Development of multidrug resistance due to overexpression of P-glycoprotein (Pgp), a cell membrane drug efflux pump, occurs commonly during in vitro selections with adriamycin (Adr). Pgp-mediated drug resistance can be overcome by the calcium channel blocker verapamil (Vp), which acts as a competitive inhibitor of drug binding and efflux. In order to identify other mechanisms of Adr resistance, we isolated an Adr-resistant subline by selecting the human breast cancer cell line MCF-7 with incremental increases of Adr in the presence of 10 μg/ml verapamil. The resultant MCF-7/AdrVp subline is 900-fold resistant to Adr, does not overexpress Pgp, and does not exhibit a decrease in Adr accumulation. It exhibits a unique cross-resistance pattern: high cross-resistance to the potent Adr analogue 3′-deamino-3′-(3-cyano-4-morpholinyl)doxorubicin, lower cross-resistance to the alkylating agent melphalan, and a sensitivity similar to the parental cell line to vinblastine. The levels of glutathione and glutathione S-transferase are similar in the parental line and the Adr-resistant subline. Topoisomerase II-DNA complexes measured by the potassium-sodium dodecyl sulfate precipitation method shows a 2-3 fold decrease in the resistant subline. The MCF-7/ AdrVp cells overexpress a novel membrane protein with an apparent molecular mass of 95 kDa. Polyclonal antibodies raised against the P-95 protein demonstrate a correlation between the level of expression and Adr resistance. Removal of Adr but not verapamil from the selection media results in a decline in P-95 protein levels that parallels a restoration of sensitivity to Adr. Immunohistochemistry demonstrates localization of the P-95 protein on the cell surface. The demonstration of high levels of the protein in clinical samples obtained from patients refractory to Adr suggests that this protein may play a role in clinical drug resistance.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jun 15 1990|
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