CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Early studies proposed that DNA methylation could have a role in regulating gene expression during development [Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9 -25]. However, some studies of DNA methylation in known tissue-specific genes during development do not support a major role for DNA methylation. In the results presented here, tissue-specific differentially methylated regions (TDMs) were first identified, and then expression of genes associated with these regions correlated with methylation status. Restriction landmark genomic scanning (RLGS) was used in conjunction with virtual RLGS to identify 150 TDMs [Matsuyama, T., Kimura, M.T., Koike, K., Abe, T., Nakao, T., Asami, T., Ebisuzaki, T., here is a history of inverse correlation between DNA methylation of CpG island promoter regions and gene expression. Almost all CpG islands on the inactive X chromosome are methylated, and monoallelic methylation of imprinted genes is associated with monoallelic gene expression (1-3). Moreover, programmed changes in DNA methylation are essential features of development, with disruption frequently resulting in aberrant development (4). Although early studies proposed that DNA methylation could have a role in regulating development (5, 6), more recent studies of DNA methylation in known tissue-specific genes during development did not support a major role for DNA methylation. Studies by Warnecke and Clark (7) found that the tissue-specific expression of the skeletal ␣-actin gene in the adult mouse does not correlate with the methylation state of the promoter. Walsh and Bestor (8) investigated the 5Ј methylation status of seven tissue-specific genes and found no correlation with tissue-specific expression. In the study presented here, rather than examining the methylation status of known tissuespecific genes, tissue-specific differentially methylated regions (TDMs) were first identified, and then genes located near the TDMs were analyzed for tissue-specific expression.Restriction landmark genomic scanning (RLGS) is a method for the two-dimensional display of end-labeled DNA restriction fragments (9-11) and can be used to scan for genomic DNA methylation (11). Because the NotI recognition site contains two CpGs and the great majority of NotI sites are within CpG islands, RLGS (with NotI as the restriction landmark) displays CpG islands and adjacent regions. If a NotI site is methylated, it will not be digested and will not be end-labeled, resulting in the absence of the spot in the RLGS profile. Global analysis of genomic DNA methylation of different tissues by using RLGS indicates that there are a relatively large number of differences in profiles suggesting TDMs (12-14). Numerous differences in the RLGS profiles of ES cells and differentiated tissues, such as kidney and brain, have been identified (13), but the DNA sequence of these genomic regions was not established, and it is not known whether these methylation differences are associated with tissue-specific gene expression.In the results presented here, RLGS was us...

Cancer cell lines are widely used in many types of cancer research, including studies aimed at understanding DNA hypermethylation of gene promoters in cancer. Hypermethylation of promoters is capable of repressing the expression of tumor suppressor genes and may play a role in the development and/or progression of cancer. Although both primary malignancies and cancer cell lines exhibit this epigenetic phenomenon, there has been no direct comparison between them. In order to address this question, we have utilized restriction landmark genomic scanning to measure the hypermethylation phenotypes of cancer cell lines and compared these data with the same analysis performed on primary malignancies. In all cases, cancer cell lines exhibit significantly higher levels of CpG island hypermethylation than the primary malignancies they represent. Colon cancer cell lines are most similar to their respective tumors, with only a 5-fold increase in hypermethylation, while head and neck squamous cell carcinoma cell lines show a 93-fold increase in hypermethylation. Furthermore, >57% of the loci methylated in cell lines are never methylated in 114 primary malignancies studied. Seventy percent of loci hypermethylated in cell lines are hypermethylated in lines from more than one type of cancer. These data indicate that most CpG island hypermethylation observed in cancer cell lines is due to an intrinsic property of cell lines as opposed to the malignant tissue from which they originated.