Ceramide induces caspase-independent apoptosis in rat hepatocytes sensitized by inhibition of RNA synthesis

Ceramide has been implicated as a second messenger in intracellular signali ng pathways leading to apoptosis in nonhepatic cells. To determine whether ceramide can mediate hepatocyte apoptosis, the cytotoxicity of ceramide was determined in rat hepatocytes. The rat hepatocyte cell line, RALA255-10G, and primary rat hepatocytes were completely resistant to toxicity from 10 t o 100 mu mol/L C-2 ceramide. Resistance was not the result of a failure to take up ceramide, because ceramide treatment did cause nuclear factor-kappa B (NF-kappa B) activation. Because ceramide may mediate cell death from tu mor necrosis factor alpha (TNF-alpha), the ability of RNA synthesis inhibit ion and NF-kappa B inactivation to sensitize hepatocytes to ceramide toxici ty was examined. RALA hepatocytes were sensitized to ceramide toxicity by c oadministration of actinomycin D (ActD), Cell death occurred by apoptosis a s determined by the presence of morphological evidence of apoptosis, caspas e activation, poly(ADP-ribose) polymerase (PARP) degradation, and DNA hypop loidy. Despite the induction of apoptosis associated with caspase activatio n, cell death from ActD/ceramide was not blocked by caspase inhibition. Inh ibition of NF-kappa B activation also sensitized RALA hepatocytes to cerami de toxicity, but to a lesser extent than for TNF-alpha. Thus, unlike many n onhepatic cell types, rat hepatocytes are resistant to cell death from cera mide because of the transcriptionally dependent up-regulation of a protecti ve gene(s), The ability of ActD and NF-kappa B inactivation to sensitize RA LA hepatocytes to ceramide toxicity suggests that ceramide may act as a dow nstream mediator of TNF-alpha toxicity.