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)

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.
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PMID:Comparison of the expression of a mutant dihydrofolate reductase under control of different internal promoters in retroviral vectors. 152 11

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

Pyrimethamine (Pyr) is commonly used for treatment of toxoplasmic encephalitis in AIDS patients; however, in two clinical studies, an increased number of deaths were observed when Pyr was coadministered with zidovudine (ZDV). The BALB/c mouse was chosen as a model to study the mechanism underlying the unexpected toxicity from coadministration of these drugs. Daily administration by oral gavage of 60 mg/kg Pyr and 240 mg/kg ZDV resulted in 100% lethality after 30 days. These dose levels produced no effect when the drugs were given individually for the same period. Administration of combinations of Pyr and ZDV resulted in macrocytic anemia and leukopenia with synergistic decreases in lymphocyte and neutrophil numbers. To examine the mechanism of this hematotoxicity at the cellular level, mouse bone marrow colony-forming unit (mCFU) assays were employed. A combination of ZDV with various concentrations of Pyr resulted in synergistic decreases in numbers of erythroid and granulocyte-macrophage precursors (mCFU-E and mCFU-GM). mCFU-GM precursors appeared more sensitive than erythroid precursors to combinations of Pyr and ZDV. Incorporation of (14)C-ZDV into cellular DNA was increased in a dose-dependent manner in the presence of increasing concentrations of Pyr in the mCFU-GM assay. This suggested that inhibition of dihydrofolate reductase by Pyr and accompanying inhibition of dTTP synthesis allows preferential incorporation of ZDV into DNA, with resulting strand breakage and cell death. (14)C-ZDV incorporation was also observed when human GM cultures were analyzed, however, incorporation was less and required higher concentrations of Pyr.
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PMID:Synergistic bone marrow toxicity of pyrimethamine and zidovudine in murine in vivo and in vitro models: mechanism of toxicity. 1203 Aug 38