Correlations of partial and extensive methylation at the p14(ARF) locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors

p14(ARF) is a putative tumor suppressor gene thought to modify the levels o f p53, CPG sites within the 5'-flanking region and exon 1 beta of p14(ARF) are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC), Here we have developed methylation-specific polyme rase chain reaction (MSPCR) methods to detect methylation of CpG sites in p 14(ARF) in CRC cell lines and primary CRC tumors, and correlated p14(ARF) m RNA expression with methylation in CRC cell lines using competitive quantit ative reverse transcription-polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methyla tion and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylate d; the other cell line, LoVo, showed partial methylation that affected exon 1 beta but not the immediate upstream CpG sites. p14(ARF) mRNA was express ed at extremely low levels in fully methylated cell lines and at 10(4)- to 10(5)-fold higher levels in unmethylated cell lines. p14(ARF) expression in the partially methylated LoVo cell line was Intermediate. Treatment of LoV o cells with 2 muM 5-aza-2'-deoxycytidine for 72 h was associated with mark ed (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14(ARF) methylation, although partial methylation was the most common patt ern observed (in 67% of methylated tumors). Methylation of p14(ARF) was oft en accompanied by p16(INK4a) methylation; however, 50% of p14(ARF) methylat ed tumors contained unmethylated p16(INK4a). Methylation at p14(ARF) was as sociated with female gender, greater age, proximal anatomic location and po or differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14(ARF) methylation in human tumors is described.