Acquisition of a Growth-inhibitory Response to Phorbol Ester Involves DNA Damage1

TPA (12-0-tetradecanoylphorbol-13-acetate), a potent tumor pro moter, has been shown to stimulate or inhibit cell growth depending on the cell type investigated. We recently found that RT101 cells, a trans formed mouse JB6 epidermal cell line, acquired a greater growth inhibi tion response to TPA during conventional subcultivation. The growth of low-passage RT101 cells was slightly inhibited by TPA in monolayer culture but stimulated in soft agar. In contrast, the growth of highpassage cells was greatly inhibited by TPA in both monolayer culture and in soft agar. Inhibition was dose dependent, directly correlated with protein kinase C-activating activities of tumor promoters, and was found to be reversible. TPA-treated high-passage cells were greatly reduced in volume, showed extensive abnormal mitoses, and were more susceptible to detachment. High-passage cells were also found to be less tumorigenic as indicated by in vivo tumorigenicity assay in nude mice. TPA treatment rendered cells still less tumorigenic in the case of both cell lines. The mechanism for acquisition of increased sensitivity to TPA of RT101 cells during subculture was investigated; it involved nonrandom DNA damage and detachment of nonviable cells. The results suggest the possibility that early-passage RT101 cells contained two subpopulations, one TPAsensitive and one TPA-resistant population. Conventional subcultivation may have selected for the former subpopulation. The sensitive subpopu lation may have been irreversibly inhibited as a result of TPA-induced cell killing, possibly apoptosis.