title: Ceramide in rafts (detergent-insoluble fraction) mediates cell death in neurotumor cell lines.
abstract: 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. [(3)H]palmitate labeling allowed the Raft fraction to be easily identified as a sharp peak of (3)H radioactivity in the 5-30% sucrose interphase. Treatment of [(3)H]palmitate-labeled cells with staurosporine (to activate caspase 8 and induce apoptosis) or exogenous sphingomyelinase specifically increased the [(3)H]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.
authors.affiliation: Departments of Pediatrics, Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 30637, USA.
authors.forename: John
authors.initials: J
authors.lastname: Kilkus
authors.forename: Rajendra
authors.initials: R
authors.lastname: Goswami
authors.forename: Fernando D
authors.initials: FD
authors.lastname: Testai
authors.forename: Glyn
authors.initials: G
authors.lastname: Dawson
mesh_terms: D016923:Cell Death / Q000502:physiology; D002460:Cell Line; D002518:Ceramides / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D009837:Oligodendroglioma / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism
chemical_list: D002518:Ceramides