ABSTRACT The mechanism of cell death triggered by C 2 -ceramide was investigated using the NB16 neuroblastoma cell line. Treatment of NB16 cells with 20 M C 2 -ceramide for 20 h resulted in approximately 75% loss of cell viability, but only 25% of cells were scored as apoptotic based on terminal deoxynucleotidyl transferase nick-end labeling. Ultrastructural analysis revealed early development of necrotic cytoplasmic vacuolization. After 20 h of treatment with C 2 -ceramide, the majority of cells possessed necrotic morphology with pronounced cytoplasmic vacuolization and without any nuclear changes, although a quarter of the cell population also exhibited clear perinuclear chromatin condensation characteristic of apoptosis. Flow cytometric analysis of cells labeled with both annexin V and propidium iodide showed the rapid accumulation of C 2 -ceramide-treated cells in the necrotic/ late apoptotic fraction. In contrast, cells treated with tumor necrosis factor ␣ plus cycloheximide (TNF␣ ϩ CHX) first appeared in the early apoptotic fraction and then accumulated in the necrotic/late apoptotic fraction. Both C 2 -ceramide and TNF␣ ϩ CHX increased caspase 8-and 3-like activities in cytosolic extracts; however, treatment of cells with the broad-spectrum caspase inhibitor Nbenzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected NB16 cells from TNF␣ ϩ CHX-induced cell death but did not prevent C 2 -ceramide cytotoxicity. Although C 2 -ceramide triggered apoptosis in a fraction of the cells, cell death in the population was primarily caused by necrosis. Thus, C
