Transcriptional Silencing of Cyclooxygenase-2 by Hyper-methylation of the 5′ CpG Island in Human Gastric Carcinoma Cells

SH Song, HS Jong, HH Choi, H Inoue, T Tanabe… - Cancer research, 2001 - AACR
SH Song, HS Jong, HH Choi, H Inoue, T Tanabe, NK Kim, YJ Bang
Cancer research, 2001AACR
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 …
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-1β 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.
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