Abshead
The present study was designed to examine the roles of p53, reactive oxygen species (ROS), and ceramide, and to determine their mutual relationships during tumor necrosis factor (TNF)-α-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-α stimulated ceramide formation via the activation of both neutral and acid sphingomyelinases (SMases), accompanied by superoxide anion (O2−•) production, and induced mitochondrial depolarization and cytochrome c release, whereas p53-deficient cells were partially resistant to TNF-α and lacked O2−• generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-α by accumulation of O2−•. z-IETD-fmk (benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone), but not z-DEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone), blocked TNF-α-induced ceramide formation through both SMases as well as O2−• generation. Caspase-8 was processed by TNF-α regardless of p53 status of cells or the presence of antioxidants. Two separate signaling cascades, p53-mediated ROS-dependent and -independent pathways, both of which are initiated by caspase-8 activation, thus contribute to ceramide formation in TNF-α-induced apoptosis of human glioma cells.
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Abbreviations
- ROS:
-
reactive oxygen species
- O2−•:
-
superoxide anion
- H2O2:
-
hydrogen peroxide
- •OH:
-
hydroxyl radical
- TNF:
-
tumor necrosis factor
- SM:
-
sphingomyelin
- SMase:
-
sphingomyelinase
- N-SMase:
-
neutral sphingomyelinase
- A-SMase:
-
acid sphingomyelinase
- HPV:
-
human papillomavirus
- CHX:
-
cycloheximide
- SR33557:
-
((2-isopropyl-1-(4- [3-N-methyl-N- (3,4-dimethoxy-beta-phenethyl) amino] propyloxy)-benzenesulfonyl))indolizine
- FB1:
-
fumonisin B1
- SOD:
-
superoxide dismutase
- HE:
-
hydroethidium
- DCFH-DA:
-
2′,7′-dichlorofluorescein diacetate
- DCF:
-
2′,7′-dichlorofluorescein
- GSH:
-
reduced glutathione
- MTp53ts:
-
temperature-sensitive human p53 val138 mutant
- Δψm:
-
mitochondrial transmembrane potential
- DiOC6(3):
-
3,3′-dihexyloxacarbocyanine iodide
- z-IETD-fmk:
-
benzyloxycarbonyl-Ile-Glu-Thr-Asp fluoromethyl ketone
- z-DEVD-fmk:
-
benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone
- CA074 Me:
-
[L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester
- NAC:
-
N-acetylcysteine
- shRNA:
-
short hairpin RNA
- DMEM:
-
Dulbecco's modified Eagle's medium
- FBS:
-
fetal bovine serum
- HPTLC:
-
high-performance thin-layer chromatography
- SDS-PAGE:
-
sodium dodecylsulfate polyacrylamide gel electrophoresis
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Acknowledgements
We are grateful to Dr. T Takahashi (Aichi Cancer Center, Japan) for wild-type human p53 plasmid, to Dr. Y Takeuchi (Chester Beatty Laboratories, ICR, UK) for FLYA13 cells, to Dr. J-P Jaffrezou (Claudius Regaud Center, France) for SR33557, and to Dr. T Ogita (Sankyo Co. Ltd, Tokyo, Japan) for scyphostatin. This work was supported in part by the Research Fellowships of the Japan Society for the Promotion of Science for M Sawada (JSPS Research Fellowships for M Sawada). This work was also supported by Grants-in-Aid for Scientific Research (B) (14370429) and Cancer Research (14026065) from The Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Sawada, M., Kiyono, T., Nakashima, S. et al. Molecular mechanisms of TNF-α-induced ceramide formation in human glioma cells:P53-mediated oxidant stress-dependent and -independent pathways. Cell Death Differ 11, 997–1008 (2004). https://doi.org/10.1038/sj.cdd.4401438
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DOI: https://doi.org/10.1038/sj.cdd.4401438
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