ABSTRACT
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 NEP expression in hormone-refractory metastatic PCs can result from hormonal therapies because NEP transcription is induced by androgens and down-regulated by androgen withdrawal. NEP is encoded by a gene that contains a 5' CpG island spanning a transcriptional regulatory region. In this 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 in vivo. DNA isolated from PC cell lines and from normal and neoplastic human prostate tissues was restriction-digested with a methylation-sensitive restriction endonuclease and analyzed by Southern blot using a 5' sequence-specific NEP probe. Methylation-specific PCR was performed using PCR primers designed to discriminate between methylated and unmethylated alleles, and reverse transcription-PCR using NEP-specific primers was performed on cDNA extracted from PC cells treated with 5-aza-2'-deoxycytidine. 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-dependent disease. Exposure of PC cells to the demethylating agent 5-aza-2'-deoxycytidine led to an increase in NEP transcripts in DU-145 and PC-3 cells. These data show that hypermethylation of the 5' CpG NEP island is associated with a loss of NEP expression in PC. Loss of NEP expression via hypermethylation of the NEP promoter may contribute to the development of neuropeptide-stimulated PCs.
Subject(s)
DNA Methylation , Neprilysin/metabolism , Promoter Regions, Genetic , Prostatic Neoplasms/metabolism , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Decitabine , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Neprilysin/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolismABSTRACT
The angiotensin I-converting enzyme (ACE) contains an insertion/deletion (I/D) polymorphism, with the DD genotype associated with benign renal diseases. The distribution frequencies of the D and I alleles, and the DD, DI and II genotypes were determined in DNA extracted from kidney tissues of 58 renal cancer patients. The observed frequencies in patients who develop renal cancer was not significantly different than the normal population.
