Kazuhiko Koike

Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.

Liver steatosis, which involves accumulation of intracytoplasmic lipid droplets, is characteristic of hepatitis C virus (HCV) infection. By use of an in vivo transgenic murine model, we demonstrate that hepatic overexpression of HCV core protein interferes with the hepatic assembly and secretion of triglyceride‐rich very low density lipoproteins (VLDL). Core expression led to reduction in microsomal triglyceride transfer protein (MTP) activity and in the particle size of nascent hepatic VLDL without affecting accumulation of MTP and protein disulfide isomerase. Hepatic human apolipoprotein AII (apo AII) expression in double‐core/apo AII transgenic mice diminished intrahepatic core protein accumulation and abrogated its effects on VLDL production. Apo AII and HCV core colocalized in human HCV‐infected liver biopsies, thus testifying to the relevance of this interaction in productive HCV infection. Our results lead us to propose a new patho‐physiological animal model for induction of viral‐related steatosis whereby the core protein of HCV targets microsomal triglyceride transfer protein activity and modifies hepatic VLDL assembly and secretion.—Perlemuter, G., Sabile, A., Letteron, P., Vona, G., Topilco, A., Chrétien, Y., Koike, K., Pessayre, D., Chapman, J., Barba, G., Bréchot, C. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral‐related steatosis FASEB J. 16, 185–194 (2002)

Molecular basis of hepatitis C virus-associated hepatocarcinogenesis: lessons from animal model studies.

Despite numerous lines of epidemiologic evidence connecting HCV infection and the development of hepatocellular carcinoma (HCC), it remains controversial whether HCV itself plays a direct role or an indirect role in the pathogenesis of HCC. Through the use of transgenic mice, it has become evident that the core protein of HCV has oncogenic potential. HCV is directly involved in hepatocarcinogenesis, albeit other factors such as inflammation and environmental factors might also play a role. The direct involvement of HCV in hepatocarcinogenesis would be achieved via 2 pathways. In one pathway, the core protein acts on the function of mitochondria, leading to the overproduction of oxidative stress, which yields genetic aberrations in cell growth-related genes. The other pathway involves the modulation of cellular gene expressions and intracellular signal transductions, such as mitogen-activated protein kinase pathway, which results in the activation of transcription factors and cell cycle machineries. The combination of these alterations would be hypothesized to provoke the development of HCC in HCV infection. This would be a mechanism for HCC development in HCV infection that is distinct from those for other cancers. The presence of the HCV core protein, to which an oncogenic potential is ascribed, might allow some of the multiple steps to be bypassed in hepatocarcinogenesis. Therefore, unlike in other cancers, HCV infection can elicit HCC in the absence of a complete set of genetic aberrations. Such a scenario, non-Vogelstein type carcinogenesis, may explain the unusually high incidence and multicentric nature of HCC development in HCV infection.

Frequent β-catenin aberration in human hepatocellular carcinoma

Abstract Recently, mutations in the β-catenin gene in hepatocellular carcinoma (HCC) have been reported: approximately 20% of HCCs had activating mutations at the glycogen synthase kinase 3β phosphorylation sites within the exon 3 of the β-catenin gene. However, changes in the level of the β-catenin protein in HCC have not been well studied. We examined, by Western blotting, the expression level of the β-catenin protein in cancerous tissues in comparison with that in adjacent non-cancerous tissues obtained from 32 cases of HCC with hepatitis C. An increase in the β-catenin protein level in cancerous tissue compared to that in adjacent non-cancerous tissue was found in 15 (46.9%) of 32 cases of HCC. Mutation in exon 3 of the β-catenin gene was found in six (18.8%) of the 32 cases, in five of which the β-catenin protein level was increased. In total, β-catenin aberration was found in 16 (50.0%) of 32 cases of HCC. It should be noted that β-catenin aberration was also found in early HCC although it was observed chiefly in advanced HCCs. These results indicate that β-catenin aberration is a frequent event in the development of HCC and may facilitate the development of HCC in the course of chronic hepatitis.

Serum lipid profile of patients with genotype 1b hepatitis C viral infection in Japan

Hepatitis C virus (HCV) infection is associated with the development of steatosis in the liver. Recently, infection with genotype 3a HCV has been reported to have a closer association with hepatic steatosis than that with genotype 1 or 2 HCV. Moreover, infection with genotype 3a HCV but not with genotype 1 has been shown to be associated with serum hypocholesterolemia or hypobetalipoproteinemia in European countries. We conducted a case control study to characterize the serum lipid profile in patients infected with genotype 1b HCV, which is the most prevalent HCV genotype in Japan. These patients had significantly lower serum cholesterol levels than those infected with HBV or genotype 2a HCV who had similar liver disease progression and body mass index. Further analysis of serum apolipoproteins revealed that not only apolipoprotein B but also apolipoprotein CII and apolipoprotein CIII levels were significantly reduced, while apolipoprotein AI, AII and E levels were similar in patients infected with genotype 1b HCV and those with HBV or genotype 2a HCV. These results indicate that, in Japan, infection with genotype 1b HCV is a cause of lipid metabolism disturbances, which may be associated with the pathogenesis of hepatitis C liver disease.

Role of hepatitis C virus in the development of hepatocellular carcinoma: transgenic approach to viral hepatocarcinogenesis.

Abstractu2003Overwhelming lines of epidemiologic evidence have indicated that chronic infection with hepatitis C virus (HCV) poses a major risk towards the development of hepatocellular carcinoma (HCC). It remains controversial whether HCV plays a direct role in the pathogenesis of HCV‐associated HCC or whether it merely serves an indirect role. Using the transgenic mouse model established by us, it has become evident that the core protein of HCV confers oncogenic potential. The findings in our studies indicate that HCV is directly involved in hepatocarcinogenesis, albeit other factors, such as continued cell death and regeneration associated with chronic hepatitis, may also play a role. Taken together, our results indicate that there could be a mechanism for the development of HCC in persistent hepatitis virus infection that is distinct from the pathogenesis of other cancers, like colorectal cancer. Thus, although accumulation of a set of genetic aberrations may also be necessary for a multistage development of HCC, HCV core protein, to which an oncogenic potential is ascribed, may allow some stages to be skipped in hepatocarcinogenesis. The possibility that infection with HCV may be capable of inducing HCC in the absence of a complete set of genetic aberrations would help explain the unusually high incidence and multicentric nature of HCC developing in chronic hepatitis C.

Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma

BackgroundSilencing of the suppressor of cytokine signaling (SOCS-1) by aberrant methylation at the CpG island in the coding region gene has been reported in hepatocellular carcinoma (HCC). However, principally, it is methylation in the 5′-noncoding region but not that in the coding region which determines the regulation of gene expression.MethodsMethylation-specific PCR was performed for the analysis of methylation status both in the 5′-noncoding region and the CpG island of SOCS-1 from 22 HCC tissue samples with adjacent non-HCC tissue samples and from two cell lines.ResultsUsing primers in the CpG island, 9 of 22 HCC samples exhibited aberrant methylation of SOCS-1, while only 1 of 22 adjacent non-HCC samples did so. The unmethylation pattern was detected in 1 of 22 HCC and in 5 of 22 non-HCC samples. Thus, aberrant methylation of SOCS-1 was significantly associated with HCC (P = 0.0076 by Fisher’s exact test). Using primers in the 5′-noncoding region, aberrant methylation was observed in 12 of 22 HCC and in 2 non-HCC samples. The unmethylated pattern was observed in 5 of 22 HCC and in 10 of 22 non-HCC samples (P = 0.0042). There was no significant correlation between the methylation status of SOCS-1 and clinicopathological findings, such as the presence or absence of cirrhosis or the histological grade of HCC.ConclusionsAberrant methylation of the SOCS-1 had a significant correlation with HCC. The rate of aberrant methylation was similar in the 5′-noncoding region and in the CpG island. Aberrant methylation of SOCS-1 may be associated with hepatocarcinogenesis, although further studies are necessary.

Emergence of the precore mutant late in chronic hepatitis B infection correlates with the severity of liver injury and mutations in the core region

OBJECTIVE:The reason that precore negative mutants (e-minus DNA) gradually become predominant in some patients during chronic hepatitis B virus infection is not clear. Theoretically, as long as both e-plus and e-minus DNA share the same epitopes in the core region, HBcAg-specific cytotoxic T lymphocytes (CTLs) cannot distinguish between the target peptides expressed by e-plus and e-minus DNA. Therefore, e-minus DNA may be accompanied by additional mutations in the core region, which may affect cytotoxic T lymphocyte recognition. To examine this possibility, the sequences of the precore and the entire core region of the hepatitis B virus genome were analyzed from paired serum samples in CH-B patients who experienced HBeAg to anti-HBe seroconversion (SC).METHODS:Patients were divided into two groups. Group A patients (n = 17) genome-converted to e-minus DNA in the precore region, which abolished HBeAg secretion within 3–4 yr after SC. Group B patients (n = 16) retained precore wild-type DNA for more than 3–4 yr after SC. To investigate the impact of the emergence of precore mutant type DNA on liver injury, alanine aminotransferase (ALT) levels were also examined.RESULTS:ALT flares were more severe among patients in group A than in group B. The average mean ALT level during the HBeAg negative phase of chronic infection was 54 ± 38 in group A and 28 ± 24 in group B. The average maximal ALT level during the HBeAg negative phase was 235 ± 249 in group A and 83 ± 106 in group B. Furthermore, all 17 patients in group A developed new core mutants during genome conversion. The average number of mutations in the core gene was 0.9 ± 1.2 before genome conversion (e-plus DNA dominant phase) and increased to 2.8 ± 1.3 for the 3–4 yr during genome conversion (e-minus DNA dominant phase). In contrast, only 56% (nine of 16) of patients in group B developed new core mutations after the loss of HBeAg. The average number of mutations in the core gene was 1.8 ± 1.3 before SC (HBeAg-positive and e-plus DNA dominant phase), and decreased to 1.1 ± 1.1 for 3–4 yr after seroconversion (anti-HBe–positive and e-plus DNA dominant phase).CONCLUSIONS:These data indicate that the emergence of a predominant precore negative genotype late in chronic hepatitis B virus infection is associated with the selection of additional mutations in the core gene, as well as with liver injury.

Survey of Hepatitis B Virus Co‐infection in Hepatitis C Virus‐infected Patients Suffering from Chronic Hepatitis and Hepatocellular Carcinoma in Japan

More than 90% of primary hepatomas in Japan are thought to be related to hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) in serum are used as a serological marker for chronic hepatitis (CH) and as a test for past HBV infection, respectively. Preliminary data recently obtained in Japan or internationally indicated that HBsAg-negative HCV-related CH or hepatocellular carcinoma (HCC) patients often showed positivity for anti-HBc and the presence of HBV DNA, suggesting co-infection of HBV. This was unexpected, as such patients had generally been considered to be free of HBV infection. Anti-HCV positivity was frequently found in blood donors presenting the HBsAg negative and the anti-HBc positive serological markers. This disposition was also observed during the screening of blood donors with anti-HBc, which was previously used as a surrogate marker for the prevention of post-transfusion nonA nonB hepatitis. We therefore surveyed the presence of HBV DNA in HBsAg negative/anti-HCV positive and anti-HBc positive CH or HCC patients in Japan, to try to clarify whether there is any involvement of HBV in the development of HCC in HCV-infected patients. Anti-HBc positivity was detected by using the commercially available standard assay system on 64-fold and/or 200-fold diluted sera from patients. A survey for HBV DNA in liver samples of patients was performed by nested polymerase chain reaction (PCR) analysis using primers for HBV DNA, 2) and by Southern blot analysis using whole and/or a specific segment of HBV DNA as a probe. Nested PCR reaction was carried out as described previously. Briefly, each sample was subjected to nested PCR reaction in a 50 μ l standard reaction mixture with combinations of primers to amplify each HBV gene as follows; HBs gene: HBS-1/HBS-R1 for 1st reaction and HBS-11/HBS-R11 for nested PCR, X gene: X-Pr/XCD-R1 for 1st reaction and XCD-1/XCDR11 for nested PCR, HBc gene: HBC-1/HBC-R1 for 1st reaction and HBC-1/HBC-R11 for nested PCR, X gene to HBc gene: XCD-1/HBC-R1 for 1st PCR and XCD-2/HBC-R11 for nested PCR. In HCV-infected CH and HCC patients with anti-HCV positive and HBsAg negative serological markers, anti-HBc positivity was found to be 41.7% in 1501 CH patients, and 58.3% in 816 HCC patients. In control, non-hepatic patients with anti-HCV negative/HBsAg negative markers, positivity of HBcAb was 18.3% in older patients (average age 64) and 11.8% in younger patients (average age 44), indicating that anti-HBc positivity in anti-HCV positive/HBsAg negative CH and HCC patients is two to three times higher than in control non-hepatic patients. These results suggested that silent HBV infection (HBsAg negative infection) frequently occurred in HCVrelated CH and/or HCC patients. The presence of HBV DNA in the liver biopsy samples of CH and HCC patients was analyzed by a quantitative nested PCR method using primers that enabled us to judge the state of HBV DNA in the cell. huH2-2 cell DNA was used as a single copy control of HBV DNA that can be detected up to 10−4 fold dilution, but not at 10−5 fold dilution in the nested PCR. When 17 CH and 62 HCC biopsy tissue samples with anti-HCV positive/HBsAg negative/anti-HBc positive markers (out of a total of 43 HCV-related CH and 149 HCV-related HCC patients) were tested, HBV DNA was detected in 58.8% (10/17) and 56.4% (35/62) of CH and Southern blot-negative HCC samples, respectively (Table I), although the copy number of HBV DNA in these samples was found to be as low as 0.1 copy per cell. Previous studies indicated that integration of HBV DNA into hepatic cells of HBV-infected patients frequently occurred between the 3′ end of the X gene and the contiguous core antigen gene, resulting in the split-up of these two genes in the HBV genome. Therefore, only closedcircular (CC) molecules without nicks or gaps in one strand can be detected using primers that amplify the HBV DNA connecting the two regions. Our analysis suggested the presence of CC HBV DNA in some cases. The clonality of HBV DNA was further tested in 65 HCC DNA samples from anti-HCVpositive patients by means of a Southern blot analysis that was sufficiently sensitive to detect 0.1

Role of hepatitis viruses in multistep hepatocarcinogenesis

K. Koike Worldwide, hepatitis B virus (HBV) and hepatitis C virus (HCV) infect hundreds of millions of people persistently and induce a spectrum of chronic liver diseases. Hence, they have an impact on society in a number of domains including medical, sociologic and economic. Hepatocellular carcinoma (HCC) has become the most frequent cause of death in individuals persistently infected with HBV or HCV. In particular, HCV has gained increasing attention because of its wide and deep penetration into the community, coupled with a very high incidence of HCC in the persistent infection. Once liver cirrhosis is established in hosts infected with HCV HCC develops at a yearly rate of 5 7% ‘. Knowledge on the mechanism by which HCC develops in chronic HCV infection is, therefore, urgently required for the prevention of HCC.

IgG1 anti‐P2 as a marker of response to interferon in patients with chronic hepatitis C

To study the relations of antibody production to long‐term outcomes after interferon (IFN) treatment in patients with chronic hepatitis C (CH‐C), we used ELISA to measure the levels of antibodies against HCV core protein and peptides. Samples from 21 complete responders and 36 non‐responders were collected before IFN therapy, soon after the end of IFN therapy and 6u2003months later. Using a set of 19 synthesized HCV core peptide antigens, we found that anti‐P2 (11–25a.a.) was the most prevalent of all IgG antibodies (93%: 39/42). Among complete responders, IgG1 anti‐P2 levels had fallen by the end of IFN therapy (from 79·8u2003±u200360·4–46·1u2003±u200344·2: Pu2003<u20030·01), and were lower still 6u2003months after the end of IFN therapy (31·0u2003±u200335·2: Pu2003<u20030·001); this change was the greatest of all antibodies studied. Among the non‐responders, there was no change within the follow‐up period. Soon after the end of IFN therapy, IgG1 anti‐P2 levels were more than 30% lower than the initial value in more than two‐thirds of the complete responders, but in only one‐third of the non‐responders (14/20 vs. 8/25: Pu2003<u20030·05). Six months after the end of IFN therapy, IgG1 anti‐P2 levels were more than 30% lower than the initial value in more than 85% of the complete responders, but in only 12% of the non‐responders (17/20 vs. 3/25: Pu2003<u20030·001). In conclusion, the changes in levels of IgG1 anti‐P2 paralleled the activity of chronic hepatitis C after IFN therapy, and IgG1 anti‐P2 levels may be markers of the efficacy of IFN therapy.