It was previously reported that maximal suppression of deoxyuridine incorporation into DNA requires exchangeable methotrexate (MTX(ex)), i.e. intracellular MTX in excess of the tightly bound fraction, (MTX(b)). Further, MTX(ex) is required to suppress 14C formate incorporation into DNA, RNA and protein as well, suggesting that in the presence of MTX(b) alone adequate tetrahydrofolate (THF) cofactor levels may be maintained. We now describe the effects of MTX(b) and MTX(ex) on the reduction of folic acid (FA) and DHF in intact cells. Cells were incubated with or without MTX to saturate high affinity sites then the MTX treated cells were divided into two portions, one incubated without MTX to allow efflux of MTX(ex) and the other continued in MTX. The three cell portions were then incubated with 3H FA or 3H DHF, sonicated, and the soluble radiolabelled components separated by DEAE cellulose chromatography. Reduction of 3H FA was markedly decreased by MTX(b) alone; although a small level of residual THF synthesis persisted, this was completely eliminated by MTX(ex). MTX(b) alone had little effect on 3H DHF reduction; but THF synthesis was abolished when MTX(ex) was 5 μM. The data indicate that MTX(ex) is required to inhibit DHF reduction in these cells in vitro. The high levels of MTX(ex) achieved with high dose infusions may contribute to the increased cytotoxicity of these regimens.
|Original language||English (US)|
|Journal||Proceedings of the American Association for Cancer Research|
|State||Published - Jan 1 1975|
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