The induction of growth arrest DNA damage-inducible gene 45 beta in human hepatoma cell lines by S-adenosylmethionine.
Down-regulation of GADD45beta, which is known to influence cell growth control, apoptosis, and cellular response to DNA damage, has been verified to be specific in hepatocellular carcinoma and consistent with the degree of malignancy. Here, we identified promoter elements for several transcriptional factors in the proximal promoter of GADD45beta using the luciferase assay. As a methyl donor for biological transmethylation reactions, S-adenosylmethionine (SAMe) could restore GADD45beta expression in HepG2 in Northern blot analyses and quantitative real-time polymerase chain reaction. Activity and binding capacity of nuclear factor (NF)-kappaB were confirmed to be specifically induced by SAMe, as evidenced by electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and a decrease of IkappaBalpha in Western blot analyses. The most upstream NF-kappaB-binding site was crucial for transcriptional activation. In contrast to NF-kappaB, although there is an E2F-1-binding site adjacent to the NF-kappaB sites, treatment with SAMe could not induce E2F-1-binding activity. Despite showing a similar GADD45beta promoter regulatory pattern as HepG2 (p53 wild type), Hep3B (p53-null) did not exhibit GADD45beta induction by SAMe, and the induction could be partially recovered on reconstituting p53 in Hep3B. Thus, our results suggest that GADD45beta induction by SAMe via NF-kappaB may represent a novel mechanism of SAMe-mediated hepatoprotection, with p53 playing an important role.
Qiu
Weihua
W
Department of Clinical and Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 91010-3000, USA.
Zhou
Bingsen
B
Chu
Peiguo G
PG
Luh
Frank
F
Yen
Yun
Y
D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D016158:Genes, p53; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012436:S-Adenosylmethionine / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D000097642:GADD45 Proteins
D000943:Antigens, Differentiation; D051550:I-kappa B Kinase; D016328:NF-kappa B; D012436:S-Adenosylmethionine; C086470:GADD45B protein, human; D000097642:GADD45 Proteins