Identification and Cloning for cisplatin Resistance gene

我们建立了卵巢癌顺铂耐药细胞系（NOS 2CR），其对顺铂的耐药性是亲本细胞系（NOS 2）的50倍。SDS-聚丙烯酰胺凝胶电泳显示，NOS 2CR中有2条明显增强，1条减弱，1条消失，但由于其他条带的污染，很难鉴定与这些条带对应的蛋白质。谷胱甘肽的去铁化作用是顺铂耐药的机制之一。因此，谷胱甘肽结合蛋白被谷胱甘肽柱洗脱并被重结晶。对NOS 2CR进行氨基酸序列分析，发现最清晰的条带是谷胱甘肽-S-转移酶，它是谷胱甘肽循环的关键酶。其他条带不能以普通方式分析，表明蛋白质被修饰。另一方面，卡铂和aqupla也可用于癌症治疗。我们进一步建立了卡铂耐药细胞系（NOS 2CBR），并检查了对铂和其他抗癌药物的敏感性，这些药物用于癌症治疗。这些细胞系显示出对铂类抗癌药物的交叉耐药性，表明在两种耐药细胞系中获得了相同的机制。

We established cisplatin resistant ovarian cancer cell line (NOS2CR), Which showed about 50-fold resistant to cisplatin compared with parental cell line (NOS2). By SDS-PAGE,2 bands were clearly augmented, 1 was weakened, and 1 was disappeared in NOS2CR.However, it was difficult to identify proteins corresponding to these bands due to contamination of other bands. Detoxification by glutathione was one of the mechanisms for cisplatin resistance. Thus, glutathione binding proteins were eluted by glutathion column and were electrophoresed. Several bands were appeared and disappeared in NOS2CR.We analyzed amino-acid sequence, but the most clear band was glutathione-S-transferase, which is a key enzyme of glutathione cycle. Other bands could not be analyzed in an ordinary way, suggesting that proteins were modified. On the other hand, carboplatin and aqupla are also available for cancer treatment. We further established carboplatin resistant cell line (NOS2CBR) and examined the sensitivity against platinum and other anticancer drugs, which were used for cancer treatment. These cell lines showed cross resistance to platinum anticancer drugs, suggesting that the same mechanisms were acquired in both resistant cell lines.