Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells. The objectives of this study are to investigate the intracellular mechanisms by which TRAIL induces apoptosis. The death receptor Fas, upon ligand binding, trimerizes and recruits the adaptor protein FADD through the cytoplasmic death domain of Fas. FADD then binds and activates procaspase-8. It is unclear whether FADD is required for TRAIL-induced apoptosis. Here we show that the signaling complex of DR4/DR5 is assembled in response to TRAIL binding. FADD and caspase-8, but not caspase-10, are recruited to the receptor, and cells de®cient in either FADD or caspase-8 blocked TRAIL-induced apoptosis. In addition, TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Dc m ), the cleavage of BID, and the redistribution of mitochondrial cytochrome c. Treatment of Jurkat cells with cyclosporin A delayed TRAIL-induced Dc m , caspase-3 activation and apoptosis. Similarly, Overexpression of Bcl-2 or Bcl-X L delayed, but did not inhibit, TRAIL-induced Dc m and apoptosis. In contrast, XIAP, cowpox virus CrmA and baculovirus p35 inhibited TRAIL-induced apoptosis. These data suggest that death receptors (DR4 and DR5) and Fas receptors induced apoptosis through identical signaling pathway, and TRAIL-induced apoptosis via both mitochondrial-dependent and -independent pathways. Oncogene (2001) 20, 2122 ± 2133.

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