title: Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene.
abstract: Hematopoietic toxicity produced by most anticancer drugs limits their potential for curative therapy. We have shown previously that the human cytidine deaminase (CD) gene can confer drug resistance in murine bone marrow cells (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the present study, as the first objective we showed that the CD gene can also render drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we investigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and level of drug resistance before transplantation. This strategy may have the potential to circumvent the problem in clinical gene therapy of low level of gene transfer and adequate long-term gene expression. Using a bicistronic retroviral vector containing the CD and the green fluorescent protein (CDiGFP), we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC, and 5-AZA-CdR were demonstrated in vitro to enrich (>95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduced primary murine BMCs cultivated at high cell density we observed that in vitro selection with ARA-C was not possible due to release of CD into the culture medium at amounts that were sufficient to inactivate the analog. The CD-containing medium produced a chemoprotective effect on mock BMCs as shown by lack of significant growth inhibition in the presence of ARA-C. However, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clonogenic assay. Selection with ARA-C was shown to significantly increase the CD enzyme activity in transduced BMC. These results suggest that CD gene has the potential to be a good selectable marker and a possible tool for chemoprotection in cancer gene therapy.
authors.affiliation: Département de pharmacologie, Université de Montréal, Centre de recherche pédiatrique, Hôpital Ste-Justine, 3175 Côte Ste-Catherine, Montréal, Quebec, Canada H3T 1C5.
authors.forename: C M
authors.initials: CM
authors.lastname: Beauséjour
authors.forename: N
authors.initials: N
authors.lastname: Eliopoulos
authors.forename: L
authors.initials: L
authors.lastname: Momparler
authors.forename: N L
authors.initials: NL
authors.lastname: Le
authors.forename: R L
authors.initials: RL
authors.lastname: Momparler
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000187:drug effects / Q000201:enzymology*; D003114:Colony-Forming Units Assay; D003561:Cytarabine / Q000494:pharmacology*; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000235:genetics*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D000077209:Decitabine; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004341:Drug Evaluation; D004351:Drug Resistance / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007938:Leukemia / Q000628:therapy; D008164:Luminescent Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D019076:Transgenes
chemical_list: D000964:Antimetabolites, Antineoplastic; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D003561:Cytarabine; D003841:Deoxycytidine; D049452:Green Fluorescent Proteins; D000077209:Decitabine; D015254:DNA Modification Methylases; D003564:Cytidine Deaminase; D001374:Azacitidine