Frequent hypermethylation of the RASSF1A gene in prostate cancer

Abstract

Recently, we have cloned and characterized the Ras association domain family 1A gene (RASSF1A) at 3p21.3, from which loss of genetic material is one of the most frequent events in several types of human solid tumors. The CpG island promoter region of this gene is highly methylated in several human cancers, most notably in small cell lung cancer, breast cancer, and renal cell carcinoma. In this study, we have analysed the methylation status of RASSF1A in primary prostate tumors and in the prostate cancer cell line LNCaP. In total, 37 out of 52 tumors (71%) were methylated at the promoter region of RASSF1A. The relative frequency of methylation was higher in more aggressive tumors compared with less malignant tumors. For instance, tumors with a Gleason score of 7 -10 (25 out of 30, 83%) were significantly more methylated compared with Gleason 4 -6 tumors (11 out of 20, 55%, P=0.032, Fisher's exact test). Coincident with a hypermethylated promoter, transcripts of RASSF1A were missing in LNCaP cells. Expression of RASSF1A was restored with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor. In conclusion, our data suggest that epigenetic inactivation of RASSF1A by methylation is a very common event in prostate cancer and might be involved in the progression of the disease. Testing for RASSF1A methylation should become useful in prostate cancer early detection and diagnosis and might aid prognosis by gauging the potential status of progression.

Methylation of the retinoid response gene TIG1 in prostate cancer correlates with methylation of the retinoic acid receptor beta gene

“…This possibility led us to investigate the methylation of two additional genes in the 50 prostate cancer samples (Table 2). Data for RASSF1A methylation were obtained from previous work (Liu et al, 2002). The methylation of RAR-beta and RASSF1A was not statistically correlated, although there was a trend for an association (r ¼ 0.26, P ¼ 0.069).…”

“…PCR products were separated on a 2% TBE agarose gel and stained with ethidium bromide, or transferred to a nylon membrane, then hybridized with a labeled probe and visualized by autoradiography. The expression of GAPDH was analysed as a control (Liu et al, 2002).…”

“…For the analysis of the methylation of the ER-beta gene, methylation-specific primers UER-beta-M (5 0 -GCGTTGGGCGTTTTTGAGATCGTC-3 0 ) and LERbeta-M (5 0 -CAATTATTTCTCGCAACCTCCGCG-3 0 ) or unmethylation-specific primers UER-beta-U (5 0 -GTGTTGGG TGTTTTTGAGATTGTT-3 0 ) and LER-beta-U (5 0 -ACAAT TATTTCTCACAACCTCCACA-3 0 ) were used. The analysis of methylation in the RASSF1A CpG island was carried out by MSP as described previously (Liu et al, 2002). For bisulfite sequencing, 100 ng of bisulfite-treated genomic DNA was amplified with TIG1 sequencing primers: TIG1seqU (5 0 -AGG AGTGGTTTTATGGGGAT-3 0 ) and TIG1seqL1 (5 0 -AACC CGAACCAAAAAA-CAAACA-3 0 ), or with the putative RARE sequencing primers: RareU (5 0 -GTTATTTGTT TAAAAA-GAGTTG-3 0 ) and RareL (5 0 -CAAACAATTC TA-CCTCAACCTC-3 0 ) in the reaction buffer (100 ml) containing dNTPs and Hotstar Taq polymerase at 951C for 15 min and then 40 cycles of 941C for 30 s, 581C (531C for RARE sequencing) for 50 s and 721C for 50 s, followed by a final extension step at 721C for 10 min.…”

“…Epigenetic inactivation of tumor suppressor genes through DNA methylation in CpGrich promoter regions contributes to tumorigenesis (Baylin and Herman, 2000;Jones and Baylin, 2002). In prostate cancer, aberrant methylation is involved in the inactivation of various important genes such as E-cadherin, CD44, RASSF1A, GSTP1, the endothelin B receptor, p16, the androgen receptor gene, the retinoic acid receptor beta (RARbeta), the estrogen receptor beta (ER-beta) and the caveolin-1 gene (Lee et al, 1994;Nelson et al, 1997;Cui et al, 2001;Kito et al, 2001;Li et al, 2000Li et al, , 2001Nakayama et al, 2001;Pao et al, 2001;Kuzmin et al, 2002;Liu et al, 2002). When tested, loss of the expression of these genes was associated with CpG island hypermethylation, and expression could be restored after treatment with 5-aza-2 0 -deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor.…”

Frequent hypermethylation of RASSF1A in early flat-type colorectal tumors

“…Somatic mutational inactivation is rare in this gene (Dammann et al, 2000(Dammann et al, , 2003bAgathanggelou et al, 2001;Burbee et al, 2001;Lo et al, 2001). However, epigenetic inactivation of RASSF1A by hypermethylation of the promoter region has been described for a variety of human carcinomas including lung, breast, kidney, prostate, ovary, melanoma, gastric and nasopharyngeal carcinoma (Dammann et al, 2000Lerman and Minna, 2000;Agathanggelou et al, 2001;Byun et al, 2001;Lo et al, 2001;Yoon et al, 2001;Liu et al, 2002;Spugnardi et al, 2003). In addition, it is considered to be one of the most commonly methylated genes in human cancer.…”