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.
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 (DHFR(L22Y)). 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 beta-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.
Belzile
Jean-Philippe
JP
Department of Human Genetics, Montreal General Hospital and Research Institute, McGill University, Montreal, Quebec, Canada. cournoyerd@muhchem.mcgill.ca
Karatzas
Antonis
A
Shiu
Hoi-Ying
HY
Létourneau
Sylvain
S
Palerme
Jean-Sébastien
JS
Cournoyer
Denis
D
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D004351:Drug Resistance; D005347:Fibroblasts / Q000187:drug effects; D005493:Folic Acid Antagonists / Q000009:adverse effects / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009588:Nitrogen Mustard Compounds / Q000009:adverse effects / Q000494:pharmacology*; D020033:Protein Isoforms / Q000737:chemistry; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D018906:Antineoplastic Agents, Alkylating; D005493:Folic Acid Antagonists; D009588:Nitrogen Mustard Compounds; D020033:Protein Isoforms; D013762:Tetrahydrofolate Dehydrogenase; D005982:Glutathione Transferase