TNFa-DEPENDENT JUN KINASE ACTIVITY MODULATES APOPTOSIS IN HUMAN HEPATOMA CELLS J. Plfimpe, C. Liedtke, C.A. Bradhatn, DA. Brenner, M.E Manns, C. Trautwein Dep. of Gastroenterology & Hepatology, Medical School Hannover, FRG. TNFc~-TNF receptor 1 (TNT-R.I) interaction activates at least three different signalling cascades after binding of adapter molecules to the intracellular domain of TNF-RI. One pathway involving the adaptor molecule FADD leads to apoptosis via activation of a caspase cascade whereas NF-kB mediates anti-apoptotic mechanisms. The third pathway leads to Jnn kinase (JNK) activation. Its role for apoptosis in liver cells is poorly understood and was therefore investigated in this study. In HepG2 and HuH 7 cells after treatment with 25 ng/ml TNFa and 1 p_g/ml Cycloheximide (Cye) apoptosis started 10 h after stimulation as shown by DAPl-staining and DNA fragmentation. This was accompanied by a 35 fold increase in INK activity 1 h after treatment while ERK activity remained unaffected. TNFc~-dapandent /NK activation was effectively blocked by infection with an adanoviral vector (adv) expressing dominant negative (DN) TAK (TGFI3 activating kinase). DN TAK infection after TNFcc/Cye stimulation resulted in a 4 hours earlier start of apoptosis. In further experiments we looked for JNK interactions with the TNFctdependent apoptosis pathway. First HUll 7 cells were cotransfectad with DN TAK and DN FADD adv showing effective blocking of apoptosis. This indicates that the FADD pathway is essential to mediate these effects. The time gap in inducing apoptosis after DN TAK infection was also evident at the caspase 3 level. We next studied the role of the mitoehondrial pathway. In cells infected with DN TAK caspase 9 was activated earlier and coinfection with a bcl2 expressing adv blocked apoptosis. These experiments indicate that mitochondria are involved in JNK related effects on apoptosis. Next we performed time course experiments using an inhibitor of Caspase 8 which is the first target downstream of FADD. These experiments revealed that Caspase 8 is activated earlier in the DN TAK group compared to the control group. In summary, our results show that TNFcc-dependant JNK activity delays apoptosis in hepatoma cells by a TNF-Rl-assoeiated mechanism upstream of caspase 8. I PIC03107 I