The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.

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Growth of Kym-1 rhabdomyosarcoma cells depends on endogenous receptor tyrosine kinase signals activated by insulin and insulin-like growth factors (IGF), as revealed from enhancement of proliferation by insulin and IGF-1 and cytostatic action of inhibitors of IR/IGFR kinases. Depending on the presence or absence of the caspase inhibitor z-VADfmk, TNF induced full growth arrest or apoptosis, respectively, indicating dominance of TNF over mitogenic signal pathways in Kym-1 cells. In accordance with a caspase-independent cytostatic action, TNF downregulated IR kinase activity and caused a profound inhibition of downstream mitogenic signals including the MAPK cascade and STAT5, key pathways of proliferation and cell survival. Removal of z-VAD-fmk after 24 h induced rapid cell death in the absence of TNF. The inhibition of survival signals concomitant with persisting proapoptotic signals may tip the balance towards an irreversible commitment of the cell to apoptosis that becomes apparent upon relief of suppression of effector caspases. Cell Death and Differentiation (2000) 7, 955 ± 965.