Methyl-CpG Binding Domain Protein 2 Represses Transcription from Hypermethylated π-Class Glutathione S-Transferase Gene Promoters in Hepatocellular Carcinoma Cells

Abstract

During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the -class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.

Quantitative promoter methylation analysis of hepatocellular carcinoma, cirrhotic and normal liver

“…35 The expression of the glutathione S-transferase gene is transcriptionally silenced by GSTP1 promoter methylation as shown in many previous studies. 36,37 For GSTP1, there is a significantly increased methylation level in HCC compared to NLT and CT (NLT 0%, CT 32%, HCC 53%). Furthermore, for GSTP1, quantitative MSP revealed significantly different methylation levels for all 3 tissue types (HCC vs. CT, p 5 0.015; HCC vs. NLT, p < 0.001; CT vs. NLT, p 5 0.012), similar to the levels shown for p16 INK4a (Fig.…”

The role of glutathione-S-transferase in anti-cancer drug resistance

“…This alteration results in the loss of GSTp expression, and is proposed to occur during pathogenesis of the disease (Lee et al, 1994). Recently, a methyl-CpG-binding domain (MBD) protein that mediates hypermethylation of the GSTp (Bakker et al, 2002). These findings provide a possible target for restoration of GSTp activity.…”

Proteins that bind methylated DNA and human cancer: reading the wrong words

“…MBD2 has also been related to GSTP1 DNA methylationdependent silencing in hepatocellular carcinoma (Bakker et al, 2002). The CpG island of this gene becomes hypermethylated during the pathogenesis of human hepatocellular carcinoma and MBD2 appears bound to GSTP1 promoter region leading to gene silencing.…”