Temporal and Quantitative Analysis of 5-Aza-2'-Deoxycytidine-Mediated Effects on Methylation and Gene Expression
Abstracts
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Introduction: Aberrant promoter methylation is associated with transciptional silencing. By inhibiting DNA methyltransferase, the chemotherapeutic agent, 5-aza-2'-deoxycytidine (5AZ) suppresses methylation and is able to reactivate methylation-mediated gene suppression. However, a quantitative relationship between 5AZ-mediated demethylation and gene expression has been difficult to establish. The Hyperplastic Polyposis Protein-1 (HPP1) is a novel gene that is frequently hypermethylated in colorectal neoplasms and in gastric cancer cell lines. Using real-time PCR, we performed a temporal and quantitative analysis of HPP1 DNA methylation and expression following treatment of AGS gastric cancer cells with 5AZ. Methods: AGS cells were exposed to 1.0 µM of 5AZ for 24 hours. Real-time methylation specific PCR (MSP) was performed on bisulfite-modifed DNA extracted from cells at pre-treatment, immediately post-treatment, and at days 2, 4 and 6. Concomitant determination of HPP1 expression was performed at the same intervals by real-time reverse transcriptase PCR. Levels of methylated HPP1 DNA and HPP1 mRNA were compared. Results: A direct temporal and quantitative correlation was demonstrated between HPP1 methylation and mRNA expression (Fig. 1). Maximal demethylation and HPP1 re-expression were noted at day 4 post-5AZ. Conclusions: Treatment of AGS cells with 5AZ results in temporally progressive HPP1 gene demethylation and is associated with re-expression of HPP1. These findings suggest that promoter methylation and associated changes in gene expression can occur in a continuous and gradual fashion. |
