Coexpression of cytidine deaminase and mutant dihydrofolate reductase by a bicistronic retroviral vector confers resistance to cytosine arabinoside and methotrexate.
The transfer of a drug resistance gene into hematopoietic cells is an approach being investigated to overcome the problem of myelosuppression produced by anticancer drugs. Chemotherapeutic agents are often given in combination in order to increase their effectiveness. Consequently, there is an advantage in designing vectors for gene transfer that are capable of expressing two drug resistance genes. We have constructed a bicistronic retroviral vector, MFG-DHFR-IRES/CD, which contains the mutated human dihydrofolate reductase (DHFR) cDNA with a phenylalanine-to-serine substitution at codon 31 (F31S) and the human cytidine deaminase (CD) cDNA. Murine fibroblast and hematopoietic cells were transduced with this vector and evaluated for their resistance to methotrexate (MTX) and cytosine arabinoside (ARA-C). The transduced fibroblast cells showed high levels of resistance to MTX and to ARA-C as determined by a clonogenic assay. Using enzymatic assays, we observed a coordinate increase in resistance to MTX and DHFR enzyme activity following an ARA-C selection. In addition, MTX selection produced an increase in CD enzyme activity and ARA-C resistance. Murine hematopoietic cells transduced with the bicistronic vector also showed drug resistance to both MTX and ARA-C. Interestingly, the double-gene construct conferred an equivalent level of drug resistance compared with single-gene vectors bearing only CD or DHFR genes in the hematopoietic cells. These results demonstrate the potential of the MFG-DHFR-IRES/CD vector to confer drug resistance to both MTX and ARA-C and may have future application in chemoprotection of normal hematopoietic cells in patients with cancer.
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Département de Pharmacologie, Université de Montréal, Centre de Recherche Pédiatrique, Hôpital Ste-Justine, Quebec, Canada.
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D016475:3T3 Cells; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D003561:Cytarabine / Q000494:pharmacology*; D003564:Cytidine Deaminase / Q000235:genetics*; D017931:DNA Primers; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D008809:Mice, Inbred C3H; D012190:Retroviridae / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014161:Transduction, Genetic
D000964:Antimetabolites, Antineoplastic; D017931:DNA Primers; D003561:Cytarabine; D013762:Tetrahydrofolate Dehydrogenase; D003564:Cytidine Deaminase; D008727:Methotrexate