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[Cancer Research 59, 2307-2312, May 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 2307-2312, May 15, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Identification of Differentially Methylated Sequences in Colorectal Cancer by Methylated CpG Island Amplification1

Minoru Toyota, Coty Ho, Nita Ahuja, Kam-Wing Jair, Qing Li, Mutsumi Ohe-Toyota, Stephen B. Baylin and Jean-Pierre J. Issa2

The Johns Hopkins Oncology Center, Baltimore, Maryland 21231

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, {alpha}-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.




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 Cancer Res.Home page
T. Ueki, M. Toyota, T. Sohn, C. J. Yeo, J.-P. J. Issa, R. H. Hruban, and M. Goggins
Hypermethylation of Multiple Genes in Pancreatic Adenocarcinoma
Cancer Res., April 1, 2000; 60(7): 1835 - 1839.
[Abstract] [Full Text]

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 Proc. Natl. Acad. Sci. USAHome page
M. Toyota, M. Ohe-Toyota, N. Ahuja, and J.-P. J. Issa
Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype
PNAS, January 18, 2000; 97(2): 710 - 715.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
M. Toyota, N. Ahuja, H. Suzuki, F. Itoh, M. Ohe-Toyota, K. Imai, S. B. Baylin, and J.-P. J. Issa
Aberrant Methylation in Gastric Cancer Associated with the CpG Island Methylator Phenotype
Cancer Res., November 1, 1999; 59(21): 5438 - 5442.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
M. Toyota, C. Ho, M. Ohe-Toyota, S. B. Baylin, and J.-P. J. Issa
Inactivation of CACNA1G, a T-Type Calcium Channel Gene, by Aberrant Methylation of Its 5' CpG Island in Human Tumors
Cancer Res., September 1, 1999; 59(18): 4535 - 4541.
[Abstract] [Full Text] [PDF]

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 Proc. Natl. Acad. Sci. USAHome page
M. Toyota, N. Ahuja, M. Ohe-Toyota, J. G. Herman, S. B. Baylin, and J.-P. J. Issa
CpG island methylator phenotype in colorectal cancer
PNAS, July 20, 1999; 96(15): 8681 - 8686.
[Abstract] [Full Text] [PDF]

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 Genome ResHome page
L. Z. Strichman-Almashanu, R. S. Lee, P. O. Onyango, E. Perlman, F. Flam, M. B. Frieman, and A. P. Feinberg
A Genome-Wide Screen for Normally Methylated Human CpG Islands That Can Identify Novel Imprinted Genes
Genome Res., April 1, 2002; 12(4): 543 - 554.
[Abstract] [Full Text] [PDF]


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