Previous studies from this laboratory established that the rapid but partial interconversion of
tetrahydrofolate cofactors to
dihydrofolate after exposure of
L1210 leukemia cells to
antifolates cannot be due to direct feedback inhibition of
thymidylate synthase by
dihydrofolate or any other endogenous folylpolyglutamates when
dihydrofolate reductase activity is abolished by
antifolates. Rather, the data suggested this preservation of
tetrahydrofolate cofactor pools is likely due to a fraction of cellular folates unavailable for oxidation to
dihydrofolate. This paper explores the role of cell cycle phase in
L1210 leukemia cells in logarithmic versus stationary phase growth as
a factor in the rate and extent of
tetrahydrofolate cofactor interconversion to
dihydrofolate after exposure of cells to the
dihydrofolate reductase inhibitor trimetrexate. The S phase fraction was reduced by inoculating
L1210 leukemia cells at high density to achieve a stationary state. Flow cytometric analysis of
DNA content indicated that log phase cultures were 53.0% S phase; this decreased to 42.1% at 24 h and 24.1% at 48 h in stationary phase cultures.
5-Bromo-2'-deoxyuridine incorporation into
DNA decreased 80 and 96%, while [3H]dUrd incorporation into
DNA declined 70 and 95% for stationary cultures at 24 and 48 h, respectively, as compared with the log phase rates. Log phase cells interconverted 28.0% of the total pool of radiolabeled folates to
dihydrofolate with a half-time of approximately 30 s. Stationary cells at 24 h interconverted 20.4% of the total
folate pool with a t1/2 of approximately 3 min, and at 48 h, net interconversion to
dihydrofolate decreased further to 12.1% with a t1/2 of approximately 6 min. The decrease in the extent of
tetrahydrofolate cofactor interconversion to
dihydrofolate in stationary phase cells was directly proportional to the decrease in the S phase fraction determined by total
DNA content. This suggests that
tetrahydrofolate cofactor depletion occurs only in S phase cells. The much larger drop in [3H]dUrd and
5-bromo-2'-deoxyuridine incorporation into
DNA in comparison with the decline in the S phase fraction measured by
DNA content along with the reduced rate of
tetrahydrofolate cofactor interconversion to
dihydrofolate indicates that the rate of
DNA synthesis is decreased in S phase cells in stationary cultures. Network thermodynamic simulations suggest that a reduction in the number of S phase cells and their
thymidylate synthase catalytic activity would account for the observed decrease in the rate and extent of interconversion of
tetrahydrofolate cofactors to
dihydrofolate after
trimetrexate in stationary phase cultures.(ABSTRACT TRUNCATED AT 400 WORDS)