The effect of the inhibition of
dihydrofolate reductase by
methotrexate on the cellular folates involved in de novo
purine and thymidylate biosynthesis has been measured in H35
hepatoma cells grown in 4 microM
folic acid or 20 nM
folinic acid. The major cellular
folate species in cells from medium with
folate or folinate is
10-formyltetrahydrofolate (approximately 5 microM), with lesser amounts of
5,10-methylenetetrahydrofolate and
tetrahydrofolate. Cultures were exposed to a pulse dose of
methotrexate, resulting in the accumulation of nearly exclusively
methotrexate polyglutamates (predominantly Glu3, Glu4, and Glu5), or a continuous exposure to the poorly glutamylated analog threo-4-fluoromethotrexate, resulting in 93% intracellular monoglutamate. At 4 hr and 18 hr after exposure to either compound there was extensive depletion of the reduced
folate coenzymes, which generally corresponded to the extent of inhibition of
glycine and
deoxyuridine incorporation. This was accompanied by an increase of the cellular
dihydrofolate and
10-formyldihydrofolate. In the H35 cells the effect of
methotrexate polyglutamates on the reduced
folate coenzyme pools was restricted to dividing cultures, because the reduced
folate coenzymes were not depleted in confluent cultures. The results demonstrate that the
methotrexate and
methotrexate polyglutamates that initially accumulate within dividing H35 cells readily inhibit
dihydrofolate reductase but are not adequate to inhibit
thymidylate synthase and prevent the depletion of reduced
folate coenzymes. Thus, inhibition of de novo
glycine and
deoxyuridine incorporation into
DNA as a result of
dihydrofolate reductase inhibitors appears to be closely related to a reduction in the intracellular concentration of
10-formyltetrahydrofolate and
5,10-methylenetetrahydrofolate, the respective
folate coenzymes for de novo
purine and thymidylate synthesis.