The role of thymidylate synthase induction in modulating p53-regulated gene expression in response to 5-fluorouracil and antifolates.
Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.
Longley
Daniel B
DB
Department of Oncology, Cancer Research Centre, Queen's University Belfast, Belfast BT9 7AB, Northern Ireland.
Boyer
John
J
Allen
Wendy L
WL
Latif
Tariq
T
Ferguson
Paul R
PR
Maxwell
Pamela J
PJ
McDermott
Ultan
U
Lynch
Maria
M
Harkin
D Paul
DP
Johnston
Patrick G
PG
D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D004790:Enzyme Induction; D005472:Fluorouracil / Q000493:pharmacokinetics / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects; D019014:fas Receptor / Q000502:physiology
D005493:Folic Acid Antagonists; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor; D013940:Thymidylate Synthase; D005472:Fluorouracil