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 p53^Val138 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.