Moran E, Cleary I, Larkin A M, Amhlaoibh R N, Masterson A, Scheper R J, Izquierdo M A, Center M, O'Sullivan F, Clynes M
National Cell and Tissue Culture Centre, BioResearch Ireland, Dublin City University, Ireland.
Eur J Cancer. 1997 Apr;33(4):652-60. doi: 10.1016/s0959-8049(96)00501-1.
Variants of the human ovarian carcinoma cell line, OAW42, exhibiting low-level intrinsic resistance (OAW42-SR) and drug-induced higher-level resistance (OAW42-A1 & OAW42-A), were studied along with a sensitive clonal population (OAW42-S) which was isolated from OAW42-SR. Expression of the MDR-associated protein P-170, the more recently discovered LRP (lung resistance-related protein) and MRP (multidrug resistance-associated protein), topoisomerase II alpha and beta, GST pi and the cytoskeletal proteins, cytokeratin 8 and vimentin, were studied (using immunocytochemistry and Western blotting techniques) in conjunction with drug (doxorubicin) accumulation and subcellular distribution. Expression of mRNA for P-170, MRP, topoisomerase 11 alpha and beta and GST pi was studied using RT-PCR (reverse transcriptase polymerase chain reaction). Results indicate differential co-expression of four MDR-associated parameters (P-170, MRP, LRP and reduced topoisomerase II alpha and beta) in the OAW42-SR and OAW42-A1 variants, whereas resistance in the OAW42-A variant appeared to be mainly P-170 mediated. Comparable amounts of MRP and greater amounts of LRP were detected in the OAW42-S cells compared to the OAW42-SR variant (which showed increased resistance compared to the OAW42-S cells), but all cell lines expressed similar low-level amounts of MRP mRNA (by RT-PCR). GST pi levels did not differ markedly between variants. Increased levels of the cytoskeletal proteins were observed with increasing levels of resistance. The relative resistance of the variants, OAW42-SR and OAW42-A1, compared with OAW42-S was seen to change during increased serial passaging of the cells. There was greater drug accumulation by the sensitive OAW42-S cell line compared with that of the resistant variants, particularly the most highly resistant OAW42-A cells. Both verapamil and cyclosporin A effectively restored the accumulation defects seen in the resistant variants, cyclosporin A being the more effective of the two. Sub-cellular location of drug was predominantly in the nucleus with maximum levels seen in the sensitive OAW42-S variant and minimum levels in the most resistant OAW42-A clone.
对人卵巢癌细胞系OAW42的变体进行了研究，这些变体包括表现出低水平固有抗性的（OAW42-SR）以及药物诱导的更高水平抗性的（OAW42-A1和OAW42-A），同时还研究了从OAW42-SR分离出的敏感克隆群体（OAW42-S）。利用免疫细胞化学和蛋白质印迹技术，结合药物（阿霉素）蓄积和亚细胞分布，研究了多药耐药相关蛋白P-170、最近发现的肺耐药相关蛋白（LRP）和多药耐药相关蛋白（MRP）、拓扑异构酶IIα和β、谷胱甘肽S-转移酶π（GST pi）以及细胞骨架蛋白细胞角蛋白8和波形蛋白的表达。使用逆转录聚合酶链反应（RT-PCR）研究了P-170、MRP、拓扑异构酶IIα和β以及GST pi的mRNA表达。结果表明，在OAW42-SR和OAW42-A1变体中，四种多药耐药相关参数（P-170、MRP、LRP以及拓扑异构酶IIα和β水平降低）存在差异共表达，而OAW42-A变体中的抗性似乎主要由P-170介导。与OAW42-SR变体（与OAW42-S细胞相比显示出更高的抗性）相比，在OAW42-S细胞中检测到相当数量的MRP和更多数量的LRP，但所有细胞系通过RT-PCR检测到的MRP mRNA水平相似且较低。各变体之间GST pi水平没有明显差异。随着抗性水平的增加，观察到细胞骨架蛋白水平升高。与OAW42-S相比，OAW42-SR和OAW42-A1变体的相对抗性在细胞连续传代增加过程中发生变化。与耐药变体相比，敏感的OAW42-S细胞系的药物蓄积更多，尤其是耐药性最强的OAW42-A细胞。维拉帕米和环孢素A均有效恢复了耐药变体中观察到的蓄积缺陷，其中环孢素A的效果更佳。药物的亚细胞定位主要在细胞核中，在敏感的OAW42-S变体中水平最高，在耐药性最强的OAW42-A克隆中水平最低。
