Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-γ

The effects of fluorouracil (5-FU) and interferon-γ (IFN-γ) on the regulation of thymidylate synthase (TS) gene expression were investigated in the human colon cancer H630 cell line. By Western immunoblot analysis, TS protein levels in H630 cells were increased 3-, 5.5-, 5-, and 2.5-fold after 8-, 16-, 24-, and 36-hr exposure to 1 μM 5-FU, respectively. When H630 cells were exposed to varying concentrations of 5-FU (0.3-10 μM) for 24 hr, increases in TS protein up to 5.5-fold were observed. A 24-hr exposure to 1 μM 5-FU resulted in a 4.5-fold increase in the level of TS protein, whereas in 5-FU/IFN-γ-treated cells TS protein was increased by only 1.8-fold, compared with control cells. IFN-γ treatment alone did not affect TS protein levels, relative to control. Northern blot analysis revealed no changes in TS mRNA levels when H630 cells were exposed either to 1 μM 5-FU for 8-36 hr, to varying concentrations of 5-FU (0.3-10 μM) for 24 hr, or to the combination of 5-FU and IFN-γ. Pulse-labeling studies with [³⁵S]methionine demonstrated a 3.5-fold increase in net synthesis of TS in cells treated with 1 μM 5-FU, whereas the level of newly synthesized TS increased only 1.5-fold in cells treated with 5-FU/IFN-γ, compared with control cells. Pulse-chase studies revealed that the half-lives of TS protein in control and 5-FU-treated cells were equivalent. These findings demonstrate that the increase in TS protein after 5-FU exposure and the subsequent inhibitory effect of IFN-γ on TS protein expression are both regulated at the post-transcriptional level.