RETINOBLASTOMA GENE-PRODUCT AS A DOWNSTREAM TARGET FOR A CERAMIDE-DEPENDENT PATHWAY OF GROWTH ARREST

Ceramide, a lipid mediator, has been most closely associated with anti proliferative activities. In this study, we examine the mechanism by w hich ceramide induces growth suppression and the role of the retinobla stoma gene product (Rb) in this process, Withdrawal of serum from the serum-dependent MOLT-4 cells resulted in significant dephosphorylation of Rb, correlating with the induction of G(0)/G(1) cell cycle arrest, Serum withdrawal resulted in marked elevation in the levels of endoge nous ceramide (3-fold at 24 h and 8-fold at 96 h) with little changes in the endogenous levels of sphingosine. The addition of exogenous C-6 -ceramide resulted in a concentration- and time-dependent dephosphoryl ation of Rb, Exogenous ceramide was active at levels comparable to end ogenous levels achieved with serum withdrawal, Peak activity of exogen ous ceramide (at 6 h) correlated with the uptake of C-6-ceramide by MO LT-4 cells, Next, a number of studies were conducted to determine whet her Rb plays a role in ceramide-induced growth suppression, (i) C-6-Ce ramide was poorly active in growth suppression of retinoblastoma cells that lack Rb, (ii) Mink lung epithelial cells in which Rb had been se questered by overexpression of large tumor antigen were resistant to t he action of ceramide compared to cells transfected with large tumor a ntigen mutated in the Rb-binding pocket, (iii) Overexpression of the E 1A adenoviral protein, which binds and sequesters Rb, resulted in prot ection from growth suppression and cell cycle arrest induced by cerami de, Thus, these studies demonstrate that Rb is a downstream target for ceramide and may function in a growth suppressor pathway resulting in cell cycle arrest.