Depletion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate by methotrexate in cultured hepatoma cells.
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.
Rhee
M S
MS
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Coward
J K
JK
Galivan
J
J
D000818:Animals; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists; D002955:Leucovorin / Q000031:analogs & derivatives* / Q000378:metabolism; D008114:Liver Neoplasms, Experimental; D008727:Methotrexate / Q000494:pharmacology*; D013763:Tetrahydrofolates / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D005493:Folic Acid Antagonists; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; C010161:10-formyltetrahydropteroylglutamic acid; D005492:Folic Acid; D002955:Leucovorin; D008727:Methotrexate