Tolner B, Hartley J A, Hochhauser D
Department of Oncology, Royal Free and University College Medical School, University College London, Gower Street Campus, London, United Kingdom.
Mol Pharmacol. 2001 Apr;59(4):699-706. doi: 10.1124/mol.59.4.699.
Topoisomerase II alpha is a critical gene involved in DNA replication and maintenance of genomic stability. Several chemotherapeutic agents target topoisomerase II and levels of expression are an important factor in chemosensitivity. Transcriptional regulation has been demonstrated to regulate topoisomerase II alpha levels under several circumstances, including cellular confluence, heat shock, and expression of oncogenes including ras and myb. Expression of topoisomerase II alpha is regulated by cellular proliferation; transcriptional down-regulation in confluent cells is modulated through sequences within the promoter. In this study, we examined DNA-protein interactions within the topoisomerase II alpha promoter in exponential and confluent phase NIH3T3 cells. Using electrophoretic mobility shift assay and in vitro DNase I footprint experiments, the involvement of NF-Y in transcriptional regulation was established. Incubation of the DNA minor groove-binding agents Hoechst 33342 and Hoechst 33258 with nuclear extracts revealed drug binding to regions surrounding the inverted CCAAT boxes within the topoisomerase II alpha promoter and displacement of proteins binding to these elements. Addition of both Hoechst 33342 and Hoechst 33258 to NIH3T3 cells at confluence resulted in increased expression of topoisomerase II alpha. In addition, MTT cytotoxicity assays in confluent cells showed an additive effect of incubation with Hoechst 33342 and the topoisomerase II alpha poison etoposide. Therefore, DNA binding drugs which block transcription factor activation of the promoter may deregulate topoisomerase II alpha and this strategy may be of value in modifying gene expression and modulating chemosensitivity.
拓扑异构酶IIα是一种参与DNA复制和维持基因组稳定性的关键基因。几种化疗药物作用于拓扑异构酶II，其表达水平是化疗敏感性的一个重要因素。在多种情况下，包括细胞汇合、热休克以及癌基因（如ras和myb）的表达，转录调控已被证明可调节拓扑异构酶IIα的水平。拓扑异构酶IIα的表达受细胞增殖调控；汇合细胞中的转录下调通过启动子内的序列进行调节。在本研究中，我们检测了指数生长期和汇合期NIH3T3细胞中拓扑异构酶IIα启动子内的DNA-蛋白质相互作用。通过电泳迁移率变动分析和体外DNase I足迹实验，确定了NF-Y参与转录调控。将DNA小沟结合剂Hoechst 33342和Hoechst 33258与核提取物一起孵育，发现药物与拓扑异构酶IIα启动子内反向CCAAT框周围的区域结合，并取代了与这些元件结合的蛋白质。在汇合期将Hoechst 33342和Hoechst 33258添加到NIH3T3细胞中，导致拓扑异构酶IIα的表达增加。此外，在汇合细胞中进行的MTT细胞毒性试验显示，Hoechst 33342与拓扑异构酶IIα毒药依托泊苷一起孵育具有相加效应。因此，阻断启动子转录因子激活的DNA结合药物可能会使拓扑异构酶IIα失调，这种策略在改变基因表达和调节化疗敏感性方面可能具有价值。
