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Osamu Yokosuka

Publications and source records attributed to Osamu Yokosuka.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine in gastric cancer cell lines.

In gastric cancer, increasing numbers of genes have been reported to be silenced by aberrant methylation. However, global analysis of epigenetic inactivation in cancer cells has rarely been performed. For screening the genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC), cDNA microarray analysis (AceGene(R), containing 30,000 genes) was performed using gastric cancer cell lines (AGS, MKN74, MKN1, MKN45 and Kato3) treated with DAC. The candidate upregulated genes were confirmed by real-time PCR, and the methylation status of 5'CpG islands was determined by bisulfite DNA sequencing or methylation-specific PCR. Among the upregulated genes considered to have CpG island in their promoter regions, we selected 5 genes (BCL2L10, DKK1, DNAJD1, GAGED2 and NMU) that exhibited a greater than 3-fold increase in at least 2 cell lines. Of these, we could determine the methylation status of 5'CpG islands of BCL2L10, DKK1 and DNAJD1. 5'CpG of BCL2L10 and DNAJD1 was hypermethylated in 4 of 5 gastric cancer cell lines, whereas 5'CpG of DKK1 was hypermethylated in only 1 cell line. MSP analysis for BCL2L10 revealed that the CpG island was demethylated after DAC treatment. In addition, we observed that overexpression of BCL2L10 could promote apoptosis and growth-inhibitory effect in gastric cancer cell lines. In conclusion, some of the genes upregulated by DAC treatment may be transcriptionally repressed by promoter hypermethylation. These genes might be related to gastric carcinogenesis. In particular, the suppression of BCL2L10, which could induce apoptosis and inhibit proliferation of cancer cells, might be one of the underlying mechanisms for gastric carcinogenesis.

Azacitidine↗
PubMed2006

Nucleotide change of codon 38 in the X gene of hepatitis B virus genotype C is associated with an increased risk of hepatocellular carcinoma.

BACKGROUND/AIMS: The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC. METHODS/RESULTS: We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89). CONCLUSION: The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV.

Biopsy↗
PubMed2006

Sequential gene expression changes in cancer cell lines after treatment with the demethylation agent 5-Aza-2'-deoxycytidine.

BACKGROUND: 5-Aza-2'-deoxycytidine (5-AzaC) is known well for its demethylation effect and is a promising anticancer agent. However, to the authors' knowledge, serial changes in gene expression over time after 5-AzaC treatment have not been studied to date. To clarify the categories of genes that are up-regulated or down-regulated after 5-AzaC treatment, the authors surveyed the genes that had expression levels changed by 5-AzaC treatment in 6 hepatoma cell lines (Hep3B, HLE, Huh7, HepG2, PLC/PRF/5, and Huh6). METHODS: Cell lines were grown in medium that contained 1 microM of 5-AzaC. Changes in messenger RNA levels were monitored from 24 hours up to 120 hours after 5-AzaC treatment using an in-house microarray that consisted of 4608 combinational DNAs. Using clustering analysis to identify the genes that had gradually changed expression levels and to exclude the substantial experimental noise by microarray analysis, the authors focused on 206 up-regulated genes and 248 down-regulated genes. RESULTS: According to their functional characterization, genes that were involved in the cytoskeleton and the extracellular matrix were enriched significantly in the up-regulated genes. Conversely, genes that were involved in metabolism were enriched significantly in the down-regulated genes. CONCLUSIONS: The current results demonstrated that 5-AzaC can regulate the expression of groups of genes with characteristic functions.

Azacitidine↗
PubMed2006

State of hepatitis C viral replication enhances activation of NF-kB- and AP-1-signaling induced by hepatitis B virus X.

We examined the effect of hepatitis B virus X (HBx) on NF-kB- and AP-1- mediated transcription in human hepatocellular carcinoma cell lines, Huh-7 with or without subgenomic hepatitis C virus (HCV) RNA. Expression of HBx in Huh-7 cells with HCV resulted in 4.9 times increased NF-kB-activation and 3.8 times AP-1-activation whereas that without HCV resulted in 2.4 times increased NF-kB-activation and 2.3 times AP-1-activation. Interestingly, the expression of the matured form of HCV core protein, Core173, did not activate NF-kB- or AP-1-transcription in either Huh7 with or without HCV replicon. HBx protein might play an important role in HCV-related hepatocarcinogenesis.

Blotting, Western↗
PubMed2006

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy.

Apoptosis↗
PubMed2006

Molecular biology of hepatitis B virus: effect of nucleotide substitutions on the clinical features of chronic hepatitis B.

Hepatitis B virus (HBV) is a major cause of liver disease worldwide. It is covered with envelope (surface antigen) proteins with the nucleocapsid (core antigen) inside. In the nucleocapsid, there is an incomplete double-stranded DNA and a DNA polymerase. Four genes, S, C, X, and P, are encoded, and these partially overlap. Mutations have been reported in each gene and in their promoter regions, and these mutations can change the efficiency of HBV replication and the clinical course of patients. In this article, we review the relationship between the molecular biology of HBV and its clinical outcome.

Base Pairing↗
PubMed2006

Serum osteopontin levels in patients with acute liver dysfunction.

OBJECTIVE: Fulminant hepatic failure (FHF) is a clinical syndrome of sudden and severe liver dysfunction accompanied by encephalopathy in a previously healthy person. In FHF, hepatocytes are severely damaged and ordinary liver regeneration is impaired. We demonstrated that the expression of osteopontin (OPN), a multifunctional cytokine, was up-regulated in mouse oval cell (a stem-cell progenitor) induction models. MATERIAL AND METHODS: Based on this finding, serum OPN levels were examined in 43 patients with FHF and in 45 patients with acute self-limited hepatitis (AH). To determine the cellular source of OPN, the expression of OPN was studied in a liver specimen derived from an FHF patient. RESULTS: The mean OPN level of patients with FHF was 2.80+/-0.48 ng/ml (log(10), +/-SD), which was significantly higher than that of the patients with AH (2.42+/-0.39 ng/ml) (p=0.003, unpaired t-test). Patients with elevated serum OPN levels had a significantly poorer prognosis than patients whose serum OPN levels were not elevated. In the FHF patient's liver, OPN protein was expressed not only in inflammatory cells but also in regenerating hepatocytes and bile ductular structures. CONCLUSIONS: Our current study indicates that serum OPN levels increased in patients with FHF and that OPN might play an important role in liver regeneration due to activation of hepatic stem cells.

Acute Disease↗
PubMed2006

Randomized, double-blind, placebo-controlled trial of bovine lactoferrin in patients with chronic hepatitis C.

Several studies have suggested that lactoferrin administration may decrease the serum level of hepatitis C virus (HCV) RNA in patients with chronic hepatitis C. The aim of the present study was to confirm the efficacy of orally administered bovine lactoferrin (bLF) in patients with chronic hepatitis C. The patients with chronic hepatitis C randomly received either oral bLF at a dose of 1.8 g daily for 12 weeks, or an oral placebo. The primary endpoint was the virologic response, defined as a 50% or greater decrease in serum HCV RNA level at 12 weeks compared with the baseline. The secondary endpoint was the biochemical response, which was defined as a 50% or greater decrease in the serum alanine aminotransferase (ALT) level at 12 weeks compared with the baseline. One hundred and ninety-eight of 199 patients were evaluable for efficacy and safety. bLF treatment was well tolerated and no serious toxicities were observed. A virologic response was achieved in 14 of 97 patients (14.4%) in the bLF group, and 19 of 101 (18.8%) in the placebo group. There was no significant difference in virologic response rates between the two groups (-4.4%, 95% confidence interval -14.8, 6.1). In addition, bLF intake did not have any favorable effect on the serum ALT level. The virologic responses were not different between two groups in any subgroup analysis. In conclusion, orally administered bLF does not demonstrate any significant efficacy in patients with chronic hepatitis C.

Administration, Oral↗
PubMed2006

Twenty-four weeks of interferon alpha-2b in combination with ribavirin for Japanese hepatitis C patients: sufficient treatment period for patients with genotype 2 but not for patients with genotype 1.

BACKGROUND: Hepatitis C virus (HCV) RNA titer and HCV genotype are two major determinants of the outcome of interferon (IFN) monotherapy. To clarify the usefulness of combination therapy with IFN and ribavirin in Japanese hepatitis C patients, we treated patients with a relatively high dose of IFN in combination with ribavirin for 24 weeks and examined the effects in relation to the viral parameters. METHODS: Two hundred and ninety-five patients were enrolled in the study. The patients received either 6 or 10 million units (MU) of interferon alpha-2b every day for 2 weeks and then three times a week for 22 weeks with a daily dose of either 600 or 800 mg of ribavirin. The treatment response and safety of this treatment were examined. RESULTS: The sustained virologic response (SVR) rates were 26.8% in genotype 1 and 76.5% in genotype 2 (P < 0.001), and 36.1% with the 6 MU group and 45.8% with the 10 MU group (P = 0.09). Multivariate analysis indicated that SVR was associated with genotype 2, HCV RNA <500 kilointernational unit/ml (kIU/ml), and HCV RNA undetectability at week 8 of treatment. CONCLUSION: Our current study showed that a 24-week course of IFN plus ribavirin combination therapy was effective with respect to virologic response in Japanese hepatitis C patients, particularly in patients with HCV genotype 2.

Adolescent↗
PubMed2006

[Hepatitis E virus infection in Japan].

Hepatitis E virus (HEV) is the major etiologic agent of enterically transmitted viral hepatitis in many developing countries. Epidemics are primarily waterborne in areas where water supplies are contaminated with HEV of human origin. There is increasing evidence, however, that HEV is also prevalent in very low numbers in non-endemic countries, including Japan. Although the source of HEV in these sporadic cases is unknown, a recently isolated swine virus is the best candidate for causing a zoonotic form of hepatitis E. The virus is serologically cross-reactive with human HEV and genetically very similar, and the human and swine strains seem to be cross-infective. Very recent evidence has also shown that swine HEV, and possibly a deer strain of HEV, may be related to avian HEV and HEV in other hosts and potential reservoirs. We examined the prevalence of anti-HEV IgM and IgG among patients serologically diagnosed with non-A, non-B, non-C acute hepatitis (n = 126) and compared with a matched control group of 76 individuals. Enzyme-linked immunosorbent assay revealed a significant difference in seroprevalence between the two groups for anti-HEV IgM (5.6% versus 0%), whereas there was no difference for anti-HEV IgG (21.4% versus 26.3%). For confirmed cases of anti-HEV IgM we also detected HEV RNA in sera by means of a sensitive polymerase chain reaction (PCR) assay. This study provides evidence of locally acquired hepatitis E in the Chiba area. Therefore, in cases of unexplained acute hepatitis, the diagnosis of hepatitis E should be considered even in the absence of foreign travel.

Animals↗
PubMed2006

[Devices for the safe performance of self-injection of interferon therapy for chronic hepatitis C].

Long-term interferon therapy is an alternative therapy for patients with chronic hepatitis C who can not tolerate to the combination of pegylated interferon and ribavirin. In 2005, self-injection of interferon for chronic hepatitis C was covered by public health insurance in Japan making the long-term interferon therapy easier to perform. For the safe performance of self-injection, education about knowledge on various adverse effects of interferon, training for actual performance of self-injection, and close contact between the patients and doctors are indispensable. In this review, we briefly summarized why self-injection is needed and how the training for safe self-injection of interferon should be performed.

Antiviral Agents↗
PubMed2005

Prolonged acute hepatitis A mimicking autoimmune hepatitis.

AIM: We report a case with a prolonged course of hepatitis A, with alanine aminotransferase (ALT) higher than 500 IU/L for more than 2 mo. METHODS: A middle-aged woman had an elevated IgG level of more than 2,000 mg/dL, positive anti-nuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA), but no evidence of persistent hepatitis A virus (HAV) infection. Liver biopsy findings were compatible with prolonged acute hepatitis, although acute onset of autoimmune hepatitis could not be ruled out. RESULTS: It was assumed that she developed a course of hepatitis similar to autoimmune hepatitis triggered by HAV infection. Ursodeoxycholic acid (UDCA) treatment was initiated and a favorable outcome was obtained. CONCLUSION: We describe a case of a middle-aged woman who showed a prolonged course of acute hepatitis A mimicking autoimmune hepatitis. Treatment with UDCA proved to be effective.

Acute Disease↗
PubMed2005

Effect of lamivudine in HBeAg-positive chronic hepatitis B: discordant effect on HBeAg and HBV DNA according to pretreatment ALT level.

AIM: To clarify differences in antiviral effect of the drug in patients with different ALT levels, we examined the changes in HBV markers in patients with high or low ALT levels with or without lamivudine treatment. METHODS: Thirty-seven HBeAg-positive patients were studied. Ten patients with ALT levels higher than 200 IU/L (group 1) and 8 patients with ALT below 200 IU/L (group 2) were treated orally with 100 mg/d of lamivudine. As untreated control, 9 patients with ALT above 200 IU/L (group 3) and 10 patients with ALT below 200 IU/L (group 4) were examined. ALT level, HBeAg/HBeAb status, and HBV DNA level were examined monthly for 11.9+/-0.4 mo. RESULTS: The ALT level normalized in all 10 patients of group 1, 7/8 of group 2, 4/9 of group 3, and 1/10 of group 4 within 6 mo (groups 1 vs 2, P = NS; groups 1 vs 3, P = 0.002; groups 1 vs 4, P<0.0001). HBV DNA fell below the detection limit in all 10 patients of group 1, 7/8 of group 2, 0/9 of group 3, and 0/10 of group 4 within 6 mo (groups 1 vs 2, P = NS). HBeAg became seronegative in 7/10 patients of group 1, 1/8 of group 2, 3/9 of group 3, and 0/10 of group 4 within 12 mo (groups 1 vs 2, P = 0.02; groups 1 vs 3, P = NS). CONCLUSION: Our data suggest that HBeAg-positive patients with higher ALT levels can be considered good candidates for lamivudine therapy, probably because lamivudine accelerates the natural seroconversion of HBeAg, accompanied by HBV DNA loss, in these patients.

Adult↗
PubMed2005

Hepatitis C virus core protein and hepatitis activity are associated through transactivation of interleukin-8.

BACKGROUND: We evaluated the association between variations in hepatitis C virus (HCV) core protein and hepatitis severity in patients with chronic HCV infection who achieved remission without viral eradication and had a biochemical response to interferon (IFN) therapy, to evaluate the effect of HCV core sequence in the absence of the influence of host factors. METHODS. Using serum from 10 patients with a biochemical response and 10 patients with no response, we measured serum levels of interleukin (IL)-1 beta , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN- gamma , and tumor necrosis factor- alpha before and after IFN therapy. Expression vectors with the core region were transfected into Huh7 cells, and cytokine induction was evaluated by reporter assay. RESULTS: In biochemical responders, only IL-8 levels decreased after IFN therapy (P=.04). Changes in the C-terminal hydrophobic region were observed more frequently in biochemical responders. Activation of the IL-8 promoter by HCV core protein was significantly decreased in biochemical responders after IFN therapy (P=.04). When 69 C-terminal amino acids from before IFN therapy were replaced with those from after IFN therapy in 3 biochemical responders, their ability to transactivate IL-8 decreased. CONCLUSIONS: Differences in amino acids in the HCV core protein correlates with hepatitis activity through the modulation of IL-8 induction in HCV-infected patients.

Adult↗
PubMed2005

Amantadine inhibits hepatitis A virus internal ribosomal entry site-mediated translation in human hepatoma cells.

The effect of six drugs (amantadine, glycyrrhizin, ribavirin, ursodeoxycholic acid, alcohol, and IFN) on HAV RNA translation from the HAV internal ribosomal entry site (IRES) was investigated using a bicistronic reporter construct containing HAV IRES as intragenic spacer. Huh-7 cells and derivatives were transfected with in vitro transcripts, and the reporter gene activity was determined. IFN suppressed both cap-dependent and HAV IRES-dependent translation, while amantadine specifically inhibited HAV IRES-dependent translation. In contrast to IFN, by reporter assay, amantadine did not activate the interferon-stimulated response element (ISRE) or interferon gamma-activated sequence (GAS)-associated pathways. Immunoblot analysis revealed that amantadine had no effect on PKR and on IFN-regulatory factor-1 (IRF-1) expression. These findings demonstrated a novel antiviral effect of amantadine against HAV with or without HCV infection.

Amantadine↗
PubMed2005

Suppression of hepatitis A virus genome translation and replication by siRNAs targeting the internal ribosomal entry site.

Small interfering RNAs (siRNAs) targeting the coding region of hepatitis A virus (HAV) were shown to specifically inhibit viral genome replication. Compared to the coding region, the HAV internal ribosomal entry site (IRES) in the 5' non-coding region is highly sequence-conserved and folds into stable secondary structures. Here, we report efficient and sustained RNA interference mediated by both RNase III-prepared siRNA (esiRNA) and vector-derived short hairpin RNAs (shRNAs) that are targeted to various domains of the HAV IRES. Using reporter constructs, and the DNA-based HAV replicon system, we found that shRNAs targeting the HAV IRES domains IIIc and V sustainably suppressed genome translation and replication whereas the IRES domains IIIa and IV were resistant to RNA interference. Our study suggests that some HAV IRES domains might be used as a universal and effective target for specific inhibition of HAV infection.

Base Sequence↗