Hypermethylation of the p14ARF Gene in Ulcerative Colitis-associated Colorectal Carcinogenesis

The p14 ARF protein directly inhibits the MDM-2 oncoprotein, which mediates degradation of the p53 protein. It has been shown that p14 ARF expression is frequently down-regulated by p14 ARF gene hypermethylation in colorectal cancer. To determine whether p14 ARF inactivation was involved in ulcerative colitis (UC)-associated carcinogenesis, the frequency and timing of p14 ARF methylation was investigated in four different histological stages of UC-associated carcinogenesis. Methylation-specific PCR and bisulfite sequencing were used to determine the prevalence of p14 ARF gene methylation. p14 ARF methylation was observed in 19 of 38 (50%) adenocarcinomas, 4 of 12 (33%) dysplasias, and 3 of the 5 (60%) nonneoplastic UC mucosae. In contrast, 3 of 40 (3.7%) normal tissues showed p14 ARF methylation (χ 2 test: P = 0.0003). Bisulfite sequencing was used to analyze 28 CpGs of p14 ARF gene in 20 samples. The number of methylated CpGs ranged from 0 to 4, 0 to 20, and 0 to 28 in the normal, dysplastic, and carcinomatous samples, respectively (Kruskall-Wallis test: P = 0.0005). Densely methylated alleles were detected only in carcinomas by bisulfite sequencing. In conclusion, our data suggest that methylation of p14 ARF is a relatively common early event in UC-associated carcinogenesis. p14 ARF offers potential as a biomarker for the early detection of cancer or dysplasia in UC. Finally, analyses of p14 ARF methylation in other organs should explore not only frank cancers but other premalignant lesions.