Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer.
BACKGROUND
Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells.


METHODS
We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method.


RESULTS
Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes.


CONCLUSIONS
These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.
Feng
Zhaohui
Z
Department of Environmental Medicine, New York University School of Medicine, Tuxedo 10987, USA.
Hu
Wenwei
W
Chen
James X
JX
Pao
Annie
A
Li
Haiying
H
Rom
William
W
Hung
Mien-Chie
MC
Tang
Moon-shong
MS
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000009:adverse effects*; D001980:Bronchi; D002273:Carcinogens / Q000009:adverse effects*; D002478:Cells, Cultured; D002874:Chromosome Mapping / Q000379:methods; D003062:Codon / Q000187:drug effects / Q000235:genetics*; D018736:DNA Adducts / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects; D029968:Escherichia coli Proteins; D005347:Fibroblasts / Q000187:drug effects; D011905:Genes, ras / Q000187:drug effects*; D006801:Humans; D009153:Mutagens / Q000009:adverse effects*; D009369:Neoplasms / Q000139:chemically induced / Q000235:genetics*; D017354:Point Mutation / Q000187:drug effects*; D016133:Polymerase Chain Reaction / Q000379:methods; D012907:Smoking / Q000009:adverse effects
D002273:Carcinogens; D003062:Codon; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D009153:Mutagens; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC