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

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Abstract

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.

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Acknowledgments

Financial support was from the Greek Ministry of Health and Social Solidarity (ΔY2β/OIK.98909/17-7-2008) and the Hellenic Society of Medical Oncology (HESMO) (1804/20-03-2009). Panagiotis K. Karkoulis is financially supported by a fellowship awarded from the Institute of Biology, NCSR “Demokritos”, Athens, Greece (Ph.D. fellowship) (Ministry of Education, Lifelong Learning and Religious Affairs) and Eumorphia G. Konstantakou is a scholarship recipient of the Hellenic State Scholarships Foundation (“IKY”) (Ph.D. fellowship).

Conflict of interest statement

We declare that we have no conflict of interest.

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Authors and Affiliations

  1. Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Zografou, 15784, Athens, Greece

    Dimitrios J. Stravopodis, Eumorphia G. Konstantakou & Lukas H. Margaritis

  2. Laboratory of Environmental Mutagenesis and Carcinogenesis, Institute of Biology, National Center for Scientific Research (NCSR) “Demokritos”, Aghia Paraskevi, 15310, Athens, Greece

    Panagiotis K. Karkoulis, Sophia Melachroinou & Gerassimos E. Voutsinas

  3. Department of Genetics and Gene Therapy, Center of Basic Research II, Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527, Athens, Greece

    Angeliki Thanasopoulou & Ema Anastasiadou

  4. 3rd Clinic of Pathology-Oncology, “Agioi Anargyroi” Oncology Hospital, 13122, Ilion, Athens, Greece

    Gerasimos Aravantinos

Authors
  1. Dimitrios J. Stravopodis
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  2. Panagiotis K. Karkoulis
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  3. Eumorphia G. Konstantakou
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  4. Sophia Melachroinou
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  5. Angeliki Thanasopoulou
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  6. Gerasimos Aravantinos
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  7. Lukas H. Margaritis
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  8. Ema Anastasiadou
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  9. Gerassimos E. Voutsinas
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Correspondence to Gerassimos E. Voutsinas.

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Stravopodis, D.J., Karkoulis, P.K., Konstantakou, E.G. et al. Thymidylate synthase inhibition induces p53-dependent and p53-independent apoptotic responses in human urinary bladder cancer cells. J Cancer Res Clin Oncol 137, 359–374 (2011). https://doi.org/10.1007/s00432-010-0891-y

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