EFFECTS OF MEMBRANE-POTENTIAL VERSUS PH(I) ON THE CELLULAR RETENTION OF DOXORUBICIN ANALYZED VIA A COMPARISON BETWEEN CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) AND MULTIDRUG-RESISTANCE (MDR) TRANSFECTANTS
Lj. Robinson et Pd. Roepe, EFFECTS OF MEMBRANE-POTENTIAL VERSUS PH(I) ON THE CELLULAR RETENTION OF DOXORUBICIN ANALYZED VIA A COMPARISON BETWEEN CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) AND MULTIDRUG-RESISTANCE (MDR) TRANSFECTANTS, Biochemical pharmacology, 52(7), 1996, pp. 1081-1095
Recently (Wei et al., Biophys J 69: 883-895, 1995), several 3T3/hu cys
tic fibrosis transmembrane conductance regulator (CFTR) transfectant c
lones were found to exhibit a low-level multidrug resistance (MDR) phe
notype. This phenotype is similar, but not identical to that found for
MDR transfectants not previously exposed to chemotherapeutic drugs. B
oth MDR and CFTR transfectants are depolarized (exhibit lower plasma m
embrane Delta Psi), but the former have alkaline pH(i) whereas the lat
ter are acidic. It has been proposed (Roepe er al., Biochemistry 32: 1
1042-11056, 1993) that both decreased Delta Psi and increased pH(i) co
ntribute to altered cellular retention of chemotherapeutic drugs in MD
R tumor cells, but the relative contribution of each to altered cellul
ar drug accumulation, drug retention, and drug efflux has not been stu
died in detail. We therefore examined doxorubicin transport for hu CFT
R and mu MDR 1 transfectants using sensitive continous monitoring of f
luorescence techniques. Both CFTR and MDR transfectants exhibited sign
ificantly reduced doxorubicin accumulation, relative to drug-sensitive
control cells. Plots of the initial rate of accumulation versus doxor
ubicin concentration were linear for the control cells and the CFTR an
d MDR transfectants between 0.1 to 0.5 mu M drug, but better fit by a
quadratic between 0.1 to 1.5 mu M drug. The slopes of these curves wer
e proportional to measured Delta Psi. Low-level selection of either CF
TR or MDR transfectants with chemotherapeutic drug did not decrease fu
rther the initial rate of drug accumulation or change Delta Psi. Accum
ulation experiments for control cells performed in the presence of var
ious concentrations of K+ further suggests that the rate of accumulati
on is related to Delta Psi. By measuring the kinetics of doxorubicin r
elease for CFTR and MDR transfectants preloaded with drug, we conclude
d 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 Delta Psi
and elevated pH(i) in altering the cellular retention of doxorubicin
in MDR tumor cells.