Fig. 3.

Hypermethylation of the AP-2alpha promoter region is associated with decreased expression of AP-2alpha in DU-145 and LNCaP prostate cancer cell lines. AP-2alpha expression can be restored by treatment of these cell lines with 5-aza-CdR. Western blot analysis of the levels of AP-2alpha and SP1 transcription factors in DU-145 and LNCaP prostate cancer cell lines: (A) the comparison with normal prostate epithelial cells RWPE-1; (B) DU-145 and LNCaP cells had been treated with 5 μM of 5-aza-CdR for 48 h followed by an additional 24 h of incubation in cell medium alone. (C) Schematic illustration of the AP-2alpha promoter region analyzed by bisulfite sequencing. The analyzed region is −654/−55 relative to the transcription start site of the indicated AP-2alpha transcript and corresponds to 10356126–10355525 nucleotide positions of the human genomic contig (NCBI accession # NT_007592.15). The NCBI genomic contig is represented schematically in the opposite direction for ease of depiction due to the fact that the AP-2alpha gene is encoded by the ‘minus’ chain. Each line crossing −654/−55 promoter region represents an individual CpG dinucleotide. (D) The bisulfite sequencing analysis of methylation of the AP-2alpha promoter region in DU-145, LNCaP and RWPE-1 cells. Each line represents an allele; each circle represents a CpG dinucleotide. Filled circles represent methylated CpG dinucleotides and clear circles represent unmethylated CpG dinucleotides. The 5′-flanking region of AP-2alpha gene is hypermethylated in DU-145 and LNCaP prostate cancer cell lines, whereas in normal prostate epithelial RWPE-1 cells, it remains unmethylated. (E) The bisulfite sequencing analysis was performed after DU-145 and LNCaP cells had been treated with 5 μM of 5-aza-CdR.