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Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells

Cancer Gene Therapy volume 8, pages 966–973 (2001)Cite this article

Abstract

Severe 5-fluorouracil (5-FU) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase (DPD) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5-FU to 5′-6′-dihydrofluorouracil, which is further metabolized to fluoro-β-alanine. We demonstrate here that overexpression of human DPD confers resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and in human CD34+-enriched hematopoietic progenitor cells. An SFG-based dicistronic retroviral vector containing human DPD cDNA, an internal ribosomal entry site (IRES), and the neomycin phosphotransferase (Neo) gene was constructed (SFG–DPD–IRES–Neo). Transduced NIH3T3 cells demonstrated a 2-fold (ED50) increase in resistance to a 4-hour exposure of 5-FU in comparison to nontransduced cells. Expression of DPD was confirmed by Northern and Western blot analyses, and DPD enzyme activity was detectable only in transduced cells. Infection of mouse bone marrow cells with this retroviral construct resulted in an increased number of 5-FU–resistant CFU-GM colonies, compared to mock-transduced bone marrow in both 4-hour and 12- to 14-day exposures. Infection of human CD34+-enriched cells with this construct and incubation with 5-FU (10−6 M) for 14 days also resulted in an increased number of 5-FU–resistant colonies. Retroviral transduction of human hematopoietic progenitor cells with a cDNA-expressing human DPD conferred resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and human CD34+-enriched cells. These results encourage the use of this gene as a method to protect patients from 5-FU myelotoxicity. Cancer Gene Therapy (2001) 8, 966–973

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Acknowledgements

We thank Michel Sadelain for advice on retroviral vector construction and Sharon Bleau for CD34+ enriched cell separations. The statistical analyses were performed by Larry Leon, MS. Supported by USPHS Grants CA 59350, CA 62164, and CA 09512.

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Author notes

  1. Naoko Takebe

    Present address: University of Maryland, Greenbaum Cancer Center, Baltimore, MD, USA

Authors and Affiliations

  1. Program of Molecular Pharmacology and Experimental Therapeutics, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Naoko Takebe, Shi-Cheng Zhao, Debabrata Banerjee & Joseph R Bertino

  2. Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Naoko Takebe & Joseph R Bertino

  3. Department of Hematology, Department of Hematology, Gulhane Military Medical Academy, School of Medicine, Etlik, Ankara, 06010, Turkey

    Ali U Ural

  4. Department of Pharmacology and Toxicology, Volker Hall Box 600, University of Alabama at Birmingham, Birmingham, 35294, Alabama, USA

    Martin R Johnson & Robert B Diasio

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  1. Naoko Takebe
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Correspondence to Joseph R Bertino.

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Takebe, N., Zhao, SC., Ural, A. et al. Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells. Cancer Gene Ther 8, 966–973 (2001). https://doi.org/10.1038/sj.cgt.7700393

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