ABSTRACT
Sequential treatment of partially (two-thirds) hepatectomized rats with diethylnitrosamine and 2-acetylaminofluorene induces the emergence of diploid hepatocytes in rat liver. These carcinogen-induced diploid cell populations are thought to contain the progenitors of hepatocellular carcinoma (HCC), i.e., initiated, cells. In the study presented here, we addressed the question of whether putative mutations in carcinogen-induced diploid hepatocytes can cooperate with activated oncogenes in the process of transformation in vitro. Both carcinogenesis in vivo and transformation in vitro have been shown to be multistep processes requiring at least two independent transforming events. Diploid and polyploid rat hepatocytes were isolated by centrifugal elutriation. The purity of the elutriated fractions was 88 +/- 3% in the diploid fraction and 84 +/- 3% in the polyploid fraction. Hepatocytes from both the elutriated cell fractions and, for comparison, hepatocytes from untreated rats were transfected by electroporation with oncogene expression vectors containing the mutated human T24 c-Ha-ras gene and of the N-myc gene. Transient expression of transfected DNA was similar in both hepatocyte populations. No cell lines could be established by using the N-myc vector. In contrast, the carcinogen-induced diploid hepatocytes, but not polyploid hepatocytes, could be converted by transfection with the ras vector into permanent anchorage-independent growing cell lines with hepatocyte-like morphology and differentiation. These cell lines expressed the myc proto-oncogene and transforming growth factor-alpha constitutively. Thus, carcinogen-induced diploid hepatocytes are sensitive to transformation by the ras oncogene, suggesting cooperation between putative preexisting mutations in the diploid cells and the ras oncogene product in hepatocellular transformation.
Subject(s)
2-Acetylaminofluorene/pharmacology , Cell Transformation, Neoplastic/drug effects , Diethylnitrosamine/pharmacology , Genes, myc , Genes, ras , Liver Neoplasms/chemically induced , Liver/cytology , Animals , Base Sequence , Cell Separation , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , In Situ Hybridization , Liver/drug effects , Male , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Ploidies , Proto-Oncogene Mas , RNA, Messenger/genetics , Rats , Rats, Wistar , Transfection , Transforming Growth Factor alpha/geneticsABSTRACT
Sequential treatment of partially hepatectomized male Wistar rats with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) induces the emergence of diploid hepatocyte populations. These carcinogen-induced hepatocytes are thought to include the precursor cells of liver carcinomas that arise later in this treatment protocol. The growth of the diploid hepatocytes is promoted by AAF and it has been suggested that the action of the arylamine may be receptor-mediated. AAF has been shown to bind specifically to the aryl hydrocarbon (Ah) receptor and the so-called 4S polycyclic aromatic hydrocarbon (PAH) binding protein. The present study addresses the question of whether the concentrations of the two binding proteins differ in diploid and polyploid hepatocytes from DEN/AAF-treated rats. Hepatocytes from carcinogen-treated rats were isolated and diploid, and tetraploid hepatocytes separated by means of centrifugal elutriation. Whereas Ah receptor concentrations in diploid hepatocytes were insignificantly lower (21.8 +/- 5.9 versus 29.2 +/- 6.6 fmol/mg cytosolic protein; n = 4; P = 0.1), levels of the 4S PAH binding protein in diploid hepatocytes were twice as high as in tetraploid hepatocytes (252.3 +/- 93.6 versus 124.0 +/- 18.5 fmol/mg cytosolic protein; n = 4; P = 0.04). We conclude from our results that the differences in growth control in polyploid and carcinogen-induced diploid hepatocytes are not associated with changes in the levels of the Ah receptor. The role of the 4S PAH binding protein in the process of hepatocarcinogenesis remains to be established.
Subject(s)
Carcinogens/analysis , Carrier Proteins/analysis , Liver/chemistry , Methyltransferases , Receptors, Drug/analysis , 2-Acetylaminofluorene , Animals , Centrifugation , Diethylnitrosamine , Glycine N-Methyltransferase , Liver/drug effects , Male , Ploidies , Polychlorinated Dibenzodioxins/metabolism , Rats , Receptors, Aryl HydrocarbonABSTRACT
Diploid hepatocytes induced by a combination of diethylnitrosamine and 2-acetylaminofluorene were isolated and separated from polyploid hepatocytes by centrifugal elutriation. The diploid and polyploid cell fractions were approximately 90% pure and contained between 1 and 1.5 x 10(7) cells. When kept in monolayer cultures both cell populations responded to the mitogenic effect of EGF and insulin. However, the percentage of labelled nuclei was higher in predominantly diploid compared to predominantly tetraploid hepatocyte cultures at all epidermal growth factor (EGF) concentrations used in this study. At 10 ng EGF/ml and 10 mU insulin/ml the labelling index was twice as high in the diploid liver cells. Further work is required to show the relevance of the stronger response of the diploid cell fraction to mitogens in the process of carcinogenesis.
