Molecular downstream events and induction of thymidylate synthase in mutant and wild-type p53 colon cancer cell lines after treatment with 5-fluorouracil and the thymidylate synthase inhibitor raltitrexed

Eur J Cancer. 2000 May;36(7):916-24. doi: 10.1016/s0959-8049(00)00026-5.

Abstract

Inhibition of the key enzyme in DNA synthesis, thymidylate synthase (TS), by 5-fluorouracil (5-FU) and the novel antifolate raltitrexed (Tomudex; ZD1694), induces dTTP depletion, resulting in DNA strand breaks, which can initiate pathways leading to an apoptotic mode of cell death. We studied 5-FU- and ZD1694-induced TS inhibition in relation to the expression of p53, p21, Bcl-2 and Bax in six colon carcinoma cell lines, two with a wild-type (wt) p53 (Lovo, LS174T) and four with a mutant (mt) p53 (WiDr, WiDr/F, HT29 and SW948) phenotype. In untreated cells, a reciprocal correlation between p53 and Bcl-2 was found: in cells with a low wt p53, Bcl-2 expression was present; whilst in cells with mt p53, Bcl-2 expression was not detectable. Exposure to 5-FU (50 and 100 microM) and ZD1694 (50 and 100 nM) for 24 and 48 h induced p53 and p21 expression in wt p53 cells, but not in mt p53 cells. TS was induced approximately 2-10-fold in all cell lines. TS induction was highest after ZD1694 exposure in the mt p53 cells HT29 and WiDr/F (6-10-fold). After 5-FU treatment, TS was present both as the free enzyme and in the ternary complex; however, predominantly as the ternary complex between TS, FdUMP and 5,10-methylenetetrahydrofolate. In wt p53 cells, both drugs increased Bax expression up to 5-fold, whereas in mt p53 cells, only a very slight induction was found. In wt p53 cells, Bcl-2 expression hardly changed after drug treatment. These results indicate a p53-independent induction of TS but a regulatory role of wt p53 in the synthesis of Bax in the colon carcinoma cell lines after TS inhibition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use*
  • Apoptosis
  • Blotting, Western
  • Colonic Neoplasms / drug therapy*
  • Colonic Neoplasms / enzymology
  • Enzyme Inhibitors / administration & dosage
  • Flow Cytometry
  • Fluorouracil / administration & dosage
  • Genes, bcl-2 / genetics
  • Genes, p53 / genetics
  • Humans
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins c-bcl-2*
  • Quinazolines / administration & dosage
  • Thiophenes / administration & dosage
  • Thymidylate Synthase / antagonists & inhibitors
  • Thymidylate Synthase / genetics
  • Thymidylate Synthase / metabolism
  • Tumor Cells, Cultured
  • bcl-2-Associated X Protein

Substances

  • BAX protein, human
  • Enzyme Inhibitors
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Quinazolines
  • Thiophenes
  • bcl-2-Associated X Protein
  • Thymidylate Synthase
  • raltitrexed
  • Fluorouracil