Identification and Cloning for cisplatin Resistance gene

我们建立了卵巢癌细胞顺铂耐药株(NOS2CR)，与亲本细胞株(NOS2)相比，NOS2CR对顺铂的耐药率约为50倍。经SDS-PAGE分析，NOS2CR有2条条带明显增强，1条条带减弱，1条条带消失，但由于受到其他条带的污染，很难鉴定出与这些条带对应的蛋白质。谷胱甘肽解毒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洗脱柱洗脱和电泳法。氨基酸序列分析表明，最清晰的条带是谷胱甘肽循环的关键酶--谷胱甘肽-S-转移酶。其他条带不能用普通方法分析，这表明蛋白质被修饰了。另一方面，卡铂和水杨酸也可用于癌症治疗。我们进一步建立了卡铂耐药细胞系(NOS2CBR)，并检测了其对铂等抗癌药物的敏感性。这些细胞株对铂类抗癌药物表现出交叉耐药，这表明两种耐药细胞株获得了相同的机制。

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.