Topoisomerase II-directed drugs and cell death

描述（由申请人提供） 接触环境化学物质或化疗剂可能会导致 DNA 损伤，从而导致毒性或致癌性。虽然在某些情况下至少部分理解了潜在的机制，但在许多其他情况下它们仍然不清楚。该提案的主要目标是了解基因组的化学损伤如何导致细胞死亡途径的激活。特别令人感兴趣的是拓扑异构酶 II (topo2) 毒物，它会导致双链 DNA 断裂，是最有效和最广泛使用的抗肿瘤药物之一，如何导致线粒体（内在）细胞凋亡途径的参与。此外，通过研究细胞如何因 Bcl-2 过度表达而对这些药物产生抗药性以逃避细胞死亡，可以获得有关潜在机制的更多信息。 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。尽管人们认为 Apaf-1 凋亡体内 caspase-9 的激活代表了该途径中的第一个或顶端 caspase 事件，但该候选者最近的发现表明 caspase-2 活性发生在线粒体细胞色素 c 释放的上游，并且 caspase-9 和 -3 响应 topo2 毒物依托泊苷而激活，表明这可能不是一般正确的。这些发现引发了这样的假设：响应 DNA 损伤剂的线粒体凋亡途径需要 caspase-2 激活，并且这种激活受 Bcl-2 调节。具体目标是： 1) 确定 caspase-2 是否最初在细胞核内响应 topo2 抑制而被激活，然后转移到细胞质，参与线粒体凋亡途径； 2) 确定 caspase-2 是否是起始 caspase 并可以独立于 Apaf-1 凋亡体和/或 caspase-3 激活诱导细胞凋亡； 3) 确定 Bcl-2 过度表达是否可以防止药物诱导的 DNA 损伤的出现或积累，从而调节 caspase-2 激活和细胞死亡。这些研究的部分内容将使用短干扰RNA (siRNA) 下调procasp-2 和apaf-1，使用流式细胞术测量细胞周期进程和凋亡标记，表达和纯化重组活性和非活性caspase-2 蛋白，以及使用共聚焦显微镜评估caspase-2 激活和再分布。这项研究的完成将提供关于 DNA 损伤剂激活细胞死亡途径的特定分子机制的重要新信息。

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.