sodium-bisulfite and Liver-Neoplasms

NAD(P)H: quinone oxydoreductase 1 (NQO1) and glutathione S-transferase P1 (GSTP1) belong to phase II xenobiotic-metabolizing enzymes. GSTP1 inactivation via CpG island hypermethylation in hepatocellular carcinoma (HCC) was previously reported, but the involvement of NQO1 in HCC is not well known. In this study, we assessed the transcription and status of methylation of NQO1 gene in human hepatoma cells and primary human HCC tissues.. NQO1 transcription and DNA hypermethylation in hepatoma cells with or without 5-aza-deoxycytidine (5-Aza-CdR) treatment were investigated by reverse-transcription PCR (RT-PCR), sodium bisulfite sequencing and methylation-specific PCR (MSP). The methylation status of NQO1 and GSTP1, and NQO1 mRNA in 44 HCC cases was also analyzed by MSP and real-time PCR, respectively.. NQO1 transcription was down-regulated and the CpG island DNA was hypermethylated in Hep3B and HuH6 cells. After treatment with 5-Aza-CdR, NQO1 transcription was restored and CpG island DNA was demethylated in these cells. MSP analysis revealed that NQO1 hypermethylation occurred in 50.0% of HCC. All of the tumors that exhibited lesser amounts of NQO1 mRNA than corresponding non-tumorous tissues showed NQO1 hypermethylation.. NQO1 transcription might be inappropriately suppressed by promoter hypermethylation in a subset of HCC, as well as GSTP1 gene.

Topics: Acetylation; Aged; Aged, 80 and over; Antioxidants; Carcinoma, Hepatocellular; Cell Line, Tumor; CpG Islands; DNA Methylation; Female; Glutathione Transferase; Histones; Humans; Liver Neoplasms; Male; Middle Aged; NAD(P)H Dehydrogenase (Quinone); Promoter Regions, Genetic; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Sulfites; Transcription, Genetic

Growth arrest DNA damage-inducible gene 45 beta (GADD45beta) has been known to regulate cell growth, apoptotic cell death, and cellular response to DNA damage. Down-regulation of GADD45beta has been verified to be specific in hepatocellular cancer (HCC) and consistent with the p53 mutant, and degree of malignancy of HCC. This observation was further confirmed by eight HCC cell lines and paired human normal and HCC tumor tissues by Northern blot and immunohistochemistry. To better understand the transcription regulation, we cloned and characterized the active promoter region of GADD45beta in luciferase-expressing vector. Using the luciferase assay, three nuclear factor-kappaB binding sites, one E2F-1 binding site, and one putative inhibition region were identified in the proximal promoter of GADD45beta from -865/+6. Of interest, no marked putative binding sites could be identified in the inhibition region between -520/-470, which corresponds to CpG-rich region. The demethylating agent 5-Aza-dC was used and demonstrated restoration of the GADD45beta expression in HepG2 in a dose-dependent manner. The methylation status in the promoter was further examined in one normal liver cell, eight HCC cell lines, eight HCC tissues, and five corresponding nonneoplastic liver tissues. Methylation-specific polymerase chain reaction and sequencing of the sodium bisulfite-treated DNA from HCC cell lines and HCC samples revealed a high percentage of hypermethylation of the CpG islands. Comparatively, the five nonneoplastic correspondent liver tissues demonstrated very low levels of methylation. To further understand the functional role of GADD45beta under-expression in HCC the GADD45beta cDNA constructed plasmid was transfected into HepG2 (p53 WT) and Hep3B (p53 null) cells. The transforming growth factor-beta was assayed by enzyme-linked immunosorbent assay, which revealed a decrease to 40% in transfectant of HepG2, but no significant change in Hep3B transfectant. Whereas, Hep3B co-transfected with p53 and GADD45beta demonstrated significantly reduced transforming growth factor-beta. The colony formation was further examined and revealed a decrease in HepG2-GADD45beta transfectant and Hep3B-p53/GADD45beta co-transfectant. These findings suggested that methylation might play a crucial role in the epigenetic regulation of GADD45beta in hepatocyte transformation that may be directed by p53 status. Thus, our results provided a deeper understanding of the molecular mechanism of

Topics: Apoptosis; Azacitidine; Binding Sites; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line, Tumor; Cloning, Molecular; CpG Islands; Decitabine; DNA; DNA Damage; DNA Methylation; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Enzyme-Linked Immunosorbent Assay; GADD45 Proteins; Genes, p53; Genes, Reporter; Genetic Vectors; Humans; Intracellular Signaling Peptides and Proteins; Liver; Liver Neoplasms; Luciferases; Mutation; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Proteins; Sulfites; Transfection; Tumor Suppressor Protein p53

The human Ras association domain family 1A gene (RASSF1A) is a newly isolated tumor suppressor gene. In this study, we analyzed the methylation status of the promoter region of RASSF1A using bisulfite sequencing and PCR-RFLP in four liver cancer cell lines (Hep3B, HepG(2), SK-HEP-1, and Huh-7) and a cohort of 43 hepatitis B virus-associated hepatocellular carcinoma (HCC) tissues and their corresponding nontumor tissue specimens.. The methylation of the CpG islands in the RASSF1A promoter was not detected in 4 samples of normal liver tissue or 10 samples of peripheral blood mononuclear cells from normal subjects. However, the CpG islands were completely methylated, and transcription of the RASSF1A was silenced in the four cell lines. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the expression of RASSF1A in the Hep3B and HepG2 cells. In 41 of 43 (95%) HCC specimens studied, the promoter region of RASSF1A was intensively methylated at its CpG sites. Although heterogeneous methylation was also detected in 16 of the 23 (70%) corresponding nontumorous tissues analyzed, the level of methylation was significantly lower than in the corresponding tumor tissues.. HCC has the highest incidence of promoter methylation of RASSF1A among all malignancies yet reported suggesting that hypermethylation of the CpG island promoter of RASSF1A may play an important pathological role in this tumor.

Topics: Adult; Aged; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cohort Studies; CpG Islands; DNA Methylation; Female; Genes, Tumor Suppressor; Hepatitis B virus; Humans; Liver Neoplasms; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sulfites; Tumor Suppressor Proteins

The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53. Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC). The methylation status of the 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. The 14-3-3 sigma protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 sigma expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 sigma expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 sigma gene in HCC. Hypermethylation and the resulting loss of expression of the 14-3-3 sigma gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC.

Topics: 14-3-3 Proteins; Azacitidine; Base Sequence; Carcinoma, Hepatocellular; CpG Islands; Cytidine Triphosphate; Cytoplasm; DNA Methylation; DNA Mutational Analysis; Gene Silencing; Humans; Immunohistochemistry; Liver; Liver Neoplasms; Polymorphism, Single-Stranded Conformational; Sulfites; Tyrosine 3-Monooxygenase