Signals for Commitment to Radiation Induces Apoptosis

描述（由申请人提供）：本研究的目的是研究电离辐射诱导细胞死亡的关键分子信号。我们的研究已经确定了一个多步骤的过程，需要完全激活细胞凋亡。Bcl-2和caspase家族蛋白代表凋亡细胞死亡的基本调节因子，并确定了其他关键分子，这些分子有助于在缺乏caspase的情况下触发细胞死亡。这些蛋白相互作用调控哺乳动物细胞死亡的确切机制尚不清楚。我们最近的研究表明，辐射诱导的细胞凋亡与两种不同类型的促凋亡bcl-2家族基因的转录激活有关，这些基因可能激活完全执行细胞凋亡过程所需的不同步骤。此外，辐照导致caspase 6的激活和其他可能参与caspase非依赖性细胞死亡的因子。为了研究它们在细胞凋亡中的独特作用，我们将使用遗传方法通过RNA干扰破坏细胞凋亡调控网络和组成基因，并使用生化方法确定等基因细胞系中不同凋亡靶点的激活机制。我们的具体目标是：1)通过cDNA阵列杂交鉴定候选基因，研究促凋亡Bcl-2家族基因的转录调控机制，研究肿瘤细胞和已知p53状态的细胞系中基因表达的调控，并利用染色质免疫沉淀法确定p53的转录激活机制；2)通过检测多结构域和仅bh3促凋亡bcl -2家族蛋白的亚细胞定位、它们之间的相互作用以及与细胞中其他已知或新型蛋白的相互作用，确定多结构域和bh3促凋亡bcl -2家族蛋白在调节线粒体事件中的作用，这些蛋白的表达被RNA干扰（RNAi）所抑制；3)通过检测半胱氨酸和丝氨酸蛋白酶的表达和调控来确定它们的作用，重点关注caspase 6和丝氨酸蛋白酶Omi/HatrA2在凋亡成分通过RNAi或显性负调节因子表达失活的细胞中的作用。这些实验将确定caspase依赖性和独立凋亡途径对细胞死亡的相对贡献。我们的研究将增加我们对关键细胞信号分子激活细胞凋亡的机制的理解，并可能为通过治疗增强促凋亡调节因子来克服放射治疗中的耐药性提供新的策略。

DESCRIPTION (provided by applicant): The objective of this research is to investigate the critical molecular signals responsible for commitment to ionizing radiation-induced cell death. Our studies have identified a multiple-step process that is required to fully activate apoptosis. The Bcl-2 and caspase-family proteins represent the basic regulators of apoptotic cell death, with additional critical molecules identified, which assist in triggering cell death in the absence of caspases. The precise mechanism by which these proteins interact to regulate cell death in mammalian cells is unclear. Our recent studies indicate that radiation-induced apoptosis is associated with transcriptional activation of two distinct classes of pro-apoptotic bcl-2 family genes which may activate distinct steps required to fully execute the apoptotic process. In addition, irradiation leads to activation of caspase 6 and additional factors that may participate in caspase-independent cell death. To examine their unique roles in apoptosis, we will use a genetic approach to disrupt apoptosis regulatory networks and component genes by RNA interference, and use biochemical methods to determine the mechanism of activation of distinct apoptotic targets in the isogenic cell lines created. Our specific aims are: 1) To determine the mechanism of transcriptional regulation of proapoptotic Bcl-2 family genes by identifying candidates using cDNA array hybridization, examining regulation of gene expression in tumor cells and cell lines with known p53 status, and determining the mechanism of transcriptional activation by p53 using chromatin immunoprecipitation assays; 2) To determine the role of multi-domain and BH3-only proapoptotic Bcl-2-family proteins in regulating mitochondrial events, by examining their sub-cellular localization, interactions between them and with other known or novel proteins in cells in which their expression is ablated by RNA interference (RNAi); and 3) To determine the role of cysteine and serine proteases, by examining their expression and regulation, with a focus on caspase 6 and the serine protease Omi/HatrA2, in cells in which the apoptotic components have been inactivated through RNAi or expression of dominant-negative regulators. These experiments will determine the relative contribution of caspase dependent and independent apoptotic pathways to cell death. Our studies will increase our understanding of the mechanism by which critical cellular signaling molecules activate apoptosis and may provide novel strategies for overcoming resistance in radiotherapy by therapeutically enhancing proapoptotic regulators.