Novel Mechanisms by which RAD18 and POLZ affect Response to Anticancer Agents

RAD18 和 POLZ 影响抗癌药物反应的新机制

基本信息

批准号：
7759548
负责人：
Christine Elizabeth Canman
金额：
$ 28.39万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-10 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7759548
关键词：
ATR protein kinase Abbreviations Address Affect Antineoplastic Agents Biological Assay Bypass Camptothecin Catalytic Domain Cell Cycle Checkpoint Cell Survival Cells Cervix carcinoma Chickens Chromosome abnormality Cisplatin Combination Drug Therapy Complex DNA DNA Double Strand Break DNA Interstrand Crosslinking DNA Intrastrand Crosslinking DNA Repair DNA biosynthesis DNA lesion DNA polymerase zeta DNA-Directed DNA Polymerase Data Double Strand Break Repair Drug usage Enzymes Event Exhibits Exposure to Frequencies Gene Conversion Genetic Recombination Genomic Instability Goals Hela Cells Human Ionizing radiation Kinetics Lead Lesion Lymphoma Malignant Epithelial Cell Malignant Neoplasms Measures Mediating Mitomycins Modeling Molecular Monoubiquitination Nuclear Pathway interactions Pharmaceutical Preparations Phenotype Play Polymerase Process Proliferating Cell Nuclear Antigen Proteins Rad30 protein Radiation Tolerance Reagent Recruitment Activity Reporter Resistance Resolution Role Single-Stranded DNA Sister Chromatid Exchange Site Small Interfering RNA Surrogate Markers TOP1 gene Testing Type I DNA Topoisomerases Ubiquitination adduct ataxia telangiectasia mutated protein base cancer cell chemotherapy coping crosslink design drug development effective therapy homologous recombination inhibitor/antagonist insight neoplastic cell novel prevent public health relevance recombinational repair repaired response therapeutic effectiveness ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Drugs which target DNA through direct crosslinking or trapping of topoisomerase I (TOP1) complexes are some of the most effective treatments for cancer. A more complete understanding of the molecular pathways that influence how cancer cells cope with these lesions may identify new targets for drug development with the goal of increasing therapeutic effectiveness through combination chemotherapy. The E3 ubiquitin ligase Rad18 plays a crucial role in postreplication repair (PPR) by monoubiquitinating PCNA in response to stalled replication forks. This event promotes lesion bypass by recruiting a group of specialized DNA polymerases that copy DNA templates containing DNA lesions through a process termed Translesion DNA synthesis (TLS). TLS is believed to be important for replicative bypass of cisplatin adducts and may be an important mechanism by which tumor cells resist therapy. To further probe into how TLS may impact chemotherapy, we used siRNA to deplete human cells of Rad18, Rev3 (the catalytic subunit of Pol ?), or two additional TLS polymerases, Rev1 and DNA Polymerase Eta, all four believed to be involved in replicating DNA containing cisplatin adducts. We found that all four gene products were necessary to prevent replication fork stalling following exposure to cisplatin consistent with a model that lesion bypass is accomplished by PCNA monoubiquitination and coordination of multiple TLS polymerases. Unexpectedly, we found that Rad18 and Rev3-depleted cells are hypersensitive to cisplatin suggesting they play additional roles in tolerance to cisplatin. We also observed that Rad18 colocalizes with DNA double strand breaks (DSBs) when cells are exposed to ionizing radiation, camptothecin, Mitomycin C, or cisplatin and that depletion of Rev3 leads to cellular phenotypes consistent with cells deficient in DSB repair. We hypothesize that Rad18 and Pol 6 possess alternative functions in addition to performing PPR. We believe these functions are important for resolution of replication-associated DSBs, a common intermediate during repair of ICLs and replication associated DSBs caused by TOP1 inhibitors. To address this hypothesis, we propose the following three specific aims: 1. Determine whether Rad18 or Pol ? promotes cell survival and prevents accumulation of chromosomal aberrations when cells are challenged with agents that induce replication-associated DSBs. 2. Determine whether Rad18 or Pol ? is necessary for the efficient repair of replication-associated DSB and whether Rad18 or Pol 6 facilitates HR triggered by camptothecin and ICL-inducing agents by measuring HR activity in cells and frequencies of drug-induced sister chromatid exchanges at the chromosomal level. 3. Determine the functional significance of localization of Rad18 to sites of DSBs by examining whether localization is dependent upon DNA replication and whether PCNA ubiquitination is associated with this event. We will also characterize the functional domains within Rad18 that are necessary for localization and whether those domains are important for cellular resistance to replication-associated DSBs and directing DNA repair. PUBLIC HEALTH RELEVANCE: Agents which target Topoisomerase I and introduce interstrand DNA crosslinks are some of the most widely used drugs used to treat cancer. We hypothesize that two enzymes, Rad18 and DNA Polymerase Zeta, are important for facilitating repair of DNA double stranded breaks associated with the therapeutic effectiveness of these agents. The studies proposed here will advance our understanding of DNA repair activities that modulate the sensitivity of cancer cells to these anticancer agents and provide new insight into how cells prevent genomic instability, a common hallmark of cancer.

描述（由申请人提供）：通过直接交联或捕获拓扑异构酶 I (TOP1) 复合物来靶向 DNA 的药物是一些最有效的癌症治疗方法。更全面地了解影响癌细胞如何应对这些病变的分子途径可能会确定药物开发的新靶点，以通过联合化疗提高治疗效果。 E3 泛素连接酶 Rad18 通过单泛素化 PCNA 来响应停滞的复制叉，从而在复制后修复 (PPR) 中发挥至关重要的作用。该事件通过招募一组专门的 DNA 聚合酶来促进病变绕过，这些聚合酶通过称为跨病变 DNA 合成 (TLS) 的过程复制包含 DNA 病变的 DNA 模板。 TLS被认为对于顺铂加合物的复制旁路很重要，并且可能是肿瘤细胞抵抗治疗的重要机制。为了进一步探讨 TLS 如何影响化疗，我们使用 siRNA 来消除人类细胞的 Rad18、Rev3（Pol 的催化亚基？）或另外两种 TLS 聚合酶 Rev1 和 DNA 聚合酶 Eta，这四种酶均被认为参与复制含有顺铂加合物的DNA。我们发现，所有四种基因产物都是防止暴露于顺铂后复制叉停滞所必需的，这与通过 PCNA 单泛素化和多个 TLS 聚合酶的协调来完成病变旁路的模型一致。出乎意料的是，我们发现 Rad18 和 Rev3 耗尽的细胞对顺铂过敏，表明它们在顺铂耐受中发挥着额外的作用。我们还观察到，当细胞暴露于电离辐射、喜树碱、丝裂霉素 C 或顺铂时，Rad18 与 DNA 双链断裂 (DSB) 共定位，并且 Rev3 的耗尽导致与 DSB 修复缺陷的细胞一致的细胞表型。我们假设 Rad18 和 Pol 6 除了执行 PPR 之外还具有替代功能。我们相信这些功能对于解决复制相关的 DSB 非常重要，复制相关的 DSB 是修复 ICL 和 TOP1 抑制剂引起的复制相关 DSB 过程中的常见中间体。为了解决这个假设，我们提出以下三个具体目标： 1. 确定 Rad18 还是 Pol ？当细胞受到诱导复制相关 DSB 的试剂攻击时，可促进细胞存活并防止染色体畸变的累积。 2. 判断Rad18还是Pol？对于复制相关 DSB 的有效修复是必要的，并且通过测量细胞中的 HR 活性和染色体水平上药物诱导的姐妹染色单体交换的频率，Rad18 或 Pol 6 是否可以促进喜树碱和 ICL 诱导剂触发的 HR。 3. 通过检查定位是否依赖于 DNA 复制以及 PCNA 泛素化是否与此事件相关，确定 Rad18 定位到 DSB 位点的功能意义。我们还将描述 Rad18 内定位所必需的功能域，以及这些域对于细胞对复制相关 DSB 的抵抗和指导 DNA 修复是否重要。公共健康相关性：靶向拓扑异构酶 I 并引入链间 DNA 交联的药物是最广泛使用的癌症治疗药物之一。我们假设两种酶，Rad18 和 DNA 聚合酶 Zeta，对于促进与这些药物的治疗效果相关的 DNA 双链断裂的修复非常重要。这里提出的研究将增进我们对调节癌细胞对这些抗癌药物敏感性的 DNA 修复活动的理解，并为细胞如何预防基因组不稳定性（癌症的常见标志）提供新的见解。