Detection of mutant Ha-ras genes in chemically initiated mouse skin epidermis before the development of benign tumors

An activated Ha-ras oncogene has been consistently found in chemically initiated benign and malignant mouse skin tumors, and an activated ras oncogene has been shown to initiate the process of mouse skin carcinogenesis. However, the exact timing of mutational activation of the Ha-ras gene relative to application of the chemical carcinogen is not known. A sensitive mutation-specific PCR technique was used to experimentally address the timing of Ha-ras gene mutational activation. This technique can detect mutant Ha-ras alleles in the presence of a very large excess of normal ras alleles. Activated Ha-ras genes with 61st codon A → T mutations were found in the epidermis of mice 1 week after topical initiation with 7,12-dimethylbenz[a]anthracene or urethane by using this assay. These results were confirmed by Xba I restriction fragment length polymorphism analysis and direct DNA sequencing. One week after initiation is 1-2 months before the appearance of benign papillomas that harbor activated Ha-ras oncogenes when the initiated mice are promoted with the tumor promoter phorbol 12-myristate 13-acetate. Our data support the hypothesis that initiated epidermal cells containing an activated Ha-ras gene can remain dormant in the skin until a tumor promoter induces regenerative hyperplasia that allows for outgrowth of these cells with an activated ras oncogene to give rise to a benign papilloma.