Differentiated Hepatoma Cell Line Research Articles

Relationship between endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase/eukaryotic initiation factor 2a/activating transcription factor 6 pathway and proliferation and migration of rat hepatoma cells

Objective To investigate the relationship between endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2a (eIF-2α)/activating transcription factor 6 (ATF6) pathway and proliferation and migration of rat hepatoma cells. Methods Rat liver cancer model was established by intermittent administration of diethylnitrosamine (DEN). Morphological changes of rat liver were observed by hematoxylin-eosin staining (HE) staining. The expression of PERK and eIF-2α protein in rat hepatocarcinoma tissues was detected by immunohistochemistry. The endoplasmic reticulum stress cell model was constructed by inducing normal liver cell LO2 and highly differentiated hepatoma cell line CBRH-7919 with absolute ethanol. Cell counting kit-8 (CCK-8) method was used to detect the proliferation of cells; the migration and invasion ability of cells were detected by scratch test; the PERK, eIF-2α, ATF6, and B lymphoma-2 were detected by Western blotting. Expression of bcl-2 and cysteine-containing aspartate proteolytic enzyme-3 (Caspase-3) protein, and statistical calculation. Results Liver cells in liver cancer were found to be irregularly arranged. Hepatocytes showed severe abnormalities, nuclear membrane depression, deep nuclear staining, and abnormal nuclear morphology of liver cells. After ethanol-induced LO2 and CBRH-7919 cells undergo endoplasmic reticulum stress, the cell migration ability of LO2 and CBRH-7919 cells was significantly decreased (tLO2 cells=8.910, P<0.01; tCBRH-7919 cells=30.010, P<0.01). The survival rates of LO2 and CBRH-7919 cells were significantly lower, and the difference was statistically significant (tLO2 cells=11.840, P<0.01; tCBRH-7919 cells=19.080, P<0.01). The expressions of PERK, eIF-2α, ATF6 and Caspase-3 in the liver cancer tissues of the experimental group were significantly increased, and the expression of bcl-2 protein was significantly decreased (tPERK=8.070, P<0.01; teIF-2α=11.790, P<0.01; tATF6= 7.750, P<0.01; tbcl-2=8.670, P<0.01; tCaspase-3=7.720, P<0.01). After ethanol-induced endoplasmic reticulum stress in LO2 cells, the expression levels of PERK, eIF-2α, ATF6, bcl-2 and Caspase-3 in LO2 cells were significantly increased, and bcl-2 protein expression was significantly decreased (tPERK=12.910, P<0.01; teIF-2α= 15.810, P<0.01; tATF6=7.690, P<0.01; tbcl-2=23.260, P<0.01; tCaspase-3=22.280, P<0.01). After the endoplasmic reticulum stress induced by absolute ethanol in CBRH-7919 cells, the expression of PERK, eIF-2α, ATF6, bcl-2 and Caspase-3 protein in CBRH-7919 cells was significantly increased, and the expression of bcl-2 protein was significantly decreased (tPERK= 9.560, P<0.01; teIF-2α= 12.400, P<0.01; tATF6=6.880, P<0.01; tbcl-2=13.840, P<0.01; tCaspase-3=9.950, P<0.01). Conclusion Activation of the PERK/eIF-2α/ATF4 pathway in endoplasmic reticulum stress significantly inhibits the proliferation and migration of rat hepatoma cells. Key words: Liver cancer; Endoplasmic reticulum stress; Protein kinase R-like endoplasmic reticulum kinase; Eukaryotic initiation factor 2α; Transcriptional activator-6

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.

The Chinese Herbal Medicine Sophora flavescens Activates Pregnane X Receptor

Sophora flavescens (SF) is an herbal medicine widely used for the treatment of viral hepatitis, cancer, viral myocarditis, gastrointestinal hemorrhage, and skin diseases. It was recently reported that SF up-regulates CYP3A expression. The mechanism of SF-induced CYP3A expression is unknown. In the current study, we tested the hypothesis that SF-induced CYP3A expression is mediated by the activation of pregnane X receptor (PXR). We used two cell lines, DPX2 and HepaRG, to investigate the role of PXR in SF-induced CYP3A expression. The DPX2 cell line is derived from HepG2 cells with the stable transfection of human PXR and a luciferase reporter gene linked with a human PXR response element identified in the CYP3A4 gene promoter. In DPX2 cells, SF activated PXR in a concentration-dependent manner. We used a metabolomic approach to identify the chemical constituents in SF, which were further analyzed for their effect on PXR activation and CYP3A regulation. One chemical in SF, N-methylcytisine, was identified as an individual chemical that activated PXR. HepaRG is a highly differentiated hepatoma cell line that mimics human hepatocytes. In HepaRG cells, N-methylcytisine significantly induced CYP3A4 expression, and this induction was suppressed by the PXR antagonist sulforaphane. These results suggest that SF induces CYP3A expression via the activation of PXR.

Hepatitis C Virus Triggers Apoptosis of a Newly Developed Hepatoma Cell Line Through Antiviral Defense System

Hepatitis C virus (HCV) has a tendency to cause chronic viral infection. Viral evasion of host immune systems plays a key role in the pathogenesis of HCV. However, the interaction between HCV and hepatocyte innate antiviral defense systems is not understood. The aim of this study was to examine how human hepatocytes respond to HCV infection. We have established a novel human hepatoma cell line, LH86, from a well-differentiated hepatocellular carcinoma tissue. An infectious HCV isolate, JFH-1, was used to infect LH86 cells. HCV replication and apoptosis after viral infection were examined. Mechanisms of HCV-induced apoptosis were determined. Type I interferon induction and the relevant signaling molecules were examined. LH86 cells permitted JFH-1 HCV infection. The viral infection caused massive apoptosis. The apoptosis was related to viral replication, because blocking viral entry with anti-CD81 or suppressing viral replication with interferon protected cells from HCV-induced apoptosis. The HCV-induced apoptosis appeared to be triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, death receptor 4 and death receptor 5, which were up-regulated in HCV infection. HCV also activated interferon response factor 3, which induced expression of interferon and TRAIL in LH86 cells. Our study showed that a specific HCV isolate, JFH-1, is cytopathic in this new hepatoma cell line. LH86 cells mount an intact innate antiviral defense through induction of interferon and triggering apoptosis of infected cells. This study reveals a novel mechanism by which host hepatocytes respond to acute HCV infection.

Reduction of PKCα decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.

Serum-free cell culture system supplemented with lipid-rich albumin for hepatitis C virus (strain O of genotype 1b) replication

HuH-7 is a highly differentiated hepatoma cell line and the only cell line that supports robust RNA replication of the hepatitis C virus (HCV). HuH-7 cells cause cell death in serum-free culture condition. However, the effect is reversed by supplementation with selenium. Serum-free cell cultures are advantageous for vaccine development and experimental reproducibility. However, HCV RNA replication in HuH-7 cells in serum-free medium had not yet been achieved. Therefore, we tried to develop a system for robust HCV RNA replication in a serum-free cell culture. Although HuH-7 cells grew in serum-free medium in the presence of selenium, HuH-7 cells under these conditions did not support HCV RNA replication in long-term culture. Among the supplements tested, serum-free medium with lipid-rich albumin (LRA) was found to yield robust HCV RNA replication. HCV proteins were detected for more than 9 months in serum-free medium supplemented with LRA. This is the first report to demonstrate a long-term, serum-free cell culture that successfully maintained robust HCV RNA replication. This cell culture system is expected to be a useful tool for vaccine development, as well as for further investigation of cellular factors that are essential for HCV RNA replication.

Increased endostatin/collagen XVIII expression correlates with elevated VEGF level and poor prognosis in hepatocellular carcinoma

Liver is the primary source for collagen XVIII, the precursor of angiogenesis inhibitor, endostatin. However, the role of endostatin/collagen XVIII expression during liver carcinogenesis remains elusive. Therefore, we studied its expression in five hepatoma cell lines and 105 hepatocellular carcinoma specimens. The poorly differentiated hepatoma cell lines exhibited increased endostatin/collagen XVIII levels compared with the well-differentiated ones. In hepatoma tissues, endostatin/collagen XVIII expression was detected in various types of liver cells and was significantly stronger in adjacent nontumor tissues than that in tumors (P<0.001). Endostatin/collagen XVIII expression in nontumor tissues correlated with tumor stages (P=0.014) and expression of vascular endothelial growth factor (P=0.007), but not the stages of hepatic fibrosis (P>0.05). Kaplan–Meier analysis showed that patients with higher endostatin/collagen XVIII expression had significantly shorter overall survival (P=0.011) and disease-free survival (P=0.0034). Moreover, endostatin/collagen XVIII level was an independent prognostic factor for tumor recurrence (P=0.034) by multivariate analysis. In conclusion, increased endostatin/collagen XVIII expression correlated with hepatoma progression and predicted poor prognosis for patients with hepatocellular carcinoma.

Comparison of rat hepatocyte and differentiated hepatoma cell line cultures as bio indicators of CYP 1A1 inducers in urban air

Cytochrome P450 1A1 CYP1A1 enzymatic activity was evaluated in cultured liver cells, and taken as a biological indicator of the presence of inducers of this isoform in urban airborne particulate matter fraction samples. It is known that CYP1A1 inducers can play an important role in the risk of mutagenesis and carcinogenesis by environmental pollution. A romatic polycyclic hydrocarbons PAH from urban air were collected in the city of Genoa Italy at two sites on two different days of the year. The objective of the study was to compare the inducibility of cultured rat hepatocytes with that of MH1C1 and FaO rat hepatoma cell lines after exposure to a PAH mixture and to a standard compound, such as benzo b fluoranthene B b F. Cytotoxic effects of the tested concentrations were evaluated by means of 3 4,5, dimenthylthyazol 2 yl 2,5 diphenyltetrazolium bromide MTT and lactate dehydrogenase release LDH tests, the potency of inducers by ethoxyresorufin O deethylase EROD assay. The results were in agreement in the three cellular systems: after exposure to the PAH mixture, an induction at low concentrations was observed; whereas no induction, but rather a decrease in activity was shown at higher concentrations; instead, the exposure to pure B b F showed a dose-response relationship in all cells, even at the highest doses. Such a difference between the toxicity of the complex mixture and that of the pure compound could be ascribed to the presence of drug metabolism inhibitors in the mixture, or to interactions between the original components and their metabolites. The finding that the cell lines responded to the CYP1A1 induction in a very efficient way gives further proof of the applicability of this system to environmental biomonitoring.

A functional hepatocyte nuclear factor 3 binding site is a critical component of the duck hepatitis B virus major surface antigen promoter.

The gene coding for the S protein, the smaller of the two envelope antigens of the duck hepatitis B virus (DHBV), is transcribed from a TATA-less promoter. In this study, we localized the promoter to a 245-bp segment of the genome that was capable of efficiently driving expression of a linked reporter gene upon transient transfection into the differentiated hepatoma cell lines LMH and HepG2. However, no measurable activity from this construct could be detected in similar assays with the dedifferentiated cell line HepG2.1 or the nonhepatic cell line HeLa. Located at position -25 relative to the transcriptional start site was a sequence conforming to the consensus binding site for hepatocyte nuclear factor 3 (HNF3). Deletion of this region reduced activity of the reporter gene to barely detectable levels in LMH cells. The results of electrophoretic mobility shift analysis (EMSA) demonstrated that a double-stranded oligonucleotide containing this sequence formed a specific complex with DNA-binding proteins from LMH and HepG2 cells but not with nuclear extracts obtained from HepG2.1 or HeLa cells. Cotransfection of HepG2.1 cells with DHBV S promoter constructs and a rat HNF3beta expression plasmid resulted in transactivation of only those constructs in which the candidate HNF3 site was present. Furthermore, EMSA using HepG2.1 nuclear extracts containing exogenously expressed HNF3 formed complexes with the same migration and competition properties as those in which the proteins were derived from the differentiated hepatoma cells. Thus, several lines of evidence suggest a critical role for HNF3 in activity from the DHBV S promoter.

Sequence of the rat PB-inducible CYP2B1 promoter

The 5′ flanking region of the rat PB-inducible CYP2B1 gene was isolated and the sequence from +27 to −3878 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 as well as an AP1, two NF-κ B and several possible STAT sites. The promoter sequence of the CYP2B1 gene was compared to that of the CYP2B1 sequence, published by Hoffman et al. ((1992) Gene Exp. 2, 353–362) and was found to be almost identical up to −2300 bp, beyond which it diverges significantly from the remaining published sequence of CYP2B1 gene. Transient transfection experiments in the differentiated hepatoma cell line, FGC4, showed that the 3.9 kb promoter was expressed, however, an increase in reporter activity was not observed in the presence of phenobarbital.

Progression through G1 and S phases of adult rat hepatocytes.

Regenerating liver, hepatocyte primary cultures and differentiated hepatoma cell lines are widely used to study the proliferation/differentiation/apoptosis equilibrium in liver. In hepatocytes, priming factors (TNF alpha, IL6) target G0/G1 transition while growth factors (HGF, EGF, TGF alpha) control a mid-late G1 restriction point. A characteristic pattern of cdk/cyclin expression is observed in hepatocytes, presumably related to their ability to proliferate a limited number of times and to undergo a reversible differentiation. Interestingly, cell-cell interactions between hepatocytes and liver biliary cells in co-cultures, result in a cell cycle arrest in mid G1 of hepatocytes which are insensitive to mitogens. Apoptosis exists in hepatocytes but is still poorly documented. However, hepatoma cell lines stimulated by TGF beta undergo cell death in a p53-independent pathway. In conclusion, the interplay of growth and apoptosis regulators and cell-cell interactions control the proliferation/differentiation/apoptosis balance which is a specific feature of hepatocytes.

A Comparison of Hepatic Enzyme Activities and their Modulation by Dexamethazone in Freshly Isolated and Cultured Hepatocytes and in the Differentiated Hepatoma Cell Line, 2sFou

Rodent hepatocytes are mitotically inhibited and lose hepatospecific functions over time in culture. In contrast, some differentiated hepatoma cell lines express stable hepatospecific functions in culture, but at much lower levels than those initially found in primary hepatocytes. A number of hepatospecific functions were measured in freshly isolated and cultured rat hepatocytes; these were compared to activities found in the differentiated Reuber hepatoma cell line, 2sFou. The effects of dexamethazone on these activities were also investigated, since dexamethazone is reported to enhance the expression of organotypic functions. The P450-related activities (ethoxyresorufin-O-deethylase activity and pentoxyresorufin-O-depenty-lase activity) and glucose-6-phosphatase activity declined in hepatocytes with increasing time in culture. The same activities in 2sFou cells were similar to those in hepatocytes which had been cultured for 72 hours. Tyrosine amino transferase (TAT) activity declined in hepatocyte cultures with time, but dexamethazone (1μM) restored activity up to freshly isolated cell values. TAT activity in hepatoma cells exceeded that in hepatocytes and was highly inducible by dexamethazone. γ-Glutamyl transpeptidase activity increased with culture time in hepatocytes and was also highly expressed in 2sFou cells. In hepatocytes, the activity of a high affinity alcohol dehydrogenase (ADH) declined with time in culture. In 2sFou cells, there was evidence of a low affinity (extra-hepatic or fetal) form of ADH, which was not evident in cultured hepatocytes.

Regulation of transcription from the hepatitis B virus large surface antigen promoter by hepatocyte nuclear factor 3

The influence of hepatocyte nuclear factor 3 (HNF3) on the level of transcriptional activity from the four hepatitis B virus promoters was investigated by transient-transfection analysis in the dedifferentiated hepatoma cell line, HepG2.1. It was found that the large surface antigen promoter and, to a much lesser extent, the nucleocapsid promoter were transactivated in the presence of HNF3. DNase I footprinting analysis demonstrated that purified recombinant HNF3 alpha protects one region of the large surface antigen promoter. Gel retardation analysis showed that a double-stranded oligonucleotide containing this HNF3-binding site formed a specific complex with DNA-binding proteins in the differentiated hepatoma cell lines, Huh7 and HepG2. The complex formed with Huh7 cell extract comigrated with exogenously expressed HNF3 beta in HeLa S3 extracts and was specifically inhibited from forming by the addition of HNF3 beta antiserum. The promoter element which appears to mediate the HNF3 transactivation was functionally mapped by mutational analysis to a region between nucleotides -65 and -54 relative to the transcriptional start site. This regulatory sequence is within the region protected from DNase I digestion by HNF3 alpha and contains 10 of 12 nucleotides homologous to the HNF3-binding-site consensus sequence. A synthetic promoter construct containing this HNF3-binding site was able to mediate transactivation by HNF3 beta. These and previous results suggest that the hepatitis B virus large surface antigen promoter is regulated by at least two liver-enriched transcription factors, HNF1 and HNF3, which together may contribute to the differentiated liver cell type specificity of this promoter.

Characterization of a Functional Hepatocyte Nuclear Factor 3 Binding Site in the Hepatitis B Virus Nucleocapsid Promoter

Mutational analysis of the hepatitis B virus (HBV) nucleocapsid promoter previously demonstrated that a regulatory sequence element (CpE) located between -72 and -56 modulated the level of transcription from this promoter in differentiated, but not dedifferentiated, hepatoma cell lines. Using gel retardation analysis, it was shown that the formation of a complex between the nucleocapsid CpE promoter sequence and the DNA-binding proteins present in the differentiated hepatoma cell line Huh7 was inhibited from forming in the presence of either the large surface antigen promoter hepatocyte nuclear factor 3 (HNF3) binding site or an HNFβ-specific antiserum. Purified recombinant HNF3α transcription factor was also shown to bind specifically to the CpE promoter sequence by gel retardation and DNase I footprinting analysis. In addition, DNase I footprinting analysis supported the suggestion that the nucleocapsid promoter region contains a second HNF3 binding site located between -112 and -86. The nucleocapsid promoter CpE regulatory element was shown to be a functional HNF3 binding site capable of mediating HNF3β-specific transcriptional transactivation in transient transfection analysis. These results suggest that the liver-enriched family of HNF3 transcription factors is involved in regulating the level of expression from the nucleocapsid promoter, in addition to the large surface antigen promoter, and is likely to be important in the coordinate regulation of HBV transcription during infection.

Constitutive expression and inducibility of O 6-methylguanine-DNA methyltransferase and N-methylpurine-DNA glycosylase in rat liver cells exhibiting different status of differentiation

We have analyzed the expression of the DNA repair genes O 6-methylguanine-DNA methyltransferase (MGMT) and N-methylpurine-DNA glycosylase (MPG) at RNA and protein activity level in primary rat hepatocytes in vitro and various rat hepatoma cell lines exhibiting different status of differentiation. The basal level of MGMT mRNA and activity correlated well with the degree of differentiation, as measured by tyrosine aminotransferase (TAT) mRNA expression. Induction of MGMT mRNA and protein activity by X-ray and Nmethyl- N′-nitro- N-nitrosoguanidine (MNNG) treatment was most pronounced in the well-differentiated hepatocytes and in various differentiated hepatoma cell lines (up to 6-fold). There was virtually no induction in H5 hepatoma cells which exhibited the lowest degree of differentiation and expressed only low amounts of MGMT. For the other hepatoma cell lines tested, MGMT induction did not clearly correlate with TAT expression. Thus, Fao cells which exhibited a high degree of differentiation responded only very weakly with respect to induction. The results indicate that the basal level of MGMT mRNA expression is dependent on liver-specific regulatory factors, whereas the inducibility is a more complex phenomenon not solely dependent on them. Contrary to MGMT, MPG was constitutively expressed at relatively high amounts in all cell lines tested and no correlation was apparent with the degree of differentiation. MPG activity was significantly induced by mutagen treatment only in H4IIE cells. The tumor promoter phenobarbital induced MGMT, but not MPG mRNA in hepatocytes. The results indicate that MGMT and MPG are not co-regulated. Hepatoma cells with low MGMT level were most sensitive to MNNG-induced cytotoxicity. On the other hand, no correlation was apparent between MPG activity and sensitivity of the cell lines to methylating agents indicating that the MPG level is not predictive for alkylating drug resistance.

Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter.

It has been demonstrated that the hepatocyte nuclear factor 1 (HNF1) binding site is critical for the majority of the hepatitis B virus (HBV) large surface antigen promoter activity in differentiated hepatoma cell lines. Examination of a series of clustered point mutations in the minimal large surface antigen promoter demonstrated that the HNF1 and TATA box binding sites are the major regulatory elements required for transcription from this promoter. Synthetic promoter constructs containing the large surface antigen promoter HNF1 binding site and TATA box element upstream of the luciferase open reading frame were tested for their transcriptional activities in HepG2. 1 cells in the absence or presence of an HNF1 expression vector. These synthetic promoter constructs displayed a similar level of transcriptional activity and induction by HNF1 in comparison with the full-length large surface antigen promoter, suggesting that additional HBV sequences are dispensable for full transcriptional activity. The distance between the HNF1 binding site and TATA box element in the synthetic promoter constructs appeared to influence the transcriptional activity modestly and in a periodic manner.

Differentiation-Specific Transcriptional Regulation of the Hepatitis B Virus Nucleocapsid Gene in Human Hepatoma Cell Lines

Transcription from the hepatitis B virus (HBV) nucleocapsid promoter is regulated in a cell-type-specific manner and can be modulated by the HBV enhancer 1 element. Mutagenesis analysis of the nucleocapsid promoter demonstrated that the two Sp1-binding sites (CpB and CpC) in the minimal promoter were the major determinants of transcriptional activity in the dedifferentiated hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3. In contrast, binding sites for transcription factors located immediately upstream (CpE) and downstream (CpF) of the two Sp1-binding sites were shown to be important determinants of nucleocapsid promoter activity in the differentiated hepatoma cell lines, Huh7 and HepG2. The role of these elements in the regulation of the nucleocapsid promoter activity correlated with the formation of specific DNA-protein complexes between Huh7 and HepG2 nuclear extracts and the CpE and CpF region sequences. Characterization of the influence of the nucleocapsid promoter mutations in the presence or absence of the enhancer 1 demonstrated that modification of individual transcription factor binding sites does not prevent enhancer-mediated activation of transcription from the nucleocapsid promoter. These results indicate that differentiated-hepatoma-specific transcription factors plus the Sp1 transcription factor interacting with the nucleocapsid promoter and the enhancer 1 regulatory region contribute to the level of transcription from this HBV promoter.

Phorbol esters inhibit the glucocorticoid-mediated stimulation of cytosolic aspartate aminotransferase gene transcription

The regulation of cytosolic aspartate aminotransferase (cAspAT) gene expression by phorbol esters was investigated in the highly differentiated hepatoma cell line Fao. Phorbol 12,13-dibutyrate (PdBu) had no effect on basal activity but partially inhibited the induction of cAspAT by dexamethasone. The extent of inhibition (40%) was similar to that obtained with insulin or vanadate. The inhibitory effects of PdBu and vanadate were additive. In the case of PdBu, the inhibitory effects could be eliminated by first incubating the cells with PdBu, which down-regulates protein kinase C. In contrast, inhibition by insulin was not modified by this treatment. The molecular mechanism of PdBu action was investigated. Northern blot analysis showed that the steady-state mRNA levels of cAspAT were decreased by PdBu in the presence of dexamethasone. In addition, the transcription rate, as measured by run-on experiments, was also decreased under the same conditions. Finally, a 2.4 kb promoter fragment driving the chloramphenicol acetyltransferase gene was stably transfected into the Fao cells. The regulation of the activity of this promoter fragment by dexamethasone and PdBu was similar to the regulation of the endogenous cAspAT activity. We conclude that PdBu acts by regulating the promoter activity of the cAsPAT gene.

Characterization of the hepatitis B virus- and nucleocapsid gene transcriptional regulatory elements

The regulatory DNA sequence elements that control the expression of the hepatitis B virus X- and nucleocapsid genes in the differentiated human hepatoma cell lines, Huh7, Hep3B, PLC/PRF/5, and HepG2, the dedifferentiated human hepatoma cell line, HepG2.1, and the human cervical carcinoma cell line, HeLa S3, were analyzed using transient transfection assays. In this system, the hepatitis B virus enhancer I located between coordinates 1071 (−239) and 1238 (−72) increases transcription from the X-gene promoter located between coordinates 1239 (−71) and 1376 (+67) more than 30-fold in the differentiated hepatoma and the HeLa S3 cell lines. In the dedifferentiated hepatoma cell line, HepG2.1, the enhancer I sequence increases the level of transcription from the X-gene promoter approximately 10-fold. The enhancer I subregion between coordinates 1117 (−193) and 1204 (−106) appears to be important for enhancer function only in the differentiated hepatoma cell lines, whereas the enhancer I subregion between coordinates 1222 (−88) and 1238 (−72) is required for enhancer activity in each of the cell lines examined. In all of the cell lines, the X-gene minimal promoter element was within a 138-nucleotide sequence located between coordinates 1239 (−71) and 1376 (+67). The enhancer I sequence increases transcription from the nucleocapsid promoter approximately 3- to 10-fold in the Huh7, Hep3B, PLC/PRF/5, and HeLa S3 cell lines, whereas it had little influence on the level of transcription from this promoter in HepG2 and HepG2.1 cells. The minimal nucleocapsid promoter element was within a 105 nucleotide sequence located between coordinates 1700 (-85) and 1804 (+20). This indicates that the levels of transcription from the X- and nucleocapsid gene promoters are determined in a cell-type-specific manner, in part, by the hepatitis B virus enhancer I and the corresponding minimal promoter sequence.

Positive and negative regulation of gene expression by insulin and glucagon: The model of L-type pyruvate kinase gene

L-type pyruvate kinase gene regulation is an excellent model of gene control by hormones and diet. In vivo and ex vivo experiments allowed us to established that thyroid hormones and glucocorticoids act on pyruvate kinase gene expression at the post-transcriptional level. In contrast, glucose and insulin together stimulate transcription of this gene while glucagon inhibits it. Insulin or glucose are individually inefficient and glucagon-dependent transcriptional inhibition seems to be dominant in insulin + glucose-dependent activation. A 14-kbp fragment encompassing the entire pyruvate kinase gene and 3.2-kbp of 5′ flanking sequences is expressed in transgenic mice exactly like the endogenous gene; the 3.2-kbp usptream region is sufficient to confer this tissue-specific and hormone/diet-regulated expression to reporter genes. In vivo, DNAse I hypersensitivity analysis revealed the presence of 3 liver-specific groups of hypersensitive sites (HSS). The proximal sites, between + 1 and −183 bp with respect to the start site of transcription, were, in addition, transcription-dependent. The nature and functional role of proteins binding to this proximal upstream sequence were analyzed by in vitro binding and cell free transcription experiments. The existence of more upstream cis-acting elements was investigated by transient transfection assays using differentiated hepatoma cell lines and hepatocytes in primary culture. These experiments permitted the detection of an extinguisher active in hepatoma Hep G 2 cells but not in hepatocytes, and of an activating element which could correspond to a distal HSS. Unfortunately, this investigation has not yet allowed us to determine with accuracy the DNA elements responsible for response to diet and hormones.