Evidence for a localized conversion of endogenous tetrahydrofolate cofactors to dihydrofolate as an important element in antifolate action in murine leukemia cells

Abstract

The inhibition of de novo nucleotide, serine, and methionine biosynthesis in mammalian cells treated with antifolates has been attributed generally to a reduction in the levels of tetrahydrofolate cofactors. In L1210 leukemia cells grown in tritiated folic acid (1μM), most of the endogenous radiolabeled folates were present as formyl-substituted tetrahydrofolates (60–73%, including 10- and 5-forrnyl and 5,10-methenyl tetrahydrofolate), with lower levels of tetrahydrofolate (including 5,10-methylene tetrahydrofolate), 5-methyl tetrahydrofolate, and non-metabolized folic acid. Trimetrexate (1μM) caused an elevation of dihydrofolate levels within 5 min following drug addition, from approximately 1 to 20% of the total folates. Whereas total reduced folates were preserved, losses in the levels of individual forms ranged from minor changes in the fonnyl tetrahydrofolates (approx. 10% decrease), to significant losses in the levels of tetrahydrofolate (approx. 60%) and 5-methyl tetrahydrofolate (95%). Under these conditions, the incorporations of [ 3H]deoxyuridine into TMP and [ 14C]glycine into purines or of [ 14C]formate into biosynthetic products were inhibited (69–95%). The majority (59–100%) of the endogenous radiolabeled folates in L1210 cells grown in various concentrations (0.2 to 3 μM) of [ 3H]folic acid was bound to soluble intracellular proteins when cell-free extracts were fractionated by rapid gel filtration or charcoal adsorption. Total intracellular folate levels increased in proportion to the changes in medium folic acid concentration; however, cofactor binding was saturable. At low concentrations, below that which supported maximal growth (less than 0.75 μM), all of the intracellular folates were protein-bound; only when maximal growth was achieved, could unbound folates be detected. Incubation with trimetrexate (1 or μM), methotrexate (10μM), or calcium leucovorin (50μM) did not alter significantly the levels of total and protein-bound [ 3H]folates in cells grown in 1μM [ 3H]folic acid. Under all conditions, formyl tetrahydrofolates were the major intracellular derivatives; however, these forms were poorly represented in the bound fraction. Conversely, all of the other intracellular folate forms were completely bound. Tetrahydrofolate was the predominant protein-bound derivative in control cells; in antifolate-treated cells, both bound tetrahydrofolate and 5-methyl tetrahydrofolate were largely replaced by protein-bound dihydrofolate. This interconversion in drug-treated cells was independent of (i) sustained levels of [ 3H]formyl tetrahydrofolates, or (ii) high extracellular concentrations of unlabeled calcium leucovorin (50μM). Hence, protein-bound tetrahydrofolates must not only be substrates for enzyme-mediated reactions (i.e. TMP synthesis) but also must slowly equilibrate with unbound cofactors. In this fashion, binding of endogenous folates to soluble proteins may function to “segregate” intracellular cofactor pools. The depletion of such a localized fraction of tetrahydrofolate cofactors in antifolate-treated cells could contribute to the inhibition of biosynthetic processes yet would not necessarily be accompanied by significant changes in the levels of other derivatives, nor the total size of the pool of reduced folates.