Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways.
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)-alpha-induced apoptosis of human glioma cells. In cells possessing wild-type p53, TNF-alpha 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-alpha and lacked O2-* generation and neutral SMase activation. Restoration of functional p53 sensitized glioma cells expressing mutant p53 to TNF-alpha 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-alpha-induced ceramide formation through both SMases as well as O2-* generation. Caspase-8 was processed by TNF-alpha 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-alpha-induced apoptosis of human glioma cells.
Sawada
M
M
Department of Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan. sawasawa@cc.gifu-u.ac.jp
Kiyono
T
T
Nakashima
S
S
Shinoda
J
J
Naganawa
T
T
Hara
S
S
Iwama
T
T
Sakai
N
N
D017209:Apoptosis; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002518:Ceramides / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008938:Mitosis; D009842:Oligopeptides / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism; D012190:Retroviridae; D015398:Signal Transduction; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; C068651:E6 protein, Human papillomavirus type 16; D004791:Enzyme Inhibitors; D018942:Macrolides; D009842:Oligopeptides; D009856:Oncogene Proteins, Viral; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; C040929:bafilomycin A1; D045304:Cytochromes c; D003513:Cycloheximide; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D002401:Cathepsin B; D005978:Glutathione; D010100:Oxygen