Activation of caspase-9, but not caspase-2 or caspase-8, is essential for heat-induced apoptosis in Jurkat cells.
Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.
Shelton
Shary N
SN
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Dillard
Cindy D
CD
Robertson
John D
JD
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000378:metabolism; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism; D053420:CRADD Signaling Adaptor Protein / Q000235:genetics / Q000378:metabolism; D053143:Caspase 2 / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism*; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D018869:Heat-Shock Response / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D019169:Jurkat Cells; D011480:Protease Inhibitors / Q000494:pharmacology; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; C494811:BCL2L1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D053420:CRADD Signaling Adaptor Protein; C104252:CRADD protein, human; D061945:Caspase Inhibitors; D011480:Protease Inhibitors; D051020:bcl-X Protein; C490360:CASP2 protein, human; C505530:CASP8 protein, human; D053143:Caspase 2; D053181:Caspase 8; D053453:Caspase 9; D003546:Cysteine Endopeptidases