Oxidative Stress Inhibits Apoptosis in Human Lymphoma Cells

Apoptosis and necrosis are two forms of cell death that are induced under different conditions and that differ in morphological and biochemical features. In this report, we show that, in the presence of oxidative stress, human B lymphoma cells are unable to undergo apoptosis and die instead by a form of necrosis. This was established using the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cell lines and by measuring classical markers of apoptotic death, including cell morphology, annexin V binding, DNA ladder formation, and caspase activation. In the presence of relatively low levels of H2O2 (75–100 μm), VP-16 and A23187 were unable to induce apoptosis in these cells. Instead, the cells underwent non-apoptotic cell death with mild cytoplasmic swelling and nuclear shrinkage, similar to the death observed when they were treated with H2O2 alone. We found that H2O2 inhibits apoptosis by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which also preserve cellular ATP levels, and can be mimicked by agents such as oligomycin, which inhibit ATP synthesis. The results show that oxidants can manipulate cell death pathways, diverting the cell away from apoptosis. The potential physiological ramifications of this finding will be discussed. Apoptosis and necrosis are two forms of cell death that are induced under different conditions and that differ in morphological and biochemical features. In this report, we show that, in the presence of oxidative stress, human B lymphoma cells are unable to undergo apoptosis and die instead by a form of necrosis. This was established using the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cell lines and by measuring classical markers of apoptotic death, including cell morphology, annexin V binding, DNA ladder formation, and caspase activation. In the presence of relatively low levels of H2O2 (75–100 μm), VP-16 and A23187 were unable to induce apoptosis in these cells. Instead, the cells underwent non-apoptotic cell death with mild cytoplasmic swelling and nuclear shrinkage, similar to the death observed when they were treated with H2O2 alone. We found that H2O2 inhibits apoptosis by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which also preserve cellular ATP levels, and can be mimicked by agents such as oligomycin, which inhibit ATP synthesis. The results show that oxidants can manipulate cell death pathways, diverting the cell away from apoptosis. The potential physiological ramifications of this finding will be discussed. poly(ADP-ribose) polymerase 3-aminobenzamide 4-hydroxyquinazoline propidium iodide fluorescein isothiocyanate 7-amino-4-trifluoromethyl coumarin fluorescence-activated cell sorter carbobenzoxy GDP dissociation inhibitor Cell death can occur through several different mechanisms, which are distinguished by unique morphological and biochemical traits and which have distinct physiological ramifications. The two most widely described forms of cell death are necrosis and apoptosis (reviewed in Ref. 1Wyllie A.H. Duvall E. McGee J.O'D. Isaacson P.J. Wright N.A. Oxford Textbook of Pathology. Oxford University Press, Oxford1992: 141-157Google Scholar). Necrosis is induced by severe environmental disturbances and is characterized by swelling of the cytoplasm and cytoplasmic organelles, early rupture of the plasma membrane, clumping of the chromatin, and usually swelling of the nucleus (1Wyllie A.H. Duvall E. McGee J.O'D. Isaacson P.J. Wright N.A. Oxford Textbook of Pathology. Oxford University Press, Oxford1992: 141-157Google Scholar). Apoptosis is regarded as an active and progressive response to physiologic and pathologic stimuli (2Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1108) Google Scholar, 3Searle J. Kerr J.F.R. Bishop C.J. Pathol. Annu. 1982; 17: 229-259PubMed Google Scholar). It is characterized by early and prominent condensation of nuclear chromatin, loss of plasma membrane phospholipid asymmetry, activation of proteases and endonucleases, enzymatic cleavage of the DNA into oligonucleosomal fragments, and segmentation of the cells into membrane-bound “apoptotic bodies.” A significant physiological consequence of cell death by apoptosis is that the apoptotic bodies can be phagocytosed by nearby cells such that the contents are degraded intracellularly (4Savill J. Br. Med. Bull. 1997; 53: 491-508Crossref PubMed Scopus (284) Google Scholar). As a result, cells dying by apoptosis cause minimal disturbance to the surrounding tissue. In contrast, the rupture of necrotic cells and the release of lysosomal and other enzymes into the surrounding tissue causes further tissue destruction and inflammation (1Wyllie A.H. Duvall E. McGee J.O'D. Isaacson P.J. Wright N.A. Oxford Textbook of Pathology. Oxford University Press, Oxford1992: 141-157Google Scholar). This report examines the effects of oxidative stress on the cell death machinery. Oxidants such as superoxide, hydrogen peroxide (H2O2), and the hydroxyl radical are generated under a variety of conditions in vivo such as during acute and chronic inflammation (5Klebanoff S. Gallin J.I. Goldstein I.M. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Raven Press, New York1988: 391-444Google Scholar). Treatment of cells in vitrowith H2O2 causes DNA strand breaks (6Schraufstätter I.U. Hyslop P.A. Hinshaw D.B. Spragg R.G. Sklar L.A. Cochrane C.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4908-4912Crossref PubMed Scopus (438) Google Scholar, 7Shacter E. Beecham E.J. Covey J.M. Kohn K.W. Potter M. Carcinogenesis. 1988; 9: 2297-2304Crossref PubMed Scopus (202) Google Scholar), oxidation of lipids (8Rice-Evans C. Burdon R. Prog. Lipid Res. 1993; 32: 71-110Crossref PubMed Scopus (445) Google Scholar) and proteins (9Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1270) Google Scholar), activation of poly(ADP)-ribosylation (10Schraufstätter I.U. Hinshaw D.B. Hyslop P.A. Spragg R.G. Cochrane C.G. J. Clin. Invest. 1986; 77: 1312-1320Crossref PubMed Scopus (436) Google Scholar), and depletion of cellular energy stores (6Schraufstätter I.U. Hyslop P.A. Hinshaw D.B. Spragg R.G. Sklar L.A. Cochrane C.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4908-4912Crossref PubMed Scopus (438) Google Scholar, 11Hyslop P.A. Hinshaw D.B. Halsey Jr., W.A. Schraufstätter I.U. Sauerheber R.D. Spragg R.G. Jackson J.H. Cochrane C.G. J. Biol. Chem. 1988; 263: 1665-1675Abstract Full Text PDF PubMed Google Scholar). Depending on the concentration of H2O2employed and the type of cell being studied, the mode of cell death induced by H2O2 has been reported to be either apoptosis or necrosis (12Lennon S.V. Martin S.J. Cotter T.G. Cell Prolif. 1991; 24: 203-214Crossref PubMed Scopus (761) Google Scholar, 13Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 17: 7-10Abstract Full Text PDF Scopus (2104) Google Scholar, 14Lee Y.-J. Shacter E. Blood. 1997; 89: 4480-4492Crossref PubMed Google Scholar), with necrosis generally being reported with higher concentrations of the oxidant (12Lennon S.V. Martin S.J. Cotter T.G. Cell Prolif. 1991; 24: 203-214Crossref PubMed Scopus (761) Google Scholar, 15Gardner A.M. Xu F.-H. Fady C. Jacoby F.J. Duffy D.C. Tu Y. Lightenstein A. Free Radical Biol. Med. 1997; 22: 73-83Crossref PubMed Scopus (335) Google Scholar). In a previous study, we found that Burkitt's lymphoma cells were highly susceptible to killing by H2O2 and that overexpression of the bcl-2 oncogene, which is known to inhibit apoptosis (16Hockenbery D. Nunez G. Milliman C. Schreiber R.D. Korsmeyer S.J. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3544) Google Scholar), did not prevent H2O2-induced cell death (14Lee Y.-J. Shacter E. Blood. 1997; 89: 4480-4492Crossref PubMed Google Scholar), contrary to earlier studies suggesting that bcl-2 protects cells from oxidant-induced killing (17Hockenbery D.M. Oltvai Z.N. Yin X. Milliman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3297) Google Scholar). It was concluded that bcl-2was unable to inhibit the cell killing by H2O2because the primary form of cell death induced was non-apoptotic. In the present studies, we investigated the effects of H2O2 on the cell death machinery and found that H2O2 actually inhibits induction of apoptosis in Burkitt's lymphoma cells. That is, in the presence of H2O2, agents that normally kill Burkitt's lymphoma cells by inducing apoptosis are no longer able to do so. H2O2 inhibited all the major steps of apoptosis. The effects of H2O2 can be explained by its ability to deplete the cells of ATP. Thus, oxidative stress can manipulate the mechanism of cell death, diverting it away from apoptosis to necrosis. The Burkitt's lymphoma cell lines JLP 119, ST-486, and BL-41 were provided by Kishor Bhatia from the laboratory of Ian Magrath (NCI, National Institutes of Health, Bethesda, MD). Cells were grown in RPMI 1640 containing 10% heat-inactivated fetal calf serum, 2 mml-glutamine, and 50 μmβ-mercaptoethanol at 37 °C in 5% CO2 in air. Exponentially growing cells were harvested by centrifugation and resuspended in fresh media to achieve a culture density of 5 × 105 cells/ml. H2O2 was added to the cell suspensions at the beginning of the experiments or after a 30-min preincubation with VP-16. The cells were then incubated for 2–24 h as indicated in the text. The poly (ADP-ribose) polymerase (PARP)1 inhibitors 3-aminobenzamide (3AB) and 4-hydroxyquinazoline (4HQ) were added to cell suspensions 30 to the experiments with oligomycin, cells were and resuspended in RPMI 1640 with 10% fetal calf serum, 2 mml-glutamine, and 50 to this for 30 cells were to Cells were with and propidium iodide and using as described (14Lee Y.-J. Shacter E. Blood. 1997; 89: 4480-4492Crossref PubMed Google Scholar). A of cells were and as cells with apoptotic cells chromatin, which is highly or necrotic cells with apoptotic cells chromatin, highly or cells into two or cellular DNA was by the of G. R. E.J. Nature. PubMed Scopus Google Scholar). DNA to × were to and was using for h at 30 was as a The apoptosis from was to the Cells were on a using was using a and was at of into the was The of and other were as described by with A. P.A. J. Med. PubMed Scopus Google Scholar). cells treated were by with or and in 10% and at a concentration of for on centrifugation at × for at were to a containing at a concentration of coumarin for caspase was added to the cell × to a concentration of 50 and the was to with at 30 °C for were by 2 of or to and the levels of were using a with at and at A of × in this The for caspase were as carbobenzoxy for for for and for and for The were all in and at cell were to and to as described (14Lee Y.-J. Shacter E. Blood. 1997; 89: 4480-4492Crossref PubMed Google Scholar). with 5% were incubated with or by a with were by using the from ATP levels were using R.G. Hinshaw D.B. Hyslop P.A. Schraufstätter I.U. Cochrane C.G. J. Clin. Invest. PubMed Scopus Google Scholar). cells × that been treated with H2O2 or were by resuspended in of at °C for and on the of the a × 105 was added to of 50 and of was in a ATP were in all experiments and were in the of ATP concentrations were at using an of oligomycin, and were from caspase were from to proteins were from for for for and for and and for were from VP-16 is a which is widely for chemotherapy Annu. Rev. Biochem. PubMed Scopus Google Scholar) and is known to kill a variety of different cells by inducing apoptosis. As in VP-16 JLP Burkitt's lymphoma cells by inducing apoptosis. The of cell killing on the concentration of drug Thus, at low concentrations of drug induction of apoptosis in of the cells by the of a of the cells show morphological of apoptosis h when treated with 5 VP-16. cells also show other classical markers of apoptosis in for the 5 such as oligonucleosomal of the DNA cleavage of the activation of the of and and cleavage of poly(ADP-ribose) polymerase that the for can also be by N.A. Biochem. Sci. 1997; 22: Full Text PDF PubMed Scopus Google Scholar), and and Annu. Rev. PubMed Scopus Google Scholar). the with this is to as In to when Burkitt's lymphoma cells are treated with H2O2, the form of cell death induced is non-apoptotic. This was by measuring DNA by and a (14Lee Y.-J. Shacter E. Blood. 1997; 89: 4480-4492Crossref PubMed Google Scholar). of cell by that most of the cells treated with the as cells and the nucleus mild nuclear condensation of the morphological induced by H2O2 can be in cells and B cells treated with these cells of the classical biochemical or morphological of apoptosis (14Lee Y.-J. Shacter E. Blood. 1997; 89: 4480-4492Crossref PubMed Google Scholar) and show classical necrotic we to as The are for the Burkitt's lymphoma cell JLP results were with two other Burkitt's lymphoma cell and into the effects of oxidative stress on cell death pathways, we the of H2O2 on apoptosis. In these H2O2 was added to the cells 30 after VP-16 and the cells were to The mode of cell death was by using cell are in and results are in A. The induction of apoptosis by VP-16 2 was inhibited by of H2O2 and the cells instead by to the mode of cell death induced by H2O2 2 In H2O2 did not with and VP-16 This was by VP-16 with H2O2 for 30 the H2O2 with and then the VP-16 to the cells. Full apoptosis inducing was In with H2O2 did not the of VP-16. The ability of H2O2 to inhibit apoptosis was using two other Burkitt's lymphoma cell and BL-41 not The effects of H2O2 on cell killing be overcome by the cells with two inhibitors of poly(ADP-ribose) polymerase were 2 and not H2O2 also inhibited induction of DNA ladder by VP-16 and this was also overcome by and not The concentrations of and in these experiments are to cause of M. M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). to the cells by 2 and inhibitors also the mode of cell death induced by H2O2 from to apoptosis 2 and for results were when the calcium ionophore A23187 was to induce apoptosis instead of VP-16 apoptosis was inhibited by of and this of H2O2 was overcome by inhibitors of A of cell death induced by H2O2 is in that, at of the cells die by apoptosis and this at a concentration 50 higher concentrations (75–100 μm), the form of cell death induced is of the markers of apoptosis is the of from the to the of the plasma membrane S.J. D.M. J. Med. PubMed Scopus Google Scholar). This can be by for of the annexin V to the cell G. Blood. 1994; PubMed Google Scholar). cells undergo this early apoptotic in the presence of H2O2, cells were treated with H2O2, or for and annexin V was by cells were in of JLP cells the levels of annexin V which with of As in VP-16 induced a induction of annexin V the of a during which of the cells morphological of apoptosis. H2O2 inhibited this by and did not by induce in annexin V of apoptosis inhibited by H2O2 is the activation of (reviewed in Ref. N.A. Biochem. Sci. 1997; 22: Full Text PDF PubMed Scopus Google Scholar). In the different caspase were by cell from treated and cells with for As in was in JLP cells and was not induced further by VP-16 and were all induced by VP-16 with being the most of the cells with H2O2 this activation in all of the activation of caspase when H2O2 was added to cells and not when it was added to cell that the oxidant inhibits activation of the enzymes in vivo and not inhibit not of caspase activation by H2O2 was by of cell from cells treated for with VP-16 for cleavage of known caspase N.A. Biochem. Sci. 1997; 22: Full Text PDF PubMed Scopus Google Scholar). The in show the results using for different caspase and and a a nuclear GDP dissociation inhibitor and H2O2 did not induce cleavage of of these with the that it cells through a non-apoptotic VP-16 induced cleavage of all of the by for of the proteins by of the that cleavage of is as loss of the the cleavage is degraded in the cells and is not by have been reported by other J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar). Treatment of cells with H2O2 is known to cause a depletion of and ATP levels, in through activation of (6Schraufstätter I.U. Hyslop P.A. Hinshaw D.B. Spragg R.G. Sklar L.A. Cochrane C.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4908-4912Crossref PubMed Scopus (438) Google I.U. Hinshaw D.B. Hyslop P.A. Spragg R.G. Cochrane C.G. J. Clin. Invest. 1986; 77: 1312-1320Crossref PubMed Scopus (436) Google Scholar). The that H2O2 inhibited steps in apoptosis and that the be overcome by inhibitors that H2O2 be by cellular energy this we the effects of H2O2 on cellular ATP levels in JLP cells using a R.G. Hinshaw D.B. Hyslop P.A. Schraufstätter I.U. Cochrane C.G. J. Clin. Invest. PubMed Scopus Google Scholar). The results are in the concentration of H2O2 no on ATP levels in the cells. 50 which and is a in ATP levels, which to levels in of and which kill cells by and inhibit apoptosis cause a 30 in which low the of the was to ATP levels after an the cells of ATP In to the of H2O2 on ATP levels is for its ability to inhibit conditions were that we either prevent the in ATP and H2O2 is able to inhibit apoptosis or the in ATP with a different to this inhibits apoptosis. As in the in cellular ATP induced by be inhibited by the cells with the inhibitor results were with not The of H2O2 on cellular ATP levels be mimicked by the cells with the ATP inhibitor in prevent ATP through these to cellular ATP levels, we found that depletion of ATP with mimicked the effects of H2O2 of cellular ATP by with the effects of H2O2, suggesting that H2O2 inhibits apoptosis by cellular ATP This was at two and H2O2 cells were treated for h with 5 VP-16 in the presence of apoptosis was that cells do not under the the of cell death observed at this was also The was by depleting cellular ATP with The ability of VP-16 to induce apoptosis in the presence of H2O2 was by the in ATP with results were when cells were after an with VP-16 that the of cell death was in the presence of H2O2 or oligomycin, the mode of cell death was from apoptosis to and this be overcome by the in ATP with The results show that H2O2 inhibits the ability of agents such as the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cells. The of H2O2 on cell death is Thus, cells treated with VP-16 or A23187 in the presence of H2O2 cells treated with H2O2 alone. Cells in the presence of H2O2 are show of the classical markers of apoptosis also are not necrotic the are instead of The biochemical steps that to this nuclear are not known be they be to apoptosis and necrosis C. A. Jacoby Tu Y. Lightenstein A. J. Res. PubMed Scopus Google Scholar, Biochem. Res. PubMed Scopus Google Scholar, M. A. S. J. Med. 1997; PubMed Scopus Google Scholar). The physiological of the of oxidative stress on the apoptotic machinery will be found in cells in the presence of H2O2 induce an they are by the surrounding apoptotic cells J. Kerr J.F.R. Bishop C.J. Pathol. Annu. 1982; 17: 229-259PubMed Google Scholar, J. Br. Med. Bull. 1997; 53: 491-508Crossref PubMed Scopus (284) Google Scholar) or they cell contents into the and induce an response necrotic cells J. Kerr J.F.R. Bishop C.J. Pathol. Annu. 1982; 17: 229-259PubMed Google Scholar). In the mechanism cell death, we found that H2O2 has such a in apoptosis it by depleting cellular energy stores by a unique or in the apoptotic machinery. Apoptosis is known to be an active ATP J. Kerr J.F.R. Bishop C.J. Pathol. Annu. 1982; 17: 229-259PubMed Google Scholar, M. S. Biochem. J. PubMed Scopus Google Scholar, C. M. A. C. PubMed Scopus Google Scholar, Hinshaw D.B. Free Radical Biol. Med. PubMed Scopus Google Scholar). In the of cells treated with levels of agents will die by necrosis instead of apoptosis M. A. S. J. Med. 1997; PubMed Scopus Google Scholar, Y. S. Y. Res. 1997; Google Scholar). In these studies, were to manipulate cellular ATP levels results that that are generated under a of in vivo Free in and Oxford University Press, Scholar) can or not cells die by apoptosis. H2O2 is a oxidant it is generated from all of oxidative to its to and low H2O2 can in and of cells and through concentrations of inflammation have been to Thus, it is that this oxidant can have such a on of apoptosis. The ability of inhibitors of to ATP levels that H2O2 causes the in ATP by This as a of the DNA breaks induced by H2O2 (6Schraufstätter I.U. Hyslop P.A. Hinshaw D.B. Spragg R.G. Sklar L.A. Cochrane C.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4908-4912Crossref PubMed Scopus (438) Google Scholar, 7Shacter E. Beecham E.J. Covey J.M. Kohn K.W. Potter M. Carcinogenesis. 1988; 9: 2297-2304Crossref PubMed Scopus (202) Google Scholar, I.U. Hinshaw D.B. Hyslop P.A. Spragg R.G. Cochrane C.G. J. Clin. Invest. 1986; 77: 1312-1320Crossref PubMed Scopus (436) Google Scholar). The depletion of that of P.A. Hinshaw D.B. Halsey Jr., W.A. Schraufstätter I.U. Sauerheber R.D. Spragg R.G. Jackson J.H. Cochrane C.G. J. Biol. Chem. 1988; 263: 1665-1675Abstract Full Text PDF PubMed Google Scholar). The is of at a ATP has been not through the steps of or through oxidative The is a in ATP H2O2 can also inhibit ATP by other mechanisms, including oxidative of ATP M. D. Free Radical Biol. Med. 24: PubMed Scopus Google Scholar) and Free Radical Biol. Med. 1997; 22: PubMed Scopus Google Scholar). This or not a in of ATP to the induction of cell death by H2O2 it is not mechanism H2O2 the of ATP concentrations with and results in cell the cells die by apoptosis instead of This finding is with earlier that H2O2 can kill cells by that do not ATP depletion or activation of Full Text PDF PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). for H2O2-induced cell death have been I.U. Hinshaw D.B. Hyslop P.A. Spragg R.G. Cochrane C.G. J. Clin. Invest. PubMed Scopus Google Scholar) and on of J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. J. Clin. 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Med. 1997; 22: 73-83Crossref PubMed Scopus (335) Google Scholar). results an for these as they to of oxidant-induced cell In the of these results to other cell it is to that different cell have different to H2O2 Thus, the concentrations of H2O2 to inhibit apoptosis will from cell to As H2O2 is to ATP levels in most it is also to inhibit apoptosis in most cells. We and for of the and for