12-O-tetradecanoylphorbol-13-acetate induces clonal expansion of potentially malignant keratinocytes in a tissue model of early neoplastic progression

Tumor promoters stimulate the selective expansion of initiated mouse kerati nocytes in the two-stage model of skin carcinogenesis. However, it is not c lear whether these promoters directly modulate the growth of initiated cell s or rather permit clonal expansion of initiated cells by modifying the env ironment of adjacent normal cells. The goal of this study was to further un derstand the mechanism of action of tumor promotion during early neoplastic progression of human stratified epithelium, To accomplish this, we have es tablished an organotypic culture model that mimics a preneoplastic: tissue and contains mixtures of genetically marked (beta-galactosidase), low-grade malignant keratinocytes (HaCaT-ras II-4) and normal human keratinocytes (N HKs) to monitor the fate and phenotype of these cells after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In submerged culture, concentra tions of 0.001-1 mu g/ml TPA were shown to limit the growth of NHKs yet had no effect on growth of II-4 cells, TPA (0.001 mu g/ml) was then added to o rganotypic cultures containing mixtures of NHK:II-4 cells at varying ratios to determine whether this agent could selectively stimulate clonal expansi on of II-4 cells in a normal epidermal background, Immunofluorescence for b eta-galactosidase demonstrated that TPA caused a significant increase in th e percentage of beta-galactosidase-positive areas in 12:1 and 4:1 mixtures, This TPA-induced expansion of II-4 cells was associated with a marked decr ease in proliferation of NHKs, suggesting that II-4 could selectively expan d because of its growth advantage relative to NHKs. Clonal expansion of tum or cells was temporally linked to the decreased expression of filaggrin and keratin 1 expression in adjacent NHKs. These findings indicate that TPA ma y enable expansion of potentially malignant cells through the epigenetic mo dification of proliferation in NHKs and differentiation of NHK and II-4 cel ls.