Signals for Commitment to Radiation Induces Apaptosis

描述(申请人提供)：这项研究的目标是调查与电离辐射诱导的细胞死亡有关的关键分子信号。我们的研究发现了一个多步骤的过程，这是充分激活细胞凋亡所必需的。Bcl2和caspase家族蛋白是细胞死亡的基本调节因子，并发现了其他关键分子，它们在缺乏caspase的情况下帮助触发细胞死亡。这些蛋白质相互作用调节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类不同的促凋亡基因bcl2家族基因的转录激活有关，这两类基因可能激活了完全执行凋亡过程所需的不同步骤。此外，辐射导致caspase6的激活，以及可能参与caspase非依赖性细胞死亡的其他因素。为了研究它们在细胞凋亡中的独特作用，我们将使用一种遗传学方法，通过RNA干扰来扰乱凋亡调控网络和组成基因，并使用生物化学方法来确定在创建的等基因细胞系中不同的凋亡靶点的激活机制。我们的具体目标是：1)通过cDNA阵列杂交确定候选基因，检测肿瘤细胞和已知P53状态的细胞系中基因表达的调节，并通过染色质免疫沉淀实验确定P53转录激活的机制，以确定促凋亡的Bcl-2家族基因的转录调控机制；2)通过检测它们的亚细胞定位、它们之间的相互作用以及它们与RNA干扰(RNAi)能够使其表达的其他已知或新的蛋白质在细胞中的表达，来确定多结构域和BH3-仅有BH3的促凋亡的BCL-2家族蛋白在调节线粒体事件中的作用；3)通过检测半胱氨酸和丝氨酸蛋白酶的表达和调控，确定半胱氨酸和丝氨酸蛋白酶的作用，重点是caspase 6和丝氨酸蛋白酶Omi/HatrA2，在通过RNAi或显性负调控基因表达使细胞中的凋亡成分失活的细胞中。这些实验将确定caspase依赖和独立的凋亡途径对细胞死亡的相对贡献。我们的研究将加深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.