Anti-MBD1 monoclonal antibody, clone AB89534

To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5' CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in I out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of IS (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P < 0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (-300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors.

Epigenetic silencing of gluthathione-S-transferase pi (GSTP1) is recognized as being a molecular hallmark of human prostate cancer. We investigated the effects of green tea polyphenols (GTPs) on GSTP1 re-expression and further elucidated its mechanism of action and long-term safety, compared with nucleoside-analog inhibitor of DNA methyltransferase (DNMT), 5-aza-2'-deoxycitidine. Exposure of human prostate cancer LNCaP cells to 1-10 mu g/ml of GTP for 1-7 days caused a concentration- and time-dependent re-expression of GSTP1, which correlated with DNMT1 inhibition. Methyl-specific-PCR and sequencing revealed extensive demethylation in the proximal GSTP1 promoter and regions distal to the transcription factor binding sites. GTP exposure in a time-dependent fashion diminished the mRNA and protein levels of MBD1, MBD4 and MeCP2; HDAC 1-3 and increased the levels of acetylated histone H3 (LysH9/18) and H4. Chromatin immunoprecipitation assays demonstrated that cells treated with GTP have reduced MBD2 association with accessible Sp1 binding sites leading to increased binding and transcriptional activation of the GSTP1 gene. Exposure of cells to GTP did not result in global hypomethylation, as demonstrated by methyl-specific PCR for LINE-1 promoter; rather GTP promotes maintenance of genomic integrity. Furthermore, exposure of cells to GIP did not cause activation of the prometaststic gene S100P, a reverse response noted after exposure of cells to 5-aza-2'deoxycitidine. Our results, for the first time, demonstrate that GTP has dual potential to alter DNA methylation and chromatin modeling, the 2 global epigenetic mechanisms of gene regulation and their lack of toxicity makes them excellent candidates for the chemoprevention of prostate cancer.