Environment hazards and stress, experimental and environmental carcinogens, environmental exposure to UV and ionizing radiation, and anti-cancer therapeutics (including chemotherapeutic drugs, radiation, and adjuvant therapeutics) cause cell death through intrinsic or mitochondrial death pathway. Abnormal apoptotic response contributes to tumor development induced by environmental hazards and carcinogens. Defects in apoptosis are also intimately associated with tumor cell resistance to anti-neoplastic agents. Elucidating the molecular mechanisms of the mitochondrial death pathway will facilitate understanding of how cells respond to environmental stresses, hazards and therapeutics. The essence of apoptosis in various signaling pathways converges on the activation of caspases. Caspase-8 acts as the most upstream caspase in apoptotic signaling initiated by Fas (CD95), TNFR, and TRAIL receptors. Normally, engagement of death receptors on the plasma membrane recruits FADD and procaspase-8/10 to the death-inducing signaling complex (DISC) to activate caspase-8, which ultimately activate caspase-3 to execute apoptosis. Recent evidence, however, indicates the presence of significant levels of caspase-8 on the mitochondria. How mitochondrially-localized caspase-8 is activated and regulated at the mitochondrial levels remains unknown. Our recent published and preliminary observations demonstrate that: 1) a variety of intrinsic apoptotic stimuli activate caspase-8 in epithelial cancer cells and mitochondria appear to function as a critical platform for FADD-mediated caspase-8 activation in these apoptotic systems, and 2) FADD on the mitochondria, upon apoptotic induction, oligomerizes to higher molecular weight protein complex. Accordingly, we hypothesize that mitochondria function as crucial signaling center to initiate FADD-mediated caspase-8 activation and DISC assembly during apoptosis induced by intrinsic stimuli. The initiation of caspase-8 activation on the mitochondria may represent the earliest event or one of the earlier events in parallel with caspase-9 activation during apoptosis induction. Elucidation of the afore-mentioned novel regulatory mechanisms should not only help us better understand how caspase activation is initiated and regulated but also lay a foundation for designing novel anti-cancer therapeutics. Two Specific Aims are proposed to test the hypothesis: 1). To establish that intrinsic apoptotic stimuli induce FADD-mediated caspase-8 activation on the mitochondria; and 2). To characterize the formation and activity of DISC-like complex on the mitochondria and its regulation by antiapoptotic proteins (i.e., Bcl-xL, c-FLIP). Environmental hazards and stress, experimental and environmental carcinogens, environmental exposure to UV and ionizing radiation cause cell death or apoptosis. Elucidating the molecular mechanisms of apoptotic cell death will facilitate the understanding of how cells respond to environmental stresses and hazards, and will be highly significant in improving human health. [unreadable] [unreadable] [unreadable]