Mutations of human topoisomerase IIα affecting multidrug resistance and sensitivity
Mutations of human topoisomerase IIα affecting multidrug resistance and sensitivity
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DOI：
10.1021/bi9909804
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发表时间：
1999-08-17
期刊：
影响因子：
2.9
通讯作者：
Liu, LF
中科院分区：
文献类型：
--
作者：
Mao, Y;Yu, C;Liu, LF
在非典型多重耐药 (at-MDR) 细胞系 CEM/VM-1 中，已鉴定出人类拓扑异构酶 II α 基因编码区的两个突变：R450Q 和 P803S，该细胞对许多结构多样的拓扑异构酶 II 靶向抗肿瘤药物（例如 VM-26、阿霉素、m-AMSA 和米托蒽醌）表现出耐药性。 R450Q突变定位在ATP利用域，而P803S突变定位在人拓扑异构酶IIα活性位点酪氨酸附近。然而，这两种突变在赋予多药耐药性方面的作用尚不清楚。为了研究这两种突变在赋予多药耐药性中的作用，我们对含有单突变或双突变的重组人 DNA 拓扑异构酶 II α 进行了表征。我们发现，R450Q 和 P803S 突变在没有 ATP 的情况下都会产生抗性。然而，在 ATP 存在的情况下，R450Q（而非 P803S）突变可以赋予多药耐药性。 R450Q 酶的酶催化作用和形成环状蛋白夹的能力均表现出 ATP 利用率受损。有趣的是，一个不相关的突变 G437E（也与 R450Q 突变位于同一结构域）在缺乏 ATP 的情况下表现出多药过敏。然而，在 ATP 存在的情况下，G437E 酶对各种拓扑异构酶 II 药物仅有最低程度的过敏。与 R450Q 酶相比，G437E 酶表现出增强的酶催化 ATP 利用率。总的来说，这些结果支持这样的观点：这些突变酶的多药耐药性和敏感性是由于酶催化过程中 ATP 利用的特定缺陷造成的。
TWO mutations, R450Q and P803S, in the coding region of the human topoisomerase II alpha gene have been identified in the atypical multidrug resistant (at-MDR) cell line, CEM/VM-1, which exhibits resistance to many structurally diverse topoisomerase II-targeting antitumor drugs such as VM-26, doxorubicin, m-AMSA, and mitoxantrone. The R450Q mutation mapped in the ATP utilization domain, while the P803S mutation mapped in the vicinity of the active site tyrosine of human topoisomerase II alpha. However, the roles of these two mutations in conferring multidrug resistance are unclear. To study the roles of these two mutations in conferring multidrug resistance, we have characterized the recombinant human DNA topoisomerase II alpha containing either single or double mutations. We show that both R450Q and P803S mutations confer resistance in the absence of ATP. However, in the presence of ATP, the R450Q, but not the P803S, mutation can confer multidrug resistance. The R450Q enzyme was shown to exhibit impaired ATP utilization both for enzyme catalysis and for its ability to form the circular protein clamp. Interestingly, an unrelated mutation, G437E, which is also located in the same domain as the R450Q mutation, exhibited multidrug hypersensitivity in the absence of ATP. However, in the presence of ATP, the G437E enzyme is only minimally hypersensitive to various topoisomerase II drugs. In contrast to the R450Q enzyme, the G437E enzyme exhibited enhanced ATP utilization for enzyme catalysis. In the aggregate, these results support the notion that the multidrug resistance and sensitivity of these mutant enzymes are due to a specific defect in ATP utilization during enzyme catalysis.