Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis.
Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.
Franklin
Emily E
EE
Department of Pharmacology, Toxicology & Therapeutics and the Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS 66160, USA.
Robertson
John D
JD
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000378:metabolism*; D053143:Caspase 2 / Q000235:genetics / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D004249:DNA Damage; D004789:Enzyme Activation; D005047:Etoposide / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism*; D008942:Mitoxantrone / Q000378:metabolism; D034622:RNA Interference
D000970:Antineoplastic Agents; D053477:Apoptotic Protease-Activating Factor 1; D005047:Etoposide; D008942:Mitoxantrone; D053143:Caspase 2; D053453:Caspase 9