Methylation of the neutral endopeptidase gene promoter in human prostate cancers

Neutral endopeptidase 24.11 (NEP) is a cell surface peptidase expressed by prostatic epithelial cells that cleaves and inactivates neuropeptide growth factors implicated in the growth of androgen-independent prostate cancer ( PC). Decreased SEP expression in hormone-refractory metastatic PCs can resu lt from hormonal therapies because NEP transcription is induced by androgen s and down-regulated by androgen withdrawal. NEP is encoded by a gene that contains a 5' CpG island spanning a transcriptional regulatory region. In t his study, we investigate whether DNA hypermethylation of the NEP promoter accompanies decreased NEP expression in PC cell lines and whether it occurs in human PC tissues irt vivo. DNA isolated from PC cell lines and from nor mal and neoplastic human prostate tissues was restriction-digested with a m ethylation-sensitive restriction endonuclease and analyzed by Southern blot using a 5' sequence-specific NEP probe. Methylation-specific PCR was perfo rmed using PCR primers designed to discriminate between methylated and unme thylated alleles, and reverse transcription-PCR using NEP-specific primers was performed on cDNA extracted from PC cells treated with 5-aza-2'-deoxycy tidine. Methylation of the NEP promoter was present in androgen-independent PC cell Lines but not in androgen-dependent or small-cell derived PC cell lines and in 3 of 21 (14%) primary PCs from patients with androgen-dependen t disease. Exposure of PC cells to the demethylating agent 5'-aza-2'-deoxyc ytidine led to an increase in NEP transcripts in DU-145 and PC-3 cells. The se data show that hypermethylation of the 5' CpG NEP island is associated w ith a loss of NEP expression in PC, Loss of NEP expression via hypermethyla tion of the NEP promoter may contribute to the development of neuropeptide- stimulated PCs.