Cellular analysis of the kinetics of alpha-fetoprotein and nuclear oncogene activation in primary cultures of adult rat hepatocytes stimulated by epidermal growth factor.
Alpha-fetoprotein ( AFP), a fetal gene normally inactivated in quiescent adult hepatocytes, is re-expressed in hepatocytes during the proliferating response induced by liver regeneration in vivo, or epidermal growth factor (EGF) in vitro. The nuclear oncogenes c-jun, c-fos and c-myc are 'immediate early' genes also activated during the proliferative response of hepatocytes. Whether AFP gene activation is linked to oncogene activation is not known. As a first step in answering this question, we have analysed the cellular kinetics of nuclear oncogene and AFP gene activation in primary cultures of adult rat hepatocytes stimulated by EGF. Gene activation was evaluated at the mRNA level by dot blot and in situ hybridization, and at the protein level by immunoperoxidase. C-jun, c-fos and c-myc mRNA steady state levels in total cellular RNA were increased from 30 min-2 h after EGF stimulation. In situ hybridization analysis showed that transcripts of the three oncogenes increased in all hepatocytes after EGF stimulation. While unstimulated cultures did not immunostain for Fos and Myc proteins. Fos immunostaining was visible in the majority of hepatocyte nuclei 1 and 2 h after EGF addition, and Myc cytoplasmic immunostaining of the majority of hepatocytes was observed at 2 h of stimulation. AFP mRNA increased in total cellular RNA 2 and 4 h after EGF stimulation, with elevated in situ hybridization signal for AFP mRNA in all hepatocytes. No hepatocytes immunostained for AFP in unstimulated cultures, but a cytoplasmic labeling of 20-30% of the hepatocytes was observed 6 h after stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)[1]References
- Cellular analysis of the kinetics of alpha-fetoprotein and nuclear oncogene activation in primary cultures of adult rat hepatocytes stimulated by epidermal growth factor. Tournier-Thurneyssen, I., Feldmann, G., Bernuau, D. Biol. Cell (1992) [Pubmed]
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