Carcinogenesis, Vol. 21, No. 1, 79-86,
January 2000
© 2000 Oxford University Press
Carcinogenesis |
Initiated rat hepatocytes in primary culture: a novel tool to study alterations in growth control during the first stage of carcinogenesis
Institut für Tumorbiologie-Krebsforschung, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria
To study growth regulation in the beginning of carcinogenesis, we established a novel ex vivo model for co-cultivation of normal and putatively initiated hepatocytes. Rats received the genotoxic hepatocarcinogen N-nitrosomorpholine (NNM). This led to the appearance of hepatocytes expressing placental glutathione S-transferase (G+ cells). These cells exhibited elevated rates of cell replication and apoptosis, as known from further advanced preneoplasia; G+ cells were considered initiated. At days 20–22 post-NNM treatment their frequency was maximal (1–2%); ~40% were still single and 60% were arranged in mini foci. At this time-point liver cells were isolated by collagenase perfusion and cultivated. G+ cells, identified by immunostaining of the culture-plates, were present at the same percentage as in vivo, excluding selective loss, enrichment or spontaneous expression of the G+ phenotype. In untreated cultures G+ hepatocytes showed significantly higher rates of replicative DNA synthesis than normal G– cells. Application of the hepatomitogen cyproterone acetate (CPA) elevated DNA replication preferentially in G+ cells. Transforming growth factor ß1 (TGF-ß1) suppressed replicative DNA synthesis which was more pronounced in G+ than in G– hepatocytes. Combined treatment with CPA and TGF-ß1 had no effect on G– cells, but considerably inhibited DNA replication in G+ cells. This suggests that the effects of TGF-ß1 predominated in G+ hepatocytes. We conclude that putatively initiated G+ hepatocytes, both in vivo and in culture, exhibit higher basal rates of DNA replication than normal G– hepatocytes and an over-response to mitogens and growth inhibitors. Therefore, G+ cells show (i) nearly identical behaviour in intact liver and in primary culture and (ii) inherent defects in growth control that are principally similar although somewhat less pronounced than in later stages of carcinogenesis. The present ex vivo system thus provides a novel and useful tool to elucidate biological and molecular changes during initiation of carcinogenesis.
Abbreviations: AB, apoptotic body; CPA, cyproterone acetate; G– cells, hepatocytes negative for placental glutathione S-transferase; G+ cells, hepatocytes positive for placental glutathione S-transferase; GST, glutathione S-transferase; LI, labeling index; NNM, N-nitrosomorpholine; PB, phenobarbital; TGF-ß1, transforming growth factor ß1.
1 To whom correspondence should be addressed Email: bettina.grasl-kraupp{at}univie.ac.at
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