Endothelin (EDN) mRNA expression analysis in breast cell lines. (a) Alignment of the primary amino acid (aa) structure of human EDN1, EDN2 and EDN3 after post-translational cleavage to the biologically active 21-aa form . The secondary structure consists of single α-helices containing two disulphide bonds that hold them in a conical spiral shape, joining cysteins at positions 1–15 and 3–11 . EDN3 structure differs mainly in the NH2-terminal region from the structure of EDN1 and EDN2. This region forms a bulge out of the basic EDN structure and has been reported to represent a major domain for binding specificity to EDN receptors and thus is critical in endothelin signalling activity . Numbers indicate consecutive aa residues, and aa residues are indicated in universal single-letter aa code. (b) Real-time polymerase chain reaction comparing EDN3 expression with EDN1 and EDN2 expression. While EDN1 is overexpressed in cancerous breast cell lines (MCF7, SKBR3, MDA-MB231 and BT20), EDN3 expression is abrogated in the same malignant cells as compared with MCF12A cells (set to 1 in each diagram).