Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity.
Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity.
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发表时间：
1986-04
期刊：
影响因子：
11.2
通讯作者：
Bonnie S. Glisson;Radhey S. Gupta;Radhey S. Gupta;S. Smallwood-Kentro;S. Smallwood-Kentro;W. Ross
中科院分区：
文献类型：
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作者：
Bonnie S. Glisson;Radhey S. Gupta;Radhey S. Gupta;S. Smallwood-Kentro;S. Smallwood-Kentro;W. Ross
最近的证据表明，II型DNA拓扑异构酶介导表鬼臼毒素诱导的DNA损伤，可能是药物抗肿瘤作用的内在因素。使用表鬼臼毒素耐药细胞系，我们现在已经进一步确定了DNA损伤和细胞死亡之间的关系，并描绘了某些药物-酶相互作用的意义。与野生型细胞相比，突变型中国仓鼠卵巢细胞系VpmR-5对依托泊苷（VP-16）的细胞毒性和DNA切割活性均表现出显著的抗性。两种细胞系中放射性标记VP-16的稳态浓度相同。野生型和VpmR-5细胞的粗核提取物中的催化活性相等，并且对VP-16的抑制同样敏感。然而，使用特异性测量32P标记的3 'DNA中5'蛋白质连接断裂的产生的测定，我们发现来自VpmR-5系的核提取物中的DNA切割活性对VP-16的刺激具有深刻的抗性。此外，VpmR-5细胞和药物敏感性EOT-3细胞的体细胞杂交系显示VP-16敏感性的恢复与DNA切割活性的重建一致。这些数据表明，刺激酶介导的DNA切割，而不是正常的拓扑异构酶功能的丧失，是表鬼臼毒素诱导的细胞毒性的原因。
Recent evidence indicates that type II DNA topoisomerases mediate epipodophyllotoxin-induced DNA damage and may be intrinsic to the drug's antitumor effects. Using an epipodophyllotoxin-resistant cell line, we have now further defined the relationship between DNA damage and cell death and delineated the significance of certain drug-enzyme interactions. When compared to wild-type cells, the mutant Chinese hamster ovary cell line, VpmR-5, exhibits marked resistance to both the cytotoxic and DNA cleavage activities of etoposide (VP-16). Steady-state concentrations of radiolabeled VP-16 are identical in both cell lines. Catalytic activity in crude nuclear extracts from wild-type and VpmR-5 cells is equal and is equally sensitive to inhibition by VP-16. However, using an assay that specifically measures generation of 5' protein-linked breaks in 32P-labeled 3' DNA, we have found that DNA cleavage activity in nuclear extract from the VpmR-5 line is profoundly resistant to stimulation by VP-16. Further, a somatic cell hybrid line of VpmR-5 cells and drug-sensitive EOT-3 cells exhibits recovery of VP-16 sensitivity in concert with reconstitution of DNA cleavage activity. These data indicate that stimulation of enzyme-mediated DNA cleavage, rather than loss of normal topoisomerase function, is responsible for epipodophyllotoxin-induced cytotoxicity.