Hyung-Bae Moon

Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein

BACKGROUND/AIMS Chronic infection with hepatitis B virus is a high-risk factor for hepatocellular carcinoma in humans. The HBV X-protein, a multi-functional viral regulator, has been suspected to play a positive role in hepatocarcinogenesis, as demonstrated by the high incidence of hepatocellular carcinoma in HBx-expressing transgenic mice, although it is still controversial. The aim of this study was to generate transgenic mice expressing the HBV X-gene under authentic promoter control and to test whether the gene products can cause hepatic tumors. METHODS Three transgenic mouse lines were generated by microinjecting the X-gene construct into hybrid (C57BL/6 x DBA) eggs. Gene expression was tested by protein and mRNA analyses. During an observation period of 18 months, mice were sacrificed and organs subjected to histologic examinations. RESULTS Grossly defined hepatocellular carcinomas reproducibly were observed in mice expressing the X-protein, which were investigated through six generations from the age of 11 to 18 months. Among 14 transgenic mice investigated from the age of 11 to 18 months, 12 were found to have hepatocellular carcinoma, grossly or microscopically. The lesion of the hepatocellular carcinoma disclosed a significant increase in the proliferating cell nuclear antigen in the nuclei. CONCLUSION The incidence of hepatocellular carcinoma (86%) in our HBV X transgenic mice may be highly significant, since, except for one case, HBV X-gene transgenic mice produced in other laboratories did not develop liver tumor or any other pathologic phenomena.

Non-structural 5A protein of hepatitis C virus induces a range of liver pathology in transgenic mice.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). However, the mechanism of HCV pathogenesis is not well understood. Our previous in vitro studies suggested that non‐structural 5A (NS5A) protein may play an important role in liver pathogenesis. To elucidate the mechanism of HCV‐induced liver pathogenesis, we investigated the histopathological changes of liver in transgenic mice harbouring the NS5A gene. We generated transgenic mice harbouring HCV NS5A gene under the control of hepatitis B virus (HBV) enhancer. Pathological changes were analysed by immunohistochemical staining and western blot analysis. Lipid composition and reactive oxygen species (ROS) production in NS5A transgenic mice were analysed. HCV NS5A transgenic mice developed extraordinary steatosis over 6 months old and induced HCC in some mice. NS5A was co‐localized with apolipoprotein A‐I in fatty hepatocytes. In addition, the extraordinarily high levels of ROS, NF‐κB and STAT3 were detected in hepatocytes of NS5A transgenic mice. These data suggest that NS5A, independent of other HCV viral proteins, may play an important role in the development of hepatic pathologies, including steatosis and hepatoceullular carcinoma in transgenic mice. Copyright

Distribution of hepatitis B virus genotypes in Korea

BACKGROUND/AIMS Considering the incidence of prevailing hepatitis B virus (HBV) genotypes in neighboring nations, the predominance of genotype C in Korea is exceptional and needs to be confirmed by nationwide investigation. METHODS A total of 510 HBsAg (+) or HBeAg (+) serum samples was collected from subjects in several cities and harbors throughout the Korean peninsula for genotype (A-G)-specific multiplex PCR analysis. Another 40 serum samples from chronic HBV carriers from Iksan city were selected for sequencing of the entire HBV genome. Phylogenetic analysis was performed with 22 whole genomic sequences of Korean HBV strains enrolled in GenBank. RESULTS An amplicon was found in 377 specimens and genotype C occupied 98.1% (370 cases); none of the other genotypes were found. A mixed pattern of genotypes B and C was seen in seven specimens (1.9%), of which five were tested using PCR targeting the X fragment; no genotype B bands were found. With the exception of 1 case, which was subgenotype A2, whole sequences of Korean HBV strains (n=62) belonged to subgenotype C2. CONCLUSIONS The prevailing HBV genotype in Korea is C2; the other genotypes occur only rarely. Future studies should include confirmation of the detection of genotypes other than C.

Proteomic analysis of liver tissue from HBx-transgenic mice at early stages of hepatocarcinogenesis

The hepatitis B virus X‐protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx‐transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2‐DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC‐MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3‐ketoacyl‐CoA thiolase, intestinal fatty acid‐binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx‐mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.

Hepatitis B Virus X Protein Regulates Hepatic Glucose Homeostasis via Activation of Inducible Nitric Oxide Synthase

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.

Association of FOXJ1 polymorphisms with systemic lupus erythematosus and rheumatoid arthritis in Korean population

The forkhead-box J1 (FOXJ1) transcription factor could suppress a spontaneous activation of T cells and B cells through an induction of IκBβ that results in repression of NF-κB activity. In Foxj1 deficiency mice, systemic autoimmune inflammation is quite common symptom. Therefore, deregulated Foxj1 is supposed to be associated with autoimmune diseases and/or other inflammatory diseases. Previously, we identified that polymorphisms of human FOXJ1 gene (g.-460C>T, g.1805G>T and g.3375G>C) are associated with allergic rhinitis in a Korean population. In present study, we compared the genotype and allele frequencies of these SNPs between healthy controls and systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) patients. We also investigated the relationships between each genotype and the expression levels of anti-nuclear antibodies in SLE patients, and rheumatoid factor and anti-cyclic citrullinated peptide in RA patients. The frequencies of haplotypes constructed by these FOXJ1 SNPs were compared between controls and SLE (or RA) patients. The results of genotype and allele analysis showed that the prevalence of polymorphism g.3375G>C was associated with the susceptibility of SLE (P = 0.0072 and 0.0042, respectively). But no significant association was found with RA. In the haplotype analysis, however, the main CGG showed a weak association between controls and RA patients (P = 0.048).

Downregulation of p38 kinase pathway by cAMP response element-binding protein protects HL-60 cells from iron chelator-induced apoptosis

The signaling mechanisms that control apoptotic events evoked by iron chelators are largely unknown. We found that cAMP response element-binding protein (CREB) is cleaved during iron chelator deferoxamine (DFO)-induced apoptosis, and that the cleavage is largely prevented by the cell-permeable analog of cAMP, dibutyryl-cAMP (dbcAMP), a known CREB activator. In addition, dbcAMP profoundly reduced DFO-induced apoptosis along with significant suppression of caspase-3 and -8 activation and inhibition of loss of mitochondrial potential. These results led us to investigate whether CREB activation is functionally connected with the MAPK family members because we previously demonstrated that p38 kinase is involved in iron chelator-induced apoptosis of HL-60 cells. dbcAMP by itself rapidly induced CREB phosphorylation but dramatically inhibited DFO-induced phosphorylation of all three MAPK family members. However, disruption of CREB expression by antisense oligodeoxyribonucleotide (AS-ODN) only restored p38 kinase activation, and simultaneously attenuated dbcAMP-induced protection of HL-60 cells from DFO-induced cell death. Conversely, inhibition of p38 kinase activity by SB203580 significantly reduced DFO-induced CREB cleavage as well as apoptosis, indicating a cross-talk between CREB and p38 kinase. Collectively, these results demonstrate that cAMP-dependent CREB activation plays an important role in protecting HL-60 cells from iron chelator-induced apoptosis, presumably through downregulation of p38 kinase.

Identifying Polymorphisms in IL-31 and Their Association with Susceptibility to Asthma.

Background Interleukin 31 (IL-31) is a T helper type 2 effector cytokine that plays an important role in the pathogenesis of atopic and allergic diseases. IL-31 may be involved in promoting allergic inflammation and in inducing airway epithelial responses such as allergic asthma. Methods Single-base extension analysis was used to detect the genotypes of IL-31 single nucleotide polymorphisms (SNPs), and we compared the genotype and allele frequencies of the IL-31 SNPs between patients with asthma and healthy controls. Results There were no significant differences in the genotype and allele frequencies of the IL-31 SNPs between patients with asthma and healthy controls. Furthermore we compared the genotype and allele frequencies of IL-31 SNPs between patients with atopic asthma, those with non-atopic asthma and healthy controls. This showed that the SNPs were not associated with the susceptibility to atopic asthma. There were no significant differences in the haplotype frequencies of IL-31 SNPs between patients with asthma and healthy controls. In patients with asthma, the IL-31 SNPs were significantly correlated with total serum levels of IgE (p=0.035). Conclusions Our results indicate that, the IL-31 SNPs may be associated with IgE production in patients with asthma.

Expression of hepatitis C virus nonstructural 4B in transgenic mice

Hepatitis C virus (HCV) is a pathogen that is of great medical significance in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Although the HCV proteins have been intensively investigated over the past decade, the biochemical functions of the NS4B protein are still largely unknown. To investigate NS4B as a potential causative agent of liver disease, transgenic mice expressing the NS4B protein in liver tissue were produced. The transgenic animals were phenotypically similar to their normal littermates for up to 18 months of age. Our results suggest that the HCV NS4B protein is not directly cytopathic or oncogenic in our transgenic mice model.

Intratumoral Injection of 188Re labeled Cationic Polyethylenimine Conjugates: A Preliminary Report

188Re(Rhenium) is easily obtained from an in-house 188W/188Re generator that is similar to the current 99Mo/99mTc generator, making it very convenient for clinical use. This characteristic makes this radionuclide a promising candidate as a therapeutic agent. Polyethylenimine (PEI) is a cationic polymer and has been used as a gene delivery vector. Positively charged materials interact with cellular blood components, vascular endothelium, and plasma proteins. In this study, the authors investigated whether intratumoral injection of 188Re labeled transferrin (Tf)-PEI conjugates exert the effect of radionuclide therapy against the tumor cells. When the diameters of the Ramos lymphoma (human Burkitts lymphoma) xenografted tumors reached approximately 1 cm, 3 kinds of 188Re bound compounds (HYNIC-PEI-Tf, HYNIC-PEI, 188Re perrhenate) were injected directly into the tumors. There were increases in the retention of 188Re inside the tumor when PEI was incorporated with 188Re compared to the use of free 188Re. The 188Re HYNIC-Tf-PEI showed the most retention inside the tumor (retention rate=approximately 97%). H&E stain of isolated tumor tissues showed that 188Re labeled HYNIC-PEI-Tf caused extensive tumor necrosis. These results support 188Re HYNIC-PEI-Tf as being a useful radiopharmaceutical agent to treat tumors when delievered by intratumoral injection.