Mechanisms for the involvement of DNA methylation in colon carcinogenesis

Abstract

C --> T transitions at CpG sites are the most prevalent mutations found in the p53 tumor suppressor gene in human colon tumors and in the germline (Li-Fraumeni syndrome). All of the mutational hot spots are methylated to 5-methylcytosine, and it has been hypothesized that the majority of these mutations are caused by spontaneous hydrolytic deamination of this base to thymine. We have previously reported that bacterial methyltransferases induce transition mutations at CpG sites by increasing the deamination rate of C --> U when the concentration of the methyl group donor S-adenosylmethionine (AdoMet) drops below its Km, suggesting an alternative mechanism to create these mutations. Unrepaired uracil pairs with adenine during replication, completing the C --> T transition mutation. To determine whether this mechanism could contribute to the development of human colon cancer, we examined the level of DNA (cytosine-5)-methyltransferase (MTase) expression, the concentration of AdoMet, and the activity of uracil-DNA glycosylase in human colon tissues, and searched for the presence of mutations in the MTase gene. Using reverse transcription-PCR methods, we found that average MTase mRNA expression levels were only 3.7-fold elevated in tumor tissues compared with surrounding normal mucosa from the same patient. Also, no mutations were found in conserved regions of the gene in 10 tumors sequenced. High-performance liquid chromatographic analysis of extracts from the same tissues showed that AdoMet concentrations were not reduced below the Km value for the mammalian enzyme, and the concentration ratio of AdoMet:S-adenosylhomocysteine, the breakdown product of AdoMet and the competitive MTase inhibitor, did not differ significantly. Finally, extracts from the tumor tissue efficiently removed uracil from DNA. Therefore, biochemical conditions favoring a mutagenic pathway of C --> U --> T were not found in a target tissue known to undergo a high rate of C --> T transitions at CpG sites.