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Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment

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Abstract

Purpose

p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents—cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed.

Methods

p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21WAF1/CIP1 and cyclin B1 proteins was examined using immunocytochemistry and western blot methods.

Results

In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis.

Conclusions

This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and on the chemotherapeutics’ modality of action.

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Acknowledgments

Support for this study by the Ministry of Science, Education and Sport of Croatia is gratefully acknowledged (Projects 098-0982464-2393, 098-0982464-2514). The authors are particularly thankful to Dr. Anamaria Brozović for exceptionally useful discussions and experimental help and Dr. Ksenija Zahradka for providing us with the BrdU chemical and anti-BrdU antibody.

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Authors and Affiliations

  1. Division of Molecular Medicine, Rudjer Bošković Institute, Bijenička cesta 54, 10000, Zagreb, Croatia

    Sandra Kraljević Pavelić, Marko Marjanović, Miroslav Poznić & Marijeta Kralj

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  1. Sandra Kraljević Pavelić
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  2. Marko Marjanović
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  3. Miroslav Poznić
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  4. Marijeta Kralj
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Correspondence to Marijeta Kralj.

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S. Kraljević Pavelić and M. Marjanović contributed equally to the preparation of this manuscript.

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Kraljević Pavelić, S., Marjanović, M., Poznić, M. et al. Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment. J Cancer Res Clin Oncol 135, 1747–1761 (2009). https://doi.org/10.1007/s00432-009-0621-5

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  • DOI: https://doi.org/10.1007/s00432-009-0621-5

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