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.Sublethal heat exposure is known to induce an evolutionarily conserved adaptive response known as the heat shock response. A key feature of this response includes the transcriptional up-regulation of several heat shock proteins that are known to confer protection against a subsequent exposure to an otherwise lethal cellular stressor, including ␥-radiation, hyperthermia, and chemotherapeutic agents (1, 2). By comparison, an initial exposure of cells to a more severe or prolonged bout of hyperthermia is known to overcome this protective heat shock response and induce apoptosis or necrosis (3). Significantly, because hyperthermia can induce apoptosis, it is currently being tested in combination with conventional anti-cancer therapy in clinical trials for advanced malignancies (4, 5). Additionally, there is a growing interest in developing ways to more selectively target tumor cells with hyperthermia for therapeutic use (6, 7).There are two distinct apoptotic pathways: (i) the mitochondria-mediated (i.e. intrinsic) pathway and (ii) the receptor-mediated (i.e. extrinsic) pathway. The extrinsic pathway is activated upon binding of a death ligand to its cognate receptor (e.g. Fas binding to the Fas receptor), which causes the receptors to move within close proximity to one another and recruit the adaptor protein Fas-associated protein with death domain (FADD), followed by the recruitment of initiator procaspase-8 or -10 (8, 9). This protein comp...
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