Staurosporine induces programmed cell death in embryonic neurons and activation of the ceramide pathway.

We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1-1.0 microM) and observed the meporphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis. N-Acylsphingosine (C2-ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [3H]-palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 microM), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that formation of ceramide from sphingomyelin is a key event in staurosporine-induced and potentially all programmed cell death.