Effect of methotrexate on intracellular folate pools in purified myeloid precursor cells from normal human bone marrow.
We investigated the effects of the antifolate methotrexate on intracellular folate pools of human myeloid precursor cells (MPCs). Immature MPCs, representing 3.2% of the original marrow population, were selected from normal human bone marrow by immune rosetting. The intracellular folate pools were labeled by incubation with 5 X 10(-8) M [3H]5-formyl-FH4 and were quantitated by high performance liquid chromatography. The predominant folates were 5-methyl-tetrahydrofolate (5-methyl-FH4) (36%), 10-formyl-FH4 (41.4%), 5-formyl-FH4 (12.3%), and FH4 (10.3%). A 12-h exposure to 1 microM methotrexate (MTX) resulted in a 34% reduction in the intracellular concentration of 10-formyl-FH4, a 61% decrease in 5-formyl-FH4, and a 62% decrease in 5-methyl-FH4, as well as the appearance and progressive expansion of the FH2 and 10-formyl-FH2 pools. These changes were maximal after 4 h of incubation with MTX. Paralleling the changes in folates, particularly the increase in FH2, were a 64% reduction in myeloid colony formation and a 77% depression of de novo purine synthesis after 4 h of MTX. We conclude that MTX does not produce quantitative depletion of 10-formyl-FH4 and that its antipurine effect may be mediated by direct inhibition of de novo purine synthesis by FH2 and, at later time points, by MTX polyglutamates.
D001854:Bone Marrow Cells; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D005575:Formyltetrahydrofolates / Q000378:metabolism; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology*; D011687:Purines / Q000096:biosynthesis; D013763:Tetrahydrofolates / Q000378:metabolism*
D005575:Formyltetrahydrofolates; D011687:Purines; D013763:Tetrahydrofolates; C005984:5-methyltetrahydrofolate; D008727:Methotrexate