Kenichi Fukai

Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells.

Objective: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. Methods: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. Results: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, α2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, α7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. Conclusions: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.

Prevalence of diabetes mellitus and insulin resistance in patients with chronic hepatitis C: comparison with hepatitis B virus-infected and hepatitis C virus-cleared patients.

Background/Aims: Our aim was to evaluate the relationship between hepatitis C virus (HCV) infection and development of diabetes mellitus (DM) or insulin resistance (IR) in comparison with hepatitis B virus (HBV) infection and eradication of HCV infection by interferon treatment.

Diagnostic Problems Caused by HBsAg Mutants – A Consensus Report of an Expert Meeting

A panel of 16 experts from 9 countries convened on April 14 at Schloss Reinhartshausen near Wiesbaden in Germany, to discuss the diagnostic significance of mutants, variants and genotypes of hepatitis B virus (HBV). Since the description of Australia antigen in 1965 and the subsequent observation that it was the envelope of the HBV and now designated hepatitis B surface antigen (HBsAg), this lipoprotein has been a mainstay in the diagnosis of HBV infections. HBsAg tests are used routinely in the diagnosis of acute and chronic liver disease, the screening of blood or organ donors and the surveillance of persons at risk to acquire or to transmit HBV. Current immunoassays for HBsAg are very specific and sensitive (both 199%) and are usually able to detect !0.5 ng HBsAg/ml serum. Their performance is validated in extensive trials before licensing and their detection limit is assayed with an International Standard for HBsAg. An immanent problem of virology is the variability of viruses. Due to the low fidelity of the viral nucleic acid polymerases and the high replication rates, virtually all nucleotide positions of a viral genome can be mutated within a relatively short time. However, the viability of the virus and its adaptation to the host allow the selection and outgrowth of only a limited number of mutants. The survival strategy of HBV in the population is mainly based on induction of immune tolerance and persistence of high viremia. In this state of infection the existing viral genome is favored whereas mutants are less fit and selected against or may be subject to an immune reaction and preferably eliminated. In fact, most of the HBsAg carriers in Germany have a very similar S gene sequence. However, under selective pressure the virus can express many different viable HBsAg mutants. Summary of the Presentations

Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma.

Purpose: Hedgehog (Hh) signaling is activated in several cancers. However, the mechanisms of Hh signaling activation in hepatocellular carcinoma (HCC) have not been fully elucidated. We analyzed the involvement of Hh-interacting protein (HHIP) gene, a negative regulator of Hh signaling, in HCC. Experimental Design: Glioma-associated oncogene homologue (Gli) reporter assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and quantitative real-time reverse transcription–PCR for the target genes of the Hh signals were performed in HHIP stably expressing hepatoma cells. Quantitative real-time PCR for HHIP was performed in hepatoma cells and 36 HCC tissues. The methylation status of hepatoma cells and HCC tissues was also analyzed by sodium bisulfite sequencing, demethylation assay, and quantitative real-time methylation-specific PCR. Loss of heterozygosity (LOH) analysis was also performed in HCC tissues. Results:HHIP overexpression induced significant reductions of Gli reporter activity, cell viability, and transcription of the target genes of the Hh signals. HHIP was hypermethylated and transcriptionally down-regulated in a subset of hepatoma cells. Treatment with a demethylating agent led to the HHIP DNA demethylation and restoration of HHIP transcription. HHIP transcription was also down-regulated in the majority of HCC tissues, and more than half of HCC tissues exhibited HHIP hypermethylation. The HHIP transcription level in HHIP-methylated HCC tissues was significantly lower than in HHIP-unmethylated HCC tissues. More than 30% of HCC tissues showed LOH at the HHIP locus. Conclusions: The down-regulation of HHIP transcription is due to DNA hypermethylation and/or LOH, and Hh signal activation through the inactivation of HHIP may be implicated in the pathogenesis of human HCC.

Methylation Status of Genes Upregulated by Demethylating Agent 5-aza-2′-Deoxycytidine in Hepatocellular Carcinoma

Background/Aims: To determine the clinical significance of gene promoter methylation in hepatocellular carcinoma (HCC), we examined in clinical samples the methylation status of those promoters that showed elevated activity in hepatoma cell lines after 5-aza-2′-deoxycytidine treatment. Methods: Regarding the genes with promoter hypermethylation in the cell lines, their expression levels and methylation status in HCC and non-HCC tissues were assessed by semiquantitive RT-PCR and methylation-specific PCR. To confirm the result, the expression levels and methylation status in 16 additional HCC and non-HCC tissues were assessed. Results: The promoter regions of caveolin 1 (CAV1), cysteine and glycine-rich protein 1 (CSRP1), Kruppel-like factor 6 (KLF6), myosin (light polypeptide 9) (MYL9), and transgelin (TAGLN) were highly methylated in the cell lines. CAV1 and CSRP1 were methylated in HCC more frequently than in non-HCC. KLF6, MYL9, and TAGLN were fully methylated in both HCC and non-HCC. Using additional clinical samples, downregulation of CAV1 and CSRP1 was observed in 38 and 56%, respectively, of the 16 HCC samples and aberrant methylation of CAV1 and CSRP1 was observed in 56% of HCC in both cases. Conclusion:CAV1 and CSRP1 were inactivated in HCC by aberrant methylation and they may serve as important biomarkers of malignancy.

Fas polymorphisms influence susceptibility to autoimmune hepatitis.

BACKGROUND AND AIMS:Genetic factors associated with autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC), immune-mediated chronic inflammatory liver diseases of unknown etiology, remain to be elucidated. Polymorphisms of the gene encoding Fas have been linked to a variety of autoimmune diseases. We hypothesized that Fas gene polymorphisms might be genetic markers for AIH and PBC.METHODS:To determine the frequency and significance of Fas polymorphisms in patients with AIH and PBC, 74 Japanese AIH patients, 98 Japanese PBC patients, and 132 ethnically matched control subjects were investigated by the use of the Taqman assay.RESULTS:We found significant differences between AIH patients and controls in allele frequencies of Fas-670 (p= 0.009), Fas IVS (intervening sequence) 2nt176 (p= 0.018), Fas IVS3nt46 (p= 0.031), and Fas IVS5nt82 (p= 0.013) polymorphisms. Haplotype analysis revealed that one of the haplotypes, GATGC, was associated with increased AIH prevalence. On the other hand, we found no statistically significant differences between PBC patients and controls in allele frequencies of the Fas polymorphisms genotyped in this study.CONCLUSIONS:These results indicate a genetic link of Fas polymorphisms to the development of AIH. Further studies are needed to determine the genetic factors contributing to the development of AIH.

Distinct expression of polycomb group proteins EZH2 and BMI1 in hepatocellular carcinoma

Polycomb gene products play a crucial role in the development of highly malignant phenotypes and aggressive cancer progression in a variety of cancers; however, their role in hepatocellular carcinoma remains unclear. First, we analyzed the impact of EZH2 and BMI1 modulation on cell growth of HepG2 cells. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays revealed marked growth inhibition after EZH2 or BMI1 knockdown. In addition, simultaneous knockdown of these 2 genes further augmented cell growth inhibitory effects. Next, we conducted immunohistochemical assessment of 86 hepatocellular carcinoma surgical specimens, evaluating the correlation between EZH2 and BMI1 protein expression and clinicopathologic features. High-level EZH2 and BMI1 expression was detected in 57 (66.3%) and 52 tumor tissues (60.5%), respectively. Among these, 48 tumor tissues (55.8%) showed colocalization of EZH2 and BMI1 in almost all tumor cells. The cumulative recurrence rate, but not survival rate, was significantly higher in patients positive for EZH2 (P = .029) and BMI1 (P = .039) than in their negative counterparts, as determined by Kaplan-Meier analysis. These data indicate that EZH2 and BMI1 may cooperate in initiation and progression of hepatocellular carcinoma.

Methylation status of p14ARF, p15INK4b, and p16INK4a genes in human hepatocellular carcinoma.

Abstract: Background: The INK4 locus consisting of three genes involved in the regulation of cell cycle, p16INK4a, p15INK4b, and p14ARF is often disrupted in human neoplasms.

Efficacy of combination therapy of antiviral and immunosuppressive drugs for the treatment of severe acute exacerbation of chronic hepatitis B

BackgroundPatients with severe exacerbation of chronic hepatitis B, sometimes developing into fulminant liver failure, are at high risk for mortality even with antiviral therapy. The efficacy of immunosuppressive therapy in clinically severe exacerbation of chronic hepatitis B has not been well demonstrated. In this study, we evaluated the efficacy of the early introduction of immunosuppressive therapy in combination with antiviral therapy in such patients.MethodsForty-two patients, 29 men and 13 women, were defined as having severe exacerbation of chronic hepatitis B based on our uniform criteria, and were enrolled in this study. Sixteen patients between 1982 and 1996 were analyzed retrospectively. We defined the criteria of severe disease in 1997, and then began to introduce sufficient doses of corticosteroids prospectively. Nucleoside analogs were administered in combination with corticosteroids after 1999. Twenty-six patients between 1997 and 2007 were analyzed prospectively.ResultsIn the retrospective study between 1982 and 1996, four of 16 (25%) patients recovered. In the prospective study between 1997 and 2007, 17 of 26 (65%) patients recovered; 15 of 17 patients treated with corticosteroids with or without antiviral drugs within 10 days after the diagnosis of severe disease recovered, none of five treated similarly but later than 10 days after the diagnosis recovered, and two of three treated with antiviral drugs recovered.ConclusionsThe early introduction of sufficient doses of corticosteroids and nucleoside analogs could be one option for reversing the potential deterioration of patients with clinically severe, life-threatening exacerbation of chronic hepatitis B.

Hepatitis B Virus e Antigen Downregulates Cytokine Production in Human Hepatoma Cell Lines

Disease activities of hepatitis B are affected by the status of hepatitis B e antigen (HBeAg). The function of the hepatitis B virus (HBV) precore or HBeAg is unknown. We assumed that HBeAg blocks aberrant immune responses, although HBeAg is not required for viral assembly, infection, or replication. We examined the interaction of HBeAg and the immune system, including cytokine production. The inflammatory cytokine TNF, IL-6, IL-8, IL-12A, IFN-α1, and IFN-ß mRNA were downregulated in HBeAg-positive HepG2, which stably expresses HBeAg, compared to HBeAg-negative HepG2 cells. The results of real-time RT-PCR-based cytokine-related gene arrays showed the downregulation of cytokine and IFN production. We also observed inhibition of the activation of NF-κB- and IFN-ß-promoter in HBeAg-positive HepG2, as well as inhibition of IFN and IL-6 production in HBeAg-positive HepG2 cell culture fluids. HBeAg might modify disease progression by inhibiting inflammatory cytokine and IFN gene expression, while simultaneously suppressing NF-κB-signaling- and IFNß-promoter activation.