Relationship between p53 status and 5-fluorouracil sensitivity in 3 cell lines.
Mouse lymphoma L5178Ytk+/- (MOLY) cells and human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. MOLY and WTK-1 cells have a p53 mutation, while TK6 cells, which were derived from the same parental line as WTK-1 cells, do not. In this study, we tested the clastogen 5-fluorouracil (5-FU) in the Tk assay and the in vitro micronucleus (MN) assay in MOLY, TK6, and WTK-1 cells to clarify whether differential responses were related to p53 gene status. We also determined the effect of 5-FU on the frequency of apoptotic cells and on cell cycle distribution in each cell line. Furthermore, we measured the activity of the 5-FU metabolizing enzymes (thymidylate synthetase (TS), dihydrouracil dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), and thymidine phosphorylase (TP)) in each cell line. We treated MOLY cells with 1.0-8.0 microg/mL 5-FU for 3 h and TK6 and WTK-1 cells with 1.56-25 and 3.13-50 microg/mL, respectively, for 4 h. In MOLY cells, the mutation frequency (MF) and MN frequency increased. In WTK-1 cells, the MN frequency but not the MF increased. In TK6 cells, neither the MF nor the MN frequency increased. Furthermore, the IC50 of 5-FU was lower in MOLY cells than in the human cells. The response to 5-FU treatment differed in other ways as well. At the same level of cytotoxicity, the frequency of apoptotic cell was highest in TK6 cells. The cell cycle was delayed just after treatment in MOLY cells while the delay appeared 24 h later in TK6 and WTK-1 cells. Nothing in our analysis, however, revealed marked differences between the cell lines that could account for the severe cytotoxic and mutagenic responses that 5-FU elicited only in MOLY cells. 5-FU is phosphorylated by OPRT and TP and detoxified by DPD. MOLY cells have higher OPRT activity and markedly lower DPD and TP activity than TK6 and WTK-1 cells. The content of TS, however, the target enzyme of 5-FU, was similar in all cell lines, suggesting that 5-FU was more readily phosphorylated and less readily detoxified in MOLY cells than in TK6 and WTK-1 cells. MOLY cells were more sensitive to 5-FU than WTK-1 cells even though both have a mutated p53 gene, suggesting that the different responses to 5-FU were due to differences in 5-FU metabolism rather than the p53 status.
Drug Safety Research Lab., Taiho Pharmaceutical Co. Ltd., 224-2 Ebisuno, Hiraishi, Kawauchi-cho, Tokushima, Japan. hiro-oka@taiho.co.jp
Hiroaki
H
Oka
Kazumasa
K
Ikeda
Hiromi
H
Yoshimura
Akinobu
A
Ohuchida
Masamitsu
M
Honma
D000818:Animals; D002452:Cell Count; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D004354:Drug Screening Assays, Antitumor; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D015162:Micronucleus Tests; D008954:Models, Biological; D013937:Thymidine Kinase / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D013940:Thymidylate Synthase; D013937:Thymidine Kinase; D005472:Fluorouracil