Abstract 869 Poster Session II, Sunday, 5/2 (poster 136)

Integrin clustering upon binding to extracellular matrix proteins initiates cell survival signaling pathways, and cell detachment results in apoptosis. The sphingolipid ceramide functions as a rheostat of cellular stress, and can be generated by sphingomyelinase (SMase) activation or de novo synthesis by ceramide synthase. We report that cells undergoing detachment-induced apoptosis contain elevated ceramide levels, and that ceramide content increases prior to the onset of apoptosis as detected by DNA degradation. Treatment with sodium orthovanadate, a phorbol ester, or transformation with an activated allele of c-Src, c-Src Y527F, protects cells from apoptosis and prevents the accumulation of cellular ceramide. Forcibly increasing intracellular ceramide levels by treating attached cells with exogenous SMase results in a marked accumulation of intracellular ceramide and subsequent apoptosis. In addition, cell detachment results in activation of acid SMase activity that precedes elevation in cellular ceramide levels. Finally, inhibition of de novo ceramide synthesis in detached cells does not affect apoptosis. Taken together, these data implicate ceramide, generated by SMase activation, as a participant in apoptosis triggered by the absence of integrin signals.

(This work was partly supported by grant K08 CA72662 to RKM).