ROLE FOR CERAMIDE IN CELL-CYCLE ARREST

The dependence of some cell types on serum factors for growth may repr esent a powerful, but poorly studied, model for antimitogenic pathways , In this study, we examine ceramide as a candidate intracellular medi ator of serum factor dependence. In Molt-4 leukemia cells, serum withd rawal caused a significant arrest in cell cycle progression (80% of ce lls in G(0)/G(1)), accompanied by a modest apoptotic cell death (12%), Serum deprivation of these cells resulted in significant sphingomyeli n hydrolysis (72%; corresponding to hydrolysis of 47 pmol/nmol phospha te), which was accompanied by a profound and progressive elevation (up to 10-15-fold) in endogenous levels of ceramide, Withdrawal of serum caused the activation of a distinct, particulate, and magnesium-depend ent sphingomyelinase. The addition of exogenous C-6-ceramide induced a dramatic arrest in the G(0)/G(1) phase of the cell cycle comparable t o the effects observed with serum withdrawal, albeit occurring much so oner, Unlike serum withdrawal, however, the addition of C-6-ceramide r esulted in more pronounced apoptosis, Because of the previously noted ability of exogenously added phorbol esters to inhibit ceramide-mediat ed apoptosis, we investigated the hypothesis that endogenous activatio n of the diacylglycerol/protein kinase C pathway may modulate the resp onse to serum withdrawal. Indeed, serum withdrawal resulted in 3-4-fol d elevation in endogenous diacylglycerol levels, The addition of exoge nous diacylglycerols resulted in selective attenuation of ceramide's e ffects on apoptosis but not on cell cycle arrest, Thus, the combinatio n of ceramide and diacylglycerol recapitulated the complex effects of serum withdrawal on cell cycle arrest and apoptosis, These studies ide ntify a novel role for ceramide in cell cycle regulation, and they may provide the first evidence for an intracellular signal transduction p athway in mammalian cells mediating cell cycle arrest, These studies a lso underscore the importance of lipid second messengers and the signi ficance of the, interplay between glycerolipid-derived and sphingolipi d-derived lipid mediators.