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. 2004 Feb;24(3):1270–1278. doi: 10.1128/MCB.24.3.1270-1278.2004

FIG. 3.

FIG. 3.

Effects of continuous zebularine treatment on methylation levels of the p16 locus. (A) Methylation levels of regions within the p16 gene locus in T24 bladder cancer cells before and after continuous treatment with zebularine as measured by Ms-SNuPE analysis. (Top) Eight regions of various CpG densities were identified upstream and downstream of the p16 promoter region (region 4) (41). Regions 1 and 2 are CpG-poor sequences (three CpGs analyzed in region 1 and four CpGs in region 2), region 3 is a CpG island containing an Alu repetitive element located upstream of the p16 promoter (three CpGs analyzed), regions 5 to 7 consist of three individual CpG dinucleotides residing in intron 1 of p16, and region 8 is the CpG island of the second exon (three CpGs analyzed). Bent arrow, transcription start site for the p16 gene; vertical arrows, specific CpG sequences analyzed by Ms-SNuPE. (Bottom) Methylation values for each region were measured by Ms-SNuPE in T24 cells, before and after continuous treatment with zebularine for 40 days. T24 cells were treated with 10−4 M zebularine for 40 continuous days and DNA and RNA were harvested immediately afterwards. (B) Levels of fully methylated (F), hemimethylated (H), and unmethylated (U) DNA at the first HpaII site of p16 intron 1 (region 5) after continuous treatment with 10−4 M zebularine. (C) Levels of fully methylated, hemimethylated, and unmethylated DNA at the first HpaII site of p16 exon 2 (region 8) after continuous treatment with 10−4 M zebularine. The ratio of H to F (H:F) represents the ratio of the percentage of hemimethylated molecules to the percentage of fully methylated molecules.