metallothionein and Hepatitis-C--Chronic

The IFNL3 genotype predicts the clearance of hepatitis C virus (HCV), spontaneously and with interferon (IFN)-based therapy. The responder genotype is associated with lower expression of interferon stimulated genes (ISGs) in liver biopsies from chronic hepatitis C patients. However, ISGs represent many interacting molecular pathways, and we hypothesised that the IFNL3 genotype may produce a characteristic pattern of ISG expression explaining the effect of genotype on viral clearance. For the first time, we identified an association between a cluster of ISGs, the metallothioneins (MTs) and IFNL3 genotype. Importantly, MTs were significantly upregulated (in contrast to most other ISGs) in HCV-infected liver biopsies of rs8099917 responders. An association between lower fibrosis scores and higher MT levels was demonstrated underlying clinical relevance of this association. As expected, overall ISGs were significantly downregulated in biopsies from subjects with the IFNL3 rs8099917 responder genotype (P=2.38 × 10(-7)). Peripheral blood analysis revealed paradoxical and not previously described findings with upregulation of ISGs seen in the responder genotype (P=1.00 × 10(-4)). The higher MT expression in responders may contribute to their improved viral clearance and MT-inducing agents may be useful adjuncts to therapy for HCV. Upregulation of immune cell ISGs in responders may also contribute to the IFNL3 genotype effect.

Topics: Genotype; Hepacivirus; Hepatitis C, Chronic; Humans; Interferon Regulatory Factors; Interferon-alpha; Interferons; Interleukins; Liver; Liver Cirrhosis; Metallothionein; Polyethylene Glycols; Polymorphism, Single Nucleotide; Recombinant Proteins; Ribavirin; Treatment Outcome; Up-Regulation; Viral Load

Gene expression profiling or karyotyping analysis has made it possible to identify novel genes with altered expressions or copy numbers that have not been previously reported in liver cancer. On the same HCC sample, we performed double array analysis, both expression profiling and karyotyping analysis using a single nucleotide polymorphism (SNP) array in an attempt to find a novel tumor suppressor gene for its prognostic marker. We conducted expression array and SNP chip array using tumor and corresponding non-tumor tissues from the resected liver specimen of a 68-year-old woman who had chronic hepatitis type C. Additionally, we performed quantitative real-time reverse transcription polymerase chain reaction (PCR) and methylation-specific PCR (MSP) for gene detection using specimens from 48 patients with HCC, and investigated their correlation with the prognosis. Metallothionein (MT) 1G gene located on 16q13 showed a decreased expression in tumor tissue. The copy number by SNP chip array revealed no loss of heterozygosity since no deletions were detected in 16q13, and HCC tissue showed AB call in both SNPs next to MT1G. In quantitative real-time PCR using 48 HCC clinical samples, mRNA expression of MT1G decreased significantly compared with that in corresponding non-cancerous liver tissues (p<0.0323). Twenty-nine (60.4%) of 48 HCCs gave a positive result in MSP, indicating a poorer prognosis than the negative group, although the difference was not significant (p<0.0978). Our results indicated that MT1G acts as a tumor suppressor gene in HCC. Moreover, findings suggested that the mechanisms of MT1G silencing are related to promoter hypermethylation.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Hepatocellular; DNA Methylation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Hepatitis C, Chronic; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Metallothionein; Oligonucleotide Array Sequence Analysis; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

An association between reactive oxygen species and liver damage has been postulated in the course of hepatitis C virus (HCV) infection. Metallothionein (MT), induced by HCV core protein and interferon (IFN), plays a role in scavenging free radicals. MT expression in liver biopsies obtained from 21 patients with chronic HCV infection before and after IFN-alpha therapy was investigated. Changes in Knodell histological activity index (HAI) scores, MT protein levels (immunohistochemistry), MT-I and MT-II messenger (m)RNA expression levels (in situ hybridization) and proliferating cell nuclear antigen (PCNA) labelling index were determined and compared in serial liver specimens. MT staining was clustered around the portal tracts with inflammatory cells and fibrosis. The pattern of MT protein before IFN-alpha therapy was similar in all patients, but was higher in IFN-sustained responders than in nonresponders after IFN-alpha therapy. HAI scores and PCNA labelling indexes were significantly reduced after IFN-alpha therapy. MT-II mRNA expression correlated positively with PCNA index before therapy and with HAI scores after therapy (P<0.05). No correlation was found between MT-I mRNA and HAI scores or PCNA index. The findings indicate that IFN-alpha-induced hepatic MT may participate in the therapeutic effects of IFN-alpha for HCV. In addition, MT-II mRNA expression may be involved in cell proliferation in the livers of patients with chronic HCV infection.

Topics: Antiviral Agents; Biopsy; Female; Gene Expression; Hepatitis C, Chronic; Humans; Immunohistochemistry; In Situ Hybridization; Interferon-alpha; Liver; Male; Metallothionein; Middle Aged; RNA, Messenger; Severity of Illness Index; Treatment Outcome

Reactive oxygen species may be involved in the pathogenesis of chronic hepatitis C virus infection. Metallothionein (MT) is an essential protein for the protection of cells against reactive oxygen species. The aim of this prospective study was to assess the influence of the hepatic level and cellular distribution of MT in hepatitis C virus (HCV) infection and in the liver disease outcome.. In liver biopsy samples of 32 patients with chronic HCV infection and of 12 control subjects, quantification of MT was performed by radioimmunoassay, MT, interleukin (IL)-1 and -6, and tumor necrosis factor (INF)-alpha mRNA by reverse transcription-polymerase chain reaction (PCR) and cellular distribution by immunohistochemistry.. In HCV-infected patients, MT liver protein level was 3-fold lower than in control specimens. A significant inverse linear regression between MT protein or mRNA expression and the Histological Activity Index, the necroinflammatory grade, and the stage of fibrosis was observed. MT immunostaining was located in the nucleus and cytoplasm in hepatocytes of control subjects, whereas it was mainly cytoplasmic in HCV-infected patients. Before interferon (IFN) therapy, the hepatic MT level in patients that were nonsustained responders was half that of sustained responders. Intrahepatic IL-6 and MT were simultaneously down-regulated, but no correlation was found between MT and intrahepatic cytokine mRNA expression in patients with chronic HCV infection.. This study shows that hepatic MT expression could reflect the severity of chronic HCV infection and could be one of the factors associated with a favorable clinical outcome in the response to interferon therapy.

Topics: Antiviral Agents; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Immunohistochemistry; Interferon-alpha; Interleukin-1; Interleukin-6; Liver; Male; Metallothionein; Middle Aged; Prospective Studies; Regression Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Viral; Tumor Necrosis Factor-alpha

Data suggesting that the hepatitis C virus (HCV) core protein influences normal cellular processes remain controversial. To determine the effects of core on cellular gene expression in hepatocytes, we developed a human hepatoma (Huh7)-derived cell line with tightly regulated core expression under the control of a tetracycline-regulated promoter. Cells expressing core did not have impaired proliferative abilities. Changes in gene expression profiles in response to core expression were determined using commercial oligonucleotide microarrays (Affymetrix GeneChip). Significant increases were observed in the abundance of mRNA-encoding members of the metallothionein (MT) family, as well as nicotinamide N-methyltransferase (NNMT) and glutathione peroxidase-like protein (GPLP). These changes did not result from removal of tetracycline from growth media, and were confirmed in reverse-transcription polymerase chain reaction (RT-PCR) assays. They suggest that core protein expression leads to intracellular oxidative stress, and that vital cellular functions are, in turn, protected by up-regulation of cellular antioxidant defense mechanisms. In conclusion, these findings can explain many potentially conflicting prior observations concerning the effects of core on cellular physiology, and are of relevance to the role of core protein in the pathogenesis of HCV-related fibrosis and hepatocellular carcinoma.

Topics: Carcinoma, Hepatocellular; Gene Expression Regulation, Viral; Hepacivirus; Hepatitis C, Chronic; Hepatocytes; Humans; Liver Neoplasms; Metallothionein; Oligonucleotide Array Sequence Analysis; Oxidation-Reduction; Peptides; Plasmids; RNA, Messenger; Tumor Cells, Cultured; Viral Core Proteins