Relationships of apoptotic signaling mediated by ceramide and TNF-alpha inU937 cells

It is commonly assumed that ceramide is a second messenger that transduces signaling leading to apoptosis, We tested this hypothesis by investigating the role of ceramide in TNF-alpha-initiated apoptotic signaling using the h istiocytic lymphoma cell line U937. We found considerable differences betwe en cell killing by TNF-alpha and by ceramide. U937 cells treated with TNF-a lpha are committed early and irreversibly to the apoptotic pathway and star t to die 90 min after treatment. U937 cells treated with ceramide start to die 12 h after the initial treatment, The cell death signaling initiated by TNF-alpha is transduced within minutes of exposure to TNF-alpha and it is irreversible. Exogenous ceramide increases the intracellular level of ceram ide rapidly, significantly, and well above the physiological levels, within minutes, but cellular commitment to death does not occur until after the f irst 6 h of incubation. Furthermore, the endogenous ceramide in U937 cells treated with TNF-alpha increases well after the commitment to the apoptotic pathway. The differences between ceramide and TNF-alpha in the kinetics an d the commitment to the apoptotic pathway suggest that, (6) ceramide is not a second messenger in the apoptotic signaling of TNF-alpha, (b) ceramide e levations, in TNF-alpha treated cells, are a consequence rather than a caus e of apoptosis and (c) exogenously added ceramide and TNF-alpha kill cells via different pathways.