Combination of low doses of de novo
pyrimidine biosynthesis inhibitors with
5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU.
Brequinar is such an inhibitor that has little clinical anti-
tumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU +/-
leucovorin (LV) +/-
brequinar and examined the effects of these treatments on
thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC50) for
brequinar, FU, and FU+LV (100 microM) were 0.4, 20, and 10 microM, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC10 (0.1 microM) of
brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 microM
brequinar and FU +/- LV did not increase the cytotoxicity of FU +/- LV. Intracellular
cytidine triphosphate (
CTP) and
uridine triphosphate (
UTP) pools, free TS binding sites, and levels of free
fluorodeoxyuridine monophosphate (
FdUMP) and
deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 microM
brequinar for 24 h alone or followed by a 2-h exposure to FU (25 microM) +/- LV (100 microM). In
brequinar-treated cells,
CTP and
UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU+brequinar. Free
FdUMP levels increased 5-fold in cells pretreated with
brequinar as compared with those treated with FU alone. The increased formation of
FdUMP was inhibited by simultaneous exposure to 100 microM
hypoxanthine and 25 microM FU. Intracellular dUMP levels were not affected by
brequinar. We conclude that a low dose of
brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to
brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of
FdUMP as a consequence of an increase in the cosubstrate
phosphoribosyl pyrophosphate (PRPP).