FIG. 4.

Effects of 5-Aza-CdR on the demethylation and transcription of the p16 promoter and exon 2, PAX-6 exon 5, c-ABL exon 11, and MYF-3 exon 3 in T24 cells. Average methylation values for specific sites within the p16, PAX-6, c-ABL, and MYF-3 exonic CpG islands were measured in T24 cells prior to drug treatment (black bars) and 72 h after treatment with 5 × 10⁻⁷ M 5-Aza-CdR (white bars). Error bars indicate the ranges of values obtained. Relative transcription levels of each gene were also estimated by comparison of band intensities (−, +, or ++) before and after drug treatment by RT-PCR analysis (data not shown). The CpG and GC contents of these regions were also analyzed to determine if these exonic sequences fulfilled the criteria of CpG islands in which a DNA sequence of ≥200 bp must have a GC content of ≥0.50 and an observed/expected CpG ratio of ≥0.60 (12). Fragments of 800 bp from each gene, all of which fulfilled the established criteria for CpG islands, were analyzed. In untreated T24 cells, transcription through the p16 promoter and exon 2 CpG islands is not initiated from the p16 promoter but is initiated from the upstream p14 promoter (38). Pro., promoter.