Long-term effect of a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, on induced differentiation of Friend leukemia cells.

A potent tumor promoter of mouse skin, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibits terminal differentiation of Friend erythroleukemia cells (FELC). The differentiation of FELC induced by hexamethylene bisacetamide (HMBA) was almost completely inhibited by the presence of 100 ng TPA per ml (1.7 x 10'' M) for a period of 8 months through 58 serial passages of the cells in a medium containing both HMBA and TPA. During this period, FELC were released periodically from the TPA blockage by being washed and transferred to fresh medium. When cells cultured with HMBA plus TPA for as long as 2.5 months were transferred to fresh medium containing HMBA alone, they were induced to differentiate to an extent similar to that for cells not previously cultured with TPA. If the cells were maintained for a longer period of time in the presence of HMBA plus TPA, however, they tended to lose their ability to differentiate when transferred to HMBA-containing medium. During early passages of FELC in HMBA plus TPA, a small percentage of cells underwent differentiation when transferred to control medium containing neither HMBA nor TPA; thus, TPA inhibits the commitment of FELC to differentiate. However, TPA-mediated inhibition of commitment is leaky, so that in creasing numbers of cells become committed during further passages in HMBA plus TPA. Since these leaked committed cells were still blocked by TPA from their expression into differentiating phenotypes, there was a gradual accumulation of committed cells in the population; up to 90% of cells became committed after 7 months of culture. For many generations, these accumulated committed cells can grow normally only in the presence of TPA maintaining their committed state. These results suggest that TPA-mediated inhibition of HMBA-induced differentiation is reversible up to 2.5 months and later becomes irreversible; they also suggest that TPA prevents FELC not only from entering into commitment but also from expressing com mitment into differentiation. Since TPA does not erase the capability of FELC in their erythroid differentiation, even after 8 months of continuous culture, and the cells grow and multiply normally, TPA appears to affect the functions specific for differentiation without changing the general growth functions of the cells.