Ceramide in rafts (detergent-insoluble fraction) mediates cell death in neurotumor cell lines

Detergent-resistant lipid microdomains (Rafts) were isolated from human oligodendroglioma (HOG), human neuroblastoma (LA-N-5), and immortalized dorsal root ganglion (F-11) cell lines by sucrose-density gradient ultracentrifugation and shown to be enriched in cholesterol, sphingomyelin, and ceramide. [H-3]palmitate labeling allowed the Raft fraction to be easily identified as a sharp peak of H-3 radioactivity in the 5-30% sucrose interphase. Treatment of [H-3] palmitate-labeled cells with staurosporine (to activate caspase 8 and induce apoptosis) or exogenous sphingomyelinase specifically increased the [H-3]ceramide content of the Raft fraction. Depletion of cholesterol with beta-methylcyclodextran decreased Raft formation and partially blocked staurosporine-induced apoptosis. Similarly, treatment of cells with Fumonisin B1 to inhibit de novo sphingolipid synthesis by 50% reduced the labeling of the Raft fraction and partially blocked staurosporine-induced apoptosis. Staurosporine treatment activated neutral sphingomyelinase but had no effect on acid sphingomyelinase activity or on other lysosomal hydrolases, such as alpha-L-fucosidase. Most of the neutral sphingomyelinase activity is in the Raft fraction, suggesting that the conversion of sphingomyelin to ceramide in Rafts is an important event in neural cell apoptosis. (C) 2003 Wiley-Liss, Inc.