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• W2000336065 abstract "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. 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. 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 γ-radiation, hyperthermia, and chemotherapeutic agents (1Parsell D.A. Taulien J. Lindquist S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1993; 339 (discussion 285–286): 279-285Crossref PubMed Scopus (161) Google Scholar, 2Mosser D.D. Morimoto R.I. Oncogene. 2004; 23: 2907-2918Crossref PubMed Scopus (443) Google Scholar). 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 (3Milleron R.S. Bratton S.B. Cell Mol. Life Sci. 2007; 64: 2329-2333Crossref PubMed Scopus (61) Google Scholar). Significantly, because hyperthermia can induce apoptosis, it is currently being tested in combination with conventional anti-cancer therapy in clinical trials for advanced malignancies (4Kouloulias V. Plataniotis G. Kouvaris J. Dardoufas C. Gennatas C. Uzunoglu N. Papavasiliou C. Vlahos L. Am. J. Clin. Oncol. 2005; 28: 91-99Crossref PubMed Scopus (39) Google Scholar, 5Wust P. Hildebrandt B. Sreenivasa G. Rau B. Gellermann J. Riess H. Felix R. Schlag P.M. Lancet Oncol. 2002; 3: 487-497Abstract Full Text Full Text PDF PubMed Scopus (1630) Google Scholar). Additionally, there is a growing interest in developing ways to more selectively target tumor cells with hyperthermia for therapeutic use (6Ito A. Honda H. Kobayashi T. Cancer Immunol. Immunother. 2006; 55: 320-328Crossref PubMed Scopus (237) Google Scholar, 7Pissuwan D. Valenzuela S.M. Cortie M.B. Trends Biotechnol. 2006; 24: 62-67Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). 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 (8Boatright K.M. Salvesen G.S. Curr. Opin. Cell Biol. 2003; 15: 725-731Crossref PubMed Scopus (1084) Google Scholar, 9Kischkel F.C. Hellbardt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1792) Google Scholar). This protein complex is termed the death-inducing signaling complex (DISC) and serves as the activating platform for initiator caspase-8 and -10 during receptor-mediated apoptosis. In so-called type I cells, there is sufficient activation of caspase-8 at the DISC to directly cleave and activate effector caspase-3, resulting in execution of apoptosis. However, in so-called type II cells, activated caspase-8 does not directly cleave and activate a sufficient amount of effector caspase-3 to execute apoptosis. In this cell type, activated caspase-8 cleaves the pro-apoptotic Bcl-2 family member Bid to truncated Bid (tBid), 2The abbreviations used are: tBidtruncated BidMOMPmitochondrial outer membrane permeabilizationApaf-1apoptotic protease activating factor-1Q-VD-OPhquinoline-Val-Asp-CH2-difluorophenoxyRAIDDreceptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domainBH3Bcl-2 homology 3ΔΨmitochondrial membrane potentialb-VAD-fmkbiotin-Val-Ala-Asp(O-methyl)-CH2FZ-VADbenzyloxycarbonyl-Val-Ala-Asp. which then engages the mitochondria-mediated pathway (10Barnhart B.C. Alappat E.C. Peter M.E. Semin. Immunol. 2003; 15: 185-193Crossref PubMed Scopus (378) Google Scholar). truncated Bid mitochondrial outer membrane permeabilization apoptotic protease activating factor-1 quinoline-Val-Asp-CH2-difluorophenoxy receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain Bcl-2 homology 3 mitochondrial membrane potential biotin-Val-Ala-Asp(O-methyl)-CH2F benzyloxycarbonyl-Val-Ala-Asp. Mitochondria-mediated apoptosis is activated following exposure to cytotoxic stressors, including DNA damage, growth factor withdrawal, and γ-radiation. During intrinsic apoptosis, caspase activation is largely regulated by the Bcl-2 family of proteins. The Bcl-2 family of proteins contains both pro- and anti-apoptotic members that function to either promote or inhibit mitochondrial outer membrane permeabilization (MOMP). MOMP leads to the release of cytochrome c from the intermembrane space of mitochondria into the cytosol where it is required for caspase activation (11Jiang X. Wang X. Annu. Rev. Biochem. 2004; 73: 87-106Crossref PubMed Scopus (1138) Google Scholar). Specifically, cytosolic cytochrome c interacts with the adaptor protein apoptotic protease activating factor-1 (Apaf-1) and dATP to form the apoptosome complex, which then recruits and activates initiator caspase-9 (11Jiang X. Wang X. Annu. Rev. Biochem. 2004; 73: 87-106Crossref PubMed Scopus (1138) Google Scholar). Activated initiator caspase-9 cleaves and activates downstream effector caspases, which then cleave various target substrates, resulting in the biochemical and morphological characteristics associated with apoptotic cell death. Although adaptive cellular responses to elevated temperatures have been studied for decades, heat-induced apoptosis has been studied to a much lesser extent, and conflicting results have emerged from these studies (3Milleron R.S. Bratton S.B. Cell Mol. Life Sci. 2007; 64: 2329-2333Crossref PubMed Scopus (61) Google Scholar, 12Bonzon C. Bouchier-Hayes L. Pagliari L.J. Green D.R. Newmeyer D.D. Mol. Biol. Cell. 2006; 17: 2150-2157Crossref PubMed Scopus (121) Google Scholar, 13Milleron R.S. Bratton S.B. J. Biol. Chem. 2006; 281: 16991-17000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 14Pagliari L.J. Kuwana T. Bonzon C. Newmeyer D.D. Tu S. Beere H.M. Green D.R. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17975-17980Crossref PubMed Scopus (157) Google Scholar, 15Cippitelli M. Fionda C. Di Bona D. Piccoli M. Frati L. Santoni A. J. Immunol. 2005; 174: 223-232Crossref PubMed Scopus (35) Google Scholar, 16Stankiewicz A.R. Livingstone A.M. Mohseni N. Mosser D.D. Cell Death Differ. 2009; 16: 638-647Crossref PubMed Scopus (52) Google Scholar, 17Tu S. McStay G.P. Boucher L.M. Mak T. Beere H.M. Green D.R. Nat. Cell Biol. 2006; 8: 72-77Crossref PubMed Scopus (174) Google Scholar). In this regard, the aim of the current study was to help determine the molecular requirements necessary for heat-induced apoptosis. Because several of the more recent studies have used Jurkat T-lymphocytes as a model system to investigate apoptosis induced by elevated temperatures, we also used a large panel of genetically modified Jurkat cells in which key steps in the intrinsic or extrinsic pathway were inhibited. In agreement with previous studies, our results indicated that heat-induced apoptosis relies heavily on the mitochondria-mediated apoptotic pathway. Significantly, although caspase-2, -8, and -9 were affinity-labeled as activated initiator caspases, subsequent experiments revealed that only the activation of caspase-9 was strictly required for heat-induced apoptosis in Jurkat cells. In addition, Bid was observed to play a role in this form of apoptosis as a regulator of MOMP, although its cleavage to tBid occurred downstream of Apaf-1, suggesting that tBid likely plays an amplification role in this process. Wild-type (clones E6.1 and A3) and caspase-8-deficient (clone I 9.2) Jurkat T-lymphocytes were cultured in RPMI 1640 complete medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT), 2% (w/v) glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 incubator. For transfected cells, 1 mg/ml Geneticin (Invitrogen) was substituted for penicillin and streptomycin. Cells were maintained in an exponential growth phase for all experiments. All cells were replated in fresh complete nonselective medium prior to apoptosis induction. Apoptosis was induced either by exposure to hyperthermia (44 °C) for 1 h followed by a 6-h incubation at 37 °C or by treatment with agonistic anti-Fas antibody (100 ng/ml) (clone CH-11, MBL International, Woburn, MA) for 6 h. The caspase inhibitor quinoline-Val-Asp-difluorophenoxymethylketone (Q-VD-OPh) (MP Biomedicals, Solon, OH) was used at a final concentration of 20 μm. Phosphatidylserine exposure on the outer leaflet of the plasma membrane was detected using the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit II (BD Pharmingen) according to the manufacturer's instructions. In brief, 106 cells were pelleted following heat or anti-Fas treatment and washed in phosphate-buffered saline (PBS). Next, cells were resuspended in 500 μl of binding buffer, and 100 μl of the suspension (2 × 105 cells) were mixed with annexin V-FITC and propidium iodide and incubated at room temperature (22 °C) for 5–10 min in the dark. Prior to flow cytometric analysis, 400 μl of binding buffer were added to the cells. For ΔΨ determination, the MitoProbe DiIC1(5) kit (Invitrogen) was used. Briefly, cells (106) were pelleted following drug treatment, washed once in PBS, and resuspended in 1 ml of warm PBS. Next, 5 μl of 10 μm DiIC1(5) were added to the cells and incubated in a humidified 5% CO2 incubator at 37 °C for 15 min. Cells were pelleted, resuspended in 500 μl of PBS, and analyzed by flow cytometry. Pelleted cells (5 × 106) were resuspended and lysed in 200 μl of ice-cold lysis buffer (10 mm Tris/HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 1 mm EDTA, 0.1% Nonidet P-40) supplemented with a mixture of protease inhibitors (Complete mini EDTA-free, Roche Applied Science). Protein concentrations were determined using the bicinchoninic acid assay (Pierce), and equal amounts were mixed with Laemmli buffer, boiled for 5 min, and subjected to 12–15% SDS-PAGE at 195 V followed by electroblotting to nitrocellulose for 1 h at 115 V. Membranes were blocked for 1 h with 5% nonfat milk in PBS at room temperature (22 °C) and subsequently probed overnight at 4 °C with primary antibody suspended in PBS containing 1% bovine serum albumin. Following overnight incubation, membranes were rinsed and incubated with a horseradish peroxidase-conjugated secondary antibody (Pierce). Following the secondary antibody incubation, membranes were rinsed, and bound antibodies were detected using enhanced chemiluminescence according to the manufacturer's instructions (GE Healthcare). The primary antibodies used were anti-β-actin (clone AC-15, Sigma), anti-caspase-2 (clone 35, BD Transduction Laboratories), anti-caspase-3 (clone 8G10, Cell Signaling), anti-caspase-8 (clone 1C12, Cell Signaling), anti-caspase-9 (Cell Signaling), anti-cytochrome c (clone 7H8.2C12, BD Pharmingen), and anti-RAIDD (StressGen). Cells (5 × 105) were pelleted and washed once with ice-cold PBS. Cells were resuspended in 25 μl of PBS, added to a microtiter plate, and combined with DEVD-aminomethylcoumarin (Peptide Institute, Osaka, Japan) dissolved in a standard reaction buffer (100 mm Hepes, pH 7.25, 10% sucrose, 10 mm dithiothreitol, 0.1% CHAPS). Cleavage of DEVD-aminomethylcoumarin was monitored by aminomethylcoumarin production in a FLx800 multi-detection microplate reader (BioTek Instruments, Winooski, Vermont) using 355 nm excitation and 460 nm emission wavelengths. Cells (2.5 × 106/ml) were incubated with 120 μm cell-permeable biotinylated form of the general caspase inhibitor VAD-fmk (b-VAD-fmk; Kamiya Biomedical Co., Seattle, Washington) for 3 h at 37 °C prior to heat (44 °C) exposure or anti-Fas treatment. Following b-VAD-fmk pretreatment, cells were diluted with RPMI 1640 complete growth medium to a density of 106/ml and either incubated with anti-Fas (100 ng/ml) for 6 h or exposed to 44 °C for 1 h followed by a 6-h incubation at 37 °C. Afterward, cells were washed with PBS and lysed with 500 μl of lysis buffer (50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1 mm DTT) supplemented with a mixture of protease inhibitors (Complete mini EDTA-free, Roche Applied Science). Lysates were incubated on ice for 30 min and subsequently centrifuged at 12,000 × g for 10 min at 4 °C. Resulting supernatants were collected and incubated with 130 μl of prewashed streptavidin-Sepharose beads (GE Healthcare) followed by gentle rocking overnight at 4 °C. Following overnight incubation, beads were washed four times with wash buffer (same as lysis buffer except that the concentration of Nonidet P-40 was 0.5%), and bound proteins were eluted with Laemmli buffer and subjected to Western blot analysis. The vector-based pSUPER RNAi system (OligoEngine, Seattle, WA) was used to suppress gene expression. The gene-specific targeting insert for RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain; RefSeq accession number NM_003805) specifies a 19-nucleotide sequence corresponding to nucleotides 957–975 (5′-GGCAGGTGTCTCATATGTA-3′) downstream of the transcription start site, which is separated by a 9-nucleotide noncomplementary spacer (TTCAAGAGA) from the reverse complement of the same 19-nucleotide sequence. The targeting sequences used to suppress APAF-1, BID, and CASP2 gene expression have been reported previously (18Franklin E.E. Robertson J.D. Biochem. J. 2007; 405: 115-122Crossref PubMed Scopus (27) Google Scholar, 19Shi M. Vivian C.J. Lee K.J. Ge C. Morotomi-Yano K. Manzl C. Bock F. Sato S. Tomomori-Sato C. Zhu R. Haug J.S. Swanson S.K. Washburn M.P. Chen D.J. Chen B.P. Villunger A. Florens L. Du C. Cell. 2009; 136: 508-520Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 20Shelton S.N. Shawgo M.E. Robertson J.D. J. Biol. Chem. 2009; 284: 11247-11255Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In each case, the sequence was ligated into the BglII and XhoI sites of the pSUPER.neo vector, which was subsequently transformed into TOP10-competent cells (Invitrogen) according to the manufacturer's instructions. Several clones were obtained, and the correct insert was verified by sequence analysis. Wild-type Jurkat T-lymphocytes (107) were transfected with 20 μg of plasmid DNA (pSUPER-neo, pSUPER-Apaf-1, pSUPER-Bid, pSUPER-Caspase-2, pSUPER-RAIDD, pSFFV-neo, pSFFV-Bcl-2, and pSFFV-Bcl-xL,) by electroporation using a Bio-Rad Gene Pulser Xcell system (0.4-cm cuvette, 300 V, and 950 microfarads). Cells were allowed to recover in RPMI 1640 complete growth medium minus antibiotics for 48 h at 37 °C in a humidified 5% CO2 incubator. Selection of transfected cells was performed in the presence of 1 mg/ml Geneticin for several weeks, at which time serial dilutions were performed to obtain single-cell clones of Apaf-1-silenced cells, Bid-silenced cells, caspase-2-silenced cells, RAIDD-silenced cells, or cells overexpressing full-length human Bcl-2 or Bcl-xL. For detection of activated Bak by flow cytometry, cells (106) were washed in PBS and fixed in 400 μl of 0.25% paraformaldehyde for 5 min, subsequently washed two times with 1% fetal bovine serum in PBS, and incubated in 50 μl of staining buffer (1% fetal bovine serum and 100 μg/ml digitonin in PBS) with a conformation-specific mouse monoclonal antibody against Bak (1:30, AM03, Calbiochem) for 30 min at room temperature (22 °C). Then, cells were washed and resuspended in 50 μl of staining buffer containing 0.25 μg of Alexa Fluor 488-labeled chicken anti-mouse for 30 min in the dark. Cells were washed again and analyzed by flow cytometry. Analysis and histogram overlays were performed using FlowJo software (Tree Star, Ashland, OR). Following heat shock, cells (106) were washed in PBS, resuspended in 50 μl of buffer (140 mm mannitol, 46 mm sucrose, 50 mm KCl, 1 mm KH2PO4, 5 mm MgCl2, 1 mm EGTA, 5 mm Tris, pH 7.4) supplemented with a mixture of protease inhibitors (Complete mini EDTA-free), and permeabilized with 3 μg of digitonin (Sigma) on ice for 10 min. Plasma membrane permeabilization was monitored by trypan blue staining, and cell suspensions were centrifuged at 12,000 × g for 10 min at 4 °C. Supernatant and pellet fractions were subjected to Western blot analysis. Previous studies have reported different mechanisms for the initiation of heat-induced apoptosis that, in some instances, are difficult to reconcile. For instance, some evidence in the literature suggests that caspase-2 is the most apical caspase activated during heat-induced apoptosis (17Tu S. McStay G.P. Boucher L.M. Mak T. Beere H.M. Green D.R. Nat. Cell Biol. 2006; 8: 72-77Crossref PubMed Scopus (174) Google Scholar, 21Bouchier-Hayes L. Oberst A. McStay G.P. Connell S. Tait S.W. Dillon C.P. Flanagan J.M. Beere H.M. Green D.R. Mol. Cell. 2009; 35: 830-840Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and functions by cleaving Bid to tBid, which, in turn, triggers Bax/Bak-dependent MOMP (12Bonzon C. Bouchier-Hayes L. Pagliari L.J. Green D.R. Newmeyer D.D. Mol. Biol. Cell. 2006; 17: 2150-2157Crossref PubMed Scopus (121) Google Scholar). However, a separate line of investigation found that caspase-2 is dispensable for heat-induced apoptosis and instead suggested that an unknown Z-VAD-inhibitable protease is the earliest protease activated in response to heat (13Milleron R.S. Bratton S.B. J. Biol. Chem. 2006; 281: 16991-17000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). According to that model, the unknown protease functions to initiate apoptosis, at least in part, by directly cleaving and activating caspase-3, suggesting that caspase-9 may not always be necessary for this form of apoptosis (13Milleron R.S. Bratton S.B. J. Biol. Chem. 2006; 281: 16991-17000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Other evidence suggests that Bax and Bak are activated directly by heat (13Milleron R.S. Bratton S.B. J. Biol. Chem. 2006; 281: 16991-17000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 14Pagliari L.J. Kuwana T. Bonzon C. Newmeyer D.D. Tu S. Beere H.M. Green D.R. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17975-17980Crossref PubMed Scopus (157) Google Scholar), an effect that can be enhanced by the ubiquitination and degradation of Mcl-1 (16Stankiewicz A.R. Livingstone A.M. Mohseni N. Mosser D.D. Cell Death Differ. 2009; 16: 638-647Crossref PubMed Scopus (52) Google Scholar). In light of the conflicting and, in some cases, contradictory nature of the reported data, we set out to better understand the molecular requirements necessary for heat-induced apoptosis. As illustrated in Fig. 1A, exposure of wild-type Jurkat cells to 1 h of hyperthermia at 44 °C followed by a 6-h incubation at 37 °C resulted in ∼32% of the cells undergoing apoptosis as determined by annexin V-FITC and propidium iodide co-staining. Heat-induced apoptosis was accompanied by the proteolytic cleavage of caspase-9, -8, -3, and -2, as well as an increase in caspase-3-like (DEVDase) activity (Fig. 1, B and C). In addition, incubation of cells with the pan-caspase inhibitor Q-VD-OPh (20 μm) for 1 h prior to heat exposure prevented cell death and caspase processing (data not shown), indicating that heat-induced apoptosis is a caspase-mediated event. Next, to determine whether the mitochondrial apoptotic pathway was involved in this form of apoptosis, we evaluated the heat sensitivity of clones of Jurkat cells that do not undergo MOMP due to the overexpression of Bcl-2 or Bcl-xL (22Shawgo M.E. Shelton S.N. Robertson J.D. J. Biol. Chem. 2008; 283: 35532-35538Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). As illustrated in Fig. 1D, Jurkat cells overexpressing either Bcl-2 or Bcl-xL were completely resistant to heat-induced apoptosis. Taken together, these data suggest that heat-induced cell death is a caspase-mediated apoptotic event that requires MOMP. Execution of apoptosis requires the activation of the caspase cascade, where active initiator caspases cleave and thereby activate downstream effector caspases that, in turn, dismantle the cell. Although proteolytic cleavage of an effector caspase is indicative of its activation, the same is not necessarily true with initiator caspases. There are currently two prevailing models for initiator caspase activation. The induced proximity model suggests that the adaptor molecules required for initiator caspase activation function to bring caspases into close proximity, leading to caspase homodimerization and subsequent activation (8Boatright K.M. Salvesen G.S. Curr. Opin. Cell Biol. 2003; 15: 725-731Crossref PubMed Scopus (1084) Google Scholar). By comparison, the induced conformation model suggests that initiator caspases undergo a conformational change upon binding to an adaptor protein that leads to their activation (23Bao Q. Shi Y. Cell Death Differ. 2007; 14: 56-65Crossref PubMed Scopus (324) Google Scholar). Neither model suggests that cleavage is an activating event, although it was recently shown that dimerization and cleavage are necessary for caspase-8 activation (24Oberst A. Pop C. Tremblay A.G. Blais V. Denault J.B. Salvesen G.S. Green D.R. J. Biol. Chem. 2010; 285: 16632-16642Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Because proteolytic cleavage of initiator caspases may not always reflect their activation and because synthetic peptide substrates are not specific for a particular caspase (25McStay G.P. Salvesen G.S. Green D.R. Cell Death Differ. 2008; 15: 322-331Crossref PubMed Scopus (258) Google Scholar), it has been technically challenging to identify apical caspases activated in response to a given insult. A recent study, however, used an innovative approach to affinity-label or ”trap“ initiator caspases as they become activated inside cells (17Tu S. McStay G.P. Boucher L.M. Mak T. Beere H.M. Green D.R. Nat. Cell Biol. 2006; 8: 72-77Crossref PubMed Scopus (174) Google Scholar). The method relies on b-VAD-fmk and immobilized streptavidin to pull down the labeled caspase(s) (26Boatright K.M. Renatus M. Scott F.L. Sperandio S. Shin H. Pedersen I.M. Ricci J.E. Edris W.A. Sutherlin D.P. Green D.R. Salvesen G.S. Mol. Cell. 2003; 11: 529-541Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). In using this approach, we initially sought to determine the extent to which b-VAD-fmk pretreatment could inhibit heat-induced apoptosis. To that end, wild-type Jurkat cells were cultured in the presence of b-VAD-fmk (120 μm) for 3 h at 37 °C prior to being exposed to hyperthermia at 44 °C for 1 h followed by a 6-h incubation at 37 °C. As illustrated in Fig. 2A, b-VAD-fmk strongly inhibited heat-induced apoptosis as determined by annexin V-FITC and propidium iodide co-staining. Subsequently, duplicate aliquots of cells were lysed and incubated in the presence of streptavidin-Sepharose beads to pull down any activated caspases. To our surprise, b-VAD-fmk affinity-labeled caspase-2, -8, and -9 as initiator caspases that are activated early in response to hyperthermia (Fig. 2B, lane 3). However, the fact that significantly more caspase-9 was pulled down as compared with caspase-2 and caspase-8 suggested to us that caspase-9 might perform a more central role as an initiator caspase in this setting. Interestingly, previous studies have reported that caspase-8 can play a role in heat-induced apoptosis through a Fas-dependent mechanism (15Cippitelli M. Fionda C. Di Bona D. Piccoli M. Frati L. Santoni A. J. Immunol. 2005; 174: 223-232Crossref PubMed Scopus (35) Google Scholar, 17Tu S. McStay G.P. Boucher L.M. Mak T. Beere H.M. Green D.R. Nat. Cell Biol. 2006; 8: 72-77Crossref PubMed Scopus (174) Google Scholar). For this reason and because caspase-8 was affinity-labeled as an activated initiator caspase in response to heat (Fig. 2B, lane 3), we first sought to determine the extent to which caspase-8 was important for heat-induced apoptosis. To do so, caspase-8-deficient Jurkat cells that have been described previously (27Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), and are completely resistant to Fas-induced apoptosis (20Shelton S.N. Shawgo M.E. Robertson J.D. J. Biol. Chem. 2009; 284: 11247-11255Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 27Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), were evaluated for their sensitivity to heat-induced apoptosis. As illustrated in Fig. 3, A and B, the results indicated that cells lacking caspase-8 underwent heat-induced apoptosis and processed caspase-9, -3, and -2 to the same extent as A3 control cells (Fig. 3, A and B), indicating that caspase-8 is not necessary in this setting. Having ruled out a significant role for caspase-8, we next investigated whether caspase-9 activation, which strictly depends on Apaf-1 (28Malladi S. Challa-Malladi M. Fearnhead H.O. Bratton S.B. EMBO J. 2009; 28: 1916-1925Crossref PubMed Scopus (100) Google Scholar, 29Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 30Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar), was necessary for heat-induced apoptosis. To do so, we used well characterized Apaf-1-deficient Jurkat cells that were shown previously to be (i) incapable of activating caspase-9 in vitro (31Shawgo M.E. Shelton S.N. Robertson J.D. J. Biol. Chem. 2009; 284: 33447-33455Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), (ii) highly resistant to mitochondria-mediated apoptosis induced by etoposide (18Franklin E.E. Robertson J.D. Biochem. J. 2007; 405: 115-122Crossref PubMed Scopus (27) Google Scholar, 22Shawgo M.E. Shelton S.N. Robertson J.D. J. Biol. Chem. 2008; 283: 35532-35538Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and (iii) partially resistant to anti-Fas-induced apoptosis due to their type II origin (31Shawgo M.E. Shelton S.N. Robertson J.D. J. Biol. Chem. 2009; 284: 33447-33455Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In stark contrast to caspase-8-deficient cells, the results showed that Apaf-1-deficient Jurkat cells were completely resistant to heat-indu" @default.
• W2000336065 created "2016-06-24" @default.
• W2000336065 creator A5027603795 @default.
• W2000336065 creator A5052912965 @default.
• W2000336065 creator A5060741300 @default.
• W2000336065 date "2010-12-01" @default.
• W2000336065 modified "2023-09-26" @default.
• W2000336065 title "Activation of Caspase-9, but Not Caspase-2 or Caspase-8, Is Essential for Heat-induced Apoptosis in Jurkat Cells" @default.
• W2000336065 cites W1547149171 @default.
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