Gastroenterology

Gastroenterology

Volume 124, Issue 3, March 2003, Pages 626-633
Gastroenterology

Clinical–Alimentary Tract
p14 methylation in human colon cancer is associated with microsatellite instability and wild-type p53,☆☆

https://doi.org/10.1053/gast.2003.50102Get rights and content

Abstract

Background & Aims: Colorectal cancers with high levels of microsatellite instability (MSI-H) have an unexplained low rate of p53 gene mutations. Most such cancers have the CpG island methylator phenotype (CIMP+) with methylation and transcriptional silencing of the mismatch repair gene MLH1. The p14 (ARF) gene on chromosome 9p is deleted and/or silenced by hypermethylation in a subset of human malignancies. There is evidence suggesting that p14 suppresses tumorigenicity by stabilizing the p53 protein. Methods: We investigated the role of p14 in colorectal cancer by determining its methylation status in cancers that were studied previously for microsatellite instability, CIMP, and mutations of p53 and K-RAS. Results: p14 methylation was present in 21 of 94 cases overall (22%) and was frequent particularly in the subgroups with MSI-H (52% [11 of 21] vs. 14% [10 of 72], P = 0.004), in CIMP+ cases (40% [19 of 48] vs. 4% [2 of 46], P < 0.001), and in cases without p53 alterations (36% [17 of 47] vs. 7% [3 of 44], P = 0.004). Of 91 fully characterized cases, 41 (45%) had p53 mutations alone, 17 (19%) had p14 methylation alone, 30 (33%) had neither, but only 3 (3%) had both p53 mutations and p14 methylation. p14 methylation is an early event in colorectal carcinogenesis, being detectable in normal aging epithelium by using sensitive assays. Conclusions: In colorectal cancer, p14 methylation is associated with the presence of microsatellite instability and with absence of p53 mutations. The results provide a possible explanation for the paucity of p53 mutations in colon cancers with microsatellite instability.

GASTROENTEROLOGY 2003;124:626-633

Section snippets

Tissue samples and cell lines

Tumor samples and corresponding adjacent tissues were collected from consenting patients in accordance with institutional policies, flash frozen, and stored at −70°C in a tumor bank. We have reported previously 3, 6, 17 on 100 colon cancers studied for the presence of CIMP, MSI (determined according to strict criteria, testing at least 5 markers, and requiring band shifts at both dinucleotide and mononucleotide tracts), p16 methylation, K-RAS codon 12 and 13 mutations (determined by the

A quantitative p14 methylation assay

Methylation assays vary in their sensitivity, which sometimes affects reproducibility of results. Bisulfite conversion coupled with PCR and restriction fragment length polymorphism (COBRA) is a robust quantitative assay that usually requires substantial methylation for detection in primary tissues, and therefore is associated strongly with gene silencing. We used a set of primers to amplify the 5' region of the p14 promoter and coupled these with restriction enzymes that digest only methylated

Discussion

Our results clearly show an inverse relationship between p14 methylation and p53 mutations in sporadic colorectal cancer, and provide additional support for the hypothesis that p14 inactivation is equivalent functionally to p53 mutations in abrogating the p53 pathway.12, 13 These results, along with the high frequency of p14 methylation in sporadic colorectal cancers with MSI-H provide an attractive explanation for the puzzling lack of p53 mutations in MSI-H cases. Thus, given that most

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    Supported by research grants from the American Cancer Society (RPG9909801MGO), the National Cancer Institute (R01 CA89245-01), the George and Barbara Bush fund for innovative cancer research, and the Yasuda Medical Research Foundation and the Nitto foundation in Japan (to Y.K.). DNA sequencing in the MDACC core sequencing facility was supported by core grant CA16672 from the National Institutes of Health.

    ☆☆

    Address requests for reprints to: Jean-Pierre Issa, M.D., Department of Leukemia, MD Anderson Cancer Center, Box 428, 1515 Holcombe, Houston, Texas 77030. e-mail: [email protected]; fax: (713) 794-4297.

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