Endotoxin regulates matrix genes increasing reactive oxygen species generation by intercellular communication between palmitate-treated hepatocyte and stellate cell.

Previous studies have shown that gut-derived bacterial endotoxins contribute in the progression of simple steatosis to steatohepatitis, although the mechanism(s) remains inaccurate to date. As hepatic stellate cells (HSC) play a pivotal role in the accumulation of excessive extracellular matrix (ECM), leading to collagen deposition, fibrosis, and perpetuation of inflammatory response, an in vitro model was developed to investigate the crosstalk between HSC and hepatocytes (human hepatoma cell) pretreated with palmitate. Bacterial lipopolysaccharide (LPS) stimulated HSC with phosphorylation of the p38 mitogen-activated protein kinase/NF-κB pathway, while several important pro-inflammatory cytokines were upregulated in the presence of hepatocyte-HSC. Concurrently, fibrosis-related genes were regulated by palmitate and the inflammatory effect of endotoxin where cells were more exposed or sensitive to reactive oxygen species (ROS). This interaction was accompanied by increased expression of the mitochondrial master regulator, proliferator-activated receptor gamma coactivator alpha, and a cytoprotective effect of the agent N-acetylcysteine suppressing ROS production, transforming growth factor-ß1, and tissue inhibitor of metalloproteinase-1. In summary, our results demonstrate that pro-inflammatory mediators LPS-induced promote ECM rearrangement in hepatic cells transcriptionally committed to the regulation of genes encoding enzymes for fatty acid metabolism in light of differences that might require an alternative therapeutic approach targeting ROS regulation.

Evaluation of genotoxicity using automated detection of γH2AX in metabolically competent HepaRG cells.

The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay.

Inflammatory cytokines promote the retrodifferentiation of tumor-derived hepatocyte-like cells to progenitor cells.

UNLABELLED: Human hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and resistance to therapies. Recent studies have reported that HCC may be derived not only from adult hepatocytes and hepatoblasts but also hepatic stem/progenitors. In this context, HepaRG cells may represent a suitable cellular model to study stem/progenitor cancer cells and the retrodifferentiation of tumor-derived hepatocyte-like cells. Indeed, they differentiate into hepatocyte- and biliary-like cells. Moreover, tumor-derived HepaRG hepatocyte-like cells (HepaRG-tdHep) differentiate into both hepatocyte- and biliary-like cells through a hepatic progenitor. In this study we report the mechanisms and molecular effectors involved in the retrodifferentiation of HepaRG-tdHep into bipotent progenitors. Gene expression profiling was used to identify genomic changes during the retrodifferentiation of HepaRG-tdHep into progenitors. We demonstrated that gene expression signatures related to a poor-prognosis HCC subclass, proliferative progenitors, or embryonic stem cells were significantly enriched in HepaRG progenitors derived from HepaRG-tdHep. HepaRG-tdHep retrodifferentiation is mediated by crosstalk between transforming growth factor beta 1 (TGFß1) and inflammatory cytokine pathways (e.g., tumor necrosis factor alpha [TNFα] and interleukin 6 [IL6]). Signatures related to TNFα, IL6, and TGFß activation pathways are induced within the first hour of retrodifferentiation. Moreover, specific activation or inhibition of these signaling pathways allowed us to determine that TNFα and IL6 contribute to the loss of hepatic-specific marker expression and that TGFß1 induces an epithelial-to-mesenchymal transition of HepaRG-tdHep. Interestingly, the retrodifferentiation process is blocked by the histone deacetylase inhibitor trichostatin A, opening new therapeutic opportunities. CONCLUSION: Cancer progenitor cells (or metastasis progenitors) may derive from tumor-derived hepatocyte-like cells in an inflammatory environment that is frequently associated with HCC.

Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells.

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation.

CYP4F3B expression is associated with differentiation of HepaRG human hepatocytes and unaffected by fatty acid overload.

Fatty acid microsomal ω-oxidation involves cytochrome P450 enzymes. Some of them belonging to the CYP4F3 family are mainly expressed in the liver, making this organ a major player in energy homeostasis and lipid metabolism. To study this important regulation pathway, we used HepaRG cells, which gradually undergo a complete differentiation process. Even at the early stage of the differentiation process, CYP4F3B generated by alternative splicing of the CYP4F3 gene represented the prevalent isoform in HepaRG cells as in the liver. Its increasing expression associated with hepatocyte differentiation status suggested a hepatic-specific control of this isoform. As in liver microsomes, the catalytic hydroxylation of the CYP4F3B substrate [1-¹4C]Z9(10)-epoxystearic acid led to major production of 18-hydroxy-9(10)-epoxystearic acid. When treated with saturated, monounsaturated, or polyunsaturated fatty acids, CYP4F3B and CYP4A11 expression remained unchanged whereas CYP4F2 and CYP4F12 expression was transiently up-regulated. A 24-h exposure of differentiated HepaRG cells to various polyunsaturated fatty acids and derivatives induced microvesicular steatosis; down-regulation of lipid metabolism gene regulators such as sterol regulatory element-binding protein-1c, fatty acid synthase, peroxisome proliferator-activated receptor γ (PPARγ), PPARα, and decreased expression of glucose-dependent metabolism genes, which could limit de novo lipogenesis. Docosahexaenoic acid seemed to be the most effective compound. These results suggest that a PPARα-independent pathway could participate to limit lipogenesis and emphasize the role of hepatocytes in the fatty acid ω-hydroxylation pathway. They also give insights on the use of HepaRG hepatocytes to open new avenues of investigations on factors mediating the lipid metabolic pathway and finding new hypolipidemic molecules.

A Minimal Subset of Seven Genes Associated with Tumor Hepatocyte Differentiation Predicts a Poor Prognosis in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a deadly cancer worldwide as a result of a frequent late diagnosis which limits the therapeutic options. Tumor progression in HCC is closely correlated with the dedifferentiation of hepatocytes, the main parenchymal cells in the liver. Here, we hypothesized that the expression level of genes reflecting the differentiation status of tumor hepatocytes could be clinically relevant in defining subsets of patients with different clinical outcomes. To test this hypothesis, an integrative transcriptomics approach was used to stratify a cohort of 139 HCC patients based on a gene expression signature established in vitro in the HepaRG cell line using well-controlled culture conditions recapitulating tumor hepatocyte differentiation. The HepaRG model was first validated by identifying a robust gene expression signature associated with hepatocyte differentiation and liver metabolism. In addition, the signature was able to distinguish specific developmental stages in mice. More importantly, the signature identified a subset of human HCC associated with a poor prognosis and cancer stem cell features. By using an independent HCC dataset (TCGA consortium), a minimal subset of seven differentiation-related genes was shown to predict a reduced overall survival, not only in patients with HCC but also in other types of cancers (e.g., kidney, pancreas, skin). In conclusion, the study identified a minimal subset of seven genes reflecting the differentiation status of tumor hepatocytes and clinically relevant for predicting the prognosis of HCC patients.

Retrodifferentiation of Human Tumor Hepatocytes to Stem Cells Leads to Metabolic Reprogramming and Chemoresistance.

Human hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and therapeutic resistance. We recently demonstrated that inflammation favors hepatocyte retrodifferentiation into progenitor cells. Here, we identify the molecular effectors that induce metabolic reprogramming, chemoresistance, and invasiveness of retrodifferentiated HCC stem cells. Spheroid cultures of human HepaRG progenitors (HepaRG-Spheres), HBG-BC2, HepG2, and HuH7 cells and isolation of side population (SP) from HepaRG cells (HepaRG-SP) were analyzed by transcriptomics, signaling pathway analysis, and evaluation of chemotherapies. Gene expression profiling of HepaRG-SP and HepaRG-Spheres revealed enriched signatures related to cancer stem cells, metastasis, and recurrence and showed that HepaRG progenitors could retrodifferentiate into an immature state. The transcriptome from these stem cells matched that of proliferative bad outcome HCCs in a cohort of 457 patients. These HCC stem cells expressed high levels of cytokines triggering retrodifferentiation and displayed high migration and invasion potential. They also showed changes in mitochondrial activity with reduced membrane potential, low ATP production, and high lactate production. These changes were, in part, related to angiopoietin-like 4 (ANGPTL4)-induced upregulation of pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of mitochondrial pyruvate dehydrogenase. Upregulation of ANGPTL4 and PDK4 paralleled that of stem cells markers in human HCC specimens. Moreover, the PDK4 inhibitor dichloroacetate reversed chemoresistance to sorafenib or cisplatin in HCC stem cells derived from four HCC cell lines. In conclusion, retrodifferentiated cancer cells develop enhanced invasion and therapeutic resistance through ANGPTL4 and PDK4. Therefore, restoration of mitochondrial activity in combination with chemotherapy represents an attractive therapeutic approach in HCC. SIGNIFICANCE: Restoring mitochondrial function in human hepatocellular carcinomas overcomes cancer resistance.

Long-term functional stability of human HepaRG hepatocytes and use for chronic toxicity and genotoxicity studies.

The human hepatoma HepaRG cells are able to differentiate in vitro into hepatocyte-like cells and to express various liver-specific functions, including the major cytochromes P450. This study was aimed to determine whether differentiated HepaRG cells retained their specific functional capacities for a long time period at confluence. We show that expression of transcripts encoding CYP1A2, 2B6, 3A4, and 2E1, several phase II and antioxidant enzymes, membrane transporters, including organic cation transporter 1 and bile salt export pump, the nuclear receptors constitutive androstane receptor and pregnane X receptor, and aldolase B remained relatively stable for at least the 4-week confluence period tested. Similarly, activities of CYP3A4 and CYP1A2 and their responsiveness to prototypical inducers were well preserved. Aflatoxin B(1), a potent hepatotoxicant and carcinogen, induced a dose-dependent and cumulative cytotoxicity. Furthermore, at a concentration as low as 0.1 microM, this mycotoxin caused a decrease in both CYP3A4 activity and intracellular ATP associated with morphological alterations, after 14 days following every 2-day exposure. Moreover, using the comet assay, a dose-dependent DNA damage was observed after a 3-h treatment of differentiated HepaRG cells with 1 to 5 microM aflatoxin B(1) in the absence of any cell damage, and this DNA damaging effect was strongly reduced in the presence of ketoconazole, a CYP3A4 inhibitor. These results bring the first demonstration of long-term stable expression of liver-specific markers in HepaRG hepatocyte cultures maintained at confluence and show that these cells represent a suitable in vitro liver cell model for analysis of acute and chronic toxicity as well as genotoxicity of chemicals in human liver.

Cytotoxic evaluation of sarcodifurines A and B, two novel dihydrofuroquinolines from Sarcomelicope follicularis (Rutaceae).

Sarcodifurines A and B are two original dihydrofuroquinolines isolated from Sarcomelicope follicularis, a New Caledonian tree. The cytotoxicity and antiproliferative activity of these two alkaloids were investigated against 8 distinct cell lines representative of the most frequent solid tumors developing in human. Cytotoxicity of sarcodifurines was low on the 8 cell lines, with, for example, less than 10% of the total cells killed after 24 h exposure at 10 microM and IC(50) approximately 7.10(-5) M (MCF-7 and MDA MB 231 cell lines). Proliferation studies confirmed that sarcodifurines had a weak effect on cancer cells growth, with less than 5% growth inhibition at 10 microM. Sarcodifurine A activity was comparable to that of Sarcodifurine B, in term of cytotoxicity and antiproliferative activity on all cell lines. In spite of the weak activity of sarcodifurines and furoquinolines, rationalized pharmacomodulations to obtain planar analogs could lead to efficient topoisomerases inhibitors and DNA intercalants.

The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics.

Although they have several important limitations primary human hepatocytes still represent the in vitro gold standard model for xenobiotic metabolism and toxicity studies. The large use of human liver cell lines either from tumoral origin or obtained by oncogenic immortalisation is prevented by the loss of various liver-specific functions, especially many cytochrome P450 (CYP)-related enzyme activities. We review here recent results obtained with a new human hepatoma cell line, named HepaRG, derived from a human hepatocellular carcinoma. These cells exhibit unique features: when seeded at low density they acquire an elongated undifferentiated morphology, actively divided and after having reached confluency formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. Moreover contrary to other human hepatoma cell lines including HepG2 cells, HepaRG cells express various CYPs (CYP1A2, 2B6, 2C9, 2E1, 3A4) and the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) at levels comparable to those found in cultured primary human hepatocytes. They also express various other functions such phase 2 enzymes, apical and canalicular ABC transporters and basolateral solute carrier transporters, albumin, haptoglobin as well as aldolase B that is a specific marker of adult hepatocytes. HepaRG cells could represent a surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies and even more, a unique model system for analysing genotoxic compounds.

TET-Catalyzed 5-Hydroxymethylation Precedes HNF4A Promoter Choice during Differentiation of Bipotent Liver Progenitors.

Understanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A.

Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line.

Functional expression of both sinusoidal and canalicular hepatic drug transporters was investigated in the highly differentiated human hepatoma HepaRG cell line and also, for comparison, in primary human hepatocytes and in the hepatoma HepG2 cell line. Using RT-qPCR assays, differentiated HepaRG cells were found to display a pattern of transporter expression close to that found in primary human hepatocytes, i.e. they exhibit substantial mRNA levels of the influx transporters OCT1, OATP-B, OATP-C and NTCP, and of the secretion transporters MRP2, MRP3, BSEP and P-glycoprotein. By contrast, expression of influx transporters was not present or very weak in HepG2 cells. Drug transport assays allowed to detect functional activities of OCT1, OATPs/OAT2, NTCP, MRPs and P-glycoprotein in differentiated HepaRG cells as in primary human hepatocytes whereas HepG2 cells only showed notable MRP and P-glycoprotein activities. In addition, expression of canalicular transporters in HepaRG cells was found to be up-regulated by known inducers of transporters such as rifampicin, phenobarbital and chenodeoxycholate acting on P-glycoprotein, MRP2 and BSEP, respectively. HepaRG cells thus exhibit functional expression of both sinusoidal and canalicular drug transporters and have retained regulatory pathways controlling transporter levels. These data, associated with the known high expression of drug metabolizing enzymes in HepaRG cells, highlight the interest of such hepatoma cells for analysing hepatic drug detoxification pathways.

A new subfamily of structurally related human F-box proteins.

F-box proteins, a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box), recruit substrates for ubiquitination and consequent degradation through their specific protein-protein interaction domains. Here, we report the identification of full-length cDNAs encoding three novel human F-box proteins named FBG3, FBG4 and FBG5 which display similarity with previously identified NFB42 (FBX2) and FBG2 (FBX6) proteins. All five proteins are characterized by an approximately 180-amino-acid (aa) conserved C-terminal domain and thus constitute a third subfamily of mammalian F-box proteins. Analysis of genomic organization of the five FBG genes revealed that all of them consist of six exons and five introns. FBG1, FBG2 and FBG3 genes are located in tandem on chromosome 1p36, and FBG4 and FBG5 are mapped to chromosome 19q13. FBG genes are expressed in a limited number of human tissues including kidney, liver, brain and muscle tissues. Expression of rat FBG2 gene was found related to differentiation/proliferation status of hepatocytes. Specifically, FBG2 mRNA was expressed in foetal liver, decreased after birth and re-accumulated in adult liver. Expression of FBG2 was strongly inhibited in hepatoma cells by okadaic acid.

Highly efficient SiRNA and gene transfer into hepatocyte-like HepaRG cells and primary human hepatocytes: new means for drug metabolism and toxicity studies.

The metabolically competent hepatocyte-like human HepaRG cells represent a suitable alternative in vitro cell model to human primary hepatocytes. Here, we describe the culture procedure required to expand progenitor HepaRG cells and to differentiate them into hepatocyte-like cells. Transient transfection of gene and siRNA into cultured cells, using nonviral strategies, is an invaluable technique to decipher gene functions. In this chapter, we detail transfection protocols for efficient transfer of plasmid DNA or siRNAs into proliferating progenitor or quiescent differentiated HepaRG cells as well as into primary hepatocytes.

Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma.

Many solid malignant tumors arise on a background of inflamed and/or fibrotic tissues, features that are found in more than 80% hepatocellular carcinomas (HCC). Activated hepatic stellate cells (HSC) play a critical role in fibrogenesis associated with HCC onset and progression, yet their functional impact on hepatocyte fate remains largely unexplored. Here, we used a coculture model to investigate the cross-talk between hepatocytes (human hepatoma cells) and activated human HSCs. Unsupervised genome-wide expression profiling showed that hepatocyte-HSC cross-talk is bidirectional and results in the deregulation of functionally relevant gene networks. Notably, coculturing increased the expression of proinflammatory cytokines and modified the phenotype of hepatocytes toward motile cells. Hepatocyte-HSC cross-talk also generated a permissive proangiogenic microenvironment, particularly by inducing VEGFA and matrix metalloproteinase (MMP)9 expression in HSCs. An integrative genomic analysis revealed that the expression of genes associated with hepatocyte-HSC cross-talk correlated with HCC progression in mice and was predictive of a poor prognosis and metastasis propensity in human HCCs. Interestingly, the effects of cross-talk on migration and angiogenesis were reversed by the histone deacetylase inhibitor trichostatin A. Our findings, therefore, indicate that the cross-talk between hepatoma cells and activated HSCs is an important feature of HCC progression, which may be targeted by epigenetic modulation.

The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays.

We report the fabrication of a 3D micropatterned agarose substrate that enables the culture of single or multiple cells. Patterning was performed on dried agarose using deep UV irradiation leading to 6-microm-deep micropatterns of 25-70 microm in diameter. Cell adhesion was facilitated by the specific grafting of ECM (extra cellular matrix) proteins such as fibronectin into the micropatterns. We show that the pattern size induced the adhesion of one or more cells, thus allowing precise control of the cell number used in the assay, and that cells proliferated similarly as in standard culture conditions. Moreover, cell polarity appeared well preserved on this substrate, so polarized cells like hepatoma HepaRG cells might maintain their differentiation status and act as primary human hepatocytes for hepatotoxicity testing. These 3D patterned culture slides have been successfully used for in situ comet assays and there is evidence that the genotoxic effects of sub-cytotoxic concentrations of drugs could be analyzed in a large number of single HeLa cells. Coupled with the parallel-based design of the 3D micropatterning, which allows automated image analysis, these results strongly indicate that this new cell array system is suitable for high-throughput cytotoxicity and genotoxicity screening applications.

Drug-metabolising enzymes are down-regulated by hypoxia in differentiated human hepatoma HepaRG cells: HIF-1alpha involvement in CYP3A4 repression.

Weak blood irrigation within solid tumours including hepatocellular carcinomas (HCCs) plays an important role in resistance to anticancer drugs by decreasing accessibility of cytotoxic agents to tumour cells. Reduced oxygen levels, or hypoxia, also contribute to drug resistance because many anticancer drugs require molecular oxygen to be cytotoxic. Our aim was to develop a new in vitro model mimicking hypoxic cells within HCCs in order to further explore the molecular responses to hypoxia, including regulation of drug-metabolising enzymes (DMEs) expression. For this purpose, we used the highly differentiated human hepatoma HepaRG cells cultured under either normoxic or hypoxic (24h at 1% O(2)) conditions. Gene and protein expressions were investigated by quantitative PCR and immunoblotting, respectively. We showed that HepaRG cells adapt to prolonged moderate hypoxia by a switch from aerobic to anaerobic glycolysis and a repression of critical genes involved in amino acid, lipid and ethanol metabolisms. Importantly, expression of several DMEs (particularly cytochromes P450 (CYPs) and phase II enzymes) and xenosensors (CAR, PXR and AhR) was down-regulated and CYPs activities (using testosterone and paclitaxel as substrates) were decreased during hypoxia. In addition, a new role for HIF-1alpha in the repression of CYP3A4 is demonstrated in cells treated with chemical inducers of HIF-1alpha, cobalt chloride or desferrioxamine, and by transfecting untreated HepaRG cells with HIF-1alpha expression vector. In conclusion, HepaRG cells cultured under hypoxia might mimic metabolic changes occurring within poorly irrigated differentiated HCCs. Furthermore, hypoxia down-regulates hepatic DMEs, a phenomenon that might compromise chemotherapy effectiveness in HCC treatment. Thus, HepaRG cells might represent a new in vitro model to test anticancer agents in hypoxic versus normoxic conditions.

Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor.

UNLABELLED: Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21(CIP1) and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of beta-catenin localization and HNF3beta expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. CONCLUSION: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors.

Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells.

Most human hepatocyte cell lines lack a substantial set of liver-specific functions, especially major cytochrome P450 (P450)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the HepaRG cells, derived from a human hepatocellular carcinoma, which exhibit a high differentiation pattern after 2 weeks at confluency to determine whether they could mimic human hepatocytes for drug metabolism and toxicity studies. We show that when passaged at low density, these cells reversed to an undifferentiated morphology, actively divided, and, after having reached confluency, formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. By contrast, when seeded at high density, hepatocyte-like clusters retained their typical differentiated morphology. Transcripts of various nuclear receptors (aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, peroxisome proliferator-activated receptor alpha), P450s (CYP1A2, 2C9, 2D6, 2E1, 3A4), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), and other liver-specific functions were estimated by reverse transcriptase-quantitative polymerase chain reaction and were found to be expressed, for most of them, at comparable levels in both confluent differentiated and high-density differentiated HepaRG cells and in cultured primary human hepatocytes. For several transcripts, the levels were strongly increased in the presence of 2% dimethyl sulfoxide. Measurement of basal activities of several P450s and their response to prototypical inducers as well as analysis of metabolic profiles and cytotoxicity of several compounds confirmed the functional resemblance of HepaRG cells to primary cultured human hepatocytes. In conclusion, HepaRG cells constitute the first human hepatoma cell line expressing high levels of the major P450s involved in xenobiotic metabolism and represent a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies.

Upregulation of the tumor suppressor gene menin in hepatocellular carcinomas and its significance in fibrogenesis.

The molecular mechanisms underlying the progression of cirrhosis toward hepatocellular carcinoma were investigated by a combination of DNA microarray analysis and literature data mining. By using a microarray screening of suppression subtractive hybridization cDNA libraries, we first analyzed genes differentially expressed in tumor and nontumor livers with cirrhosis from 15 patients with hepatocellular carcinomas. Seventy-four genes were similarly recovered in tumor (57.8% of differentially expressed genes) and adjacent nontumor tissues (64% of differentially expressed genes) compared with histologically normal livers. Gene ontology analyses revealed that downregulated genes (n = 35) were mostly associated with hepatic functions. Upregulated genes (n = 39) included both known genes associated with extracellular matrix remodeling, cell communication, metabolism, and post-transcriptional regulation gene (e.g., ZFP36L1), as well as the tumor suppressor gene menin (multiple endocrine neoplasia type 1; MEN1). MEN1 was further identified as an important node of a regulatory network graph that integrated array data with array-independent literature mining. Upregulation of MEN1 in tumor was confirmed in an independent set of samples and associated with tumor size (P = .016). In the underlying liver with cirrhosis, increased steady-state MEN1 mRNA levels were correlated with those of collagen alpha2(I) mRNA (P < .01). In addition, MEN1 expression was associated with hepatic stellate cell activation during fibrogenesis and involved in transforming growth factor beta (TGF-beta)-dependent collagen alpha2(I) regulation. In conclusion, menin is a key regulator of gene networks that are activated in fibrogenesis associated with hepatocellular carcinoma through the modulation of TGF-beta response.