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Increased resistance to nitrogen mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione S-transferase A3 and a mutant dihydrofolate reductase

Cancer Gene Therapy volume 10, pages 637–646 (2003)Cite this article

Abstract

We have constructed a retroviral bicistronic vector, MFG/GID, that transduces the expression of both the A3 isoform of the rat glutathione S-transferase (GST A3), and the tyr-22 variant of the human dihydrofolate reductase (DHFRL22Y). Transduction of murine 3T3 fibroblasts with this vector increased their in vitro resistance to chlorambucil (1.8-fold) and trimetrexate (TMTX) (748-fold). TMTX selection of a mixed population of 20% GID-transduced NIH 3T3 cells and 80% control cells resulted in a marked increase in the GST peroxidase activity associated with the GST A3 isoform (17.7-fold). MFG/GID-transduced primary clonogenic murine hematopoietic progenitor cells were likewise more resistant to TMTX and chlorambucil than control β-gal-transduced cells. Selecting GID-transduced hematopoietic cells with a combination of TMTX and a nucleoside transport inhibitor resulted in a marked increase in resistance upon re-exposure to TMTX (99% survival). Similarly, GID-transduced hematopoietic cells selected with TMTX were more resistant to chlorambucil, with 40% survival at a drug concentration that killed practically all control cells. These results suggest that antifolate-mediated selection of MFG/GID-transduced hematopoietic cells could be used as a mean to enrich the population of transduced cells prior to or following transplantation, thus potentially conferring in vivo chemoprotection to nitrogen mustards and antifolates.

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Acknowledgements

We thank Dr Jacques Galipeau and Dr Brian P Sorrentino for the gift of the tyrosine 22 variant of DHFR and derived plasmids. We thank Dr Terry Chow, Dr Harry L Goldsmith, and Alexandre I Belenkov for technical advices and helpful suggestions. This work was supported by a grant from The Cancer Research Society Inc.

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Authors and Affiliations

  1. Department of Human Genetics, Montreal General Hospital and Research Institute, McGill University, Montreal, Quebec, Canada

    Jean-Philippe Belzile, Antonis Karatzas, Hoi-Ying Shiu & Denis Cournoyer

  2. Department of Medicine, Divisions of Haematology and Experimental Medicine, Montreal General Hospital and Research Institute, McGill University, Montreal, Quebec, Canada

    Sylvain Létourneau, Jean-Sébastien Palerme & Denis Cournoyer

  3. Department of Oncology, Montreal General Hospital and Research Institute, McGill University, Montreal, Quebec, Canada

    Denis Cournoyer

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  1. Jean-Philippe Belzile
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Belzile, JP., Karatzas, A., Shiu, HY. et al. Increased resistance to nitrogen mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione S-transferase A3 and a mutant dihydrofolate reductase. Cancer Gene Ther 10, 637–646 (2003). https://doi.org/10.1038/sj.cgt.7700619

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