Immortalized Hepatocyte Cell Line Research Articles

Replication and Injury Associated With SARS-CoV-2 in Cultured Hepatocytes.

Liver dysfunction is one of the hallmarks of SARS-CoV-2 infection. The mechanism(s) of hepatic injury in SARS-CoV-2 infection remains controversial with some reporting viral replication and cellular injury and others suggesting lack of replication and injury due to non-cytopathogenic etiologies. To investigate this further, we evaluated SARS-CoV-2 replication in immortalized hepatic cell lines and primary hepatocytes, examined whether cell injury was associated with apoptotic pathways, and also determined the effect of the antiviral remdesivir on these processes. Immortalized hepatocyte cell lines (HepG2 and Huh7.5), as well as primary human hepatocytes, were exposed to SARS-CoV-2 at a multiplicity of infection of 0.1 PFU/mL. Viral replication was evaluated by plaque assays, immunohistochemical staining for the viral spike protein, and caspase-3 expression evaluated with and without exposure to remdesivir. All hepatocyte cell lines and primary hepatocytes supported active replication of SARS-CoV-2. Significant cytopathic effect was observed by light microscopy, and caspase-3 staining supported activation of apoptotic pathways. Remdesivir abrogated infection in a dose-dependent fashion and was not independently associated with hepatocyte injury. Hepatocytes appear to be highly permissive of SARS-CoV-2 replication which leads to rapid cell death associated with activation of apoptotic pathways. Viral replication and hepatocytes injury are abrogated with remdesivir. We conclude that active viral replication is most likely a key contributor to liver enzyme abnormalities observed in the setting of acute SARS-CoV-2 infection.

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

BackgroundMany drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI).MethodsTo generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations.ResultsThe hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen.ConclusionsiPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

Potential for Isolation of Immortalized Hepatocyte Cell Lines by Liver-Directed In Vivo Gene Delivery of Transposons in Mice.

Isolation of hepatocytes and their culture in vitro represent important avenues to explore the function of such cells. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferate in vitro. Immortalization of isolated hepatocytes is thus an important step toward continuous in vitro culture. For cellular immortalization, integration of relevant genes into the host chromosomes is a prerequisite. Transposons, which are mobile genetic elements, are known to facilitate integration of genes of interest (GOI) into chromosomes in vitro and in vivo. Here, we proposed that a combination of transposon- and liver-directed introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods.

Proteomic responses of HepG2 cell monolayers and 3D spheroids to selected hepatotoxins

Despite the importance of hepatotoxicity testing in the development of new potential pharmaceuticals, standardized methods for preclinical in vitro hepatotoxicity is complicated by the perceived adequacy of approach, diversity of origin of cells, and the ability to retain a satisfactory hepatocellular phenotype. Additionally, the confidence with which cells mimic in vitro hepatocytes is dictated by the spatial dynamics of the cell culture microenvironment. This study sought to compare the proteome of conventional monolayer cultures of an immortalized hepatocyte cell line (HepG2) with more complex three-dimensional spheroid cultures to ascertain whether changes in culture technique better mimic the phenotype of hepatocytes and thereby improve responses to in vivo hepatotoxins. The proteome was assayed using isobaric tagging from six independent experiments, yielding relative quantitation of over 4600 proteins per multiplexed set. Approximately 34% of proteins present in all replicates differed between monolayer and 3D spheroid cultures. These data suggest that the cellular transition from an exponential to an equilibrium growth phase is inconsistent across biological replicates during spheroid formation which then variably alters the proteome from a stable phenotype in monolayers. Continuous exposure to hepatotoxins, did not implicate specific subsets of proteins in describing the associated mechanisms of toxicity of each drug. However, dynamic changes in HepG2 cells cultured as 3D spheroids were described. These data suggest that the duration of spheroid culture could be essential to reconcile the differences observed in the spheroid proteome to achieve reproducible proteomic transitions to a stable 3D spheroid phenotype.

Berberine induces miR-373 expression in hepatocytes to inactivate hepatic steatosis associated AKT-S6 kinase pathway

Berberine is a Chinese herbal medicine extracted from rhizoma coptidis that functions to improve insulin resistance, hyperlipidemia, hepatosteatosis and inflammation. Berberine can modify the activity of cell metabolism and signaling pathways by regulating expression of genes. However, the roles and effects of differential microRNA (miRNA) expression induced by berberine treatment are largely unexplored. It is believed that miRNAs expression modified by berberine contributes to its therapeutic effects to diseases such as hepatosteatosis and non-alcoholic fatty liver disease. By identifying novel miRNAs and their putative gene targets associated with abnormal hepatic lipid deposition, the underlying mechanism of these diseases could be established and effective therapies against the diseases could be developed. Here, we used the immortalized hepatocyte cell line MIHA as a model to study the effect of berberine on global miRNA expression profile of hepatocytes. Global miRNA expression levels were measured in berberine-treated MIHA cells by quantitative reverse transcription PCR miRNA panel, and the potential berberine regulated miRNAs were then validated in MIHA and HepG2 cells. MicroRNA-373 (MiR-373) was consistently upregulated in both cell lines upon berberine treatments. Gene expression microarray showed that berberine upregulated Early Growth Response 1 (EGR1) level which functioned to transactivate miR-373 expression. Subsequently, we showed that upregulation of miR-373 depleted its target gene AKT serine/threonine kinase 1 (AKT1) mRNA level, which led to the inhibition of AKT-mTOR-S6K signaling pathway in hepatocytes that was critical in the development of hepatosteatosis. Study of the therapeutic effect of manipulating miR-373 against abnormal lipid deposition in liver is warranted.

Ex Vivo Cell-Based Screening Platform for Modulators of Hepatosteatosis.

Nonalcoholic fatty liver disease (NAFLD) is the result of the ectopic accumulation of lipids in hepatic cells and is the early stage of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. While some mechanisms of aberrant lipid storage are understood, unbiased phenotypic drug screening holds the potential to identify new therapeutic small molecule mechanisms that reverse lipid accumulation in hepatic cells and prevent disease progression. Immortalized hepatocyte cell lines are often used as in vitro models of hepatocyte function, including in the study of lipid accumulation. However, mechanisms and therapeutic agents studied in these systems suffer from poor translation to primary cells and animal models of disease. Herein, we report an ex vivo high-throughput screening platform using primary mouse hepatocytes with a physiologically relevant lipid-laden phenotype isolated from mice that are administered a choline-methionine deficient diet. This screening platform using primary diseased hepatocytes may help to overcome a major hurdle in liver disease drug discovery and could lead to the development of new therapeutics for hepatosteatosis.

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC.

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.

Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18

Keratins 8/18 (K8/18) are phosphoglycoproteins and form the major intermediate filament network of simple epithelia. The three O-GlcNAcylation (Ser(29), Ser(30), and Ser(48)) and two phosphorylation (Ser(33) and Ser(52)) serine sites on K18 are well characterized. Both of these modifications have been reported to increase K18 solubility and regulate its filament organization. In this report, we investigated the site-specific interplay between these two modifications in regulating the functional properties of K18, like solubility, stability, and filament organization. An immortalized hepatocyte cell line (HHL-17) stably expressing site-specific single, double, and triple O-GlcNAc and phosphomutants of K18 were used to identify the site(s) critical for regulating these functions. Keratin 18 mutants where O-GlcNAcylation at Ser(30) was abolished (K18-S30A) exhibited reduced phosphorylation induced solubility, increased stability, defective filament architecture, and slower migration. Interestingly, K18-S30A mutants also showed loss of phosphorylation at Ser(33), a modification known to regulate the solubility of K18. Further to this, the K18 phosphomutant (K18-S33A) mimicked K18-S30A in its stability, filament organization, and cell migration. These results indicate that O-GlcNAcylation at Ser(30) promotes phosphorylation at Ser(33) to regulate the functional properties of K18 and also impact cellular processes like migration. O-GlcNAcylation and phosphorylation on the same or adjacent sites on most proteins antagonize each other in regulating protein functions. Here we report a novel, positive interplay between O-GlcNAcylation and phosphorylation at adjacent sites on K18 to regulate its fundamental properties.

A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies

Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate losing their hepatocyte functions and metabolic competence. We have developed a three-dimensional in vitro model using extracellular matrix-based hydrogel for long-term culture of the human hepatoma cell line HepG2. HepG2 cells cultured in this model stop proliferating, self-organize and differentiate to form multiple polarized spheroids. These spheroids re-acquire lost hepatocyte functions such as storage of glycogen, transport of bile salts and the formation of structures resembling bile canaliculi. HepG2 spheroids also show increased expression of albumin, urea, xenobiotic transcription factors, phase I and II drug metabolism enzymes and transporters. Consistent with this, cytochrome P450-mediated metabolism is significantly higher in HepG2 spheroids compared to monolayer cultures. This highly differentiated phenotype can be maintained in 384-well microtiter plates for at least 28days. Toxicity assessment studies with this model showed an increased sensitivity in identifying hepatotoxic compounds with repeated dosing regimens. This simple and robust high-throughput-compatible methodology may have potential for use in toxicity screening assays and mechanistic studies and may represent an alternative to animal models for studying DILI.

Surface Coatings Protect against the In vitro Toxicity of Zinc Oxide Nanoparticles in Human Hepatic Stellate Cells

Coatings applied to the surfaces of zinc oxide (ZnO) nanoparticles have been shown to decrease the capacity of uncoated nanoparticles to generate free radicals, as well as to reduce their solubility, and may be utilized in safetyby- design approaches to decrease ZnO nanoparticle toxicity. The liver is a target organ for ZnO nanoparticles, or ionic zinc released from ZnO nanoparticles, whether the route of entry is by dermal absorption, ingestion, injection or inhalation. Liver injury and fibrosis have been demonstrated in vivo in response to ZnO nanoparticle treatment, and cell injury has also been shown in vitro using immortalized hepatocyte cell lines. Despite their activation being a critical step for liver fibrosis, however, the response of human hepatic stellate cells (hHSCs) to ZnO nanoparticles has not yet been reported. Here, hHSCs were treated with four types of commercially-available ZnO nanoparticles suspended in cell culture medium. Two types were uncoated nanoparticles of different sizes, and the other two types with similar particle size carried different surface coatings. Cells were also treated with pre-dissolved ZnSO4 to provide a comparison of the cellular responses induced by ionic zinc. A systems biology approach was utilized whereby the activation of cell signaling pathways, changes in the transcriptome, and alterations in cellular function were assessed over a 24 h period. Treatment with both types of uncoated ZnO nanoparticles mobilized pathways and responses centered on cellular stress, survival, and apoptosis. Both surface coatings conferred almost complete protection against ZnO nanoparticle-induced cytotoxicity. Ionic zinc had little effect on these cells indicating that toxicity is mainly induced by the nanoparticles. The results presented here support the use of surface coatings in commercial applications of ZnO nanoparticles where there is human exposure.

SIRT2 overexpression in hepatocellular carcinoma mediates epithelial to mesenchymal transition by protein kinase B/glycogen synthase kinase-3β/β-catenin signaling

Sirtuin 1 (SIRT1) has been implicated in telomere maintenance and the growth of hepatocellular carcinoma (HCC). Nevertheless, the role of other sirtuins in the pathogenesis of HCC remains elusive. We found that sirtuin 2 (SIRT2), another member of the sirtuin family, also contributes to cell motility and invasiveness of HCC. SIRT2 is up-regulated in HCC cell lines and in a subset of human HCC tissues (23/45). Up-regulations of SIRT2 in primary HCC tumors were significantly correlated with the presence of microscopic vascular invasion (P = 0.001), a more advanced tumor stage (P = 0.004), and shorter overall survival (P = 0.0499). Functional studies by short hairpin RNA-mediated suppression of SIRT2 expression in HCC cell lines revealed significant inhibition of motility and invasiveness. Depletion of SIRT2 also led to the regression of epithelial-mesenchymal transition (EMT) phenotypes, whereas the ectopic expression of SIRT2 in the immortalized hepatocyte cell line L02 promoted cell motility and invasiveness. Mechanistic studies revealed that SIRT2 regulates the deacetylation and activation of protein kinase B, which subsequently impinges on the glycogen synthase kinase-3β/β-catenin signaling pathway to regulate EMT. Our findings have uncovered a novel role for SIRT2 in HCC metastasis, and provide a rationale to explore the use of sirtuin inhibitors in HCC therapy. (HEPATOLOGY 2013;).

SIRT2 Overexpression in Hepatocellular Carcinoma Mediates Epithelial to Mesenchymal Transition via Akt/GSK‐3β/β‐catenin Signaling.

The yeast silent information regulator 2 (SIR2) is a histone deacetylase that utilizes NAD+ as a cofactor for its functions. In mammals, SIR2 is represented by seven homologues (SIRT1–7). We found that Sirtuin 2 (SIRT2) contributes to cell motility and invasiveness of HCC. SIRT2 is upregulated in HCC cell lines and in a subset of human HCC tissues (23/45). Up‐regulations of SIRT2 in primary HCC tumors were significantly correlated with the presence of microscopic vascular invasion (p=0.001), more advanced tumor stage (p=0.004) and shorter overall survival (p=0.0499). Functional studies by shRNA‐mediated suppression of SIRT2 expression in HCC cell lines revealed significant inhibition of motility and invasiveness. Depletion of SIRT2 also led to the regression of epithelial‐mesenchymal transition (EMT) phenotypes, whereas the ectopic expression of SIRT2 in the immortalized hepatocyte cell line L02 enhanced the expression of EMT markers, and promoted cell motility and invasiveness. Mechanistic studies revealed that SIRT2 regulates the deacetylation and activation of Akt, which subsequently impinges on GSK‐3β/β‐catenin signaling pathway to regulate EMT. Concordantly, we showed that there is a positive correlation between the level of SIRT2 and β‐catenin in human HCC. Our findings uncover a novel role of SIRT2 in HCC metastasis, and provide a rationale to explore the use of sirtuin inhibitors in HCC therapy.

Human hepatocyte carcinogenesis

Hepatocellular carcinoma is the third most frequent cause of cancer-related death worldwide; and its incidence rate is increasing. Clinical and molecular medical analyses have revealed substantial information on hepatocarcinogenesis. Hepatocarcinogenesis is a stepwise process during which multiple genes are altered. Genetic changes and their biological consequences in human HCC can be divided into at least 4 groups: i) tumor suppressor genes (p53, retinoblastoma, phosphatase tensin homolog and runt-related transcription factor 3), ii) oncogenes (myc, K-ras, BRAF), iii) reactivation of developmental pathways (Wnt, hedgehog), and iv) growth factors and their receptors (transforming growth factor-α, insulin-like growth factor-2 receptor). An experimental model of human hepatocarcinogenesis such as in vitro neoplastic transformation of human hepatocytes has not been successfully achieved yet, but several immortalized human hepatocyte cell lines have been established. These immortalized human hepatocytes will become useful tools for the elucidation of hepatocarcinogenesis, especially for the initial step of multistep hepatocarcinogenesis.

S-Adenosylmethionine-dependent Protein Methylation Is Required for Expression of Selenoprotein P and Gluconeogenic Enzymes in HepG2 Human Hepatocytes

Cellular methylation processes enable expression of gluconeogenic enzymes and metabolism of the nutrient selenium. Selenium status has been proposed to relate to type II diabetes risk, and plasma levels of selenoprotein P (SEPP1) have been positively correlated with insulin resistance. Increased expression of gluconeogenic enzymes glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) has negative consequences for blood glucose management in type II diabetics. Transcriptional regulation of SEPP1 is directed by the same transcription factors that control the expression of G6PC and PCK1, and these factors are activated by methylation of arginine residues. We sought to determine whether expression of SEPP1 and the aforementioned glucoconeogenic enzymes are regulated by protein methylation, the levels of which are reliant upon adequate S-adenosylmethionine (SAM) and inhibited by S-adenosylhomocysteine (SAH). We treated a human hepatocyte cell line, HepG2, with inhibitors of adenosylhomocysteine hydrolase (AHCY) known to increase concentration of SAH before analysis of G6PC, PCK1, and SEPP1 expression. Increasing SAH decreased 1) the SAM/SAH ratio, 2) protein-arginine methylation, and 3) expression of SEPP1, G6PC, and PCK1 transcripts. Furthermore, hormone-dependent induction of gluconeogenic enzymes was reduced by inhibition of protein methylation. When protein-arginine methyltransferase 1 expression was reduced by siRNA treatment, G6PC expression was inhibited. These findings demonstrate that hepatocellular SAM-dependent protein methylation is required for both SEPP1 and gluconeogenic enzyme expression and that inhibition of protein arginine methylation might provide a route to therapeutic interventions in type II diabetes.

Hepatitis C viral protein NS5A induces EMT and participates in oncogenic transformation of primary hepatocyte precursors

Apicobasal polarity, which is essential for epithelial structure and function, is targeted by several tumour-related pathogens and is generally perturbed in the course of carcinogenesis. Hepatitis C virus (HCV) infection is associated with a strong risk of hepatocellular carcinoma, typically preceded by dysplastic alterations of cell morphology. We investigated the molecular mechanisms and the functional consequences of HCV-driven perturbations of epithelial polarity. We used biochemical, genetic, and cell biology approaches to assess the impact of hepatitis C viral protein NS5A on the polarity and function of hepatocytes and hepatic progenitors. Transgenic animals and xenograft models served for in vivo validation of the results obtained in cell culture. We found that expression of HCV-NS5A in primary hepatic precursors and in immortalized hepatocyte cell lines gave rise to profound modifications of cell polarity, leading to epithelial to mesenchymal transition (EMT). NS5A, either alone or in the context of the full complement of viral proteins in the course of infection, acted through activating Twist2, a transcriptional regulator of EMT. The effects of NS5A were additive to those of TGF-β, a cytokine abundant in diseased liver and highly relevant to HCV-related pathology. Moreover, NS5A cooperates with oncogenic Ras, giving rise to transformed, invasive cells that are highly tumorigenic in vivo. Our data suggest that in the context of HCV infection, NS5A favors formation of preneoplastic lesions by disrupting cell polarity and additional oncogenic events cooperate with the viral protein to give rise to motile and invasive tumour cells.

Chemerin in hepatocelluar carcinoma: expressions and prognostic values

Objective To study the expressions of Chemerin in hepatocellular carcinoma (HCC) and HCC cell lines,and to demonstrate the relationship between the expressions and prognosis.Methods The expressions of Chemerin protein in normal hepatocellular tissues,HCC and their paired tissues were detected by immunohistochemical staining of SABC; the expressions of Chemerin protein in HCC,cell lines and immortalized hepatocyte cell lines were detected by RT-PCR.Results The positive expression rates of Chemerin were 56.67％,90.00％,100％ in HCC,HCC cell line and normal hepatocellular tissues respectively,and the differences were significant (P＜0.05).The expressions of Chemerin mRNA in HCC paired tissues were higher than in HCC (P＜0.05).There was a higher expression of Chemerin mRNA in immortalized hepatocyte cell line LO2 ; but a lower expression in HCC cell lines.The expressions of Chemerin were related to lymph node metastasis,portal vein tumour thrombi,differentiation and TNM stage but not related to sex,age,tumour size,HBsAg and AFP.Conclusions Down-regulated expression of Chemerin may play an important role in the development,progression and metastasis of HCC.It may be a molecular marker for prognosis of HCC. Key words: Chemerin; Hepatocellular carcinoma; Prognosis

Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD50 values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC50 data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD50 data (mg compound/mg cellular protein) showed that the variation in LD50 values was generally less than that suggested by the original LC50 data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD50 values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo.

Block of proliferation 1 (BOP1) plays an oncogenic role in hepatocellular carcinoma by promoting epithelial-to-mesenchymal transition

Genomic amplification of regional chromosome 8q24 is a common event in human cancers. In hepatocellular carcinoma (HCC), a highly aggressive malignancy that is rapidly fatal, recurrent 8q24 gains can be detected in >50% of cases. In this study, attempts to resolve the 8q24 region by way of array comparative genomic hybridization for affected genes in HCC revealed distinctive gains of block of proliferation 1 (BOP1). Gene expression evaluation in an independent cohort of primary HCC (n = 65) revealed frequent BOP1 up-regulation in tumors compared with adjacent nontumoral liver (84.6%; P < 0.0001). Significant associations could also be drawn between increased expressions of BOP1 and advance HCC staging (P = 0.004), microvascular invasion (P = 0.006), and shorter disease-free survival of patients (P = 0.02). Examination of expression of C-MYC, a well-known oncogene located in proximity to BOP1, in the same series of primary HCC cases did not suggest strong clinicopathologic associations. Functional investigations by small interfering RNA-mediated suppression of BOP1 in HCC cell lines indicated significant inhibition on cell invasion (P < 0.005) and migration (P < 0.05). Overexpression of BOP1 in the immortalized hepatocyte cell line L02 showed increase cellular invasiveness and cell migratory rate (P < 0.0001). In both gene knockdown and ectopic expression assays, BOP1 did not exert an effect on cell viability and proliferation. Evident regression of the epithelial-mesenchymal transition (EMT) phenotype was readily identified in BOP1 knockdown cells, whereas up-regulation of epithelial markers (E-cadherin, cytokeratin 18, and γ-catenin) and down-regulation of mesenchymal markers (fibronectin and vimentin) were seen. A corresponding augmentation of EMT was indicated from the ectopic expression of BOP1 in L02. In addition, BOP1 could stimulate actin stress fiber assembly and RhoA activation. Our findings underline an important role for BOP1 in HCC invasiveness and metastasis potentials through inducing EMT and promoting actin cytoskeleton remodeling.

Gene expression profile of Li23, a new human hepatoma cell line that enables robust hepatitis C virus replication: Comparison with HuH‐7 and other hepatic cell lines

Aim: Human hepatoma cell line HuH‐7‐derived cells are currently the only cell culture system used for robust hepatitis C virus (HCV) replication. We recently found a new human hepatoma cell line, Li23, that enables robust HCV replication. Although both cell lines had similar liver‐specific expression profiles, the overall profile of Li23 seemed to differ considerably from that of HuH‐7. To understand this difference, the expression profile of Li23 cells was further characterized by a comparison with that of HuH‐7 cells.Methods: cDNA microarray analysis using Li23 and HuH‐7 cells was performed. Li23‐derived ORL8c cells and HuH‐7‐derived RSc cells, in which HCV could infect and efficiently replicate, were also used for the microarray analysis. For the comparative analysis by reverse transcription polymerase chain reaction (RT–PCR), human hepatoma cell lines (HuH‐6, HepG2, HLE, HLF and PLC/PRF/5) and immortalized hepatocyte cell line (PH5CH8) were also used.Results: Microarray analysis of Li23 versus HuH‐7 cells selected 80 probes to represent highly expressed genes that have ratios of more than 30 (Li23/HuH‐7) or 20 (HuH‐7/Li23). Among them, 17 known genes were picked up for further analysis. The expression levels of most of these genes in Li23 and HuH‐7 cells were retained in ORL8c and RSc cells, respectively. Comparative analysis by RT–PCR using several other hepatic cell lines resulted in the classification of 17 genes into three types, and identified three genes showing Li23‐specific expression profiles.Conclusion: Li23 is a new hepatoma cell line whose expression profile is distinct from those of frequently used hepatic cell lines.

Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy

Hepatitis B virus X protein (pX), implicated in hepatocarcinogenesis, induces DNA damage because of re-replication and allows propagation of damaged DNA, resulting in partial polyploidy and oncogenic transformation. The mechanism by which pX allows cells with DNA damage to continue proliferating is unknown. Herein, we show pX activates Polo-like kinase 1 (Plk1) in the G(2) phase, thereby attenuating the DNA damage checkpoint. Specifically, in the G(2) phase of pX-expressing cells, the checkpoint kinase Chk1 was inactive despite DNA damage, and protein levels of claspin, an adaptor of ataxia telangiectasia-mutated and Rad3-related protein-mediated Chk1 phosphorylation, were reduced. Pharmacologic inhibition or knockdown of Plk1 restored claspin protein levels, Chk1 activation, and p53 stabilization. Also, protein levels of DNA repair protein Mre11 were decreased in the G(2) phase of pX-expressing cells but not with Plk1 knockdown. Interestingly, in pX-expressing cells, Mre11 co-immunoprecipitated with transfected Plk1 Polo-box domain, and inhibition of Plk1 increased Mre11 stability in cycloheximide-treated cells. These results suggest that pX-activated Plk1 by down-regulating Mre11 attenuates DNA repair. Importantly, concurrent inhibition of Plk1, p53, and Mre11 increased the number of pX-expressing cells with DNA damage entering mitosis, relative to Plk1 inhibition alone. By contrast, inhibition or knockdown of Plk1 reduced pX-induced polyploidy while increasing apoptosis. We conclude Plk1, activated by pX, allows propagation of DNA damage by concurrently attenuating the DNA damage checkpoint and DNA repair, resulting in polyploidy. We propose this novel Plk1 mechanism initiates pX-mediated hepatocyte transformation.