology and Therapeutics Program, Raymond & Beverly Sackler Foundation Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Roepe
P D
PD
D000818:Animals; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials / Q000502:physiology*; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004317:Doxorubicin