REGULATION OF DNA IN CAMPTOTHECIN TREATED CELLS

喜树碱（CPT）及其类似物是拓扑异构酶I（Topo I）抑制剂，并显示出针对已经被治疗的实体瘤的功效。 历史上对大多数癌症化疗剂具有抗性。 的 本申请的长期目标是阐明分子生物学的分子生物学特征。 CPT处理的细胞中DNA复制抑制的机制。 的 CPT的细胞毒性主要作用于S期细胞， 抑制DNA复制 这种DNA复制的抑制 被认为是由顺式作用过程介导的， 前进的复制叉与可裂解的CPT-Topo的碰撞 I-DNA复合体 在这个实验室里进行的工作表明， 除了顺式作用的抑制过程，也有反式作用的 CPT处理的细胞中的活性抑制过程。 研究者 假设CPT产生的受损/修饰的DNA是介体， 招募RPA进行修复并抑制其在DNA复制中的功能。 是 进一步假设Ku可能与RPA竞争结合到 受损/修饰的DNA，或分解或重塑RPA-DNA复合物， 释放RPA。 因此，RPA被允许再次启动DNA复制， 因此抑制降低。 分子信息 这种DNA复制调节的决定因素可能提供了新的方法， 在诊所进行干预，以实现CPT敏化。 具体 目的是集中研究RPA和Ku作为负责 CPT处理的细胞中DNA复制的调节 以上模型。 目标是：1. 要表征 RPA的性质与DNA复制抑制有关， CPT处理的细胞和2. 研究Ku对DNA复制抑制的影响 在CPT处理的细胞中，以及它通过何种机制与细胞相互作用并调节细胞的生长， RPA的活动。 基于猿猴病毒40（SV 40）的体外DNA复制 将在拟定实验中使用该测定。 拟议的研究是 基于初步结果的信息，并结合当前的知识 对DNA复制和CPT细胞毒性的影响，以表征 CPT处理的细胞中的DNA复制。

DESCRIPTION: Camptothecin (CPT) and its analogs are topoisomerase I (Topo I) inhibitors and show efficacy against solid tumors which have been historically resistant to most cancer chemotherapeutic agents. The long-term objective of the present application is to elucidate the molecular mechanism of DNA replication inhibition in CPT-treated cells. The cytotoxicity of CPT is mainly exerted on S-phase cells and is associated with an inhibition of DNA replication. This inhibition of DNA replication is thought to be mediated by cis-acting processes as a result of the collision of the advancing replication fork with the cleavable CPT-Topo I-DNA complex. Work carried out in this laboratory suggests that in addition to cis-acting inhibitory processes, there are also trans-acting inhibitory processes active in CPT-treated cells. The investigator hypothesizes that damaged/modified DNA produced by CPT is a mediator that recruits RPA for repair and inhibits its function in DNA replication. It is further hypothesized that Ku may either compete with RPA for binding to damaged/modified DNA, or disassemble or remodel the RPA-DNA complex to release RPA. Thus RPA is allowed to initiate DNA replication again, and therefore the inhibition is reduced. Information on the molecular determinants of this regulation of DNA replication may offer new ways of intervention in the clinic for achieving CPT sensitization. The Specific Aims focus on studying RPA and Ku as the activities responsible for the regulation of DNA replication in CPT-treated cells in the framework of the above model. The goals are: 1. To characterize modifications in the properties of RPA that are related to DNA replication inhibition in CPT-treated cells and 2. To study how Ku affects DNA replication inhibition in CPT-treated cells, and by what mechanism it interacts and modulates the activity of RPA. The Simian virus 40 (SV40) based in vitro DNA replication assay will be used in the proposed experiments. The proposed research is based on information from preliminary results and combines current knowledge on the DNA replication and CPT cytotoxicity to characterize the modulation of DNA replication in CPT-treated cells.