Claudin‐1, miR‐122 and apolipoprotein E transductions improve the permissivity of SNU‐182, SNU‐398 and SNU‐449 hepatoma cells to hepatitis C virus

Hepatitis C virus (HCV) is a human hepatotropic virus, but many hepatoma cell lines are not permissive to this virus. In a previous study, we observed that SNU‐182, SNU‐398 and SNU‐449 hepatoma cell lines were nonpermissive to HCV. To understand the nonpermissivity, we evaluated the ability of each cell line to support the different steps of HCV life cycle (entry, replication and production of infectious particles). Using retroviral pseudoparticles pseudotyped with HCV envelope proteins and recombinant HCV produced in cell culture, we observed that low level or absence of claudin‐1 (CLDN1) expression limited the viral entry process in SNU‐182 and SNU‐398 cells, respectively. Our results also showed that supplementation of the three cell lines with miR‐122 partly restored the replication of a JFH1 HCV replicon. Finally, we observed that expression of apolipoprotein E (ApoE) was very low or undetectable in the three cell lines and that its ectopic expression permits the production of infectious viral particles in SNU‐182 and SNU‐398 cells but not in SNU‐449 cells. Nevertheless, the supplementation of SNU‐182, SNU‐398 and SNU‐449 cells with CLDN1, miR‐122 and ApoE was not sufficient to render these cells as permissive as HuH‐7 cells. Thus, these cell lines could serve as cell culture models for functional studies on the role of CLDN1, miR‐122 and ApoE in HCV life cycle but also for the identification of new restriction and/or dependency host factors essential for HCV infection.     

alpha-fetoprotein-producing hepatoma cell lines share common expression profiles of genes in various categories demonstrated by cDNA microarray analysis

Liver carcinogenesis is a multistep process involving various genetic alterations. cDNA microarray containing 1,080 elements (930 unique genes) was used to comprehensively analyze the genetic alterations in hepatoma cell lines, and clustering analysis was used to analyze the relatedness of the gene-expression profiles. Among 7 hepatoma cell lines analyzed, 5-alpha-fetoprotein (AFP)-producing hepatoma cell lines (HepG2, Huh7, Hep3B, PLC/PRF/5, and Huh6) were shown to have common gene-expression profiles compared with those of AFP-negative hepatoma cell lines (HLE and SK-Hep1) and cancer cell lines of nonhepatocyte origin (HeLa and KMBC). Furthermore, HepG2, Huh7, and Hep3B had higher expressions of AFP and shared a common gene-expression profile even when compared with other AFP-producing cells. Analysis of the genes with a common expression profile among these 3 AFP-positive cells revealed 254 genes across various categories. We found that 18 of these genes consistently showed altered levels of expression (more than 3-fold changes) in the 3 AFP-producing hepatoma cell lines (11 up-regulated and 7 down-regulated). In these 18 genes, 5 genes, including that for AFP, were previously reported to be involved in HCC and 6 genes involved only in other types of cancer. Our study showed that AFP-producing hepatoma cell lines shared a distinct expression profile of genes in various categories. An understanding of a causal relationship of this particular expression profile of genes to AFP-positive and AFP-negative hepatocellular carcinoma (HCC) may contribute to more rational therapy in future.     

Effect of hepatitis C virus core shadow protein expressed in human hepatoma cell line on human gene expression profiles

BACKGROUND AND AIMS: The hepatitis C virus (HCV) C region has been reported to have overlapping genes or regions, and may encode a core shadow protein that has a role in HCV self-replication, pathogenesis and carcinogenesis. The aim of this study was to identify the effect of HCV core shadow protein expressed in a human hepatoma (Huh-7) cell line on human gene expression profiles. METHODS: Recombinants for expression of HCV genotype 1b core shadow protein and genotype 1b core protein were constructed, and an Huh-7 cell line was established that could express the shadow protein and the core protein constitutively. Affymetrix human gene chip, HG-U133 A and B microarray analysis and semiquantitative RT-PCR were employed to identify the expression profiles of two kinds of core proteins in the Huh-7 cell line. RESULTS: The microarray analysis showed that the core shadow protein caused expression of more genes to be up/down-regulation than the core protein, including signal transduction, protease activity, molecular transport and, particularly, immune responses genes. Surprisingly, the core shadow protein could increase/decrease expression of apoptosis and anti-apoptosis genes simultaneously. The expression profiles of three up-regulated genes were confirmed by semiquantitative RT-PCR, with results similar to the microarray analysis. CONCLUSIONS: Hepatitis C virus core shadow protein may play an important role in inhibiting or stimulating host cells apoptosis processing and carcinogenesis, which is useful for the understanding of HCV core shadow protein biological functions in vivo and in vitro.     

Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma

BACKGROUND & AIMS: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. METHODS: Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. RESULTS: We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. CONCLUSIONS: Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.     

Gene expression associated with interferon alfa antiviral activity in an HCV replicon cell line

Interferon alfa (IFN-alpha)-based treatment is the only therapeutic option for chronic hepatitis C viral infection. However, the molecular mechanisms of IFN-alpha antiviral activity are not completely understood. The recent development of an HCV replicon cell culture system provides a feasible experimental model to investigate the molecular details of IFN-induced direct antiviral activity in hepatocytes. In this report, we show that IFN-alpha can effectively inhibit HCV subgenomic RNA replication and suppress viral nonstructural protein synthesis. Using cDNA microarray analysis, we also show that the replicon cells have different gene expression profile compared with the parental hepatoma cells (Huh7). IFN-alpha can induce a number of responsive genes in the replicon cells. One of the genes, 6-16 (G1P3), can enhance IFN-alpha antiviral efficacy. In addition, we demonstrate that IFN-alpha can significantly activate STAT3 in hepatoma cells, suggesting that this pathway plays a role in IFN-alpha signaling. In conclusion, our results indicate that IFN-alpha antiviral activity is associated with activation of STAT3-signaling pathway and intracellular gene activation. Our results also suggest that IFN-alpha-induced target genes may play an important role in IFN-alpha anti-HCV activity.     

Gene expression profile of Huh‐7 cells expressing hepatitis C virus genotype 1b or 3a core proteins

Background: The liver disease expression in chronic hepatitis C patients is variable and may partially depend on the sequence of the infecting viral genotype. Aim: To identify some hepatitis C virus (HCV) genotype‐specific virus–host interactions potentially leading to clinically significant consequences. Methods: We compared the gene expression profile of Huh‐7 cells transiently expressing the core protein of HCV genotype 1b and 3a using microarray technology. Results: Thirty‐two genes were overexpressed in Huh‐7 transfected with the HCV genotype 1b core protein and 57 genes in cells transfected with the genotype 3a core protein. On the other hand, we found 20 genes downregulated by core 1b and 31 genes by core 3a. These included genes involved in lipid transport and metabolism, cell cycle, immune response and insulin signalling. Conclusion: The expression of HCV core proteins of different genotypes leads to a specific gene expression profile. This may account for the variable disease expression associated with HCV infection.     

Serum‐derived hepatitis C virus 1a infection of human astrocyte cell line SVG

Neuroinvasion of hepatitis C virus (HCV) is evidenced by recent clinical studies. In this study, serum‐derived HCV infection of astrocytes was analysed. Astrocytes were infected with HCV‐positive serum, and viral replication was assessed on different days postinfection. RT‐PCR was positive for HCV‐negative strand on 5th and 7th day postinfection in the HCV‐positive serum‐infected astrocytes. Real‐time RNA count in the cell culture supernatant was steadily increasing from day 3 to day 7. To reconfirm the viral replication, astrocytes were treated with an antiviral before the serum infection, and the antiviral treatment significantly reduced the viral RNA count. Further, the virus‐infected cells stained positive for the presence of viral core protein. Electron microscopy revealed the presence of HCV‐like particles in the astrocyte cell culture supernatant. In conclusion, serum‐derived HCV replicates in human astrocyte cell line SVG.     

Ephrin-A1 expression contributes to the malignant characteristics of {alpha}-fetoprotein producing hepatocellular carcinoma

BACKGROUND AND AIMS: alpha-Fetoprotein (AFP), a tumour marker for hepatocellular carcinoma (HCC), is associated with poor prognosis. Using cDNA microarray analysis, we previously found that ephrin-A1, an angiogenic factor, is the most differentially overexpressed gene in AFP producing hepatoma cell lines. In the present study, we investigated the significance of ephrin-A1 expression in HCC. METHODS: We examined ephrin-A1 expression and its effect on cell proliferation and gene expression in five AFP producing hepatoma cell lines, three AFP negative hepatoma cell lines, and 20 human HCC specimens. RESULTS: Ephrin-A1 expression levels were lowest in normal liver tissue, elevated in cirrhotic tissue, and further elevated in HCC specimens. Ephrin-A1 expression was strongly correlated with AFP expression (r = 0.866). We showed that ephrin-A1 induced expression of AFP. This finding implicates ephrin-A1 in the mechanism of AFP induction in HCC. Ephrin-A1 promoted the proliferation of ephrin-A1 underexpressing HLE cells, and an ephrin-A1 antisense oligonucleotide inhibited the proliferation of ephrin-A1 overexpressing Huh7 cells. Thus ephrin-A1 affects hepatoma cell growth. cDNA microarray analysis showed that ephrin-A1 induced expression of genes related to the cell cycle (p21), angiogenesis (angiopoietin 1 and thrombospondin 1), and cell-cell interactions (Rho, integrin, and matrix metalloproteinases) in cultured hepatoma cells. These ephrin-A1 induced genes are also activated in HCC tissues that overexpress AFP. CONCLUSION: These findings suggest that the poor prognosis of patients with AFP producing HCC is partially caused by ephrin-A1 expression, which induces expression of genes related to tumour cell growth, angiogenesis, invasion, and metastasis.     

Effect of heptitis C virus core protein on cellular gene expression: specific inhibition of cyclooxygenase 2

Hepatitis C virus (HCV) core protein plays a significant role in the alteration of cellular gene expression. We expressed HCV core protein using a tetracycline-inducible expression system in HeLa cell lines. Profiles of gene expression in cells expressing the HCV core protein were compared with those in control cells by use of microarray analysis. Cells expressing the HCV core protein showed 86 down-regulated and 41 up-regulated genes, compared with control cells. One gene affected was cyclooxygenase 2 (COX-2). Levels of both COX-2 RNA and the Cox-2 protein were significantly inhibited after the expression of HCV core protein in HeLa cells. Similar results were obtained in hepatoma cells and in a functional assay that measured the production of the Cox-2 protein in response to a mitogenic stimulus. The inhibition of the Cox-2 protein could serve as a means of muting the cellular inflammatory response during HCV infection. Correlation of these findings with analysis of clinical specimens from chronically infected patients should lend further significance to the down-regulation of the inflammatory response via Cox-2.     

Modulation of RANTES expression by HCV core protein in liver derived cell lines

Background

Hepatitis C virus (HCV) infection is associated with high percentage of chronicity which implies the ability of the virus to evade or modulate host cell immune system. Modulation of chemokines, such as RANTES may be part of the virus induced pathogenicity. We examined the effect of core and structural proteins of HCV on RANTES expression in two liver derived cell lines, HepG2 and Chang Liver (CHL).

Methods

HepG2 and Chang Liver (CHL) cell lines were established and selected for constitutive expression of HCV core and structural genes. Flow cytometry and quantitative RT-PCR analysis were performed to examine the effect of HCV core protein on RANTES expression. Luciferase analysis after RANTES-Luc-promoter transfection of established cell lines was assayed by luminometer measurements (RLU) of RANTES promoter activity. IRF-1 and IRF-7 expression was then examined by immunoblotting analysis.

Results

Results of flow cytometry and RT-PCR analysis indicated that RANTES is differentially regulated by HCV core protein in the two cell lines examined as its expression was inhibited in HepG2 cells, by a reduction of RANTES promoter activity. Conversely, RANTES protein and mRNA were induced by the core protein in CHL cells, through the induction of the promoter. Since HCV genome modulates IRF-1 and IRF-7 in replicon system and IRF-1, IRF-3 and IRF-7 have been reported to regulate RANTES promoter in various cell systems, analysis of the mechanism underlying RANTES modulation by the core protein revealed that IRF-1 expression was induced in HepG2 cells by the core protein, whereas in CHL cells it was expressed at a very low level that was not influenced by transfection with the core protein construct. This suggested that IRF-1 level may mediate the expression of RANTES in cell lines of liver origin. The effect of the core protein on RANTES promoter was countered by co-transfection with NF90, a double-stranded-RNA binding protein that activates some interferon response genes and acts as a component of cell defense against viral infection.

Conclusion

HCV core protein have opposite effects on the expression of RANTES in different cell types in vitro, possibly reflecting a similar scenario in different microenvironments in vivo.     

Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells

Background

Hepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naïve Huh7 cells.

Methods

Huh7 cell lines that stably expressed HCV genotype 1b or 2a replicon were used. The cured cells were established by treating HCV replicon cells with interferon-alpha. Expression of 54,675 cellular genes was analyzed by GeneChip DNA microarray. The data were analyzed by using the KEGG Pathway database.

Results

Hierarchical clustering analysis showed that the gene-expression profiles of each cell group constituted clear clusters of naïve, HCV replicon-expressed, and cured cell lines. The pathway process analysis between the replicon-expressing and the cured cell lines identified significantly altered pathways, including MAPK, steroid biosynthesis and TGF-beta signaling pathways, suggesting that these pathways were affected directly by HCV replication. Comparison of cured and naïve Huh7 cells identified pathways, including steroid biosynthesis and sphingolipid metabolism, suggesting that these pathways were required for efficient HCV replication. Cytoplasmic lipid droplets were obviously increased in replicon-expressing and cured cells as compared to naïve cells. HCV replication was significantly suppressed by peroxisome proliferator-activated receptor (PPAR)-alpha agonists but augmented by PPAR-gamma agonists.

Conclusion

Comprehensive gene expression and pathway analyses show that lipid biosynthesis pathways are crucial to support proficient virus replication. These metabolic pathways could constitute novel antiviral targets against HCV.     

Transcriptomic comparison of human hepatoma Huh-7 cell clones with different hepatitis C virus replication efficiencies

Hepatitis C virus (HCV) infection represents a major public health problem throughout the world. The establishment of viral replicons has enhanced our understanding of the mechanism underlying HCV replication. However, the specific virus-host cell interactions involved in HCV RNA replication are not well understood. In the present study, we isolated several human hepatoma Huh-7-derived subclones with a range of HCV RNA replication efficiencies by end-point dilution. Of these, the clones HuhTe4 and HuhTe6 were observed to proliferate at the same rate; however, HuhTe6 supported a significantly greater degree of viral RNA replication. Using cDNA microarray analysis, a total of 36 genes (0.4%) demonstrated variable expression, with a >or=2-fold difference in expression noted between HuhTe4 and HuhTe6. Among genes that are implicated in a variety of functional categories, a subset of these differentially-expressed genes has a role in signal transduction and cell communication, including thioredoxin-interacting protein, Rab6B, sorting nexin 16 and UDP-galactose:ceramide glycosyltransferase. The genes identified in this study should be examined further to determine their roles in HCV RNA replication. The Huh-7 subclones identified in this study provide a tool for identifying novel host factors involved in viral replication.     

Characteristic gene expression induced by polyurethane foam/spheroid culture of hepatoma cell line, Hep G2 as a promising cell source for bioartificial liver

BACKGROUND/AIMS: Polyurethane foam (PUF)/ spheroid-culture can improve liver-specific functions of hepatoma cell line, Hep G2. Therefore, gene expression profile in the PUF/spheroid culture is hypothesized to be different from that in the monolayer culture. The aim of this study is to clarify the characteristic gene expression in PUF/spheroid-cultured Hep G2 cells, as a cell source for bioartificial liver (BAL), using microarray analysis. METHODOLOGY: Morphological change and liver specific functions of ammonia removal rate and albumin synthesis rate of Hep G2 were compared between in a monolayer or PUF/spheroid culture. Microarray analysis was performed using cDNA microarrays made in Hitachi Software Engineering Co., Ltd., (Yokohama, Japan), which contains a total of 1,281 cDNA clones. RESULTS: The ammonia removal rate of Hep G2 spheroids increased to 369%, and the albumin synthesis rate of Hep G2 spheroids also increased 311% when compared with monolayer culture. In addition, the ammonia removal capacity of primary human hepatocytes in the PUF/spheroid culture was superior to that in the monolayer culture. The microarray analysis demonstrated that the PUF/spheroid-cultured Hep G2 cells expressed 39 up-regulated (more than 3.0-fold) and 31 down-regulated (less than 0.333-fold) genes. Among the 70 genes differentially expressed in PUF/spheroid cultured Hep G2 cells, subsets of glutathione S-transferase- and angio-tensin-related genes were drastically up-regulated, on the other hand, subsets of assigned for growth factor, glucocorticoid, and stress response, were down-regulated. CONCLUSIONS: Hepatoma cell line, Hep G2 cells in the PUF/spheroid culture is a promising hepatocyte source for BAL. Microarray analysis revealed a number of characteristic genes altered by the PUF/spheroid.