Chen Y N, Mickley L A, Schwartz A M, Acton E M, Hwang J L, Fojo A T
Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jun 15;265(17):10073-80.
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.
多药耐药性的产生是由于细胞膜药物外排泵P-糖蛋白（Pgp）的过度表达，这在阿霉素（Adr）的体外筛选过程中很常见。Pgp介导的耐药性可被钙通道阻滞剂维拉帕米（Vp）克服，维拉帕米作为药物结合和外排的竞争性抑制剂发挥作用。为了确定阿霉素耐药的其他机制，我们通过在含有10微克/毫升维拉帕米的情况下用递增剂量的阿霉素筛选人乳腺癌细胞系MCF-7，分离出了一个阿霉素耐药亚系。所得的MCF-7/AdrVp亚系对阿霉素的耐药性提高了900倍，不过度表达Pgp，且阿霉素蓄积没有减少。它表现出独特的交叉耐药模式：对强效阿霉素类似物3'-脱氨基-3'-(3-氰基-4-吗啉基)阿霉素有高度交叉耐药性，对烷化剂美法仑有较低交叉耐药性，对长春碱的敏感性与亲代细胞系相似。亲代细胞系和阿霉素耐药亚系中的谷胱甘肽和谷胱甘肽S-转移酶水平相似。通过十二烷基硫酸钠钾沉淀法测定的拓扑异构酶II-DNA复合物在耐药亚系中减少了2-3倍。MCF-7/AdrVp细胞过度表达一种表观分子量为95 kDa的新型膜蛋白。针对P-95蛋白产生的多克隆抗体表明表达水平与阿霉素耐药性之间存在相关性。从筛选培养基中去除阿霉素而不是维拉帕米会导致P-95蛋白水平下降，这与对阿霉素敏感性的恢复平行。免疫组织化学显示P-95蛋白定位于细胞表面。在从对阿霉素难治的患者获得的临床样本中检测到该蛋白的高水平，这表明该蛋白可能在临床耐药中起作用。
