Regulation of the p14ARF promoter by DNA methylation

Abstract

The INK4a/ARF locus encodes p14ARF which plays an important role in the p53 pathway. Interestingly, methylation of the INK4a/ARF locus is a common event in carcinogenesis. In this study we analyzed the effect of epigenetic alteration on the p14ARF promoter and its direct link to the expression of the p14ARF mRNA and protein. The osteosarcoma cell line U2OS was used as a model to study the regulation of the ARF promoter by DNA methylation. Treatment of U2OS cells with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) showed a marked induction of the p14ARF mRNA and protein. A novel quantitative method described here, using restriction enzyme digestion followed by real time PCR, allowed the analysis of the level of methylation over a defined region of DNA on the p14ARF promoter. The change in the methylation level of the promoter closely corresponded to the increase in the transcription of p14ARF mRNA and protein. Upon removal of 5-aza-CdR the methylation pattern on the p14ARF promoter was re-laid with a concomitant decrease in the levels of p14ARF mRNA and protein. The increase in the levels of p14 ARF was concomitant with an induction of G1-G2 cell cycle arrest and an induction of p21 protein. No increase in the levels of p53 was observed. However, induction of p14ARF upon treatment with 5-aza-CdR led to the sequestering of MDM2 to the nucleolus. Additionally, we could show a dependency between the demethylation of the p14ARF promoter, the induction of p14ARF mRNA and protein and the effect of 5-aza-CdR on cell cycle. ©2008 Landes Bioscience.