Quantitation of primary in vitro clonogenic keratinocytes from normal adult murine epidermis, following initiation, and during promotion of epidermal tumors

Abstract

A quantitative in vitro assay for clonogenic epidermal cells from adult mice has been designed to focus on the cells from normal epidermis that are potential targets for carcinogens. Dorsal epidermal cells were isolated by trypsinization from groups of three to six CD-1 female mice with yields of 1.31 +/- 6.84 X 10(6) (average of n = 7; SD) viable epidermal cells per square centimeter of skin. Suspensions of single epidermal cells were plated at clonal density onto irradiated Swiss 3T3 cells. The cultures were maintained in high calcium SPRD-105 medium designed to support concomitant proliferation and terminal differentiation of keratinocytes from normal as well as carcinogen-exposed mice. Two weeks later, the dishes were fixed and stained with rhodanile blue, and epidermal colonies were counted. The average number of colonies from normal epidermis was 45 +/- 8.5 (n = 11; SD) per 10(4) cells plated for mice 9 to 69 weeks of age. To determine the effects of initiation on the number of clonogenic epidermal cells, groups of mice 8 weeks of age were treated topically with 200 nmol of 7,12-dimethylbenz(a)anthracene or acetone alone. The number of colonies in both treatment groups remained within the control (untreated) range at all intervals from 7 to 61 weeks after initiation. In contrast, the number of clonogenic cells from control as well as initiated epidermis remained elevated at 1 month following multiple in vivo treatments of skin with 12-O-tetradecanoylphorbol-13-acetate (TPA). The increase in the number of clonogens was always greater from initiated epidermis treated with TPA than from control epidermis treated with TPA. These results suggest that an increase in the clonogenic population was a consequence of promotion rather than initiation and are in agreement with a concomitant carcinogenesis experiment confirming the apparent irreversibility of initiation, the veritable absence of tumors in the absence of promotion, and the similarity of the tumor responses regardless of the age of the animal at initiation or the length of the delay interval between initiation and promotion.