Thymidylate synthase inhibition induces p53-dependent and p53-independent apoptotic responses in human urinary bladder cancer cells.
OBJECTIVE
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.
Stravopodis
Dimitrios J
DJ
Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Zografou, 15784 Athens, Greece.
Karkoulis
Panagiotis K
PK
Konstantakou
Eumorphia G
EG
Melachroinou
Sophia
S
Thanasopoulou
Angeliki
A
Aravantinos
Gerasimos
G
Margaritis
Lukas H
LH
Anastasiadou
Ema
E
Voutsinas
Gerassimos E
GE
D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D050687:E2F1 Transcription Factor / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005472:Fluorouracil / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*
D000964:Antimetabolites, Antineoplastic; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D004791:Enzyme Inhibitors; D016159:Tumor Suppressor Protein p53; D013940:Thymidylate Synthase; D020169:Caspases; D005472:Fluorouracil