The mechanism of action of methotrexate. III. Requirement of free intracellular methotrexate for maximal suppression of [¹⁴C]formate incorporation into nucleic acids and protein

Recent studies from this laboratory suggest that association of methotrexate with high afffinity intracellular binding sites has little effect upon the incorporation of [³H]deoxyuridine into DNA, while relatively high intracellular levels of methotrexate in excess of the tightly bound fraction are required for inhibition. In this report the effects of different components of intracellular methotrexate on [¹⁴C]formate uptake and incorporation into nucleic acids and proteins in cultured L cell mouse fibroblasts are evaluated. [¹⁴C]Formate is rapidly taken up by these cells and metabolized to molecular forms which do not penetrate the cell membrane. After 30 min about 60% of the ¹⁴C remains in the acid soluble fraction. Of the radiolabel in the acid precipitate approximately 55% is associated with RNA, 11% with DNA, and 33% with protein. Intracellular methotrexate sufficient only to saturate high affinity sites causes only slight inhibition of [¹⁴C]formate uptake by intact cells or of [¹⁴C]formate incorporation into RNA, DNA, and protein. However, intracellular methotrexate in excess of this fraction markedly inhibits these processes, with 50% inhibition of [¹⁴C]formate incorporation into DNA, RNA, and protein at intracellular methotrexate levels of 0.3, 1.0, and 3 μM, respectively. These data suggest that intracellular methotrexate in excess of that necessary to saturate high affinity sites is required to suppress nucleic acid and protein synthesis, an effect that may be related to an interaction between methotrexate and a dihydrofolate reductase form with a low affinity for this agent.