MECHANISMS OF SUPPRESSING CAMPTOTHECIN TOXICITY

描述：（申请人的摘要）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）所例示的，这些药物通过增加Top1的表达来靶向Top1。 共价酶-DNA中间体的稳定性。在S阶段， 前进的复制叉与Cpt-酶-DNA复合物的碰撞导致 在DNA损伤的形成，信号细胞周期停滞和细胞死亡。 在酵母和哺乳动物细胞中的研究表明，top1的过表达是 固有的细胞毒性，而Top1中的突变稳定了共价结合， 复合物概括了Cpt处理的表型结果。采取 总之，这些数据支持一种模型，其中增加的共价键浓度 复合物，作为药物作用，酶浓度或突变的结果， 导致潜在致命的DNA损伤的产生。然而， 了解产生的损伤的性质和修复过程 为解决所需。不太清楚的是修复过程的保真度 或治疗后继发性恶性肿瘤的潜在诱导 使用Top1毒药。这项申请旨在填补我们 通过研究Top1毒物的细胞毒性作用， 参与抑制Cpt的细胞毒性作用的细胞过程， Top1毒物诱导的DNA损伤可能促进肿瘤形成。 在酵母中，泛素异肽酶Ubp 11的过表达抑制了 Cpt的细胞毒性作用，对Top1活性几乎没有影响。进一步 Ubp 11功能的表征将定义所涉及的细胞过程 Cpt诱导的DNA损伤的形成和修复。Top1突变体 证明Top1中毒的不同机制将被用于研究 不同DNA损伤的细胞毒性和致突变潜力。的能力 Ubp 11（或人类同系物）抑制这些Top1毒物的作用， 也被调查，沿着的后果，酵母和哺乳动物细胞 存活率对突变率和细胞增殖的影响。为了定义致癌的 潜在的Top1-DNA损伤，Top1诱导的肿瘤转化将是 在未转化的成纤维细胞和转基因小鼠模型中评估。这些 研究将提供对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.