Molecular mechanisms of TNF-α-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways

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)-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. © 2004 Nature Publishing Group All rights reserved.