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• W2018017071 abstract "The death receptor ligand TRAIL arouses much interest for clinical application. We found that TRAIL receptor could induce cytochrome c (Cyt c) release from mitochondria in cells that failed to respond to CD95. Therefore, we examined whether these two closely related death receptors use different intermediates to convey the apoptotic signal to mitochondria. Dominant negative FADD, FLIPL, or a Bid mutant lacking cleavage sites for caspase-8/10 completely inhibited Cyt crelease in response to either receptor. Depletion of Bid from TRAIL- or CD95-activated cytosols blocked their capacity to mediate Cytc release from mitochondria in vitro, whereas Bax depletion reduced it. We conclude that FADD, caspase-8/10, and caspase-cleaved Bid are required for TRAIL receptor and CD95 signaling to mitochondria, whereas Bax is a common accessory. In vitro, caspase-8 treatment of cytosol from CD95-resistant cells permitted generation of truncated Bid and its association with mitochondria. However, this cytosol impaired the ability of truncated Bid to liberate Cyt c from exogenous mitochondria. We conclude that the TRAIL receptor can bypass or neutralize the activity of cytosolic factor that blocks truncated Bid function. This may benefit the capacity of TRAIL to break apoptosis resistance in tumor cells. The death receptor ligand TRAIL arouses much interest for clinical application. We found that TRAIL receptor could induce cytochrome c (Cyt c) release from mitochondria in cells that failed to respond to CD95. Therefore, we examined whether these two closely related death receptors use different intermediates to convey the apoptotic signal to mitochondria. Dominant negative FADD, FLIPL, or a Bid mutant lacking cleavage sites for caspase-8/10 completely inhibited Cyt crelease in response to either receptor. Depletion of Bid from TRAIL- or CD95-activated cytosols blocked their capacity to mediate Cytc release from mitochondria in vitro, whereas Bax depletion reduced it. We conclude that FADD, caspase-8/10, and caspase-cleaved Bid are required for TRAIL receptor and CD95 signaling to mitochondria, whereas Bax is a common accessory. In vitro, caspase-8 treatment of cytosol from CD95-resistant cells permitted generation of truncated Bid and its association with mitochondria. However, this cytosol impaired the ability of truncated Bid to liberate Cyt c from exogenous mitochondria. We conclude that the TRAIL receptor can bypass or neutralize the activity of cytosolic factor that blocks truncated Bid function. This may benefit the capacity of TRAIL to break apoptosis resistance in tumor cells. Tumor necrosis factor (TNF) 1The abbreviations used are: TNF, tumor necrosis factor; Cyt c , cytochrome c ; DISC, death-inducing signaling complex; DR, death receptor; mAb, monoclonal antibody; FLIP, FLICE-inhibitory protein; cFLIPL, cellular FLIPL; tBid, truncated Bid; dnFADD, dominant negative FADD; PIPES, 1,4-piperazinediethanesulfonic acid; MIB, mitochondrion incubation buffer. receptor family members share homology in the extracellular domain, which interacts with homotrimeric, TNF-related ligands (1Locksley R.M. Killeen N. Lenardo M. Cell. 2001; 104: 487-501Google Scholar). Within this family, the death receptors (DR) share the capacity to induce apoptosis, which impinges on a cytoplasmic death domain (2Ashkenazi A. Dixit V.M. Opin. Cell Biol. 1999; 11: 255-260Google Scholar). Presently six human DR are known, including TNF receptor-1, CD95 (APO-1/Fas), and two receptors for TNF-related apoptosis-inducing ligand, also known as TRAIL (3Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.-P. Nicholl J.K. Sutherland G.R. Davis Smith T. Rauch C. Smith C.A. Goodwin R.G. Immunity. 1995; 3: 673-682Google Scholar). The mouse genome encodes only one TRAIL receptor, suggesting closely related functions for the two human receptors (4Wu G.S. Burns T.F. Zhan Y. Alnemri E.S. El-Deiry W.S. Cancer Res. 1999; 59: 2770-2775Google Scholar). All DR ligands, with the exception of TNFα, are transmembrane molecules (1Locksley R.M. Killeen N. Lenardo M. Cell. 2001; 104: 487-501Google Scholar). Various soluble recombinant ligand forms retain biological activity and have been explored for clinical use. Toxicity of TNFα and CD95 ligand (CD95L) precludes their use for systemic therapy. TRAIL, however, is very promising. In vitro, it killed the majority of malignantly transformed cell lines tested but not normal cells. Both in mouse and monkey preclinical models, TRAIL was not toxic to normal tissues (5Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A., Le, T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Google Scholar, 6Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumen I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Invest. 1999; 104: 155-162Google Scholar). DR are interesting candidates for anti-cancer therapy, because they can bypass certain forms of apoptosis resistance that frequently occur in tumor cells. DR-induced apoptosis is not affected by loss of functional alleles of the p53 transcription factor, which is the most common mutation in human cancer (7El-Deiry W.S. Cell Death Differ. 2001; 8: 1066-1075Google Scholar). Also, in a number of tumor cell lines, CD95 and TRAIL receptors could bypass the inhibitory action of Bcl-2 proto-oncogene products (8Scaffidi C. Fulda S. Srinivasan A. Friesen C., Li, F. Tomaselli K.J. Debatin K.M. Peter M. EMBO J. 1998; 17: 1675-1687Google Scholar, 9Walczak H. Bouchon A. Stahl H. Krammer P.H. Cancer Res. 2000; 60: 3051-3057Google Scholar, 10Knight M.J. Riffkin C.D. Muscat A.M. Ashley D.M. Hawkins C.J. Oncogene. 2001; 20: 5789-5798Google Scholar). Moreover, DR are promising candidates for combination therapy with radiation or chemotherapeutic drugs, in the case of tumors with wild-type p53; TRAIL receptor-2, also called DR5, is a bona fide p53 target gene (11Sheikh M.S. Burns T.F. Huang Y., Wu, G.S. Amundson S. Brooks K.S. Fornace A.J. El-Deiry W.S. Cancer Res. 1998; 58: 1593-1598Google Scholar, 12Takimoto R. El-Deiry W.S. Oncogene. 2000; 19: 1735-1743Google Scholar). Accordingly, TRAIL proved synergistic with radiation and chemotherapeutic drugs in cell death induction (13Chinnaiyan A.M. Prasad U. Shankar S. Hamstra D.A. Shanaiah M. Chenevert T.L. Ross B.D. Rehemtulla A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1754-1759Google Scholar, 14Lacour S. Hamman A. Wotawa A. Corcos L. Solary E. Dimanche-Boitrel M.T. Cancer Res. 2001; 61: 1645-1651Google Scholar). The majority of apoptotic stimuli act via the intrinsic mitochondrial pathway. In this pathway, proapoptotic Bcl-2 family members induce permeability of the mitochondrial outer membrane, permitting release of cytochrome c (Cyt c). Binding of cytosolic Cytc and dATP to the scaffold protein Apaf-1 allows recruitment and activation of the inducer caspase-9, followed by effector caspase activation and execution of the apoptotic program. In addition, release of Smac/Diablo alleviates blockade of effector caspases by the inhibitor of apoptosis proteins (15Hengartner M.O. Nature. 2000; 407: 770-776Google Scholar). Inhibitory Bcl-2 family members prevent mitochondrial permeability (15Hengartner M.O. Nature. 2000; 407: 770-776Google Scholar, 16Wang X. Genes Dev. 2001; 15: 2922-2933Google Scholar) and consistently inhibit apoptosis induced by DNA damaging (anti-cancer) regimens (17Strasser A. Harris A.W. Jacks T. Cory S. Cell. 1994; 79: 329-339Google Scholar). DR also signal to mitochondria and induce Cyt c release. However, in various cell types, DR can bypass a blockade at the mitochondria imposed by inhibitory Bcl-2 family members. This is explained by their capacity to recruit and activate caspase-8 or -10, which can directly cleave effector caspases (8Scaffidi C. Fulda S. Srinivasan A. Friesen C., Li, F. Tomaselli K.J. Debatin K.M. Peter M. EMBO J. 1998; 17: 1675-1687Google Scholar). The CD95 signaling pathway has been explored by biochemical and genetic approaches. Upon ligand binding, CD95 recruits the FADD adaptor through homotypic death domain interactions. FADD in turn recruits caspase-8 through homotypic death effector domain interactions. Within the death-inducing signaling complex (DISC) thus formed at the receptor tail, caspase-8 zymogens are activated by proteolytic self-processing and released into the cytosol, where they can activate effector caspases (18Peter M.E. Krammer P.H. Curr. Opin. Immunol. 1998; 10: 545-551Google Scholar). The second death effector domain-containing caspase, caspase-10, has also been demonstrated in the endogenous CD95 DISC (19Kischkel F. Lawrence D.A. Tinel A. LeBlanc H. Virmani A. Schow P. Gazdar A. Blenis J. Arnott D. Ashkenazi A. J. Biol. Chem. 2001; 276: 46639-46646Google Scholar). Recently, FADD, caspase-8, and caspase-10 were all demonstrated in native TRAIL receptor complexes (19Kischkel F. Lawrence D.A. Tinel A. LeBlanc H. Virmani A. Schow P. Gazdar A. Blenis J. Arnott D. Ashkenazi A. J. Biol. Chem. 2001; 276: 46639-46646Google Scholar, 20Sprick M.R. Weigand M.A. Rauch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Google Scholar, 21Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Google Scholar, 22Bodmer J.-L. Holler N. Reynard S. Vinciguerra P. Schneider P. Juo P. Blenis J. Tschopp J. Nat. Cell Biol. 2000; 2: 241-243Google Scholar). That FADD is required for apoptosis induction by both TRAIL receptors followed from the use of FADD-deficient cells (23Kuang A.A. Diehl G.E. Zhang J. Winoto A. J. Biol. Chem. 2000; 275: 25065-25068Google Scholar). Involvement of caspase-8 or -10 was first suggested by the finding that TRAIL receptor-induced apoptosis is blocked by FLICE-inhibitory proteins (FLIPs), which have a death effector domain and compete with caspase-8/10 for FADD binding (24Irmler M. Thome M. Hahne M. Schneider P. Hofmann K. Steiner V. Bodmer J.-L. Schroter M. Burns K. Mattman C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Google Scholar). Moreover, in caspase-8-deficient cells, either caspase-8 or -10 could restore TRAIL receptor and CD95 signaling (19Kischkel F. Lawrence D.A. Tinel A. LeBlanc H. Virmani A. Schow P. Gazdar A. Blenis J. Arnott D. Ashkenazi A. J. Biol. Chem. 2001; 276: 46639-46646Google Scholar). These data indicate that both CD95 and TRAIL receptors induce apoptosis via FADD and either caspase-8 or -10. Caspase-8 cleaves and activates effector caspases but also processes the BH3 domain-only Bcl-2 family member Bid to generate a proapoptotic carboxyl-terminal fragment termed truncated Bid (tBid) (25Li H. Zhu H., Xu, C.J. Yuan J. Cell. 1998; 94: 491-501Google Scholar, 26Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Google Scholar). tBid translocates to mitochondria, inserts in the outer membrane, and, in cooperation with Bax or Bak, brings about Cyt c release, by a yet incompletely resolved mechanism (27Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Google Scholar, 28Wei M.C. Zong W.-X. Cheng E.H.-Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Google Scholar). It has been established that CD95 conveys the apoptotic signal to mitochondria via Bid (26Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Google Scholar). TRAIL receptors also signal to mitochondria, as evidenced by Cytc release and mitochondrial depolarization (9Walczak H. Bouchon A. Stahl H. Krammer P.H. Cancer Res. 2000; 60: 3051-3057Google Scholar, 29Thomas W.D. Zhang X.D. Franco A.V. Nguyen T. Hersey P. J. Immunol. 2000; 165: 5612-5620Google Scholar, 30Rokhlin O.W. Guseva N. Tagiyev A. Knudson C.M. Cohen M.B. Oncogene. 2001; 20: 2836-2843Google Scholar). However, the exact sequence of events in the TRAIL receptor pathway has not been established. Bid processing has been observed, but it is not clear whether it is instrumental in TRAIL-induced Cyt crelease (30Rokhlin O.W. Guseva N. Tagiyev A. Knudson C.M. Cohen M.B. Oncogene. 2001; 20: 2836-2843Google Scholar, 31MacFarlane M. Merrison W. Dinsdale Cohen G.M. J. Cell Biol. 2000; 148: 1239-11254Google Scholar, 32Yamada H. Tada-Oikawa S. Uchida A. Kawanishi S. Biochem. Biophys. Res. Commun. 1999; 265: 130-133Google Scholar). TRAIL receptor signaling pathways are far from elucidated. TRAIL receptors have been reported to recruit the TRADD adaptor, either downstream or independent of FADD, as well as the RIP kinase (33Chaudhary P.M. Eby M. Jasmin A. Bookwalter A. Murray J. Hood L. Immunity. 1997; 7: 821-830Google Scholar, 34Schneider P. Thome M. Burns K. Bodmer J.-L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Google Scholar). Both TRAIL receptors activate NF-κB (33Chaudhary P.M. Eby M. Jasmin A. Bookwalter A. Murray J. Hood L. Immunity. 1997; 7: 821-830Google Scholar, 34Schneider P. Thome M. Burns K. Bodmer J.-L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Google Scholar) and c-Jun kinase. According to certain authors, this proceeds through two distinct cascades emerging from the TRAF2 adaptor protein (35Hu W.-H. Johnson H. Shu H.-B. J. Biol. Chem. 1999; 274: 30603-30610Google Scholar), whereas others link RIP to the NF-κB response (36Lin Y. Devin A. Cook A. Keane M.M. Kelliher M. Lipkowitz S. Liu Z.G. Mol. Cell. Biol. 2000; 20: 6638-6645Google Scholar). DAP3, a GTP-binding adaptor protein, associates with the TRAIL receptor death domains in the yeast two-hybrid system. DAP3 was suggested to link FADD to the receptor tails (37Myazaki T. Reed J.C. Nat. Immunol. 2001; 2: 493-500Google Scholar). However, all of these findings are based on use of transient overexpression systems and may not reflect the endogenous situation. For instance, endogenous TRAIL receptor complexes in BJAB cells did not contain TRADD or RIP, in contrast to TNF receptor-1 complexes (21Kischkel F.C. Lawrence D.A. Chuntharapai A. Schow P. Kim K.J. Ashkenazi A. Immunity. 2000; 12: 611-620Google Scholar). Our studies were prompted by the observation that variant Jurkat T cells, which failed to display Cyt c release in response to CD95 stimulation, did so in response to TRAIL. We delineate here the pathway employed by TRAIL receptor-2 to convey the death signal to mitochondria. The pathway requires FADD, caspase-8/10, and caspase-cleaved Bid, whereas Bax facilitates the process. We find that in this respect, the TRAIL receptor is indiscernible from CD95 in its mechanism of action. However, TRAIL receptor signaling to the mitochondria is not affected by a cytosolic factor, which impedes the CD95 pathway downstream from tBid. Mouse anti-human CD95 monoclonal antibody (mAb) 7C11 was obtained from Immunotech (Marseille, France), and soluble human recombinant TRAIL and enhancer were from Alexis (Laufelfingen, Switzerland). Recombinant human caspase-8 and anti-Cyt c mAb 7H8.2C12 were obtained from PharMingen. Anti-actin mAb C4 was from Roche Molecular Biochemicals, and anti-CD3 mAb OKT3 was originally from Ortho Pharmaceuticals (Raritan, NJ). Polyclonal rabbit anti-Bid serum was raised in our laboratory against a fusion protein of glutathione S-transferase and full-length Bid. It detects full-length Bid and tBid, as established in transfection experiments and in vitro (38Werner A.B. de Vries E. Tait S.W.G. Bontjer I. Borst J. J. Biol. Chem. 2002; 277: 22781-22788Google Scholar). Rabbit anti-Bax serum SC-493 was from Santa Cruz Biotechnology, and anti-Bax mAb B8429 was from Sigma. Horseradish peroxidase-conjugated rabbit anti-mouse Ig and swine anti-rabbit Ig were obtained from DAKO A/S (Glostrup, Denmark). Protein A- and G-Sepharose beads and the ECL kit for standard immunoblotting procedures were purchased from Amersham Biosciences. SuperSignal West Dura extended duration substrate for chemiluminescence was from Pierce. The J16 clone was derived from the human T-acute lymphoblastic leukemia cell line Jurkat by limiting dilution and selected for CD95 sensitivity (39Boesen-de Cock J.G.R. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1999; 274: 14255-14261Google Scholar, 40Tepper A.D. Ruurs P. Wiedmer T. Sims P. Borst J. van Blitterswijk W.J. J. Cell Biol. 2000; 150: 155-164Google Scholar). CD95-resistant JA variant clones were derived by limiting dilution from the parental Jurkat line after 5 weeks of selection for cells resistant to anti-CD95 mAb (39Boesen-de Cock J.G.R. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1999; 274: 14255-14261Google Scholar). Cells were cultured in Iscove's modified Dulbecco's medium, supplemented with 8% FCS, 2 mm glutamine, and antibiotics. Before stimulation, cells were transferred to serum-free Yssel's medium (41Yssel H. de Vries J.E. Koken M. van Blitterswijk W.J. Spits H. J. Immunol. Methods. 1984; 72: 219-227Google Scholar) and seeded at 1 × 106/ml, 200 μl/well, in round bottom 96-well plates for apoptosis assays and at 5–10 × 106/ml in 24-well culture plates for Cyt crelease assays. Cells were stimulated with medium, anti-CD95 mAb, recombinant TRAIL plus a 10-fold excess of enhancer, or coated anti-CD3 mAb at the indicated concentrations and incubated for various time periods at 37 °C, 5% CO2. pET15b/Bid vector containing human full-length Bid cDNA was a gift from Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX). pET15b/tBid (nucleotides 181–588) was made by introducing suitable restriction sites by PCR into this Bid cDNA. Point mutations in the caspase-8 and Granzyme-B cleavage sites of Bid (pET15b/BidD59E/D75E) were introduced by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) as described (26Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Google Scholar). Human FADD and mouse cellular FLIPL(cFLIPL) cDNAs were kindly provided by Dr. J. Tschopp (Institute of Biochemistry, Epalinges, Switzerland). A cDNA encoding dominant negative FADD (dnFADD), lacking amino acids 2–77, was generated by PCR. All sequences were verified by dideoxynucleotide sequencing. dnFADD, cFLIPL, and BidD59E/D75E cDNAs were cloned into the retroviral vector LZRS-MS-IRES-eGFP, and human Bcl-2 cDNA was cloned into LZRS-MS-IRES-Zeo/pBR, both derivatives of LZRS-pBMN-lacZ (42Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Google Scholar), which was provided by Dr. G. Nolan (Stanford University School of Medicine, San Francisco, CA). To produce retrovirus, LZRS-dnFADD-IRES-eGFP, LZRS-cFLIPL-IRES-eGFP, LZRS-BidD59E/D75E-IRES-eGFP, LZRS-Bcl-2-IRES-Zeo/pBR, or empty vectors were transfected into the 293T human embryonic kidney cell-derived packaging line Phoenix Ampho (42Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Google Scholar), using Fugene-6 transfection reagent, according to instructions of the manufacturer (Roche Molecular Biochemicals). Transfected cells were selected with 1 μg/ml puromycin (Clontech).Virus-containing supernatants were harvested after 2–5 days and stored at −80 °C until further use. J16 cells were seeded on dishes coated with RetroNectin (Takara) and transduced with 1 ml of virus-containing supernatant/0.5 × 106cells. Supernatants were removed after overnight incubation, and cells were cultured in fresh medium. Transduced cells were selected for enhanced green fluorescent protein expression using a MoFlo high speed cell sorter (Cytomation, Fort Collins, CO) or selected from 48 h after transduction for growth in the presence of 200 μg/ml Zeocin (Invitrogen). To measure nuclear fragmentation (subdiploid DNA content), cells were lysed in 0.1% Triton X-100, 0.1% sodium citrate, 50 μg/ml propidium iodide (43Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Google Scholar) as described earlier (39Boesen-de Cock J.G.R. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1999; 274: 14255-14261Google Scholar, 40Tepper A.D. Ruurs P. Wiedmer T. Sims P. Borst J. van Blitterswijk W.J. J. Cell Biol. 2000; 150: 155-164Google Scholar). Fluorescence intensity of propidium iodide-stained DNA was determined on a FACScan flow cytometer (Becton Dickinson, San Jose, CA), and data were analyzed using CellQuest software. Full-length Bid and tBid were expressed from the pET15b vector in Escherichia coli. Recombinant proteins, containing an amino-terminal tag of six histidines encoded by the pET15b vector, were recovered from bacteria lysed in 50 mm Tris-HCl, pH 8.0, 20% sucrose, 10 mm β-mercaptoethanol, 0.2 mm sodium metabisulfite, with protease inhibitors. Proteins were purified by Q-Sepharose column chromatography, followed by Talon metal affinity resin, according to the instructions of the manufacturer (Clontech). Recombinant protein was eluted from this column with 100 mmimidazole in 50 mm Tris-HCl, pH 8.0, 10% sucrose, 300 mm KCl, 10 mm sodium metabisulfite and stored at −80 °C. His-tagged full-length Bid and tBid were also made byin vitro transcription/translation in the presence of [35S]methionine/cysteine. They were produced from the appropriate pET15b vectors with the TNT Quick Coupled Transcription/Translation for genes cloned downstream from the T7 RNA polymerase promoter, according to instructions supplied by the manufacturer (Promega). After incubation with the appropriate stimuli (100 ng/ml anti-CD95 mAb or 200 ng/ml TRAIL plus enhancer), cells were washed twice with ice-cold PBS. They were suspended in 100 μl of extraction buffer (50 mm PIPES-KOH, pH 7.4, 220 mmmannitol, 68 mm sucrose, 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, 1 mm dithiothreitol, and protease inhibitors) and allowed to swell on ice for 30 min (44Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Google Scholar). Cells were homogenized by passing the suspension through a 25-gauge needle (10 strokes). Homogenates were centrifuged in a Beckman Airfuge at 100,000 × g for 15 min at 4 °C, and supernatants were harvested and stored at −80 °C. Protein content in cytosols was determined by the Bio-Rad protein assay. For analysis of Cyt c release, 10 μg of cytosolic protein was loaded per lane. For immunodepletion, cytosols were incubated with anti-Bid or a combination of both anti-Bax antibodies and Protein A-Sepharose beads five times for 2 h at 4 °C. Proteins were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose sheets, which were blocked for 1 h in PBS, 0.05% Tween 20 with 5% dry milk. Blots were probed in PBS, 0.05% Tween 20, with anti-Cyt c mAb (1:1000), anti-actin mAb (1:10,000), SC-493 anti-Bax serum (1:250), or anti-Bid serum (1:1000) and as secondary antibodies horseradish peroxidase-conjugated rabbit anti-mouse Ig or swine anti-rabbit Ig (1:7500), as appropriate. Immunostained proteins were visualized by ECL. Mouse liver cells were lysed by Dounce homogenization in mitochondrion incubation buffer (MIB): 250 mm mannitol, 0.5 mm EGTA, 5 mmHepes, pH 7.2, 0.1% (w/v) bovine serum albumin, 1 μg/ml leupeptin, and 0.1 mm phenylmethylsulfonyl fluoride (45Petit P.X. O'Connor J.E. Grunwald D. Brown S.C. Eur. J. Biochem. 1990; 194: 389-397Google Scholar). Nuclei and debris were removed by centrifugation at 600 × g for 5 min at 4 °C. Mitochondria were pelleted by centrifugation at 10,000 × g for 10 min at 4 °C. The pellet was suspended in MIB and layered on a gradient, consisting of layers of 10, 18, 30, and 70% Percoll in 25 mm Hepes, pH 7.2, 225 mm mannitol, 0.5 mm EGTA, and 0.1% (w/v) bovine serum albumin. Purified mitochondria were collected at the 30%/70% interface after centrifugation in an SW-41 rotor at 13,500 × g for 35 min at 4 °C. The harvested fraction was diluted in MIB, at least 5-fold, and centrifuged at 6,300 × g for 10 min at 4 °C. After two more washes in MIB, mitochondria were suspended to a protein concentration of 5 mg/ml in Wang buffer B (20 mm Hepes, pH 7.5, 220 mm mannitol, 68 mm sucrose, 100 mmKCl, 1.5 mm MgCl2, 1 mmNa2EDTA, 1 mm Na2EGTA, 1 mm dithithreitol, and 0.1 mmphenylmethylsulfonyl fluoride) (30Rokhlin O.W. Guseva N. Tagiyev A. Knudson C.M. Cohen M.B. Oncogene. 2001; 20: 2836-2843Google Scholar). Purified mitochondria (25 μg/sample) were incubated in the presence or absence of defined protein amounts of cytosol or recombinant proteins in a final volume of 30 μl of Wang buffer B at 30 °C for 1 h or for the time periods indicated and then centrifuged for 10 min at 10,000 ×g at 4 °C. Mitochondrial pellets, corresponding to 12.5 μg of protein, and the corresponding volume of supernatant fractions were solubilized in SDS sample buffer and separated by 13% SDS-PAGE gels. Cyt c immunoblotting was performed as described above. Where appropriate, ECL signals were quantified using a Fluorchem 8000 chemoluminescence imager (Alpha Innotech Corp., San Leandro, CA). Jurkat T leukemic cells are a useful model system for apoptosis, since they are sensitive to a variety of stimuli, including CD95 triggering and DNA damage (39Boesen-de Cock J.G.R. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1999; 274: 14255-14261Google Scholar, 40Tepper A.D. Ruurs P. Wiedmer T. Sims P. Borst J. van Blitterswijk W.J. J. Cell Biol. 2000; 150: 155-164Google Scholar). They are also sensitive to TRAIL, which acts via TRAIL receptor-2 (DR5) in these cells (20Sprick M.R. Weigand M.A. Rauch C.T. Juo P. Blenis J. Krammer P.H. Walczak H. Immunity. 2000; 12: 599-609Google Scholar). We have previously characterized CD95-resistant clones selected from the Jurkat cell line. These variant cells displayed cross-resistance to DNA-damaging anti-cancer regimens (i.e.treatment with the topoisomerase inhibitor etoposide or γ-radiation). We found that resistance was due to a blockade in Cyt crelease (39Boesen-de Cock J.G.R. Tepper A.D. de Vries E. van Blitterswijk W.J. Borst J. J. Biol. Chem. 1999; 274: 14255-14261Google Scholar). Here, we show that the variant Jurkat cells are sensitive to TRAIL. In J16 wild-type cells, anti-CD95 mAb 7C11 (Fig.1 A), natural CD95L as induced by stimulation of the T cell receptor-CD3 complex (46Dhein J. Walczak H. Baumler C. Debatin K.-M. Krammer P.H. Nature. 1995; 373: 438-441Google Scholar) (Fig.1B), and recombinant TRAIL (Fig. 1 C) effectively induced apoptosis, as read out by nuclear fragmentation. A representative JA variant clone was resistant to both anti-CD95 mAb and natural CD95L (Fig. 1, A and B). In response to TRAIL, however, JA cells underwent apoptosis at wild-type levels (Fig. 1 C). Both CD95- and TRAIL-induced apoptosis were severely impeded by retrovirus-mediated overexpression of Bcl-2 in J16 cells (Fig.1 D). This indicates that, in these cells, apoptotic execution downstream from either receptor is strongly facilitated by a mitochondrial contribution. Immunoblot analysis of mitochondrion-free cytosols prepared from control and stimulated cells showed that CD95 stimulation could give rise to Cyt c release in wild-type J16 cells but failed to do so in JA cells (Fig. 1 E). TRAIL, however, could induce Cyt c release in variant JA cells as effectively as in wild-type J16 cells. In conclusion, the TRAIL receptor, but not CD95, can activate mitochondria in JA cells, which subsequently allows for apoptotic execution. The finding that TRAIL receptor, but not CD95, could induce Cyt c release in JA cells suggests that these receptors use different signaling components to convey the death signal to mitochondria. To examine this, we first assessed whether FADD and capase-8/-10, which are recruited to both receptor types, were required to induce Cyt c release. J16 cells were retrovirally transduced with vectors encoding either a dominant negative FADD mutant or cFLIPL and tested for Cytc release and apoptosis. In both dnFADD- and cFLIPL-overexpressing cells, CD95- and TRAIL-induced apoptosis were completely blocked (Fig.2, A and B), indicating the effectiveness of these proteins in preventing caspase-8/10 activation. Inhibition of caspase-8, -3, and -7 processing was demonstrated by immunoblotting (data not shown). Fig.2 C shows that Cyt c release was also completely blocked in dnFADD- and cFLIPL-overexpressing cells, whereas it proceeded effectively in J16 cells transduced with empty vector. We conclude that the TRAIL receptor, like CD95, uses FADD and caspase-8/10 to signal to mitochondria. Next, we explored whether Bid is involved in the TRAIL receptor pathway upstream from mitochondria. J16 and JA cells were retrovirally transduced with a Bid cDNA containing point mutations that delete the caspase-8 (D59E) and granzyme B (D75E) cleavage sites (26Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Google Scholar). Overexpression of this noncleavable Bid mutant inhibited both CD95- and TRAIL-induced apoptosis in J16 wild-type cells. The effect of this Bid mutant on TRAIL-induced apoptosis was comparable in J16 and JA cells (Fig. 3, A and B). Immunoblotting of mitochondrion-free cytosols, derived from CD95- and TRAIL-stimulated J16 cells, showed that the BidD59E/D75E mutant impeded Cyt c release in both cases (Fig. 3 C). In conclusion, like CD95, the TRAIL receptor requires FADD, caspase-8/10, and caspase-cleaved Bid to convey the apoptotic signal to mitochondria. We used an in vitro assay to examine whether lack of Cyt c release in CD95-resistant JA cells was due to an impediment in the cytosol or intrinsic to mitochondria. Cytosols derived from wild-type J16 cells or variant JA cells were activated in vitro with recombinant caspase-8 and incubated with mouse liver mitochondria. Subsequently, mitochondria were assayed for the presence of Cyt c. In case mitochondria were incubated with activated cytosol from wild-type J16 cells, Cytc w" @default.
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• W2018017071 title "TRAIL Receptor and CD95 Signal to Mitochondria via FADD, Caspase-8/10, Bid, and Bax but Differentially Regulate Events Downstream from Truncated Bid" @default.
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