EDN3 promoter methylation analysis in breast cell lines. (a) Prediction of CpG islands in EDN family genes. A 2-kb genomic nucleotide sequence of EDN1, EDN2 and EDN3 was analysed with MethPrimer software . A region of particularly high CpG density (red vertical bars) in the EDN3 nucleotide sequence proximal to the transcription start site (TSS) was identified as a CpG island (blue shaded). (b) Schematic representation of the EDN3 gene fragment that has been analysed for methylation. Arrows indicate hybridisation sites of methylation-specific polymerase chain reaction (MSP) primers, +1 indicates TSS, and vertical bars depict CpG dicnucleotides. (c) A dilution series of in vitro poly-methylated DNA with unmethylated DNA demonstrates the sensitivity of the applied MSP primers, which detect at least 1% of methylated DNA (M) in a background of unmethylated DNA in MSPs. (d) The EDN3 promoter is unmethylated in HMEC and non-malignant tissues. (e) EDN3 methylation analysis in breast cancer cell lines before (-) and after (+) treatment with demethylating (DAC) and histone reacetylating (TSA) drugs. In cell lines originally showing methylated EDN3 promoter alleles (MCF12A, MDA-MB231, MCF7 and BT20), a conversion of methylation was achieved as indicated by a gain of signal strength for non-methylation (U) and loss of signal strength for methylation (M), whereas in originally unmethylated cells (MCF10A and SKBR3), the treatment showed no effect. (f) EDN3 mRNA expression analysis as determined by real-time polymerase chain reaction before (-) and after (+) the demethylating treatment illustrates strong re-expression in those cell lines that were substantially demethylated (MCF7: 28-fold; MDA-MB231: 47-fold) as compared with cell lines showing weaker demethylation (BT20: 3-fold; MCF12A: 5-fold) or unmethylated cells (MCF10A: 1.6-fold; SKBR3: 1.0-fold). bp, base pairs; CpG, cytosine-phosphate-guanine dinucleotide; DAC, 5-aza-2'-deoxycytidine; EDN, endothelin; GC, guanine-cytosine content; HMEC, human mammary epithelial cells; PBL, peripheral blood lymphocyte; Plac, placental tissue; TSA, trichostatin A.