Effect of methotrexate on intracellular folate pools in purified myeloid precursor cells from normal human bone
Abstract:
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.
Insights
Methotrexate (MTX) alters folate pools in myeloid cells, decreasing key folates and increasing dihydrofolate forms. This impacts purine synthesis and myeloid colony formation.
Area of Science:
- Biochemistry
- Hematology
- Pharmacology
Background:
- Methotrexate (MTX) is an antifolate drug used in various therapies.
- Understanding MTX's effects on intracellular folate metabolism is crucial for its clinical application.
- Human myeloid precursor cells (MPCs) are critical for blood cell development.
Purpose of the Study:
- To investigate the impact of MTX on intracellular folate pools in human MPCs.
- To determine the specific folate species affected by MTX exposure.
- To correlate changes in folate pools with effects on myeloid cell function and purine synthesis.
Main Methods:
- Isolation of immature human MPCs from bone marrow using immune rosetting.
- Labeling of intracellular folate pools with [3H]5-formyl-tetrahydrofolate ([3H]5-formyl-FH4).
- Quantification of folate pools using high-performance liquid chromatography (HPLC) after MTX exposure.
- Assessment of myeloid colony formation and de novo purine synthesis.
Main Results:
- MTX significantly reduced intracellular concentrations of 10-formyl-FH4, 5-formyl-FH4, and 5-methyl-FH4.
- MTX induced the appearance and expansion of dihydrofolate (FH2) and 10-formyl-FH2 pools.
- These folate changes, particularly FH2 increase, correlated with reduced myeloid colony formation and de novo purine synthesis.
Conclusions:
- MTX does not cause quantitative depletion of 10-formyl-FH4.
- The antifolate effect of MTX on purine synthesis may involve direct inhibition by FH2.
- MTX polyglutamates might contribute to antifolate effects at later time points.
