p21WAF1/CIP1 gene is inactivated in metastatic prostatic cancer cell lines by promoter methylation.
BACKGROUND
p21WAF1/CIP1 may act as a tumour suppressor gene (TSG) and loss of the p21WAF1/CIP1 gene has been reported in several solid tumours. The aim of this study was to see whether p21WAF1/CIP1 was expressed in metastatic prostate cancer cell lines and to determine if there was methylation of the p21WAF1/CIP1 promoter.


METHODS
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). p21WAF1/CIP1 mRNA expression was analysed by RT-PCR. DNA from untreated cell lines was modified with sodium bisulphite and promoter sequencing was performed.


RESULTS
p21WAF1/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 5' 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 p21WAF1/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 p21WAF1/CIP1 TSG in prostate cancer.
Bott
S R J
SR
Prostate Cancer Research Centre, Institute of Urology, University College London, London, UK. simonrjbott@doctors.org.uk
Arya
M
M
Kirby
R S
RS
Williamson
M
M
D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D006801:Humans; D008297:Male; D009362:Neoplasm Metastasis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D004247:DNA