Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis

The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 μm C6-ceramide induced significant H2O2 production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect. Furthermore, no H2O2 production was observed in C6-ceramide-treated U937 ρ° cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-κB and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H2O2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor). H2O2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramide-induced oxidative stress. Finally, C6-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis. The key events implicated in ceramide-triggered apoptosis remain unknown. In this study we show that 25 μm C6-ceramide induced significant H2O2 production within 60 min, which increased up to 180 min in human myeloid leukemia U937 cells. Inactive analogue dihydro-C6-ceramide had no effect. Furthermore, no H2O2 production was observed in C6-ceramide-treated U937 ρ° cells, which are mitochondrial respiration-deficient. We also present evidence that ceramide-induced activation of the transcription factors NF-κB and AP-1 is mediated by mitochondrial derived reactive oxygen species. Both H2O2 production, transcription factor activation as well as apoptosis could be inhibited by rotenone and thenoyltrifluoroacetone (specific mitochondrial complexes I and II inhibitors) and antioxidants, N-acetylcysteine and pyrrolidine dithiocarbamate. These effects could be potentiated by antimycin A (specific complex III mitochondrial inhibitor). H2O2 production was also inhibitable by ruthenium red, suggesting a role of mitochondrial calcium homeostasis alterations in ceramide-induced oxidative stress. Finally, C6-ceramide had no influence on mitochondrial membrane potential within the first 6 h. Altogether, our study points to reactive oxygen species, generated at the ubiquinone site of the mitochondrial respiratory chain, as an early major mediator in ceramide-induced apoptosis. Ceramide has emerged as a potentially important mediator of a number of natural or pharmacological agents that affect cell growth, viability, and differentiation (1Kolesnick R.N. Prog. Lipid Res. 1991; 30: 1-38Crossref PubMed Scopus (271) Google Scholar, 2Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1515) Google Scholar). This lipid second messenger is the breakdown product of sphingomyelin (SPM)1 generated by the activation of a neutral and/or an acidic sphingomyelinase. Agonists of the SPM-ceramide pathway include: cytokines or growth factors such as tumor necrosis factor-α (TNF-α) (3Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1671) Google Scholar), interleukin-1β (4Andrieu N. Salvayre R. Levade T. Biochem. J. 1994; 270: 24518-24524Google Scholar), γ-interferon (5Kim M.-Y. Linardic C. Obeid L. Hannun Y. J. Biol. 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The observation that cell-permeant ceramides or natural ceramide (generated by treating cells with bacterial sphingomyelinase) could mimic the biological effects of most SPM-ceramide cycle agonists has provided significant weight as to the role of ceramide in signal transduction. Finally, ceramide has been shown to exert a wide range of biological effects, depending on the cellular model, including cell activation, mitogenic signaling, survival promoting effect, growth inhibition, and apoptosis. As an example, ceramide has been described to induce growth inhibition in Molt-4 cells (14Jayadev S. Liu B. Bielawaska A.E. Lee J.Y. Nazaire F. Pushkareva M.Y. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), proliferation in fibroblasts (15Olivera A. Buckley N.E. Spiegel S. J. Biol. 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Chem. 1992; 267: 5048-5051Abstract Full Text PDF PubMed Google Scholar) and/or a proline-directed protein kinase termed ceramide-activated protein kinase (18Mathias S. Dressler K.A. Kolesnick R.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10009-10013Crossref PubMed Scopus (385) Google Scholar), which, in turn, could activate the mitogen-activated protein kinase cascade in U937 cells (19Yao B. Zhang Y. Delikat S. Mathias S. Basu S. Kolesnick R.N. Nature. 1995; 378: 307-310Crossref PubMed Scopus (304) Google Scholar). Ceramide has also been described to activate stress-activated protein kinases (SAPK/c-Jun kinase), which may be more closely related to ceramide-induced apoptosis in U937 and in endothelial cells (9Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1724) Google Scholar). In addition, ceramide has been shown to activate transcription factors such as NF-κB (20Schütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Krönke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (1034) Google Scholar) and AP-1, which could also play an important role since, for example, inhibitors of AP-1 activation or antisense oligonucleotides for c-jun prevented ceramide-induced apoptosis of HL-60 cells (21Sawai H. Okazaki T. Yamamoto H. Okano H. Takeda Y. Tashima M. Sawada H. Okuma M. Ishikura H. Umehara H. Domae N. J. Biol. Chem. 1995; 270: 27326-27331Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Nevertheless, to what extent these various signaling proteins are directly or indirectly involved within the SPM-ceramide pathway remains elusive. Despite the characterization of various biochemical changes associated with the SPM-ceramide signaling, such as apoptosis, a consensus on the sequence of cellular events has not been reached. However, there is mounting evidence that radical oxygen species (ROS) may be central (22Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2123) Google Scholar). Indeed, ROS are involved in apoptosis induced by agents such as TNF-α (23Larrick J.W. Wright S.C. FASEB J. 1990; 4: 3215-3223Crossref PubMed Scopus (514) Google Scholar), UV light (24Devary Y. Rosette C. DiDonato J.A. Karin M. Science. 1993; 261: 1442-1445Crossref PubMed Scopus (607) Google Scholar), ionizing radiation (25Manome Y. Datta R. Taneja N. Shafman T. Bump E. Hass R. Weichselbaum R. Kufe D. Biochemistry. 1993; 32: 10607-10613Crossref PubMed Scopus (73) Google Scholar), and anthracyclines (26Quillet-Mary A. Mansat V. Duchayne E. Come M.G. Allouche M. Bailly J.D. Bordier C. Laurent G. Leukemia. 1996; 10: 417-425PubMed Google Scholar), which have been all documented to activate the sphingomyelin cycle. Furthermore, ROS themselves, such as low doses of H2O2, or prooxidant conditions, such as UV or γ-irradiation, activate signaling pathways and transcription factors that have been involved in ceramide-induced apoptosis such as mitogen-activated protein kinase (27Stevenson M.A. Pollock S.S. Coleman N. Calderwood S.K. Cancer Res. 1994; 54: 12-15PubMed Google Scholar) or stress-activated protein kinases cascades (28Shafman T.D. Saleem A. Kyriakis J. Weichselbaum R. Kharbanda S. Kufe D.W. Cancer Res. 1995; 55: 3242-3245PubMed Google Scholar) as well as AP-1 and NF-κB activation (for review, see Ref. 29Weichselbaum R.R. Hallahan D. Fuks Z. Kufe D. Int. J. Radiat. Oncol. Biol. Phys. 1994; 30: 229-234Abstract Full Text PDF PubMed Scopus (176) Google Scholar). Moreover, oxidant production has been shown to be accompanied by cell death triggered by a cell permeant ceramide (C2-ceramide) in lymphoid B cells (30Fang W. Rivard J.J. Ganser J.A. LeBien T.W. Nath K.A. Mueller D.L. Behrens T.W. J. Immunol. 1995; 155: 66-75PubMed Google Scholar). Altogether, these observations suggest ROS as a common mediator in ceramide-induced apoptosis. Here we show that C6-ceramide induces intracellular H2O2 production followed by DNA fragmentation in U937 cells whereas the inactive analogue (dihydro-C6 ceramide) was ineffective. Ceramide-induced apoptosis was inhibited by ROS scavengers such as dithiocarbamates (PDTC) and N-acetylcysteine, a thiol antioxidant and a glutathione (GSH) precursor. In this study, we provide evidence that H2O2 is produced at the ubiquinone site of the mitochondrial respiratory chain with mitochondrial Ca2+ homeostasis alterations followed by disregulation of mitochondrial membrane potential (ΔΨm). Finally, we show that C6-ceramide could activate the transcriptional factor NF-κB and AP-1 via mitochondrial ROS production. The following reagents were purchased from Sigma: N-hexanoyl-d-sphingosine (C6-ceramide), carbonyl cyanide m-chlorophenyl hydrazone,N-acetylcystein, PDTC, rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, ruthenium red (RR). Dihydro-C6-ceramide was a generous gift from Dr. Salem Chouaib (CJF INSERM 9411, Villejuif, France). C2938 (6-carboxy-2′,7′ dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester)) and DiOC6 (3,3′-dihexyloxacarbocyanine iodide) were purchased from Molecular Probes (Interchim, France). Stock solutions of the reagents were routinely prepared in phosphate-buffered saline (PBS), dimethyl sulfoxide or ethanol as appropriate. [methyl-3H]Thymidine (79 Ci/mmol) was purchased from Amersham (Les Ulis, France). TNF-α was purchased from PeproTech-Tebu (Le Perray en Yvelines, France), and daunorubicin (Cerubidine®) was from Laboratoire Roger Bellon (Neuilly-sur -Seine, France). The human leukemia cell line U937 (monocytic) was obtained from the ATCC (Rockville, MD) and grown in RPMI 1640 at 37 °C in 5% CO2. Culture medium was supplemented with 10% heat-inactivated fetal calf serum, 2 mml-glutamine, and antibiotics, streptomycin (100 μg/ml) and penicillin (200 units/ml). All experiments with C6-ceramide were done in RPMI 1640 supplemented with 1% fetal calf serum,l-glutamine, and antibiotics. Cell stocks were screened routinely for Mycoplasma by the polymerase chain reaction method (Stratagene Mycoplasma PCR kit, La Jolla, CA). The U937 ρ° subline was maintained in the same medium as U937 cells but supplemented with glucose (4.5 mg/ml), pyruvate (0.1 mg/ml), and uridine (50 μg/ml) (31Gamen S. Anel A. Montoya J. Marzo I. Pineiro A. Naval J. FEBS Lett. 1995; 376: 15-18Crossref PubMed Scopus (35) Google Scholar). For some experiments U937 cells were grown in RPMI 1640 without glucose supplemented with 10% heat-inactivated fetal calf serum dialyzed against PBS. Production of ROS was detected with C2938 fluorescent probe. C2938 is an uncharged cell-permeant molecule. Inside the cells, this probe is cleaved by nonspecific esterases, formed carboxydichlorofluroscein which is oxidized in the presence of H2O2. Exponentially growing cells (5 × 105 cells/ml) were labeled with 0.5 μm C2938 for 1 h and then incubated in the absence or presence of C6-ceramide at 37 °C for various periods of time. The cells were washed in PBS, and cell fluorescence was determined using flow cytometry on a FACScan (Becton Dickinson). The presence of ethanol (final concentration 0.25%) in the culture medium did not affect the fluorescence of C2938. Additional experiments were performed by preincubating the cells with nontoxic concentrations of specific mitochondrial inhibitors (rotenone, TTFA, antimycin A, RR) or by coincubation with antioxidant (PDTC). The optimal effective nontoxic concentrations for 3 h continuous incubation of these inhibitors were determined by dose-effect studies with rotenone 1–5 μm, TTFA 10–50 μm, and antimycin A 5–10 μm (data not shown). Exponentially growing cells (5 × 105 cells/ml) were preincubated with or without various inhibitors before the addition of C6-ceramide. Cells were harvested by centrifugation onto glass slides and stained with May-Grünwald-Giemsa stain. Changes in cell morphology were examined by light microscopy using a Zeiss microscope. Apoptotic cells were scored and expressed as the number of cells exhibiting morphology typical of apoptosis (chromatin condensation and fragmentation, and cytoplasmic volume reduction) per 200 cells counted (5–10 fields). For photography, cells were fixed in 4% paraformaldehyde, washed in PBS. Cells were then stained with 0.1 μg/ml 4′,6′-diamidino-2-phenylindol for 1 h, washed, and mounted for fluorescence microscopy (Leica model Diaplan). DNA fragmentation was quantified as described previously (26Quillet-Mary A. Mansat V. Duchayne E. Come M.G. Allouche M. Bailly J.D. Bordier C. Laurent G. Leukemia. 1996; 10: 417-425PubMed Google Scholar). Exponentially growing cells (5 × 105 cells/ml) were labeled with 0.5 μCi/106 cells of [methyl-3H]thymidine for 24 h and washed three times with nonradioactive fresh medium. Labeled cells were exposed to C6-ceramide for 6- or 24-h continuous exposure. In additional experiments, cells were preincubated with various inhibitors before the addition of C6-ceramide. Cells were harvested by centrifugation, and the pellets were suspended in lysis buffer containing Tris 15 mm, EDTA 20 mm, Triton X-100 0.5%, pH 8.0. After 30 min on ice, samples were centrifugated at 20,000 × g for 30 min, and the pellets were resuspended in lysis buffer. The radioactivity present in the supernatant (detergent-soluble low molecular weight DNA) and in the pellet (intact chromatin DNA or large chromatin fragments, >50 kilobase pairs) was determined by liquid scintillation counting. In control cells DNA fragmentation with [3H]thymidine alone was below 2–3%. ICE activation was measured as described previously (32Los M. Van de Caren M. Penning L.C. Schenk H. Westendorp M. Bauerle P.A. Dröge M. Krammer P. Fiers W. Schulze-Osthoff K. Nature. 1995; 375: 81-83Crossref PubMed Scopus (652) Google Scholar). Briefly, after treatment with C6-ceramide, cells were made permeable with 20 μm digitonin for 5 min. Cells were washed in PBS and resuspended in 100 mm HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mm dithiothreitol, and 0.1 mg/ml ovalbumin at 25 °C and incubated 30 min with 20 μm of the fluorogenic ICE substrate DABC-YL-YVADAP-EDANS (BACHEM, Voisins le Bretonneux, France). The fluorescence was analyzed by spectrofluorometry using an excitation wavelength of 340 nm and an emission wavelength of 490 nm (33Pennington M.W. Thornberry N.A. Pept. Res. 1994; 7: 72-76PubMed Google Scholar). To evaluate ΔΨm, exponentially growing cells (5 × 105 cells/ml) were incubated with C6-ceramide for various periods of time. 15 min before the end of incubation, cells were labeled with DiOC6 (40 nm in PBS) at 37 °C (34Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1443) Google Scholar). After washing, cells were analyzed by flow cytometry. Control experiments were performed in the presence of carbonyl cyanidem-chlorophenyl hydrazone, an uncoupling agent that abolishes the ΔΨm, at 50 μm for 15 min at 37 °C. Extracts were prepared as described previously (19Yao B. Zhang Y. Delikat S. Mathias S. Basu S. Kolesnick R.N. Nature. 1995; 378: 307-310Crossref PubMed Scopus (304) Google Scholar). Cells (5 × 106) were incubated with or without C6-ceramide in the presence or absence of different inhibitors. Cells were then washed twice with ice-cold PBS and resuspended in 10 mm HEPES, pH 7.8, 10 mmKCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 μm pepstatin A, 0.6 μm leupeptin, 1 μg/ml aprotinin, and 0.6% Nonidet P-40. After 15 min on ice, the nuclear pellet was recovered after centrifugation at 1200 × gand resuspended in 20 mm HEPES, pH 7.9, 0.4 mNaCl, 1 mm EDTA, 1 mm EGTA. Aliquots were then incubated at 4 °C for 30 min and centrifuged at 21,000 ×g, and supernatants containing nuclear proteins were collected. Protein concentrations were determined according to Smithet al. (35Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (19432) Google Scholar) using bicinchoninic acid (Sigma). Labeling of NF-κB (5′-AGTTGAGGGGACTTTCCCAGGC-3′) and AP-1 (5′-CGCTTGATGAGTCAGCCGGAA-3′) consensus oligonucleotides (binding sites are underlined) was performed using T4 polynucleotide kinase and [γ-32P]ATP (specific activity, 5000 Ci/mmol, Amersham, Les Ulis, France). Binding reactions were carried out in 2 mm HEPES (pH 7.5), 50 mm NaCl, 0.5 mm EDTA, 1 mm MgCl2, 2% glycerol, 0.5 mm dithiothreitol, 1 μg poly(dI-dC), and 2 μg of bovine serum albumin. Typical reactions contained 50,000 cpm of end-labeled NF-κB or AP-1 consensus oligonucleotide (Promega, Madison, WI) with 2–6 μg of nuclear extract. After 20 min of incubation, the mixture was electrophoresed through a low ionic strength 4% polyacrylamide gel (acrylamide:bisacrylamide ratio 80:1) containing 6.7 mm Tris-HCl (pH 7.9), 3.3 mmsodium acetate, 2 mm EDTA. The gel was preelectrophoresed for 90 min at 10 V/cm. Electrophoresis was carried out at the same voltage for 3 h at room temperature with buffer recirculation. The gel was then dried and autoradiographed with intensifying screens at −70 °C. Quantification of bands was performed by densitometry and by radioactivity counting of excised bands. Band specificity was determined by competition experiments using 100-fold excess unlabeled NF-κB or AP-1 consensus oligonucleotide, as well as supershift assays (data not shown) for NF-κB using p65-, p55-, and c-Rel-specific antibodies generously provided by Dr. J. Imbert (INSERM U119, Marseille). The fluorescence distribution of the C2938 dye, revealing the presence of hydrogen peroxide, was measured by flow cytometry in the viable cell population. Fig. 1 A shows the increase in the mean C2938 fluorescence in 25 μmC6-ceramide-treated cells relative to untreated cells. The mean fluorescence increased as a function of time, reflecting H2O2 generation in U937 cells induced by C6-ceramide. Within 60 min, significant H2O2was detected, and this level increased up to 180 min. At low doses (5 μm and 10 μm) we observed only a low level of H2O2 generation at 180 min (Fig. 1 A). In comparison, Fig. 1 B presents the H2O2 produced, after 60 min, by SPM-ceramide agonists daunorubicin and TNF-α. Treatment of U937 cells by TNF-α generated comparable H2O2 production, whereas daunorubicin induced significantly higher H2O2generation. The mean fluorescence emitted by C2938 in cells treated with exogenous H2O2 is presented, as well as dihydro-C6-ceramide, an inactive ceramide analogue, which had no significant effect. In addition, glucose deprivation and addition of deoxyglucose (2.5 mm) did not change H2O2 production in U937 cells treated with C6-ceramide (data not shown). These results suggest that, in this model, H2O2 production is independent of the pentose cycle. To further investigate the role of mitochondria in the generation of H2O2 induced by C6-ceramide, we tested several classic specific mitochondrial respiratory chain inhibitors (36Boveris A. Cadenas E. Stroppani A.O.M. Biochem. J. 1976; 156: 435-445Crossref PubMed Scopus (553) Google Scholar, 37Cadenas E. Boveris A. Biochem. J. 1980; 188: 31-37Crossref PubMed Scopus (243) Google Scholar, 38Konstantinov A.A. Peskin A.V. Popova E.Y. Khomutov G.B. Ruuge E.K. Biochim. Biophys. Acta. 1987; 894: 1-10Crossref PubMed Scopus (85) Google Scholar, 39Cino M. Del Maestro R.F. Arch. Biochem. Biophys. 1989; 269: 623-638Crossref PubMed Scopus (129) Google Scholar). Two sites of the mitochondrial respiratory chain have been identified as sources of ROS. One depends on the autooxidation of complex I, whereas the other is dependent on the autooxidation of complex III (ubiquinone site) (40Turrens J.F. Alexandre A. Lehninger A.L. Arch. Biochem. Biophys. 1985; 237: 408-414Crossref PubMed Scopus (1087) Google Scholar). When U937 cells were pretreated with rotenone (a specific inhibitor of complex I that interferes with the electron flow from NADH-linked substrates and NADH dehydrogenase to the ubiquinone pool), or TTFA (a specific inhibitor of complex II that interferes with the electron transport flow from succinate dehydrogenase to the ubiquinone pool) followed by treatment with C6-ceramide, we observed an almost complete inhibition of H2O2 generation (Fig. 2). On the other hand, pretreatment by antimycin A (a specific inhibitor of complex III, which inhibits the electron flow from ubiquinone to complex IV), increased by approximately 2-fold H2O2 production induced by C6-ceramide (Fig. 2). Furthermore, we tested H2O2 production in U937 ρ° cells. These cells have been selected in the presence of ethidium bromide and present mitochondrial DNA depletion and a reduced complex III activity in the mitochondrial respiratory chain (31Gamen S. Anel A. Montoya J. Marzo I. Pineiro A. Naval J. FEBS Lett. 1995; 376: 15-18Crossref PubMed Scopus (35) Google Scholar). We did not observe significant H2O2 production in U937 ρ° cells treated for 1 h with 25 μmC6-ceramide (Fig. 2, inset). These results strongly support the role of complex III in C6-ceramide-induced H2O2 production. In addition, since calcium has been shown to play a role in apoptosis in certain experimental systems (41Richter C. Kass G.E.N. Chem.-Biol. Interact. 1991; 77: 1-23Crossref PubMed Scopus (290) Google Scholar), we tested the contribution of calcium to the production of H2O2 induced by C6-ceramide. U937 cells were pretreated with ruthenium red (an inhibitor of the mitochondrial calcium uptake) (42Hennet T. Richter C. Peterhans E. Biochem. J. 1993; 289: 587-592Crossref PubMed Scopus (266) Google Scholar) and then analyzed for the production of H2O2 generated by C6-ceramide. As shown in Fig. 2, ruthenium red inhibited C6-ceramide induced H2O2 generation by 60–70%. We tested the relationship between H2O2 production and apoptosis induced by C6-ceramide in U937 cells. U937 cells were treated in kinetics experiments with different doses of C6-ceramide and analyzed for typical morphological features of apoptosis (Fig. 3 A) as well as DNA fragmentation using the [3H]thymidine release assay. At 6 h, approximately 10% of U937 cells treated by 25 μm C6-ceramide presented typical apoptotic features. This population grew to approximately 30% at 24 h (Fig. 3 B). Analysis of DNA fragmentation also reflected a similar effect (8% at 6 h; 37% at 24 h) and we observed that C6-ceramide dose-dependent DNA fragmentation correlated closely with H2O2 production (data not shown). In addition, cleavage of ICE-substrate (32Los M. Van de Caren M. Penning L.C. Schenk H. Westendorp M. Bauerle P.A. Dröge M. Krammer P. Fiers W. Schulze-Osthoff K. Nature. 1995; 375: 81-83Crossref PubMed Scopus (652) Google Scholar) was observed as early as 4 h after 25 μm C6-ceramide treatment (Fig. 3 C). We tested the influence of different ROS scavengers on apoptosis and DNA fragmentation induced by C6-ceramide. U937 cells were pretreated with different concentrations of N-acetylcysteine, the thiol antioxidant and GSH precursor (22Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2123) Google Scholar), or PDTC, the oxygen radical scavenger and iron chelator (43Schreck R. Meier B. Männel D. Dröge W. Bauerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1453) Google Scholar), in combination with 6 h (data not shown) or 24 h of continuous exposure to 25 μmC6-ceramide. C6-Ceramide induced-apoptosis (data not shown) and DNA fragmentation were inhibited by both N-acetylcysteine (Fig. 4 A) and PDTC (Fig. 4 B) in a dose-dependent manner. While confirming that C6-ceramide is able to induce apoptosis in U937 cells, these results implicated ROS within this process, since PDTC was also able to inhibit ROS production induced by C6-ceramide (Fig. 4 B,inset). To further substantiate the role of ROS in ceramide-mediated apoptosis, we evaluated the impact of both rotenone and TTFA on this cell death process. Both mitochondrial respiratory chain inhibitors significantly inhibited C6-ceramide-triggered apoptosis (not shown) and DNA fragmentation (Fig. 5). In addition, pretreatment of U937 cells with antimycin A greatly increased the apoptotic population induced by C6-ceramide (not shown) as well as DNA fragmentation (Fig. 5). The effective nontoxic concentrations for 6 h of continuous exposure of these inhibitors were determined by dose-effect studies (data not shown).Figure 5Differential effects of mitochondrial respiratory chain inhibitors on C6-ceramide-induced DNA fragmentation. Cells were pretreated 30 min with rotenone (1 μm), TTFA (10 μm), or antimycin A (5 μm) before treatment with C6-ceramide during 6 h of continuous exposure. DNA fragmentation was analyzed as described under “Experimental Procedures.” Results represent the mean (±S.D.) of three separate experiments. *p < 0.05 as compared with C6-ceramide alone evaluated by Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been described that a reduction in ΔΨm precedes apoptosis and may represent an early signaling event (34Zamzami N. Marchetti P. Castedo M. Decaudin D. Macho A. Hirsh T. Susin S.A. Petit P.X. Mignotte B. Kroemer G. J. Exp. Med. 1995; 182: 367-377Crossref PubMed Scopus (1443) Google Scholar). Using the DiOC6 fluorescent probe, we analyzed the ΔΨm of U937 treated with C6-ceramide. Cells were exposed to DiOC6 probe at 40 nm, 15 min before the end of incubation with C6-ceramide. As described previously, under these conditions, only mitochondria are labeled as confirmed by confocal microscopy experiments (data not shown). In addition, treatment with carbonyl cyanide m-chlorophenyl hydrazone (an uncoupling agent that abolishes the ΔΨm) and DiOC6 showed a drastic reduction of ΔΨm, whereas incubation in the presence of high concentration of KCl (120 mm) showed no difference, confirming that, under these conditions, only mitochondrial membrane potential is measured (data not shown). In our hands, C6-ceramide had no significant influence on mitochondrial membrane potential at 2 h, and only 5% of cells showed a ΔΨm reduction at 6 h. However, after 20 h of incubation with C6-ceramide, 20% of cells showed a drastic reduction of the ΔΨm (Fig. 6), suggesting that