THE FORMATION OF CERAMIDE FROM SPHINGOMYELIN IS ASSOCIATED WITH CELLULAR APOPTOSIS

The apoptotic response of the immature E-cell to the cross-linking of surface IgM receptors provides a good model for cell death and we show in WEHI-231 B-cells that the time course of apoptosis corresponds to the increased formation of ceramide, as measured either by mass (using the diacylglycerol kinase method) or radiolabelling with [H-3]palmita te. inhibitors of sphingosine biosynthesis have no effect on cell deat h induced by anti-IgM in WEHI-231 but inhibitors of ceramidase acceler ate apoptosis, suggesting that activation of sphingomyelinase is the k ey event in apoptosis. We have demonstrated this by in vitro assay of neutral sphingomyelinase. Apoptosis is also important in normal brain development and neuronal survival is dependent upon phosphatidylinosit ol 3-kinase (PI3-kinase) activation by growth factors (insulin, nerve growth factor etc.). Withdrawal of these growth factors or inhibition of PI3-kinase with wortmannin or LY294002 activated the pro-apoptotic CPP32 (Yama/Apopain/caspase 3, EC 3.4.22), activated neutral sphingomy elinase and increased ceramide formation in an immortalized dorsal roo t ganglion cell line F-ll. Protection against apoptosis can be achieve d by overexpression of the bcl2 family of proteins or addition of drug s which elevate cAMP levels, cAMP protects against apoptosis induced b y either wortmannin or staurosporine. The specificity for cAMP was con firmed by showing protection with the specific agonist (Sp)cAMPS and i ncreased killing with the antagonist (Rp)cAMPS. However, cAMP did not protect against ceramide killing, suggesting that there are at least t wo major pathways of apoptosis in neuronal cells.