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Carcinogenesis, Vol. 21, No. 11, 2057-2064, November 2000
© 2000 Oxford University Press

Carcinogenesis

Correlations of partial and extensive methylation at the p14ARF locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors

Shichun Zheng, Pengchin Chen, Alex McMillan, Amalia Lafuente, Maria Jose Lafuente, Antonio Ballesta, Manuel Trias and John K. Wiencke

Laboratory for Molecular Epidemiology, Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA 94143-0560, USA,
1 Department of Clinical Biochemistry, Hospital Clinic, University of Barcelona and
2 Department of Surgery, Hospital St Pablo, Barcelona, Spain

p14ARF is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1ß of p14ARF are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14ARF in CRC cell lines and primary CRC tumors, and correlated p14ARF mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription–polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1ß but not the immediate upstream CpG sites. p14ARF mRNA was expressed at extremely low levels in fully methylated cell lines and at 104- to 105-fold higher levels in unmethylated cell lines. p14ARF expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 µM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14ARF methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14ARF was often accompanied by p16INK4a methylation; however, 50% of p14ARF methylated tumors contained unmethylated p16INK4a. Methylation at p14ARF was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14ARF methylation in human tumors is described.


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