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.
To test the concept that protection of bone marrow progenitor cells via introduction of a drug resistance gene would allow larger and curative doses of chemotherapy to be administered, we used mice bearing a transplanted breast cancer as a model system. Mice bearing the E0771 tumor were treated with lethal doses of cyclophosphamide (CPA) and rescued from toxicity by administration of bone marrow transduced with a mutant dihydrofolate reductase (DHFR) cDNA (Ser-31) in a retroviral construct. Animals receiving marrow not transduced with mutant DHFR cDNA died from methotrexate (MTX) toxicity, whereas mice transduced with mutant DHFR cDNA containing marrow were able to tolerate MTX treatment post-transplant; 44% of the mice had no demonstrable tumor when sacrificed on day 52. Another control group of mice treated with CPA and transplanted but not treated with MTX post-transplant succumbed to tumor regrowth. These data provide a strong rationale for the use of drug resistance genes to protect marrow from drug toxicity because the increase in dose tolerated may result in an improved cure rate of chemosensitive tumors.
To determine the effect of different promoters on the expression of an altered dihydrofolate reductase (DHFR) gene conferring methotrexate (MTX) resistance in different cell types, double-copy retroviral vectors were constructed carrying a murine mutant DHFR under the control of five different promoters, i.e., human adenosine deaminase (ADA), simian virus 40 (SV40), thymidine kinase (TK), human beta-actin, and cytomegalovirus (CMV). Their expression was compared in NIH-3T3 cells, three human leukemia cell lines, and mouse bone marrow. The variant DHFR is readily expressed from these various promoters in retroviral vectors at a selectable level. In 3T3 cells, the DHFR constructs containing the SV40 promoter conferred the highest levels of resistance to MTX. In K562 and Raji cells, the construct with the TK promoter produced the highest level of resistance. However granulocyte-macrophage colony-forming unit (CFU-GM) colonies from mouse marrow were more resistant to MTX when infected with vectors containing the SV40 promoter and ADA promoter as compared to the other promoter constructs. These studies show that mouse fibroblast cell lines such as NIH-3T3 do not predict the effectiveness of retroviral-mediated gene transfer for marrow progenitor cells, and that the activity of retroviral vector-encoded promoters vary in an unpredictable manner from cell type to cell type. Possible implications for basic gene transfer studies and clinical applications are discussed.
Severe 5 -fluorouracil ( 5 -FU ) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase ( DPD ) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5 -FU to 5 0 -6 0 -dihydrofluorouracil, which is further metabolized to fluoro --alanine. We demonstrate here that overexpression of human DPD confers resistance to 5 -FU in
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