AbstracthTERT, which encodes the catalytic subunit of telomerase and is expressed in most immortalized and cancer cells, has been reported to have increased DNA methylation in its promoter region in many cancers. This pattern is inconsistent with observations that DNA methylation of promoter CpG islands is typically associated with gene silencing. Here, we provide a comprehensive analysis of promoter DNA methylation, chromatin patterns, and expression of hTERT in cancer and immortalized cells. Methylation-specific PCR and bisulfite sequencing of the hTERT promoter in breast, lung, and colon cancer cells show that all cancer cell lines retain alleles with little or no methylation around the transcription start site despite being densely methylated in a region 600 bp upstream of the transcription start site. By real-time reverse transcription-PCR, all cancer cell lines express hTERT. Chromatin immunoprecipitation (ChIP) analysis reveals that both active (acetyl-H3K9 and dimethyl-H3K4) and inactive (trimethyl-H3K9 and trimethyl-H3K27) chromatin marks are present across the hTERT promoter. However, using a novel approach combining methylation analysis of ChIP DNA, we show that active chromatin marks are associated with unmethylated DNA, whereas inactive marks of chromatin are associated with methylated DNA in the region around the transcription start site. These results show that DNA methylation patterns of the hTERT promoter (À150 to +150 around the transcription start) are consistent with the usual dynamics of gene expression in that the absence of methylation in this region and the association with active chromatin marks allow for the continued expression of hTERT. [Cancer Res 2007;67(1):194-201]

Genetic and biochemical studies indicate that the evolutionarily conserved Swi/Snf complex acts at a subset of genes to help transcriptional activators function on chromatin templates. The mechanism by which this complex is targeted to specific chromosomal loci remains unknown. We show that Swi/Snf is required for expression of the yeast histone HTA1-HTB1 locus because of the role of Hir1p and Hir2p corepressors in negatively regulating transcription. Snf5p, Snf2p/Swi2p, and Swi3p, three components of the yeast Swi/Snf complex, coimmunoprecipitate with each Hir protein, and Snf5p is maximally associated with the HTA1-HTB1 promoter when the Hir-based repression system is intact and the Swi/Snf complex is functional. The data support a role for the Hir repressors in the gene-specific targeting of Swi/Snf.

Inhibitors of DNA methyltransferases (DNMT) and histone deacetylases can reactivate epigenetically silenced tumor suppressor genes and thereby decrease tumor cell growth. Little, however, is known on the effects of these compounds in endothelial cell biology and tumor angiogenesis. Here, we show that the DNMT inhibitors 5-aza-2V -deoxycytidine and zebularine markedly decrease vessel formation in different tumor models. We show that DNMT inhibitors are antiproliferative for tumor-conditioned endothelial cells, without affecting endothelial cell apoptosis and migration. Furthermore, these compounds inhibit angiogenesis in vitro and in vivo as shown by inhibition of endothelial cells sprouting in a three-dimensional gel and inhibition of microvessel formation in the chorioallantoic membrane, respectively. 5-Aza-2V -deoxycytidine, as well as the histone deacetylase inhibitor trichostatin A, reactivates the growth-inhibiting genes TSP1, JUNB, and IGFBP3, which are suppressed in tumor-conditioned endothelial cells. Despite enhanced DNMT activity and increased overall genomic methylation levels in tumorconditioned endothelial cells, silencing of these genes seemed not to be regulated by direct promoter hypermethylation. For IGFBP3, gene expression in endothelial cells correlated with histone H3 acetylation patterns. In conclusion, our data show that DNMT inhibitors have angiostatic activity in addition to their inhibitory effects on tumor cells. This dual action of these compounds makes them promising anticancer therapeutics. [Mol Cancer Ther 2006;5(2):467 -75]