MECHANISMS OF SUPPRESSING CAMPTOTHECIN TOXICITY

描述：(申请人摘要)近年来，DNA拓扑异构酶I(Top1) 已经成为越来越多的抗肿瘤药物的细胞靶点。 如喜树碱(Cpt)所示，这些药物通过增加Top1的 共价酶-DNA中间体的稳定性。在S阶段， 超前复制叉与Cpt-酶-DNA复合体的碰撞结果 在DNA损伤的形成中发出细胞周期停滞和细胞死亡的信号。 在酵母和哺乳动物细胞中的研究表明，top1的过度表达是 固有的细胞毒性，而Top1中稳定共价的突变 复合体概括了Cpt治疗的表型后果。已被占用 总而言之，这些数据支持一个模型，在这个模型中，共价分子浓度增加 作为药物作用、酶浓度或突变的结果， 导致潜在致命性DNA损伤的产生。然而，几乎没有什么是 了解产生的损伤的性质和修复过程 才能解决问题。不太清楚的是修复过程的保真度 或在治疗后潜在地诱发继发性恶性肿瘤 TOP1毒药的应用。这个应用程序旨在填补我们在 通过对Top1毒物的研究了解Top1毒物的细胞毒作用 参与抑制Cpt和Cpt细胞毒作用的细胞过程 Top1毒物诱导DNA损伤促进肿瘤形成的可能性。 在酵母中，泛素异肽酶Ubp11的过表达抑制了 Cpt具有细胞毒作用，对Top1活性影响不大。进一步 Ubp11功能的特征将定义所涉及的细胞过程 在Cpt诱导的DNA损伤形成和修复中起重要作用。Top1突变体 将使用演示Top1中毒的不同机制来调查 不同DNA损伤的细胞毒性和致突变潜能。的能力 Ubp11(或人类同系物)抑制这些Top1毒物的作用将 以及酵母菌和哺乳动物细胞的后果 生存依赖于突变率和细胞增殖。为了定义致癌的 潜在的Top1-DNA损伤，Top1诱导的肿瘤转化 在未转化的成纤维细胞和转基因小鼠模型中进行了评估。这些 研究将深入了解Top1靶向药物诱导 儿童和成人人群中的肿瘤。

DESCRIPTION: (Applicant's Abstract) In recent years, DNA topoisomerase I (Top1) has emerged as the cellular target of an increasing number of antitumor agents. As exemplified by camptothecin (Cpt), these drugs target Top1 by increasing the stability of the covalent enzyme-DNA intermediate. During S-phase, the collision of advancing replication forks with Cpt-enzyme-DNA complexes results in the formation of DNA lesions that signal cell cycle arrest and cell death. Studies in yeast and mammalian cells indicate that overexpression of top1 is inherently cytotoxic, while mutations in Top1 that stabilize the covalent complex recapitulate the phenotypic consequences of Cpt treatment. Taken together, these data support a model where increased concentrations of covalent complexes, as a consequence of drug action, enzyme concentration or mutation, result in the production of potentially lethal DNA lesions. However, little is known about the nature of the lesions produced and the repair processes required for their resolution. Less clear is the fidelity of repair processes or the potential induction of secondary malignancies following the therapeutic application of Top1 poisons. This application aims to fill the void in our understanding of the cytotoxic action of Top1 poisons by investigating the cellular processes involved in suppressing the cytotoxic action of Cpt and the potential for DNA lesions induced by Top1 poisons to promote tumor formation. In yeast, overexpression of the ubiquitin isopeptidase, Ubp11, suppresses the cytotoxic action of Cpt, with little effect on Top1 activity. Further characterization of Ubp11 function will define the cellular processes involved in the formation and repair of DNA lesions induced by Cpt. Top1 mutants demonstrating distinct mechanisms of Top1 poisoning will be used to investigate the cytotoxic and mutagenic potential of different DNA lesions. The ability of Ubp11 (or human homologues) to suppress the action of these Top1 poisons will also be investigated, along with the consequences of yeast and mammalian cell survival on mutation rates and cell proliferation. To define the oncogenic potential of Top1-DNA lesions, Top1-induced neoplastic transformation will be assessed in untransformed fibroblasts and in a transgenic mouse model. These studies will provide insight into the ability of Top1-targeted drugs to induce tumors in pediatric and adult populations.