Quantitative assay for carcinogen altered differentiation in mouse epidermal cells

Basal epidermal cells can be selectively maintained as a monolayer in culture medium containing a low ionic calcium concentration of 0.01-0.10 mM. Cessation of proliferation, maturation and shedding of squamous sheets can be induced in this population by increasing the calcium concentration above 0.1 mM. Since alterations in the regulation of proliferation and differentiation are associated with epidermal carcinogenesis in vivo, it appeared reasonable that changes in the phenotypic response to calcium might follow exposure to carcinogens in vitro. Support for this hypothesis was provided by the observation that malignant epidermal cells continued to proliferate when switched from low to high calcium medium, and could thus be selected from a mixture of such cells and a large excess of normal cells which did not survive after induced differentiation. Normal primary epidermal cells were plated in low calcium medium, treated on day 3 with a chemical carcinogen, maintained for 3-9 weeks in low calcium (0.02 mM) and then switched to high calcium medium (1.4 mM). After an additional 4 weeks, surviving epithelial colonies were fixed, stained with rhodamine and counted. Treatment of cultures with 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine yielded 4-10 fold more colonies than solvent controls. Colony number was proportional to carcinogen dose for both agents, and increased with time in low calcium prior to selection by calcium increase. Cells obtained from colonies in treated cultures demonstrated characteristic epidermal morphology and keratinization, and could be subcultured, but did not grow in agar or produce tumors in syngeneic hosts. This model system represents a quantitative assay for carcinogen altered epithelial cell differentiation and may select for an early property of preneoplastic epidermal cells.