title: Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester.
abstract: Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O6-methyldeoxyguanosine (O6-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O6-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O6-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 mumol O6-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O6-MedG in the DNA from both their tumor and normal tissue. Ki-ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC-->AT transitions: 28% and 8% were GC-->TA and CG-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC-->TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O6-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC-->AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype.
authors.affiliation: Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, United Kingdom.
authors.forename: P E
authors.initials: PE
authors.lastname: Jackson
authors.forename: C N
authors.initials: CN
authors.lastname: Hall
authors.forename: A F
authors.initials: AF
authors.lastname: Badawi
authors.forename: P J
authors.initials: PJ
authors.lastname: O'Connor
authors.forename: D P
authors.initials: DP
authors.lastname: Cooper
authors.forename: A C
authors.initials: AC
authors.lastname: Povey
mesh_terms: D001483:Base Sequence; D003106:Colon / Q000737:chemistry; D015179:Colorectal Neoplasms / Q000235:genetics*; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004739:England; D011905:Genes, ras; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D017354:Point Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*
chemical_list: D017931:DNA Primers; D006147:Guanine; C008449:O-(6)-methylguanine; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)