Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein.
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 microgram/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 correaltion 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.
Chen
Y N
YN
Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.
Mickley
L A
LA
Schwartz
A M
AM
Acton
E M
EM
Hwang
J L
JL
Fojo
A T
AT
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents / Q000494:pharmacology; D001943:Breast Neoplasms; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D004317:Doxorubicin; D004250:DNA Topoisomerases, Type II