Human Molecular Genetics Advance Access originally published online on July 15, 2003
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Human Molecular Genetics, 2003, Vol. 12, No. 17 2209-2219
DOI: 10.1093/hmg/ddg226
© 2003 Oxford University Press
Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases
1Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Centre (CNIO), Madrid 28029, Spain, 2Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri School of Medicine, Columbia 65203, USA, 3Cytogenetics Unit, Spanish National Cancer Center (CNIO), Madrid 28029, Spain and 4Research Institute of Oncology (IRO), L'Hospitalet 08907, Catalonia, Spain
Received May 15, 2003; Accepted July 5, 2003
Hypermethylation associated silencing of the CpG islands of tumor suppressor genes is a common hallmark of human cancer. Here we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using two molecular screenings for differentially methylated loci [differential methylation hybridization (DMH) and amplification of inter-methylated sites (AIMS)], we found that DKO cells, but not the single DNMT1 or DNMT3b knockouts, have a massive loss of hypermethylated CpG islands that induces the re-activation of the contiguous genes. We have characterized a substantial number of these CpG island associated genes with potentially important roles in tumorigenesis, such as the cadherin member FAT, or the homeobox genes LMX-1 and DUX-4. For other genes whose role in transformation has not been characterized, such as the calcium channel 
1I or the thromboxane A2 receptor, their re-introduction in DKO cells inhibited colony formation. Thus, our results demonstrate the role of DNMT1 and DNMT3b in CpG island methylation associated silencing and the usefulness of genetic disruption strategies in searching for new hypermethylated loci.
* To whom correspondence should be addressed at: Cancer Epigenetics Laboratory, Spanish National Cancer Centre (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spain. Tel: +34 912246940; Fax: +34 912246923; Email: mesteller{at}cnio.es
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