Susceptibility of nonpromoter CpG islands to de novo methylation in normaland neoplastic cells

Background: Many cancers display alterations in methylation patterns of CpG islands-stretches of DNA rich in CpG dinucleotides often associated with g ene promoters that are involved in initiation of gene transcription. This m ethylation may perturb expression of genes critical to the regulation of ce ll proliferation. Aberrant methylation is not limited to a few genes or to promoter regions but has been found on a genome-wide scale in a variety of neoplasias, including colorectal cancer and acute myelogenous leukemia. Our goal was to characterize, in a quantitative manner, the profiles of abnorm ally methylated genes that may be specific for different cancers. Methods: Using a quantitative assay, methylation-sensitive single nucleotide primer extension (MS-SNuPE), we have analyzed the methylation levels of promoter a nd exonic (coding region) CpG islands of two cyclin-dependent kinase inhibi tors [p15(INK4B) and p16(INK4A)] and the PAX6 gene, which encodes a transcr iptional factor involved in neuronal proliferation, in DNA samples taken fr om patients with chronic myelogenous leukemia, acute myelogenous leukemia, myelodysplastic syndrome, and colorectal cancer. Results: De novo methylati on of all three exonic loci in tumors-relative to baseline levels found in nontumor tissue or blood-was observed in hematologic neoplasias and in soli d tumors as well as in normal colonic tissue. However, methylation of promo ter regions was more limited. Moreover, two different patterns of promoter methylation distinguished the leukemias from colorectal cancer: ply promote r hypermethylation was found only in the leukemias, and p16 promoter hyperm ethylation occurred only in colon tumors. However, we did not address this issue prospectively; therefore, such an observation is only hypothesis gene rating. Conclusions: The methylation patterns that we observed suggest that exonic CpG islands are more susceptible to de novo methylation than promot er islands and that methylation may be seeded in exonic regions, from which it can spread to other islands, including promoter regions. Subsequent sel ection of cells with a growth advantage conferred by spread of methylation into and inactivation of a particular promoter might then contribute to the genesis of a specific type of cancer.