Yutaka Aoyagi

Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture

ABSTRACT 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—β-carotene, vitamin D2, 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, β-carotene, vitamin D2, 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.

Overexpression of extracellular signal-regulated protein kinase and its correlation with proliferation in human hepatocellular carcinoma

Abstract: Background: Dysregulation of cell proliferation is one of the most important features of human cancers including hepatocellular carcinoma (HCC). However, the molecular basis underlying the proliferation of HCC has not been fully clarified. Because a previous study reported that overexpression of extracellular signal‐regulated protein kinase (ERK), which transduces extracellular growth stimuli to the nuclei, was frequently observed in several human cancers, this study was performed to analyze the expression of ERK in human HCC and its correlation with HCC proliferation.

Lack of Bcl11b tumor suppressor results in vulnerability to DNA replication stress and damages

Bcl11b/Rit1 is involved in T-cell development and undergoes chromosomal rearrangements in human T-cell leukemias. Thymocytes of Bcl11b−/− newborn mice exhibit apoptosis at a certain developmental stage when thymocytes re-enter into the cell-cycle. Here, we show that Bcl11b-knockdown T-cell lines, when exposed to growth stimuli, exhibited apoptosis at the Su2009phase with concomitant decreases in a cell-cycle inhibitor, p27 and an antiapoptotic protein, Bcl-xL, owing to transcriptional repression. This repression was a likely consequence of the impairment of Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase associating with Bcl11b. Activation of the apoptotic process cleaved the mediator protein, Claspin, and inhibited phosphorylation of cell-cycle checkpoint kinase 1 (Chk1) that plays a central role in sensing and responding to incomplete replication. Bcl11b−/− thymocytes also failed to phosphorylate Chk1 when UV irradiated. These results implicate Bcl11b in the remedy for DNA replication stress and maintenance of genomic integrity.

MEK/ERK signaling is a critical mediator for integrin-induced cell scattering in highly metastatic hepatocellular carcinoma cells.

The human hepatocellular carcinoma (HCC)-derived cell line KYN-2 is thought to provide a good model for studying the molecular basis of invasion and metastasis of human HCC, because it often shows cell scattering in vitro and intrahepatic metastasis in vivo. We previously found that integrin-mediated extracellular signals inactivated E-cadherin in KYN-2, and caused loss of cell–cell contact with gain of cell motility, which is considered to be a critical step in the process of cancer cell invasion and metastasis. To further understand molecular mechanisms involved in biological aggressiveness of HCC, we investigated intracellular signaling involved in integrin-mediated scattering of KYN-2 cells. Cultured KYN-2 cells formed trabecular aggregates in suspension, but when adhering to integrin-stimulating substrata, they scattered according to phosphorylation of extracellular signal-regulated kinase (ERK). Upon treatment with ERK kinase (MEK) inhibitor PD98059, adhered KYN-2 cell scattering was inhibited, tight cell-to-cell contact was recovered, and both E-cadherin and actin filaments accumulated in the area of intercellular contact zone. In contrast, constitutively active MEK1-transfected KYN-2 cells showed reduced E-cadherin and actin filaments in the intercellular contact zone, showing a flattened phenotype with broad lamellipodia. Enforced signaling of MEK-ERK pathway in KYN-2 cells suppressed cadherin-mediated homotypic adhesion and increased the potential of cell motility. An antibody-based protein microarray analysis revealed that the cytoplasmic protein c-Cbl was significantly downregulated in MEK1-transfected KYN-2 cells, suggesting that c-Cbl might be a candidate downstream mediator of integrin/MEK/ERK-mediated cell scattering. In conclusion, cell scattering of the highly metastatic cell line KYN-2 is regulated through the integrin-MEK-ERK signaling cascade, suggesting that this molecular pathway may be critical in intrahepatic metastasis of human HCC.

Comparative study of genotype B and C hepatitis B virus-induced chronic hepatitis in relation to the basic core promoter and precore mutations.

Background: The clinicopathological profiles and outcome of chronic hepatitis B can differ by hepatitis B virus (HBV) genotypes. In Japan, genotype B and C are two major HBV genotypes. The basic core promoter and precore mutations are other known viral factors for disease activity, although the relationship between HBV genotypes and these mutations is not fully understood.

Long‐Term Culture of Postnatal Mouse Hepatic Stem/Progenitor Cells and Their Relative Developmental Hierarchy

Few studies on the long‐term culture of postnatal mouse hepatic stem/progenitor cells have been reported. We successfully adapted a serum‐free culture system that we employed previously to expand fetal mouse hepatic stem/progenitor cells and maintained them in culture over long periods. The expanded postnatal cells contained immature α‐fetoprotein‐positive cells along with hepatocytic and cholangiocytic lineage‐committed cells. These cells expressed CD49f but not CD45, CD34, Thy‐1, c‐kit, CD31, or flk‐1, and oncostatin M induced their differentiation. This heterogeneous population contained side population (SP) cells, which express the ATP‐binding cassette transporter ABCG2, and sca‐1+ cells. As mice aged, the frequency of SP and sca‐1+ cells decreased along with the ability of cultured cells to expand. Approximately 20%–40% of the SP cells expressed sca‐1, but only a few sca‐1+ cells were also SP cells. Analysis of colonies derived from single SP or sca‐1+ cells revealed that, although both cells had dual differentiation potential and self‐renewal ability, SP cells formed colonies more efficiently and gave rise to SP and sca‐1+ cells, whereas sca‐1+ cells generated only sca‐1+ progeny. Thus, SP cells are more characteristic of stem cells than are sca‐1+ cells. In regenerating livers, ABCG2+ cells and sca‐1+ cells were detected around or in the portal area (the putative hepatic stem cell niche). The expanded cells share many features of fetal hepatic stem/progenitor cells or oval cells and may be useful in determining the mechanisms whereby hepatic stem cells self‐renew and differentiate.

Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1 (Mtf-1 )

Genetic predisposition to cancers is significant to public health because a high proportion of cancers probably arise in a susceptible human subpopulation. Using a mouse model of γ-ray-induced thymic lymphomas, we performed linkage analysis and haplotype mapping that suggested Mtf-1, metal-responsive transcription factor-1 (Mtf-1), as a candidate lymphoma susceptibility gene. Sequence analysis revealed a polymorphism of Mtf-1 that alters the corresponding amino acid at position 424 in the proline-rich domain from a serine in susceptibility strains to proline in resistant strains. The transcriptional activity of Mtf-1 encoding serine and proline was compared by transfecting the DNA to Mtf-1-null cells, and the change to proline conferred a higher metal responsiveness in transfections. Furthermore, the resistant congenic strains possessing the Mtf-1 allele of proline type exhibited higher radiation inducibility of target genes than susceptible background strains having the Mtf-1 allele of serine type. Since products of the targets such as metallothionein are able to suppress cellular stresses generated by irradiation, these results suggest that highly inducible strains having Mtf-1 of proline type are refractory to radiation effects and hence are resistant to lymphoma development.

The fucosylation index of serum α-fetoprotein as useful prognostic factor in patients with hepatocellular carcinoma in special reference to chronological changes

Aim of this study was to establish fucosylation index (FI) of alpha-fetoprotein (AFP) before and after initial treatment as a useful prognostic factor in patients with hepatocellular carcinoma (HCC). One hundred ninety-seven patients with HCC from 1990 to 2000, in whom an increment of serum AFP concentrations more than 30 ng/ml was observed before treatment, were examined in the present study. Enrolled patients with HCC underwent transcatheter arterial embolization, chemoembolization, percutaneous ethanol injection and/or percutaneous microwave coagulation therapy. The current patients status was confirmed as of the end of March 2001. FI was determined by crossed immunoaffinoelectrophoresis in the presence of Lens culinaris agglutinin (LCA). FI of AFP was defined as the percentage of the LCA-reactive species in total AFP (same as L3 fraction). When the tentative discriminating line of FI was set at 18%, the mean survival rate in the HCC group, whose FI-1 (before treatment) was higher than 18% (high FI), was significantly lower than that in another HCC group, whose FI-1 was equal to or less than 18% (low FI) by the generalized Wilcoxon test and the log rank test (P<0.0001). There were statistical significant differences of survival rate when FI-2 (2 months after treatment) and FI-3 (at the time of HCC recurrence or 2 years after treatment in the case of no recurrence) were introduced in the same analysis. Additionally, statistical significant differences of survival rates were obtained between HCC groups with high and low FI-1 when the patient stage was limited to II, III, IVA or IVB. The HCC group, FI-1, FI-2 and FI-3 of which were persistently equal to or less than 18%, showed considerably better prognosis than the group, whose FI-1, FI-2 and FI-3 were persistently higher than 18%. The univariate analysis in the prognostic factor by the Coxs proportional hazards model showed that FI-1, FI-2 and FI-3 were independent prognostic factors. The present study indicates that measuring FI from the sera before and after the treatment serves as a new prognostic indicator and may improve prognostic estimates and appraisal of therapeutic outcome in patients with HCC.

Attenuation of mouse acute colitis by naked hepatocyte growth factor gene transfer into the liver

Hepatocyte growth factor (HGF) has multiple biological effects on a wide variety of cells. It modulates intestinal epithelial proliferation and migration, and critically regulates intestinal wound healing.

Bidirectional gastric differentiation in cellular mucin phenotype (foveolar and pyloric) in serrated adenoma and hyperplastic polyp of the colorectum

This study examined whether gastric pyloric gland‐type mucin is expressed in serrated adenoma (SA) and in hyperplastic polyp (HP) of the colorectum, and whether cellular position‐based gastric differentiation is observed in these lesions as previously hypothesized. Immunostaining was performed for MUC6 and α‐linked GlcNAc residue (pyloric gland‐type mucin markers), human gastric mucin (HGM; foveolar‐type mucin marker) and Ki‐67 (proliferating cell marker) for 31 SA, 22 HP, 21 traditional tubular adenoma (TA) and 20 hyperplastic nodule (HN). MUC6 showed varying expression in SA, 22/31 (71.0%); HP, 15/22 (68.2%); TA, 2/21 (9.5%); and HN, 0/20 (0%) with significantly higher frequencies in SA and HP compared to those in TA and HN. The α‐linked GlcNAc residue was found only in SA (3/31, 9.7%) and in HP (2/22, 9.1%). In SA and HP, HGM was typically expressed in the entire crypt length, but some reduction in expression was shown in the basal crypt portion below the proliferative zone. MUC6 and α‐linked GlcNAc residues were expressed in the basal crypt portion below or below and including proliferative zone. These data demonstrate that SA and HP show bidirectional gastric (foveolar and pyloric gland) differentiation with respect to mucin cellular phenotype and the potential for cellular position‐based differentiation, which mimics the gastric antral mucosa.