RESISTANCE TO FAS-MEDIATED APOPTOSIS IN HUMAN HEPATOMA-CELLS

CTLs- and lymphokine-induced apoptosis of infected hepatocytes during the course of chronic viral hepatitis is thought to be important for b oth disease termination and prevention of hepatocellular transformatio n, We therefore studied apoptosis induced by Pas (APO-1 or CD95) - a w idely expressed cell surface receptor whose ligand is involved in lymp hocyte cytotoxicity - in a set of human hepatoma cell lines. As normal hepatocytes, all of the human hepatoma cell lines tested do express d etectable amounts of Fas on their surface. Nevertheless, only PLC/PRF/ 5 cells undergo apoptosis following treatment with anti-Fas. Systemati c cloning and sequence analysis of the Pas cDNA did not show mutations in the Pas gene in any of the cells lines tested, However, due to alt ernative splicing, 5 to 10% of the Fas cDNAs are deleted of 63 interna l nucleotides corresponding to the transmembrane domain, thus encoding for a soluble and secreted form of Fas (Fas Delta TM), potentially ab le to neutralize anti-Fas or Fas-Ligand. Although we could not demonst rate a direct correlation between resistance of different hepatoma cel l lines to Pas mediated death and endogenous expression of this transc ript, we show that PLC/PRF 5 stable transfectants overexpressing Fas D elta TM are less sensitive to anti-Pas than control cells, Tn three di fferent cell lines, resistance to anti-Pas was overcome by treatment w ith the protein synthesis inhibitor cyclohexymide, Although this could suggest the existence of short-lived repressors of the Fas-activated apoptotic signalling pathway(s), we show that translational inhibition is not required for the synergistic effect of cycloheximide to take p lace, and that resistant hepatoma cells can be sensitized to anti-Pas by subinhibitory concentrations of this protein synthesis inhibitor. S ince cycloheximide is able to activate intracellular signalling indepe ndently on its effects on protein sysnthesis, we suggest that it might provide a costimulatory signal that cooperates with Fas in the induct ion of cell death and that, at least in the cells we tested, resistanc e to Fas is not an active process involving gene transciption and tran slation but only the consequence of an inadequate apoptotic stimulatio n.