Assessment of growth-limiting events caused by 5-fluorouracil in mouse cells and in human cells.

Abstract

The purpose of this study was to assess the significance of thymidylate synthetase (dTMP synthetase) inhibition in growth inhibition caused by 5-fluorouracil (FUra). Three fibroblastic mouse cell lines (Sarcoma 180, MMT, L-cells), three human carcinoma cell lines (HeLa, KB, Hep-2), and a human fibroblast line (U-cells) were incubated for 3 hr with varied concentrations of FUra, and the following was determined: ( a ) subsequent growth (5 or 6 days) in FUra-free medium and in “rescue” medium i.e. , in a medium supplemented with 30 µm thymidine (dThd) or 1 mm 2′-deoxyuridine (dUrd; ( b ) molarity of FUra and its metabolites in cell water and its incorporation into RNA; ( c ) the specific activity of dTMP synthetase in cell extracts; ( d ) the incorporation of 5 µm [2-14C]dUrd into DNA and the acid-soluble nucleotides derived therefrom. The specific activities of dTMP synthetase in untreated cells related as Sarcoma 180 < MMT < Hep-2 < U-cells < L-cells < KB < HeLa, being 4.5 times higher in HeLa cells than in Sarcoma 180 cells. The enzyme was inhibited to a similar degree in all cells after 1 to 10 µm FUra, while the acid-soluble 5-fluoro-2′-deoxyuridine 5′-monophosphate remained below detection. The half-life of free 5-fluoro-2′-deoxyuridine 5′-monophosphate (formed from 5-fluoro-2′-deoxyuridine) was very short in Sarcoma 180, MMT, L-, and U-cells (1 to 3 min), 10 times longer in HeLa, KB, and Hep-2, while the half-life of deoxyuridine 5′-monophosphate (dUMP) was within 1 and 4 min in all cells. Mouse cells could be partially rescued with dThd, and the dose-response curves for the inhibition of dUrd incorporation and the dTMP synthetase activity were similar and paralleled the growth inhibition curve. In these cells, the level of labeled deoxythymidine triphosphate formed from [2-14C]dUrd was reduced by 80 to 90%, while the level of [2-14C]dUMP was hardly affected. A limited, spontaneous recovery of the capacity to incorporate dUrd occurred in Sarcoma 180 up to 6 hr following FUra, and 1 mm dUrd rescued these cells as well as did 30 µm dThd. These observations are consistent with the conclusion that in Sarcoma 180, MMT, L-, and U-cells dTMP synthetase inhibition is the growth-limiting event at low concentrations of FUra (5 to 20 µm). After higher concentrations (15 to 70 µm), even these cells could not be rescued with dThd (second site). In contrast, HeLa, KB, and Hep-2 cells, which require higher concentrations of FUra for growth inhibition (50 to 200 µm), were rescued poorly or not at all by dThd, and the inhibition of [2-14C]dUrd incorporation into DNA paralleled growth inhibition, not inhibition of dTMP synthetase. In these cells, the level of labeled deoxythymidine triphosphate derived from [14C]dUrd was hardly affected by FUra, while the labeled dUMP in cell water rose from undetectable concentrations to 200 µm. Thus, in spite of the fact that dTMP synthetase was inhibited by concentrations of FUra similar to those in mouse cells, the ability of these cells to accumulate high levels of dUMP may prevent dTMP synthetase inhibition from becoming critical for growth. At the second site, all mouse and human cells incorporated 5 to 17 nmol, of FUra per mg of RNA, i.e. , 1 to 2% replacement of RNA uracil by FUra, and the cell water contained 200 to 600 µm 5-fluoro-2′-deoxyuridine triphosphate and 50 to 100 µm FUra. It is concluded that low levels of FUra cause growth inhibition of Sarcoma 180, MMT, L-, and U-cells by inhibiting dTMP synthetase, while at high levels of FUra the incorporation into RNA seems to be growth limiting. In contrast, the growth inhibition of human HeLa, KB, and Hep-2 cells by FUra is due to its incorporation into RNA.