Many tumor suppressor genes are known to be inactivated by epigenetic modifications including DNA methylation and histone deacetylation. RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase (MMP) activity and inhibit tumor metastasis. Our previous study demonstrated that oncogenic ras inhibited RECK expression via Sp1 binding site in the RECK promoter by histone deacetylation mechanism. In this study, we tried to characterize the molecular pathway that mediates the inhibitory effect of ras on RECK. Methylation specific PCR (MSP) indicated that RECK gene promoter is hypermethylated in ras-activated 2-12 cells. We also tested whether ras activation induced the binding of DNA methyltransferases (DNMTs) to Sp1 to repress RECK expression. Our data showed Sp1-associated DNMT3b in cells was increased after ras induction. By using DNA affinity precipitation assay (DAPA) and chromatin immunoprecipitation (ChIP) , we found that induction of oncogenic ras enhanced the binding of DNMT3b to the Sp1 site in the RECK promoter in vitro and in vivo. Additionally, treatment of DNMT inhibitor 5'-azacytidine led to the re-expression of RECK in ras-activated 2-12 cells. The signaling pathway by which ras suppresses RECK was also addressed. Chemical inhibitor of ERK signaling pathway U0126 reversed the methylation of RECK promoter and up-regulated RECK expression in ras-activated 2-12 cells. More importantly, 5'-azacytidine and DNMT3b siRNA may suppress the invasive ability of 2-12 cells. Taken together, our results suggest that oncogenic ras inhibit the metastasis suppressor gene RECK via a DNA methylation mechanism and provide valuable insights for the understanding of the mechanisms by which ras promotes tumor invasion and metastasis
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0721106-102443 |
| Date | 21 July 2006 |
| Creators | Cho, Chun-yu |
| Contributors | Huang-chou Chang, Ming-Hong Tai¡@, Wen- chun Hung |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0721106-102443 |
| Rights | withheld, Copyright information available at source archive |
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