Transforming growth factor beta 1 partially suppresses the transformed phenotype of ras-transformed hepatocytes.

The effect of transforming growth factor beta type 1 (TGF-beta 1) on DNA synthesis, anchorage-dependent and anchorage-independent proliferation, cytoskeletal organization, and gene expression in ras-transformed simian virus 40 (SV40)-immortalized hepatocyte cell lines was measured. An SV40-immortalized cell line (CWSV1), a control neo-transfected and selected cell line (N1), and neo+ras-transfected and selected cell lines (NR3 and NR4) were used for this study. CWSV1 and N1 cells do not grow in soft agarose and are not tumorigenic. The ras-transformed hepatocytes NR3 and NR4 grow in soft agar and are tumorigenic. TGF-beta 1 treatment did not inhibit DNA synthesis or anchorage-dependent growth in the SV40-immortalized hepatocyte cell line CWSV1 or in the ras-transformed hepatocytes. TGF-beta 1 treatment inhibited anchorage-independent growth, increased actin cytoskeleton organization, and altered the morphology of ras-transformed hepatocytes; that is, with regard to all three of these properties, TGF-beta 1-treated ras-transformed hepatocytes more closely resembled the immortalized parent cell line. c-Ha-ras and c-myc RNA levels were not altered in TGF-beta 1-treated NR4 cells. TGF-beta 1 treatment did alter expression of some genes in NR4 cells. The level of expression of alpha 1 integrin RNA was higher in CWSV1 cells than in NR4 cells and increased in NR4 cells when they were treated with TGF-beta 1. Similarly, the levels and profiles of integrins on the cell surface of CWSV1 cells compared to NR4 cells, as determined by cell surface protein iodination, differed and in TGF-beta 1-treated NR4 cells more closely resembled the surface integrin profile for CWSV1 cells.     

Transformation of isolated rat hepatocytes with simian virus 40

Rat hepatocytes were transformed by simian virus 40 (SV40). Hepatocytes from two different strains of rats and a temperature-sensitive mutant (SV40tsA 1609), as well as wild-type virus were used. In all cases, transformed cells arose from approximately 50% of the cultures containing hepatocytes on collagen gels or a collagen gel-nylon mesh substratum. Cells did not proliferate in mock-infected cultures. SV40-transformed hepatocytes were epithelial in morphology, retained large numbers of mitochondria, acquired an increased nucleus to cytoplasm ratio, and contained cytoplasmic vacuoles. Evidence that these cells were transformed by SV40 came from the findings that transformants were 100% positive for SV40 tumor antigen expression, and that SV40 was rescued when transformed hepatocytes were fused with monkey cells. All SV40-transformed cell lines tested formed clones in soft agarose. Several cell lines transformed by SV40tsA 1609 were temperature dependent for colony formation on plastic dishes. Transformants were diverse in the expression of characteristic liver gene functions. Of eight cell lines tested, one secreted 24% of total protein as albumin, which was comparable to albumin production by freshly plated hepatocytes; two other cell lines produced 4.2 and 5.7%, respectively. Tyrosine aminotransferase activity was present in five cell lines tested but was inducible by dexamethasone treatment in only two. We conclude from these studies that adult, nonproliferating rat hepatocytes are competent for virus transformation.     

Liver-specific physiology of immortal,functionally differentiated hepatocytes and of deficient hepatocyte-like variants

Summary Five different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This research completes a previous study by Klocke and coworkers in 2002, using gene expression analysis of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202), consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/ enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg/h, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP-U value was found for the thre cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP-U ratio characteristic for continuous cell lines. This study demonstrates that the four transgenic and the p53-knockout, hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue-specific functions in immortalized cells.     

Transformation of differentiated rat hepatocytes with adenovirus and adenovirus DNA

Primary cultures of hepatocytes isolated by collagenase perfusion of adult rats were transformed by infection with adenovirus type 5 or transfection with adenovirus DNA. Total virion DNA or recombinant plasmid DNA containing the adenovirus E1A and E1B genes transformed hepatocytes at comparable frequencies. No foci of replicating hepatocytes were detected after transfection with a plasmid containing the E1A gene alone. The frequency of transformation by the adenovirus E1A and E1B genes was dependent on the composition of the culture medium. Transformation occurred at a low frequency when the transfected hepatocytes were maintained in a chemically defined medium (CDM), but the frequency was enhanced 8- to 10-fold when the cells were maintained in (i) serum-supplemented medium or (ii) CDM supplemented with epidermal growth factor. Cell lines derived from the adenovirus-transformed colonies of hepatocytes expressed adenovirus E1A and E1B RNAs. When hepatocytes were maintained in CDM supplemented with dimethyl sulfoxide and transfected with plasmids containing the E1A and E1B genes, it was possible to derive cell lines that retained the ability to express several liver-specific genes, including albumin, transferrin, hemopexin, and the third component of complement. The amount of albumin secreted per cell varied from 1 to 5 pg per cell per 24 h, and in one cell line it was below detectable levels by passage 9. Adenovirus-transformed hepatocytes were not tumorigenic when inoculated subcutaneously into neonatal syngeneic rats. We conclude that the adenovirus E1A and E1B genes are capable of transforming adult rat hepatocytes, a differentiated epithelial cell type.     

Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1.     

Isolation and characterization of mouse hepatocyte lines carrying a lethal albino deletion.

Mice homozygous for chromosomal deletions at or around the albino locus on chromosome 7 express reduced levels of a group of liver genes, including tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), and generally die perinatally. Sequences within the deleted region are thought to encode a regulatory factor(s) that affects expression of these genes in trans. To facilitate study of the putative factors, we immortalized hepatocytes derived from newborn cch wild-type and c14CoS deletion homozygous mice as well as cch/c14CoS heterozygous mice using a SV40 temperature-sensitive A255 mutant virus. Three c14CoS deletion homozygous hepatocyte lines were characterized and compared with the homozygous wild-type and heterozygous lines. The SV40 tsA255 mutant-transformed hepatocyte lines were temperature-sensitive for maintenance of transformation and expressed many liver-specific genes. In agreement with in vivo studies, hepatocyte lines derived from mice homozygous for the deletion expressed reduced mRNA levels of a number of liver genes including TAT, PEPCK, X1, X2, and X7 in comparison with heterozygous and wild-type cell lines. Similar mRNA levels of transferrin and albumin, genes whose expression is unaffected by the mutation in vivo, were observed in all cell lines. The expression of two genes, X5 and metallothionein, reported to be reduced in newborn mutant mice, did not differ appreciably among cell lines. TAT and PEPCK have been shown to respond poorly to glucocorticoids and cAMP in newborn mutant mice. Interestingly, all affected liver genes tested were responsive to glucocorticoids and dibutyryl cAMP in deletion homozygous cell lines as well as in wild-type and heterozygote-derived cell lines. This may suggest that effects of the deletion on expression of liver-specific genes do not cause loss of responsiveness to glucocorticoids and cAMP. These immortalized hepatocyte lines, which express most, if not all, liver-specific genes, should provide a useful means for further investigation of the effects of the albino lethal deletion.     

Effect of simian virus 40 infection on albumin production by hepatocytes cultured in chemically defined medium and plated on collagen and non-collagen attachment surfaces

Simian virus 40 (SV40)-infected rat hepatocytes were tested for albumin production (a marker of differentiated hepatocyte function) by growth in serum-free medium for 30-40 days. We observed a biphasic peak of albumin production, with the first peak at 4-6 and the second at 10-20 days after plating. A 2- to 3-fold increase in the second peak was observed when albumin production by SV40-infected cultures was compared with that by uninfected cultures. No increase in cell number in SV40-infected cultures was observed during this 20-day period. By 25-35 days, albumin production by uninfected cultures ceased but was detected in 50-60% of SV40-infected cultures. SV40-induced stimulation and persistence occurred regardless of the attachment surface and did not require collagen. Transformation occurred regardless of whether stimulation or persistence of albumin production was seen; however, stimulation and persistence were necessary to yield transformed cultures that produced albumin.     

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Five simian virus 40 (SV40)-hepatocyte cell lines were examined for tumorigenicity and the effect of in vitro passage on the expression of four liver-specific genes (albumin, transferrin, alpha 1-antitrypsin, and phosphoenolpyruvate carboxykinase), two oncogenes (c-Ha-ras and c-raf), and two genes associated with hepatocarcinogenesis (alpha-fetoprotein and placental-type glutathione-S-transferase). At low passage (12 to 22), all five cell lines expressed the four liver-specific genes at levels similar to those in the liver and were not tumorigenic or were weakly tumorigenic. At high passage (33 to 61), the cell lines formed carcinomas, and four out of five cell lines produced primary tumors that metastasized. At least two cell lines produced well-differentiated hepatocellular carcinomas that expressed liver-specific RNAs. Levels of expression of liver-specific genes changed with time in culture. Some of the changes in liver-specific gene expression in the tumor tissue (such as for the phosphoenolpyruvate carboxykinase gene) paralleled those that occurred with in vitro passage, while other changes (such as for the albumin gene) did not parallel those that occurred with in vitro passage. Correlations between enhanced expression of c-Ha-ras and tumorigenic potential and between the process of SV40 immortalization and induced expression of c-raf and glutathione-S-transferase-P were observed. Induction of alpha-fetoprotein was detected with in vitro and in vivo passage only in the CWSV14 cell line and was paralleled by diminished albumin expression. In conclusion, we developed a model system with five SV40-hepatocyte cell lines, tumors induced by them, and tumor cell lines to examine changes in gene expression that accompany the progression from a normal cell to a hepatocellular carcinoma. Because the SV40-hepatocyte cell lines and tumor cell lines remain highly differentiated and vary in the magnitude of expression of specific genes, they can be used to study the molecular mechanisms regulating gene expression, in particular those regulating specific genes associated with differentiation.     

Immortalization of chimpanzee hepatocytes with an amphoteric retrovirus encoding simian virus 40 T antigen.

Primary chimpanzee (Pan troglodytes) hepatocyte cultures were maintained in a serum-free medium containing hormones and growth factors and exhibited the de novo synthesis and secretion of numerous liver-specific plasma proteins for over 3 weeks in vitro. The long-term maintenance of differentiated, primate hepatocytes in this serum-free medium allowed for subsequent immortalization events to occur after infection with the amphoteric retrovirus U19, which encodes the simian virus 40 large T antigen oncogene. Several hepatocyte cell lines were selected and examined for the expression of liver-specific plasma proteins and the capacity to synthesize apolipoproteins. Several cell lines expressed a majority of the plasma proteins investigated, including apolipoproteins A1 and E. These results demonstrate the ability of this serum-free medium to maintain long-term differentiated primate hepatocytes, allowing for the experimental immortalization of this cell type in vitro and the maintenance of differentiated functions in the established cell lines. This methodology should be amenable to the study of the liver and its related diseases.     

Establishment of a human hepatocyte line (OUMS-29) having CYP 1A1 and 1A2 activities from fetal liver tissue by transfection of SV40 LT

Summary Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.     

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses.     

Finite life span of hybrids formed by fusion of different simian virus 40-immortalized human cell lines.

Simian virus 40 (SV40) genes are able to induce immortalization of normal human cells after a culture crisis during which unknown cellular genetic changes presumably occur. To determine whether these genetic changes are always identical, we performed somatic cell hybridization analysis of an SV40-immortalized human bronchial epithelial cell line, BET-1A. Fusion of BET-1A with an SV40-immortalized fibroblast cell line resulted in hybrids that senesced, indicating that these cell lines are in different complementation groups for immortalization.     

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter-based cell sorting. ES cell-derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell-derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell-derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.     

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.     

Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes

Hepatocytes of transgenic mouse fetuses harboring SV40 virus transforming gene sequences in the SV delta e-MGH fusion gene construct 202 driven by the mouse metallothionein (MT-I) enhancer [R. D. Palmiter, H. Y. Chen, A. Messing, and R. L. Brinster (1985) Nature (London) 316, 457-460] were cultured at Day 19 of gestation and established as a differentiated line expressing albumin and alpha-fetoprotein (AFP) mRNAs. Hepatocyte line FMH-202 contains integrated SV40 sequences, expresses SV40 T-antigen genes, and exhibits unlimited growth potential because it has been cultured 18 months without apparent decrease in cell viability or in growth rate that could suggest the occurrence of a crisis period. Immortalized cells multiply in chemically defined medium deficient in arginine with transferrin plus insulin, whereas EGF, insulin, and transferrin are obligatory requirements for fetal or newborn mouse hepatocyte multiplication in primary cultures. Cells did not grow in agar and were not tumorigenic in nude mice. Their immortalized, nonmalignant phenotype was further documented by low saturation densities of confluent monolayers showing no overgrowth, and by growth arrest in the absence of insulin with subsequent induction of DNA synthesis and resumption of cell growth in response to insulin. Thus, it appears that immortalized SV40 T-antigen-expressing hepatocytes are present in the liver of the transgenic mice. However, at later points in liver development the transforming activity of T-antigen becomes apparent and leads to hepatocellular carcinoma formation in vivo.     

Switching from differentiation to growth in hepatocytes: control by extracellular matrix.

Studies were carried out to analyze how different extracellular matrix (ECM) molecules regulate hepatocyte growth and differentiation. Freshly isolated rat hepatocytes were cultured on non-adhesive plastic dishes that were pre-coated with defined densities of either laminin, fibronectin, type I collagen, or type IV collagen. Sparse cell plating densities were used to minimize cell-cell contact formation and all studies were carried out in chemically defined medium that contained a saturating amount of soluble growth factors. Dishes coated with a low ECM density (1 ng/cm2) supported hepatocyte attachment, but did not promote cell spreading or growth. Computerized image analysis confirmed that over 80% of cells remained free of contact with other cells under these conditions. Yet, these round cells maintained high levels of albumin gene expression as well as elevated secretion rates for multiple liver-specific proteins (albumin, transferrin, and fibrinogen), regardless of the type of ECM molecule used for cell attachment. When ECM coating densities were raised from 1 to 1,000 ng/cm2, cell spreading, expression of histone mRNA, DNA synthesis, and cell proliferation all increased in parallel. Activation of growth by high ECM densities was also accompanied by a concomitant down-regulation of differentiated functions and again, dishes coated with all four types of ECM molecules produced similar effects. Thus, the ability to switch hepatocytes from differentiation to growth (i.e., between different genetic programs) is not limited to a single ECM molecule, a distinct three dimensional ECM geometry, or due to alteration of cell-cell interactions. Rather, the regulatory signals conveyed by immobilized ECM molecules depend on the density at which they are presented and thus, on their ability to either prohibit or support cell spreading.