Reduced Activity of Anabolizing Enzymes in 5-Fluorouracil-resistant Human Stomach Cancer Cells

The mechanism of resistance to 5-fluorouracil (5-FU) was studied with NUGC-3/5FU/L, a human stomach cancer cell line which had acquired resistance as a consequence of repeated 5-day exposures to stepwise-increasing concentrations of 5-FU in vitro . NUGC-3/5FU/L was 200-fold and over 16-fold resistant to 96-h and 1-h exposures to 5-FU, respectively. NUGC-3/5FU/L incorporated less 5-FU into RNA, indicating resistance to the RNA-directed action of 5-FU. On the other hand, NUGC-3/5FU/L also showed resistance to in situ thymidylate synthase (TS) inhibition by 5-FU. Polymerase chain reaction-single-strand conformation polymorphism analysis of TS cDNA and a FdUMP ligand binding assay showed that quantitative and qualitative alterations of TS are not responsible for this resistance. In contrast, the ability to metabolize 5-FU to its active metabolites, FUTP and FdUMP, was reduced in NUGC-3/5FU/L. We found that not only the activities of uridine phosphorylase/kinase and orotate phosphoribosyl-transferase (OPRT), but also the level of phosphoribosyl pyrophosphate, a cosubstrate for OPRT, were significantly lower in NUGC-3/5FU/L than in the parent NUGC-3. These results indicated that resistance to 5-FU in NUGC-3/5FU/L is due to reduced activities of 5-FU-anabolizing enzymes, but not to an alteration of TS. 2'-Deoxyinosine effectively enhanced TS inhibition by 5-FU in the resistant cells, thus markedly sensitizing them to 5-FU.