Noritoshi Koh

p16 Promoter Hypermethylation in Human Hepatocellular Carcinoma with or without Hepatitis Virus Infection

Background: Epigenetic alteration through methylation is one of the most important steps in carcinogenesis. However, the relation between hepatitis virus infection and epigenetic alterations is poorly understood. Methods: Sixteen patients without hepatitis B virus (HBV) and hepatitis C virus (HCV) and 35 patients with HBV or HCV who underwent liver resection for hepatocellular carcinoma (HCC) were studied. Mutation of p53 was detected by direct sequencing. Methylation status of p16 was evaluated in tumor and noncancerous liver tissues by methylation-specific polymerase chain reaction. Results: In HCC without HBV and HCV, p53 mutations were detected in 5 (31%) of 16 HCCs. Methylation of p16 promoter was detected in 2 (25%) of 8 moderately differentiated HCCs, 6 (75%) of 8 poorly differentiated HCCs, and none of 16 noncancerous tissue specimens. In HCC with HBV or HCV, p53 mutations were detected in 8 (23%) of 35 HCCs. Methylation of p16 promoter was detected in 2 (100%) of 2 well-differentiated HCCs, 13 (76%) of 17 moderately differentiated HCCs, 12 (75%) of 16 poorly differentiated HCCs, and 9 (26%) of 35 noncancerous liver tissue specimens. Conclusions: Our results suggest that hepatitis viruses might induce methylation of p16 promoter in liver with chronic inflammation, before appearance of HCC.

Correlation between clinical characteristics and mitochondrial D-loop DNA mutations in hepatocellular carcinoma.

BackgroundMitochondrial DNA (mtDNA) mutations are found in many kinds of human cancer. The aim of this study was to evaluate the relationship between mtDNA mutations in the liver and human hepatocarcinogenesis.MethodsDirect sequencing of mtDNA was done in 54 hepatocellular carcinomas (HCCs) and 47 surrounding liver tissue samples, obtained from 54 patients with HCC, and in 5 liver samples without inflammation, obtained from 5 patients with metastatic liver tumors. We also examined p53 mutations in the 54 HCCs to examine the correlation between nuclear DNA mutations and mtDNA mutations.ResultsMutations of mtDNA in the D-loop region were found in both HCC and noncancerous liver tissue. In normal liver without chronic inflammation, no mtDNA mutation was detected. In every case, the number of mtDNA mutations in HCC correlated with that in noncancerous liver tissue. Twelve of 52 mutation sites in the D-loop region of mtDNA were specific for HCC. The mean number of mtDNA mutations was 1.7 in well-differentiated HCC, as compared with 4.5 in moderately differentiated HCC and 4.6 in poorly differentiated HCC. The frequency of mtDNA mutations was thus higher in less differentiated HCC. We detected p53 mutations in 15 (28%) of 54 HCCs. The mean number of mtDNA mutations was 5.3 in HCC with p53 mutations and 3.8 in HCC with wild-type p53 (P = 0.024).ConclusionsA higher frequency of mtDNA mutations was found in less differentiated HCCs, and it is also possible that mtDNA mutations are related to nuclear DNA mutations in HCC. The accumulation of mtDNA mutations is a useful predictor of hepatocarcinogenesis.

Identification of genes differentially expressed by putrescine in HepG2 hepatoblastoma cells

In order to identify genes differentially expressed by putrescine, a polyamine, which play important roles in the regulation of cell proliferation and the development of cancer, we performed mRNA differential display analysis using total RNA extracted from HepG2 cells (human hepatoblastoma cell line) treated with a specific inhibitor of polyamine biosynthesis, alpha-difluorometylornithine (DFMO). A total of 25 genes were up-regulated and 32 genes down-regulated by putrescine. Of the genes differentially expressed by putrescine, we chose three that were related to the respiratory chain and oxidative phosphorylation and analyzed them by Northern blot analysis. Cytochrome oxidase subunit 1, low molecular mass ubiquinone-binding protein, and NADH dehydrogenase subunit 2 were found to be down-regulated by putrescine. We examined intracellular ATP level in HepG2 cells, and found that ATP level in DFMO-treated cells was increased by exogenous putrescine.