INDUCTION OF PROGRAMMED CELL-DEATH IN HUMAN RETINOBLASTOMA Y79 CELLS BY C-2-CERAMIDE

C-2-ceramide, a cell-permeable analogue of ceramide, induced significa nt, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characte rized by microscopic evidence of cell shrinkage, membrane blebbing, nu clear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexi n V-FITC. Apoptosis was also assessed by monitoring changes in cell gr anularity by staining with the combined fluorescent dyes acridine oran ge and ethidium bromide. C-2-ceramide induced these morphological chan ges without a concomitant production of oligonucleosomal fragments res ponsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C-2-ceramide, as C-2-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydroly sis were ineffective. The effect also depended on mRNA and protein syn thesis as it was markedly inhibited by actinomycin D and cycloheximide . Sphingomyelinase and interleukin-1 beta, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induc ed apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.