Chang Jang-Yang, Liu Jin-Fen, Juang Shin-Hun, Liu Tsang-Wu, Chen Li-Tzong
Division of Cancer Research, National Health Research Institutes, 100 Taipei, Taiwan, Republic of China.
Cancer Res. 2002 Jul 1;62(13):3716-21.
To identify mechanisms of camptothecin (CPT) resistance and the relationship between CPT-resistant cells and other anticancer agents, a CPT-resistant cell line (CPT30) and its partial revertant cell line (CPT30R) were established from a human nasopharyngeal carcinoma cell line (HONE-1). CPT30 and CPT30R cells displayed a 14- and 3.5-fold resistance to CPT compared with HONE-1 cells, respectively. The resistant and partial revertant cell lines showed cross-resistance to topotecan and increased sensitivity to cisplatin, carboplatin, and 1,3-bis(chloroethyl)-1-nitrosurea. The topoisomerase (Top) I catalytic activity of CPT30 and CPT30R cells was 30% and 200%, respectively, compared with that of HONE-1 cells. The expression of Top I protein and mRNA levels in CPT30 cells was 40% and 30% less than that in HONE-1 cells, respectively, whereas in CPT30R cells, the levels of Top I protein and mRNA were 50% and 20% higher, respectively, than that in HONE-1 cells. Both the resistant and revertant cell line whole-cell lysates demonstrated different levels of sensitivity to CPT in in vitro assays in comparison with that of HONE-1 cells. Furthermore, CPT exhibited 15- and 7-fold better binding affinity in stabilizing protein-linked DNA breaks in HONE-1 cells than in CPT30 and CPT30R cells, respectively. Direct DNA sequencing of the reverse transcription-PCR product and genomic DNA revealed a point mutation resulting in E418K mutation in the Top I of both CPT30 and CPT30R cells. Wild-type Top I RNA and genomic DNA were also detected in these two cell lines. A yeast system was used to examine whether this mutation could be responsible for CPT resistance. Our results showed that a single amino acid change (E418K) resulted in CPT resistance. Therefore, quantitative and qualitative changes in Top I were responsible for CPT resistance in CPT30 cells. CPT resistance in CPT30R cells was caused by mutation of Top I.
为了确定喜树碱（CPT）耐药机制以及CPT耐药细胞与其他抗癌药物之间的关系，从人鼻咽癌细胞系（HONE-1）中建立了CPT耐药细胞系（CPT30）及其部分回复细胞系（CPT30R）。与HONE-1细胞相比，CPT30和CPT30R细胞对CPT的耐药性分别提高了14倍和3.5倍。耐药和部分回复细胞系对拓扑替康表现出交叉耐药性，对顺铂、卡铂和1,3-双（氯乙基）-1-亚硝基脲的敏感性增加。与HONE-1细胞相比，CPT30和CPT30R细胞的拓扑异构酶（Top）I催化活性分别为30%和200%。CPT30细胞中Top I蛋白表达和mRNA水平分别比HONE-1细胞低40%和30%，而在CPT30R细胞中，Top I蛋白和mRNA水平分别比HONE-1细胞高50%和20%。与HONE-1细胞相比，耐药和回复细胞系的全细胞裂解物在体外试验中对CPT表现出不同程度的敏感性。此外，在稳定HONE-1细胞中蛋白质连接的DNA断裂方面，CPT分别比CPT30和CPT30R细胞表现出高15倍和7倍的结合亲和力。逆转录-PCR产物和基因组DNA的直接DNA测序显示，CPT30和CPT30R细胞的Top I中均出现导致E418K突变的点突变。在这两种细胞系中也检测到野生型Top I RNA和基因组DNA。使用酵母系统来检测这种突变是否可能导致CPT耐药。我们的结果表明，单个氨基酸变化（E418K）导致CPT耐药。因此，Top I的定量和定性变化是CPT30细胞中CPT耐药的原因。CPT30R细胞中的CPT耐药是由Top I突变引起的。
