A flow cell assay for evaluation of whole cell drug efflux kinetics: Analysis of paclitaxel efflux in CCRF-CEM leukemia cells overexpressing P-glycoprotein

P-glycoprotein (Pgp) mediates drug accumulation defects in malignant cells in vitro. It confers resistance to multiple drugs including paclitaxel, an agent useful in treating malignancies including acute leukemia. Pgp-mediate d drug resistance appears to be due to primary active drug-transport as wel l as other effects on membrane permeability, but the relative contribution of each is unclear. Flow cells are useful for differentiating transport-med iated efflux from altered membrane permeability, but their utility is limit ed to attached cells. We developed a novel flow cell to study drug efflux k inetics in suspension culture cells and examined paclitaxel efflux in resis tant CEM/VLB100 leukemia cells, which overexpress Pgp, compared with its se nsitive CEM parent line. Paclitaxel efflux from both cell lines was describ ed by bi-exponential kinetics. The predominant initial rapid component incr eased linearly with paclitaxel concentration, consistent with passive efflu x, and was faster in CEM/VLB100 than CEM cells. The slow terminal component of efflux was also more rapid for CEM/VLB100 than CEM, and was saturable ( V-max = 9.1 +/- 1.1 versus 3.5 +/- 0.3 pmol/min/10(7) cells, respectively) at a lower paclitaxel concentration than the parental CEM cells (k(m) = 63 +/- 46 nM versus 144 +/- 56 nM, respectively). In CEM/VLB100 cells, this sa turable component was inhibited by verapamil and was temperature-sensitive, consistent with Pgp-mediated transport. Verapamil also inhibited the rapid component of efflux, suggesting additional effects on membrane permeabilit y. Our studies show that the present technique is useful for studying drug transport and that effects of Pgp on membrane permeability contribute signi ficantly to the net drug-accumulation defect.