Mechanisms of viral induced caspase activation in human hepatoma cell lines

For a growing number of liver diseases like viral induced hepatitis or fulminant liver failure, apoptosis induction in hepatocytes plays a key role in the course of the disease. In the effector phase of apoptotic cell death a family of proteases, called caspases, is organized in a hierachial order, thus most members of this cascade are activated by other members being more upstream in the pathway. Therefore, investigation of the very first mechanisms leading to the activation of a so called initiator caspase like caspases-S or -9, are important steps in molecular diagnosis and treatment of various diseases. We have looked for new caspase activation pathways in various cell lines, including human hepatoma lines by infection in our model system with the paramyxovirus Sendai virus (SeV). We could show signs of apoptosis 15 to 20 h after SeV infection by TUNEL assay or FACS analysis of infected cells (e.g. HepG2). A direct influence of caspases on this process could be demonstrated by a complete inhibition of apoptosis by incubation of infected cells with the broad caspase inhibitor z-VADfmk. Subsequently we could not only detect the activation of caspase-3 which is regarded as an effector caspase being downstream of initiating events, but we could also demonstrate caspase-8 and -9 cleavage by immunoblotting in these cell lines. Previous work demonstrated caspase-S as the first caspase in the signal transduction via death receptors, whereas caspase-9 activation is regarded as the first activated caspase mediated by chemotherapeutic agents causing cytochrome c release from the mitochondria. Surprisingly neither the involvement of the death receptors CD95 or TNF-RI (inhibition experiments by decoy proteins), nor the release of cytochrome c (immunoblot, immunofluorescence) could be shown, indicating the existence of an alternative way of caspase-8 and/or caspase-9 activation. To further characterize this pathway we are currently investigating the involvement of other proteases, death receptors or even viral proteins in the activation of initiator caspases.