Tomicic Maja T, Christmann Markus, Kaina Bernd
Institute of Toxicology, University of Mainz, Mainz, Germany.
Cancer Res. 2005 Oct 1;65(19):8920-6. doi: 10.1158/0008-5472.CAN-05-0266.
The anticancer drug topotecan belongs to the group of topoisomerase I (topo I) inhibitors. In the presence of topotecan, topo I cleaves the DNA but is unable to religate the single-strand break. This leads to stabilization of topo I-DNA-bound complexes and the accumulation of DNA strand breaks that may interfere with DNA replication. The molecular mechanism of controlling the repair of topo I-DNA covalent complexes and its impact on sensitivity of cells to topotecan is largely unknown. Here, we used mouse embryonic fibroblasts expressing wild-type p53 and deficient in p53, in order to elucidate the role of p53 in topotecan-induced cell death. We show that p53-deficient mouse embryonic fibroblasts are significantly more sensitive to topotecan than wild-type cells, displaying a higher frequency of topotecan-induced apoptosis and DNA strand breaks. Treatment of p53 wild-type cells with pifithrin-alpha, an inhibitor of the trans-activating activity of p53, caused reversal of the phenotype, making wild-type cells more sensitive to topotecan. Upon topotecan treatment, topo I was degraded in wild-type but not in p53-deficient cells. Topo I degradation was attenuated by the proteosomal inhibitor MG132. Similar data were obtained with human glioblastoma cells. U138 cells (p53 mutated) were significantly more sensitive to topotecan than U87 cells (p53 wild-type). Furthermore, U87 cells showed significant degradation of topo I upon topotecan treatment, whereas in U138 cells, this response was abrogated. Topo I degradation was again attenuated by pifithrin-alpha. The data suggests that p53 causes resistance of cells to topo I inhibitors due to stimulation of topotecan-triggered topo I degradation which may impact topotecan-based cancer therapy.
抗癌药物拓扑替康属于拓扑异构酶I（topo I）抑制剂类。在拓扑替康存在的情况下，topo I切割DNA但无法重新连接单链断裂处。这导致topo I-DNA结合复合物的稳定以及DNA链断裂的积累，可能会干扰DNA复制。控制topo I-DNA共价复合物修复的分子机制及其对细胞对拓扑替康敏感性的影响在很大程度上尚不清楚。在此，我们使用表达野生型p53和p53缺陷的小鼠胚胎成纤维细胞，以阐明p53在拓扑替康诱导的细胞死亡中的作用。我们发现，p53缺陷的小鼠胚胎成纤维细胞对拓扑替康的敏感性明显高于野生型细胞，表现出更高频率的拓扑替康诱导的凋亡和DNA链断裂。用pifithrin-α（一种p53反式激活活性抑制剂）处理p53野生型细胞导致表型逆转，使野生型细胞对拓扑替康更敏感。在拓扑替康处理后，topo I在野生型细胞中降解，但在p53缺陷细胞中未降解。蛋白酶体抑制剂MG132减弱了topo I的降解。用人胶质母细胞瘤细胞也获得了类似的数据。U138细胞（p53突变）对拓扑替康的敏感性明显高于U87细胞（p53野生型）。此外，U87细胞在拓扑替康处理后显示出topo I的明显降解，而在U138细胞中，这种反应被消除。pifithrin-α再次减弱了topo I的降解。数据表明，p53由于刺激拓扑替康触发的topo I降解而导致细胞对topo I抑制剂产生抗性，这可能会影响基于拓扑替康的癌症治疗。
