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A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein

Cancer Gene Therapy volume 12, pages 497–508 (2005)Cite this article

Abstract

Virus-directed enzyme prodrug therapy (VDEPT) is an emerging strategy against cancer. Our approach is a P450-based VDEPT that consists of using cyclophosphamide (CPA) as a prodrug and a Cytochrome P450 2B6/NADPH cytochrome P450 reductase fusion protein (CYP2B6/RED) as a prodrug-activating enzyme. Due to the heterogenous expression of proteins in tumor cells, basal reductase activity may not be sufficient to supply CYP2B6 with electrons, the fusion protein should enable the expression of both proteins at high levels in tumor cells. CYP/RED fusion proteins have never been previously expressed in mammalian cells, to enable expression the fusion protein was cloned into an adenoviral vector and subsequently several pulmonary tumor cell lines were infected. The CYP2B6/RED fusion protein was detected by Western blot, its mRNA by Northern blot, and its heme incorporation into an active form by spectral analysis. Infection with the fusion gene increased RED activity in microsomes by a factor of 3 compared to the control. After infection and treatment with CPA, in cell lines with low endogenous RED, the fusion protein mediated significantly higher CPA-induced cytotoxicity compared to cells expressing solely CYP2B6. In conclusion, the fusion protein is functional for VDEPT by providing one protein for higher levels of CPA metabolism.

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Acknowledgements

We would like to thank Dr Marie-Agnes Sari for her advice and help, Dr P Urban for kindly providing the pUC18/RED plasmid. Dr EMGillam for her support and feedback on the manuscript. This work was supported by the INSERM, the Université Paris V René Descartes, the Region Ile de France (SESAME), the Ligue contre le Cancer, the Association de Research contre le Cancer (ARC), the Fondation pour la Research Médicale (FRM) and Oncodesign S.A.

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Authors and Affiliations

  1. INSERM UMRS 490, Molecular Toxicology, Université René Descartes, 45, rue des Saints-Pères, 75006, Paris, France

    Marina Tychopoulos, Philippe Beaune & Isabelle de Waziers

  2. INSERM U517, 7, Bld. Jeanne d'Arc, 21000, Dijon, France

    Laurent Corcos

  3. Oncodesign S.A, Parc Technologique, 28, rue Louis de Broglie, 21076, Dijon Cedex, France

    Philippe Genne

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  1. Marina Tychopoulos
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Correspondence to Isabelle de Waziers.

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Tychopoulos, M., Corcos, L., Genne, P. et al. A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein. Cancer Gene Ther 12, 497–508 (2005). https://doi.org/10.1038/sj.cgt.7700817

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