Silencing of tumor suppressor genes is an important and prevalent mechanism for human carcinogenesis. We developed the methylation-sensitive representational difference analysis (MS-RDA) method to search for differences in methylation in any two given DNA (1). Analyzing human lung, breast, and stomach cancers by using MS-RDA, we identified two, two, and nine genes that were silenced in these cancers, respectively.
In the analysis of lung cancers, 34 DNA fragments that were hypermethylated in two lung cancer cell lines were isolated. Nineteen of them were associated with CpG islands that met a traditional criterion of Gardiner-Garden and colleagues (2), and 14 of those were associated with gene-expressed sequence tags. After analysis by RT-PCR and demethylation experiments with 5-aza-2'-deoxycytidine (aza-dC), only two of the genes were found to have typical features of gene silencing: methylation of a CpG island in the promoter region, complete loss of expression, and remarkable restoration of gene expression by aza-dC treatment (3). The two silenced genes had dense and large CpG islands. Similar kinetics regarding the number of positive clones were observed in human breast and stomach cancers.
On the basis of the literature and the above analysis, sizable numbers of methylation differences seem to be present in human cancers, but only a fraction of them are in CpG islands under the traditional criterion. Even when in CpG islands, most of them were under loose regulation of DNA methylation, and only a minor fraction of them were involved in gene silencing. Instead, when a new criterion for CpG islands was used (4), the fraction involved in gene silencing increased significantly although the number of eligible genomic regions decreased. It seems to be critical, when analyzing DNA methylation, to pay attention to how important a genomic region being analyzed is from the viewpoint of gene regulation.
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