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Biochem J. Jul 15, 2001; 357(Pt 2): 407–416.
PMCID: PMC1221967

Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis.

Abstract

To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the response kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly sensitive Jurkat cell line, early caspase-8 activation, observed from 2 h after treatment, was chronologically associated with an acute depletion of glutathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PARP), followed by a progressive fall in the mitochondrial transmembrane potential (Delta(psi)m), between 4 and 48 h after treatment. Ceramide levels began to increase 2 h after the addition of anti-Fas (with no increase during the first hour), and increased continuously to 640% of control cells at 48 h. In the moderately sensitive SCC61 adherent cells, comparable results were observed, though with lower levels of ceramide and a delay in the response kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2 h, and a sustained level of activation until 48 h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. This was confirmed by a lack of ceramide generation and mitochondrial changes, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhibition of caspase processing, and amplification of endogenous ceramide signalling by pharmacological agents, allowed us to establish the order of cellular events, locating ceramide release after caspase-8 activation and before caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provide strong arguments for the role of endogenous ceramide as a key executor of apoptosis, rather than as a consequence of membrane alterations.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996 Jun 14;85(6):803–815. [PubMed]
  • Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH, Peter ME. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 1995 Nov 15;14(22):5579–5588. [PMC free article] [PubMed]
  • Schlegel J, Peters I, Orrenius S, Miller DK, Thornberry NA, Yamin TT, Nicholson DW. CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem. 1996 Jan 26;271(4):1841–1844. [PubMed]
  • Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 2000 Sep;10(9):369–377. [PubMed]
  • Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998 Mar 16;17(6):1675–1687. [PMC free article] [PubMed]
  • Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 Nov 14;91(4):479–489. [PubMed]
  • Liu G, Kleine L, Hébert RL. Advances in the signal transduction of ceramide and related sphingolipids. Crit Rev Clin Lab Sci. 1999 Dec;36(6):511–573. [PubMed]
  • Tepper AD, Cock JG, de Vries E, Borst J, van Blitterswijk WJ. CD95/Fas-induced ceramide formation proceeds with slow kinetics and is not blocked by caspase-3/CPP32 inhibition. J Biol Chem. 1997 Sep 26;272(39):24308–24312. [PubMed]
  • Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. Ordering of ceramide formation, caspase activation, and mitochondrial changes during CD95- and DNA damage-induced apoptosis. J Clin Invest. 1999 Apr;103(7):971–978. [PMC free article] [PubMed]
  • Gudz TI, Tserng KY, Hoppel CL. Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J Biol Chem. 1997 Sep 26;272(39):24154–24158. [PubMed]
  • García-Ruiz C, Colell A, Marí M, Morales A, Fernández-Checa JC. Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione. J Biol Chem. 1997 Apr 25;272(17):11369–11377. [PubMed]
  • Quillet-Mary A, Jaffrézou JP, Mansat V, Bordier C, Naval J, Laurent G. Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis. J Biol Chem. 1997 Aug 22;272(34):21388–21395. [PubMed]
  • Ghafourifar P, Klein SD, Schucht O, Schenk U, Pruschy M, Rocha S, Richter C. Ceramide induces cytochrome c release from isolated mitochondria. Importance of mitochondrial redox state. J Biol Chem. 1999 Mar 5;274(10):6080–6084. [PubMed]
  • Raisova M, Bektas M, Wieder T, Daniel P, Eberle J, Orfanos CE, Geilen CC. Resistance to CD95/Fas-induced and ceramide-mediated apoptosis of human melanoma cells is caused by a defective mitochondrial cytochrome c release. FEBS Lett. 2000 May 4;473(1):27–32. [PubMed]
  • Rodriguez-Lafrasse C, Rousson R, Pentchev PG, Louisot P, Vanier MT. Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders. Biochim Biophys Acta. 1994 May 25;1226(2):138–144. [PubMed]
  • Merrill AH, Jr, Wang E, Mullins RE, Jamison WC, Nimkar S, Liotta DC. Quantitation of free sphingosine in liver by high-performance liquid chromatography. Anal Biochem. 1988 Jun;171(2):373–381. [PubMed]
  • Neuschwander-Tetri BA, Roll FJ. Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-orthophthalaldehyde adduct. Anal Biochem. 1989 Jun;179(2):236–241. [PubMed]
  • Boesen-de Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation. J Biol Chem. 1999 May 14;274(20):14255–14261. [PubMed]
  • van den Dobbelsteen DJ, Nobel CS, Schlegel J, Cotgreave IA, Orrenius S, Slater AF. Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody. J Biol Chem. 1996 Jun 28;271(26):15420–15427. [PubMed]
  • Ekert PG, Silke J, Vaux DL. Caspase inhibitors. Cell Death Differ. 1999 Nov;6(11):1081–1086. [PubMed]
  • Lee L, Abe A, Shayman JA. Improved inhibitors of glucosylceramide synthase. J Biol Chem. 1999 May 21;274(21):14662–14669. [PubMed]
  • Albouz S, Vanier MT, Hauw JJ, Le Saux F, Boutry JM, Baumann N. Effect of tricyclic antidepressants on sphingomyelinase and other sphingolipid hydrolases in C6 cultured glioma cells. Neurosci Lett. 1983 Apr 29;36(3):311–315. [PubMed]
  • Michael JM, Lavin MF, Watters DJ. Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling. Cancer Res. 1997 Aug 15;57(16):3600–3605. [PubMed]
  • Chmura SJ, Nodzenski E, Beckett MA, Kufe DW, Quintans J, Weichselbaum RR. Loss of ceramide production confers resistance to radiation-induced apoptosis. Cancer Res. 1997 Apr 1;57(7):1270–1275. [PubMed]
  • Tepper CG, Jayadev S, Liu B, Bielawska A, Wolff R, Yonehara S, Hannun YA, Seldin MF. Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8443–8447. [PMC free article] [PubMed]
  • Cifone MG, De Maria R, Roncaioli P, Rippo MR, Azuma M, Lanier LL, Santoni A, Testi R. Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J Exp Med. 1994 Oct 1;180(4):1547–1552. [PMC free article] [PubMed]
  • Perry DK, Hannun YA. The use of diglyceride kinase for quantifying ceramide. Trends Biochem Sci. 1999 Jun;24(6):226–227. [PubMed]
  • Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science. 1996 Dec 13;274(5294):1855–1859. [PubMed]
  • Zhang J, Alter N, Reed JC, Borner C, Obeid LM, Hannun YA. Bcl-2 interrupts the ceramide-mediated pathway of cell death. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5325–5328. [PMC free article] [PubMed]
  • Gamen S, Anel A, Piñeiro A, Naval J. Caspases are the main executioners of Fas-mediated apoptosis, irrespective of the ceramide signalling pathway. Cell Death Differ. 1998 Mar;5(3):241–249. [PubMed]
  • Laouar A, Glesne D, Huberman E. Involvement of protein kinase C-beta and ceramide in tumor necrosis factor-alpha-induced but not Fas-induced apoptosis of human myeloid leukemia cells. J Biol Chem. 1999 Aug 13;274(33):23526–23534. [PubMed]
  • Tepper AD, Diks SH, van Blitterswijk WJ, Borst J. Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens. J Biol Chem. 2000 Nov 3;275(44):34810–34817. [PubMed]
  • Cock JG, Tepper AD, de Vries E, van Blitterswijk WJ, Borst J. CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingomyelinase. J Biol Chem. 1998 Mar 27;273(13):7560–7565. [PubMed]
  • Lin T, Genestier L, Pinkoski MJ, Castro A, Nicholas S, Mogil R, Paris F, Fuks Z, Schuchman EH, Kolesnick RN, et al. Role of acidic sphingomyelinase in Fas/CD95-mediated cell death. J Biol Chem. 2000 Mar 24;275(12):8657–8663. [PubMed]
  • Gulbins E, Bissonnette R, Mahboubi A, Martin S, Nishioka W, Brunner T, Baier G, Baier-Bitterlich G, Byrd C, Lang F, et al. FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway. Immunity. 1995 Apr;2(4):341–351. [PubMed]
  • Brenner B, Ferlinz K, Grassmé H, Weller M, Koppenhoefer U, Dichgans J, Sandhoff K, Lang F, Gulbins E. Fas/CD95/Apo-I activates the acidic sphingomyelinase via caspases. Cell Death Differ. 1998 Jan;5(1):29–37. [PubMed]
  • Lozano J, Menendez S, Morales A, Ehleiter D, Liao WC, Wagman R, Haimovitz-Friedman A, Fuks Z, Kolesnick R. Cell autonomous apoptosis defects in acid sphingomyelinase knockout fibroblasts. J Biol Chem. 2001 Jan 5;276(1):442–448. [PubMed]
  • Bezombes C, Ségui B, Cuvillier O, Bruno AP, Uro-Coste E, Gouazé V, Andrieu-Abadie N, Carpentier S, Laurent G, Salvayre R, et al. Lysosomal sphingomyelinase is not solicited for apoptosis signaling. FASEB J. 2001 Feb;15(2):297–299. [PubMed]
  • Chiba T, Takahashi S, Sato N, Ishii S, Kikuchi K. Fas-mediated apoptosis is modulated by intracellular glutathione in human T cells. Eur J Immunol. 1996 May;26(5):1164–1169. [PubMed]
  • Hentze H, Künstle G, Volbracht C, Ertel W, Wendel A. CD95-Mediated murine hepatic apoptosis requires an intact glutathione status. Hepatology. 1999 Jul;30(1):177–185. [PubMed]
  • Liu B, Hannun YA. Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione. J Biol Chem. 1997 Jun 27;272(26):16281–16287. [PubMed]
  • Decaudin D, Geley S, Hirsch T, Castedo M, Marchetti P, Macho A, Kofler R, Kroemer G. Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res. 1997 Jan 1;57(1):62–67. [PubMed]
  • Bossy-Wetzel E, Green DR. Caspases induce cytochrome c release from mitochondria by activating cytosolic factors. J Biol Chem. 1999 Jun 18;274(25):17484–17490. [PubMed]
  • Watson RW, O'Neill A, Brannigan AE, Brannigen AE, Coffey R, Marshall JC, Brady HR, Fitzpatrick JM. Regulation of Fas antibody induced neutrophil apoptosis is both caspase and mitochondrial dependent. FEBS Lett. 1999 Jun 18;453(1-2):67–71. [PubMed]
  • Grullich C, Sullards MC, Fuks Z, Merrill AH, Jr, Kolesnick R. CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis. J Biol Chem. 2000 Mar 24;275(12):8650–8656. [PubMed]
  • Borner C, Monney L. Apoptosis without caspases: an inefficient molecular guillotine? Cell Death Differ. 1999 Jun;6(6):497–507. [PubMed]
  • Juo P, Woo MS, Kuo CJ, Signorelli P, Biemann HP, Hannun YA, Blenis J. FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth Differ. 1999 Dec;10(12):797–804. [PubMed]

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