Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method.
One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10,000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7-9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.
Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha 68198-6805, USA.
D
D
Chakravarti
P
P
Mailander
J
J
Franzen
S
S
Higginbotham
E L
EL
Cavalieri
E G
EG
Rogan
D000818:Animals; D001483:Base Sequence; D001580:Benzopyrenes / Q000633:toxicity*; D002273:Carcinogens / Q000633:toxicity*; D003062:Codon; D017931:DNA Primers; D005260:Female; D011905:Genes, ras; D051379:Mice; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D011230:Precancerous Conditions / Q000139:chemically induced / Q000235:genetics*; D012878:Skin Neoplasms / Q000139:chemically induced / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000633:toxicity
D001580:Benzopyrenes; D002273:Carcinogens; D003062:Codon; D017931:DNA Primers; C041517:dibenzo(a,l)pyrene; D013755:Tetradecanoylphorbol Acetate