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.