Hepatocytes sensitized to tumor necrosis factor-α cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-α toxicity by repression of NF-κB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-α cytotoxicity by similar mechanisms, TNF-α-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IκB, that blocks NF-κB activation or following cotreatment with actinomycin D (ActD). TNF-α treatment of Ad5IκB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-α induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IκB/TNF-α- induced cell death but did not inhibit ActD/TNF-α-induced apoptosis. A Fas- associated protein with death domain (FADD) dominant negative decreased Ad5IκB/TNF-αand ActD/TNF-α-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IκB/TNF-α but not ActD/TNF-α treatment caused mitochondrial cytochrome c release. These results suggest that NF-κB inactivation and inhibition of RNA synthesis sensitize RALA255- 10G hepatocytes to TNF-α toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-α cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.