Occurrence of hepatocellular carcinoma was not a rare event during and immediately after antiviral treatment in Japanese HCV-positive patients.

Advanced chronic hepatitis C patients with sustained virolological response by antivirals remain at risk for hepatocellular carcinoma (HCC). We investigated the incidence of HCC during and immediately after peginterferon-alfa-2a and ribavirin (RBV) treatment in patients with chronic hepatitis C in Japan. HCC was detected in 8 of 238 patients during and after these treatments (mean follow-up period: 572 ± 252 days). In conclusion, occurrence of HCC is not a rare event during and immediately after peginterferon-alfa-2a plus RBV treatment. In cases with cirrhosis, higher α-fetoprotein levels, old age, or a previous history of HCC treatment, clinicians should be especially alert for the possible development of HCC during and immediately after peginterferon-alfa-2a and RBV treatment. Clinicians should regularly check for the possible development of HCC even in chronic hepatitis C patients under treatment.

Long-term follow-up of patients with hepatitis B e antigen negative chronic hepatitis B.

BACKGROUND AND AIM: After hepatitis B virus (HBV) e antigen (HBeAg) seroconversion, HBV-DNA continues to replicate, and HBeAg-negative patients still face the risk of liver disease progression. We investigated the predictive factors for alanine aminotransferase (ALT) elevation, antiviral drug use, and hepatocellular carcinoma (HCC) occurrence in HBeAg-negative patients. METHODS: Age, sex, ALT, platelet counts, HBV-DNA levels, genotype, antidiabetic drug use, body mass index, smoking, and alcohol consumption were analyzed for a total of 244 HBV carriers who were HBeAg-negative. RESULTS: Of 244 HBeAg-negative patients, 158 (64.8%) showed normal ALT levels at baseline. Multivariate Cox hazard regression analysis identified high HBV-DNA levels and high ALT at baseline as independent risk factors for ALT elevation in the patients with normal ALT at baseline. The threshold ALT and HBV-DNA levels were determined to be 31 IU/L and 5.3 log copies/mL, respectively. Seventeen (7.0%) patients used antiviral drugs. Multivariate Cox hazard regression analysis identified high HBV-DNA levels (threshold, 5.7 log copies/mL), the use of antidiabetic drugs, and daily alcohol consumption at baseline as an independent risk factor for the use of antiviral drugs in HBeAg-negative patients. In 10 patients (4.1%), HCC was detected, and a low platelet count (threshold, 10.0×10(4)/mm(3)) was associated with the occurrence of HCC. CONCLUSION: This study identified predictors of future active liver disease in HBeAg-negative patients, i.e. ALT elevation, unavoidable use of antiviral drugs, and occurrence of HCC.

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.

[Prevention of hepatitis B virus reactivation in B-cell lymphoma patients receiving chemotherapy with rituximab].

Reactivation of hepatitis B virus (HBV) has been recognized as one of the most serious complications in patients receiving chemotherapy with rituximab. From October 2007 to December 2008, rituximab was administered to 123 B-cell lymphoma patients in our institute. Four patients with positive hepatitis B surface antigen (HBsAg) received preemptive entecavir, and none of them developed HBV reactivation. For 26 patients whose hepatitis B surface antibody (HBsAb) and/or hepatitis B core antibody (HBcAb) were positive, HBV-DNA was monitored for one year after completion of chemotherapy. During this period, HBV reactivation was observed in two patients. Hepatitis was prevented in one patient by the administration of entecavir at the time HBV-DNA turns positive. Another developed de novo hepatitis B due to failure of monitoring. Preemptive entecavir for HBsAg positive patients and HBV-DNA monitoring for HBsAb and/or HBcAb positive patients seem to be effective.

BCL2L10 is frequently silenced by promoter hypermethylation in gastric cancer.

In gastric cancer, several tumor suppressor and tumor-related genes are silenced by aberrant methylation. Previously, we demonstrated that BCL2L10, which belongs to the pro-apoptotic Bcl-2 family, was transcriptionally repressed by promoter hypermethylation and that its overexpression caused apoptosis and growth inhibition of gastric cancer cells. In this study, we investigated the methylation status of BCL2L10 and its expression in 21 gastric cancer tissues and corresponding non-neoplastic mucosae along with the methylation status of p16, RUNX3, and hMLH1 genes by using methylation specific PCR. In addition, we examined the association between the methylation status of each gene and the expression of EZH2, which was associated with DNA methylation of its target genes. As a result, aberrant methylation of BCL2L10 was detected in 38% of gastric cancer and in 24% of corresponding non-neoplastic mucosae and correlated with low expression of BCL2L10. Methylation of p16, RUNX3, and hMLH1 was found in gastric cancer and in corresponding non-neoplastic mucosae at almost similar frequencies as previous reports. Expression of EZH2 was detected more frequently in tumors (48%) as compared to corresponding non-neoplastic mucosae (10%) (p=0.006), however, no significant difference was found between expression of EZH2 and the methylation frequency of each gene. In conclusion, our data suggest that silencing of BCL2L10 by aberrant methylation is a common feature in gastric cancer and its inactivation may be involved in the early steps of gastric carcinogenesis.

Risk of hepatocellular carcinoma in patients with chronic hepatitis B virus infection.

OBJECTIVE: To determine the risk factors for the occurrence of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) infection. MATERIAL AND METHODS: A total of 620 patients who tested positive for hepatitis B surface antigen and were referred to Chiba University Hospital between February 1985 and March 2008 were included in the study and the following characteristics were analyzed: age, gender, status of hepatitis B e antigen, alanine aminotransferase level, HBV DNA level, and number of platelets (PLTs). RESULTS: HCC was detected in 30 cases during the follow-up period (5.4 +/- 5.1 years). Multivariate analysis revealed that age > 40 years [compared with patients aged < 40 years; odds ratio (OR) = 4.28; 95% confidence interval (CI) = 1.68-10.9] and PLT level < 206,000/microl (compared with patients with a higher PLT level; OR = 8.50; 95% CI = 1.98-36.2) were predictive factors for HCC occurrence. In patients aged > 40 years, the HBV DNA level (compared with < 5.0 log copies/ml; OR = 4.22, 95% CI = 1.13-15.8) and PLT level (compared with patients with > 196,000/microl PLTs; OR = 15.6, 95% CI = 2.06-118.3) were predictive factors for HCC occurrence. CONCLUSIONS: Advanced age and low PLT level were risk factors for HCC occurrence in patients with HBV infection. In patients aged > 40 years, viral load was also a risk factor for HCC.

Association between mutations in the core region of hepatitis C virus genotype 1 and hepatocellular carcinoma development.

BACKGROUND & AIMS: To determine whether amino acid mutations in the core region of hepatitis C virus (HCV) genotype 1 are associated with response to interferon (IFN) therapy and development of hepatocellular carcinoma (HCC). METHODS: We followed up 361 patients (median duration, 121 months), and IFN monotherapy was administered to 275 (76%) [sustained virological response (SVR) rate, 26.5%]. Using pretreatment sera, mutations at core residues 70 and 91 were analyzed [double wild (DW)-type amino acid pattern: arginine, residue 70; leucine, residue 91]. RESULTS: A low aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio and low HCV load were independently associated with SVR, but core mutations were not. During follow-up, 12 of 81 (14.8%) patients with the DW-type pattern and 52 of 216 (24.1%) patients with non-DW-type pattern developed HCC (p=0.06, Breslow-Gehan-Wilcoxon test). Multivariate analysis with the Cox proportional-hazards model revealed the following independent risk factors for HCC: male gender [p<0.0001; risk ratio (RR), 3.97], older age (p<0.05; RR, 2.08), advanced fibrosis (p<0.0001; RR, 5.75), absence of SVR (p<0.01; RR, 10.0), high AST level (p<0.01; RR, 2.08), high AST/ALT ratio (p<0.01; RR, 2.21), and non-DW-type pattern (p<0.05; RR, 1.96). In patients with F0-F2 fibrosis at entry, non-DW-type was likely to lead to cirrhosis (p=0.051). CONCLUSIONS: In HCV genotype 1 patients, HCC risk could be predicted by studying core mutations, response to IFN, and host factors like age, gender, and liver fibrosis.

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.

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.

[Hepatitis B virus reactivation after cessation of prophylactic lamivudine therapy in B-cell lymphoma patients treated with rituximab combined CHOP therapy].

Here we report three cases of hepatitis B virus (HBV) reactivation after cessation of preemptive lamivudine therapy in B-cell lymphoma patients treated with rituximab plus CHOP (R-CHOP). Two patients received eight cycles of R-CHOP, and one received two cycles of R-CHOP followed by two courses of rituximab. As all the patients were HBV surface antigen (HBsAg) positive, lamivudine was administered simultaneously with R-CHOP to prevent virus reactivation. All the patients developed hepatitis due to HBV reactivation 6, 8 and 13 months after completion of chemotherapy, and 4, 2 and 2 months after cessation of lamivudine, respectively. They were treated with either lamivudine or entecavir and all achieved full recovery. When HBV carriers undergo immunosuppressive anticancer treatment, prophylactic antiviral therapy is well recognized as effective. However, the optimal method of prophylaxis has not yet been established. Since the introduction of rituximab, new problems such as delayed HBV reactivation from HBsAg positive patients and de novo hepatitis B from HBsAg negative patients have emerged. Guidelines for prophylactic antiviral therapy in the era of rituximab need to be established.

Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma.

PURPOSE: Hedgehog (Hh) signaling is activated in several cancers. However, the mechanisms of Hh signaling activation in hepatocellular carcinoma (HCC) have not been fully elucidated. We analyzed the involvement of Hh-interacting protein (HHIP) gene, a negative regulator of Hh signaling, in HCC. EXPERIMENTAL DESIGN: Glioma-associated oncogene homologue (Gli) reporter assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and quantitative real-time reverse transcription-PCR for the target genes of the Hh signals were performed in HHIP stably expressing hepatoma cells. Quantitative real-time PCR for HHIP was performed in hepatoma cells and 36 HCC tissues. The methylation status of hepatoma cells and HCC tissues was also analyzed by sodium bisulfite sequencing, demethylation assay, and quantitative real-time methylation-specific PCR. Loss of heterozygosity (LOH) analysis was also performed in HCC tissues. RESULTS: HHIP overexpression induced significant reductions of Gli reporter activity, cell viability, and transcription of the target genes of the Hh signals. HHIP was hypermethylated and transcriptionally down-regulated in a subset of hepatoma cells. Treatment with a demethylating agent led to the HHIP DNA demethylation and restoration of HHIP transcription. HHIP transcription was also down-regulated in the majority of HCC tissues, and more than half of HCC tissues exhibited HHIP hypermethylation. The HHIP transcription level in HHIP-methylated HCC tissues was significantly lower than in HHIP-unmethylated HCC tissues. More than 30% of HCC tissues showed LOH at the HHIP locus. CONCLUSIONS: The down-regulation of HHIP transcription is due to DNA hypermethylation and/or LOH, and Hh signal activation through the inactivation of HHIP may be implicated in the pathogenesis of human HCC.

Body mass index in Japanese patients with autoimmune liver disease: overweight patients with primary biliary cirrhosis tend to be asymptomatic.

BACKGROUND/AIMS: The aim of this study was to investigate the effects of being overweight on autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) patients. METHODOLOGY/RESULTS: 44 AIH and 95 PBC patients were enrolled in this study. Body weight and body mass index (BMI) of AIH (57.6 +/- 10.4 kg and 23.8 +/- 2.9 kgm(-2), respectively) were higher than those of PBC (51.6 +/- 7.0 kg and 22.0 +/- 2.6 kgm(-2), respectively) (P < 0.001). The prevalence of overweight patients in AIH was also higher than those in PBC (P < 0.005). Being overweight and having 25 < or = BMI < 30 did not affect the progression of hepatic fibrosis in AIH and PBC. In comparison with the non-overweight with PBC, overweight patients with PBC tended not to be symptomatic, such as having itching or fatigue (P = 0.027). CONCLUSIONS: Clinicians should be aware that not only non-alcoholic fatty liver disease but also PBC patients might be included among the overweight hepatic disease patients with unknown etiology.

Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis and hepatocellular carcinoma.

Hepatitis B virus (HBV) genotype C and the basic core promoter (BCP) mutations were reported to be associated with the development of hepatocellular carcinoma (HCC). In this study the full sequences of HBV genomes were analyzed in order to find the other predictors of HCC development. We determined the full sequences of HBV genomes in 24 genotype C carriers who developed HCC (HCC group) at the beginning of follow-up and at the time of HCC diagnosis, and 20 patients who did not develop HCC (non-HCC group) served as a control. The number of nucleotide and amino acid substitutions in most regions was higher in the HCC group than in the non-HCC group, and the following substitutions and deletions were found more frequently in the HCC group than in the non-HCC group: G1317A and T1341C/A/G in the X promoter region were detected in 13 and six of the HCC cases, four and none of the non-HCC cases, respectively; and pre-S2 deletion was detected in eight HCC and none of the non-HCC cases. Compared with the wild type X promoter, the mutant type X promoters, M1 (G1317A), M2 (T1341C), and M4 (T1341G) showed increases in activity of 2.3, 3.8, and 1.4 times, respectively, in HepG2 cells. Substitutions and deletion of nucleotides of the HBV genome, especially the pre-S2 deletion and G1317A and T1341C/A/G mutations may be useful markers for predicting the development of HCC.

Inhibition of subgenomic hepatitis C virus RNA replication in HeLa cells.

BACKGROUND/AIMS: Antiviral therapy such as combination interferon and ribavirin can eradicate hepatitis C virus (HCV) RNA by up to 40-50%. However, many patients still remain non-responders to this treatment for various reasons. The aim of this study was to evaluate the effect of interferon or ribavirin treatment on subgenomic HCV RNA replication in 'non-hepatic' HeLa cells. METHODOLOGY: Huh-7 or HeLa cells harboring HCV replicon were constructed by using cellular RNA of Huh-7 harboring HCV replicon RNAs, named as C13-3 cells. We also tested whether interferon or ribavirin can suppress HCV RNA in HeLa cells. RESULTS: Huh-7 or HeLa cells harboring HCV replicon RNAs were constructed by using cellular RNA of C13-3 cells than using in vitro-transcribed RNA. Ribavirin at 1 microg/mL or 10 microg/mL did not suppress colony formation in HeLa cells, but at 100 microg/mL suppression was observed. Interferon-alpha 2b suppressed HCV replication even at 1 U/mL. CONCLUSIONS: HeLa cells harboring HCV replicon RNAs also might be useful for the development of antiviral drugs.

Procaine inhibits the proliferation and DNA methylation in human hepatoma cells.

PURPOSE: Transcriptional silencing of tumor suppressor genes associated with DNA hypermethylation has been known as a hallmark of human cancer. In this study, we revealed that a local anesthetic, procaine (PCA), possessed growth-inhibitory and demethylating effect on human hepatoma cells in vitro and in vivo. METHODS: The viability of PCA-treated cells with or without trichostatin A (TSA) was investigated. To clarify the mechanism of the antiproliferating effect of PCA, TUNEL assay, FACS analysis, and morphological observation of PCA-treated cells were performed. The expression levels and epigenetic alterations of 4 genes inactivated by DNA hypermethylation in hepatocellular carcinoma (HCC) were examined in hepatoma cells with or without PCA treatment. The growth-inhibitory and demethylating effect of PCA in vivo was tested in nude mice bearing xenograft. RESULTS: The viability of HLE, HuH7, and HuH6 cells was significantly decreased by PCA treatment. In these cells, the combination treatment with TSA and PCA exhibited stronger reduction of the viability. Inhibition of S/G2/M transition, morphological changes such as vacuolation and no increase in apoptosis rate were observed in the PCA-treated HLE cells. All the genes transcriptionally suppressed by DNA hypermethylation were demethylated and reactivated with PCA treatment. PCA treatment led to partial demethylation and significant reduction in tumor volume in vivo. CONCLUSIONS: These data indicated that PCA had growth-inhibitory and demethylating effects on human hepatoma cells in vitro and in vivo. PCA may be a candidate agent for future therapies for HCC.

Nucleotide change of codon 38 in the X gene of hepatitis B virus genotype C is associated with an increased risk of hepatocellular carcinoma.

BACKGROUND/AIMS: The hepatitis B virus (HBV) genotype C is associated with the development of hepatocellular carcinoma (HCC). In addition, the HBV X gene, which encodes the pleiotropic transactivator HBx, has also been associated with the development of HCC. In this study, we investigated whether nucleotide changes in the X gene of genotype C are associated with the development of HCC. METHODS/RESULTS: We sequenced the X gene in age- and sex-matched 39 HBV-infected patients with HCC and 36 HBV-infected patients without HCC. A novel nucleotide change that resulted in a proline to serine substitution at codon 38 in HBx (codon-38 change) was preferentially found in patients with HCC. Then, sera were collected from a new group of age- and sex-matched 52 patients with HCC and 51 patients without HCC. In this cohort also, the codon-38 change was associated with HCC. Multiple logistic regression analysis showed the prevalence of the codon-38 change was significantly associated with HCC in all patients (P=0.001, odds ratio: 4.89). CONCLUSION: The codon-38 change in genotype C is an independent risk factor for the development of HCC and may serve as a useful molecular marker for predicting the clinical outcomes in patients infected with HBV.

Sequential gene expression changes in cancer cell lines after treatment with the demethylation agent 5-Aza-2'-deoxycytidine.

BACKGROUND: 5-Aza-2'-deoxycytidine (5-AzaC) is known well for its demethylation effect and is a promising anticancer agent. However, to the authors' knowledge, serial changes in gene expression over time after 5-AzaC treatment have not been studied to date. To clarify the categories of genes that are up-regulated or down-regulated after 5-AzaC treatment, the authors surveyed the genes that had expression levels changed by 5-AzaC treatment in 6 hepatoma cell lines (Hep3B, HLE, Huh7, HepG2, PLC/PRF/5, and Huh6). METHODS: Cell lines were grown in medium that contained 1 microM of 5-AzaC. Changes in messenger RNA levels were monitored from 24 hours up to 120 hours after 5-AzaC treatment using an in-house microarray that consisted of 4608 combinational DNAs. Using clustering analysis to identify the genes that had gradually changed expression levels and to exclude the substantial experimental noise by microarray analysis, the authors focused on 206 up-regulated genes and 248 down-regulated genes. RESULTS: According to their functional characterization, genes that were involved in the cytoskeleton and the extracellular matrix were enriched significantly in the up-regulated genes. Conversely, genes that were involved in metabolism were enriched significantly in the down-regulated genes. CONCLUSIONS: The current results demonstrated that 5-AzaC can regulate the expression of groups of genes with characteristic functions.

Analysis of genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine in gastric cancer cell lines.

In gastric cancer, increasing numbers of genes have been reported to be silenced by aberrant methylation. However, global analysis of epigenetic inactivation in cancer cells has rarely been performed. For screening the genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC), cDNA microarray analysis (AceGene(R), containing 30,000 genes) was performed using gastric cancer cell lines (AGS, MKN74, MKN1, MKN45 and Kato3) treated with DAC. The candidate upregulated genes were confirmed by real-time PCR, and the methylation status of 5'CpG islands was determined by bisulfite DNA sequencing or methylation-specific PCR. Among the upregulated genes considered to have CpG island in their promoter regions, we selected 5 genes (BCL2L10, DKK1, DNAJD1, GAGED2 and NMU) that exhibited a greater than 3-fold increase in at least 2 cell lines. Of these, we could determine the methylation status of 5'CpG islands of BCL2L10, DKK1 and DNAJD1. 5'CpG of BCL2L10 and DNAJD1 was hypermethylated in 4 of 5 gastric cancer cell lines, whereas 5'CpG of DKK1 was hypermethylated in only 1 cell line. MSP analysis for BCL2L10 revealed that the CpG island was demethylated after DAC treatment. In addition, we observed that overexpression of BCL2L10 could promote apoptosis and growth-inhibitory effect in gastric cancer cell lines. In conclusion, some of the genes upregulated by DAC treatment may be transcriptionally repressed by promoter hypermethylation. These genes might be related to gastric carcinogenesis. In particular, the suppression of BCL2L10, which could induce apoptosis and inhibit proliferation of cancer cells, might be one of the underlying mechanisms for gastric carcinogenesis.

Methylation status of genes upregulated by demethylating agent 5-aza-2'-deoxycytidine in hepatocellular carcinoma.

BACKGROUND/AIMS: To determine the clinical significance of gene promoter methylation in hepatocellular carcinoma (HCC), we examined in clinical samples the methylation status of those promoters that showed elevated activity in hepatoma cell lines after 5-aza-2'-deoxycytidine treatment. METHODS: Regarding the genes with promoter hypermethylation in the cell lines, their expression levels and methylation status in HCC and non-HCC tissues were assessed by semiquantitive RT-PCR and methylation-specific PCR. To confirm the result, the expression levels and methylation status in 16 additional HCC and non-HCC tissues were assessed. RESULTS: The promoter regions of caveolin 1 (CAV1), cysteine and glycine-rich protein 1 (CSRP1), Kruppel-like factor 6 (KLF6), myosin (light polypeptide 9) (MYL9), and transgelin (TAGLN) were highly methylated in the cell lines. CAV1 and CSRP1 were methylated in HCC more frequently than in non-HCC. KLF6, MYL9, and TAGLN were fully methylated in both HCC and non-HCC. Using additional clinical samples, downregulation of CAV1 and CSRP1 was observed in 38 and 56%, respectively, of the 16 HCC samples and aberrant methylation of CAV1 and CSRP1 was observed in 56% of HCC in both cases. CONCLUSION: CAV1 and CSRP1 were inactivated in HCC by aberrant methylation and they may serve as important biomarkers of malignancy.

Evaluation of clinical usefulness of second-generation HCV core antigen assay: comparison with COBAS AMPLICOR HCV MONITOR assay version 2.0.

BACKGROUND: Hepatitis C virus (HCV) is an important etiologic agent for chronic liver diseases. METHODS: The aim of this study was to evaluate the clinical usefulness of second-generation HCV core antigen assay by comparing the results of the assay with those of the COBAS AMPLICOR HCV MONITOR version 2.0 (COBAS v2.0). RESULTS: HCV core antigen was detectable by this assay in 142/149 (95.3%) of serotype 1 (3821+/-322 fmol/l; mean+/-SD), in 56/58 (96.6%) of serotype 2 (2589+/-449 fmol/l), and in 6/6 (100%) of serotypes 1+2 (1240+/-548 fmol/l). The HCV core antigen levels measured by this assay correlated well with the HCV RNA levels by COBAS v2.0 (r=0.848, P<0.0001). In relation to the outcome of interferon monotherapy, the pretreatment HCV core antigen levels of sustained and non-sustained virological responders were 659+/-189 and 4904+/-376 fmol/l in serotype 1, 1993+/-740 and 3145+/-519 fmol/l in serotype 2. The cutoff values with the best accuracy for HCV core Ag levels to discriminate between sustained and non-sustained virological response were 699 fmol/l for serotype 1 and 292 fmol/l for serotype 2, respectively, by receiver operating characteristic curve analysis. CONCLUSION: This new assay was considered to be useful in evaluating the HCV levels in patients with chronic hepatitis C.