Genome-Wide Transcriptional Effects of the Anti-Cancer Agent Camptothecin

The anti-cancer drug camptothecin inhibits replication and transcription by trapping DNA topoisomerase I (Top1) covalently to DNA in a “cleavable complex”. To examine the effects of camptothecin on RNA synthesis genome-wide we used Bru-Seq and show that camptothecin treatment primarily affected transcription elongation. We also observed that camptothecin increased RNA reads past transcription termination sites as well as at enhancer elements. Following removal of camptothecin, transcription spread as a wave from the 5’-end of genes with no recovery of transcription apparent from RNA polymerases stalled in the body of genes. As a result, camptothecin preferentially inhibited the expression of large genes such as proto-oncogenes, and anti-apoptotic genes while smaller ribosomal protein genes, pro-apoptotic genes and p53 target genes showed relative higher expression. Cockayne syndrome group B fibroblasts (CS-B), which are defective in transcription-coupled repair (TCR), showed an RNA synthesis recovery profile similar to normal fibroblasts suggesting that TCR is not involved in the repair of or RNA synthesis recovery from transcription-blocking Top1 lesions. These findings of the effects of camptothecin on transcription have important implications for its anti-cancer activities and may aid in the design of improved combinatorial treatments involving Top1 poisons.抗癌药物喜树碱（camptothecin）可通过将DNA拓扑异构酶I（DNA topoisomerase I，Top1）以“切割复合物（cleavable complex）”的形式共价捕获于DNA分子之上，进而抑制DNA复制与转录过程。为探究喜树碱对全基因组RNA合成的影响，本研究采用Bru-Seq技术开展实验，结果显示喜树碱处理主要干扰转录延伸过程。研究同时观察到，喜树碱会使转录终止位点（transcription termination sites）下游以及增强子元件（enhancer elements）区域的RNA测序读段数量显著增多。去除喜树碱后，转录以波状形式从基因的5'端向外扩散，而滞留在基因本体内部的RNA聚合酶并未介导转录功能的恢复。由此，喜树碱会优先抑制大型基因的表达，例如原癌基因（proto-oncogenes）与抗凋亡基因（anti-apoptotic genes）；而小型核糖体蛋白基因（ribosomal protein genes）、促凋亡基因（pro-apoptotic genes）以及p53靶基因（p53 target genes）的表达水平则相对更高。转录偶联修复（transcription-coupled repair，TCR）缺陷的科凯恩综合征B型成纤维细胞（Cockayne syndrome group B fibroblasts，CS-B），其RNA合成恢复特征与正常成纤维细胞相似，这表明TCR并未参与转录阻滞型Top1损伤的修复或RNA合成恢复过程。本研究关于喜树碱对转录的影响的相关发现，对其抗癌活性具有重要指导意义，同时可为优化基于拓扑异构酶I毒剂（Top1 poisons）的联合治疗方案设计提供参考。