Thymidylate synthase inhibition induces p53-dependent and p53-independent apoptotic responses in human urinary bladder cancer cells

J Cancer Res Clin Oncol. 2011 Feb;137(2):359-74. doi: 10.1007/s00432-010-0891-y. Epub 2010 Apr 28.


Purpose: In search for more effective clinical protocols, the antimetabolite drug 5-fluorouracil (5-FU) has been successfully included in new regimens of bladder cancer combination chemotherapy. In the present study, we have investigated the effects of 5-FU treatment on apoptosis induction in wild-type and mutant p53 urinary bladder cancer cells.

Methods: We have used MTT-based assays, FACS analysis, Western blotting and semi-quantitative RT-PCR in RT4 and RT112 (grade I, wild-type p53), as well as in T24 (grade III, mutant p53) and TCCSUP (grade IV, mutant p53) human urinary bladder cancer cell lines.

Results: In the urothelial bladder cancer cell lines RT4 and T24, 5-FU-induced TS inhibition proved to be associated with cell type-dependent (a) sensitivity to the drug, (b) Caspase-mediated apoptosis, (c) p53 stabilization and activation, as well as Rb phosphorylation and E2F1 expression and (d) transcriptional regulation of p53 target genes and their cognate proteins, while an E2F-dependent transcriptional network did not seem to be critically engaged in such type of responses.

Conclusions: We have shown that in the wild-type p53 context of RT4 cells, 5-FU-triggered apoptosis was prominently efficient and mainly regulated by p53-dependent mechanisms, whereas the mutant p53 environment of T24 cells was able to provide notable levels of resistance to apoptosis, basically ascribed to E2F-independent, and still unidentified, pathways. Nevertheless, the differential vulnerability of RT4 and T24 cells to 5-FU administration could also be associated with cell-type-specific transcriptional expression patterns of certain genes critically involved in 5-FU metabolism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimetabolites, Antineoplastic / pharmacokinetics
  • Antimetabolites, Antineoplastic / pharmacology*
  • Apoptosis / drug effects*
  • Blotting, Western
  • Caspases / metabolism
  • E2F1 Transcription Factor / metabolism
  • Enzyme Inhibitors / pharmacology
  • Fluorouracil / pharmacokinetics
  • Fluorouracil / pharmacology*
  • Humans
  • Reverse Transcriptase Polymerase Chain Reaction
  • Thymidylate Synthase / antagonists & inhibitors*
  • Thymidylate Synthase / metabolism
  • Tumor Cells, Cultured
  • Tumor Suppressor Protein p53 / metabolism*
  • Urinary Bladder Neoplasms / enzymology
  • Urinary Bladder Neoplasms / metabolism*
  • Urinary Bladder Neoplasms / pathology*


  • Antimetabolites, Antineoplastic
  • E2F1 Transcription Factor
  • E2F1 protein, human
  • Enzyme Inhibitors
  • Tumor Suppressor Protein p53
  • Thymidylate Synthase
  • Caspases
  • Fluorouracil