Hepatocytes sensitized to tumor necrosis factor-alpha cytotoxicity undergoapoptosis through caspase-dependent and caspase-independent pathways

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappa B activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicit y by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepato cytes was examined following infection with an adenovirus, Ad5I kappa B, th at blocks NF-kappa B activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5I kappa B-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by a poptosis and was associated with caspase activation, although caspase activ ation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhib itors blocked Ad5I kappa B/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death doma in (FADD) dominant negative decreased Ad5I kappa B/TNF-alpha- and ActD/TNF- alpha-induced cell death by 81 and 47%, respectively, However, downstream e vents differed, since Ad5I kappa B/TNF-alpha but not ActD/TNF-alpha treatme nt caused mitochondrial cytochrome c release. These results suggest that NF -kappa B inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways tha t diverge below the level of FADD, ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independen t pathway of apoptosis.