Epidermal growth factor (EGF) and insulin in combination have previously been shown to initiate S-phase in primary cultures of adult rat hepatocytes. We here describe the detailed time course and dose-dependency of the effects of EGF and insulin on DNA synthesis in cultured hepatocytes. The DNA synthesis was assessed either biochemically or autoradiographically with a fairly good correlation between the two methods. DNA synthesis started 24-30 h after plating of the cells and peaked at approximately 70 h. Up to 70% of the cells entered DNA synthesis during this period. EGF and insulin acted synergistically on the DNA synthesis. Dexamethasone raised the DNA synthesis slightly, maximal effect occurred at concentrations above 2.5 nM and this agent was routinely used in the experiments with EGF and insulin. In the presence of 0.4 microM insulin from the time of plating, EGF dose-dependently increased the DNA synthesis with maximal effect at 5-15 nM. When added in combination with 1.7 nM EGF, insulin enhanced the DNA synthesis over the concentration range from 0.1 to 3 nM. These studies show that primary cultures of hepatocytes are useful in assessing the quantitative aspects of the interactions between the growth stimulating effects of hormones.

DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [3H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [3H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination. The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [3H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [3H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G1-fractions was only 2 and 7%, respectively.

FEBS LettersVolume 120, Issue 1 p. 89-93 Full-length articleFree Access Increased effect of adrenaline on cyclic AMP formation and positive β-adrenergic modulation of DNA-synthesis in regenerating hepatocytes Gunnar Brønstad, Gunnar Brønstad Institute of Pharmacology, University of Oslo, PO Box 1057, Blindern, Oslo 3, NorwaySearch for more papers by this authorThoralf Christoffersen, Thoralf Christoffersen Institute of Pharmacology, University of Oslo, PO Box 1057, Blindern, Oslo 3, NorwaySearch for more papers by this author Gunnar Brønstad, Gunnar Brønstad Institute of Pharmacology, University of Oslo, PO Box 1057, Blindern, Oslo 3, NorwaySearch for more papers by this authorThoralf Christoffersen, Thoralf Christoffersen Institute of Pharmacology, University of Oslo, PO Box 1057, Blindern, Oslo 3, NorwaySearch for more papers by this author First published: October 20, 1980 https://doi.org/10.1016/0014-5793(80)81053-2Citations: 49AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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