p21WAF1/CIP1 gene is inactivated in metastatic prostatic cancer cell lines by promoter methylation

Introduction: p21(WAF1/CIP1) may act as a tumour suppressor gene (TSG) and loss of the p21(WAF1/CIP1) gene has been reported in several solid tumours. The aim of this study was to see whether p21(WAF1/CIP1) was expressed in metastatic prostate cancer cell lines and to determine if there was methylation of the p21(WAF1/CIP1) promoter. Method: PC3, LNCaP and DU145 metastatic prostate cancer cell lines, 1542NP normal prostate, and RD rhabdomyosarcoma cell lines were cultured in the demethylating agent 5-Aza-2 deoxycytidine (5-Aza-CdR). p21(WAF1/CIP1) mRNA expression was analysed by RT-PCR. DNA from untreated cell lines was modified with sodium bisulphite and promoter sequencing was performed. Results: p21(WAF1/CIP1) was expressed at low or undetectable levels in metastatic prostate cancer cell lines but expression was reactivated by treatment with 5-Aza-CdR. Sequence analysis of the promoter region revealed several sites of methylation at the 50 end of a CpG island in the PC3, LNCaP and DU145 cell line DNA but not in the normal prostate control DNA. Most notably the Sis-inducible element (SEI)-1 - a STAT1-binding site, was methylated. Conclusions: In this study, we show that p21(WAF1/CIP1) expression in metastatic prostate cancer cell lines is enhanced as a result of demethylation of the DNA. Furthermore, several cytosine residues in the promoter region are methylated, including critical binding sites. The inhibition of the STAT1-signalling pathway by methylation of the promoter may inactivate the p21(WAF1/CIP1) TSG in prostate cancer.