Abstract
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 (Vmax= 9.1 ± 1.1 versus 3.5 ± 0.3 pmol/min/107 cells, respectively) at a lower paclitaxel concentration than the parental CEM cells (km = 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.
Footnotes
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Send reprint requests to: James Lin, M.D., Dept. of Internal Medicine, Division of Hematology/Oncology, 301 University Boulevard, University of Texas Medical Branch, Galveston, TX 77555-0565. E-mail: jlin{at}utmb.edu
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↵1 Current address: United States Environmental Protection Agency, Research Triangle Park, NC 27711.
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↵2 Current address: University of Texas Arlington, Department of Chemistry and Biochemistry, P.O. Box 19065, Arlington, TX 76019.
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This work was supported in part by National Institutes of Health Grant CA63660 (to S.A.).
- Abbreviations used are::
- MDR
- multidrug resistance
- conA
- concanavalin A
- conA-seph
- concanavalin A linked to Sepharose-4B
- HBSS
- Hanks' balanced salt solution
- Pgp
- P-glycoprotein
- Received May 12, 2000.
- Accepted October 18, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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