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The Intracellular Concentration Dependence of Antifolate Inhibition of DNA Synthesis in L1210 Leukemia Cells¹

Memorial Sloan-Kettering Cancer Center, New York, New York 10021

The relationship between the accumulation of methotrexate (MTX) in L1210 cells and effects on DNA synthesis, as measured by the incorporation of uridine-³H into DNA, was examined in vivo during therapy and in vitro using a system supporting metabolic activity. Effects on DNA synthesis in vitro were not observed until the intracellular drug level approached the equivalence of the dihydrofolate reductase content. At approximately 1 equivalence, DNA synthesis is inhibited 20%. Further inhibition of synthesis required the accumulation of MTX to levels well in excess of enzyme content. The 50% inhibitory dose for [MTX]_internal was 1 x 10^-6 M. Effects on DNA synthesis in vivo were not observed until the intracellular drug level exceeded 60% of the equivalent dihydrofolate reductase. However, at approximately 1 equivalence, DNA synthesis was completely inhibited. Effects of MTX on DNA synthesis in vivo displays a delayed time course. Approximately 2 hr were required before the effect on synthesis fully reflected the [MTX]_internal. Thymidine-³H incorporation into DNA in vitro was stimulated by MTX. Stimulation of thymidine-³H incorporation was also observed in vivo but was transitory and followed by a short period of inhibition which was dose dependent.

The results of the studies in vivo are in complete agreement with the notion of a high-affinity dihydrofolate reductase as the primary target of antifolate drugs. The discrepancy observed in vitro on the dose dependence of drug-related effects on DNA synthesis, namely the requirement for high levels of free (exchangeable) MTX well in excess of that required to completely inhibit dihydrofolate reductase, most probably reflects an aberrant metabolic state under conditions that do not allow normal proliferative activity. Other results suggest that high levels of free drug in vitro achieve inhibition of DNA synthesis via a direct effect on thymidylate synthetase.

¹ This work was supported in part by Grant CA-08748-09 from the National Cancer Institute and by Grant BC-108 from the American Cancer Society.

Received 7/22/74. Accepted 9/ 9/74.