Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors Journal Article

Authors: Takebe, N.; Nakahara, S.; Zhao, S. C.; Adhikari, D.; Ural, A. U.; Iwamoto, M.; Banerjee, D.; Bertino, J. R.
Article Title: Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors
Abstract: We previously reported the protection of hematopoietic cells from methotrexate (MTX) toxicity using an N2-based double copy vector containing serine 31 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whether the use of SFG-based dicistronic vectors will lead to improvement in gene transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells, the 50% effective dose values of MTX conferred by the SFG vector were 8-fold higher than those obtained with the DC/SV6 vector. DHFR mRNA levels were 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respectively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was the same. These results indicate that the in vitro transduction efficiency and transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors.
Keywords: controlled study; protein expression; leukemia; human cell; dose response; nonhuman; methotrexate; animal cell; mouse; animals; mice; reverse transcription polymerase chain reaction; bone marrow; in vitro study; drug resistance; tumor cells, cultured; gene transfer; animalia; genetic vectors; transduction, genetic; cancer resistance; stem cell; blotting, western; reverse transcriptase polymerase chain reaction; gene therapy; hematopoietic cell; hematopoietic stem cells; dna primers; dihydrofolate reductase; tetrahydrofolate dehydrogenase; blotting, northern; moloney murine leukemia virus; complementary dna; 3t3 cells; dna, complementary; virus vector; gene expression regulation, enzymologic; granulocyte-macrophage colony-stimulating factor; promoter regions (genetics); blotting, southern; humans; human; male; priority journal; article; gene transfer efficiency; mutated dihydrofolate reductase cdna; n2-based double copy vector; sfg-based dicistronic vector
Journal Title: Cancer Gene Therapy
Volume: 7
Issue: 6
ISSN: 0929-1903
Publisher: Nature Publishing Group  
Date Published: 2000-06-01
Start Page: 910
End Page: 919
Language: English
PUBMED: 10880023
PROVIDER: scopus
DOI: 10.1038/sj.cgt.7700199
Notes: Export Date: 18 November 2015 -- Source: Scopus
Citation Impact
MSK Authors
  1. Debabrata Banerjee
    136 Banerjee
  2. Naoko Takebe
    25 Takebe
  3. Joseph Bertino
    363 Bertino
  4. Shi-Cheng Zhao
    42 Zhao