Topoisomerase II-directed drugs and cell death

描述（由申请人提供） 暴露于环境化学品或化疗剂可导致DNA损伤，从而导致毒性或致癌性。虽然在某些情况下，基本机制至少得到部分理解，但在许多其他情况下，这些机制仍不清楚。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（topo 2）毒药如何导致线粒体（内在）凋亡途径的参与，拓扑异构酶II（topo 2）毒药导致双链DNA断裂，并且是最有效和最广泛规定的凋亡剂之一。此外，通过研究细胞如何由于Bcl-2的过表达而对这些药剂产生抗性，从而逃避细胞死亡，将获得关于潜在机制的额外信息。 众所周知，线粒体凋亡途径的激活伴随着细胞色素c的释放和半胱天冬酶-9的激活。尽管人们认为Apaf-1线粒体内caspase-9的激活代表了该途径中的第一个或顶端caspase事件，但候选人最近的研究结果表明，caspase-2活性发生在线粒体细胞色素c释放的上游，caspase-9和-3激活响应于topo 2毒物依托泊苷，这表明这可能不是普遍正确的。这些发现导致了这样的假设，即响应于DNA损伤剂的线粒体凋亡途径的参与需要半胱天冬酶-2活化，并且这种活化受Bcl-2调节。具体目标是：1）确定胱天蛋白酶-2是否最初在细胞核内响应topo 2抑制而被激活，然后易位到胞质溶胶中，在胞质溶胶中它参与线粒体凋亡途径; 2）确定胱天蛋白酶-2是否是起始胱天蛋白酶，并且可以独立于Apaf-1溶酶体和/或胱天蛋白酶-3激活而诱导凋亡;和3）确定Bcl-2过表达是否阻止药物诱导的DNA损伤的出现或积累，从而调节半胱天冬酶-2活化和细胞死亡。这些研究将进行，部分，使用短干扰RNA（siRNA）下调procasp-2和apaf-1，流式细胞术测量细胞周期进程和凋亡标志物，表达和纯化的重组活性和非活性caspase-2蛋白，共聚焦显微镜，以评估caspase-2的激活和再分布。这项研究的完成将提供有关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.