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Glutathione Peroxidase-1 Protects from CD95-induced Apoptosis

2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês

10.1074/jbc.m203067200

1083-351X

Valérie Gouazé‐Andersson, Nathalie Andrieu‐Abadie, Olivier Cuvillier, Sophie Malagarie‐Cazenave, Marie‐Françoise Frisach, Marc-Édouard Mirault, Thierry Levade,

Bioactive Compounds and Antitumor Agents

Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis. Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis. CD95 ligand l-buthionine-(S,R)-sulfoximine N-acetylcysteine glutathione peroxidase glutathione reactive oxygen species 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 4′,6-diamidino-2-phenylindole benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone Ac-Asp-Glu-Val-Asp-aminomethylcoumarin benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethyl ketone phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid extracellular signal-regulated protein kinase 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) The CD95 (CD95/APO-1) receptor/CD95 ligand (CD95L)1 system is a key signal pathway involved in the regulation of apoptosis in various human cells including myeloid, T-lymphoid, fibroblast, and malignant glioma cells (1Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3980) Google Scholar, 2Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar). CD95 is 36-kDa transmembrane type I receptor, which belongs to the nerve growth factor/tumor necrosis factor receptor superfamily (3Locksley R.M. Killeen N. Lenardo M.J. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3038) Google Scholar). Upon binding of CD95L or agonistic anti-CD95 antibodies, an adapter molecule, called FADD, is instantly recruited to the so-called Death Domain of CD95. This results in the binding and activation of procaspase-8, leading to its processing to generate an active molecule capable of cleaving and activating downstream effector caspases, ultimately resulting in cell dismantling (4Krammer P.H. Nature. 2000; 407: 789-795Crossref PubMed Scopus (1407) Google Scholar). Numerous studies (5Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2104) Google Scholar, 6Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1148) Google Scholar) have implicated oxidative stress in apoptosis and most particularly in CD95-induced cell death. Indeed, various antioxidants such as thioredoxin (7Matsuda M. Masutani H. Nakamura H. Miyajima S. Yamauchi A. Yonehara S. Uchida A. Irimajiri K. Horiuchi A. Yodoi J. J. Immunol. 1991; 147: 3837-3841PubMed Google Scholar), catalase (8Kasahara Y. Iwai K. Yachie A. Ohta K. Konno A. Seki H. Miyawaki T. Taniguchi N. Blood. 1997; 89: 1748-1753Crossref PubMed Google Scholar), Cu,Zn-dependent superoxide dismutase (9Jayanthi S. Ordonez S. McCoy M.T. Cadet J.L. Brain Res. Mol. Brain Res. 1999; 72: 158-165Crossref PubMed Scopus (37) Google Scholar),N-acetylcysteine (NAC) (10Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 11Gulbins E. Brenner B. Schlottmann K. Welsch J. Heinle H. Koppenhoefer U. Linderkamp O. Coggeshall K.M. Lang F. Immunology. 1996; 89: 205-212Crossref PubMed Scopus (72) Google Scholar, 12Wedi B. Straede J. Wieland B. Kapp A. Blood. 1999; 94: 2365-2373Crossref PubMed Google Scholar), GSH (13Um H.D. Orenstein J.M. Wahl S.M. J. Immunol. 1996; 156: 3469-3477PubMed Google Scholar, 14Watson R.W. Rotstein O.D. Jimenez M. Parodo J. Marshall J.C. Blood. 1997; 89: 4175-4181Crossref PubMed Google Scholar, 15Laouar A. Glesne D. Huberman E. J. Biol. Chem. 1999; 274: 23526-23534Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), andN-t-butyl-phenylnitrone (11Gulbins E. Brenner B. Schlottmann K. Welsch J. Heinle H. Koppenhoefer U. Linderkamp O. Coggeshall K.M. Lang F. Immunology. 1996; 89: 205-212Crossref PubMed Scopus (72) Google Scholar) can prevent CD95-mediated apoptosis in several cell types. Reports showing that CD95 ligation results in ROS generation in human B-lymphoma (16Suzuki Y. Ono Y. Hirabayashi Y. FEBS Lett. 1998; 425: 209-212Crossref PubMed Scopus (135) Google Scholar), T-leukemia (10Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 11Gulbins E. Brenner B. Schlottmann K. Welsch J. Heinle H. Koppenhoefer U. Linderkamp O. Coggeshall K.M. Lang F. Immunology. 1996; 89: 205-212Crossref PubMed Scopus (72) Google Scholar,17Kohno T. Yamada Y. Hata T. Mori H. Yamamura M. Tomonaga M. Urata Y. Goto S. Kondo T. J. Immunol. 1996; 156: 4722-4728PubMed Google Scholar), or glioma (9Jayanthi S. Ordonez S. McCoy M.T. Cadet J.L. Brain Res. Mol. Brain Res. 1999; 72: 158-165Crossref PubMed Scopus (37) Google Scholar) cells, and monocytes (13Um H.D. Orenstein J.M. Wahl S.M. J. Immunol. 1996; 156: 3469-3477PubMed Google Scholar) provide additional evidence that ROS are key molecules in CD95-induced cell death. Conversely, oxidative stress has also been shown to promote CD95 or CD95L expression in different cell types. Indeed, addition of pro-oxidants such as H2O2 (18Bauer M.K. Vogt M. Los M. Siegel J. Wesselborg S. Schulze-Osthoff K. J. Biol. Chem. 1998; 273: 8048-8055Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 19Kwon D. Choi C. Lee J. Kim K.O. Kim J.D. Kim S.J. Choi I.H. J. Neuroimmunol. 2001; 113: 1-9Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), paraquat (20Vogt M. Bauer M.K. Ferrari D. Schulze-Osthoff K. FEBS Lett. 1998; 429: 67-72Crossref PubMed Scopus (124) Google Scholar), or hypoxia and subsequent reoxygenation (20Vogt M. Bauer M.K. Ferrari D. Schulze-Osthoff K. FEBS Lett. 1998; 429: 67-72Crossref PubMed Scopus (124) Google Scholar) have been reported to be potent inducers of CD95 and/or CD95L. Further support that oxidative stress and CD95 are intimately associated in apoptosis came from the observation that intracellular GSH levels modulate CD95-induced cell death. Depletion of GSH levels in Jurkat cells through overexpression of transaldolase rendered these cells highly susceptible to anti-CD95 antibodies (10Banki K. Hutter E. Colombo E. Gonchoroff N.J. Perl A. J. Biol. Chem. 1996; 271: 32994-33001Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Reciprocally, CD95-resistant variants of T-leukemia cells exhibit higher GSH content than original cells (17Kohno T. Yamada Y. Hata T. Mori H. Yamamura M. Tomonaga M. Urata Y. Goto S. Kondo T. J. Immunol. 1996; 156: 4722-4728PubMed Google Scholar, 21Chiba T. Takahashi S. Sato N. Ishii S. Kikuchi K. Eur. J. Immunol. 1996; 26: 1164-1169Crossref PubMed Scopus (149) Google Scholar) and become sensitive to CD95 when incubated with l-buthionine-(S,R)-sulfoximine (BSO) or in GSH-free/cysteine-free medium to deplete GSH (21Chiba T. Takahashi S. Sato N. Ishii S. Kikuchi K. Eur. J. Immunol. 1996; 26: 1164-1169Crossref PubMed Scopus (149) Google Scholar). Similarly, increasing the GSH concentration in activated human peripheral T-cells resulted in total protection against CD95-induced apoptosis (22Deas O. Dumont C. Mollereau B. Metivier D. Pasquier C. Bernard-Pomier G. Hirsch F. Charpentier B. Senik A. Int. Immunol. 1997; 9: 117-125Crossref PubMed Scopus (74) Google Scholar). Moreover, CD95 ligation has been shown to decrease intracellular GSH content in Jurkat T-lymphocytes by stimulating the efflux of GSH (23van den Dobbelsteen D.J. Nobel C.S. Schlegel J. Cotgreave I.A. Orrenius S. Slater A.F. J. Biol. Chem. 1996; 271: 15420-15427Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). However, acute GSH depletion has also been reported to prevent CD95-induced apoptosis in some instances (see Ref. 24Hentze H. Schmitz I. Latta M. Krueger A. Krammer P.H. Wendel A. J. Biol. Chem. 2002; 277: 5588-5595Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar and references therein), whereas chronic depletion enhances apoptosis (25Haouzi D. Lekehal M. Tinel M. Vadrot N. Caussanel L. Letteron P. Moreau A. Feldmann G. Fau D. Pessayre D. Hepatology. 2001; 33: 1181-1188Crossref PubMed Scopus (65) Google Scholar). Thus, how GSH is connected with CD95-induced cell death signaling pathways still remains to be clarified. The relationship between oxidative stress, GSH levels, and CD95-induced apoptosis prompted us to examine the role of glutathione peroxidase (GPx), one of the major enzymes responsible for ROS detoxification in mammalian cells, in CD95 apoptotic signaling. Four forms of human GPx have been described, including the classical cytosolic/mitochondrial selenium-dependent GPx1, the gastrointestinal GPx2, the plasma enzyme GPx3, and the phospholipid hydroperoxide GPx4 (26Brigelius-Flohe R. Free Radic. Biol. Med. 1999; 27: 951-965Crossref PubMed Scopus (883) Google Scholar). Nomura and co-workers (27Nomura K. Imai H. Koumura T. Arai M. Nakagawa Y. J. Biol. Chem. 1999; 274: 29294-29302Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) have reported that the latter GPx could suppress apoptosis induced by several stresses including etoposide and UV irradiation through the inhibition of cytochrome crelease and caspase-3 activation. Regarding GPx1, we recently reported that overexpression of this GPx can abolish doxorubicin-induced sphingolipid signaling, a phenomenon accompanied by inhibition of ROS formation and partial protection against doxorubicin-induced apoptosis (28Gouazé V. Mirault M.E. Carpentier S. Salvayre R. Levade T. Andrieu-Abadie N. Mol. Pharmacol. 2001; 60: 488-496PubMed Google Scholar). To assess the yet undetermined role of GPx1 in CD95-induced cell death, we used human breast carcinoma T47D cells, which were stably transfected with a cDNA encoding human GPx1 (29Mirault M.E. Tremblay A. Beaudoin N. Tremblay M. J. Biol. Chem. 1991; 266: 20752-20760Abstract Full Text PDF PubMed Google Scholar). Generally, breast cancer cell lines are known to resist CD95-mediated apoptosis, but T47D cells may represent an exception inasmuch as they express high levels of CD95 (30Keane M.M. Ettenberg S.A. Lowrey G.A. Russell E.K. Lipkowitz S. Cancer Res. 1996; 56: 4791-4798PubMed Google Scholar). The susceptibility of GPx1-overexpressing cells to CD95, in terms of toxicity and cell signaling, was compared with that of parental cells, which are characterized by low endogenous GPx activity. Here we show that not only could overexpression of GPx1 strongly inhibit CD95-induced ROS formation, caspase activation, and apoptosis but also partially protect mice from the lethal effect of anti-CD95. These findings suggest that GPx plays a critical role in CD95 signaling by regulating effector caspase activation. N-Acetylcysteine,l-buthionine-(S,R)-sulfoximine, 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and sodium selenite were supplied from Sigma. Z-Val-Ala-dl-Asp-fluoromethyl ketone, Z-Asp-Glu-Val-Asp-chloromethyl ketone (DEVD), and Ac-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC) were from Bachem (Voisins-Le-Bretonneux, France). 6-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (DCFH-DA) was from Molecular Probes (Leiden, The Netherlands). Anti-CD95 (CH-11 antibody) was from Beckman Coulter (Marseille, France). RPMI 1640 Glutamax, hygromycin, G418, and antibiotics were from Invitrogen; fetal calf serum was from BioMedia (Boussens, France). The human breast cancer T47D cell line, a differentiated epithelial substrain of ductal carcinoma origin, was transfected with pML-Hygro or pML-Hygro-HCMV-GPx plasmids, which contains part of a cDNA clone encoding human GPx1 (29Mirault M.E. Tremblay A. Beaudoin N. Tremblay M. J. Biol. Chem. 1991; 266: 20752-20760Abstract Full Text PDF PubMed Google Scholar). Empty vector and HCMV-GPx-transfected cells are designated T47D/H3 and T47D/GPx, respectively. These cells were grown in a humidified 5% CO2 atmosphere at 37 °C in RPMI 1640 medium containing Glutamax (2 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), hygromycin B (150 μg/ml), sodium selenite (1 μm), and heat-inactivated fetal calf serum (10%). The breast carcinoma MCF-7 sublines, stably transfected to express the CD95 receptor (denoted MCF-7/CD95) or both CD95 and Bcl-xL (denoted MCF-7/CD95/Bcl-xL), were kindly provided by Dr. V. Dixit (Genentech Inc., San Francisco, CA). They were grown in RPMI medium supplemented with G418 (200 μg/ml) and hygromycin (150 μg/ml). The wild-type (A3) and caspase-8 mutant (I9–2) Jurkat cells were kindly provided by Dr. J. Blenis (Harvard Medical School, Boston), and cultured in RPMI 1640 medium (31Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). Cell viability was evaluated by using the tetrazolium-based MTT assay (32Denizot F. Lang R. J. Immunol. Methods. 1986; 89: 271-277Crossref PubMed Scopus (4357) Google Scholar). Adherent cells were detached by incubation for 10 min with EDTA (5 mm) in phosphate-buffered saline (PBS). After washing with PBS, cells were incubated for 30 min at 4 °C in the presence of phycoerythrin-labeled anti-CD95 antibody (20 μl, Beckman Instruments), washed again, and analyzed on a FACScan (BD Biosciences) cytometer. After treatment with anti-CD95, T47D cells were washed twice in PBS, lysed for 20 min at 4 °C in 0.5 ml of lysis buffer (0.5% Triton X-100 v/v, 20 mm EDTA, and 5 mm Tris-HCl, pH 8.0), and then centrifuged for 20 min at 27,000 × g in order to separate the DNA fragments from the chromatin pellet. The DNA content of pellet (resuspended in 1 ml of 1 mm EDTA in 10 mm Tris-HCl, pH 8.0 buffer) and supernatant was determined by the fluorometric DAPI procedure (33Andrieu-Abadie N. Jaffrezou J.P. Hatem S. Laurent G. Levade T. Mercadier J.J. FASEB J. 1999; 13: 1501-1510Crossref PubMed Scopus (162) Google Scholar). ROS production was assessed using DCFH-DA. This probe is an uncharged cell-permeant molecule, which is cleaved by nonspecific esterases once inside the cell, and releases carboxydichlorofluorescein that is oxidized in the presence of ROS. Exponentially growing cells were labeled with 10 μmDCFH-DA for 30 min at 37 °C before the reaction was stopped. Cells were washed three times with PBS. Cell pellets were suspended in 1 ml of distilled water and sonicated at 4 °C. The cell-associated fluorescence was recorded at excitation and emission wavelengths of 495 and 525 nm, respectively, using a Jobin-Yvon 3D fluorometer. After incubation with anti-CD95, cells were sedimented. Cell pellets were homogenized in 10 mm HEPES, pH 7.4, 42 mm KCl, 5 mm MgCl2, 0.5% CHAPS, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 2 μg/ml leupeptin. Reaction mixtures contained 100 μl of cell lysates and 100 μl of 40 μm Ac-DEVD-AMC. After 30 min of incubation at room temperature, the amount of the released fluorescent product aminomethylcoumarin was determined at 351 and 430 nm for the excitation and emission wavelengths, respectively. Analysis of caspase cleavage was assessed by Western blot using the cell lysates prepared for DEVD cleavage enzyme assay. Samples were loaded onto a 15% SDS-polyacrylamide gel, electrophoresed, and transferred to a nitrocellulose membrane. Caspase-3 and its cleaved fragments were detected by using a rabbit polyclonal antiserum (a kind gift of Dr. D. Nicholson, Merck-Frosst, Pointe-Claire, Quebec, Canada); caspase-7 was detected with a rabbit polyclonal antiserum (Oncogene, France Biochem, Meudon, France); caspase-8 was detected using a rabbit polyclonal antiserum (a kind gift of Dr. G. Cohen, Leicester, UK) (34Sun X.M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) and a goat anti-rabbit secondary antibody (Bio-Rad). Western blot was also used to analyze the expression of Bcl-2 (using a monoclonal antibody, Dako, Trappes, France), Bcl-xL, and Mcl-1 (using antibodies from Pharmingen), Bid (using a rabbit antibody kindly provided by Dr. X Wang, Dallas, TX), ERK (using a rabbit polyclonal antibody, Santa Cruz Biotechnology), phospho-ERK (using a monoclonal antibody, Cell Signaling Technology, Beverly, MA), and usurpin/FLIP (using a rabbit polyclonal antibody kindly provided by Dr. D. Nicholson). An anti-β-actin (Sigma) was used as a control for protein loading. Mitochondrial preparations were carried out as described previously (35Cuvillier O. Nava V.E. Murthy S.K. Edsall L.C. Levade T. Milstien S. Spiegel S. Cell Death Differ. 2001; 8: 162-171Crossref PubMed Scopus (119) Google Scholar). Briefly, cell samples were washed once with ice-cold PBS and resuspended with ice-cold buffer A (20 mmHEPES, pH 7.5, 0.1% bovine serum albumin, 1 mm sodium EDTA, 1 mm dithiothreitol, 0.1 mmphenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 μg/ml pepstatin A) containing 250 mmsucrose. After swelling on ice for 5 min, the cells were homogenized with 15–20 strokes of a Kontes Dounce homogenizer with the B pestle (Kontes Glass Company, Vineland, NJ), and the homogenates were centrifuged at 750 × g for 5 min at 4 °C. The supernatants were then pelleted at 10,000 × g for 15 min at 4 °C. Resulting pellets containing mitochondria were resuspended in cold buffer A. The resulting supernatants ("cytosolic" extracts) were further cleared at 20,000 ×g for 30 min at 4 °C. For Western blot analysis, equal amounts of mitochondrial and cytosolic proteins were separated on 15% SDS-PAGE and then transblotted to nitrocellulose. Monoclonal antibodies used against cytochrome c and cytochrome oxidase subunit II were from Pharmingen and Molecular Probes, respectively. Age-matched female GPx6 and GPx13 transgenic mice (36Mirault M.E. Tremblay A. Furling D. Trepanier G. Dugre F. Puymirat J. Pothier F. Ann. N. Y. Acad. Sci. 1994; 738: 104-115Crossref PubMed Scopus (36) Google Scholar) and control mice (B6C3F1, obtained from Charles River Laboratories) of 10–16 weeks of age were intraperitoneally injected with 0.36 μg Jo2 antibody (Pharmingen) per g of body weight in a total volume of 200 μl of sterile saline. Mice were checked for mortality every 30 min. For in vitro assays, Student's t test was used for statistical analysis. For animal studies, Kaplan-Meier survival rates were compared using the Logrank test. As CD95-mediated apoptosis has been shown to implicate the production of ROS, we investigated the effect of GPx1 overexpression on cell death induced by an agonistic anti-CD95 antibody. This was examined in cells overexpressing GPx (T47D/GPx) as compared with cells transfected with an empty vector (T47D/H3), which express similar levels of CD95 antigen (Fig. 1 A). As illustrated in Fig. 1 B, the CD95-induced time-dependent cytotoxic effect was considerably reduced in GPx1-expressing cells (after 72 h of exposure to the anti-CD95, the viability of T47D/GPx cells exceeded 80%, whereas that of control cells approximated 50%). The resistance of T47D/GPx cells was further confirmed by examining the rate of DNA fragmentation following 48 h of treatment with anti-CD95. As shown in Fig. 1 C, only a small (1.2 times) increase in DNA fragmentation was observed in the GPx1-overexpressing cells (as compared with a 2.5-fold elevation in control cells). To substantiate the role of the antioxidant enzyme GPx1 in CD95-induced cell death, control experiments were carried out by manipulating the intracellular levels of GSH. Whereas the GSH precursor and ROS scavenger NAC completely protected T47D/H3 cells from CD95-induced cell death, the GSH-depleting agent BSO sensitized both control and GPx1-expressing cells to anti-CD95 (Fig. 1 C). These compounds have been shown previously (28Gouazé V. Mirault M.E. Carpentier S. Salvayre R. Levade T. Andrieu-Abadie N. Mol. Pharmacol. 2001; 60: 488-496PubMed Google Scholar) to increase or decrease, respectively, the GSH levels in T47D cells, suggesting that the observed variations in cell viability are indeed related to GSH levels. The resistance of T47D/GPx1 cells to the lethal effect of anti-CD95 was linked to a protection from apoptosis, as indicated by the suppression of DNA fragmentation in T47D/H3 conferred by the rather non-selective caspase inhibitors ZVAD and DEVD (37Garcia-Calvo M. Peterson E.P. Leiting B. Ruel R. Nicholson D.W. Thornberry N.A. J. Biol. Chem. 1998; 273: 32608-32613Abstract Full Text Full Text PDF PubMed Scopus (849) Google Scholar) (Fig. 1 C). Consistent with these results, activation of effector and initiator caspase processing was found in T47D cells upon CD95 ligation (Fig.2). Caspase activity, as measured by the cleavage of the fluorogenic tetrapeptide substrate Ac-DEVD-AMC, increased in T47D/H3 cells within 6 h, peaking at 24 h post-treatment (Fig. 2 A). Only a very moderate, progressive increase (less than 1.5-fold) was noted in GPx1-overexpressing cells. Moreover, Western blot analysis confirmed that executioner caspase-3 (Fig. 2 B) and caspase-7 (not shown) processing was reduced in T47D/GPx cells. Processing of the initiator caspase-8, which was detected as early as 15–30 min (data not shown), appeared essentially unaffected by GPx1 overexpression (Fig. 2 B). Finally, CD95-induced cytochrome c release from mitochondria was diminished in GPx1-expressing cells (Fig. 2 C). Because CD95-mediated apoptosis has been described to be accompanied by the production of ROS, we next investigated the effect of GPx1 overexpression on intracellular ROS levels measured in anti-CD95-treated T47D cells. Fig.3 A shows that CD95 ligation induced a rapid increase in ROS (probably mostly peroxide) levels as measured by dichlorofluorescein fluorescence. This phenomenon was detectable within the first 15 min of incubation and peaked around 60 min; no increase in ROS levels was observed at later points,e.g. at 24 h (data not shown). In contrast, anti-CD95 failed to produce any detectable ROS increase in T47D/GPx cells, suggesting that in these cells GPx1 overexpression strongly reduced DCF-detected ROS accumulation. Of interest is the finding that caspase inhibitors suppressed the CD95-induced ROS increase in control cells (Fig. 3 B), indicating a protease-dependent mechanism in ROS generation. To explore further the molecular events that lead to ROS production after CD95 ligation, and because this production was found to be dependent on caspase activation (Fig.3 B), we investigated the role of initiator caspases. To this end, ROS production was monitored in human leukemia Jurkat cells expressing or not expressing caspase-8 (31Juo P. Kuo C.J. Yuan J. Blenis J. Curr. Biol. 1998; 8: 1001-1008Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar); the caspase-8 mutant cell line expresses very low levels of caspase-8 (see Fig.4 C) and is resistant to anti-CD95-triggered cell death (data not shown). Although wild-type and caspase-8 mutant Jurkat cells expressed comparable levels of CD95 (Fig.4 A), CD95 ligation was not accompanied by early ROS production in the caspase-8 mutant cell line (Fig. 4 B). In the control cell line, generation of ROS was detectable at 60 min, that is concomitantly to the first measurable cleavage of caspase-8 (Fig.4 C). Caspase-3 processing occurred no earlier than 60 min. These data indicate that CD95-induced ROS production immediately follows activation of the initiator caspase-8. Complementary studies further showed that caspase-8 cleavage precedes ROS generation. Indeed, incubation of Jurkat cells with antioxidants (NAC, pyrrolidinedithiocarbamate, or butylated hydroxyanisole) did not affect CD95-induced proteolytic processing of caspase-8 (Fig.4 D). Because different proteins, including FLIP/usurpin (38Rasper D.M. Vaillancourt J.P. Hadano S. Houtzager V.M. Seiden I. Keen S.L. Tawa P. Xanthoudakis S. Nasir J. Martindale D. Koop B.F. Peterson E.P. Thornberry N.A. Huang J. MacPherson D.P. Black S.C. Hornung F. Lenardo M.J. Hayden M.R. Roy S. Nicholson D.W. Cell Death Differ. 1998; 5: 271-288Crossref PubMed Scopus (280) Google Scholar), members of the Bcl-2 family (39Medema J.P. Scaffidi C. Krammer P.H. Peter M.E. J. Biol. Chem. 1998; 273: 3388-3393Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar,40Armstrong R.C. Aja T. Xiang J. Gaur S. Krebs J.F. Hoang K. Bai X. Korsmeyer S.J. Karanewsky D.S. Fritz L.C. Tomaselli K.J. J. Biol. Chem. 1996; 271: 16850-16855Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar), and ERK (41Wilson D.J. Alessandrini A. Budd R.C. Cell. Immunol. 1999; 194: 67-77Crossref PubMed Scopus (51) Google Scholar, 42Holmstrom T.H. Chow S.C. Elo I. Coffey E.T. Orrenius S. Sistonen L. Eriksson J.E. J. Immunol. 1998; 160: 2626-2636PubMed Google Scholar), are known to counteract the proapoptotic action of CD95 ligation, we examined whether the expression level of these proteins was affected by GPx1 overexpression. As shown in Fig.5, similar levels of FLIP/usurpin, Bcl-xL, Mcl-1, Bid, and ERK (either the total or active, phosphorylated forms) were noted in T47D/H3 and T47D/GPx cells, excepted for Bcl-2 that was less expressed in GPx1-overexpressing cells. These data indicate that the resistance of T47D/GPx cells is not due to an increased expression of these proteins. Interestingly, the lower level of Bcl-2 in GPx1-expressing cells is reminiscent of a previous observation reporting a decreased amount of Bcl-xL in murine fibrosarcoma cells overexpressing another antioxidant enzyme, Mn-superoxide dismutase (43Kiningham K.K. Oberley T.D. Lin S. Mattingly C.A. St Clair D.K. FASEB J. 1999; 13: 1601-1610Crossref PubMed Scopus (92) Google Scholar). Because CD95-mediated apoptosis is known to involve mitochondria, and because expression of members of the Bcl-2 family are able to suppress the mitochondrial events, leading to protection from apoptosis in some cell types (see "Discussion"), we investigated whether overexpression of these proteins could alter the above detected production of ROS. To this end, we employed MCF-7 cells, another cell line derived from a human breast carcinoma, that overexpress CD95 and the anti-apoptotic molecule Bcl-xL (44Srinivasan A., Li, F. Wong A. Kodandapani L. Smidt Jr., R. Krebs J.F. Fritz L.C., Wu, J.C. Tomaselli K.J. J. Biol. Chem. 1998; 273: 4523-4529Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). As published previously (35Cuvillier O. Nava V.E. Murthy S.K. Edsall L.C. Levade T. Milstien S. Spiegel S. Cell Death Differ. 2001; 8: 162-171Crossref PubMed Scopus (119) Google Scholar, 44Srinivasan A., Li, F. Wong A. Kodandapani L. Smidt Jr., R. Krebs J.F. Fritz L.C., Wu, J.C. Tomaselli K.J. J. Biol. Chem. 1998; 273: 4523-4529Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 45Jaattela M. Benedict M. Tewari M. Shayman J.A. Dixit V.M. Oncogene. 1995; 10: 2297-2305PubMed Google Scholar), Bcl-xL overexpression (Fig.6 A) resulted in protection of MCF-7 cells from anti-CD95-induced cell death (Fig. 6 B) and caspase-7 proteolytic cleavage (Fig. 6 C). As the MCF-7 cell line is dev