Induction of p73 in primary rat hepatocytes after treatment with hepatocarcinogen N-nitrosomorpholin (NNM)

S Tudzarova-Trajkovska, W. Parzefall PhD, J Wesierska-Gadek, PhD

Institute of Cancer Research, University of Vienna, VIENNA, AUSTRIA

AIMS: In vivo administration of NNM causes severe hepatotoxicity associated with apoptosis and necrosis. We reported recently that NNM treatment of rats resulted in an induction of p73 and p53 tumor suppressors. Interestingly, increase of p73 level preceded that of p53. In the present work we tried to dissect the role of p73 and p53 activation in response to NNM treatment. METHODS: Expression and intracellular localization of distinct proteins was analysed by immunoblotting and immunocytochemistry, respectively. Apoptosis was evaluated by scoring of hepatocytes showing changes characteristic for early and late apoptosis as determined by Hoechst 33258 staining and by examination of caspases status. RESULTS: p73 but not p53 protein was induced in primary hepatocytes after application of NNM. An increase of p73 protein level was dose-dependent and preceded an onset of apoptosis. Following the upregulation of p73, GADD-45 was induced. Analysis of pulse-chase labeled proteins revealed a significant increase of the p73 half-life. p53 protein was activated in primary hepatocytes in response to UV- and gamma-radiation. CONCLUSIONS: These results show that treatment of primary hepatocytes by NNM results in the induction of p73 protein due to protein stabilization and that primary hepatocytes depending on type of stress stimuli activate distinct cellular early emergency programs.

KEY WORDS: tumor suppressors, p53, p73 stability, apoptosis, activated caspases, .

For more information, contact

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Apoptosis - Molecular Mechanisms.