Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification

Minoru Toyota, Coty Ho, Nita Ahuja, Kam Wing Jair, Qing Li, Mutsumi Ohe-Toyota, Stephen B. Baylin, Jean Pierre J. Issa

Research output: Contribution to journalArticle

Abstract

CpG island methylation has been linked to tumor suppressor gene inactivation in neoplasia and may serve as a useful marker to clone novel cancer-related genes. We have developed a novel PCR-based method, methylated CpG island amplification (MCA), which is useful for both methylation analysis and cloning differentially methylated genes. Using restriction enzymes that have differential sensitivity to 5-methyl-cytosine, followed by adaptor ligation and PCR amplification, methylated CpG rich sequences can be preferentially amplified. In a model experiment using a probe from exon 1 of the p16 gene, signal was detected from MCA products of a colorectal cancer cell line but not in normal colon mucosa. To identify novel CpG islands differentially methylated in colorectal cancer, we have applied MCA coupled with representational difference analysis to the colon cancer cell line Caco2 as a tester and normal colon mucosa as a driver. Using this strategy, we isolated 33 differentially methylated DNA sequences, including fragments identical to several known genes (PAX6, Versican, α-tubulin, CSX, OPT, and rRNA gene). The association of hypermethylation of the clones obtained and transcriptional suppression in colorectal cancer was confirmed by examining the Versican gene, which we found to be silenced in methylated cell lines and reactivated by the methylation inhibitor 5-aza-2'-deoxycytidine. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.

Original languageEnglish (US)
Pages (from-to)2307-2312
Number of pages6
JournalCancer Research
Volume59
Issue number10
StatePublished - May 15 1999

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ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Toyota, M., Ho, C., Ahuja, N., Jair, K. W., Li, Q., Ohe-Toyota, M., Baylin, S. B., & Issa, J. P. J. (1999). Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Research, 59(10), 2307-2312.