Taki T, Ohnishi T, Arita N, Hiraga S, Hayakawa T
Department of Neurosurgery, Osaka University Medical School, Suita, Japan.
J Neurooncol. 1998 Jan;36(1):41-53. doi: 10.1023/a:1005718912236.
We have established an in vivo etoposide-resistant glioma cell line (C6/VP) from C6 rat glioma cells by stepwise exposure to increasing doses of etoposide. The C6/VP cells were 10 times more resistant to etoposide than the parental C6 cells. In addition C6/VP cells demonstrated cross-resistance to vincristine and vinblastine, but not to ADM or m-AMSA. Interestingly, the cells had collateral sensitivity to ACNU, cisDDP and Ara-C. The C6/VP cells did not express the MDR gene or p-glycoprotein, while they showed 16 times less topoisomerase II catalytic activity compared to the C6 cells. Although there was no significant difference between C6 and C6/VP cells in amounts of topoisomerase II in nuclear extracts, the C6/VP cells had 2.9 times higher amounts of the enzyme than C6 cells in nuclear scaffold prepared from a relatively low-salt buffer (0.5 M NaCl). Northern blot analysis demonstrated that mRNAs of topoisomerase IIalpha isoforms were expressed both in C6 and C6/VP cells, and that the amounts of topoisomerase IIalpha in C6/VP cells were 14 times greater than in C6 cells. The total uptake of etoposide in tumor tissues derived from C6/VP cells was 3 times less than those derived from parental C6 cells. These results indicate that the C6/VP acquired a multi-drug resistance phenotype by a reduction of the catalytic activity of topoisomerase II and/or diminished accumulation of drugs. This phenotype did not involve the p-glycoprotein. Alterations of topoisomerase II in the C6/VP cells also were accompanied by an increased amount of the topoisomerase IIalpha isoform, most of which was localized in the nuclear scaffold (matrix). This suggests that altered binding of topoisomerase II to topologically organized DNAs in the nuclear scaffold may be the molecular basis of this multi-drug resistance phenotype.
我们通过逐步增加依托泊苷的剂量，从C6大鼠胶质瘤细胞建立了一种体内依托泊苷耐药的胶质瘤细胞系（C6/VP）。C6/VP细胞对依托泊苷的耐药性是亲代C6细胞的10倍。此外，C6/VP细胞对长春新碱和长春花碱表现出交叉耐药性，但对阿霉素或米托蒽醌不耐药。有趣的是，这些细胞对阿糖胞苷、顺铂和阿糖胞苷具有协同敏感性。C6/VP细胞不表达多药耐药基因或P-糖蛋白，而与C6细胞相比，它们的拓扑异构酶II催化活性低16倍。尽管C6和C6/VP细胞核提取物中拓扑异构酶II的量没有显著差异，但在由相对低盐缓冲液（0.5M NaCl）制备的核支架中，C6/VP细胞中该酶的量比C6细胞高2.9倍。Northern印迹分析表明，拓扑异构酶IIα亚型的mRNA在C6和C6/VP细胞中均有表达，且C6/VP细胞中拓扑异构酶IIα的量比C6细胞高14倍。源自C6/VP细胞的肿瘤组织中依托泊苷的总摄取量比源自亲代C6细胞的肿瘤组织少3倍。这些结果表明，C6/VP通过降低拓扑异构酶II的催化活性和/或减少药物积累获得了多药耐药表型。这种表型不涉及P-糖蛋白。C6/VP细胞中拓扑异构酶II的改变还伴随着拓扑异构酶IIα亚型量的增加，其中大部分定位于核支架（基质）。这表明拓扑异构酶II与核支架中拓扑组织化DNA的结合改变可能是这种多药耐药表型的分子基础。
