P53-DEPENDENT CERAMIDE RESPONSE TO GENOTOXIC STRESS

Both p53 and ceramide have been implicated in the regulation of growth suppression, p53 has been proposed as the ''guardian of the genome'' and ceramide has been suggested as a ''tumor suppressor lipid.'' Both molecules appear to regulate cell cycle arrest, senescence, and apopto sis. In this study, we investigated the relationship between p53 and c eramide. We found that treatment of Molt-4 cells with low concentratio ns of actinomycin D or gamma-irradiation, which activate p53-dependent apoptosis, induces apoptosis only in cells expressing normal levels o f p53. In these cells, p53 activation was followed by a dose- and time -dependent increase in endogenous ceramide levels which was not seen i n cells lacking functional p53 and treated similarly. Similar results were seen in irradiated L929 cells whereby the p53-deficient clone was significantly more resistant to irradiation and exhibited no ceramide response. However, in p53-independent systems, such as growth suppres sion induced by TNF-alpha or serum deprivation, ceramide accumulated i rrespective of the upregulation of p53, indicating that p53 regulates ceramide accumulation in only a subset of growth-suppressive pathways. Finally, ceramide did not increase p53 levels when used at growth-sup pressive concentrations. Also, when cells lacking functional p53, eith er due to mutation or the expression of the E6 protein of human papill oma virus, were treated with exogenous ceramide, there was equal growt h suppression, cell cycle arrest, and apoptosis as compared with cells expressing normal p53. These results indicate that p53 is unlikely to function ''downstream'' of ceramide. Instead, they suggest that, in s ituations where p53 performs a critical regulatory role, such as the r esponse to genotoxic stress, it functions ''upstream'' of ceramide. Th ese studies begin to define a relationship between these two pathways of growth inhibition.