Biomedical subjects

Hiromichi Dansako

Publications and source records attributed to Hiromichi Dansako.

15 recordsLinked to original sources

PubMed2006

Epigenetic silencing of interferon-inducible genes is implicated in interferon resistance of hepatitis C virus replicon-harboring cells.

BACKGROUND/AIMS: We previously established hepatitis C virus (HCV) replicon-harboring cell lines possessing two interferon (IFN)-resistant phenotypes: a partially resistant phenotype (alphaR series) and a severely resistant phenotype (betaR series). We recently found that the severe IFN resistance of the betaR-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 alphaR-series cells. METHODS: alphaR-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 1alphaR cells, which belong to the alphaR 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 alphaR-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 1alphaR 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.

Antiviral Agents↗

PubMed2006

Different anti-HCV profiles of statins and their potential for combination therapy with interferon.

We recently developed a genome-length hepatitis C virus (HCV) RNA replication system (OR6) 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 C, 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 the 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 micromol/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.

Antiviral Agents↗

PubMed2005

Hepatitis C virus NS5B delays cell cycle progression by inducing interferon-beta via Toll-like receptor 3 signaling pathway without replicating viral genomes.

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.

Cell Cycle↗

PubMed2005

Hepatitis C virus proteins exhibit conflicting effects on the interferon system in human hepatocyte cells.

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 or 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-beta, 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.

Cell Line↗

PubMed2005

Mizoribine inhibits hepatitis C virus RNA replication: effect of combination with interferon-alpha.

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 microM) 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 microM 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.

Antiviral Agents↗

PubMed2005

Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system.

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.

Amino Acid Substitution↗

PubMed2005

cDNA microarray analysis to compare HCV subgenomic replicon cells with their cured cells.

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-alpha. 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 cells, and it allowed us to compile the first list of genes that the replicons transcriptionally regulate.

Base Sequence↗

PubMed2005

Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture.

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.

Cell Culture Techniques↗

PubMed2005

Interferon resistance of hepatitis C virus replicon-harbouring cells is caused by functional disruption of type I interferon receptors.

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.

Drug Resistance, Viral↗

PubMed2004

cDNA microarray analysis of lactoferrin expression in non-neoplastic human hepatocyte PH5CH8 cells.

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 up-regulated (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 LFs, 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.

Animals↗

PubMed2004

Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype.

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 (betaR 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 betaR 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 betaR 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.

Antiviral Agents↗

PubMed2004

Promotion of microsatellite instability by hepatitis C virus core protein in human non-neoplastic hepatocyte cells.

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 non-neoplastic 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.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro.

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 (50-1) 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-alpha, interferon-beta, 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.

Cells, Cultured↗

PubMed2003

Differential activation of interferon-inducible genes by hepatitis C virus core protein mediated by the interferon stimulated response element.

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.

2',5'-Oligoadenylate Synthetase↗

PubMed2002

Specificity of polymerase chain reaction-based clonality analysis of immunoglobulin heavy chain gene rearrangement for the detection of bone marrow infiltrate in B-cell lymphoma-associated haemophagocytic syndrome.

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.

Aged↗