title: Transcriptional silencing of Cyclooxygenase-2 by hyper-methylation of the 5' CpG island in human gastric carcinoma cells.
abstract: It has been well established that overexpression of Cyclooxygenase-2 (Cox-2) in epithelial cells inhibits apoptosis and increases the invasiveness of malignant cells, favoring tumorigenesis and metastasis. However, the molecular mechanism that regulates Cox-2 expression has not been well defined in gastric carcinoma. In this study, we examined whether the Cox-2 expression could be regulated by hyper-methylation of the Cox-2 CpG island (spanning from -590 to +186 with respect to the transcription initiation site) in human gastric carcinoma cell lines. By Southern analysis, we found that three gastric cells (SNU-601, -620, and -719) without Cox-2 expression demonstrated hyper-methylation at the Cox-2 CpG island. A detailed methylation pattern using bisulfite sequencing analysis revealed that all of the CpG sites were completely methylated in SNU-601. Treatment with demethylating agents effectively reactivated the expression of Cox-2 and restored IL-1beta sensitivity in the previously resistant SNU-601. By transient transfection experiments, we demonstrate that constitutively active Cox-2 promoter activities were exhibited even without an exogenous stimulation in SNU-601. Furthermore, when the motif of the nuclear factor for interleukin-6 expression site, the cyclic AMP response element, or both was subjected to point mutation, the constitutive luciferase activity was markedly reduced. In addition, Cox-2 promoter activity was completely blocked by in vitro methylation of all of the CpG sites in the Cox-2 promoter region with SssI (CpG) methylase in SNU-601. Taken together, these results indicate that transcriptional repression of Cox-2 is caused by hyper-methylation of the Cox-2 CpG island in gastric carcinoma cell lines.
authors.affiliation: Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799, Korea.
authors.forename: S H
authors.initials: SH
authors.lastname: Song
authors.forename: H S
authors.initials: HS
authors.lastname: Jong
authors.forename: H H
authors.initials: HH
authors.lastname: Choi
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: T
authors.initials: T
authors.lastname: Tanabe
authors.forename: N K
authors.initials: NK
authors.lastname: Kim
authors.forename: Y J
authors.initials: YJ
authors.lastname: Bang
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015139:Blotting, Southern; D002277:Carcinoma / Q000201:enzymology / Q000235:genetics; D002874:Chromosome Mapping; D018899:CpG Islands; D051546:Cyclooxygenase 2; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D004790:Enzyme Induction; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008565:Membrane Proteins; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics*; D013274:Stomach Neoplasms / Q000201:enzymology* / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D007527:Isoenzymes; D008565:Membrane Proteins; D000077209:Decitabine; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D001374:Azacitidine