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）复合物靶向DNA的药物是癌症的一些最有效的治疗方法。对影响癌细胞如何应对这些病变的分子途径的分子途径有了更全面的了解，可能会确定药物发育的新靶标，目的是通过组合化疗提高治疗有效性。 E3泛素连接酶RAD18通过单液化PCNA响应停滞的复制叉，在脊椎修复（PPR）中起着至关重要的作用。该事件通过募集一组专门的DNA聚合酶来促进病变旁路，该聚合酶通过称为Translesion DNA合成（TLS）的过程复制含有DNA病变的DNA模板。据信TLS对于顺铂加合物的复制搭桥术很重要，并且可能是肿瘤细胞抗治疗的重要机制。为了进一步探究TLS如何影响化疗，我们使用siRNA耗尽了RAD18，REV3（POL的催化亚基？）或两个其他TLS聚合酶，Rev1和DNA聚合酶ETA的人体细胞，据信所有四种据信与复制含有Cisplatin添加的DNA有关。我们发现，所有四种基因产物都是为了防止在接触顺铂的复制叉降低的必要条件，该模型与PCNA单次素养和多种TLS聚合酶的配位来实现病变旁路的模型一致。出乎意料的是，我们发现RAD18和REV3耗尽的细胞对顺铂表明它们在耐受性方铂发挥了额外的作用。我们还观察到，当细胞暴露于电离辐射，凸轮皮霉素，丝霉素C或顺铂时，RAD18与DNA双链断裂（DSB）共定位，REV3的耗竭会导致与DSB维修中缺乏细胞一致的细胞表型。我们假设Rad18和Pol 6除了执行PPR外，还具有替代功能。我们认为，这些功能对于分辨复制相关的DSB很重要，复制相关的DSB是ICL修复过程中的常见中间体以及由TOP1抑制剂引起的复制相关的DSB。为了解决这一假设，我们提出以下三个特定目的：1。确定RAD18还是POL？促进细胞的存活，并防止当细胞受到诱导复制相关的DSB的质挑战时染色体畸变的积累。 2.确定是Rad18还是POL？对于有效地修复复制相关的DSB是必要的，而RAD18或POL 6是否通过测量铬粒水平的药物诱导的姐妹染色质交换的细胞和频率来促进由凸轮皮甲基蛋白和ICL诱导剂触发的HR。 3。通过检查定位是否取决于DNA复制以及PCNA泛素化是否与此事件相关，确定RAD18定位到DSB的位点的功能显着性。我们还将表征RAD18内的功能域，这些功能域对于定位所必需，以及这些结构域对于对复制相关的DSB和指导DNA修复是否重要。公共卫生相关性：靶向拓扑异构酶I并引入链间DNA交联的药物是用于治疗癌症的一些最广泛使用的药物。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有效性相关的DNA双链断裂的修复很重要。此处提出的研究将提高我们对DNA修复活性的理解，该活动调节癌细胞对这些抗癌剂的敏感性，并提供有关细胞如何预防基因组不稳定性的新见解，这是癌症的常见标志。