Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retroviral gene transfer into human myeloid precursor cells allows introduction of marker genes as well as genes conferring resistance to chemotherapeutic drugs. We transduced a human mutant dihydrofolate reductase (DHFR) cDNA into CD34 antigen-positive peripheral blood cells from patients with breast or ovarian cancer obtained after treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). This mutant DHFR has been shown to confer resistance to methotrexate (MTX) in murine bone marrow. We established a transduction protocol that permitted ex vivo expansion and selection of transduced early progenitor cells. The number of progenitor cells from transduced CD34-positive cells increased 50-fold after cytokine prestimulation with interleukin-1 (IL-1), c-kit ligand (KL; stem cell factor), and IL-3 and 2 weeks in liquid culture. Transduced colony-forming unit-granulocyte-macrophage (CFU-GM), assayed directly after the transduction procedure, were protected completely against 2 x 10(-8) mol/L MTX, a concentration that significantly reduced the CFU-GM detected in the control population. Gene transfer of the mutant DHFR led to a twofold selective advantage for a pre-CFU population after exposure to MTX in liquid culture (P < .001). Polybrene, in contrast with protamine, significantly inhibited the expansion of progenitors. The presence of proviral DNA was monitored by polymerase chain reaction (PCR) and was detected in greater than 80% of CFU-GM and ex vivo expanded pre-CFU. We have demonstrated that human hematopoietic precursor cells can be expanded extensively after retroviral gene transfer. The same population of early progenitors can be selected ex vivo with low-dose MTX. As long-term expression of transduced genes in human hematopoietic cells remains a problem in vivo, these results may have implications for future clinical trials, especially for the introduction of nonselectable genes.
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PMID:Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer. 752 65

Previous studies have shown that dihydrofolate reductase (DHFR) gene transfer protects marrow from methotrexate (MTX)-mediated toxicity; however, MTX treatment in vivo has not convincingly been shown to enrich DHFR-transduced progenitors or stem cells. Experiments were performed to better characterize the hematological effects of MTX, and maneuvers were tested with the aim of improving the utility of MTX as an agent for in vivo selection. Progenitors were assayed as colony forming unit cells in culture (CFU-C) and in the spleens of irradiated mice (day 11 CFU-S). A single injection of MTX at doses up to 250 mg/kg (more than three times the LD10) failed to reduce CFU-C numbers significantly in the femur or spleen assayed 1-3 days later. However, consistent declines in the number of mononuclear cells per femur reflected a significant depletion of nonclonogenic precursor cells. Preceding administration of pegylated stem cell factor (SCF), 100 micrograms/kg per day, increased CFU-C killing by a single dose of 5-fluorouracil (5-FU) 15- to 65-fold in the femur, and 5- to 15-fold in the spleen, consistent with previous reports. In contrast, despite preceding SCF administration there was no significant progenitor killing by MTX. Similar results were obtained using a second folate analog, trimetrexate. These results suggest that the mechanism by which folate analogs exert their hematological toxicity is through the depletion of relatively mature, nonclonogenic precursor cells, and not by killing progenitors. This information is relevant to the use of DHFR in gene therapy protocols, and suggests that folate analogs are poorly suited agents for selection at the level of clonogenic progenitor cells in vivo.
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PMID:The hematological effects of folate analogs: implications for using the dihydrofolate reductase gene for in vivo selection. 893 21

To protect bone marrow cells from the toxicity of chemotherapy, a multidrug resistant gene or a dihydrofolate reductase gene has been introduced into stem cells. These genes, however, are not capable of conferring refractoriness to alkylating agents (AA), which are some of the most commonly used agents in chemotherapy regimens. In the present study, an attempt was made to endow human stem cell (CD34+ cells) with resistance to cyclophosphamide, a well-known AA, and adriamycin (ADM) by transducing the glutathione-S-transferase pi (GST-pi) gene whose product is thought to detoxify AA by conjugating them with glutathione and to remove a toxic peroxide formed by ADM. The gene transduction was carried out retrovirally with a virus titer of 1 x 10(5) FFU/ml, employing a recombinant fibronectin fragment; transduction efficiency was extremely low without the fragment. Incubation with interleukin-6 and stem cell factor enhanced the expression of fibronectin ligands VLA4 and VLA5 on CD34+ cells. This enhanced expression of VLA4 and VLA5 was considered to facilitate a close contact of the CD34+ cell to the retroviral vector via fibronectin fragments and the subsequent transduction process. The GST-pi gene-transduced CD34+ cells formed almost 3- and 2.5-fold more CFU-GM than neo gene-transduced CD34+ cells in the presence of 2.5 microg/ml of 4-hydroperoxycyclophosphamide (4-HC), an active form of cyclophosphamide, and 30 ng/ml ADM, respectively. The transfectants formed an appreciable number of colonies, even at higher concentrations of these drugs (5.0 microg/ml of 4-HC, 50 ng/ml of ADM) whereas neo gene-transduced or nontransduced CD34+ cells formed no colonies at all, indicating the possibility of selecting out the transfectants by exposing them to these anticancer drugs. Thus, we were able to demonstrate that transduction of the GST-pi gene confers resistance to cyclophosphamide as well as to ADM, and therefore this approach can be applied clinically for high-dose chemotherapy.
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PMID:Fibronectin fragment-facilitated retroviral transfer of the glutathione-S-transferase pi gene into CD34+ cells to protect them against alkylating agents. 938 56

Attempts to expand repopulating hematopoietic cells ex vivo have yielded only modest amplification in stem cell numbers. We now report that expression of an exogenous human multi-drug resistance 1 (MDR1) gene enables dramatic ex vivo stem cell expansion in the presence of early acting hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the MDR1 gene or a variant of human dihydrofolate reductase (DHFR), and then expanded for 12 days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor. When these cells were injected into nonirradiated mice, high levels of long-term engraftment were only seen with MDR1-transduced grafts. To verify that expansion of MDR1-transduced repopulating cells had occurred, competitive repopulation assays were performed using MDR1 expanded grafts. These experiments showed progressive expansion of MDR1-transduced repopulating cells over the expansion period, with a 13-fold overall increase in stem cells after 12 days. In all of the experiments, mice transplanted with expanded MDR1-transduced stem cells developed a myeloproliferative disorder characterized by high peripheral white blood cell counts and splenomegaly. These results show that MDR1-transduced stem cells can be expanded in vitro using hematopoietic cytokines without any drug selection, but enforced stem cell self-renewal divisions can have adverse consequences.
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PMID:Transduction of murine bone marrow cells with an MDR1 vector enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferative syndrome in transplanted mice. 974 64

Ex vivo expansion of hematopoietic stem cells would be useful for bone marrow transplantation and gene therapy applications. Toward this goal, we have investigated whether retrovirally-transduced murine stem cells could be expanded in culture with hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the human multidrug resistance 1 gene (HaMDR1), a variant of human dihydrofolate reductase (HaDHFR), or both MDR1 and DHFR in an internal ribosomal entry site (IRES)-containing bicistronic vector (HaMID). Cells were then expanded for 15 days in cultures stimulated with interleukin (IL)-3, IL-6, and stem cell factor. When very low marrow volumes were injected into lethally irradiated recipient mice, long-term reconstitution with 100% donor cells was seen in all mice injected with HaMDR1- or HaMID-transduced cells. By contrast, engraftment with HaDHFR- or mock-transduced cells ranged from partial to undetectable despite injection of significantly larger marrow volumes. In addition, mice transplanted with expanded HaMDR1- or HaMID-transduced stem cells developed a myeloproliferative disorder that was characterized by an increase in abnormal peripheral blood leukocytes. These results show that MDR1-transduced stem cells can be expanded in vitro with hematopoietic cytokines, but indicate that an increased stem cell division frequency can lead to stem cell damage.
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PMID:Effects of retroviral-mediated MDR1 expression on hematopoietic stem cell self-renewal and differentiation in culture. 1037 17

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4(+), CD8(+), CD20(+), and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452)
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PMID:High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells. 1062 48