STEREOSELECTIVITY OF INDUCTION OF THE RETINOBLASTOMA GENE-PRODUCT (PRB) DEPHOSPHORYLATION BY D-ERYTHRO-SPHINGOSINE SUPPORTS A ROLE FOR PRB IN GROWTH SUPPRESSION BY SPHINGOSINE

Sphingosine has been shown to inhibit cell growth in many cell lines although the mechanism of this effect remains obscure. More recently, D-eythro-sphingosine has been shown to act as an early inducer of dephosphorylation of the retinoblastoma gene product (pRb) in the lymphoblastic leukemia cell Line MOLT-LF [Chao, R., Khan, W., and Hannun, Y. A. (1992) J. Biol. Chem., 267, 23459-23462]. In the current study, the role of the natural D-erythro-sphingosine in regulation of cell growth and pRb dephosphorylation was evaluated using chemically synthesized pure isomers of sphingosine. Of the four possible stereoisomers of sphingosine, D-erythro-sphingosine was most active in inducing dephosphorylation of pRb protein with an EC(50%) of 0.6 mu M whereas its enantiomer L-erythro-sphingosine was 8-fold less potent with an EC(50%) of 5 mu M. The dose responses for inhibition of cell growth were nearly identical to the EC(50%) for pRb dephosphorylation with D-erythro-sphingosine causing 50% inhibition at 0.6 mu M whereas L-erythro-sphingosine was 5-6-fold less potent. All of the stereoisomers were taken up by the cells, and the greater potency of D-erythro-sphingosine was not due to differences in cellular uptake. The metabolism of D-erythro-sphingosine was also studied to evaluate the possible role of sphingosine metabolites on regulation of retinoblastoma protein. Evidence is provided against a role for ceramide or sphingosine I-phosphate as mediators of the effects of sphingosine on pRb dephosphorylation. These results support a specific role for D-erythro-sphingosine in regulation of phosphorylation of pRb and provide evidence for a role of pRb dephosphorylation in mediating the growth inhibitory effects of sphingosine.