Detection of codon 12 point mutations of the K-ras gene from mouse lung adenocarcinoma by 'enriched' PCR.
OBJECTIVE
Recent studies have shown that chemical carcinogens induce a high frequency of point mutations in the K-ras oncogene from mouse lung tumours at codons 12, 13 and 61. These experiments were performed to identify K-ras mutations in tissues from control and radiation-exposed mice.


METHODS
By modifying the technique of the 'enriched' polymerase chain reaction (PCR), it was possible to detect point mutations at codon 12 of the K-ras oncogene from 25-year-old paraffin-embedded normal lungs and lung adenocarcinomas from mice exposed to radiation. Together, a total of 120 lung tissues were screened for point mutations at codon 12 of the K-ras oncogene in this study.


RESULTS
A significant increase in K-ras codon 12 point mutations was observed in the normal lungs from mice exposed to 24 once-weekly neutron irradiations (100%), compared with normal lungs from mice with sham-irradiation (50%) (p<0.05). Lung adenocarcinomas from mice receiving 24 once-weekly neutron irradiations also had a significantly higher frequency of K-ras codon 12 point mutations (100%) than the lung adenocarcinomas of mice receiving 24 or 60 once-weekly gamma-ray irradiations (50%), but the higher frequency was not significantly different from that in spontaneous lung adenocarcinomas from mice (75%; p > 0.05). The validity of the technique was confirmed by sequencing two of the mutants. In doing so, a K-ras 13(Asp) point mutation was observed.


CONCLUSIONS
The data suggest that high-linear energy transfer (LET) neutron radiation was more effective than low-LET gamma-rays in inducing K-ras point mutations at codon 12 in the lungs of B6CF1 mice.
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, IL 60439-4833, USA.
Y
Y
Zhang
G E
GE
Woloschak
D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D005720:Gamma Rays / Q000009:adverse effects; D017353:Gene Deletion; D011905:Genes, ras / Q000528:radiation effects*; D018499:Linear Energy Transfer / Q000502:physiology; D008168:Lung / Q000528:radiation effects*; D008175:Lung Neoplasms / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D009502:Neutrons / Q000009:adverse effects; D016612:Paraffin Embedding; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA