In a previous publication, we were able to show that irradiation of KupVer cells, the liver resident macrophages, leads to an increased TNF-concentration in the culture medium. The pathomechanisms underlying this phenomenon, however, remained to be elucidated. Here, we show that following irradiation of KupVer cells, the apoptosis rate increased drastically within 48 h. At the same time, the total TNF-concentration in cell lysates of KupVer cells attached to the culture plate decreased. However, normalization of the TNF-concentration with respect to cell number revealed that TNF-concentration per attached cell remained constant during the observation period. Western blot analysis showed that heat shock protein 27 (Hsp27) is strongly downregulated and bax is upregulated in irradiated KupVer cells as compared to sham-irradiated cells. Overexpression of Hsp27 in KupVer cells was shown to prevent the eVect of irradiation on bax expression, apoptosis and, at the same time, on increase of TNF-concentration in the KupVer cell medium. We conclude that irradiation of KupVer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression. Furthermore, we suggest that apoptosis of KupVer cells leads to an increase of TNFconcentration in the culture medium which may be due to cell death rather than active release or synthesis.

This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF--mediated apoptosis. I B was upregulated in irradiated hepatocytes. Administration of I B antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by I B antisense oligonucleotides was mediated by suppression of caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes to TNF--mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF--administration depend on radiation-induced upregulation of I B, thus demonstrating an apoptosis permitting eVect of I B.

In a previous publication, we were able to show