Vol. 29, Issue 2, 103-110, February 2001

A Flow Cell Assay for Evaluation of Whole Cell Drug Efflux Kinetics: Analysis of Paclitaxel Efflux in CCRF-CEM Leukemia Cells Overexpressing P-Glycoprotein

James T. Lin, Rashmi Sharma,¹ James J. Grady, and Sanjay Awasthi²

Departments of Internal Medicine (J.T.L., R.S., S.A.) and Biostatistics (J.J.G.), University of Texas Medical Branch, Galveston, Texas

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-mediated drug resistance appears to be due to primary active drug-transport as well as other effects on membrane permeability, but the relative contribution of each is unclear. Flow cells are useful for differentiating transport-mediated efflux from altered membrane permeability, but their utility is limited to attached cells. We developed a novel flow cell to study drug efflux kinetics in suspension culture cells and examined paclitaxel efflux in resistant CEM/VLB100 leukemia cells, which overexpress Pgp, compared with its sensitive CEM parent line. Paclitaxel efflux from both cell lines was described by bi-exponential kinetics. The predominant initial rapid component increased linearly with paclitaxel concentration, consistent with passive efflux, 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/10⁷ 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 saturable 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 permeability. Our studies show that the present technique is useful for studying drug transport and that effects of Pgp on membrane permeability contribute significantly to the net drug-accumulation defect.