Formation of topoisomerase II alpha complexes with nascent DNA is related to VM-26-induced cytotoxicity.
Formation of topoisomerase II alpha complexes with nascent DNA is related to VM-26-induced cytotoxicity.
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拓扑异构酶 II α 与新生 DNA 复合物的形成与 VM-26 诱导的细胞毒性有关。
DOI：
10.1021/bi9619637
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Fernandes,DJ
中科院分区：
文献类型：
--
作者：
Qiu,J;Catapano,CV;Fernandes,DJ
已知几种临床活性抗癌药物干扰DNA拓扑异构酶II活性。然而，单个α（170 kDa）和β（180 kDa）同工酶作为拓扑异构酶II活性药物靶点的重要性尚不清楚。为了解决这个问题，人CCRF-CEM白血病细胞与溴脱氧尿苷一起孵育，并通过用抗溴脱氧尿苷抗体免疫沉淀来纯化新生DNA或未进行复制的大量DNA。通过Western印迹分析与新生DNA或散装DNA共沉淀的拓扑异构酶II同工酶。在VM-26或米托蒽醌预处理的细胞中，α同工酶与新生DNA形成复合物，而β同工酶仅与本体DNA结合。在中等细胞毒性浓度下，VM-26增强拓扑异构酶IIα与新生DNA的结合，与大量DNA相比至少5.2倍。然而，在与等毒性浓度的VM-26孵育的VM-26抗性CEM/VM-1细胞中，与新生DNA形成的拓扑异构酶IIα复合物与大量DNA相比至少减少5.5倍。在CEM/VM-1细胞中，药物诱导的拓扑异构酶IIβ与本体DNA的结合与细胞毒性无关。总之，这些结果表明，VM-26稳定的拓扑异构酶IIα与新生DNA的复合物的形成对于细胞毒性的发展至关重要，并且CEM/VM-1细胞对VM-26的抗性与这些复合物的形成受损有关。这一结果也为拓扑异构酶IIα参与DNA复制提供了间接证据。
Several clinically active anticancer drugs are known to interfere with DNA topoisomerase II activity. However, the importance of the individual α (170 kDa) and β (180 kDa) isozymes as targets of topoisomerase II-active drugs is not clear. To address this question, human CCRF-CEM leukemia cells were incubated with bromodeoxyuridine, and either the nascent DNA or bulk DNA not undergoing replication was purified by immunoprecipitation with an anti-bromodeoxyuridine antibody. The topoisomerase II isozymes that coprecipitated with either the nascent DNA or bulk DNA were analyzed by Western blotting. The α isozyme formed complexes with nascent DNA in cells pretreated with either VM-26 or mitoxantrone, while the β isozyme was only bound to bulk DNA. At moderately cytotoxic concentrations, VM-26 enhanced the binding of topoisomerase IIα to nascent DNA at least 5.2-fold compared to bulk DNA. However, in VM-26 resistant CEM/VM-1 cells incubated with equitoxic concentrations of VM-26, topoisomerase IIα complex formation with nascent DNA was decreased at least 5.5-fold compared to bulk DNA. Drug-induced binding of topoisomerase IIβ with bulk DNA in CEM/VM-1 cells did not correlate with cytotoxicity. Collectively, these results indicate that the formation of VM-26 stabilized complexes of topoisomerase IIα with nascent DNA are critical to the development of cytotoxicity, and that resistance of CEM/VM-1 cells to VM-26 is related to impaired formation of these complexes. The results also provide indirect evidence that topoisomerase IIα is involved in DNA replication.