Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Apoptosis
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Blotting, Western
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Brain Neoplasms / metabolism
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Caspase 8
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Caspases / metabolism
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Cathepsin B / metabolism
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Cathepsin B / pharmacology
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Cell Line, Tumor
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Cell Nucleus / metabolism
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Ceramides / metabolism*
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Chromatography, High Pressure Liquid
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Cycloheximide / pharmacology
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Cysteine Proteinase Inhibitors / pharmacology
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Cytochromes c / metabolism
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Cytosol / metabolism
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Electrophoresis, Polyacrylamide Gel
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Enzyme Activation
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Enzyme Inhibitors / pharmacology
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Glioma / metabolism*
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Glutathione / metabolism
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Humans
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Macrolides / pharmacology
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Microscopy, Fluorescence
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Mitochondria / metabolism
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Mitosis
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Oligopeptides / pharmacology
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Oncogene Proteins, Viral / metabolism
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Oxidation-Reduction
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Oxidative Stress
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Oxygen / metabolism
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Protein Synthesis Inhibitors / pharmacology
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RNA, Small Interfering / metabolism
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Reactive Oxygen Species
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Recombinant Proteins / chemistry
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Repressor Proteins / metabolism
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Retroviridae
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Signal Transduction
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Temperature
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Time Factors
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Transfection
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Tumor Necrosis Factor-alpha / metabolism*
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Tumor Suppressor Protein p53 / metabolism*
Substances
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Ceramides
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Cysteine Proteinase Inhibitors
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E6 protein, Human papillomavirus type 16
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Enzyme Inhibitors
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Macrolides
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Oligopeptides
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Oncogene Proteins, Viral
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Protein Synthesis Inhibitors
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RNA, Small Interfering
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Reactive Oxygen Species
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Recombinant Proteins
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Repressor Proteins
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Tumor Necrosis Factor-alpha
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Tumor Suppressor Protein p53
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benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone
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benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone
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bafilomycin A1
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Cytochromes c
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Cycloheximide
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CASP8 protein, human
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Caspase 8
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Caspases
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Cathepsin B
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Glutathione
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Oxygen