Initiated rat hepatocytes in primary culture: a novel tool to study alterations in growth control during the first stage of carcinogenesis

Abstract

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%); approximately 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 beta1 (TGF-beta1) suppressed replicative DNA synthesis which was more pronounced in G(+) than in G(-) hepatocytes. Combined treatment with CPA and TGF-beta1 had no effect on G- cells, but considerably inhibited DNA replication in G(+) cells. This suggests that the effects of TGF-beta1 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.

New cellular tools reveal complex epithelial–mesenchymal interactions in hepatocarcinogenesis

“…The new BLC cell lines produce TNFa, TNFb, TGFb1, IL-6, and presumably IL-10 (Table 1; Rochford et al, 1997). In recent studies, TNFa and IL-6 had no impact on DNA replication of premalignant hepatocytes, whereas TGFb1 even suppressed the growth (Löw-Baselli et al, 2000;Drucker et al, 2006). In this work, 0.7 or 1 ng of TNFb ml À1 medium lowered DNA-replication of hepatocytes in primary culture by 57 and 76%, respectively (data not shown).…”

Inherent growth advantage of (pre)malignant hepatocytes associated with nuclear translocation of pro-transforming growth factor α

“…Phenobarbital (PB) was admixed to the powder diet and was fed to a subgroup of rats from day 4 to 17 months post-NNM. Concentrations of PB were adjusted every 14 days to provide a daily dose of 50 mg kg À1 body weight (Löw-Baselli et al, 2000b). Animals were killed by decapitation under CO 2 asphyxation.…”

“…After 21 days, livers were perfused with collagenase as described (Parzefall et al, 1989;Löw-Baselli et al, 2000b).…”

“…Treatment of primary hepatocyte cultures Cells were seeded and kept under serum-free conditions, as described (Parzefall et al, 1989;Löw-Baselli et al, 2000b). Treatment commenced 4 h after plating (time point 0).…”

“…Recently, preneoplastic rat liver cells have become available for investigation in an ex vivo culture model (Löw-Baselli et al, 2000b). We applied this model, in combination with studies on human livers, and found that proTGFa-positive nuclei increased in the course of hepatocarcinogenesis.…”