Enhancement of Carcinogenesis in Cultured Respiratory Tract Epithelium by 12-O-Tetradecanoylphorbol-13-Acetate

Abstract

The enhancement of chemical carcinogenesis was demonstrated in vitro in rat tracheal epithelium exposed in organ culture to N′-methyl-N′-nitro-N-nitroso-guanidine (MNNG) followed by multiple exposures to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The explants were exposed to 0 or 0.0001 μg MNNG per ml for 6 h on days 3 and 6 of culture, and then to 0 or 1.0 μg TPA per ml for l h on days 9, 15, 21, and 27 of culture. Primary cell cultures were then established from epithelial outgrowths of these explants. Morphologically altered cells were seen in the primary cell cultures derived from MNNG + TPA-exposed explants on an average of 130 days after MNNG exposure. In primary cultures derived from explants exposed to either TPA or MNNG alone, morphologically altered cells were seen at 190 days and 210 days, respectively. No morphologically altered cells were seen in solvent control primary cell cultures. Only cultures containing morphologically altered cells could be repeatedly subcultured. Cell lines established from these cultures were inoculated i.m. into immunosuppressed isogeneic hosts at 150, 200, 250, 310, and 365 days following carcinogen exposure. All five explants exposed to both MNNG and TPA produced at least one cell line which formed carcinomas when inoculated 365 days following exposure, while only two of the five explants produced tumorigenic cell lines in the MNNG-only group. Cell lines from explants exposed to both MNNG and TPA were tumorigenic an average of 90 days earlier than cell lines from explants exposed to MNNG alone. None of the cell lines obtained from the explants exposed only to TPA were tumorigenic. These studies show that, following carcinogen exposure, TPA can enhance the oncogenesis of cultured respiratory tract epithelium in terms of increasing the frequency of transformation and decreasing the time at which transformation could first be observed.