BCL-2 INTERRUPTS THE CERAMIDE-MEDIATED PATHWAY OF CELL-DEATH

Ceramide, a product of sphingomyelin turnover, has been proposed as a novel lipid second messenger with specific roles in mediating antiprol iferative responses including apoptosis and cell cycle arrest. In this study, we examine the relationship between the ceramide-mediated path way of growth suppression and the bcl-2 protooncogene. In ALL-697 leuk emia cells, the addition of the chemotherapeutic agent vincristine res ulted in a time-dependent growth suppression characterized by marked a poptosis. The effects of vincristine on cell death were preceded by a prolonged and sustained accumulation of endogenous ceramide levels rea ching approximate to 10.4 pmol ceramide/nmol phospholipids at 12 hr fo llowing the addition of vincristine-an increase of 220% over vehicle-t reated cells. Overexpression of bcl-2 resulted in near total protectio n of cell death in response to vincristine. However, the ceramide resp onse to vincristine was not modulated by overexpression of bcl-2, indi cating that bcl-2 does not interfere with ceramide formation. Overexpr ession of bcl-2 prevented apoptosis in response to ceramide, suggestin g that bcl-2 acts at a point downstream of ceramide. On the other hand , bcl-2 did not interfere with the ability of ceramide to activate the retinoblastoma gene product or to induce cell cycle arrest, suggestin g that the effects of ceramide on cell cycle arrest can be dissociated from the effects on apoptosis. These studies suggest that ceramide an d bcl-2 partake in a common pathway of cell regulation. The results al so cast ceramide as a gauge of cell injury rather than an ''executor'' of cell death with clearly dissociable biological outcomes of its act ion depending on downstream factors.