SIGNALS FOR COMMITMENT TO RADIATION INDUCED APOPTOSIS

电离辐射（IR）和所有常用于治疗癌症的化疗药物被认为通过触发一种称为细胞凋亡的细胞死亡形式最终杀死肿瘤细胞。因此，控制细胞凋亡的基因的调节改变可能在确定肿瘤的相对放射抗性中起重要作用。 本研究的目的是研究IR诱导细胞死亡的关键分子信号。 Bcl-2/CED-9家族蛋白、ICE/CED-3家族蛋白酶（半胱天冬酶）和CED 4/Apa-f1蛋白代表细胞凋亡的基本调节剂。然而，这些蛋白质相互作用以调节哺乳动物细胞中细胞死亡的确切机制尚不清楚。我们的初步研究表明，IR诱导的细胞凋亡与多种半胱天冬酶的激活有关，这些半胱天冬酶通过蛋白水解切割细胞蛋白，包括Bcl-2。特别地，Bcl-2的异位表达阻止半胱天冬酶的激活和其自身的切割。 本提案将解决Bcl-2的双重功能：i）作为与半胱天冬酶和Apaf-1相互作用的对接蛋白，或ii）作为介导该细胞器中的凋亡变化的线粒体相关蛋白。 作为一个模型系统，我们将使用多发性骨髓瘤细胞系与差异辐射敏感性和半胱天冬酶激活动力学。具体目的1：研究半胱天冬酶3对Bcl-2裂解的机制：该目的将检查：（i）具有改变的裂解的bcl-2突变的影响，（ii）该裂解的一般性，和（iii）Bcl-2蛋白水解裂解的机制。具体目的2：确定Bcl-2和蛋白质之间的相互作用对细胞凋亡的承诺至关重要。 这些相互作用将在没有去污剂的情况下进行检查，在这种情况下，蛋白质结构被保留，使用Bcl-2体内切割。 将通过免疫定位和免疫沉淀结合免疫印迹分析来检查蛋白质-蛋白质直接相互作用。 Bcl-2的相互作用也将在工程化以诱导表达Bcl-2的细胞和其中Bcl-2已经靶向特定亚细胞位置的细胞中进行检查。 具体目标3：为了确定Bcl-2在调节细胞凋亡所必需的线粒体事件中的作用：这些实验将评估：（i）细胞色素c释放和胞质复合物活化，以及（ii）线粒体膜电位和线粒体渗透性孔转换在介导来自IR和Bcl-2的信号中的作用。我们的长期目标是确定导致细胞凋亡的半胱天冬酶激活所必需的关键细胞信号。 通过了解IR触发的信号和细胞凋亡的临界点，我们最终可能能够特异性地增强caspase激活和细胞死亡，以克服放射治疗中的放射抗性。

Ionizing radiation (IR), and all chemotherapeutic drugs commonly used in the treatment of cancer, are thought to ultimately kill tumor cells by triggering a form of cell death called apoptosis. Consequently, altered regulation of the genes that control apoptosis may play an important role in determining the relative radioresistance of tumors. The objective of this research is to investigate the critical molecular signals responsible for the commitment to IR-induced cell death. Bcl-2/CED-9 family proteins, ICE/CED-3 family proteases (caspases), and the CED4/Apa-f1 protein represent the basic regulators of apoptosis. However, the precise mechanism by which these proteins interact to regulate cell death in mammalian cells is unclear. Our preliminary studies indicate that IR-induced apoptosis is associated with activation of multiple caspases, which proteolytically cleave cellular proteins, including Bcl-2. Paradoxically, ectopic expression of Bcl-2 prevents caspase activation and its own cleavage. The present proposal will address the dual functions of Bcl-2: i) as a docking protein interacting with caspases and Apaf-1, or ii) as a mitochondrial associated protein that mediates apoptotic changes in this organelle. As a model system we will use multiple myeloma cell lines with both differential radiation sensitivity and kinetics of caspase activation. There are three specific aims of the current proposal: SPECIFIC AIM 1: To study the mechanism of Bcl-2 cleavage by caspase 3: This aim will examine: (i) the effect of bcl-2 mutations with altered cleavage, (ii) the generality of this cleavage, and (iii) the mechanism of Bcl-2 proteolytic cleavage. SPECIFIC AIM 2: To determine the interactions between Bcl-2 and proteins critical for commitment to apoptosis. These interactions will be examined in the absence of detergents, conditions in which the protein structures are preserved, using Bcl-2 cleavage in vivo. Direct protein-protein interactions will be examined by immunolocalization and immunoprecipitations combined with immunoblot analyses. The interactions of Bcl-2 will be also examined in cells engineered to express Bcl-2 inducibly and cells in which Bcl-2 has been targeted to specific subcellular locations. SPECIFIC AIM 3: To determine the role of Bcl-2 in regulating mitochondrial events necessary for commitment to apoptosis: These experiments will evaluate: (i) cytochrome c release and cytosolic complex activation, and (ii) the role of mitochondrial membrane potential and mitochondrial permeability pore transition in mediating signals from IR and Bcl-2. Our long-term goals are to define the critical cellular signals essential for activation of caspases leading to apoptosis. By understanding the IR-triggered signals and the critical point of cellular commitment to apoptosis we may ultimately be able to specifically enhance caspase activation and cell death to overcome radioresistance in radiotherapy.