Quantitation of early clonal expansion of two mutant 61st codon c-Ha-ras alleles in DMBA/TPA treated mouse skin by nested PCR/RFLP.
It has been hypothesized that tumor promotion in mouse skin involves clonal expansion of initiated cells with activated c-Harvey (Ha)-ras oncogene to give rise to benign tumors. We have used the two stage mouse skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiator and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as the tumor promoter to quantitate the number of mutated c-Ha-ras alleles in mouse epidermal DNA. Epidermal samples were harvested over a 12-week period before the appearance of papillomas. Three 61st codon (i.e. CAA) c-Ha-ras mutations, CTA (T2), CGA (G2) and CAT (T3) were quantitated by newly developed nested PCR/RFLP assays. During TPA promotion the number of T2 mutant copies showed a progressive increase starting at 4 weeks after initiation and the number of T3 mutant alleles showed an increase starting at 6 weeks. By 12 weeks after initiation, TPA-promoted mouse epidermis averaged approximately 8x10(5) T2 mutant alleles per epidermis while the number of T3 mutant alleles averaged 3x10(4) per epidermis. The best-fit lines for the quantitation of mutant alleles derived from DMBA/TPA-treated mice from 4 to 12 weeks after initiation were exponential. These results were consistent with clonal expansion of epidermal cells carrying these mutations during tumor promotion. The slopes of the best-fit lines for the mutant copies indicated a trend in which cells with the T2 mutations had a growth advantage during TPA promotion over cells with the T3 mutation.
Finch
J S
JS
Department of Radiation Oncology, University of Arizona Cancer Center, Tucson 85724-5024, USA.
Albino
H E
HE
Bowden
G T
GT
D015127:9,10-Dimethyl-1,2-benzanthracene; D000483:Alleles; D000818:Animals; D003062:Codon; D005260:Female; D011905:Genes, ras; D051379:Mice; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012878:Skin Neoplasms / Q000139:chemically induced* / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate
D003062:Codon; D015127:9,10-Dimethyl-1,2-benzanthracene; D013755:Tetradecanoylphorbol Acetate