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• W2045810049 abstract "Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202–325) and a C-terminal death domain (DD) (amino acids 326–413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80–205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80–205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1–111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway. Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202–325) and a C-terminal death domain (DD) (amino acids 326–413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80–205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80–205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1–111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway. tumor necrosis factor cytokine response modifier A butylated hydroxyanisole death domain death effector domain dihydrorhodamine 123 Fas-associated death domain p55 human tumor necrosis factor receptor propidium iodide receptor interacting protein reactive oxygen intermediates p55 tumor necrosis factor receptor tumor necrosis factor receptor-associated death domain benzyloxycarbonyl-Val-Ala-Asp- (OMe)-fluoromethylketone fluorescence-activated cell sorter Tumor necrosis factor (TNF)1 can induce cell death by necrosis or apoptosis, depending on the cell line (1Grooten J. Goossens V. Vanhaesebroeck B. Fiers W. Cytokine. 1993; 5: 546-555Crossref PubMed Scopus (88) Google Scholar, 2Laster S.M. Wood J.G. Gooding L.R. J. Immunol. 1988; 141: 2629-2635PubMed Google Scholar, 3Fiers W. Beyaert R. Boone E. Cornelis S. Declercq W. Decoster E. Denecker G. Depuydt B. De Valck D. De Wilde G. Goossens V. Grooten J. Haegeman G. Heyninck K. Penning L. Plaisance S. Vancompernolle K. Van Criekinge W. Vandenabeele P. Vanden Berghe W. Van de Craen M. Vandevoorde V. Vercammen D. J. Inflamm. 1996; 47: 67-75Google Scholar, 4Fiers W. Beyaert R. Declercq W. Vandenabeele P. Oncogene. 1999; 18: 7719-7730Crossref PubMed Scopus (760) Google Scholar) and/or the intracellular ATP concentration (5Leist M. Single B. Castoldi A.F. Kuhnle S. Nicotera P. J. Exp. Med. 1997; 185: 1481-1486Crossref PubMed Scopus (1651) Google Scholar). Apoptosis is morphologically characterized by membrane blebbing, shrinking of the cell and its organelles, and internucleosomal degradation of DNA (6Kroemer G. Petit P. Zamzami N. Vayssiere J.L. Mignotte B. FASEB J. 1995; 9: 1277-1287Crossref PubMed Scopus (967) Google Scholar). Finally, the cell disintegrates and apoptotic bodies are cleared by phagocytosis, in most cases without any detrimental effects on the surrounding tissue (7Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12927) Google Scholar, 8Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6728) Google Scholar). In contrast, cell death by necrosis is often accompanied by inflammation due to massive release of the cytoplasmic cell content. Necrosis is characterized by swelling of the cell and its organelles and an immediate loss of the plasma membrane integrity (1Grooten J. Goossens V. Vanhaesebroeck B. Fiers W. Cytokine. 1993; 5: 546-555Crossref PubMed Scopus (88) Google Scholar). A key step in the pathway to apoptosis is activation of procaspases. Activation of these cysteinyl aspartate-specific proteases is initiated by formation of a death-inducing signaling complex (DISC) after oligomerization of the p55 TNF receptor (TNF-R55) or the Fas receptor by the respective ligands (9Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (798) Google Scholar, 10Kischkel 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). Both death receptors contain a homologous C-terminal cytoplasmic death domain (DD) involved in apoptosis (11Tartaglia L.A. Ayres M. Wong G.H.W. Goeddel D.V. Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1169) Google Scholar, 12Itoh N. Yonehara S. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-243Abstract Full Text PDF PubMed Scopus (2678) Google Scholar). After binding of TNF to TNF-R55, the clustered DDs recruit the TNF-R55-associated DD-containing protein TRADD (13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar, 14Jones S.J. Ledgerwood E.C. Prins J.B. Galbraith J. Johnson D.R. Pober J.S. Bradley J.R. J. Immunol. 1999; 162: 1042-1048PubMed Google Scholar, 15Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Crossref PubMed Scopus (345) Google Scholar). TRADD in turn recruits Fas-associated DD protein (FADD) by DD-DD interaction (13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar). In contrast, the DD of Fas does not require the TRADD adaptor protein but serves as a direct docking surface for FADD (16Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 17Chinnaiyan A.M. O'Rourke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2165) Google Scholar, 18Varfolomeev E.E. Boldin M.P. Goncharov T.M. Wallach D. J. Exp. Med. 1996; 183: 1271-1275Crossref PubMed Scopus (108) Google Scholar). Besides its C-terminal DD, FADD contains a death effector domain (DED) implicated in the recruitment of procaspase-8 (19Boldin M.P. Goncharov T.M. Goltsev Y.V. Wallach D. Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2113) Google Scholar, 20Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2743) Google Scholar). Oligomerization of procaspase-8 leads to proximity-induced autocatalytic activation followed by direct or indirect downstream activation of executionary caspases, which cleave substrates involved in apoptotic morphology (21Muzio M. Stockwell B.R. Stennicke H.R. Salvesen G.S. Dixit V.M. J. Biol. Chem. 1998; 273: 2926-2930Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar, 22Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar). The initial intracellular molecular events responsible for necrosis are less well understood. Leist and co-workers (5Leist M. Single B. Castoldi A.F. Kuhnle S. Nicotera P. J. Exp. Med. 1997; 185: 1481-1486Crossref PubMed Scopus (1651) Google Scholar) proposed a model in which low cellular ATP concentrations give rise to a necrotic cell death process, whereas in the presence of high ATP concentrations the apoptotic, caspase-dependent pathway becomes apparent. On the other hand, it was shown that mitochondria are crucial in the necrotic process (reviewed in Ref. 4Fiers W. Beyaert R. Declercq W. Vandenabeele P. Oncogene. 1999; 18: 7719-7730Crossref PubMed Scopus (760) Google Scholar). Depletion of mitochondria protects L929sA cells from necrotic cell death (23Schulze-Osthoff K. Beyaert R. Vandevoorde V. Haegeman G. Fiers W. EMBO J. 1993; 12: 3095-3104Crossref PubMed Scopus (549) Google Scholar), and TNF induces the production of mitochondrial reactive oxygen intermediates (ROI) (24Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (558) Google Scholar). This oxidative phosphorylation-linked ROI production is required for TNF-induced necrotic cell death, since addition of butylated hydroxyanisole (BHA), an oxygen radical scavenger and inhibitor of oxidative phosphorylation (25Fones E. Amigo H. Gallegos K. Guerrero A. Ferreira J. Biochem. Pharmacol. 1989; 38: 3443-3451Crossref PubMed Scopus (25) Google Scholar), blocks TNF cytotoxicity (24Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (558) Google Scholar). Recently, we demonstrated that inhibition of caspase activity by the caspase inhibitors zVAD-fmk or CrmA resulted in enhanced ROI formation and considerably increased the sensitivity to TNF-induced necrosis (26Vercammen D. Beyaert R. Denecker G. Goossens V. Van Loo G. Declercq W. Grooten J. Fiers W. Vandenabeele P. J. Exp. Med. 1998; 187: 1477-1485Crossref PubMed Scopus (751) Google Scholar), suggesting a modulator role for caspases in the oxidative metabolism. In this paper we demonstrate that the DD of TNF-R55 as such is sufficient for mediating necrotic signaling pathways of TNF. We show that the membrane-proximal region, upstream of the DD, is required neither for necrosis nor for formation of mitochondrial ROI. Furthermore, the strong sensitization of TNF-induced necrosis in the presence of caspase inhibitors is also confined to the DD. In contrast to apoptotic systems, overexpression of FADD-(80–205) lacking the DED is cytotoxic for L929sA cells in a CrmA-insensitive way, while overexpression of a FADD-(1–111) mutant containing the DED is cytotoxic in a CrmA-inhibitory way. This indicates that the death domain of FADD might be implicated in TNF-R55-mediated necrosis. The mouse fibrosarcoma cells L929sA (27Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F.M. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar), the mouse fibrosarcoma cells 24T2.5 (Hans Schreiber, Chicago), and the human HeLa H21 cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 5% newborn calf serum and 5% fetal calf serum, penicillin (100 units/ml), streptomycin (0.1 mg/ml), andl-glutamine (0.03%). Stable transfected clones of these cells were generated as described previously (28Boone E. Vandevoorde V. De Wilde G. Haegeman G. FEBS Lett. 1998; 441: 275-280Crossref PubMed Scopus (42) Google Scholar) and maintained under selection by adding 400 μg/ml G418 (Life Technologies, Inc., Paisley, United Kingdom) to the medium. Recombinant murine TNF was produced in our laboratory and was purified to at least 99% homogeneity. The specific activity amounted to 2.2 × 108 IU/mg, as determined in a standardized cytotoxicity assay on L929sA cells. htr1 and htr9 are agonistic mouse monoclonal antibodies directed against the extracellular domain of the p55 human tumor necrosis factor receptor (hTNF-R55) and was generously provided by Dr. M. Brockhaus (Hoffmann-La Roche, Basel, Switzerland) (27Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F.M. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar). The agonistic anti-human Fas antibody, clone 2R2, was purchased from Cell Diagnostica (Münster, Germany). BHA (Sigma) was dissolved in ethanol and used at 100 μm. zVAD-fmk (Enzyme Systems Products, Dublin, CA) was dissolved in ethanol and used at 25 μm. Propidium iodide (PI; Sigma) was prepared in phosphate-buffered saline (3 mm) and was used at 30 μm. Dihydrorhodamine 123 (DHR123; Molecular Probes, Eugene, OR) was dissolved at 5 mm in dimethyl sulfoxide and was used at 1 μm. Constitutive expression of hTNF-R55 and various mutants thereof were obtained by cloning the cDNA after the early SV40 promoter in pSV25S as described previously (29Vandenabeele P. Declercq W. Vanhaesebroeck B. Grooten J. Fiers W. J. Immunol. 1995; 154: 2904-2913PubMed Google Scholar). pSV2neo, containing the neomycin resistance gene, was used as a selection vector. Mutants were generated by standard cloning procedures and subsequently verified by sequence analysis. For transient transfection assays, receptor variants were cloned into pCDM8 (Invitrogen, Carlsbad, CA). The expression vectors for CrmA (cDNA was a gift from D. Pickup, Durham, NC) and human Fas (the cDNA was a gift from S. Nagata, Department of Genetics, Osaka University Medical School, Suita, Japan) have been described previously (30Vercammen D. Brouckaert G. Denecker G. Van de Craen M. Declercq W. Fiers W. Vandenabeele P. J. Exp. Med. 1998; 188: 919-930Crossref PubMed Scopus (473) Google Scholar). Mouse RIP and procaspase-8 (31Van de Craen M. Vandenabeele P. Declercq W. Vandenbrande I. Van Loo G. Molemans F. Schotte P. Van Criekinge W. Beyaert R. Fiers W. FEBS Lett. 1997; 403: 61-69Crossref PubMed Scopus (186) Google Scholar) were cloned via reverse transcription-polymerase chain reaction and introduced into the mammalian expression vector pCDNA1 and pCDNA3 (Invitrogen, Carlsbad, CA), respectively. The human FADD and TRADD genes were also picked up by reverse transcription-polymerase chain reaction and cloned into pCDNA1. FADD-(80–205), encoding a DED-deficient FADD molecule and reported as a dominant negative molecule in many apoptotic assays (32Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar), FADD-(1–111), containing the entire DED but lacking the DD, were made by standard cloning procedures. All sequences were verified by sequence analysis. Cells were cultured in uncoated 24-well suspension plates. At day 1, cells were seeded at 5 × 105/well and incubated at 37 °C in a humidified air incubator. 1 × 106 cells were incubated on ice for 1 h with 200 μl of primary anti-hTNF-R55 antibody solution (htr9 at 1 ng/μl). Fluorescein isothiocyanate-conjugated goat anti-mouse Ig (Harlan Sera-Lab, Crawley Down, UK) was used as secondary antibody. Fluorescein isothiocyanate fluorescence intensity (measured at 525 nm) was analyzed on a FACScalibur flow fluorocytometer (Becton Dickinson, Sunnyvale, CA), equipped with a 488 nm argon ion laser. Cells were seeded on day 1 at 2 × 104/well in 96-well plates. The next day, cells were treated with inhibitors, cytokines, and/or antibodies, as mentioned. Generally, cells were pretreated for 2 h with inhibitor, followed by 18–24-h treatment with TNF or htr1 agonistic antibody. Next, medium was removed by flicking the microtiter plate to discard detached dead cells. Crystal violet staining on the remaining adherent cells was used to monitor the extent of cell viability. The percentage of cell survival was calculated as follows: (A 595treated cells − A 595 blank well)/(A 595 untreated cells −A 595 blank well) × 100. In case of combined addition of TNF or htr1 with zVAD-fmk, the percentage of cell survival was compared with the condition with caspase inhibitor alone. To obtain L929sA cells in suspension, cells were cultured in bacterial-grade Petri dishes or uncoated 24-well plates. At day 1, cells were seeded at 5 × 105/ml and incubated overnight at 37 °C in a humidified air incubator. DHR123 was added together with TNF, and samples were taken at different time points. Simultaneously, PI fluorescence (excitation at 488 nm and detection at 610 nm) was measured to exclude interference by dead cells. Rhodamine 123 fluorescence, as a result from DHR123 oxidation, was excited at 488 nm and was detected at 525 nm on 3000 PI-negative cells. Changes in rhodamine 123 fluorescence are shown by subtracting the basal mean fluorescence of untreated cells from the fluorescence of treated cells at a given time point (24Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (558) Google Scholar). L929sA, 24T2.5, or HeLa H21 cells were seeded 24 h before transfection at 40,000 cells/24-well plate. Transient transfection was done using the LipofectAMINE PLUS transfection system (Life Technologies, Inc.). The cytotoxicity in transient transfection assays with TNF-R55 and FADD constructs was evaluated by cotransfecting the pUT651 reporter gene construct, containing the β-galactosidase gene fused to a nuclear localization signal under control of the cytomegalovirus promoter (Cayla, Toulouse, France). The total amount of DNA per 24-well plate was 400 ng. Immediately after removal of the transfection mix, cells were either left untreated or treated with 100 ng/ml htr1 for 24 h, after which cells were lysed to measure β-galactosidase activity by chemiluminescence using the Galacto Light kit according to manufacturer's instructions (Tropix, Bedford, MA). The cells dying from overexpression of cell death-inducing molecules detach from the bottom surface of the well and are removed during the washing steps prior to the lysis of the remaining cells. This results in a reduced β-galactosidase activity in case of cell death. Percentage cell survival was calculated as follows: [(light units sample) − (light units of blank)]/[(light units only pUT651 transfected cells) − (light units of blank)] × 100%. Blank is the amount of light units obtained in cells transfected with control vectors not expressing β-galactosidase, whereas 100% cell survival corresponded to the amount of light units in pUT651 transfected cells only. Transfection was carried out in triplicate for each condition. L929sA cells were stably transfected with cDNAs encoding different hTNF-R55 variants (Fig.1 A) and a pSV2neo selection plasmid. After selection, several clones were screened for plasma membrane expression of hTNF-R55 and the different mutants. FACS analysis revealed constitutive cell membrane expression of full-length hTNF-R55, of the deletion mutants hR55Δ203–304, hR55Δ327–426, and hR55Δ243–383, as well as of the hR55-L351A mutant mimicking the DD-inactivating lprcg mutation originally found in Fas (Fig. 1, A and B).Figure 1A, schematic representation of the different hTNF-R55 proteins. EC, extracellular domain;TM, transmembrane domain; MPR, membrane-proximal region; DD, death domain; E, E-tag;link, 18-amino acid long linker with the sequence GGS(G4S)3. The L351A mutation corresponds to the inactivating lpr mutation in the DD of Fas (48Watanabe-Fukanaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2737) Google Scholar). B, FACS analysis of membrane-expressed wild type and mutant hTNF-R55 receptors in L929sA cells.View Large Image Figure ViewerDownload (PPT) Specific triggering of the membrane-associated hTNF-R55 mutants was achieved by treatment of the cells with the agonistic antibody htr1. The L929sA transfectants expressing those two receptor variants containing an active DD, viz. hTNF-R55 and hR55Δ203–304, displayed htr1-dependent death (Fig.2 A). In contrast, oligomerization of hR55Δ327–426, hR55-L351A, or hR55Δ243–383 with htr1 revealed the inability of TNF-R55 variants lacking the death domain to trigger cell death (Fig. 2 A). Next, we excluded the possibility of having selected for TNF-resistant L929sA cell clones (27Vanhaesebroeck B. Van Bladel S. Lenaerts A. Suffys P. Beyaert R. Lucas R. Van Roy F.M. Fiers W. Cancer Res. 1991; 51: 2469-2477PubMed Google Scholar). Therefore, we treated these murine cells with human TNF, which interacts both with human and murine TNF-R55, and with agonistic anti-murine TNF-R55 polyclonal antibodies. We found that triggering of endogenous TNF-R55 was still cytotoxic (data not shown). Microscopic evaluation of treated cells revealed that both hTNF-R55 and hR55Δ203–304 induced characteristic necrotic swelling of the cell, resulting in loss of membrane integrity and finally cell lysis (Fig.2 B). Staining with PI further demonstrated the absence of nuclear condensation (Fig. 2 B). Triggering of hR55Δ327–426, hR55Δ243–383, or hR55-L351A did not result in cell death or in any morphological changes (data not shown). Hence, the DD of TNF-R55 is required and sufficient for TNF-R55-mediated necrosis. Recently, we demonstrated that overexpression of CrmA, which is a specific inhibitor of caspase-1 and caspase-8 (33Zhou Q. Snipas S. Orth K. Muzio M. Dixit V.M. Salvesen G.S. J. Biol. Chem. 1997; 272: 7797-7800Crossref PubMed Scopus (486) Google Scholar), strongly increased TNF-induced necrosis in L929sA cells, instead of blocking it. A similar observation was made when cells were pretreated with zVAD-fmk (26Vercammen D. Beyaert R. Denecker G. Goossens V. Van Loo G. Declercq W. Grooten J. Fiers W. Vandenabeele P. J. Exp. Med. 1998; 187: 1477-1485Crossref PubMed Scopus (751) Google Scholar). To elucidate the mechanism of zVAD-fmk-induced synergy, the different TNF-R55 mutants were triggered in the presence of this caspase inhibitor. As shown in Fig. 3 A, htr1-induced necrosis by clustering hTNF-R55 was 100-fold sensitized in the presence of zVAD-fmk. In contrast, the necrotic inactive mutants hR55-L351A, hR55Δ327–426, or hR55Δ243–383 remained insensitive to htr1 treatment, even in the presence of zVAD-fmk (Fig. 3, B,D, and E). However, these clones retained the ability to respond to a combined treatment of human TNF and zVAD-fmk, indicating that the endogenous zVAD-fmk-sensitive pathway(s) were still intact in these cells (data not shown). Necrotic cell death induced by hR55Δ203–304, on the other hand, was enhanced as strongly by zVAD-fmk as the full-length receptor (Fig. 3 C). This demonstrates that the synergistic action of caspase inhibitors to necrotic cell death occurs independently of the membrane-proximal region of hTNF-R55. The production of mitochondrial ROI by TNF has been shown to be crucial for necrotic cell death of L929sA cells (24Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (558) Google Scholar). Nevertheless, it is still not clear which signaling pathways originating from TNF-R55 are involved in the formation of ROI. When hTNF-R55 was triggered by htr1, the generation of ROI could be detected by the accumulation of oxidized DHR123 in PI-negative cells (Fig.4 A). Simultaneously, necrotic cell death was monitored by the uptake of PI (Fig. 4 B). After 3 h of incubation, about 50% of the cells were dead, whereas the remaining plasma membrane-intact cells produced twice as much ROI. Aggregation of hR55Δ203–304 resulted in a delayed cell death, as reported previously (34Devos K. Goossens V. Boone E. Vercammen D. Vancompernolle K. Vandenabeele P. Haegeman G. Fiers W. Grooten J. J. Biol. Chem. 1998; 273: 9673-9680Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, the extent of ROI production in PI-negative cells at 50% cell death was exactly the same as with full-length receptor. Treatment of cells expressing hR55Δ327–426 or hR55-L351A did not result in the production of ROI (data not shown). Thus the DD alone is sufficient to generate a full oxidative response. To examine whether an increase in ROI is required for necrotic cell death, cells were incubated in the presence of the hydrophobic radical scavenger and inhibitor of oxidative phosphorylation BHA (25Fones E. Amigo H. Gallegos K. Guerrero A. Ferreira J. Biochem. Pharmacol. 1989; 38: 3443-3451Crossref PubMed Scopus (25) Google Scholar). Fig.5 shows that BHA strongly delayed the formation of PI-positive cells, both in cells expressing hTNF-R55 and hR55Δ203–304. Furthermore, BHA abrogated almost completely the synergistic effect of zVAD-fmk, confirming the involvement of mitochondrial ROI production in zVAD-fmk-synergized necrotic cell death (26Vercammen D. Beyaert R. Denecker G. Goossens V. Van Loo G. Declercq W. Grooten J. Fiers W. Vandenabeele P. J. Exp. Med. 1998; 187: 1477-1485Crossref PubMed Scopus (751) Google Scholar). The TNF-R55 adapter molecules TRADD and FADD have been shown to be required for TNF-R55 induced apoptosis (13Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar). Also RIP is recruited in the TNF-R55 complex and its overexpression induces apoptotic cell death (35Stanger B.Z. Leder P. Lee T.-H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (869) Google Scholar). We examined the influence of FADD-(80–205), a dominant negative mutant for TNF-R55-induced apoptosis (32Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar), on TNF-R55, TRADD, RIP, and FADD cytotoxicity in the necrotically dying L929sA cells. Surprisingly, in L929sA cells transient expression of FADD-(80–205) alone resulted already in massive cell death (Fig.6 A). As a control, overexpression of FADD-(80–205) in 24T2.5 (Figs. 6 B and Fig. 7 B) or HeLa H21 cells (Fig. 8C) prevented TNF-R55-, TRADD-, and RIP-induced apoptosis. Overexpression of hTNF-R55 or hR55Δ203–304 induced already substantial cell death in both L929sA and 24T2.5 cells (Fig. 6, A and B). Addition of htr-1 agonistic antibody further enhanced cell death in this transfection system. Receptors lacking an active death domain (hR55-L351A and hR55Δ327–426) were incapable of activating any cell death program in both cell lines. Thus, the transient transfection cytotoxic assays with TNF-R55 mutants reflect the data obtained in stable transfected cell lines (Fig. 2 A). The strong cytotoxic effect of FADD-(80–205), that lacks the DED and is not able to recruit procaspase-8, prompted us to distinguish whether FADD is at the bifurcation of necrotic or apoptotic cell death in L929sA cells. Therefore, we tested whether cell death by transient overexpression of human TNF-R55 mutants, TRADD, FADD, and RIP was affected by cotransfection with the caspase-8 inhibitor CrmA. Clearly, overexpression of CrmA is not able to block TNF-R55-, hR55-link-326–426-, TRADD-, and RIP-induced cell death in L929sA cells (Fig. 8 A). Furthermore, also FADD-induced cell death is insensitive to CrmA-mediated inactivation of caspases. This indicates that FADD-induced cell death occurs despite the presence of CrmA. To elaborate further on the ability of FADD to induce cell death in the presence of a caspase-8 inhibitor, we tested the influence of CrmA overexpression on the cytotoxicity by FADD mutants that either lacked the DED domain or the DD domain, FADD-(80–205) and FADD-(1–111), respectively. As shown in Fig. 8 B, FADD- and FADD-(80–205)-induced cell death is not affected by the presence of CrmA. In contrast, cell death induced by FADD-(1–111) in the same cells is blocked by the presence of CrmA, indicating a role for caspase-8. As a control, the same constructs were transfected in HeLa H21 cells (Fig. 8 C). In these cells both FADD- and FADD-(1–111)-induced cell death is counteracted by cotransfecting CrmA, whereas FADD-(80–205) has no killing capacity at all, as expected (Fig. 8 C).Figure 8CrmA is unable to block TNF-R55 -, TRADD -, FADD -, or RIP-induced cell death in L929sA cells. CrmA blocks cell death induced by FADD-(1–111) but not by FADD-(80–205) in L929sA cells. L929sA cells (A, B) and HeLa H21 cells (C) were transiently transfected with 300 ng of β-galactosidase expression vector, 20 ng of expression vectors for receptors, adapter molecules, or mutants thereof and, where mentioned, 100 ng of CrmA expression plasmid. 24 h after transfection cells were lysed and assayed for β-galactosidase activity. Percentage cell survival was calculated as described under “Material and Methods.” Transfection in HeLa H21 functions as a control for apoptotic cell death.View Large" @default.
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