Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publishing type：Research paper (scientific journal)  

Intracranial metastases are common in nonsmall-cell lung cancer (NSCLC) patients, whose prognosis is very poor. In addition, intracranial progression is common during systemic treatments due to the inability to penetrate central nervous system (CNS) barriers, whereas the intracranial effects of cancer immunotherapies remain unclear. We analyzed clinical data to evaluate the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies compared with those treated without PD-1 blockade therapies, and found that the frequency of intracranial progression in advanced NSCLC patients treated with PD-1 blockade therapies was significantly lower than that in patients treated with cytotoxic chemotherapies. In murine models, intracranial rechallenged tumors after initial rejection by PD-1 blockade were suppressed. Accordingly, long-lived memory precursor effector T cells and antigen-specific T cells were increased by PD-1 blockade in intracranial lesions. However, intracranial rechallenged different tumors are not suppressed. Our results indicate that cancer immunotherapies can prevent intracranial progression, maintaining long-term effects intracranially as well as systemically. If intracranial recurrence occurs during the treatment with PD-1 blockade therapies, aggressive local therapies could be worthwhile.

DOI： 10.1002/ijc.34700

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Hydrogen peroxide (H2 O2 ) induces oxidative stress and cytotoxicity, and can be used for treating cancers in combination with radiotherapy. A product comprising H2 O2 and sodium hyaluronate has been developed as a radiosensitizer. However, the effects of H2 O2 on antitumor immunity remain unclear. To investigate the effects of H2 O2 , especially the abscopal effect when combined with radiotherapy (RT), we implanted murine tumor cells simultaneously in two locations in mouse models: the hind limb and back. H2 O2 mixed with sodium hyaluronate was injected intratumorally, followed by irradiation only at the hind limb lesion. No treatment was administered to the back lesion. The H2 O2 /RT combination significantly reduced tumor growth at the noninjected/nonirradiated site in the back lesion, whereas H2 O2 or RT individually did not reduce tumor growth. Flow cytometric analyses of the tumor-draining lymph nodes in the injected/irradiated areas showed that the number of dendritic cells increased significantly with maturation in the H2 O2 /RT combination group. In addition, analyses of tumor-infiltrating lymphocytes showed that the number of CD8+ (cluster of differentiation 8) T cells and the frequency of IFN-γ+ (interferon gamma) CD8+ T cells were higher in the noninjected/nonirradiated tumors in the H2 O2 /RT group compared to those in the other groups. PD-1 (programmed death receptor 1) blockade further increased the antitumor effect against noninjected/nonirradiated tumors in the H2 O2 /RT group. Intratumoral injection of H2 O2 combined with RT therefore induces an abscopal effect by activating antitumor immunity, which can be further enhanced by PD-1 blockade. These findings promote the development of H2 O2 /RT therapy combined with cancer immunotherapies, even for advanced cancers.

DOI： 10.1111/cas.15911

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Combination therapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and anti-programmed death-1 (PD-1) monoclonal antibodies (mAbs) has dramatically improved the prognosis of patients with multiple types of cancer, including renal cell carcinoma (RCC). However, more than half of RCC patients fail to respond to this therapy. Regulatory T cells (Treg cells) are a subset of highly immunosuppressive CD4+ T cells that promote the immune escape of tumors by suppressing effector T cells in the tumor microenvironment (TME) through various mechanisms. CTLA-4 is constitutively expressed in Treg cells and is regarded as a key molecule for Treg cell-mediated immunosuppressive functions, suppressing antigen-presenting cells by binding to CD80/CD86. Reducing Treg cells in the TME with an anti-CTLA-4 mAb with antibody-dependent cellular cytotoxicity (ADCC) activity is considered an essential mechanism to achieve tumor regression. In contrast, we demonstrated that only CTLA-4 blockade without ADCC activity enhanced CD28 costimulatory signaling pathways in Treg cells and promoted Treg-cell proliferation in mouse models. CTLA-4 blockade also augmented CTLA-4-independent immunosuppressive functions, including cytokine production, leading to insufficient antitumor effects. Similar results were also observed in human peripheral blood lymphocytes and tumor-infiltrating lymphocytes from patients with RCC. Our findings highlight the importance of Treg-cell depletion to achieve tumor regression in response to CTLA-4 blockade therapies.

DOI： 10.1111/cas.15756

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Hepatitis B virus (HBV) is a human hepatotropic pathogen causing hepatocellular carcinoma. We recently obtained HBV-susceptible immortalized human hepatocyte NKNT-3 by exogenously expressing NTCP and its derived cell clones, #28.3.8 and #28.3.25.13 exhibiting different levels of HBV susceptibility. In the present study, we showed that HBV infection activated the ATM-Chk2 signaling pathway in #28.3.25.13 cells but not in #28.3.8 cells. Both the cell culture supernatant and extracellular vesicles (EVs) derived from HBV-infected #28.3.25.13 cells also activated the ATM-Chk2 signaling pathway in naïve #28.3.25.13 cells. Interestingly, EVs derived from HBV-infected #28.3.25.13 cells included higher level of mitochondrial DNA (mtDNA) than those from HBV-infected #28.3.8 cells. Based on our results, we propose the novel model that EVs mediate the activation of ATM-Chk2 signaling pathway by the intercellular transfer of mtDNA in HBV-infected human hepatocyte.

DOI： 10.1096/fj.202002678r

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1096/fj.202002678R

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We previously found that N-89 and its derivative, N-251, which are being developed as antimalarial compounds, showed multiple antiviral activities including hepatitis C virus (HCV). In this study, we focused on the most characterized anti-HCV activity of N-89(N-251) to clarify their antiviral mechanisms. We first prepared cells exhibiting resistance to N-89(N-251) than the parental cells by serial treatment of HCV-RNA-replicating parental cells with N-89(N-251). Then, we newly generated HCV-RNA-replicating cells with the replacement of HCV-RNAs derived from N-89(N-251)-resistant cells and parental cells. Using these cells, we examined the degree of inhibition of HCV-RNA replication by N-89(N-251) and found that the host and viral factors contributed almost equally to the resistance to N-89(N-251). To further examine the contribution of the host factors, we selected several candidate genes by cDNA microarray analysis and found that the upregulated expression of at least RAC2 and CKMT1B genes independently and differently contributed to the acquisition of an N-89(N-251)-resistant phenotype. For the viral factors, we selected several mutation candidates by the genetic comparative analysis of HCV-RNAs and showed that at least one M414I mutation in the HCV NS5B contributed to the resistance to N-89. Moreover, we demonstrated that the combination of host factors (RAC2 and/or CKMT1B) and a viral factor (M414I mutation) additively increased the resistance to N-89. In summary, we identified the host and viral factors contributing to the acquisition of N-89(N-251)-resistance in HCV-RNA replication. These findings will be useful for clarification of the antiviral mechanism of N-89(N-251).

DOI： 10.1096/fba.2020-00107

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Glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) is a rate-limiting enzyme catalyzing triglyceride synthesis. Recently, we demonstrated that the anti-viral drug ribavirin (RBV) reduces GPAM expression by downregulating CCAAT/enhancer-binding protein α (C/EBPα). However, the precise mechanisms of GPAM suppression have remained unclear. Here, we found that RBV suppressed GPAM expression by downregulating not only C/EBPα, but also sterol regulatory element-binding protein-1c (SREBP-1c). We also found that cis-elements regulated by C/EBPα and SREBP-1c functioned as distal and proximal enhancers, respectively, to express hepatocyte- and adipocytes-specific GPAM variants. These results imply that RBV disrupts formation of the enhancer machineries on the GPAM genome by downregulating both transcription factors. Our findings may contribute to the development of treatments for fatty liver diseases caused by aberrant triglyceride synthesis.

DOI： 10.1016/j.bbrc.2020.08.112

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The most characteristic feature of the hepatitis C virus (HCV) genome in patients with chronic hepatitis C is its remarkable variability and diversity. To better understand this feature, we performed genetic analysis of HCV replicons recovered from two human hepatoma HuH-7-derived cell lines after 1, 3, 5, 7, and 9 years in culture: The cell lines 50-1 and sO harbored HCV 1B-1 and O strain-derived HCV replicons established in 2002 and 2003, respectively. The results revealed that genetic variations in both replicons accumulated in a time-dependent manner at a constant rate despite the maintenance of moderate diversity (less than 1.8% difference) between the clones and that the mutation rate in the 50-1 and sO replicons was 2.5 and 2.9 × 10-3 base substitutions/site/year, respectively. We found that the genetic distance of both replicons increased from 7.9% to 10.5% after 9 years in culture. In addition, we observed that the guanine + cytosine (GC) content of both replicon RNAs increased in a time-dependent manner, as observed in our previous studies. Finally, we demonstrated that the high sensitivity of both replicons to direct-acting antivirals was maintained even after 9 years in culture. Our results suggest that long-term cultured HCV replicon-harboring cells are a useful model for understanding the variability and diversity of the HCV genome and the drug sensitivity of HCV in patients with chronic hepatitis C.

DOI： 10.1007/s00705-019-04461-0

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Hepatitis B virus (HBV) infection, which increases the risk of cirrhosis and hepatocellular carcinoma and requires lifelong treatment, has become a major global health problem. However, host factors essential to the HBV life cycle are still unclear, and the development of new drugs is needed. Cells derived from the human hepatoma cell line HepG2 and engineered to overexpress sodium taurocholate cotransporting polypeptide (NTCP: a receptor for HBV), termed HepG2/NTCP cells, are widely used as the cell-based HBV infection and replication systems for HBV research. We recently found that human hepatoma cell line Li23-derived cells overexpressing NTCP (A8 cells subcloned from Li23 cells), whose gene expression profile was distinct from that of HepG2/NTCP cells, were also sensitive to HBV infection. However, the HBV susceptibility of A8 cells was around 1/100 that of HepG2/NTCP cells. Since we considered that plural cell assay systems will be needed for the objective evaluation of anti-HBV reagents, as we previously demonstrated in hepatitis C virus research, we here attempted to develop a new Li23 cell-derived assay system equivalent to that using HepG2/NTCP cells. By repeated subcloning of A8 cells, we successfully established a new cell line (A8.15.78.10) exhibiting high HBV susceptibility equal to that of HepG2/NTCP cells. Characterization of A8.15.78.10 cells revealed that the increase of HBV susceptibility was correlated with increases in the protein and glycosylation levels of NTCP, and with decreased expression of STING, a factor contributing to innate immunity. Finally, we performed a comparative evaluation of HBV entry inhibitors (cyclosporin A and rosiglitazone) by an HBV/secNL reporter assay using A8.15.78.10 cells or HepG2/NTCP cells. The results confirmed that cyclosporin A exhibited anti-HBV activity in both cell lines, as previously reported. However, we found that rosiglitazone did not show the anti-HBV activity in A8.15.78.10 cells, although it worked in HepG2/NTCP cells as previously reported. This suggested that the difference in anti-HBV activity between cyclosporin A and rosiglitazone was due to the different types of cells used for the assay. In conclusion, plural assay systems using different types of cells are required for the objective and impartial evaluation of anti-HBV reagents.

DOI： 10.1016/j.bbrc.2019.05.126

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Publishing type：Research paper (scientific journal)   Publisher：Portland Press Ltd.  

<title>Abstract</title>
Recently, we demonstrated that the anti-viral drug ribavirin (RBV) had the ability to suppress lipogenesis through down-regulation of retinoid X receptor α (RXRα) under the control of the intracellular GTP-level and AMP-activated protein kinase-related kinases, especially microtubule affinity regulating kinase 4 (MARK4). RXRα-overexpression attenuated but did not abolish lipogenesis suppression by RBV, implying that additional factor(s) were involved in this suppressive effect. In the present study, we found that the protein level, but not the mRNA level, of CCAAT/enhancer-binding protein α (C/EBPα) was down-regulated by RBV in hepatic cells. Treatment with proteasome inhibitor attenuated RBV-induced down-regulation of C/EBPα, suggesting that RBV promoted degradation of C/EBPα protein via the ubiquitin–proteasome pathway. Depletion of intracellular GTP through inosine monophosphate dehydrogenase inhibition by RBV led to down-regulation of C/EBPα. In contrast, down-regulation of C/EBPα by RBV was independent of RXRα and MARK4. Knockdown of C/EBPα reduced the intracellular neutral lipid levels and the expression of genes related to the triglyceride (TG) synthesis pathway, especially glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), which encodes the first rate-limiting TG enzyme. Overexpression of C/EBPα yielded the opposite results. We also observed that RBV decreased GPAM expression. Moreover, overexpression of GPAM attenuated RBV-induced reduction in the intracellular neutral lipid levels. These data suggest that down-regulation of C/EBPα by RBV leads to the reduction in GPAM expression, which contributes to the suppression of lipogenesis. Our findings about the mechanism of RBV action in lipogenesis suppression will provide new insights for therapy against the active lipogenesis involved in hepatic steatosis and hepatocellular carcinomas.

DOI： 10.1042/BCJ20180680

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)  

DOI： 10.1096/fba.1022

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Hepatitis B virus (HBV) causes hepatic diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. These diseases are closely associated with persistent HBV infection. To prevent the progression of hepatic diseases, it is thus important to suppress persistent HBV infection. Daunorubicin (DNR), a topoisomerase II (Top II) poison, is a clinically used anticancer agent with a wide spectrum of activity against malignancies. DNR was recently reported to cause DNA damage-dependent interferon (IFN)-β induction through exogenous cyclic GMP-AMP synthetase (cGAS) and subsequently to suppress Ebola virus replication. In the present study, we demonstrated that DNR caused the inhibition of cell proliferation, but not cell death, through the DNA damage response in immortalized human hepatocyte NKNT-3/NTCP cells. Interestingly, DNR triggered the endogenous cGAS-dependent innate immune response and subsequently suppressed viral production of HBV in NKNT-3/NTCP cells. Top II poisons may be anti-HBV drug candidates.

DOI： 10.1016/j.bbrc.2018.08.195

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The chemically synthesized endoperoxide compound N-89 and its derivative N-251 were shown to have potent antimalarial activity. We previously demonstrated that N-89 and N-251 potently inhibited the RNA replication of hepatitis C virus (HCV), which belongs to the Flaviviridae family. Since antimalarial and anti-HCV mechanisms have not been clarified, we were interested whether N-89 and N-251 possessed the activity against viruses other than HCV. In this study, we examined the effects of N-89 and N-251 on other flaviviruses (dengue virus and Japanese encephalitis virus) and hepatitis viruses (hepatitis B virus and hepatitis E virus). Our findings revealed that N-89 and N-251 moderately inhibited the RNA replication of Japanese encephalitis virus and hepatitis E virus, although we could not detect those anti-dengue virus activities. We also observed that N-89 and N-251 moderately inhibited the replication of hepatitis B virus at the step after viral translation. These results suggest the possibility that N-89 and N-251 act on some common host factor(s) that are necessary for viral replications, rather than the possibility that N-89 and N-251 directly act on the viral proteins except for HCV. We describe a new type of antiviral reagents, N-89 and N-251, which are applicable to multiple different viruses.

DOI： 10.1016/j.bbrep.2018.05.007

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley Blackwell  

Natural killer (NK) cells through their NK group 2 member D (NKG2D) receptors recognize NKG2D ligands such as UL16-binding proteins (ULBPs) on virus-infected cells and subsequently trigger the host innate immune response. In the present study, we demonstrated that hepatitis C virus (HCV) induced the cell surface expression of ULBP1 in human immortalized hepatocyte PH5CH8 cells and human hepatoma HuH-7 cell-derived RSc cells. Interestingly, NK cell line NK-92 induced cytotoxicity and interferon-γ mRNA expression and subsequently reduced the levels of HCV RNA replication during co-culture with HCV-infected RSc cells. From these results, we conclude that ULBP1 is a target of the NK cell-mediated innate immune response in HCV-infected human hepatocytes.

DOI： 10.1002/2211-5463.12373

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DOI： 10.1002/hep4.1065

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a major global health problem. Recently developed treatments with direct-acting antivirals (DAAs) have largely improved the sustained virologic response rate of patients with chronic hepatitis C. However, this approach is still hindered by its great expense and the problem of drug resistance. Using our cell-based HCV assay systems, we reported that the preclinical antimalarial drugs N-89 and N-251 exhibited potent anti-HCV activities. In this study we used our assay systems to evaluate the anti-HCV activities of six kinds of DAAs individually or in combination with N-89 or N-251. The results showed that the DAAs had potent anti-HCV activities and N-89 or N-251 contributed additive or synergistic effect. Using DAA-resistant HCV-RNA-replicating cells, which were prepared by continuous treatment with each DAA, we demonstrated that N-89 and N-251 could overcome all of the DAA-resistant HCVs. These preclinical drugs would have been potential as components of a therapeutic regimen that also included combinations of various DAAs. In addition, sequence analysis of the NS3-NS5B regions of the DAA-resistant HCV genomes newly found several amino acid (aa) substitutions that were suggested to contribute to DAA-resistance in addition to the aa substitutions already known to cause DAA-resistance. Among these new aa substitutions, we found that two substitutions in the NS3 region (D79G and S174Y) contributed to simeprevirand/or asunaprevir-resistance.

DOI： 10.1016/j.virusres.2017.03.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OKAYAMA UNIV MED SCHOOL  

Membrane transport probably participates in the lifecycle of hepatitis C virus (HCV). Rab proteins are essential host factors for HCV RNA replication, but these proteins' roles in other steps of the HCV lifecycle are not clear. The tight junction (TJ) plays a key role in HCV infection. Rab13 regulates the endocytic recycling of the TJ-associated proteins. Here we investigated whether Rab13 is involved in the HCV entry step. We used HuH-7-derived RSc cells and Li23-derived D7 cells. To evaluate the effect of Rab13 in HCV infection, we transfected the cells with siRNA targeting Rab13 before HCV infection. The down-regulation of Rab13 inhibited HCV infection. The D7 cells had showed a greater inhibitory effect against HCV infection compared to that in the RSc cells by Rab13 knockdown. Next, to evaluate the effect of Rab13 after infection, we inoculated the cells with HCV before transfection of the siRNA. The down-regulation of Rab13 did not show any effects after HCV infection. We further examined whether Rab13 would influence HCV RNA replication by using HCV replicon-harboring cells. The results revealed that Rab13 did not affect the step of HCV RNA replication. These results suggest that Rab13 plays an important role in the step of HCV entry.

DOI： 10.18926/AMO/54190

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OKAYAMA UNIV MED SCHOOL  

The mechanisms of hepatitis C virus (HCV)-associated hepatocarcinogenesis and disease progression are unclear. We previously observed that the expression level of carboxypeptidase B2 (CPB2) gene was remarkably suppressed by persistent HCV RNA replication in human hepatoma cell line Li23-derived cells. The results of the present study demonstrated that the CPB2 expression in patients with chronic hepatitis C was inversely correlated with several risk factors of hepatic fibrosis or steatosis, although ectopic CPB2 expression did not suppress the expression of fibrogenic or lipogenic genes. The suppressed CPB2 expression was restored by treatment with 5-azacytidine. To clarify the mechanism underlying this phenomenon, we analyzed the CPB2 promoter, and the results revealed that (1) hepatocyte nuclear factor 1 (HNF1), especially HNF1 alpha, was essential for the CPB2 promoter, and (2) CPB2 promoter was not methylated by persistent HCV RNA replication. The expression levels of HNF1 alpha and HNF1 beta were also not changed by persistent HCV RNA replication. These results suggest the existence of 5-azacytidine-inducible or -reducible unknown factor(s) that can control the CPB2 expression. To evaluate this idea we performed a microarray analysis, and several gene candidates corresponding to the suggested factor(s) were identified.

DOI： 10.18926/AMO/54186

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

During viral replication, the innate immune response is induced through the recognition of viral replication intermediates by host factor(s). One of these host factors, cyclic GMP-AMP synthetase (cGAS), was recently reported to be involved in the recognition of viral DNA derived from DNA viruses. However, it is uncertain whether cGAS is involved in the recognition of hepatitis B virus (HBV), which is a hepatotropic DNA virus. In the present study, we demonstrated that HBV genome-derived double-stranded DNA induced the innate immune response through cGAS and its adaptor protein, stimulator of interferon genes (STING), in human hepatoma Li23 cells expressing high levels of cGAS. In addition, we demonstrated that HBV infection induced ISG56 through the cGAS-STING signaling pathway. This signaling pathway also showed an antiviral response towards HBV through the suppression of viral assembly. From these results, we conclude that the cGAS-STING signaling pathway is required for not only the innate immune response against HBV but also the suppression of HBV assembly. The cGAS-STING signaling pathway may thus be a novel target for anti-HBV strategies.

DOI： 10.1111/febs.13563

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OKAYAMA UNIV MED SCHOOL  

Persistent infection with hepatitis C virus (HCV) often causes chronic hepatitis, and then shows a high rate of progression to liver cirrhosis and hepatocellular carcinoma. To clarify the mechanism of the persistent HCV infection is considered to be important for the discovery of new target(s) for the development of anti-HCV strategies. In the present study, we found that the expression level of annexin A1 (ANXA1) in human hepatoma cell line Li3-derived D7 cells was remarkably lower than that in parental Li23 cells, whereas the susceptibility of D7 cells to HCV infection was much higher than that of Li23 cells. Therefore, we hypothesized that ANXA1 negatively regulates persistent HCV infection through the inhibition of viral RNA replication. The results revealed that HCV production was significantly inhibited without a concomitant reduction in the amount of lipid droplets in the D7 cells stably expressing exogenous ANXAL Further, we demonstrated that ANXA1 negatively regulated the step of viral RNA replication rather than that of viral entry in human hepatocytes. These results suggest that ANXA1 would be a novel target for the development of anti-HCV strategies.

DOI： 10.18926/AMO/53335

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Hepatitis B virus (HBV) replication is controlled by liver-enriched transcriptional factors, including forkhead box protein A (FOXA) members. Here, we found that FOXA members are directly and indirectly involved in HBV replication in human hepatic cells. HBV replication was elevated in HuH-7 treated with individual FOXA members-specific siRNA. Reciprocally, the downregulation of HBV replication was observed in FOXA-induced HuH-7. However, the mechanism of downregulation is different among FOXA members at the level of HBV RNA transcription, such as precore/pg RNA and 2.1 kb RNA. In addition, FOXA1 and FOXA2 suppressed nuclear hormone receptors, such as HNF4 alpha, that are related to HBV replication. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2015.03.022

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Authorship：Lead author   Language：Japanese  

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Other Link： http://search.jamas.or.jp/link/ui/2015112459

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background
Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.
Methodology/Principal Findings
By repetitive RBV (100 mu M) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 mu M). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.
Conclusions/Significance
These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.

DOI： 10.1371/journal.pone.0118313

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

During persistent infection of HCV, the HCV core protein (HCV-JFH-1 strain of genotype 2a) is recruited to lipid droplets (LDs) for viral assembly, but the mechanism of recruitment of the HCV core protein is uncertain. Here, we demonstrated that one of the Ras-related small GTPases, Rab18, was required for trafficking of the core protein around LDs. The knockdown of Rab18 reduced intracellular and extracellular viral infectivity, but not intracellular viral replication in HCV-JFH-1-infected RSc cells (an HuH-7-derived cell line). Exogenous expression of Rab18 increased extracellular viral infectivity almost two-fold. Furthermore, Rab18 was co-localized with the core protein in HCV-JFH-1-infected RSc cells, and the knockdown of Rab18 blocked recruitment of the HCV-JFH-1 core protein to LDs. These results suggest that Rab18 has an important role in viral assembly through the trafficking of the core protein to LDs. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2014.05.017

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Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although the sustained virologic response rate in the treatment of genotype 1 using new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has been improved by more than 70%, several severe side effects such as skin rash/ageusia and advanced anemia have become a problem. Under these circumstances, a new type of anti-HCV oral drug with few side effects is needed. Our recently developed HCV drug assay systems, including the HuH-7 cell line-derived OR6 and AH1R, and the Li23 cell line derived ORL8 and ORL11, allow genome-length HCV RNAs (several strains of genotype 1b) encoding renilla luciferase to replicate efficiently. Using these systems as anti-HCV candidates, we have identified numerous existing medicines that can be used against HCV with few side effects, such as statins and teprenon. To obtain additional anti-HCV candidates, we evaluated a number of oral health supplements, and found that the capsule but not the liquid form of Cordyceps militaris (CM) (Ascomycotinanorth, North Chinese caterpillar fungus), which is used as a Chinese herbal medicine, exhibited moderate anti-HCV activity. In combination with interferon-a or ribavirin, CM exhibited an additive inhibitory effect. Among the main components of CM, cordycepin, but not ergosterol, contributed to the anti-HCV activity of CM. In consideration of all these results, we suggest that CM would be useful as an oral anti-HCV agent in combination with interferon-alpha and/or ribavirin. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2014.03.150

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Background: The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells.
Methodology/Principal Findings: Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5'-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a timedependent manner, and the mutation rates in the 5'-terminus-NS2 and NS3-NS5B regions were 4.0-9.0x10(-3) and 2.7-4.0x10(-3) base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents.
Conclusions/Significance: Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.

DOI： 10.1371/journal.pone.0091156

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DOI： 10.21769/BioProtoc.1061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.
Methodology/Principal Findings: Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-alpha demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-alpha-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-alpha and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.
Conclusions/Significance: We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.

DOI： 10.1371/journal.pone.0072519

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Background: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.
Methodology/Principal Findings: Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-alpha demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-alpha-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-alpha and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.
Conclusions/Significance: We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.

DOI： 10.1371/journal.pone.0072519

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Persistent infections with hepatitis C virus (HCV) may result in life-threatening liver disease, including cirrhosis and cancer, and impose an important burden on human health. Understanding how the virus is capable of achieving persistence in the majority of those infected is thus an important goal. Although HCV has evolved multiple mechanisms to disrupt and block cellular signaling pathways involved in the induction of interferon (IFN) responses, IFN-stimulated gene (ISG) expression is typically prominent in the HCV-infected liver. Here, we show that Toll-like receptor 3 (TLR3) expressed within uninfected hepatocytes is capable of sensing infection in adjacent cells, initiating a local antiviral response that partially restricts HCV replication. We demonstrate that this is dependent upon the expression of class A scavenger receptor type 1 (MSR1). MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. RNAi-mediated knockdown of MSR1 expression blocks TLR3 sensing of HCV in infected hepatocyte cultures, leading to increased cellular permissiveness to virus infection. Exogenous expression of Myc-MSR1 restores TLR3 signaling in MSR1-depleted cells with subsequent induction of an antiviral state. A series of conserved basic residues within the carboxy-terminus of the collagen superfamily domain of MSR1 are required for binding and transport of dsRNA, and likely facilitate acidification-dependent release of dsRNA at the site of TLR3 expression in the endosome. Our findings reveal MSR1 to be a critical component of a TLR3-mediated pattern recognition receptor response that exerts an antiviral state in both infected and uninfected hepatocytes, thereby limiting the impact of HCV proteins that disrupt IFN signaling in infected cells and restricting the spread of HCV within the liver. © 2013 Dansako et al.

DOI： 10.1371/journal.ppat.1003345

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2012.11.108

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

We developed a new cell culture drug assay system (AH1R), in which genome-length hepatitis C virus (HCV) RNA (AH1 strain of genotype 1b derived from a patient with acute hepatitis C) efficiently replicates. By comparing the AH1R system with the OR6 assay system that we developed previously (O strain of genotype 1b derived from an HCV-positive blood donor), we demonstrated that the anti-HCV profiles of reagents including interferon-gamma and cyclosporine A significantly differed between these assay systems. Furthermore, we found unexpectedly that rolipram, an anti-inflammatory drug, showed anti-HCV activity in the AH1R assay but not in the OR6 assay, suggesting that the anti-HCV activity of rolipram differs depending on the HCV strain. Taken together, these results suggest that the AH1R assay system is useful for the objective evaluation of anti-HCV reagents and for the discovery of different classes of anti-HCV reagents.

DOI： 10.1007/s11262-012-0712-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE LONDON  

Postmenopausal women with chronic hepatitis C exhibited a poor response to interferon (IFN) therapy compared to premenopausal women. Osteoporosis is the typical complication that occurs in postmenopausal women. Recently, it was reported that an osteoporotic reagent, vitamin D3, exhibited anti-hepatitis C virus (HCV) activity. Therefore, we investigated whether or not another osteoporotic reagent, raloxifene, would exhibit anti-HCV activity in cell culture systems. Here, we demonstrated that raloxifene inhibited HCV RNA replication in genotype 1b and infection in genotype 2a. Raloxifene enhanced the anti-HCV activity of IFN-alpha . These results suggest a link between the molecular biology of osteoporosis and the HCV life cycle. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.fob.2012.08.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Background
Previously we reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, statins, inhibited hepatitis C virus (HCV) RNA replication. Furthermore, recent reports revealed that the statins are associated with a reduced risk of hepatocellular carcinoma and lower portal pressure in patients with cirrhosis. The statins exhibited anti-HCV activity by inhibiting geranylgeranylation of host proteins essential for HCV RNA replication. Geranylgeranyl pyrophosphate (GGPP) is a substrate for geranylgeranyltransferase. Therefore, we examined the potential of geranyl compounds with chemical structures similar to those of GGPP to inhibit HCV RNA replication.
Methods
We tested geranyl compounds [geranylgeraniol, geranylgeranoic acid, vitamin K(2) and teprenone (Selbex)] for their effects on HCV RNA replication using genome-length HCV RNA-replicating cells (the OR6 assay system) and a JFH-1 infection cell culture system. Teprenone is the major component of the anti-ulcer agent, Selbex. We also examined the anti-HCV activities of the geranyl compounds in combination with interferon (IFN)-alpha or statins.
Results
Among the geranyl compounds tested, only teprenone exhibited anti-HCV activity at a clinically achievable concentration. However, other anti-ulcer agents tested had no inhibitory effect on HCV RNA replication. The combination of teprenone and IFN-alpha exhibited a strong inhibitory effect on HCV RNA replication. Although teprenone alone did not inhibit geranylgeranylation, surprisingly, statins&apos; inhibitory action against geranylgeranylation was enhanced by cotreatment with teprenone.
Conclusions
The anti-ulcer agent teprenone inhibited HCV RNA replication and enhanced statins&apos; inhibitory action against geranylgeranylation. This newly discovered function of teprenone may improve the treatment of HCV-associated liver diseases as an adjuvant to statins.

DOI： 10.1111/j.1478-3231.2011.02499.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Ribavirin (RBV) is a potential partner of interferon (IFN)-based therapy for patients with chronic hepatitis C. However, to date, its anti-hepatitis C virus (HCV) mechanism remains ambiguous due to the marginal activity of RBV on HCV RNA replication in HuH-7-derived cells, which are currently used as the only cell culture system for robust HCV replication. We investigated the anti-HCV activity of RBV using novel cell assay systems. The recently discovered human hepatoma cell line, Li23, which enables robust HCV replication, and the recently developed Li23-derived drug assay systems (ORL8 and ORL11), in which the genome-length HCV RNA (O strain of genotype 1b) encoding renilla luciferase efficiently replicates, were used for this study. At clinically achievable concentrations, RBV unexpectedly inhibited HCV RNA replication in ORL8 and ORL11 systems, but not in OR6 (an HuH-7-derived assay system). The anti-HCV activity of RBV was almost cancelled by an inhibitor of equilibrative nucleoside transporters. The evaluation of the anti-HCV mechanisms of RBV proposed to date using ORL8 ruled out the possibility that RBV induces error catastrophe, the IFN-signaling pathway or oxidative stress. However, we found that the anti-HCV activity of RBV was efficiently cancelled with guanosine, and demonstrated that HCV RNA replication was notably suppressed in inosine monophosphate dehydrogenase (IMPDH)-knockdown cells, suggesting that the antiviral activity of RBV is mediated through the inhibition of IMPDH. In conclusion, we demonstrated for the first time that inhibition of IMPDH is a major antiviral target by which RBV at clinically achievable concentrations inhibits HCV RNA replication. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2011.02.005

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Background: Recently, lipid droplets have been found to be involved in an important cytoplasmic organelle for hepatitis C virus (HCV) production. However, the mechanisms of HCV assembly, budding, and release remain poorly understood. Retroviruses and some other enveloped viruses require an endosomal sorting complex required for transport (ESCRT) components and their associated proteins for their budding process.
Methodology/Principal Findings: To determine whether or not the ESCRT system is needed for HCV production, we examined the infectivity of HCV or the Core levels in culture supernatants as well as HCV RNA levels in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, expressing short hairpin RNA or siRNA targeted to tumor susceptibility gene 101 (TSG101), apoptosis-linked gene 2 interacting protein X (Alix), Vps4B, charged multivesicular body protein 4b (CHMP4b), or Brox, all of which are components of the ESCRT system. We found that the infectivity of HCV in the supernatants was significantly suppressed in these knockdown cells. Consequently, the release of the HCV Core into the culture supernatants was significantly suppressed in these knockdown cells after HCV-JFH1 infection, while the intracellular infectivity and the RNA replication of HCV-JFH1 were not significantly affected. Furthermore, the HCV Core mostly colocalized with CHMP4b, a component of ESCRT-III. In this context, HCV Core could bind to CHMP4b. Nevertheless, we failed to find the conserved viral late domain motif, which is required for interaction with the ESCRT component, in the HCV-JFH1 Core, suggesting that HCV Core has a novel motif required for HCV production.
Conclusions/Significance: These results suggest that the ESCRT system is required for infectious HCV production.

DOI： 10.1371/journal.pone.0014517

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Background: Studies on patients with hepatitis C virus (HCV) of genotype 1b have suggested that amino acids (aa) 70 and/or 91 of the HCV core protein affect the outcome of interferon (IFN)-alpha and ribavirin (RBV) therapy, although there are no clear supporting data in vitro. Aims: This study was designed to determine the differences among the antiviral activities of HCV core proteins with various substitutions at aa70 and/or aa91. Methods: The retroviral vectors expressing the HCV core proteins with substitutions of arginine/leucine, arginine/methionine, glutamine/leucine or glutamine/methionine at aa70/aa91 were transiently transfected or stably transducted into an immortalized hepatocyte line (PH5CH8), hepatoma cell lines and an HCV-RNA replicating cell line (sOR) to evaluate antiviral responses to IFN-alpha or IFN-alpha/RBV. Sequence analysis was performed using genome-length HCV-RNA replicating cells (OR6 and AH1) to evaluate HCV core mutations during IFN-alpha treatment. Results: The promoter activity levels of IFN-stimulated genes in the transiently transfected cells or the mRNA levels of 20-50-oligoadenylate synthetase in the stably transducted PH5CH8 cells were not associated with the HCV core aa70 and/or aa91 substitutions during IFN-alpha treatment. Antiviral responses to IFN-alpha or IFN-alpha/RBV treatment were enhanced in sOR cells stably transducted with the HCV core, although there were no differences in antiviral responses among the cells expressing different core types. Sequence analysis showed no aa mutations after IFN-alpha treatment. Conclusions: Antiviral activities were enhanced by HCV core transduction, but they were not associated with the HCV core aa70 and/or aa91 substitutions by in vitro analysis.

DOI： 10.1111/j.1478-3231.2010.02299.x

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：Japanese   Publisher：岡山医学会  

HCV感染の機序として、DNAゲノムを保持するウイルスが感染細胞内のDNA損傷応答経路を活性化し、ウイルスの自己複製に利用していることが知られている。また、RNAゲノムしか保持しないHCVであっても、その感染により宿主細胞に二重鎖DNA切断を誘導し、宿主ゲノムを変異させていることが報告されている。今回、RNAゲノムしか保持していないHCVの複製にもDNA損傷応答経路が関与しているか否かを検討した結果、関与していることが示唆された。すなわち、DNA損傷センサーであるATMとChk2はHCV RNA複製に必要な宿主因子であることが明らかになった。

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2010&ichushi_jid=J00175&link_issn=&doc_id=20100420290002&doc_link_id=10.4044%2Fjoma.122.9&url=https%3A%2F%2Fdoi.org%2F10.4044%2Fjoma.122.9&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a serious global health problem. Cell culture-based persistent HCV RNA replication systems and infectious HCV production systems are widely used in HCV research. However, persistent HCV production systems have been developed only for HuH-7 hepatoma cells. Here we found a new human hepatoma cell line, Li23, that enables persistent HCV production and anti-HCV reagent assay. Li23&apos;s cDNA expression profile differed from HuH-7&apos;s, although the two cells had similar liver-specific expression profiles. We used HCV RNA with a specific combination of adaptive mutations to develop an HCV replicon system and genome-length HCV RNA replicating systems including a reporter assay system. Finally, Li23-derived cells persistently produced infectious virus of an HCV strain. Li23-derived cells are potentially useful for understanding the HCV life cycle and for finding antiviral targets. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2009.08.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Cyclosporine A (CsA) is a well-characterized anti-HCV reagent. Recently it was reported that the genotype 2a JFH-1 strain was more resistant than genotype 1 HCV strains to CsA in a cell culture system. However, the JFH-1 responsible region for the resistance to CsA remains unclear. It was also demonstrated that in genotype 1b HCVs, NS5B interacts with cyclophilin (CyP). To clarify whether or not NS5B of JFH-1 is significant for CsA resistance, we developed a chimeric replicon with NS5B of JFH-1 in the genotype 1b backbone. The chimeric replicon was more resistant to CsA than the parental genotype 1b replicon. Furthermore, reduction of CyPA had a greater effect on HCV RNA replication and sensitivity to CsA than reduction of CyPB. Here, we demonstrated that NS5B of JFH-1 contributed to this strain&apos;s CsA-resistant phenotype. NS5B and CyPA are significant for determining HCV&apos;s sensitivity to CsA.

DOI： 10.1007/s00705-009-0502-x

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Aim:
The cure rate of current interferon (IFN) therapy is limited to approximately 50% and most of the relapses after therapy are caused by genotype-1. To develop a relapse model in cell culture, we attempted to obtain genome-length hepatitis C virus ribonucleic acid (HCV RNA) harboring cells possessing the IFN-alpha-resistance phenotype from previously established OR6 cells, which enabled the luciferase reporter assay for monitoring of HCV RNA replication.
Methods:
The IFN-alpha-resistant HCV RNA-harboring cells and control cells were obtained by the treatment of OR6 cells with and without IFN-alpha, respectively. Then, we examined the relapse of HCV in IFN-alpha-resistant HCV RNA-harboring cells.
Results:
Only type I IFN (alpha and beta) showed significantly different anti-HCV activity between IFN-alpha-resistant HCV RNA-harboring cells and control cells. There was no significant difference in the anti-HCV activity of IFN-gamma, fluvastatin, or cyclosporine A between the two types of cells. Furthermore, we showed that fluvastatin or cyclosporine A in combination with IFN-alpha could prevent the relapse after therapy in the IFN-alpha-resistant HCV RNA-harboring cells.
Conclusion:
We developed a HCV relapse model in cell culture using IFN-alpha-resistant HCV RNA-harboring cells. Thus anti-HCV reagents, which have a mechanism different from IFN-alpha, were shown to be useful for preventing a relapse of IFN-alpha-resistant HCV.

DOI： 10.1111/j.1872-034X.2009.00525.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Recently, we reported that beta-carotene, vitamin D(2), and linoleic acid inhibited hepatitis C virus (HCV) RNA replication in hepatoma. cells. Interestingly, in the course of the study, we found that the antioxidant vitamin E negated the anti-HCV activities of these nutrients. These results suggest that the oxidative stress caused by the three nutrients is involved m their anti-HCV activities. However, the molecular mechanism by which oxidative stress induces anti-HCV status remains unknown. Oxidative stress is also known to activate extracellular signal-regulated kinase (ERK). Therefore, we hypothesized that oxidative stress induces anti-HCV status via the mitogen activated protein kinase (MAPK)/ERK kinase (MEK)-ERK1/2 signaling pathway. In this study, we found that the MEK1/2-spedfic inhibitor U0126 abolished the anti-HCV activities of the three nutrients in a dose-dependent manner. Moreover, U0126 significantly attenuated the anti-HCV activities of polyunsaturated fatty acids, interferon-gamma, and cyclosporine A, but not statins. We further demonstrated that, with the exception of the statins, all of these anti-HCV nutrients and reagents actually induced activation of the MEK-ERK1/2 signaling pathway, which was inhibited or reduced by treatment not only with U0126 but also with vitamin E We also demonstrated that phosphorylation of ERK1/2 by cyclosporine A was attenuated with N-acetylcysteine treatment and led to the negation of inhibition of HCV RNA replication. We propose that a cellular process that follows ERK1/2 phosphorylation and is specific to oxidative stimulation might lead to down-regulation of HCV RNA replication. Conclusion: Our results demonstrate the involvement of the MEK-ERK1/2 signaling pathway in the anti-HCV status induced by oxidative stress in a broad range of anti-HCV reagents. This intracellular modulation is expected to be a therapeutic target for the suppression of HCV RNA replication. (HEPATOLOGY 2009;50: 678-688.)

DOI： 10.1002/hep.23026

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

We previously demonstrated that hepatitis C virus (HCV) serine protease NS3-4A was unable to cleave TRIF (adaptor protein of Toll-like receptor 3), resulting in a lack of suppression of the TRIF-mediated pathway, whereas NS3-4A cleaved Cardif (adaptor protein of retinoic acid-inducible gene I or melanoma differentiation-associated gene-5), resulting in an interruption of the Cardif-mediated pathway in non-neoplastic human hepatocyte PH5CH8 cells. To elucidate these observations, we examined the cleavage potential of NS3-4A for TRIF in PH5CH8 cells, genome-length HCV RNA-replicating O cells, and HCV-infected cells, and we demonstrated that NS3-4A lacked the ability to cleave endogenous TRIF, regardless of HCV strains derived from patients with different stages of hepatic disease. Furthermore, we demonstrated that inflammatory cytokine production by NF-kappa B activation via the TRIF-mediated pathway also remained unsuppressed by NS3-4A. These results suggest that the inhibitory effects of NS3-4A on antiviral signaling pathways are limited to the Cardif-mediated pathway in human hepatocytes.

DOI： 10.1007/s00705-009-0375-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Oncostatin M (OSM), a member of the interleukin-6 family, possesses various functions, including hepatocyte differentiation and suppression of melanoma cell growth. Here, we report anti-hepatitis C virus (HCV) activity of OSM as a new function of this cytokine. OSM possessed marked anti-HCV activity (50% effective concentration: 0.71 ng/ml) in an HCV RNA replication cell culture system. The most striking finding is that OSM exhibited synergistic inhibitory activity on interferon (IFN)-alpha even at a low concentration with weak anti-HCV activity, such as 25 pg/ml. OSM is a candidate anti-HCV reagent and may improve the current IFN therapy for patients with chronic hepatitis C. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2009.03.054

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Half of the population of genotype 1 HCV is resistant to current pegylated-interferon-alpha (PEG-IFN-alpha) and ribavirin therapy. The resistance to IFN therapy is an urgent problem, especially in patients with genotype I HCV infection. However, sensitivities among HCV strains to anti-HCV reagents including IFNs have not been thoroughly addressed. Here, we established three different subgenomic replicons (1B-4, 1B-5, and KAH5 strains) in addition to our previously established replicon (0 strain). We comparatively examined the sensitivities of four replicons to IFN-alpha, IFN-gamma, IFN-lambda, cyclosporine A, and fluvastatin. Among the replicons, the 1B-4 and KAH5 replicons were the most sensitive and resistant, respectively to IFN-lambda (EC(50): 1.50 ng/ml vs. 8.50 ng/ml) and fluvastatin (EC(50): 2.82 mu M vs. 7.87 mu M), although these replicons possessed similar features in terms of genetic distance from the 0 strain, HCV RNA expression levels, and sensitivity to IFN-alpha (EC(50): 1.44 IU/ml vs. 1.37 IU/ml) and cyclosporine A (EC(50): 0.71 mu g/ml vs. 0.96 mu g/ml). These replicons are thus useful tools for examining the mechanism of anti-HCV activity, especially in IFN-lambda and statins. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.antiviral.2009.01.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Arsenic trioxide (ATO), a therapeutic reagent used for the treatment of acute promyelocytic leukemia, has recently been reported to increase human immunodeficiency virus type 1 infectivity. However, in this study, we have demonstrated that replication of genome-length hepatitis C virus (HCV) RNA (O strain of genotype 1b) was notably inhibited by ATO at submicromolar concentrations without cell toxicity. RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were also inhibited by ATO after the HCV infection. To clarify the mechanism of the anti-HCV activity of ATO, we examined whether or not PML is associated with this anti-HCV activity, since PML is known to be a target of ATO. Interestingly, we observed the cytoplasmic translocation of PML after treatment with ATO. However, ATO still inhibited the HCV RNA replication even in the PML knockdown cells, suggesting that PML is dispensable for the anti-HCV activity of ATO. In contrast, we found that N-acetyl-cysteine, an antioxidant and glutathione precursor, completely and partially eliminated the anti-HCV activity of ATO after 24 h and 72 h of treatment, respectively. In this context, it is worth noting that we found an elevation of intracellular superoxide anion radical, but not hydrogen peroxide, and the depletion of intracellular glutathione in the ATO-treated cells. Taken together, these findings suggest that ATO inhibits the HCV RNA replication through modulation of the glutathione redox system and oxidative stress.

DOI： 10.1128/JVI.01840-08

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Hepatitis C virus (HCV) is known to circulate persistently in vivo as a complex population of different but closely related viral variants. To understand the quasispecies nature of HCV, we performed genetic analysis of intracellular HCV RNAs obtained in long-term cell culture of genome-length HCV-RNA-replicating cells. The results revealed that genetic mutations in HCV RNAs accumulated in a time-dependent manner, and that the mutation rates of HCV RNAs were 3.5-4.8 x 10(-3) base substitutions/site/year. The mutation rates of nonstructural regions that are essential for RNA replication were lower than those of structural regions. The genetic diversity of HCVs was also enlarged in a time-dependent manner. Furthermore, we found that the GC content of HCV RNA was increased in a time-dependent manner. These results suggest that an HCV-RNA-replicating cell culture system would be useful for analysis of the evolutionary dynamics and variations of HCV.

DOI： 10.1007/s00705-008-0282-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Cellular responses to DNA damage are crucial for maintaining genome integrity, virus infection, and preventing the development of cancer. Hepatitis C virus (HCV) infection and the expression of the HCV nonstructural protein NS3 and core protein have been proposed as factors involved in the induction of double-stranded DNA breaks and enhancement of the mutation frequency of cellular genes. Since DNA damage sensors, such as the ataxia-telangiectasia mutated kinase (ATM), ATM-and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2), play central roles in the response to genotoxic stress, we hypothesized that these sensors might affect HCV replication. To test this hypothesis, we examined the level of HCV RNA in HuH-7-derived cells stably expressing short hairpin RNA targeted to ATM, ATR, PARP-1, or Chk2. Consequently, we found that replication of both genome-length HCV RNA (HCV-O, genotype 1b) and the subgenomic replicon RNA were notably suppressed in ATM-or Chk2-knockdown cells. In addition, the RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were suppressed in these knockdown cells after inoculation of the cell culture-generated HCV. Consistent with these observations, ATM kinase inhibitor could suppress the HCV RNA replication. Furthermore, we observed that HCV NS3-NS4A interacted with ATM and that HCV NS5B interacted with both ATM and Chk2. Taken together, these results suggest that the ATM signaling pathway is critical for HCV RNA replication and may represent a novel target for the clinical treatment of patients with chronic hepatitis C.

DOI： 10.1128/JVI.00351-08

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We previously developed a cell-based luciferase reporter assay system for monitoring genome-length hepatitis C virus (HCV) RNA replication (OR6 assay system). Here. we aimed to develop a new living cell-based reporter assay system using enhanced green fluorescent protein (EGFP). Genome-length HCV RNAs encoding EGFP were introduced into a subline of HuH-7 cells and G418 selection was performed. One cloned cell line, OGF7, was successfully selected from among the several G418-resistant cell lines obtained, and the robust expression of HCV RNA and proteins in OGF7 cells was confirmed. The fluorescent intensity of OGF7 cells was decreased by interferon-alpha treatment in a dose-dependent manner, and it correlated well with the HCV RNA concentration. We demonstrated that the interferon-alpha sensitivity in the OGF7 assay system measuring the fluorescent intensity was equivalent to that of the OR6 assay system, and that the OGF7 assay system Was useful for quantitative evaluation of anti-HCV reagents. The OGF7 assay system is expected to be the most time-saving and inexpensive assay system for high-throughput screening of anti-HCV reagents. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2008.06.001

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publisher：日本癌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We report for the first time a new RNA replication system with a hepatitis C virus (HCV) strain (AHI) derived from a patient with acute hepatitis C. Using an HCV replicon RNA library constructed with the AH1 strain (genotype 1b), we first established a cloned cell line, sAH1, harboring the HCV replicon. Cured cells obtained with interferon treatment of sAH1 cells were used for transfection with genome-length HCV RNA possessing four mutations found in sAH1 replicon. Consequently, one cloned cell line, AH1, supporting efficient replication of genome-length HCV RNA was obtained. By the comparison of AH1 cells with the O cells supporting genome-length HCV RNA (HCV-O strain) replication, we found different anti-HCV profiles of interferon-gamma and cyclosporine A between AH1 and O cells. Reporter assay analysis suggests that the diverse effects of interferon-gamma are due to the difference in HCV strains, but not the cellular environment. (c) 2008 Elsevier Inc. All rights reserved

DOI： 10.1016/j.bbrc.2008.04.005

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.

DOI： 10.1128/JVI.01517-07

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Toll-like receptors and RNA helicase family members [retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene-5 (MDA5)] play important roles in the induction of interferon-P as a major event in innate immune responses after virus infection. TRIF (adaptor protein of Toll-like receptor 3)-mediated and Cardif (adaptor protein of RIG-I or MDA5)-mediated signaling pathways contribute rapid induction of interferon-P through the activation of interferon regulatory factor-3 (IRF-3). Previously, it has been reported that the hepatitis C virus NS3-4A serine protease blocks virus-induced activation of IRF-3 in the human hepatoma cell line HuH-7, and that NS3-4A cleaves TRIF and Cardif molecules, resulting in the interruption of antiviral signaling pathways. On the other hand, it has recently been reported that non-neoplastic human hepatocyte PH5CH8 cells retain robust TRIF- and Cardif-mediated pathways, unlike HuH-7 cells, which lack a TRIF-mediated pathway. In the present study, we further investigated the effect of NS3-4A on antiviral signaling pathways. Although we confirmed that PH5CH8 cells were much more effective than HuH-7 cells for the induction of interferon-P, we obtained the unexpected result that NS3-4A could not suppress the interferon-P production induced by the TRIF-mediated pathway, although it suppressed the Cardif-mediated pathway by cleaving Cardif at the Cys508 residue. Using PH5CH8, HeLa, and HuH-7-derived cells, we further showed that NS3-4A could not cleave TRIF, in disagreement with a previous report describing the cleavage of TRI F by NS3-4A. Taken together, our findings suggest that the blocking of the interferon production by NS3-4A is not sufficient in HCV-Infected hepatocyte cells.

DOI： 10.1111/j.1742-4658.2007.05942.x

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Language：English   Publisher：日本癌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

To date, only a limited number of studies have reported finding an influence of ordinary nutrients on hepatitis C virus (HCV) RNA replication. However, the effects of other nutrients on HCV RNA replication remain largely unknown. We recently developed a reporter assay system for genome-length HCV RNA replication in hepatoma-derived HuH-7 cells (OR6). Here, using this OR6 assay system, we comprehensively examined 46 nutrients from four nutrient groups: vitamins, amino acids, fatty acids, and salts. We found that three nutrients-beta-carotene, vitamin D-2, and linoleic acid-inhibited HCV RNA replication and that their combination caused additive and/or synergistic effects on HCV RNA replication. In addition, combined treatment with each of the three nutrients and interferon alpha or beta or fluvastatin inhibited HCV RNA replication in an additive manner, while combined treatment with cyclosporine synergistically inhibited HCV RNA replication. In contrast, we found that vitamin E enhanced HCV RNA replication and negated the effects of the three anti-HCV nutrients and cyclosporine but not those of interferon or fluvastatin. These results will provide useful information for the treatment of chronic hepatitis C patients who also take anti-HCV nutrients as an adjunctive therapy in combination with interferon. In conclusion, among the ordinary nutrients tested, beta-carotene, vitamin D-2, and linoleic acid possessed anti-HCV activity in a cell culture system, and these nutrients are therefore considered to be potential candidates for enhancing the effects of interferon therapy.

DOI： 10.1128/AAC.01426-06

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

HuH-7 is a highly differentiated hepatoma cell line and the only cell line that supports robust RNA replication of the hepatitis C virus (HCV). HuH-7 cells cause cell death in serum-free culture condition. However, the effect is reversed by supplementation with selenium. Serum-free cell cultures are advantageous for vaccine development and experimental reproducibility. However, HCV RNA replication in HuH-7 cells in serum-free medium had not yet been achieved. Therefore, we tried to develop a system for robust HCV RNA replication in a serum-free cell culture. Although HuH-7 cells grew in serum-free medium in the presence of selenium, HuH-7 cells under these conditions did not support HCV RNA replication in long-term culture. Among the supplements tested, serum-free medium with lipid-rich albumin (LRA) was found to yield robust HCV RNA replication. HCV proteins were detected for more than 9 months in serum-free medium supplemented with LRA. This is the first report to demonstrate a long-term, serum-free cell culture that successfully maintained robust HCV RNA replication. This cell culture system is expected to be a useful tool for vaccine development, as well as for further investigation of cellular factors that are essential for HCV RNA replication. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2006.12.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We recently established a genome-length HCV RNA-replicating cell line (O strain of genotype 1b; here called O cells) using cured cells derived from sO cells, in which HCV subgenomic replicon RNA with an adaptive NS5A mutation (S2200R) is replicated. Characterization of the O cells revealed a second adaptive NS3 mutation (K1609E) required for genome-length HCV RNA replication. To clarify the role of adaptive mutation in genome-length HCV RNA replication, we newly established one and three kinds of genome-length HCV RNA-replicating cell lines possessing the cell background of so and 0 cells, respectively, and found additional adaptive NS3 mutations (Q11 12R, P1115L, and E1202G) required for the robust replication of genome-length HCV RNA. We further found that specific combinations of adaptive NS3 mutations drastically enhanced HCV RNA replication, regardless of the cell lines examined. These findings suggest that specific viral factors may affect the replication level of genome-length HCV RNA. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2006.12.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

Previously, we found that bovine and human lactoferrin (LF) specifically inhibited hepatitis C virus (HCV) infection in cultured non-neoplastic human hepatocyte-derived PH5CH8 cells, and we identified 33 amino acid residues (termed C-s3-33; amino acid 600-632) from human LF that were primarily responsible for the binding activity to the HCV E2 envelope protein and for the inhibiting activity against HCV infection. Since the anti-HCV activity of C-s3-33 was weaker than that of human LF, we speculated that an increase of E2 protein-binding activity might contribute to the enhancement of anti-HCV activity. To test this possibility, we made two repeats [(C-0-33)(2)] and three repeats [(C-s3-33)(3)] of C-s3-33 and characterized them. Far-Western blot analysis revealed that the E2 protein-binding activities of (C-s3-33), and (C-ws3-33)(3) became stronger than that of the C-0-33, and that the binding activity of (C-s3-33)(3) was stronger than that of (C-s3-33)(3). Using an HCV infection system in PH5CH8 cells, we demonstrated that the anti-HCV activities of (C-s3-33)2 and (C-s3-33)(3) became stronger than that of the C-0-33. Furthermore, using a recently developed infection system with a VSV pseudotype harboring the green fluorescent protein gene and the native E1 and E2 genes, we demonstrated that the antiviral activities of (C-s3-33)(2) and (C-s3-33)(3) were stronger than that of C-s3-33. These results suggest that tandem repeats of LF-derived anti-HCV peptide are useful as anti-HCV reagents.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

We recently developed a genome-length hepatitis C virus (HCV) RNA replication system (0116) with luciferase as a reporter. The OR6 assay system has enabled prompt and precise quantification of HCV RNA replication. Pegylated interferon (IFN) and ribavirin combination therapy is the world standard for chronic hepatitis Q but its effectiveness is limited to about 55% of patients. Newer therapeutic approaches are needed. In the present study, we used the OR6 assay system to evaluate die anti-HCV activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, called statins, and their effects in combination with IFN-alpha. Five types of statins (atorvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin) were examined for their anti-HCV activities. Fluvastatin exhibited the strongest anti-HCV activity (IC50: 0.9 mu mol/ L), whereas atorvastatin and simvastatin showed moderate inhibitory effects. However, lovastatin, reported recently as an inhibitor of HCV replication, was shown to exhibit the weakest anti-HCV activity. The anti-HCV activities of statins were reversed by the addition of mevalonate or geranylgeraniol. Surprisingly, however, pravastatin exhibited no anti-HCV activity, although it worked as an inhibitor for HMG-CoA reductase. The combination of IFN and the statins (except for pravastatin) exhibited strong inhibitory effects on HCV RNA replication. In combination with IFN, fluvastatin also exhibited a synergistic inhibitory effect. In conclusion, statins, especially fluvastatin, could be potentially useful as new anti-HCV reagents in combination with IFN.

DOI： 10.1002/hep.21232

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Background/Aims: We previously established hepatitis C virus (HCV) replicon-harboring cell lines possessing two interferon (IFN)-resistant phenotypes: a partially resistant phenotype (alpha R series) and a severely resistant phenotype (OR series). We recently found that the severe IFN resistance of the OR-series cells is caused by the functional disruption of type I IFN receptors. Here, we aimed to clarify the mechanism(s) underlying the partial IFN resistance of the alpha R-series cells.
Methods: alpha R-series cells were pre-treated with 5-azacytidine to evaluate the effects of DNA demethylation on IFN resistance. cDNA microarray analysis was carried out in order to compare 1 alpha R cells, which belong to the alpha R series, treated with both 5-azacytidine and IFN-alpha with cells treated with 5-azacytidine or IFN-alpha alone.
Results: We found that the IFN-resistant phenotype of alpha R-series cells was impaired by treatment with 5-azacytidine. cDNA microarray analysis identified seven IFN-stimulated genes, which were up-regulated by 5-azacytidine treatment. We demonstrated here that the ectopic expression of each of these seven genes in 1 alpha R cells frequently weakened the IFN resistance of these cells.
Conclusions: The present results suggest that the epigenetic silencing of IFN-stimulated genes is implicated in the acquisition of a partially IFN-resistant phenotype of HCV replicon-harboring cells. (c) 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jhep.2006.01.030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

To clarify the pathogenesis of hepatitis C virus (HCV), we have studied the effects of HCV proteins using human hepatocytes. Here, we found that HCV NS5B, an RNA-dependent RNA polymerase, delayed cell cycle progression through the S phase in PH5CH8 immortalized human hepatocyte cells. Since treatment with anti-interferon (IFN)-beta neutralizing antibody restored the cell cycle delay, IFN-beta was deemed responsible for the cell cycle delay in NS5B-expressing PH5CH8 cells. The induction of IFN-beta and the cell cycle delay were overridden by the down-regulation of Toll-like receptor 3 (TLR3) through RNA interference in NS5B-expressing PH5CH8 cells. Moreover, the NS5B full form was required for the cell cycle delay, the induction of IFN-beta, and the activation of the IFN-beta signaling pathway. Our findings revealed that NS5B induced IFN-beta through the TLR3 signaling pathway in immortalized human hepatocytes even without replicating viral genomes. (C) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2005.10.023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Hepatitis C virus (HCV) replicon-harbouring cell lines possessing interferon (IFN)-resistant phenotypes have recently been established. These were divided into two classes: partially IFN resistant and highly IFN resistant. Here, the viral and cellular factors contributing to the IFN resistance of HCV replicon-harbouring cells were evaluated. The results revealed that cellular factors rather than viral factors contributed to a highly IFN-resistant phenotype. The possibility of genetic abnormality of the factors involved in IFN signalling was investigated. As a result, nonsense mutations and deletions in type I IFN receptor genes (IFNAR1 and IFNAR2c) were found in replicon-harbouring cells showing a highly IFN-resistant phenotype, but rarely appeared in cells showing a partially IFN-resistant phenotype. Furthermore, similar genetic alterations were also found in IFN-resistant phenotype, replicon-harbouring cell lines obtained additionally by IFN-beta treatment. Moreover, it was shown that ectopic expression of wild-type IFNAR1 in IFN-resistant phenotype, replicon-harbouring cells possessing the IFNAR1 mutant restored type I IFN signalling.

DOI： 10.1099/vir.0.81124-0

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We previously found that hepatitis C virus (HCV) core protein (Core) activated the interferon (IFN)-inducible 40/46 kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene through an IFN-stimulated response element (ISRE) in non-neoplastic human hepatocyte PH5CH8 cells. Here, we found that Core and NS5B synergistically enhanced the 2'-5'-OAS gene promoter activity through ISRE. Further analysis revealed that amino acid positions 12 and/or 13 of Core and RNA-dependent RNA polymerase activity of NS5B were essential for the activation of the 2'-5'-OAS gene promoter. Interestingly, we observed that the activation by Core of NS5B was still partially enhanced by even the NS5B or Core mutant lacking the activating ability, respectively, suggesting an indirect interaction between Core and NS5B. Furthermore, we showed that the activation by NS5B could be explained by NS5B's induction of IFN-P, however, IFN-beta was not induced by Core. Moreover, we showed that the synergistic effect of Core and NS5B was not invalidated by NS3-4A, although NS3-4A significantly inhibited the activation by combination of Core and NS5B. Taken together, our findings reveal that NS5B/Core and NS3-4A exhibit conflicting effects (activation and inhibition) on the IFN system in PH5CH8 cells, and suggest that such effects may promote the distraction of the host defense system to lead to persistent infection. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.08.112

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Interferon (IFN)-alpha monotherapy, as well as the more effective combination therapy of IFN-alpha and ribavirin, are currently used for patients with chronic hepatitis C caused by hepatitis C virus (HCV) infection, although the mechanisms of the antiviral effects of these reagents on HCV remain ambiguous, and side effects such as anemia due to the administration of ribavirin present a problem for patients who are advanced in years. Using a recently developed reporter assay system in which genome-length dicistronic HCV RNA encoding Renilla luciferase gene was found to replicate efficiently, we found that mizoribine, an imidazole nucleoside, inhibited HCV RNA replication. The anti-HCV activity of mizoribine (IC50: approximately 100 mu M) was similar to that of ribavirin. Using this genome-length HCV RNA replication monitor system, we were the first to demonstrate that the combination of IFN-alpha and ribavirin exhibited more effective anti-HCV activity than the use of IFN-alpha alone, Moreover, we found that the anti-HCV activity of mizoribine in co-treatment with IFN-alpha was at least equivalent to that of ribavirin. This effect was apparent in the presence of at least 5 mu M mizoribine. Since mizoribine is currently used in several clinical applications and has not been associated with severe side effects, mizoribine is considered to be of potential use as a new anti-HCV reagent in combination with IFN-alpha. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.03.062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Recently we reported a subgenomic hepatitis C virus (HCV) replicon derived from HCV (HCV-O strain) infected in non-neoplastic human hepatocyte PH5CH8. In this study, we developed a genome-length dicistronic HCV RNA from HCV-O. A cured HuH-7 cell line (sOc) was obtained from a cloned subgenomic replicon cell line (sO) by interferon (IFN) treatment and used for transfection with genome-length HCV RNA. One cloned cell line, O, was successfully selected by G418 treatment following the introduction of genome-length HCV RNA into sOc cells, and the robust expression of HCV RNA and proteins was confirmed. Oc, a cured cell line, was also obtained from the cloned cell line (O) by IFN treatment. The number of colonies increased drastically when genome-length HCV RNA was introduced into Oc cells. However, the cloned cured cell lines, sOc and Oc, differed in their colony formation efficiency despite their common origin. This result suggests that even a cloned cell line can change its characteristics during cell culture. Sequence analysis of HCV RNA from the O cells revealed an amino acid substitution in the NS3 helicase region (K1609E). This substitution worked as an adaptive mutation in transient reporter and colony formation assays. Using the advantages of this adaptive mutation and of Oc cells in colony formation, we established the first cell line in which genome-length dicistronic HCV RNA encoding a luciferase gene replicated efficiently. This culture system is useful tool for the study of HCV replication and mass screening for anti-HCV reagents. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.02.138

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Hepatitis C virus (HCV) genomic sequences are known to vary widely among HCV strains, but to date there have been few reports on the genetic variations and dynamics of HCV in an experimental system of HCV replication. In this study, a genetic analysis of HCV replicons obtained in long-term culture of two HCV replicon cells (50-1 and 1B-2R1), which were established from two HCV strains, 1B-1 and 1B-2, respectively, was performed. One person cultured 50-1 cells for 18 months, and two people independently cultured 50-1 cells for 12 months. 1B-2R1 cells were also cultured for 12 months. The whole nucleotide sequences of the three independent replicon RNA clones obtained at several time points were determined. It was observed that genetic mutations in both replicons accumulated in a time-dependent manner, and that the mutation rates of both replicons were approximately 3.0 x 10(-3) base substitutions/site/year. The genetic diversity of both replicons was also enlarged in a time-dependent manner. The colony formation assay by transfection of total RNAs isolated from both replicon cells at different time points into naive HuH-7 cells revealed that the genetic mutations accumulating with time in both replicons apparently improved colony formation efficiency. Taken together, these results suggest that the HCV replicon system is useful for the analysis of evolutionary dynamics and variations of HCV. Using this replicon cell culture system, it was demonstrated further that neither ribavirin nor its derivative mizoribine accelerated the mutation rate or the increase in the genetic diversity of HCV replicon.

DOI： 10.1099/vir.0.80479-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The hepatitis C virus (HCV) replicon system carrying autonomously replicating HCV subgenomic RNA in human hepatocyte cells is a potent tool for basic studies of HCV, such as viral replication and drug development. Recently, we developed two HCV subgenomic replicons (50-1 and 1B-2R1) derived from two HCV strains, 1B-1 and 1B-2, respectively. Since the expression of HCV proteins is thought to affect the host cells' gene expression profiles, we attempted to identify target genes of HCV proteins using microarray analysis (9970 genes) by comparing 50-1 and 1B-2R1 replicon cells with their "cured cells", from which the replicons had been eliminated by prolonged treatment with interferon-a. The results showed that HCV replicons could have a variety of expression profiles in human hepatocytes. The results also showed that 2 and 6 genes were commonly up-regulated (more than 2.0-fold) and down-regulated (less than 0.50-fold), respectively in both 50-1 and 1B-2R1 replicon cells compared with their cured cells. The differential expression profiles of genes selected by the microarray analysis Were confirmed with standard RT-PCR and real-time LightCycler PCR. It was noteworthy that the commonly down-regulated genes contained large multifunctional proteases 2 and 7, which are known as catalytic subunits of immunoproteasome. and serine proteinase inhibitor clade C. Our microarray analysis demonstrated that HCV subgenomic replicons can change the gene expression profiles of host and it allowed us to compile the first list of genes that the replicons transcriptionally regulate. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2004.06.013

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DOI： 10.1016/j.bbagen.2004.10.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Lactoferrin (LF), a milk protein belonging to the iron transporter transferrin family, is known as a primary defense protein against pathogenic microorganisms. Previously, we found that bovine and human LFs prevented hepatitis C virus infection in cultured human hepatocytes by a direct interaction with the virus. Since LF is proposed to have transcriptional regulatory activity in addition to its antimicrobial function, we sought to identify the target genes that these two types of LF have in common. To this end, we were the first to perform microarray analysis (9970 genes) using human hepatocytes that expressed bovine or human LF by retrovirus-mediated gene transfer. In the results, LF could give a variety of expression profiles in the human hepatocytes, and showed that 9 and 19 genes were commonly upregulated (more than 2.0-fold) and down-regulated (less than 0.50-fold), respectively, in both bovine and human LF-expressing cells compared with control cells. Among these genes, we found that gamma-aminobutyric acid (GABA)-B receptor 2 was transcriptionally down-regulated by bovine and human Us, but not by human transferrin. Furthermore, we obtained the suggestive result that LF may modulate the level of intracellular cAMP. This modulation is one of the cellular responses that the GABA-B receptor modifies. This is the first report of microarray analysis applied to search inclusively for the target genes of LF. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbagen.2004.10.003

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To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-alpha and then transfecting the total RNA derived from the IFN-alpha-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-alpha (200 IU/ml). These cloned cells were further treated with IFN-alpha or IFN-beta (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (alphaR series) possessing the IFN-alpha-resistant phenotype and four replicon cell lines (PR series) possessing the IFN-beta-resistant phenotype. Furthermore, we obtained an additional replicon cell line (alphaRmix) possessing the IFN-alpha-resistant phenotype by two rounds of prolonged treatment with IFN-alpha and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the PR series were highly resistant to both IFN-alpha and IFN-beta, although the alphaR series containing alphaRmix were only partially resistant to both IFN-alpha and IFN-beta. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the PR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2004.08.091

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Hepatitis C virus proteins exert an effect on a variety of cellular functions, including gene expression, signal transduction, and apoptosis, and because they possess oncogenic potentials, they have also been suggested to play an important role in hepatocarcinogenesis. Although the mechanisms of hepatocarcinogenesis remain poorly understood, we hypothesized that the disease may arise because of a disturbance of the DNA repair system by hepatitis C virus proteins. To test this hypothesis, we developed a reproducible microsatellite instability assay system for mismatch-repair using human-cultured cells transducted with pCXpur retrovirus expression vector, in which the puromycin resistance gene was rendered out-of-frame by insertion of a (CA)(17) dinucleotide repeat tract immediately following the ATG start codon. Using several human cancer cell lines known to be replication error positive or negative, we demonstrated that this assay system was useful for monitoring the propensity for mismatch-repair in the cells. This assay system was applicable to nonneoplastic human PH5CH8 hepatocytes, which could support hepatitis C virus replication. Using PH5CH8 cells, in which hepatitis C virus proteins were stably expressed by the retrovirus-mediated gene transfer, we found that the core protein promoted microsatellite instability in PH5CH8 cells. Interestingly, such promotion by the core protein only occurred in cells having the core protein belonging to genotype 1b or 2a and did not occur in cells having the core protein belonging to genotype 1a, 2b, or 3a. This is the first report to demonstrate that the core protein may disturb the DNA repair system.

DOI： 10.1158/0008-5472.CAN-03-2992

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We previously found that hepatitis C virus (HCV) core protein, which possesses the consensus sequence of genotype 1b, transcriptionally activates the interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene in human hepatocyte cells. To clarify the mechanism of this activation, we further characterized the core protein as an activator of the 2'-5'-OAS gene. We demonstrated that the activation of the 2'-5'-OAS gene by the core protein is a general phenomenon, regardless of HCV genotype and strain. We showed that the 20 N-terminal amino acids (aa) of the core protein were important to the activation of the 2'-5'-OAS gene, although this N-terminal region did not have any effect on the subcellular localization of the core protein. We demonstrated that the core protein was able to activate all promoters possessing the IFN-stimulated response element (ISRE) examined. However, we found that the level of activation of the 2'-5'-OAS gene promoter possessing a particular variant type of ISRE was significantly higher than that of other IFN-inducible gene promoters. This phenomenon was confirmed using synthetic promoters possessing five repeats of the consensus or a 2'-5'-OAS-type ISRE. In addition, we showed that gene activation induced by the core protein is mediated by the ISRE. These results imply that the core protein prefers a subclass of IFN-inducible genes, the promoters of which possess the 2-5'-OAS-type ISRE. Accordingly, we found that the IFN-inducible double-stranded RNA-specific adenosine deaminase gene promoter, possessing a 2'-5'-OAS-type ISRE sequence, was also efficiently activated by the core protein. The exact mechanism by which the core protein enhances gene expression was not determined, but we could find no effects of core protein on gene expression and phosphorylation status of the components of the JAK-STAT signaling transduction pathway. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S0168-1702(03)00218-1

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The hepatitis C virus (HCV) replicon system is a potent tool for understanding the mechanisms of HCV replication and proliferation, and for the development of treatments for patients with HCV. Recently, we established an HCV subgenomic replicon (501) using HCV genome RNA obtained from the cultured human T cell line MT-2C infected with HCV (isolate 1B-1) in vitro. In order to further obtain other HCV replicons without difficulty, we generated a replicon RNA library derived from human non-neoplastic hepatocytes infected with HCV (isolate 1B-2) in vitro. Upon transfection of the generated RNA library to "cured cells," from which the 50-1 subgenomic replicon was eliminated by prolonged treatment with interferon-alpha, we successfully established a new HCV subgenomic replicon, 1B-2R1. We characterized 1B-2R1 replicon in terms of efficiency of replication, HCV sequence, and sensitivity to interferons. The results revealed that the replication level of the 1B-2R1 replicon was comparable to that of the 50-1 replicon. We also found that the 1B-2R1 replicon possessed an HCV sequence distinct from those of other replicons established to date, and that the 1B-2R1 replicon was sensitive to interferon-a, interferon-P, and interferon-gamma. Taken together, present results indicate that the replicon RNA library generated using an in vitro HCV infection system is useful for the establishment of an HCV subgenomic replicon. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(03)01047-7

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

We report a case of therapy-related myelodysplastic syndrome (t-MDS) with t(10;16)(q22;p13), in which novel fusion transcripts of the MORF and CBP genes were detected. In one MORF-CBP fusion transcript, exon 15 of the MORF gene was fused in frame with exon 5 of the CBP gene. In a reciprocal CBP-MORF transcript, exon 4 of the CBP gene was fused in frame with exon 16 of the MORF gene. This is the first reported case of t-MDS associated with t(10;16), and provides molecular evidence that the novel MORF-CBP and/or CBP-MORF fusion protein(s) might play an important role in the development of t-MDS.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

As a wide range of disorders underlie haemophagocytic syndrome, a rapid distinction between benign polyclonal and malignant monoclonal lymphoid proliferations is critical. We investigated whether polymerase chain reaction ( PCR) amplification of immunoglobulin heavy chain gene rearrangement could efficiently detect clonal B-cell populations in non-diagnostic marrow for B-cell lymphoma-associated haemophagocytic syndrome (B-LAHS). On amplifying two DNA samples per biopsy, no reproducible monoclonal PCR result was found in reactive haemophagocytic marrows. In contrast, four out of nine assessable B-LAHS patients with histomorphologically and immunohistochemically lymphoma-free bone marrow showed a reproducible monoclonal immunoglobulin heavy chain gene rearrangement. At the molecular level, two B-LAHS patients had lymphoma-free marrow as demonstrated by patient-specific PCR, suggesting that haemophagocytic marrow is not always associated with lymphoma involvement. PCR-based demonstration of clonal B-cell populations in marrow would add an extra dimension to B-LAHS diagnosis.

DOI： 10.1046/j.1365-2141.2002.03866.x

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

An 80-year-old woman developed therapy-related myelodysplastic syndrome with translocation (8;21), which was successfully treated with an acute myeloid leukemia oriented chemotherapy. Five years before admission she had received cyclophosphamide, epirubicin, and carboplatin for endometrial cancer. The leukemia cell morphology alerted us to the possibility of the presence of t(8;21) before cytogenetic results were obtained, and AML1/ETO fusion transcript was detected by reverse transcription polymerase chain reaction. She achieved complete remission after one course of idarubicin and cytosine arabinoside. She has remained in complete remission for 6 months. Our experience suggests that recognition of typical morphological features for de novo M2 acute myeloid leukemia with t(8;21) would be important in diagnosis of therapy related myelodysplastic syndrome/acute myeloid leukemia with this translocation, which could respond to an intensive chemotherapy.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Combined deficiency of factor V and factor VIII is a distinct clinical entity and is an autosomal recessive disorder. Recently identification of the gene, the endoplasmic reticulum-Golgi intermediate compartment (ERGIC-53), responsible for combined factor V-factor VIII deficiency and mutations of the ERGIC-53 gene in affected patients have been reported. In this report we analyzed two Japanese patients with combined factor V-factor VIII deficiency by genomic polymerase chain reaction and sequencing analysis. In one patient we found a point mutation of C to T at nucleotide 604 in exon 5, resulting in a transition of arginine to stop codon, which was reported in previous reports. The DdeI digestion study demonstrated that this patient is homozygous for this nonsense mutation. In the other patient we found no mutation in the ERGIC-53 gene in analysis of the entire coding region and the intron/exon junctions, which is also consistent with the previous reports, suggesting the possibility of defects at other genetic loci.

DOI： 10.1007/s002770000283

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Carden Jennings Publishing Co. Ltd  

We studied the molecular basis of type I antithrombin (AT) deficiency in 2 Japanese families, in which affected persons had histories of recurrent venous thrombosis and low (about 50% of normal) levels of AT protein according to measurements by both functional and antigen assays. Southern blotting of DNA isolated from peripheral leukocytes revealed no abnormalities in all the cases examined. Direct sequencing of the polymerase chain reaction (PCR) products from case 1 suggested a novel heterozygous nonsense mutation in exon 4 (GAG→TAG at nucleotide position 7627, leading to Glu306 stop). The sequencing of the subclones of the patient's exon 4 products confirmed the nonsense mutation. No other sequence abnormalities were detected in the rest of the PCR products. The same mutation was detected in this patient's brother, who had a history of recurrent venous thrombosis and a reduced level of AT activity. In case 2, the direct sequencing of PCR products suggested a novel heterozygous 9-bp deletion in exon 3a(-CACTTC at nucleotide position 5354-5362, leading to the deletion of 3 amino acids, His120, Phe121, and Phe122). The 9-bp deletion mutation in the region of a unique quasi palindrome was confirmed by sequencing several of the subclones of the patient's exon 3a from the PCR products. No other mutations were found by direct sequencing of the rest of the coding regions. The 2 mutations found in this study are novel. The use of PCR and the sequencing of the PCR product subclones has simplified and confirmed the detection and characterization of the various AT muta-tions. © 2001 The Japanese Society of Hematology.

DOI： 10.1007/BF02982095

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

We report a case of Philadelphia-positive chronic myelogenous leukaemia in blastic phase with the additional translocation (8:21)(q22;q22), which is frequent in acute myeloid leukaemia but not in chronic myelogenous leukaemia. The t(8;21) was not detected in the chronic phase, and was the only additional chromosomal anomaly in the blastic clone. Reverse transcription-polymerase chain reaction revealed the AML1/ETO fusion transcript in the cells of blastic phase but not in those of chronic phase. Regarding t(9:22). the breakpoint on chromosome 22 occurred in the CL-BCR region of the BCR gene, resulting in hybrid BCR/ABL mRNA with an e19a2 junction. Our findings provided molecular evidence that t(8;21) can occur as an additional genetic change in Philadelphia-positive chronic myelogenous leukaemia.

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Language：English   Publishing type：Research paper (scientific journal)  

Kininogens are multifunctional plasma glycoproteins. There are two forms of human kininogen: low molecular weight kininogen (LK) and high molecular weight kininogen (HK). Both are derived from the same gene by alternative splicing. Some patients with kininogen deficiency have been reported to be deficient only in HK while others are deficient in both HK and LK (total kininogen deficiency). We analyzed three Japanese patients with total kininogen deficiency by the Csp45I digestion study of exon 5 as previously reported in Williams trait and found that two had the same point mutation of C to T at base 22 of exon 5, resulting in a transition of CGA (Are) codon to TGA (Stop) codon.This is the first report of molecular characterization of total kininogen deficiency in the Japanese population. © 1999 The Japanese Society of Hematology.

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PubMed

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(株)日本メディカルセンター  

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Other Link： https://search.jamas.or.jp/default/link?pub_year=2014&ichushi_jid=J01937&link_issn=&doc_id=20140526200003&doc_link_id=10.19020%2FJ01937.2014242754&url=https%3A%2F%2Fdoi.org%2F10.19020%2FJ01937.2014242754&type=%E5%8C%BB%E6%9B%B8.jp_%E3%82%AA%E3%83%BC%E3%83%AB%E3%82%A2%E3%82%AF%E3%82%BB%E3%82%B9&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00024_2.gif

J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：English   Publisher：(一社)日本癌学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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Language：Japanese   Publisher：(一社)日本肝臓学会  

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JOHN WILEY & SONS INC  

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Language：English   Publisher：AMER ASSOC CANCER RESEARCH  

Gene targeting studies in mice have shown that the transcription factor Ikaros plays an essential role in lymphoid development and as a tumor suppressor in T cells, whereas the related gene Aiolos functions as a tumor suppressor in B cells. We analyzed the expression levels of the Ikaros gene family, Ikaros and Aiolos, in human bone marrow samples from patients with adult acute lymphoblastic leukemia [ALL (n = 46; B-cell ALL = 41; T-cell ALL = 5)]. Overexpression of the dominant negative isoform of Ikaros gene Ik-6 was observed in 14 of 41 B-cell ALL patients by reverse transcription-PCR, and the results were confirmed by sequencing analysis and immunoblotting, None of the other dominant negative isoforms of the Ikaros gene were detected by reverse transcription-PCR analysis, Southern blotting analysis with PstI digestion revealed that those patients with the dominant negative isoform Ik-6 might have small mutations in the Ikaros locus. We did not detect any overexpression of dominant negative isoforms of Aiolos in adult ALL patients. These results suggest that Ikaros plays a key role in human B-cell malignancies through the dominant negative Isoform Ik-6.

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Event date： 2019.6.29 - 2019.6.30

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2019.6.29 - 2019.6.30

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.10.28 - 2018.10.30

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2018.10.28 - 2018.10.30

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2018.10.28 - 2018.10.30

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2018.10.28 - 2018.10.30

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2018.10.28 - 2018.10.30

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2018.10.28 - 2018.10.30

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2018.10.3 - 2018.10.6

Language：English   Presentation type：Poster presentation  

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Event date： 2018.8.29 - 2018.8.31

Language：English   Presentation type：Poster presentation  

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Event date： 2018.6.23 - 2018.6.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.6.23 - 2018.6.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.6.23 - 2018.6.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.6.23 - 2018.6.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2018.6.23 - 2018.6.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2015.11.22 - 2015.11.24

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2015.6.27 - 2015.6.28

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2014.11.10 - 2014.11.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2014.9.7 - 2014.9.11

Language：English   Presentation type：Poster presentation  

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Event date： 2014.6.28 - 2014.6.29

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：山口大学  

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Event date： 2013.12.3 - 2013.12.6

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2013.12.3 - 2013.12.6

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2013.11.10 - 2013.11.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.11.10 - 2013.11.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.11.10 - 2013.11.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.11.10 - 2013.11.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.11.10 - 2013.11.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.11.10 - 2013.11.12

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.10.9 - 2013.10.10

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2013.10.6 - 2013.10.10

Language：English   Presentation type：Poster presentation  

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Event date： 2013.10.6 - 2013.10.10

Language：English   Presentation type：Poster presentation  

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Event date： 2013.10.6 - 2013.10.10

Language：English   Presentation type：Poster presentation  

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Event date： 2013.10.6 - 2013.10.10

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Event date： 2013.10.6 - 2013.10.10

Language：English   Presentation type：Poster presentation  

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Event date： 2013.10.3 - 2013.10.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2013.6.22 - 2013.6.23

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.6.22 - 2013.6.23

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.6.22 - 2013.6.23

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.6.22 - 2013.6.23

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.6.22 - 2013.6.23

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.6.22 - 2013.6.23

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2013.6.6 - 2013.6.7

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2013.6.6 - 2013.6.7

Language：Japanese   Presentation type：Oral presentation (keynote)  

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Event date： 2013.6.6 - 2013.6.7

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2012.11.13 - 2012.11.15

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2012.11.13 - 2012.11.15

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2012.10.5 - 2012.10.9

Language：English   Presentation type：Poster presentation  

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Event date： 2012.10.5 - 2012.10.9

Language：English   Presentation type：Poster presentation  

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Event date： 2012.9.19 - 2012.9.21

Language：English   Presentation type：Poster presentation  

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Event date： 2012.9.19 - 2012.9.21

Language：English   Presentation type：Poster presentation  

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Event date： 2012.6.23 - 2012.6.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2011.9.8 - 2011.9.12

Language：English   Presentation type：Poster presentation  

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Event date： 2010.12.7 - 2010.12.10

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2010.11.7 - 2010.11.9

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2010.11.7 - 2010.11.9

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2010.11.7 - 2010.11.9

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2010.10.13

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2010.10.13

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2010.9.22 - 2010.9.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2010.9.22 - 2010.9.24

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2010.9.10 - 2010.9.14

Language：English   Presentation type：Poster presentation  

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Event date： 2010.9.10 - 2010.9.14

Language：English   Presentation type：Poster presentation  

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Event date： 2010.6.26 - 2010.6.27

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2010.6.26 - 2010.6.27

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Event date： 2009.10.25 - 2009.10.27

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Event date： 2009.10.25 - 2009.10.27

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Event date： 2009.10.25 - 2009.10.27

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Event date： 2009.10.25 - 2009.10.27

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Event date： 2009.10.25 - 2009.10.27

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2009.10.14 - 2009.10.16

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2009.10.3 - 2009.10.7

Language：English   Presentation type：Oral presentation (general)  

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Event date： 2009.10.3 - 2009.10.7

Language：English   Presentation type：Poster presentation  

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Event date： 2009.10.3 - 2009.10.7

Language：English   Presentation type：Poster presentation  

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Event date： 2009.10.3 - 2009.10.7

Language：English   Presentation type：Poster presentation  

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Event date： 2009.10.3 - 2009.10.7

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Event date： 2009.10.3 - 2009.10.7

Language：English   Presentation type：Poster presentation  

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Event date： 2009.10.3 - 2009.10.7

Language：English   Presentation type：Poster presentation  

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Event date： 2009.7.4 - 2009.7.5

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2009.7.4 - 2009.7.5

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2009.7.4 - 2009.7.5

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Event date： 2009.6.4 - 2009.6.5

Language：Japanese   Presentation type：Poster presentation  

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Event date： 2007.10.21 - 2007.10.23

Presentation type：Poster presentation  

Venue：札幌市  

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Event date： 2007.10.21 - 2007.10.23

Presentation type：Oral presentation (general)  

Venue：札幌市  

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Event date： 2007.10.21 - 2007.10.23

Presentation type：Oral presentation (general)  

Venue：札幌市  

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Event date： 2007.10.21 - 2007.10.23

Presentation type：Poster presentation  

Venue：札幌市  

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Event date： 2007.10.21 - 2007.10.23

Presentation type：Oral presentation (general)  

Venue：札幌市  

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Event date： 2007.10.21 - 2007.10.23

Presentation type：Poster presentation  

Venue：札幌市  

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Event date： 2007.10.3 - 2007.10.5

Presentation type：Poster presentation  

Venue：横浜市  

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Event date： 2007.10.3 - 2007.10.5

Presentation type：Oral presentation (general)  

Venue：横浜市  

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Event date： 2007.10.3 - 2007.10.5

Presentation type：Oral presentation (general)  

Venue：横浜市  

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Event date： 2007.9.9 - 2007.9.13

Presentation type：Poster presentation  

Venue：Glasgow, Scotland UK  

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Event date： 2007.9.9 - 2007.9.13

Presentation type：Poster presentation  

Venue：Glasgow, Scotland UK  

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Event date： 2007.9.9 - 2007.9.13

Presentation type：Poster presentation  

Venue：Glasgow, Scotland UK  

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Event date： 2007.9.9 - 2007.9.13

Presentation type：Poster presentation  

Venue：Glasgow, Scotland UK  

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Event date： 2007.9.9 - 2007.9.13

Presentation type：Poster presentation  

Venue：Glasgow, Scotland UK  

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Event date： 2007.7.5 - 2007.7.6

Presentation type：Poster presentation  

Venue：京都市  

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Event date： 2007.6.16 - 2007.6.17

Presentation type：Oral presentation (general)  

Venue：松山市  

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Event date： 2007.6.16 - 2007.6.17

Presentation type：Oral presentation (general)  

Venue：松山市  

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Event date： 2007.6.16 - 2007.6.17

Presentation type：Oral presentation (general)  

Venue：松山市  

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Event date： 2007.5.31 - 2007.6.1

Presentation type：Oral presentation (general)  

Venue：東京  

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Event date： 2006.11.19 - 2006.11.21

Venue：名古屋市  

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Event date： 2006.11.19 - 2006.11.21

Venue：名古屋市  

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Event date： 2006.11.19 - 2006.11.21

Venue：名古屋市  

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Event date： 2006.11.19 - 2006.11.21

Venue：名古屋市  

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Event date： 2006.11.19 - 2006.11.21

Venue：名古屋市  

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Event date： 2006.11.19 - 2006.11.21

Venue：名古屋市  

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Event date： 2006.9.28 - 2006.9.30

Venue：横浜市  

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Event date： 2006.9.28 - 2006.9.30

Venue：横浜市  

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Event date： 2006.9.28 - 2006.9.30

Venue：横浜市  

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Event date： 2006.8.27 - 2006.8.31

Venue：Cairns, Australia  

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Event date： 2006.8.27 - 2006.8.31

Venue：Cairns, Australia  

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Event date： 2006.8.27 - 2006.8.31

Venue：Cairns, Australia  

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Event date： 2006.8.27 - 2006.8.31

Venue：Cairns, Australia  

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Event date： 2006.8.27 - 2006.8.31

Venue：Cairns, Australia  

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Event date： 2006.6.10 - 2006.6.11

Venue：鳥取市  

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Event date： 2006.6.10 - 2006.6.11

Venue：鳥取市  

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Event date： 2006.6.10 - 2006.6.11

Venue：鳥取市  

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Event date： 2005.11.20 - 2005.11.22

Venue：横浜市  

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Event date： 2005.11.20 - 2005.11.22

Venue：横浜市  

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Event date： 2005.11.20 - 2005.11.22

Venue：横浜市  

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Event date： 2005.11.20 - 2005.11.22

Venue：横浜市  

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Event date： 2005.11.20 - 2005.11.22

Venue：横浜市  

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Event date： 2005.10.2 - 2005.10.6

Venue：Montreal, Canada  

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Event date： 2005.10.2 - 2005.10.6

Venue：Montreal, Canada  

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Event date： 2005.10.2 - 2005.10.6

Venue：Montreal, Canada  

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Event date： 2005.10.2 - 2005.10.6

Venue：Montreal, Canada  

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Event date： 2005.9.14 - 2005.9.16

Venue：札幌市  

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Event date： 2005.9.14 - 2005.9.16

Venue：札幌市  

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Event date： 2005.6.18 - 2005.6.19

Venue：岡山県倉敷市  

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Event date： 2005.6.18 - 2005.6.19

Venue：岡山県倉敷市  

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Event date： 2004.11.21 - 2004.11.23

Venue：横浜市  

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Event date： 2004.11.21 - 2004.11.23

Venue：横浜市  

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Event date： 2004.11.21 - 2004.11.23

Venue：横浜市  

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Event date： 2004.10.3 - 2004.10.7

Venue：Heidelberg, Germany  

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Event date： 2004.10.3 - 2004.10.7

Venue：Heidelberg, Germany  

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Event date： 2004.10.3 - 2004.10.7

Venue：Heidelberg, Germany  

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Event date： 2004.10.3 - 2004.10.7

Venue：Heidelberg, Germany  

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Event date： 2004.6.26 - 2004.6.27

Venue：高知市  

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Event date： 2004.6.26 - 2004.6.27

Venue：高知市  

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Event date： 2003.12.2 - 2003.12.6

Venue：Kyoto, Japan  

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Event date： 2003.12.2 - 2003.12.6

Venue：Kyoto, Japan  

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Event date： 2003.12.2 - 2003.12.6

Venue：Kyoto, Japan  

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Event date： 2003.12.2 - 2003.12.6

Venue：Kyoto, Japan  

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Event date： 2003.10.27 - 2003.10.29

Venue：京都市  

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Event date： 2003.10.27 - 2003.10.29

Venue：京都市  

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Event date： 2003.10.27 - 2003.10.29

Venue：京都市  

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Event date： 2003.10.27 - 2003.10.29

Venue：京都市  

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Event date： 2003.9.25 - 2003.9.27

Venue：名古屋市  

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Event date： 2003.9.25 - 2003.9.27

Venue：名古屋市  

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Event date： 2002.11.11 - 2002.11.14

Venue：横浜市  

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Event date： 2002.10.16 - 2002.10.18

Venue：札幌市  

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Event date： 2002.10.1 - 2002.10.3

Venue：東京  

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Event date： 2002.5.25 - 2002.5.26

Venue：岡山市  

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Event date： 2001.12

Venue：横浜市  

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Event date： 2001.12

Venue：横浜市  

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Event date： 2001.8

Venue：Tokyo, Japan  

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Event date： 2001.4

Venue：名古屋市  

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Event date： 2000.3

Venue：福岡市  

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