Activation of Caspase-9, but Not Caspase-2 or Caspase-8, Is Essential for Heat-induced Apoptosis in Jurkat Cells

• Published in: The Journal of biological chemistry 2010-12, Vol.285 (52), p.40525-40533
• Main Authors: Shelton, Shary N., Dillard, Cindy D., Robertson, John D.
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - drug effects; Apoptosis - physiology; Apoptotic Protease-Activating Factor 1 - genetics; Apoptotic Protease-Activating Factor 1 - metabolism; bcl-X Protein - genetics; bcl-X Protein - metabolism; BH3 Interacting Domain Death Agonist Protein - genetics; BH3 Interacting Domain Death Agonist Protein - metabolism; Caspase; Caspase 2 - genetics; Caspase 2 - metabolism; Caspase 8 - genetics; Caspase 8 - metabolism; Caspase 9 - genetics; Caspase 9 - metabolism; Caspase Inhibitors; Cell Biology; Cell Death; CRADD Signaling Adaptor Protein - genetics; CRADD Signaling Adaptor Protein - metabolism; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Cytochrome c; Death Protease; Enzyme Activation - drug effects; Enzyme Activation - physiology; Heat Shock; Heat-Shock Response - drug effects; Heat-Shock Response - physiology; Humans; Jurkat; Jurkat Cells; Mitochondrial Apoptosis; Protease Inhibitors - pharmacology; shRNA
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.167635

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-xL 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)-CH2F) 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.