Transformation Of Liver Cells Research Articles

Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. The Wnt-beta-catenin pathway is activated in colon cancers and some melanoma cell lines, but has not yet been investigated in HCC. We have examined the status of the beta-catenin gene in different transgenic mouse lines of HCC obtained with the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the beta-catenin gene similar to those found in colon cancers and melanomas. These alterations in the beta-catenin gene (point mutations or deletions) lead to a disregulation of the signaling function of beta-catenin and thus to carcinogenesis. We then analyzed human HCCs and found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations led to the accumulation of beta-catenin in the nucleus. Thus alterations in the beta-catenin gene frequently are selected for during liver tumorigenesis and suggest that disregulation of the Wnt-beta-catenin pathway is a major event in the development of HCC in humans and mice.

Differential expression of key enzymes of energy metabolism in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholine and dehydroepiandrosterone.

Preneoplastic liver foci and neoplasms of different morphological phenotypes were induced in rats with N-nitrosomorpholine (NNM; 120 mg/l in drinking water for 7 weeks) and the peroxisome proliferator dehydroepiandrosterone (DHEA; 0.6% in the diet for up to 84 weeks). Preneoplastic glycogen storage foci (GSF) occurred mainly upon treatment with NNM, and amphophilic cell foci (APF) were mainly observed in rats treated with DHEA alone or in combination with NNM. The 2 types of lesions belong to 2 different cellular lineages, the glycogenotic/basophilic lineage and the amphophilic lineage, which are characterized by distinct patterns of alterations in key enzymes of energy metabolism. Whereas in GSF enzymes of glucose metabolizing pathways were modified (increase in glucose-6-phosphate dehydrogenase and pyruvate kinase, decrease in glucose-6-phosphatase), APF mainly demonstrated alterations in mitochondrial enzymes (increase in cytochrome c oxidase, succinate dehydrogenase and glycerol-3-phosphate dehydrogenase) and, to a lower extent, in peroxisomal enzymes (increase in peroxisomal hydratase and acyl-CoA oxidase). The alterations in enzyme expression reflect an insulinomimetic effect in GSF and a thyromimetic effect in APF. Neoplasms resulting from APF show a more differentiated phenotype than those arising from GSF. We suggest that the different and in many aspects opposite effects of the 2 carcinogens on key enzymes of distinct pathways of energy metabolism modulate the process of neoplastic liver cell transformation and result in phenotypically different preneoplasias and neoplasias reflecting different cellular lineages.

Chemical transformation of mouse liver cells results in altered cyclin D-CDK protein complexes.

Dysregulated cell proliferation is one phenotypic change associated with neoplasia. Key protein complexes involved in regulating cell division are composed of cyclins, cyclin-dependent kinases (CDK) and CDK inhibitors (CDI). Many virally transformed cells in culture exhibit disrupted cyclin-CDK-CDI complexes, suggesting that such changes may play a mechanistic role in viral transformation. To determine whether similar alterations may be involved in chemical carcinogenesis we characterized cyclin D1-CDK-CDI protein complexes in a non-tumorigenic mouse liver cell line and investigated whether complexes were altered after transformation with the genotoxic carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 3-methylcholanthrene (MC). In non-tumorigenic mouse liver cells cyclin D1 associated with CDK6, CDK4 or CDK2 to form binary (cyclin D1-CDK), tertiary (cyclin D1-CDK-p27KIP1) or quaternary (cyclin D1-CDK-p21WAF1-PCNA) complexes. After chemical transformation of mouse liver cells with either MC or MNNG, select cyclin D1-CDK-CDI protein complexes were altered. In MC-transformed cells formation of various binary, tertiary and quaternary cyclin D1-CDK-(CDI) protein complexes was reduced, resulting in decreased CDK4 kinase activity. Interestingly, CDK6 kinase activity was dramatically elevated due to high levels of cyclin D3 in association with CDK6. In MNNG-transformed cells select cyclin D1-CDK6-CDI and cyclin D1-CDK2-CDI protein complexes were altered but CDK6 and CDK4 kinase activity remained unaffected. Distinct changes in cyclin D1-CDK-CDI complexes found between the two chemically transformed mouse liver cell lines suggest that each cell line harbored unique mutations or alterations that differentially contributed to stabilization of cyclin D1-CDK-CDI holoenzymes. p53 gene mutations were not detected in the MC- or MNNG-transformed mouse liver cell lines and thus were not involved in disrupting cyclin D1-CDK-CDI protein complexes. In summary, this study presents evidence that D-type CDK protein complexes can be altered physically and functionally after chemical transformation with genotoxic carcinogens, suggesting that components of the cell cycle machinery can be targeted during chemical carcinogenesis.

Suppressive effect of oestradiol on chemical hepatocarcinogenesis in rats

Aims —To examine the effects of oestradiol and testosterone on the early carcinogenic changes expressed in rat liver from the diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF), partial hepatectomy (PH) model of hepatocarcinogenesis. Methods —Preneoplastic liver lesions were evaluated using immunohistochemical analysis of glutathione-S-transferase placental form (GST-P) expression; oestrogen and androgen receptor levels were measured by radioimmunoassay. Results—Oestradiol administration to non-castrated DEN-AAF-PH treated males resulted in a decrease in the area of GST-P positive foci, while testosterone increased the serum oestradiol level and reduced the area. In males, castration alone and castration with oestradiol replacement significantly reduced the GST-P positive area, and increased the hepatic oestrogen receptor level. In DEN-AAF-PH treated females, castration with testosterone replacement was associated with a significant increase in the GST-P positive area and the hepatic androgen receptor level. Conclusion —These findings suggest that exogenous and endogenous oestradiol can suppress chemical hepatocarcinogenesis. It appears that oestrogen receptors may be involved in the inhibition of malignant transformation of preneoplastic liver cells, while androgens and androgen receptors are involved in hepatocarcinogenesis.

Suppressive effect of oestradiol on chemical hepatocarcinogenesis in rats

Aims—To examine the effects of oestradiol and testosterone on the early carcinogenic changes expressed in rat liver from the diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF), partial hepatectomy (PH) model of hepatocarcinogenesis.Methods—Preneoplastic liver...

Hepatitis B viruses and cancerogenesis

Hepatitis B virus (HBV) is a small, enveloped DNA virus which primarily infects liver cells and causes acute and persistent liver disease. Chronic HBV infection, frequently associated with cirrhosis and eventually hepatocellular carcinoma (HCC), represents a major health problem in the world. HBV is the prototype member of the hepadnavirus family, which includes several related mammalian viruses also implicated in liver carcinogenesis in the host. Although epidemiological evidence has clearly linked HBV infection with HCC development, the precise role of the virus and the molecular mechanisms of liver cell transformation remain elusive. Here we discuss potential oncogenic strategies of HBV, ranging from indirect mechanisms related to chronic necroinflammatory disease and to the effects of viral gene products on cell proliferation and apoptosis, to direct insertional activation of cellular (onco)genes. Presently, vaccination of high risk populations represents a major way to prevent the development of HBV-related liver cancer.

A specific distal promoter controls gamma-glutamyl transpeptidase gene expression in undifferentiated rat transformed liver cells.

In rat undifferentiated hepatoma cells, the gamma-glutamyl transpeptidase (GGT) gene is transcribed into a 2.3 and a 2.6 kb mRNA which, in contrast with other rat GGT transcripts, are not detected in more differentiated liver cells or adult tissues. Analysis of the cDNA sequences obtained from H5 hepatoma cells reveals that these two transcripts differ from other GGT mRNAs by a 312-nt unique untranslated leader sequence; this sequence maps on the gene in a single exon 10 kb upstream from the GGT promoter IV transcription start site. We established that the 2.6 kb mRNA V-1 and the 2.3 kb GGT mRNA V-2 derive, by alternate splicing, from a primary transcript initiated on a distal promoter on the rat GGT gene. This gene appears to be transcribed from five promoters, and the specific expression of this new distal promoter in undifferentiated hepatoma cells requires binding of activator protein-1 and hepatic nuclear factor 3 specific transcription factors to a composite cis-element in the proximal region of the promoter. The distal GGT promoter, specifically expressed in undifferentiated liver cells, might reflect the expression of that gene in liver precursor cells before they differentiate in the hepatocytic or biliary lineage.

Comprehensive allelotyping of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world, however the molecular mechanisms underlying liver cell transformation remain obscure. A genome-wide scan of loss of heterozygosity (LOH) in tumors provides a powerful tool to search for genes involved in neoplastic processes. To identify recurrent genetic alterations in liver tumors, we examined DNAs isolated from 120 HCCs and their adjacent non tumorous parts for LOH using a collection of 195 microsatellite markers located roughly every 20 cM throughout 39 autosomal arms. The mean heterozygosity was 73%. Our findings provide additional support that LOH for loci on chromosomal arms 1p, 4q, 6q, 8p, 13q and 16p is significantly elevated in HCC. The highest percentage of LOH is found for a locus in 8p23 (42% of informative csaes). This corresponds to one of the most common genetic abnormalities reported to date in these tumors. In addition, high ratio of LOH (> or = 35%) is observed on chromosome arms which had not been implicated in previous studies, notably on 1q, 2q and 9q. No correlation was found between LOH of specific chromosomal regions and etiologic factors such as chronic infections with hepatitis B or C viruses. This first report of an extensive allelotypic analysis of HCC should help in identifying new genes whose loss of function contributes to the development of liver cancer.

The Stereoisomeric Fjord-Region Benzo[c]phenanthrene-3,4-Dihydrodiol 1,2-Oxides Malignantly Transform Rat Liver Epithelial Cells

Abstract The rat liver oval cell line OC/CDE 22 was exposed to each of the four optically active fjord-region dihydrodiol epoxides of benzo[c]phenanthrene to investigate their capacity for malignant transformation of liver cells. All four configurational isomers malignantly transformed OC/CDE 22 cells, resulting in a similar colony-forming efficiency in soft agar. Inoculation of the transformed cells into newborn syngeneic rats produced an extremely high incidence of carcinomas with a short latency period. The induced carcinomas displayed cholangiocellular, adenoid and solid growing structures. Thus, the fjord-region dihydrodiol epoxides of benzo[c]phenanthrene are able to efficiently transform rat liver cells. Furthermore, experimental evidence is presented that proliferation plays a central role in liver cell transformation mediated by polycyclic aromatic hydrocarbons.

Precore/core mutations of hepatitis B virus in hepatocellular carcinomas developed on noncirrhotic livers

BACKGROUND & AIMS: Hepatitis B virus genomes have been detected in hepatocellular carcinoma developed without the pretumoral step of cirrhosis; this finding is consistent with a direct effect of the virus in liver cell transformation. The aim of this study was to investigate the molecular bases for the hepatitis B virus persistence. Three hepatitis B surface antigen-positive and 2 hepatitis B surface antigen- negative patients with hepatocellular carcinoma and without cirrhosis were studied for precore/core mutations in tumorous and nontumorous tissues. METHODS: The precore/core region was amplified and analyzed both by cloning and sequencing and by direct sequencing. RESULTS: Nonsynonymous mutations were identified selectively in tumorous tissues. Clusters of mutations were identified in the viral encapsidation (epsilon) signal, in immunodominant epitopes of the core protein, and in the polymerase. Common associated point mutations were found in 3 patients infected with hepatitis B virus genotype A in the epsilon signal and in the polymerase gene. CONCLUSIONS: Precore/core mutations located in domains involved in hepatitis B virus replication and immune response to the virus were identified. The selective presence of some of these mutations in tumorous tissues is consistent with their role in the persistence of the virus in neoplastic cells and, possibly, in liver oncogenesis. (Gastroenterology 1996 Sep;111(3):691-700)

LDL versus apolipoprotein B responses to variable proportions of selected amino acids in semipurified diets fed to rabbits and in the media of HepG2 cells

Rabbits fed amino acid diets containing high proportions of several essential amino acids (EAA), mainly Lys and Met, develop hypercholesterolemia associated with elevated low density lipoprotein (LDL) cholesterol and apoprotein B (apo B) levels. This effect is partially prevented by feeding a high proportion of Arg. We previously demonstrated that down-regulation of hepatic LDL receptors contributes to elevation of LDL produced by selected EAA. Additionally, our earlier studies and recent observation in transformed liver cells HepG2 suggested that amino acids can alter apo B production, and that direct interaction between amino acids and hepatic cells is likely to be involved in the mechanism. Presently, these hypotheses were further examined. Our experiment in vivo demonstrated that rates of incorporation of 14C-Lys into LDL apoprotein were elevated in rabbits fed hypercholesterolemic Lys- and Met-enriched diet compared with normocholesterolemic animals fed diets enriched with Lys and Leu. This suggested that increase in hepatic apo B production contributed to hypercholesterolemic response induced by the experimental diet. In vitro, we used HepG2 cells to investigate whether exposure of these cells to amino acid mixtures similar to those tested in rabbits can induce changes in apo B consistent with LDL responses found in vivo. These results showed that apo B responses in cell culture corresponded with LDL changes observed in rabbits in relation to effects of Arg, but not to those of hypercholesterolemic EAA. This suggests that HepG2 cells provide a useful model for studying the mechanism of regulation of apo B metabolism by dietary Arg but not of the regulation by other dietary amino acids.

Glutamine transport in isolated human hepatocytes and transformed liver cells

The transport of l-glutamine was examined in isolated adult and fetal human hepatocytes as well as in the human hepatoma cell lines HepG2 and SK-Hep. In all cells studied, glutamine uptake was at least 85% Na +-dependent. Kinetic analysis of the Na +-dependent component indicated mediation by a single transporter in three human hepatocyte preparations and in SK-Hep cells, whereas two transporters appeared to be responsible for glutamine uptake in HepG2 cells and in hepatocytes from the liver of one male patient. Amino acid inhibition analysis showed primary mediation by System N in fetal and adult hepatocytes, whereas System ASC was principally responsible for glutamine uptake in transformed cells. Similar to the rat transporter, human System N was pH-sensitive, stereospecific, and responsive to treatment with steroid hormones. Although the human carrier was less tolerant of Li +- for Na + substitution, glutamine transport in primary human hepatocytes was stimulated by treatment with hypotonic buffer (cell swelling), as reported in rat parenchymal cells. In contrast, glutamine transport in hepatoma cells was relatively insensitive to changes in extracellular pH and failed to show enhanced activity in response to hypoosmotic challenge. Collectively, the data suggest that markedly distinct plasma membrane transporters mediate the concentrative uptake of glutamine in normal and transformed human hepatocytes, and that the salient properties of System N have been largely conserved from rat to man.

Glutamine transport in isolated human hepatocytes and transformed liver cells

The transport of L-glutamine was examined in isolated adult and fetal human hepatocytes as well as in the human hepatoma cell lines HepG2 and SK-Hep. In all cells studied, glutamine uptake was at least 85% Na(+)-dependent. Kinetic analysis of the Na(+)-dependent component indicated mediation by a single transporter in three human hepatocyte preparations and in SK-Hep cells, whereas two transporters appeared to be responsible for glutamine uptake in HepG2 cells and in hepatocytes from the liver of one male patient. Amino acid inhibition analysis showed primary mediation by System N in fetal and adult hepatocytes, whereas System ASC was principally responsible for glutamine uptake in transformed cells. Similar to the rat transporter, human System N was pH-sensitive, stereospecific, and responsive to treatment with steroid hormones. Although the human carrier was less tolerant of Li(+)- for Na+ substitution, glutamine transport in primary human hepatocytes was stimulated by treatment with hypotonic buffer (cell swelling), as reported in rat parenchymal cells. In contrast, glutamine transport in hepatoma cells was relatively insensitive to changes in extracellular pH and failed to show enhanced activity in response to hypoosmotic challenge. Collectively, the data suggest that markedly distinct plasma membrane transporters mediate the concentrative uptake of glutamine in normal and transformed human hepatocytes, and that the salient properties of System N have been largely conserved from rat to man.

Malignant transformation of the liver tumour precursor cell line OC/CDE 22 by the four stereoisomeric fjord region 3,4-dihydrodiol 1,2-epoxides of benzo[c]phenanthrene.

In previous work we established the rat liver oval cell line OC/CDE 22 in order to study in vitro mechanisms of liver cell transformation. We have now exposed OC/CDE 22 cells to each of the four optically active fjord region dihydrodiol epoxides of benzo[c]phenanthrene to investigate their capacity for malignant transformation of liver cells. All four configurational isomers, which are among the most potent carcinogenic metabolites of polycyclic aromatic hydrocarbons tested in murine tumour models, malignantly transform OC/CDE 22 cells at a 2 microM dose level, resulting in a similar colony-forming efficiency in soft agar. Inoculation of the transformed cells into newborn syngeneic rats produced an extremely high incidence of carcinomas with a short latency period. The induced carcinomas displayed cholangiocellular, adenoid and solid growing structures. Neither cell growth in soft agar nor induction of tumor formation in newborn rats were achieved if confluent OC/CDE 22 cell cultures were exposed to each of the four stereoisomers and left in the confluent state for 4 weeks. In contrast, if confluent cells were exposed to the four stereoisomers, immediately split and then subcultured as usual, full transformation was accomplished. Our results indicate that the fjord region dihydrodiol epoxides of benzo[c]phenanthrene are highly efficient transforming agents for rat liver cells and that proliferation plays a pivotal role in the liver cell transformation process induced by polycylic aromatic hydrocarbons.

Expression of thec-myc protooncogene product in cells infected with the hepatitis delta virus

The intrahepatic accumulation of the c-myc protooncogene product was observed on immunofluorescence in each of six patients with chronic hepatitis delta virus infection who exhibited the hepatitis D antigen in their livers. The c-myc product was stained in the same nuclei that contained the hepatitis D antigen. C-myc was not observed in acute hepatitis D or in cases of chronic hepatitis delta virus infection without expression of the hepatitis D antigen. The protooncogene product was detected in only 1 of 32 viral and nonviral liver disorders unrelated to hepatitis delta virus. To confirm these observations, we transfected HBsAg-positive (PCL/PRF/5) and HBsAg-negative (HepG2) transformed liver cell lines with a plasmid containing a hepatitis delta virus cDNA trimer under the control of the SV40 early enhancer/promoter sequences. Whereas baseline c-myc expression was barely detectable in mock-transfected PLC/PRF/5 or HepG2 cells, strong c-myc nuclear fluorescence was observed when these same cells were transfected with the hepatitis D antigen expression vector. Similar results were obtained after infection of HeLa cells with a recombinant vaccinia virus expressing the hepatitis D antigen. Detection of c-myc mRNA sequences by means of in situ hybridization suggested that the c-myc product accumulation was not due to increased amounts of its mRNA. The c-myc protein accumulates selectively in the livers of patients with chronic hepatitis delta virus infection and in the same nuclei that contain the hepatitis D antigen. The expression of c-myc in hepatitis D antigen-containing cells does not require the presence of hepatitis B virus infection.

Evidence for long-range oncogene activation by hepadnavirus insertion.

Insertional mutagenesis of host genes, a common oncogenic strategy of slow transforming retroviruses, has recently been described for a DNA virus of the hepadnavirus group: the woodchuck hepatitis virus. This virus causes insertional activation of myc genes, mainly the intronless N-myc2 oncogene, in > 50% of woodchuck liver tumours. In most remaining tumours, N-myc2 is overexpressed without any apparent genetic alteration. To elucidate the role of the virus in such cases, we have cloned and analysed single integration sites in four woodchuck tumours carrying wild-type myc alleles. All sites were clustered within < 20 kb in a single locus, in which scarce unique sequences showed no detectable transcriptional activity. By fluorescent in situ hybridization, N-myc2 and the new locus (win) were localized to the same region of the long arm of the woodchuck X chromosome, and a 150-180 kb intervening distance was deduced from pulse-field gel analysis. The detection of viral integrations in win in additional tumours that produced abundant N-myc2 transcripts further substantiates the link between these two loci in woodchuck tumorigenesis. We propose that efficient activation of the N-myc2 promoter by the hepadnavirus enhancer acting over a long distance might operate in liver cell transformation.

Functional characterization of the rat gamma-glutamyl transpeptidase promoter that is expressed and regulated in the liver and hepatoma cells.

gamma-Glutamyl transpeptidase (GGT) is an enzyme encoded by multiple mRNAs (mRNAI to mRNAIV) that, in the rat, are transcribed from a single copy gene in a tissue-specific manner. In the liver, GGT expression is up-regulated in transformed cells, and this induction is the most widely used marker of liver cell transformation. We characterized the GGT mRNA species expressed in the liver (mRNAIII), and we report that this mRNA differs from the other GGT mRNA species by a 275-base alternate 5'-end sequence. Its transcription occurs on a specific promoter (promoter III) that maps on the GGT gene upstream of the two promoters coding for the GGT mRNAI and mRNAII. In hepatoma cells, mRNAIII expression is related to the differentiation state of the cells. We have shown that, in Reuber H-35-derived cell lines, the GGT mRNAIII is transcribed in cells that express a differentiated phenotype (Fao), but not in the dedifferentiated C2 and H5 variants. Moreover, we observed a reexpression of the GGT mRNAIII species in the C2 Rev7 variant, which has reverted from C2 toward a differentiated hepatocyte phenotype. In the proximal promoter III region, we identified a sequence that strongly enhances transcriptional activity in Fao and C2 Rev7 cells, but not in the dedifferentiated C2 variant. This motif interacts with nuclear proteins belonging to the NF-1 and NF-Y families that govern GGT promoter III expression in differentiated hepatoma cells.

Alterations of the p53 tumor-suppressor gene in transformed mouse liver cells.

Mutational inactivation of p53, a potential tumor-suppressor gene, has been found in many tumors of humans as well as rodents. The p53 status in normal and transformed mouse liver cell lines has, however, not been investigated. We examined possible point mutations and compared mRNA and protein expression of the p53 gene in normal vs. transformed mouse liver cells. The transformed cells studied included lines spontaneously transformed by sub-culture, virally transformed by simian virus 40 (SV40), and chemically transformed by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) or methylcholanthrene epoxide (MC). A heterozygous G-->A point mutation at codon 241, position 1, of p53 was detected in MNNG-transformed cells after screening of 5 evolutionarily conserved regions where mutation hot-spots are clustered. The mutation causes a gly-->arg substitution. No mutations were found in normal or other transformed cells. The steady-state levels of p53 mRNA were decreased in chemically transformed (both MNNG- and MC-transformed) cells. Elevated levels of p53 protein were found in spontaneously transformed and SV40-transformed cells, an observation that may reflect a longer half-life of the protein, as has been shown in other transformed lines. The low level of the p53 protein in MC-transformed cells may result from transcriptional depression of the p53 gene. We conclude from these data that abnormal p53 status, such as point mutation or altered expression, may play a role during the malignant transformation of mouse liver cells.

The expression of human plasma cholesteryl-ester-transfer protein in HepG2 cells is induced by sodium butyrate. Quantification of low mRNA levels by polymerase chain reaction.

Although human plasma cholesteryl-ester-transfer protein (CETP) is primarily synthesized in the liver, its expression in a number of transformed liver cell lines is very low. However the use of the human hepatoma cell line HepG2 as a model system for the regulation of CETP on mRNA level is facilitated by a quantitative reverse-transcribed polymerase chain reaction. We demonstrate a time-dependent and concentration-dependent 3-4-fold induction of CETP mRNA by sodium butyrate. CETP mass in the medium is also augmented; however, the effect on protein level is less pronounced.

The mitogenic potential of trichloroethylene in B6C3F1 mice

Male and female B6C3F1 mice were given TCE by gavage for 10 days. No histopathologic changes in the livers from control male and female mice were found. Moderate changes around central veins were noted in male or female mice that received 1000 mg/kg/body weight TCE. Histopathological changes included an increase in cytoplasmic eosinophilic staining and apoptoses around the central veins. Mitosis and DNA synthesis were examined using incorporation of [ 3H]thymidine into liver cells. Incorporation of [ 3H]thymidine was significantly increased in the DNA of animals receiving TCE. Total liver DNA extracted from TCA-treated mice was not significantly different than those of control groups. Autoradiographic examination of liver sections showed that the incorporation of label in control animals was primarily in perisinusoidal cells. The majority of radiolabel incorporation in TCE-treated mice was found in intermediate zone cells that appeared to be mature hepatocytes. No outstanding differences in the distribution of radiolabel in the liver from male or female mice were noted. When incorporation of [ 3H] thymidine was quantified by enumeration of labeled hepatocytes following autoradiography, incorporation of the radiolabel into hepatocytes increased proportionally to the applied dose of TCE, but did not increase in peri-sinusoidal cells. Increased mitotic figures in intermediate zone cells resembling mature hepatocytes were found in all mice treated with TCE. These results suggest that liver cell DNA synthesis and mitosis are stimulated by TCE and that these effects may be in part responsible for transformation of liver cells in these mice.