Reduced activity of anabolizing enzymes in 5-fluorouracil-resistant human stomach cancer cells.
The mechanism of resistance to 5-fluorourcil (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-3/5/5FU/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.
Inaba
M
M
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan.
Mitsuhashi
J
J
Sawada
H
H
Miike
N
N
Naoe
Y
Y
Daimon
A
A
Koizumi
K
K
Tsujimoto
H
H
Fukushima
M
M
D000964:Antimetabolites, Antineoplastic / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics; D004351:Drug Resistance; D005472:Fluorouracil / Q000378:metabolism*; D005796:Genes; D006801:Humans; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D018807:Polymorphism, Single-Stranded Conformational; D012334:RNA, Neoplasm / Q000737:chemistry; D013274:Stomach Neoplasms / Q000201:enzymology*; D013940:Thymidylate Synthase / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D010754:Phosphoribosyl Pyrophosphate; D013940:Thymidylate Synthase; D005472:Fluorouracil