Fig. 5.

EGCG induces demethylation at E2F-1 binding sites in the hTERT promoter and increases E2F-1 binding capacity. MCF-7 treated cells were assayed for hTERT promoter methylation. Sodium bisulfite conversion was used to study 37 CpG sites within the hTERT promoter (indicated by boxes). Nested PCR using primers specific for converted hTERT DNA were used: first round sense, 5′-ATTTGGAGGTAGTTTTGGGTT-3′; first round anti-sense, 5′-AACCACCAACTCCTTCAAA-3′. For second round sense, 5′-GTTTTTAGGGTTTTTATATTATGG-3′; second round anti-sense, 5′-CACACCAAACACTAAACCACCA-3′. A: As shown, EGCG caused hypomethylation at both the distal and proximal E2F-1 binding sites containing two and three CpG sites, respectively. The gray shaded boxes represent undetermined methylation status and possibly result from partial methylation at these sites. B: Gel electrophoresis showing an increase in E2F-1 binding at the hTERT promoter. C: Histogram illustrating the % binding of E2F-1.