Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8 -10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 M) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10 -15 M inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.Cellular senescence is defined as the limited capacity of cells to undergo population doublings (1); consequently, cells have a finite life span beyond which they can no longer proliferate. This finite life span correlates with the age of the organism and with the life expectancy of the species from which the cells were obtained; such that the older the age or the shorter the life span, the less the ability of the cells to undergo population doubling (1).Several important observations have been made in understanding the senescent phenotype. Senescence is a dominant process as demonstrated by cell fusion experiments demonstrating that the resultant heterokaryons have a finite life span (2) and by the presence of factors from senescent cells that inhibit DNA synthesis in young cells (3). Senescence also appears to be an "irreversible" process, although it may be overridden by DNA tumor viruses leading to proliferation (4). Several known biochemical parameters of senescence are beginning to shed light on the underlying mechanisms involved in this developmental program. These include lack of c-fos transcription (5) and AP-1 activation (6), presence of the Rb protein in a predominantly dephosphorylated form (7), and the occurrence of several alterations in cell cycle pr...

The novel lipid second messenger, ceramide, specifically induced poly(ADP-ribose) polymerase cleavage through activation of the protease prICE. Over-expression of Bcl-2 inhibited ceramide-induced poly(ADP-ribose) polymerase proteolysis and protected cells from ceramide-induced death. These data provide the first insight into the mechanism by which ceramide mediates apoptosis and suggest a mechanism by which Bel-2 protects from cell death.

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 apoptosis. In this study, we investigated the relationship between p53 and ceramide. We found that treatment of Molt-4 cells with low concentrations of actinomycin D or ␥ -irradiation, which activate p53-dependent apoptosis, induces apoptosis only in cells expressing normal levels of p53. In these cells, p53 activation was followed by a dose-and time-dependent increase in endogenous ceramide levels which was not seen in 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 suppression induced by TNF-␣ or serum deprivation, ceramide accumulated irrespective 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-suppressive concentrations. Also, when cells lacking functional p53, either due to mutation or the expression of the E6 protein of human papilloma virus, were treated with exogenous ceramide, there was equal growth 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 situations where p53 performs a critical regulatory role, such as the response to genotoxic stress, it functions "upstream" of ceramide. These studies begin to define a relationship between these two pathways of growth inhibition. ( J. Clin. Invest. 1998. 102: 329-339.)