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p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells

Abstract

The present study was designed to elucidate the relationship between p53 and ceramide, both of which are involved in apoptotic signaling. Treatment of human glioma cells with etoposide caused apoptosis only in cells expressing functional p53. p53 activation was followed by the formation of reactive oxygen species (ROS), superoxide anion (O2−•) measured by hydroethidium oxidation into ethidium and hydrogen peroxide (H2O2) measured by oxidation of 2′,7′-dichlorofluorescin (DCFH) into 2′,7′-dichlorofluorescein (DCF), which was accompanied with ceramide generation through the activation of neutral, but not acid, sphingomyelinase. Superoxide dismutase (SOD), a selective antioxidant for O2−•, had no effects on p53 expression but inhibited ceramide generation and apoptotic cell death caused by etoposide. However, catalase, a specific antioxidant for H2O2, only weakly inhibited and sodium formate, a hydroxyl radical ( OH) scavenger, unaffected etoposide-induced apoptosis. Like etoposide-induced cell death, treatment of glioma cells with the O2−•-releasing agent, pyrogallol, induced typical apoptosis and ceramide generation even in the presence of catalase. In contrast, human glioma cells lacking functional p53, either due to mutation or the expression of E6 protein of human papillomavirus, were highly resistant to etoposide and exhibited no significant change in the ceramide level. Moreover, expression of functional p53 protein in glioma cells expressing mutant p53 using a temperature-sensitive human p53Val138 induced ceramide accumulation by the activation of neutral sphingomyelinase which was dependent on the generation of O2−•. Taken together, these results suggest that p53 may modulate ceramide generation by activation of neutral sphingomyelinase through the formation of O2−•, but not its downstream compounds H2O2 or  OH.

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Abbreviations

A-SMase:

acid sphingomyelinase

DCF:

2′,7′-dichlorofluorescein

DCFH-DA:

2′,7′-dichlorofluorescin diacetate

DMEM:

Dulbecco's modified Eagle's medium

FBS:

fetal bovine serum

GSH:

reduced glutathione

H2O2:

hydrogen peroxide

HE:

hydroethidium

HPTLC:

high performance thin-layer chromatography

HPV:

human papillomavirus

MTp53ts:

temperature-sensitive human p53 val138 mutant

NAC:

N-acetylcysteine

N-SMase:

neutral and magnesium-dependent sphingomyelinase

O2−•:

superoxide anion

 OH:

hydroxyl radical

PDTC:

pyrrolidinedithiocarbamate

ROS:

reactive oxygen species

SDS–PAGE:

sodium dodecylsulfate polyacrylamide gel electrophoresis

SM:

sphingomyelin

SOD:

superoxide dismutase

TNF:

tumor necrosis factor.

References

  • Borek C . 1987 Br. J. Cancer 8: S74–S86

  • Brune B, Gotz C, Messmer UK, Sandau K, Hirvonen MR, Lapetina EG . 1997 J. Biol. Chem. 272: 7253–7258

  • Busciglio J, Yankner BA . 1995 Nature 378: 776–779

  • Buttke TM, Sandstrom PA . 1994 Immunol. Today 15: 7–10

  • Cifone MG, De Maria R, Roncaioli P, Rippo MR, Azuma M, Lanier LL, Santoni A, Testi R . 1994 J. Exp. Med. 180: 1547–1552

  • Cosset FL, Takeuchi Y, Battini JL, Weiss RA, Collins MKL . 1995 J. Virol. 69: 7430–7436

  • Dbaibo GS, Pushkareva MY, Rachid RA, Alter N, Smyth MJ, Obeid LM, Hannun YA . 1998 J. Clin. Invest. 102: 329–339

  • El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B . 1993 Cell 75: 817–825

  • Frankel RH, Bayona W, Koslow M, Newcomb EW . 1992 Cancer Res. 52: 1427–1433

  • Gomez-Manzano C, Fueyo J, Kyritsis AP, Steck PA, Roth JA, McDonnell TJ, Steck KD, Levin VA, Yung WKA . 1996 Cancer Res. 56: 694–699

  • Haimovitz-Friedman A, Kan CC, Ehleiter D, Persaud RS, McLoughlin M, Fuks Z, Kolesnick RN . 1994 J. Exp. Med. 180: 525–535

  • Halbert CL, Demers GW, Galloway DA . 1991 J. Virol. 65: 473–478

  • Hannun YA . 1996 Science 274: 1855–1859

  • Hartwell LH, Kastan MB . 1994 Science 266: 1821–1828

  • Hollstein M, Sidransky D, Vogelstein B, Harris CC . 1991 Science 253: 49–53

  • Huang C, Zhang Z, Ding M, Li J, Ye J, Leonard SS, Shen H-M, Butterworth L, Lu Y, Costa M, Rojanasakul Y, Castranova V, Vallyathan V, Shin X . 2000 J. Biol. Chem. 275: 32516–32522

  • Jacobson MD . 1996 Trend Biochem. Sci. 21: 83–86

  • Jaffrézou J, Levade T, Bettaïeb A, Andrieu N, Bezombes C, Maestre N, Vermeersch S, Rousse A, Laurent G . 1996 EMBO J. 15: 2417–2424

  • Jayadev S, Liu B, Bielawska AE, Lee JY, Nazaire F, Pushkareva MYU, Obeid LM, Hannun YA . 1995 J. Biol. Chem. 270: 2047–2052

  • Johnson TM, Yu ZX, Ferrans VJ, Lowenstein RA, Finkel T . 1996 Proc. Natl. Acad. Sci. USA 93: 11848–11852

  • Kiyono T, Hiraiwa A, Fujita M, Hayashi Y, Akiyama T, Ishibashi M . 1997 Proc. Natl. Acad. Sci. USA 94: 11612–11616

  • Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ . 1998 Nature 396: 84–88

  • Klingelhutz AJ, Foster SA, McDougall JK . 1996 Nature 380: 79–82

  • Kramer W, Drutsa V, Jansen HW, Kramer B, Pflugfelder M, Fritz HJ . 1984 Nucl. Acids Res. 12: 9441–9456

  • Kroemer G, Zamzami N, Susin SA . 1997 Immunol. Today 18: 45–51

  • Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB . 1992 Proc. Natl. Acad. Sci. USA 89: 7491–7495

  • Lang D, Miknyoczki SJ, Huang L, Ruggeri BA . 1998 Oncogene 16: 1593–1602

  • Lennon SV, Martin SJ, Cotter TG . 1991 Cell Prolif. 24: 203–214

  • Levine AJ . 1997 Cell 88: 323–331

  • Li PF, Dietz R, von Harsdorf R . 1999 EMBO J. 18: 6027–6036

  • Lin D, Shields MT, Ullrich SJ, Appella E, Mercer WE . 1992 Proc. Natl. Acad. Sci. USA 89: 9210–9214

  • Liu B., Andrieu-Abadie N, Levade T, Zhang P, Obeid L, Hannun YA . 1998 J. Biol. Chem. 273: 11313–11320

  • Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T . 1993 Nature 362: 847–849

  • Michalovitz D, Halevy O, Oren M . 1990 Cell 62: 671–680

  • Moreno-Manzano V, Ishikawa Y, Lucio-Cazana J, Kitamura M . 2000 J. Biol. Chem. 275: 12684–12691

  • Narayanan PK, Goodwin EH, Lehnert BE . 1997 Cancer Res. 57: 3963–3971

  • Okazaki T, Bieloawska A, Bell RM, Hannun YA . 1990 J. Biol. Chem. 265: 15823–15831

  • Polyak K, Waldman T, He TC, Kinzler KW, Vogelstein B . 1996 Genes Dev. 10: 1945–1952

  • Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B . 1997 Nature 389: 300–305

  • Ramakrishnan N, Catravas GN . 1992 J. Immunol. 148: 1817–1821

  • Rothe G, Valet G . 1990 J. Leukoc. Biol. 47: 440–448

  • Sawada M, Nakashima S, Banno Y, Yamakawa H, Takenaka K, Shinoda J, Nishimura Y, Sakai N, Nozawa Y . 2000a Oncogene 19: 3508–3520

  • Sawada M, Nakashima S, Banno Y, Yamakawa H, Hayashi K, Takenaka K, Nishimura Y, Sakai N, Nozawa Y . 2000b Cell Death Differ. 7: 761–772

  • Scheffner M, Huibregtse JM, Vierstra RD, Howley PM . 1993 Cell 75: 495–505

  • Schuler M, Bossy-Wetzel E, Goldstein JC, Fitzgerald P, Green DR . 2000 J. Biol. Chem. 275: 7337–7342

  • Schwandner R, Wiegmann K, Bernardo K, Kreder D, Kronke M . 1998 J. Biol. Chem. 273: 5916–5922

  • Tepper AD, Cock JG, de Vries E, Borst J, van Blitterswijk WJ . 1997 J. Biol. Chem. 272: 24308–24312

  • Tepper AD, de Vries E, van Blitterswijk WJ, Borst J . 1999 J. Clin. Invest. 103: 971–978

  • Vayssiere JL, Petit PX, Risler Y, Mignotte B . 1994 Proc. Natl. Acad. Sci. USA 91: 11752–11756

  • Venable ME, Lee JY, Smyth MJ, Bielawska A, Obeid LM . 1995 J. Biol. Chem. 270: 30701–30708

  • Walker PR, Smith C, Youdale T, Leblanc J, Whitfield JF, Sikorska M . 1991 Cancer Res. 51: 1078–1085

  • Xie YW, Kaminski PM, Wolin MS . 1998 Circ. Res. 82: 891–897

  • Yoshimura S, Sakai H, Ohguchi K, Nakashima S, Banno Y, Nishimura Y, Sakai N, Nozawa Y . 1997 J. Neurochem. 69: 713–720

  • Yoshimura S, Banno Y, Nakashima S, Takenaka K, Sakai H, Nishimura Y, Sakai N, Shimizu S, Eguchi Y, Tsujimoto Y, Nozawa Y . 1998 J. Biol. Chem. 273: 6921–6927

  • Yoshimura S, Banno Y, Nakashima S, Hayashi K, Yamakawa H, Sawada M, Sakai N, Nozawa Y . 1999 J. Neurochem. 73: 675–683

  • Zhang P, Liu B, Jenkins GM, Hannun YA, Obeid LM . 1997 J. Biol. Chem. 272: 9609–9612

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Acknowledgements

We are grateful to Dr T Takahashi (Aichi Cancer Center, Japan) for wild-type human p53 plasmid and to Dr Y Takeuchi (Chester Beatty Laboratories, ICR, UK) for FLYA13 cells. We also thank F Yang for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from The Ministry of Education, Science, Sports and Culture of Japan, The Naito Foundation and ONO Medical Research Foundation.

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Sawada, M., Nakashima, S., Kiyono, T. et al. p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells. Oncogene 20, 1368–1378 (2001). https://doi.org/10.1038/sj.onc.1204207

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