Folate analogs that inhibit
dihydrofolate reductase result in only partial interconversion of
tetrahydrofolate cofactors to
dihydrofolate with preservation of the major portion of reduced cellular
folate cofactors in
L1210 leukemia cells. One possible explanation for this phenomenon is that low levels of
dihydrofolate polyglutamates that accumulate in the presence of
antifolates block
thymidylate synthase to prevent depletion of reduced
folate pools. This paper correlates biochemical analyses of rapid interconversions of radiolabeled folates and changes in
purine and
pyrimidine biosynthesis in L1210 murine
leukemia cells exposed to
antifolates with network thermodynamic computer modeling to assess this hypothesis. When cells are exposed to 1 microM
trimetrexate there is an almost instantaneous inhibition of [3H]
deoxyuridine or [14C]
formate incorporation into
nucleotides which is maximal within 5 min. This is associated with a rapid rise in cellular
dihydrofolate (t1/2 approximately 1.5 min), which reaches a steady state that represents only 27.9% of the total
folate pool. Pretreatment of cells with
fluorodeoxyuridine, to inhibit
thymidylate synthase by about 95% followed by
trimetrexate only slows the rate of
folate interconversion (t1/2 approximately 25 min) but not the final
dihydrofolate level achieved. This is consistent with computer simulations which predict that direct inhibition of
thymidylate synthase by 97, 98, and 99% should increase the half-time of
dihydrofolate rise after
trimetrexate to 40, 60, and 124 min, respectively, but the final level achieved is always the same as in cells with normal
thymidylate synthase activity. The data reflect the high degree of catalytic activity of
thymidylate synthase relative to
tetrahydrofolate cofactor pools in the cells and the enormous extent of inhibition of this
enzyme that is necessary to slow the rate of
folate interconversions after addition of
antifolates. The model predicts, and the data demonstrate, that virtually any residual
thymidylate synthase activity will permit the interconversion of all
tetrahydrofolate cofactors available for oxidation to
dihydrofolate when
dihydrofolate reductase activity is abolished, but the rate of interconversion will be slowed. Additional simulations indicate that the time course of cessation of
tetrahydrofolate-dependent
purine and
pyrimidine biosynthesis after
antifolates in these cells can be accounted for solely on the basis of
tetrahydrofolate cofactor depletion alone. These data exclude the possibility that direct inhibition of
thymidylate synthase by
dihydrofolate polyglutamates, or any other intracellular folates that accumulate in cells after
antifolates, can account for the rapid but partial interconversion of reduced
folate cofactors to
dihydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)