Superoxide in Apoptosis
1998; Elsevier BV; Volume: 273; Issue: 19 Linguagem: Inglês
10.1074/jbc.273.19.11401
ISSN1083-351X
Autores Tópico(s)ATP Synthase and ATPases Research
ResumoActivation of apoptosis is associated with generation of reactive oxygen species. The present research shows that superoxide is produced by mitochondria isolated from apoptotic cells due to a switch from the normal 4-electron reduction of O2 to a 1-electron reduction when cytochromec is released from mitochondria. Bcl-2, a protein that protects against apoptosis and blocks cytochrome c release, prevents superoxide production when it is overexpressed. The switch in electron transfer provides a mechanism for redox signaling that is concomitant with cytochrome c-dependent activation of caspases. The block of cytochrome c release provides a mechanism for the apparent antioxidant function of Bcl-2. Activation of apoptosis is associated with generation of reactive oxygen species. The present research shows that superoxide is produced by mitochondria isolated from apoptotic cells due to a switch from the normal 4-electron reduction of O2 to a 1-electron reduction when cytochromec is released from mitochondria. Bcl-2, a protein that protects against apoptosis and blocks cytochrome c release, prevents superoxide production when it is overexpressed. The switch in electron transfer provides a mechanism for redox signaling that is concomitant with cytochrome c-dependent activation of caspases. The block of cytochrome c release provides a mechanism for the apparent antioxidant function of Bcl-2. Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; cyt c, cytochrome c; SOD, superoxide dismutase; NAC, N-acetylcysteine; TNF, tumor necrosis factor; MnSOD, manganese superoxide dismutase; DHR, dihydrorhodamine;Eh, GSH redox potential; MPT, mitochondrial permeability transition. are important regulators of apoptosis. Pro-oxidants and redox cycling agents, such as H2O2 (1Gardner A.M. Xu F.H. Fady C. Jacoby F.J. Duffey D.C. Tu Y. Lichtenstein A. Free Radical Biol. Med. 1997; 22: 73-83Crossref PubMed Scopus (338) Google Scholar), diamide (2Sato N. Iwata S. Nakamura K. Hori T. Mori K. Yodoi J. J. Immunol. 1995; 154: 3194-3203PubMed Google Scholar), etoposide (3Bustamante J. Tovar B.A. Montero G. Boveris A. Arch. Biochem. Biophys. 1997; 337: 121-128Crossref PubMed Scopus (57) Google Scholar), and semiquinones (4Sun X. Ross D. Chem. Biol. Interact. 1996; 100: 267-276Crossref PubMed Scopus (23) Google Scholar), can induce apoptosis. Other apoptotic stimuli, such as treatment with TNFα (5Albrecht H. Tschopp J. Jongeneel C.V. FEBS. Lett. 1994; 351: 45-48Crossref PubMed Scopus (98) Google Scholar), lipopolysaccharide (6Wang J.H. Redmond H.P. Watson R.W. Bouchier-Hayes D. Am. J. Physiol. 1997; 272: C1543-C1551Crossref PubMed Google Scholar), ceramide (7Garcia-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar), growth factor withdrawal (8Atabay C. Cagnoli C.M. Kharlamov E. Ikonomovic M.D. Nanev H. J. Neurosci. Res. 1996; 43: 465-475Crossref PubMed Scopus (143) Google Scholar), and human immunodeficiency virus infection (9Dobmeyer T.S. Findhammer S. Dobmeyer J.M. Klein S.A. Raffel B. Hoelzer D. Helm E.B. Kabelitz D. Rossol R. Free Radical Biol. Med. 1997; 22: 775-785Crossref PubMed Scopus (106) Google Scholar), can stimulate the ROS production. Studies with TNFα and ceramide have shown that these agents activated ROS generation by mitochondria (7Garcia-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar). The recent proposed model of p53-induced apoptosis also placed ROS as a central signaling event (10Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Voglestein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2270) Google Scholar, 11Johnson T.M. Yu Z.X. Ferrans V.J. Lowenstein R.A. Finkel T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11848-11852Crossref PubMed Scopus (529) Google Scholar). Antioxidants and thiol reductants, such as NAC (12Mayer M. Noble M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7496-7500Crossref PubMed Scopus (352) Google Scholar), overexpression of thioredoxin (13Iwata S. Hori T. Sata N. Hirota K. Sasada T. Mitsui A. Hirakawa T. Yodoi J. J. Immunol. 1997; 158: 3108-3117PubMed Google Scholar), and MnSOD (14Wong G.H. Elwell J.H. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (796) Google Scholar), can block or delay apoptosis. On the other hand, ROS can provide protective mechanisms under some conditions, such as the activation of NFκB by TNF-induced ROS production (15Chu Z.L. McKinsey T.A. Liu L. Gentry J.J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (829) Google Scholar, 16Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2376) Google Scholar). Thus ROS, and the resulting cellular redox change, can be part of the signal transduction pathway during apoptosis. Yet the mechanisms of ROS generation and its relationship with the well studied caspase activation have not been resolved. A role of ROS during apoptosis was initially proposed based upon the observation that Bcl-2, a general inhibitor of apoptosis in mammalian cells, has an apparent antioxidant function (17Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliiman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3325) Google Scholar, 18Kane D.J. Sarafian R. Anton H. Butler G.J. Selverstone V.T. Bredesen D.E. Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1624) Google Scholar). Bcl-2 is largely localized to the mitochondrial outer membrane, and when overexpressed, it protects cells from lipid peroxidation and thiol oxidation induced by menadione and hydrogen peroxide (17Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliiman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3325) Google Scholar, 18Kane D.J. Sarafian R. Anton H. Butler G.J. Selverstone V.T. Bredesen D.E. Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1624) Google Scholar). Bcl-2 also protects ρ0 cells from apoptosis under conditions that ROS generation is limited (19Jacobson M.D. Burne J.F. King M.P. Miyashita T. Reed J.C. Raff M.C. Nature. 1993; 361: 365-369Crossref PubMed Scopus (715) Google Scholar, 20Shimizu S.Y. Eguchl H. Kosaka W. Kamiike H. Masuda H. Tsujimoto Y. Nature. 1995; 374: 811-813Crossref PubMed Scopus (629) Google Scholar). Thus, its anti-apoptotic function is not limited to the antioxidant property. Nonetheless, this apparent antioxidant property of Bcl-2 has not been explained. When human myeloid leukemia HL 60 and U-937 cells undergo apoptosis induced by staurosporine (21Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar), etoposide (21Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar), cytosine β-d-arabinofuranoside (22Kim C.N. Wang X. Huang Y. Ibrado A.M. Liu L. Fang G. Bhalla K. Cancer Res. 1997; 57: 3115-3120PubMed Google Scholar), and ionizing radiation (23Kharbanda S. Pandey P. Schefield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6932-6942Crossref Scopus (373) Google Scholar), there is an early release of cyt c from mitochondria, and the released cyt c participates in the activation of caspase 3. Bcl-2 family proteins prevent cyt c from entering the cytosol either by blocking cyt c release (21Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar, 22Kim C.N. Wang X. Huang Y. Ibrado A.M. Liu L. Fang G. Bhalla K. Cancer Res. 1997; 57: 3115-3120PubMed Google Scholar) or by binding directly with cyt c (23Kharbanda S. Pandey P. Schefield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z-M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6932-6942Crossref Scopus (373) Google Scholar). Cyt c is localized in the mitochondrial intermembrane space and is part of the mitochondrial electron transport chain. A variety of mitochondrial poisons are well known to stimulate ROS generation by a mechanism that involves inhibition of electron transfer, accumulation of reducing equivalents in the middle portion of the electron transfer chain, and direct one-electron transfer to O2 to produce superoxide (for review, see Ref. 24Turrens J.F. Biosci. Rep. 1997; 17: 3-8Crossref PubMed Scopus (763) Google Scholar). This switch appears to occur principally at the level of coenzyme Q (ubiquinone/ubiquinol) and thus could also occur as a consequence of cyt c loss. In this study, we used staurosporine-treated HL 60 cells to study the mechanism of ROS generation during apoptosis. Results show that the cellular redox change occurred following mitochondrial release of cytochrome c and was in parallel to the caspase activation. The release of cytochrome c was associated with inhibited mitochondrial respiration and stimulated mitochondrial superoxide production, and overexpression of Bcl-2 inhibited all of these processes. HL 60 cells transfected with bcl-2(bcl-2) or vector alone (neo) were cultured as described (21Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar). Overexpression of Bcl-2 was monitored by Western blot. Cells were passaged every 2–3 days and seeded at 0.25 × 106/ml, and apoptosis was induced by adding 1 μmstaurosporine. After treatment, cells were pelleted and resuspended in 5% perchloric acid/saturated boric acid, supplemented with 5 μm γ-glutamylglutamate as internal standard. Intracellular GSH and GSSG were measured with high pressure liquid chromatography as described (25Jones D.P. Maellaro E. Jiang S. Slater A.F. Orrenius S. Immunol. Lett. 1995; 205: 45-51Google Scholar), and the amount of acid-insoluble protein was measured by the Bradford method. Cell volume and pH were measured radiochemically (26Andersson B.S. Aw T.Y. Jones D.P. Am. J. Physiol. 1987; 254: C349-C355Crossref Google Scholar), and the redox potential was calculated using the Nernst equation with E 0 adjusted to cell pH (−59 mV/pH unit). E0 at pH 7.0 was taken as −240 mV (27Rost J. Rapoport S. Nature. 1964; 201: 185Crossref PubMed Scopus (141) Google Scholar). Mitochondrial GSH and GSSG were measured similarly, except cells were first permeabilized with digitonin as described below. After treatment with 1 μm staurosporine for 4 h, cells were stained with 15 μm dihydrorhodamine (DHR) for 20 min, washed with phosphate-buffered saline, and loaded onto a Becton-Dickinson FACStation. FL-1 fluorescence was recorded, and cell debris was electronically gated out based on the forward light scatter. 300 μm tert-butylhydroperoxide treatment was used as a positive control. The DHR fluorescence was also recorded with confocal microscopy. Cells double-stained with 60 nmDiOC6 and 15 μm DHR were washed and resuspended in 15 μl of phosphate-buffered saline and placed on a no. 1 thickness coverslip. The green and red fluorescence were simultaneously recorded with a Bio-Rad MRC 1024 laser scanning confocal microscopy system, with the excitation wavelength set at 488 nm. Images were acquired and processed with Laser-Sharp software, using identical background and gain settings. Six to eight areas were randomly picked, and representative ones are presented in Fig. 2. After treatment, cells were loaded with either 2 μm rhodamine 123 (28Lemaster J.J. Nieminen A.L. Chacon E. Bond J.M. Harper I. Reece J.M. Herman B. Methods Toxicol. 1994; 1B: 438-455Google Scholar) or 60 nmDiOC6 (29Marchetti P. Castedo M. Susin S.A. Zamzami N. Hirsh T. Macho A. Haeffner A. Hirsch F. Geuskens M. Kroeamer G. J. Exp. Med. 1996; 184: 1155-1160Crossref PubMed Scopus (788) Google Scholar) for 10 min, and mtΔψ was measured by FACScan. After treatment with staurosporine for the indicated periods of time, 2 × 106 cells were washed with phosphate-buffered saline and resuspended in mitochondrial respiration buffer (30Krippner A. Matsuno-Yagi A. Gottlieb R.A. Babior B.M. J. Biol. Chem. 1996; 271: 21629-21636Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). O2 consumption was measured in a 2.0-ml chamber with a Clark-type oxygen electrode (Yellow Springs) calibrated with air and sodium dithionite (30Krippner A. Matsuno-Yagi A. Gottlieb R.A. Babior B.M. J. Biol. Chem. 1996; 271: 21629-21636Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Cells were permeabilized with 0.01 mg/ml digitonin for 1 min, and substrate-stimulated respiration was recorded by adding 2.5 mm succinate and 0.25 mmADP. The substrate stimulated O2 consumption rate over the rate after digitonin permeabilization was calculated as the respiratory control ratio. Mitochondria were isolated from neo and bcl-2 cells with nagarse-digitonin treatment (31Lash L.H. Sall J.M. Methods Toxicol. 1993; 2: 8-28Google Scholar). Measurements were performed in a SLM/Aminco DW-2000 spectrophotometer at 30 °C. 40 μmacetylated cytochrome c was included in the incubation buffer, and the absorbance change was monitored at 550 nm (32Kakinuma K. Minakami S. Biochim. Biophys. Acta. 1978; 538: 50-59Crossref PubMed Scopus (122) Google Scholar). Reaction was initiated by adding 3 mm succinate. KCN (0.5 mm) was used to inhibit normal electron flow and stimulate superoxide production (positive control), and bovine erythrocyte superoxide dismutase (SOD) (Sigma; 100 units) was added to verify that the reduction of acetylated cyt c was due to superoxide generation. Mitochondrial and cytosolic fractions were prepared with digitonin-nagarse treatment. Each lane was loaded with 50 μg of protein, and Western blot was performed as described (21Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar), using a gift antibody from Dr. R. Jemmerson (University of Minnesota). To determine whether an oxidant signal precedes or follows mitochondrial release of cyt c, we measured the redox state of the intracellular GSH pool as a function of time following treatment of neo cells with 1 μm staurosporine (Fig.1). Under these conditions, cytc release was detectable already at 1 h (Fig.1 B), but oxidation of cellular GSH/GSSG did not occur until after 2 h (Fig. 1 A). By 6 h, the redox potentialEh was oxidized by 72 mV (from −239 ± 6 to −167 ± 9 mV), a change similar to the 86 mV oxidation in apoptotic HT29 cells (data not shown). In cells overexpressing Bcl-2, cyt c release was blocked (Fig. 1 B), and the redox change was inhibited (Fig. 1 A). The measured mitochondrial GSH/GSSG ratio changed with a similar time course (Fig.1 A, inset). Thus, cyt c loss preceded the generation of an oxidant signal and as previously reported, Bcl-2 blocked cyt c release (21Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar) and had an apparent antioxidant function (17Hockenbery D.M. Oltvai Z.N. Yin X.M. Milliiman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3325) Google Scholar, 18Kane D.J. Sarafian R. Anton H. Butler G.J. Selverstone V.T. Bredesen D.E. Science. 1993; 262: 1274-1277Crossref PubMed Scopus (1624) Google Scholar), as measured by Eh of the GSH/GSSG pool and the mitochondrial GSH/GSSG ratio. Antioxidants, such as NAC, can inhibit apoptosis in some systems (12Mayer M. Noble M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7496-7500Crossref PubMed Scopus (352) Google Scholar). Although the staurosporine-induced caspase activation and subsequent DNA fragmentation could be completely inhibited by overexpression of Bcl-2, they were not sensitive to NAC (Fig. 1 C). Thus in staurosporine-induced apo-ptosis, the caspase activation seems to be redox-insensitive, and the effects of Bcl-2 cannot be attributed to its apparent antioxidant function. Because the redox signal was not observed until after cyt crelease, we then examined whether the mitochondrion is a possible source of the redox signal. For this, we used DHR, a non-fluorescent compound that accumulates in the mitochondria and can be oxidized to the fluorescent rhodamine (33Rothe G. Emmendorffer A. Oser A. Roesler J. Valet G. J. Immunol. Methods. 1991; 138: 133-135Crossref PubMed Scopus (175) Google Scholar). FACScan showed that DHR was oxidized in staurosporine-treated neo cells but not in cells overexpressing Bcl-2 (Fig. 2, A and B). Confocal microscopy confirmed that this fluorescence was mainly localized to the mitochondria, together with the mtΔψ-driven dye DiOC6 (Fig. 2 C). Thus, the results suggest that ROS may be generated in the mitochondria upon cyt c release and that this is prevented by Bcl-2 in association with its inhibition of cyt c release. Babior and co-workers (30Krippner A. Matsuno-Yagi A. Gottlieb R.A. Babior B.M. J. Biol. Chem. 1996; 271: 21629-21636Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 34Adachi S. Cross A.R. Babior B.M. Gottlieb R.A. J. Biol. Chem. 1997; 272: 21878-21882Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) found that substrate-dependent mitochondrial O2consumption was inhibited with loss of cyt c in a Fas-activated model of apoptosis. Measurements in mitochondria of neo cells treated with staurosporine also showed that substrate-stimulated O2 consumption was inhibited (Fig.3 A). Thus, the loss of cytc is associated with an interruption of normal electron flow that could divert electron transfer to the generation of superoxide. Activation of the mitochondrial permeability transition (MPT) is a major controlling mechanism in some apoptotic systems (for review, see Ref. 35Hrish T. Marzo I. Kroemer G. Biosci. Rep. 1997; 17: 67-76Crossref PubMed Scopus (194) Google Scholar), and this could also contribute to cyt c release (data not shown) and superoxide production (36Backway K.L. McCulloch E.A. Chow S. Hedley D.W. Cancer Res. 1997; 57: 2446-2451PubMed Google Scholar, 37Marchetti P. Decaudin D. Macho A. Zamzami N. Hirsch T. Susin S.A. Kroemer G. Eur. J. Immunol. 1997; 27: 289-296Crossref PubMed Scopus (198) Google Scholar). To determine whether the MPT had occurred in neo cells treated with staurosporine, we used FACS analysis with rhodamine 123 or DiOC6. Results showed that Δψ was largely retained under conditions where cytc was released and the redox signal was generated (Fig. 3,B and C). At later times, the Δψ was lost (Ref. 21Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T-I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4452) Google Scholar and data not shown), indicating that the redox change preceded the MPT and was not a result of MPT. To determine whether mitochondria that have lost cyt cduring apoptotic signaling have increased substrate-dependent superoxide production, we measured stimulated superoxide production in mitochondria isolated from staurosporine-treated cells. Results showed that superoxide generation was enhanced in mitochondria isolated from staurosporine-treated neo cells. By 4 h, the rate was 5.28 ± 0.33 nmol/mg protein per min (Fig. 4 A). This was dependent upon respiratory substrate and was inhibited by superoxide dismutase. To determine whether superoxide production could be blocked by preventing cytochrome c release, superoxide production by mitochondria from staurosporine-treated bcl-2 cells was measured. Results showed that mitochondria from cells overexpressing Bcl-2 had no increase in superoxide production following staurosporine treatment (Fig. 4 B). Addition of cyanide, a direct mitochondrial electron transport inhibitor, resulted in superoxide generation in mitochondria from Bcl-2-overexpressing cells at a rate similar to that from neo cells. Thus, the results show that the effect of Bcl-2 was not due to a direct electron-scavenging or superoxide-metabolizing activity of Bcl-2 itself but rather to the prevention of O⨪2production. These observations demonstrate that the antioxidant activity of Bcl-2 is indirect and clarify an important issue concerning Bcl-2 function. However, this finding also raises a fundamental question about the redox signaling in cells. The redox state of the GSH/GSSG pool is substantially oxidized (−239 mV) compared with the NADPH pool (−394 mV) (38Sies H. Sies H. Metabolic Compartmentation. Academic Press, London1982: 205-231Google Scholar) that drives GSSG reduction. If the steady stateEh of GSH/GSSG reflects a balance between the rate of GSH oxidation by mitochondrially derived oxidants (e.g. superoxide and H2O2) and the rate of GSSG reduction by NADPH, then it places Bcl-2 at a pivotal point in regulation of the cellular thiol disulfide redox state by controlling the rate of mitochondrial generation of ROS (Fig. 5). The present demonstration of a mechanism for ROS generation during apoptosis does not resolve whether ROS has a role in apoptosis signaling. Despite the fact that an oxidant signal occurs during apoptosis by diverse stimuli, so far the available pieces of evidence suggest that caspase activation is irrelevant to the redox state.In vitro data using cell-free systems indicated that caspase 3 activation was independent of the redox state of cyt c(39Kluck R.M. Martin S.J. Hoffman B.M. Zhou J.S. Green D.R. Newmeyer D.D EMBO J. 1997; 16: 4639-4649Crossref PubMed Scopus (361) Google Scholar, 40Hampton M.B. Zhivotovsky B. Slater A.F.G. 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The superoxide generated could function as a mechanism to inactivate caspases and prevent their destruction of cells that phagocytose apoptotic bodies (41Hampton M.B. Orrenius S. FEBS. Lett. 1997; 414: 552-556Crossref PubMed Scopus (599) Google Scholar). ROS may also counteract the lethal effects of caspases in non-apoptotic cells. Caspase 3 has recently been found to function in the processing of pro-interleukin 16, a non-apoptotic function that would appear to require some low level of constitutive activation (43Zhang Y. Center D.M. Wu M.H. Cruikshank W.W. Yuan J. Andrews D.W. Kornfeld H. J. Biol. Chem. 1998; 273: 1144-1149Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). If low levels of cyt c release are involved in this process, a concomitant superoxide signal may activate protective mechanisms, such as the activation of the redox-sensitive transcription factor NF-κB (44Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2460) Google Scholar, 45Liu Z.-G. Hsu H. Goeddel D.V. Michael K. Cell. 1996; 87: 565-576Abstract Full Text Full Text PDF PubMed Scopus (1788) Google Scholar). In TNF-α-induced apoptosis, NF-κB has been shown to control the expression of several protective genes, such as c-IAP2 and MnSOD (13Iwata S. Hori T. Sata N. Hirota K. Sasada T. Mitsui A. Hirakawa T. Yodoi J. J. Immunol. 1997; 158: 3108-3117PubMed Google Scholar,14Wong G.H. Elwell J.H. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (796) Google Scholar). Despite the phosphorylation of I-κB, the intracellular redox state is known to be another regulatory mechanism of NF-κB (46Staal F.J. Roederer M. Herzenberg L.A. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9943-9947Crossref PubMed Scopus (904) Google Scholar). Alternatively, the switch of mitochondrial electron transport from its normal pathway through cyt c, which does not result in free radical production, to one in which electrons are transferred to O2 with production of superoxide, may provide a fail-safe mechanism that complements cyt c-dependent caspase activation for the execution of cell death. The dramatic 75-mV oxidation of the cellular GSH/GSSG pool (Fig. 1 A) is a sufficient signal to result in a 500-fold change in the function of systems regulated by vicinal dithiols with Eo in an appropriate range (47Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar). Thus, global oxidation due to the electron transfer switch triggered by cyt c release could provide a general backup mechanism for a redox-activated molecular machinery of apoptosis. One example of such a mechanism could be the activation of MPT. The MPT pore has critical thiols, which upon oxidation can trigger its opening (48Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2211) Google Scholar) and release the apoptosis-inducing factor (35Hrish T. Marzo I. Kroemer G. Biosci. Rep. 1997; 17: 67-76Crossref PubMed Scopus (194) Google Scholar, 49Susin S.A. Zamzami N. Castedo M. Hirsh T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1038) Google Scholar). Although not purified yet, apoptosis-inducing factor seems also to be a cysteine protease and can induce nuclear apoptosis in cell-free systems. Oxidants, such as diamide andtert-butylhydroperoxide, can induce its release from isolated mitochondria (49Susin S.A. Zamzami N. Castedo M. Hirsh T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1038) Google Scholar). The time course of mtΔψ change (Fig. 3) indicated that MPT does not contribute significantly to the generation of superoxide in staurosporine-induced apoptosis in HL 60 cells, but rather superoxide production may subsequently activate the MPT in a final common pathway of cell death. We thank Prof. Sten Orrenius for critical reading and helpful comments; Dr. Ronald Jemmerson (University of Minnesota) for kindly providing the anti-cytochrome cantibody; Nyoka Roberts for manuscript preparation; and Dr. Jie Yang and Kasey Nelson for technical support.
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