Topoisomerase II-directed drugs and cell death

描述(由申请人提供) 暴露在环境化学品或化疗药物中会造成DNA损伤，从而导致毒性或致癌。虽然在某些情况下至少部分了解了基本机制，但在许多其他情况下，这些机制仍然不清楚。这项提议的广泛目标是了解化学对基因组的损伤如何导致细胞死亡途径的激活。尤其令人感兴趣的是拓扑异构酶II(TopO2)毒物是如何导致线粒体(内在)凋亡途径参与的。拓扑异构酶II毒物导致双链DNA断裂，是最有效和最广泛使用的抗肿瘤药物之一。此外，关于潜在机制的更多信息将通过调查由于Bcl-2的过度表达而使细胞对这些药物产生抗药性而逃脱细胞死亡的方式获得。众所周知，线粒体凋亡途径的激活伴随着细胞色素c的释放和caspase-9的激活。尽管Apaf-1凋亡体中caspase-9的激活被认为是该途径中的第一个或顶端caspase事件，但候选人最近的发现表明，caspase-2活性发生在线粒体细胞色素c释放的上游，caspase-9和caspase-3的激活响应于Topo2毒药依托泊苷，这表明这可能不是普遍正确的。这些发现导致了一种假设，即caspase-2的激活是线粒体在DNA损伤剂作用下参与凋亡途径所必需的，而这种激活是由Bcl-2调节的。其具体目的是：1)确定caspase-2是否最初在细胞核内被激活以响应TopO2的抑制，然后转移到参与线粒体凋亡途径的胞浆中；2)确定caspase-2是否是caspase的起始者，是否可以独立于APAF-1凋亡体和/或caspase-3的激活而诱导细胞凋亡；以及3)确定Bcl-2过表达是否阻止了药物诱导的DNA损伤的出现或积累，从而调节caspase-2的激活和细胞死亡。这些研究将部分进行，使用短干扰RNA(SiRNA)下调proasp-2和APAF-1，流式细胞术检测细胞周期进程和凋亡标志物，重组活性和非活性caspase-2蛋白的表达和纯化，以及共聚焦显微镜评估caspase-2的激活和重新分布。这项研究的完成将为DNA损伤剂激活细胞死亡途径的具体分子机制提供关键的新信息。

DESCRIPTION (provided by applicant) Exposure to environmental chemicals or chemotherapeutic agents can cause DNA damage that results in toxicity or carcinogenicity. While the underlying mechanisms are in some cases at least partially understood, in many other instances they remain unclear. The broad objective of this proposal is to understand how chemical injury to the genome leads to activation of cell death pathways. Of particular interest is how topoisomerase II (topo2) poisons, which cause double-stranded DNA breaks and are among the most effective and widely prescribed antineoplastic agents, lead to the engagement of the mitochondrial (intrinsic) apoptotic pathway. Further, additional information on the underlying mechanisms will be obtained by investigating how cells made resistant to these agents due to an overexpression of Bcl-2 escape cell death. It is well known that activation of the mitochondrial apoptotic pathway is accompanied by the release of cytochrome c and activation of caspase-9. Although it is believed that activation of caspase-9 within the Apaf-1 apoptosome represents the first or apical caspase event in this pathway, the candidate's recent findings demonstrating that caspase-2 activity occurs upstream of mitochondrial cytochrome c release and caspase-9 and -3 activation in response to the topo2 poison etoposide suggest that this may not be generally true. These findings have led to the hypothesis that caspase-2 activation is required for engagement of the mitochondrial apoptotic pathway in response to DNA-damaging agents, and this activation is regulated by Bcl-2. The specific aims are: 1) to determine whether caspase-2 is initially activated within the nucleus in response to topo2 inhibition and then translocates to the cytosol where it engages the mitochondrial apoptotic pathway; 2) to determine if caspase-2 is an initiator caspase and can induce apoptosis independently of the Apaf-1 apoptosome and/or caspase-3 activation; and 3) to determine if Bcl-2 overexpression prevents the appearance or accumulation of drug-induced DNA damage and thereby regulates caspase-2 activation and cell death. These studies will be carried out, in part, using short interfering RNA (siRNA) to down-regulate procasp-2 and apaf-1, flow cytometry to measure cell cycle progression and apoptotic markers, expression and purification of recombinant active and inactive caspase-2 proteins, and confocal microscopy to evaluate caspase-2 activation and redistribution. Completion of this research will provide critical new information about the specific molecular mechanisms whereby DNA-damaging agents activate cell death pathways.