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

To study growth regulation in the beginning of carcinogenesis, we establish ed a novel ex vivo model for co-cultivation of normal and putatively initia ted hepatocytes, Rats received the genotoxic hepatocarcinogen N-nitrosomorp holine (NNM). This led to the appearance of hepatocytes expressing placenta l glutathione S-transferase (G(+) cells). These cells exhibited elevated ra tes of cell replication and apoptosis, as known from further advanced prene oplasia; G(+) cells were considered initiated. At days 20-22 post NNM treat ment their frequency was maximal (1-2%); similar to 40 % were still single and 60 % were arranged in mini foci, At this time-point liver cells were is olated by collagenase perfusion and cultivated. G(+) cells, identified by i mmunostaining 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 signific antly higher rates of replicative DNA synthesis than normal G(-) cells. App lication of the hepatomitogen cyproterone acetate (CPA) elevated DNA replic ation preferentially in G(+) cells. Transforming growth factor beta 1 (TGF- beta 1) suppressed replicative DNA synthesis which was more pronounced in G (+) than in G(-) hepatocytes, Combined treatment with CPA and TGF-beta 1 ha d no effect on G- cells, but considerably inhibited DNA replication in G(+) cells. This suggests that the effects of TGF-beta 1 predominated in G(+) h epatocytes, We conclude that putatively initiated G(+) hepatocytes, both il l vivo and in culture, exhibit higher basal rates of DNA replication than n ormal G(-) hepatocytes and an over-response to mitogens and growth inhibito rs, Therefore, G(+) cells show (i) nearly identical behaviour in intact liv er and in primary culture and (ii) inherent defects in growth control that are principally similar although somewhat less pronounced than in later sta ges of carcinogenesis. The present ex vivo system thus provides a novel and useful tool to elucidate biological and molecular changes during initiatio n of carcinogenesis.