Phenotypic resistance to methotrexate and N-phosphonacetyl L-aspartate is induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA).

Three different 3T6 mouse fibroblast cell clones with intrinsically different sensitivities to methotrexate (MTX) have been isolated from an originally heterogeneous population. When these 3 different clones were exposed to MTX in the presence of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) the degree of enhancement of recovery of methotrexate resistant (MTXR) colonies was greatest in the most sensitive clone. MTXR colonies isolated and cultured in the presence and absence of MTX and TPA were analysed for dihydrofolic acid reductase (dhfr) levels by flow cytometry after binding of fluorescent methotrexate. None of the 58 clones showed major changes in dhfr levels. Dot-blot analysis of 12 clones indicated no increases in dhfr gene copy number or mRNA levels consistent with gene amplification. Southern analysis of 6 further clones indicated that only 1 clone isolated by multi-step selection had amplified dhfr sequences. TPA-enhanced mouse 3T6 N-phosphonacetyl-L-aspartate (PALA)-resistant colony recovery and mouse 3T3 MTXR colony recovery were also shown, by dot-blot analysis, not to be due to gene amplification. The data indicate that TPA can have a profound effect on drug-resistant colony recovery by mechanisms other than induction of gene amplification.