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 (1 microM), most of the endogenous radiolabeled folates were present as formyl-substituted
tetrahydrofolates (60-73%, including 10- and 5-formyl and 5,10-methenyl
tetrahydrofolate), with lower levels of
tetrahydrofolate (including 5,10-methylene
tetrahydrofolate), 5-methyl
tetrahydrofolate, and non-metabolized
folic acid.
Trimetrexate (1 microM) 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 formyl
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 microM) 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 microM), all of the intracellular folates were
protein-bound; only when maximal growth was achieved, could unbound folates be detected. Incubation with
trimetrexate (1 or 10 microM),
methotrexate (10 microM), or
calcium leuvovorin (50 microM) did not alter significantly the levels of total and
protein-bound [3H]folates in cells grown in 1 microM [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 (50 microM). Hence,
protein-bound
tetrahydrofolates must not only be substrates for
enzyme mediated reactions (i.e.
TMP synthesis) but also must slowly equilibrate with unbound cofactor. In this fashion, binding of endogenous folates to soluble
proteins may function to "segregate' intracellular cofactor pools.(ABSTRACT TRUNCATED AT 400 WORDS)