CELLULAR RESPONSE TO TOPOISOMERASE I

细胞对拓扑异构酶 I 的反应

• 批准号: 7313995
• 负责人: MARY-ANN BJORNSTI
• 金额: $ 24.75万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7313995
• 关键词: Affect; Biological Models; CDC45L gene; Camptothecin; Cell Cycle Arrest; Cell Death; Cell Survival; Cells; Childhood; Chromatin; Clinical; Complex; DNA; DNA Adducts; DNA Damage; DNA Topoisomerases; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Data; Development; Down-Regulation; Drug Delivery Systems; Drug resistance; Enzymes; Fiber; Fire - disasters; Frequencies; Genetic; Genetic Recombination; Genetic Transcription; Genotoxic Stress; Human; Induced Mutation; Label; Libraries; Malignant Neoplasms; Mediating; Modeling; Mutagenesis; Mutation; Natural Killer Cells; New Agents; Pathway interactions; Pharmaceutical Preparations; Phase; Play; Poison; Protein phosphatase; Proteins; RNR1 gene; Rate; Refractory; Replication Origin; Resistance; Ribonucleotide Reductase; Role; Signal Pathway; Signal Transduction; Sirolimus; Small Interfering RNA; Staining method; Stains; Stream; Topoisomerase; Topotecan; Toxic effect; Type I DNA Topoisomerases; United States Food and Drug Administration; Yeasts; analog; base; chromatin immunoprecipitation; cytotoxic; density; human CDC45L protein; human FRAP1 protein; human TOP1 protein; insight; research study; response; small hairpin RNA; therapeutic target; yeast genetics

DMA topoisomerase I (Topi) plays important roles in DMAreplication, transcription and recombination and is also the target of camptothecin (CRT), FDA approved analogs of which are effective new agents in the treatment of human cancers. CRT poisons Topi by reversibly stabilizing a covalent enzyme-DNA complex. During S-phase, the collision of replication forks with CPT-Top1-DNA adducts produces DMAlesions that signal cell cycle arrest and cell death. Although it is generally accepted that Topi targeted drugs induce DNA damage in S-phase, it is clear that signaling pathways activated in response to damage ultimately dictate cellular fate. Using yeast as a model system, conserved components of the replication machinery, CDC45 and DPB11(TopBP1), protect cells from Topi damage. Rapamycin-sensitive TOR signaling also protects yeast cells from cytotoxic DNA lesions during S-phase. Our data support a model whereby TOR acts as a survival pathway in response to genotoxic stress by maintaining replication fork stability and the dNTP pools necessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response to DNA damaging agents coincides with increased mutation rates, which may contribute to the acquisition of drug resistance. Three specific aims are proposed to investigate conserved aspects of the replication machinery and TOR signaling that maintain cell survival in response to cytoxic agents, suca at CRT. In Aim 1, a combination of yeast genetics and chromatin immunoprecipitates to query high-density tiling arrays (ChlP-chip experiments) will investigate the mechanism by which rapamycin-sensitive TOR signaling maintains replication fork stability and regulates DNA damage-induced mutagenesis. Aim 2 proposes to determine if rapamycin- sensitive mTOR signaling regulates human cell sensitivity to cyotoxic chemotherapeutics and the acquisition of drug resistance. A DNA fiber labeling strategy will determine if rapamycin treatment affects replication fork progression and stability in the presence of DNA damage, while the extent of DNA damage induced will be defined by yH2AX staining. siRNA-based approaches will determine if S-phase checkpoint function is required for the protective function of mTOR. In Aim 3, an analysis of synthetic lethal interactions will define pathway interactions of the conserved human DNA replication proteins, CDC45 and TopBPI, in regulating cell sensitivity to CRT and rapamycin. These studies will provide critical insights into the function of the TOR pathway in modulating cellular responses to DNA damage, while will impact the clinical development of rapamycin in combination with topoisomerase l-targeted therapeutics. The potential to block drug-induced mutations that confer resistance represents a unique application of rapamycins with clinical importance for the treatment of pediatric malignancies.

DMA拓扑异构酶I（TOPI）在Dmareplication，转录和重组中起重要作用 FDA批准的类似物也是Camptothecin（CRT）的目标 治疗人类癌症。 CRT毒药TOPI通过可逆地稳定共价酶-DNA复合物。 在S期间，复制叉与CPT-TOP1-DNA加合物的碰撞会产生DMalesions 信号细胞周期停滞和细胞死亡。尽管普遍认为TOPI靶向药物会诱导DNA S期损坏，很明显，响应损害而激活的信号通路最终决定 细胞命运。使用酵母作为模型系统，复制机制的保守组件，cdc45 和DPB11（TOPBP1），保护细胞免受TOPI损伤。雷帕霉素敏感的TOR信号也可以保护 S期间细胞毒性DNA病变的酵母细胞。我们的数据支持TOR充当的模型 通过维持复制叉稳定性和DNTP库来应对遗传毒性应激的生存途径 对于容易出错的跨性别DNA聚合酶所必需的。因此，响应于TOR依赖性细胞存活 DNA损害剂与突变率的增加一致，这可能有助于获取 耐药性。 提出了三个具体目标来研究复制机制和TOR的保守方面 响应细胞毒性剂的信号传导，在CRT处进行Suca。在AIM 1中，组合 酵母遗传学和染色质免疫沉淀物可查询高密度平铺阵列（CHLP-CHIP实验） 将研究雷帕霉素敏感的TOR信号传导复制叉的机制 稳定性并调节DNA损伤诱导的诱变。 AIM 2建议确定雷帕霉素是否是否 敏感的MTOR信号传导调节人类细胞对细胞毒性化疗的敏感性和采集 耐药性。 DNA纤维标记策略将确定雷帕霉素治疗是否影响复制叉 在存在DNA损伤的情况下的进展和稳定性，而DNA损伤的程度将是 由YH2AX染色定义。基于siRNA的方法将确定S期检查点功能是否为 MTOR的保护功能所需。在AIM 3中，对合成致命相互作用的分析将定义 保守的人DNA复制蛋白Cdc45和TopBPI的途径相互作用在调节中 细胞对CRT和雷帕霉素的敏感性。这些研究将为TOR的功能提供关键的见解 调节细胞对DNA损伤的反应的途径，而将影响 雷帕霉素与拓扑异构酶L靶向治疗剂结合使用。阻止药物引起的 赋予抗性的突变代表了雷帕霉素具有临床重要性的独特应用 小儿恶性肿瘤的治疗。