MECHANISMS OF SUPPRESSING CAMPTOTHECIN TOXICITY

DNA螺旋的两条链的缠绕构成了许多 拓扑问题，通过催化DNA拓扑结构的变化， 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， 保存破碎的能量 磷酸二酯键。 一些重要的治疗药物， 包括有效的逆转剂喜树碱， 稳定这些共价复合物。 喜树碱特异性靶向 真核生物DNA拓扑异构酶1，其在其结构上高度保守， 一级氨基酸序列、作用机制和药物敏感性。 的 喜树碱的细胞毒性是S期依赖性的，这是由于 由DNA复制叉碰撞产生的双链DNA断裂 用药物稳定的酶-DNA加合物 然而， 关于这些细胞的加工和修复机制 潜在的致命病变，或药物所需的信号通路， 诱导细胞杀伤。 本申请提出定义喜树碱诱导的细胞凋亡的途径。 细胞致死性，通过筛选酵母和人类基因产物， 在酿酒酵母中的过表达可以保护这些细胞 药物介导的细胞死亡。 由于表型的后果， 喜树碱治疗忠实地重申在酵母，细胞 涉及将药物稳定的复合物转化为致命的 病变可以通过实验解决在这个遗传上易处理的 系统 这些高拷贝抑制基因（HCS）的后续鉴定 基因及其细胞功能，无论是在酵母和哺乳动物细胞， 将阐明喜树碱诱导的细胞凋亡所需的细胞过程。 DNA损伤和凋亡。这些基因抑制相关基因表达的能力 细胞死亡的机制也将在酵母和哺乳动物细胞中进行研究 表达致死性DNA拓扑异构酶1突变体，其模拟细胞毒性 喜树碱的作用。 这些研究将进一步加深我们对 药物致死的机制，也将导致更大的 了解正常的细胞功能是如何被干扰的原因 细胞死亡 由于几种喜树碱类似物已经进入 治疗卵巢癌、乳腺癌、结肠癌和非小细胞肺癌的临床试验 细胞肺癌，HCS基因功能的表征将有 更广泛的应用在设计和开发新的 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.