Signals for Commitment to Radiation Induces Apoptosis

辐射诱导细胞凋亡的信号

• 批准号: 6897116
• 负责人: Alexandru Almasan
• 金额: $ 22.95万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6897116
• 关键词: BCL2 gene /protein; apoptosis; cell line; clinical research; confocal scanning microscopy; cysteine endopeptidases; enzyme activity; enzyme mechanism; gene expression; genetic transcription; human tissue; immunoprecipitation; ionizing radiation; mass spectrometry; protein protein interaction; radiation genetics

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.

描述（由申请人提供）：本研究的目的是研究导致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完全激活细胞凋亡所需的多步骤过程。 Bcl-2 和 caspase 家族蛋白代表细胞凋亡的基本调节因子，并鉴定出其他关键分子，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 bcl-2 家族基因的转录激活有关，这可能激活完全执行细胞凋亡过程所需的不同步骤。 此外，辐射还会激活 caspase 6 和其他可能参与 caspase 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节网络和组成基因，并使用生化方法确定所创建的同基因细胞系中不同细胞凋亡靶标的激活机制。 我们的具体目标是： 1) 通过使用 cDNA 阵列杂交鉴定候选基因，检查肿瘤细胞和已知 p53 状态的细胞系中基因表达的调控，并使用染色质免疫沉淀测定确定 p53 转录激活的机制，确定促凋亡 Bcl-2 家族基因的转录调节机制； 2) 通过检查多结构域和仅 BH3 促凋亡 Bcl-2 家族蛋白在调节线粒体事件中的作用，通过检查它们的亚细胞定位、它们之间以及它们与细胞中其他已知或新蛋白的相互作用，在细胞中它们的表达被 RNA 干扰 (RNAi) 消除； 3) 通过检查半胱氨酸和丝氨酸蛋白酶的表达和调节来确定半胱氨酸和丝氨酸蛋白酶的作用，重点关注半胱天冬酶 6 和丝氨酸蛋白酶 Omi/HatrA2，在细胞凋亡成分已通过 RNAi 或显性失活调节因子的表达失活的细胞中。 这些实验将确定半胱天冬酶依赖性和独立的细胞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机制的理解，并可能通过治疗性增强促凋亡调节因子来提供克服放疗耐药性的新策略。