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 Journal Article

  

Authors:	Takebe, N.; Zhao, S. C.; Ural, A. U.; Johnson, M. R.; Banerjee, D.; Diasio, R. B.; Bertino, J. R.
Article Title:	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
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.
Keywords:	controlled study; protein expression; human cell; fluorouracil; nonhuman; animal cell; mouse; animals; mice; cd34 antigen; gene expression; antimetabolites, antineoplastic; animal experiment; drug resistance; enzyme activity; transfection; genetic transduction; animalia; genetic vectors; peripheral blood stem cell; western blotting; gene therapy; hematopoietic stem cells; murinae; virus infection; bone marrow cell; hematopoietic stem cell; oxidoreductases; fluoropyrimidine; immunosuppressive agents; cell strain 3t3; complementary dna; bone marrow toxicity; drug exposure; retrovirus; retroviridae; colony forming unit gm; 3t3 cells; dna, complementary; gene construct; northern blotting; virus vector; retroviral vector; dihydropyrimidine dehydrogenase; unidentified retrovirus; humans; human; male; priority journal; article; cd34+ cells; dihydrouracil dehydrogenase (nadp); kanamycin kinase; dpd
Journal Title:	Cancer Gene Therapy
Volume:	8
Issue:	12
ISSN:	0929-1903
Publisher:	Nature Publishing Group
Date Published:	2001-12-01
Start Page:	966
End Page:	973
Language:	English
DOI:	10.1038/sj.cgt.7700393
PUBMED:	11781659
PROVIDER:	scopus
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-0035213225&partnerID=40&md5=51a9be1baa8e815cec19c76adf0d05ad
Notes:	Export Date: 21 May 2015 -- Source: Scopus

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