FREQUENCY OF KI-RAS MUTATIONS AND DNA ALKYLATION IN COLORECTAL TISSUEFROM INDIVIDUALS LIVING IN MANCHESTER

Most human colorectal cancers arise through the accumulation of a seri es of genetic alterations such as point mutations within the Ki-ras an d p53 genes, but the chemical carcinogens that may be implicated in th ese events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O-6-methyldeoxyguanosine ( O-6-MedG), a promutagenic lesion arising from exposure to as yet unide ntified methylating agents. To address whether such exposure may resul t in oncogene activation in human colorectal tumors, we examined anoth er series of paired normal and tumor DNA samples from the lower intest inal tract for the presence of O-6-MedG in DNA (as a marker of exposur e) and for mutations within the Ki-ras gene. After isolation by high p ressure liquid chromatography, O-6-MedG was quantified by a radioimmun oassay with a limit of detection of 0.01 mu mol O-6-MedG/mol dG. The f requencies 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 detec table O-6-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. Th e frequencies of mutations in tumor DNA isolated from the cecum, sigmo id colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolate d from macroscopically normal tissue was found to contain Ki-ras mutat ions in 14% of sigmoid colon samples and 12% of rectal samples. Most b ase mutations were in codon 12 (72%), and 64% were GC-->AT transitions : 28% and 8% were GC-->TA and GC-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 exce pt 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 o f O-6-MedG in DNA from either normal or tumor tissue or both normal an d tumor tissue and the incidence of Ki-ras mutations or GC-->AT transi tions in mutated Ki-ras genes. It remains to be determined, however, w hether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O-6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational a ctivation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susce ptibility 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 e mergence of the mutator phenotype. (C) 1996 Wiley-Liss, Inc.