Yutaka Yonemitsu

The Polycomb Gene Product BMI1 Contributes to the Maintenance of Tumor-Initiating Side Population Cells in Hepatocellular Carcinoma

Side population (SP) cell analysis and sorting have been successfully applied to hepatocellular carcinoma (HCC) cell lines to identify a minor cell population with cancer stem cell properties. However, the molecular mechanisms operating in SP cells remain unclear. The polycomb gene product BMI1 plays a central role in the self-renewal of somatic stem cells in a variety of tissues and organs and seems to be implicated in tumor development. In this study, we determined the critical role of BMI1 in the maintenance of cancer stem cells with the SP phenotype in HCC cell lines. BMI1 was preferentially expressed in SP cells in Huh7 and PLC/PRF/5 HCC cells compared with the corresponding non-SP cells. Lentiviral knockdown of BMI1 considerably decreased the number of SP cells in both Huh7 and PLC/PRF/5 cells. Long-term culture of purified SP cells resulted in a drastic reduction in the SP subpopulation upon the BMI1 knockdown, indicating that BMI1 is required for the self-renewal of SP cells in culture. More importantly, the BMI1 knockdown abolished the tumor-initiating ability of SP cells in nonobese diabetic/severe combined immunodeficiency mice. Derepression of the INK4A and ARF genes that are major targets for BMI1 was not necessarily associated with impaired self-renewal of SP cells caused by BMI1 knockdown. In conclusion, our findings define an important role for BMI1 in the maintenance of tumor-initiating SP cells in HCC. BMI1 might be a novel therapeutic target for the eradication of cancer stem cells in HCC.

Distinct expression of polycomb group proteins EZH2 and BMI1 in hepatocellular carcinoma

Polycomb gene products play a crucial role in the development of highly malignant phenotypes and aggressive cancer progression in a variety of cancers; however, their role in hepatocellular carcinoma remains unclear. First, we analyzed the impact of EZH2 and BMI1 modulation on cell growth of HepG2 cells. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assays revealed marked growth inhibition after EZH2 or BMI1 knockdown. In addition, simultaneous knockdown of these 2 genes further augmented cell growth inhibitory effects. Next, we conducted immunohistochemical assessment of 86 hepatocellular carcinoma surgical specimens, evaluating the correlation between EZH2 and BMI1 protein expression and clinicopathologic features. High-level EZH2 and BMI1 expression was detected in 57 (66.3%) and 52 tumor tissues (60.5%), respectively. Among these, 48 tumor tissues (55.8%) showed colocalization of EZH2 and BMI1 in almost all tumor cells. The cumulative recurrence rate, but not survival rate, was significantly higher in patients positive for EZH2 (P = .029) and BMI1 (P = .039) than in their negative counterparts, as determined by Kaplan-Meier analysis. These data indicate that EZH2 and BMI1 may cooperate in initiation and progression of hepatocellular carcinoma.

Autoimmune fulminant liver failure in adults: Experience in a Japanese center

Aim:  After the establishment of the international criteria of autoimmune hepatitis (AIH) in 1999 and the recognition of acute onset AIH, the diagnosis of patients with fulminant type of AIH came to be made. We diagnosed autoimmune fulminant liver failure based on the criteria, and discussed the etiology of fulminant hepatitis (FH) and late onset hepatic failure (LOHF), and the characteristics of autoimmune fulminant liver failure.

Efficacy of combination therapy of antiviral and immunosuppressive drugs for the treatment of severe acute exacerbation of chronic hepatitis B

BackgroundPatients with severe exacerbation of chronic hepatitis B, sometimes developing into fulminant liver failure, are at high risk for mortality even with antiviral therapy. The efficacy of immunosuppressive therapy in clinically severe exacerbation of chronic hepatitis B has not been well demonstrated. In this study, we evaluated the efficacy of the early introduction of immunosuppressive therapy in combination with antiviral therapy in such patients.MethodsForty-two patients, 29 men and 13 women, were defined as having severe exacerbation of chronic hepatitis B based on our uniform criteria, and were enrolled in this study. Sixteen patients between 1982 and 1996 were analyzed retrospectively. We defined the criteria of severe disease in 1997, and then began to introduce sufficient doses of corticosteroids prospectively. Nucleoside analogs were administered in combination with corticosteroids after 1999. Twenty-six patients between 1997 and 2007 were analyzed prospectively.ResultsIn the retrospective study between 1982 and 1996, four of 16 (25%) patients recovered. In the prospective study between 1997 and 2007, 17 of 26 (65%) patients recovered; 15 of 17 patients treated with corticosteroids with or without antiviral drugs within 10 days after the diagnosis of severe disease recovered, none of five treated similarly but later than 10 days after the diagnosis recovered, and two of three treated with antiviral drugs recovered.ConclusionsThe early introduction of sufficient doses of corticosteroids and nucleoside analogs could be one option for reversing the potential deterioration of patients with clinically severe, life-threatening exacerbation of chronic hepatitis B.

Hepatitis B Virus e Antigen Downregulates Cytokine Production in Human Hepatoma Cell Lines

Disease activities of hepatitis B are affected by the status of hepatitis B e antigen (HBeAg). The function of the hepatitis B virus (HBV) precore or HBeAg is unknown. We assumed that HBeAg blocks aberrant immune responses, although HBeAg is not required for viral assembly, infection, or replication. We examined the interaction of HBeAg and the immune system, including cytokine production. The inflammatory cytokine TNF, IL-6, IL-8, IL-12A, IFN-α1, and IFN-ß mRNA were downregulated in HBeAg-positive HepG2, which stably expresses HBeAg, compared to HBeAg-negative HepG2 cells. The results of real-time RT-PCR-based cytokine-related gene arrays showed the downregulation of cytokine and IFN production. We also observed inhibition of the activation of NF-κB- and IFN-ß-promoter in HBeAg-positive HepG2, as well as inhibition of IFN and IL-6 production in HBeAg-positive HepG2 cell culture fluids. HBeAg might modify disease progression by inhibiting inflammatory cytokine and IFN gene expression, while simultaneously suppressing NF-κB-signaling- and IFNß-promoter activation.

Efficacy of high‐dose corticosteroid in the early stage of viral acute liver failure

Acute liver failure (ALF) is a worldwide problem despite its rare incidence because of its extremely high mortality. There are no beneficial therapies except for emergency liver transplantation for ALF. However, in Japan where the problem of a shortage of donor livers still remains, therapies other than transplantation must be further investigated for patients with ALF. Our aim was to elucidate the efficacy of high‐dose corticosteroid (CS) in decreasing liver enzyme levels in the early stage of ALF.

Importance of computed tomography imaging features for the diagnosis of autoimmune acute liver failure

Aim:  The diagnosis of acute liver failure due to autoimmune hepatitis is often difficult because of atypical clinicopathological features. Patients with autoimmune acute liver failure are sometimes resistant to immunosuppressive therapy and have poor prognosis. Although their survival rates are especially poor (5–20%) without liver transplantation in Japan, their clinicopathological features have remained uncertain. A major problem is that there is no gold standard for making the diagnosis of acute onset autoimmune hepatitis. If there are diagnosing tools supporting clinicopathological features, they are of benefit to the patients. We examined computed tomography (CT) imaging features of autoimmune acute liver failure to clarify the usefulness of imaging for the diagnosis.

Advanced histology and impaired liver regeneration are associated with disease severity in acute-onset autoimmune hepatitis.

Fujiwara K, Nakano M, Yasui S, Okitsu K, Yonemitsu Y & Yokosuka O
(2011) Histopathology58, 693–704
Advanced histology and impaired liver regeneration are associated with disease severity in acute‐onset autoimmune hepatitis

Quantification of hepatitis C virus in patients treated with peginterferon-alfa 2a plus ribavirin treatment by COBAS TaqMan HCV test.

Summary.  Extremely low levels of serum hepatitis C virus (HCV) RNA can be detected by COBAS TaqMan HCV test. To investigate whether the COBAS TaqMan HCV test is useful for measuring rapid virological response (RVR) and early virological response (EVR) to predict sustained virological response (SVR), we compared the virological response to PEG‐IFN‐alfa 2a plus RBV in 76 patients infected with HCV genotype 1 when undetectable HCV RNA by the COBAS TaqMan HCV test was used, with those when below 1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test was used, which corresponded to the use of traditional methods. Among the 76 patients, 28 (36.8%) had SVR, 13 (17.1%) relapsed, 19 (25.0%) did not respond, and 16 (21.0%) discontinued the treatment due to side effects. The positive predictive values for SVR based on undetectable HCV RNA by COBAS TaqMan HCV test at 24 weeks after the end of treatment [10/10 (100%) at week 4, 21/23 (91.3%) at week 8 and 26/33 (78.7%) at week 12] were superior to those based on <1.7 log IU/mL HCV RNA [17/19 (89.4%) at week 4, 27/38 (71.0%) at week 8, and 27/43 (62.7%) at week 12]. The negative predictive values for SVR based on <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test [46/57 (80.7%) at week 4, 37/38 (97.3%) at week 8, and 32/33 (96.9%) at week 12] were superior to those based on undetectable HCV RNA [48/66 (72.7%) at week 4, 46/53 (86.7%) at week 8, and 41/43 (95.3%) at week 12]. The utilization of both undetectable RNA and <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test is useful and could predict SVR and non‐SVR patients with greater accuracy.

Quantification of hepatitis C amino acid substitutions 70 and 91 in the core coding region by real-time amplification refractory mutation system reverse transcription-polymerase chain reaction.

Objective. The effects of hepatitis C virus (HCV) sequence variations on the success of antiviral therapy or the development of hepatocellular carcinoma (HCC) are complex for many reasons. Recently, there have been several reports on the effects of genotype 1b HCV core amino acid substitutions 70 and/or 91 on the outcome of antiviral therapies and the clinical course. The purpose of this study was to establish real-time amplification refractory mutation system (ARMS) reverse transcription (RT)-polymerase chain reaction (PCR) assays for easy detection of these HCV mutations. Material and methods. Plasmids p-core-W, including the wild-type HCV core coding region (70R and 91L), and p-core-M, including the mutant-type HCV core (70Q and 91M), were constructed by cloning and PCR-based mutagenesis for control vector of the wild-type core and that of the mutant core, respectively. Using serially diluted forms of these vectors, SyBr Green-based real-time ARMS RT-PCR detection with each of the specific primer pairs was performed. Results. Each primer could clearly distinguish the difference between p-core-W and p-core-M at the same copy numbers. Concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-W versus p-core-M could be distinguished, while for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. Conclusions. This method could be a useful alternative for the detection of genotype 1b HCV core amino acid substitutions 70 and 91 and be reliably applied for rapid screening.