Hoffman M M, Wei L Y, Roepe P D
Program in Molecular Pharmacology and Therapeutics, Raymond and Beverly Sackler Foundation Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
J Gen Physiol. 1996 Oct;108(4):295-313. doi: 10.1085/jgp.108.4.295.
Multidrug resistance (MDR) mediated by overexpression of the MDR protein (P-glycoprotein) has been associated with intracellular alkalinization, membrane depolarization, and other cellular alterations. However, virtually all MDR cell lines studied in detail have been created via protocols that involve growth on chemotherapeutic drugs, which can alter cells in many ways. Thus it is not clear which phenotypic alterations are explicitly due to MDR protein overexpression alone. To more precisely define the MDR phenotype mediated by hu MDR 1 protein, we co-transfected hu MDR 1 cDNA and a neomycin resistance marker into LR73 Chinese hamster ovary fibroblasts and selected stable G418 (geneticin) resistant transfectants. Several clones expressing different levels of hu MDR 1 protein were isolated. Unlike previous work with hu MDR 1 transfectants, the clones were not further selected with, or maintained on, chemotherapeutic drugs. These clones were analyzed for chemotherapeutic drug resistance, intracellular pH (pHi), membrane electrical potential (Vm), and stability of MDR 1 protein overexpression. LR73/hu MDR 1 clones exhibit elevated pHi and are depolarized, consistent with previous work with LR73/mu MDR 1 transfectants (Luz, J.G. L.Y. Wei, S. Basu, and P.D. Roepe. 1994. Biochemistry. 33:7239-7249). The extent of these perturbations is related to the level of hu MDR 1 protein that is expressed. Cytotoxicity experiments with untransfected LR73 cells with elevated pHi due to manipulating percent CO2 show that the pHi perturbations in the MDR 1 clones can account for much of the measured drug resistance. Membrane depolarization in the absence of MDR protein expression is also found to confer mild drug resistance, and we find that the pHi and Vm changes can conceivably account for the altered drug accumulation measured for representative clones. These data indicate that the MDR phenotype unequivocally mediated by MDR 1 protein overexpression alone can be fully explained by the perturbations in Vm and pHi that accompany this overexpression. In addition, MDR mediated by MDR protein overexpression alone differs significantly from that observed for MDR cell lines expressing similar levels of MDR protein but also exposed to chemotherapeutic drugs.
由多药耐药蛋白（P-糖蛋白）过表达介导的多药耐药（MDR）与细胞内碱化、膜去极化及其他细胞改变有关。然而，几乎所有经过详细研究的MDR细胞系都是通过涉及在化疗药物上生长的方案构建的，这会以多种方式改变细胞。因此，尚不清楚哪些表型改变是仅由MDR蛋白过表达明确导致的。为了更精确地定义由人MDR 1蛋白介导的MDR表型，我们将人MDR 1 cDNA和新霉素抗性标记共转染到LR73中国仓鼠卵巢成纤维细胞中，并筛选出稳定的对G418（遗传霉素）耐药的转染子。分离出了几个表达不同水平人MDR 1蛋白的克隆。与先前对人MDR 1转染子的研究不同，这些克隆没有进一步用化疗药物筛选或在化疗药物上维持培养。对这些克隆进行了化疗药物抗性、细胞内pH（pHi）、膜电位（Vm）以及MDR 1蛋白过表达稳定性的分析。LR73/人MDR 1克隆表现出pHi升高和膜去极化，这与先前对LR73/鼠MDR 1转染子的研究结果一致（Luz, J.G., L.Y. Wei, S. Basu, and P.D. Roepe. 1994. Biochemistry. 33:7239 - 7249）。这些扰动的程度与所表达的人MDR 1蛋白水平相关。对因调节二氧化碳百分比而导致pHi升高的未转染LR73细胞进行的细胞毒性实验表明，MDR 1克隆中的pHi扰动可以解释大部分测得的耐药性。在没有MDR蛋白表达的情况下，膜去极化也被发现可赋予轻度耐药性，并且我们发现pHi和Vm的变化可以想象地解释代表性克隆中测得的药物蓄积改变。这些数据表明，仅由MDR 1蛋白过表达明确介导的MDR表型可以完全由伴随这种过表达的Vm和pHi扰动来解释。此外，仅由MDR蛋白过表达介导的MDR与在表达相似水平MDR蛋白但也暴露于化疗药物的MDR细胞系中观察到的MDR有显著差异。
