Journal Article

Quantitation of early clonal expansion of two mutant 61st codon c-Ha-<i>ras</i> alleles in DMBA/TPA treated mouse skin by nested PCR/RFLP

Joanne S. Finch, H.Emanuel Albino and G.Tim Bowden

in Carcinogenesis

Volume 17, issue 12, pages 2551-2557
Published in print December 1996 | ISSN: 0143-3334
Published online December 1996 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/17.12.2551
Quantitation of early clonal expansion of two mutant 61st codon
                        c-Ha-ras alleles in DMBA/TPA treated mouse skin by
                    nested PCR/RFLP

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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 carcino-genesis model using 7, 12-dimethylbenz[a]anthracene (DMBA) as the initiator and 12-0-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 ∼8×105 T2 mutant alleles per epidermis while the number of T3 mutant alleles averaged 3×104 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.

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Subjects: Clinical Cytogenetics and Molecular Genetics

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