title: Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene.
abstract: The genetic transfer of drug resistance to hematopoietic cells is an attractive approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors. We constructed a retroviral vector containing both a human cytosolic ALDH-1 cDNA and a human doubly mutated DHFR cDNA (Phe22/Ser31; termed F/S in the description of constructs) to generate increased resistance to both CTX and MTX. Infection of NIH3T3 cells resulted in increased resistance to both 4-hydroperoxycyclophosphamide (4HC) (1.9 +/- 0.1-fold) and MTX (73 +/- 2.8-fold). Transduced human CD34(+) enriched hematopoietic progenitor cells were also resistant to both 4HC and MTX by CFU-GM readout. Lethally irradiated mice transplanted with SFG-ALDH-IRES-F/S or mock-transduced bone marrow cells were treated with high-dose pulse CTX or high-dose CTX/MTX. Animals receiving marrow not transduced with ALDH-1 or mutated DHFR cDNA died from CTX or CTX/MTX toxicity, whereas mice transduced with ALDH-1 and mutated DHFR cDNA-containing marrow were able to tolerate the same doses of CTX or CTX/MTX treatment posttransplant. These data taken together indicate that ALDH-1 overexpression and mutant DHFR increased both 4HC and MTX resistance in vitro and in the in vivo mouse model. This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression.
authors.affiliation: Department of Medicine, Program of Molecular Pharmacology and Experimental Therapeutics, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue No. 78, New York, New York 10021, USA.
authors.forename: N
authors.initials: N
authors.lastname: Takebe
authors.forename: S C
authors.initials: SC
authors.lastname: Zhao
authors.forename: D
authors.initials: D
authors.lastname: Adhikari
authors.forename: S
authors.initials: S
authors.lastname: Mineishi
authors.forename: M
authors.initials: M
authors.lastname: Sadelain
authors.forename: J
authors.initials: J
authors.lastname: Hilton
authors.forename: M
authors.initials: M
authors.lastname: Colvin
authors.forename: D
authors.initials: D
authors.lastname: Banerjee
authors.forename: J R
authors.initials: JR
authors.lastname: Bertino
mesh_terms: D016475:3T3 Cells; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D015153:Blotting, Western; D016026:Bone Marrow Transplantation; D003520:Cyclophosphamide / Q000031:analogs & derivatives / Q000494:pharmacology*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D018014:Gene Transfer Techniques; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013997:Time Factors; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34; D000964:Antimetabolites, Antineoplastic; D018076:DNA, Complementary; D012313:RNA; D003520:Cyclophosphamide; D000444:Aldehyde Dehydrogenase; D013762:Tetrahydrofolate Dehydrogenase; C011272:perfosfamide; D008727:Methotrexate