Phorbol ester dramatically increases incidence of methotrexate-resistant mouse cells: possible mechanisms and relevance to tumor promotion.

I have tested the hypothesis that at least some of the known tumor promoters may act by facilitating gene amplification. A series of single-step selections for resistance to methotrexate, a specific inhibitor of dihydrofolate reductase (DHFR), was carried out with 3T6 cells in the presence and in the absence of a potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Incidence of methotrexate-resistant, colony-forming 3T6 cells is increased up to 100 fold if selection is carried out in the presence of TPA. For a major portion of this new TPA effect to be observed, it is sufficient to add TPA simultaneously with methotrexate. The effect of TPA on the incidence of methotrexate resistance is detectable at less than 20 nM TPA and is maximal at about 200 nM TPA. Phorbol (a nonpromoting analog of TPA), thymidine and dimethylsulfoxide each fail to produce any TPA-like effect in this system. DHFR gene copy numbers per cell in clones resistant to 100, 200 and 300 nM methotrexate are approximately 3, 10 and 16 times higher, respectively, than the DHFR gene copy number in the parental 3T6 cells. These numbers do not depend on the presence or absence of TPA during methotrexate selection.