MECHANISMS OF SUPPRESSING CAMPTOTHECIN TOXICITY

DNA螺旋的两条链缠绕在一起，形成了许多 通过催化DNA拓扑结构的改变来解决拓扑问题 DNA链断裂和重新连接的协同机制。这个过程是 伴随着共价酶-DNA中间体的形成，这 保存破碎者的能量 磷酸二酯键。一些治疗上很重要的药物， 包括强效抗肿瘤药物喜树碱，可逆性 稳定这些共价络合物。喜树碱专门针对 真核DNA拓扑异构酶1，高度保守的 初级氨基酸序列、作用机制和药物敏感性。这个 喜树碱的细胞毒性是S时相依赖的，其原因是 DNA复制叉碰撞产生的双链DNA断裂 药物稳定的酶-DNA加合物。然而，人们对此知之甚少 关于参与处理和修复这些信息的机制 潜在的致命损伤，或药物所需的信号通路- 诱导细胞死亡。 这项申请建议定义喜树碱诱导的途径 细胞致死性，通过筛选酵母和人类基因产物 在酵母中的过表达对这些细胞具有保护作用 来自药物介导的细胞死亡。因为病毒的表型后果 喜树碱的治疗在酵母细胞中得到了忠实的重申 将药物稳定的复合体转化为致命性化合物的过程 损伤可以在实验中解决，在这个遗传上容易处理的 系统。随后鉴定这些高复制抑制子(Hcs) 酵母和哺乳动物细胞中的基因及其细胞功能， 将阐明喜树碱诱导所需的细胞过程 DNA损伤和细胞凋亡。这些基因抑制相关基因的能力 细胞死亡的机制也将在酵母和哺乳动物细胞中进行研究。 表达模拟细胞毒作用的致死DNA拓扑异构酶1突变体 喜树碱的作用。这些研究将加深我们对 而药物致死的机制，也会导致更大的 理解正常的细胞功能如何会受到某种原因的干扰 细胞死亡。因为已经有几个喜树碱类似物进入 卵巢、乳腺、结肠及非小细胞癌治疗的临床研究 细胞性肺癌中，HCS基因功能的表征将具有 在新的设计和开发中有更广泛的应用 治疗学。

The intertwining of the two strands of a DNA helix poses a number of topological problems by catalyzing changes in DNA topology through a concerted mechanism of DNA strand breakage and rejoining. This process is accompanied by the formation of covalent enzyme-DNA intermediates, which conserve the energy of the broken phosphodiester linkages. A number of therapeutically important drugs, including the potent antineoplastic agent camptothecin, reversibly stabilize these covalent complexes. Camptothecin specifically targets eukaryotic DNA topoisomerase 1, which is highly conserved in terms of its primary amino acid sequence, mechanism of action and drug sensitivity. The cytotoxicity of camptothecin is S-phase dependent, resulting from the double-strand DNA breaks produced by the collision of DNA replication forks with the drug-stabilized enzyme-DNA adducts. However, little is known about the mechanisms involved in the processing and repair of these potentially lethal lesions, or the signaling pathways required for drug- induced cell killing. This application proposes to define the pathway of camptothecin-induced cell lethality, by screening for yeast and human gene products whose overexpression in the yeast Saccharomyces cerevisiae protects these cells from drug-mediated cell death. Since the phenotypic consequences of camptothecin treatment are faithfully reiterated in yeast, the cellular processes involved in converting the drug-stabilized complexes into lethal lesions can be experimentally addressed in this genetically tractable system. The subsequent identification of these high copy suppressor (HCS) genes and their cellular functions, both in yeast and in mammalian cells, will elucidate athe cellular processes required for camptothecin-induced DNA damage and apoptosis. The ability of these genes to suppress related mechanisms of cell death will also be examined in yeast and mammalian cells expressing lethal DNA topoisomerase 1 mutants that mimic the cytotoxic action of camptothecin. These studies will further our understanding of the mechanism of drug-induced lethality, and will also lead to greater understanding of how normal cellular functions can be perturbed to a cause cell death. As several camptothecin analogs have been entered into clinical trials for the treatment of ovarian, breast, colon and non small cell lung cancers, the characterization of HCS gene functions will have much broader applications in the design and development of new therapeutics.