Identification and characterization of a mutation in the dihydrofolate reductase gene from the methotrexate-resistant Chinese hamster ovary cell line Pro-3 MtxRIII.

  title={Identification and characterization of a mutation in the dihydrofolate reductase gene from the methotrexate-resistant Chinese hamster ovary cell line Pro-3 MtxRIII.},
  author={A. Dicker and M. Volkenandt and B. Schweitzer and D. Banerjee and J. Bertino},
  journal={The Journal of biological chemistry},
  volume={265 14},
A methotrexate-resistant Chinese hamster ovary cell line (Pro-3 MtxRIII), resistant due to a low-level amplified, altered target enzyme, dihydrofolate reductase (DHFR), has been characterized on the molecular level. The cDNA and coding regions of all six DHFR exons were amplified in vitro using Taq polymerase and directly sequenced. Analysis of the Pro-3 MtxRIII DHFR cDNA demonstrated a C----T base transition at nucleotide 67 that results in the substitution of phenylalanine for leucine at… Expand
Effect of codon 22 mutations on substrate and inhibitor binding for human dihydrofolate reductase.
Results suggest that codon 22 may be a “hot spot” which readily mutates to impart a MTX-resistant phenotype to its host cell. Expand
A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line.
Results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells. Expand
Saturation mutagenesis at dihydrofolate reductase codons 22 and 31. A variety of amino acid substitutions conferring methotrexate resistance.
It was observed that codon 22 variants were generally more resistant to MTX, but codon 31 variants retained substantially more catalytic activity at a given level of MTX resistance, which has important implications for the function of different DHFR variants as mediators of drug resistance. Expand
Gene amplification and enzyme modification are responsible for the methotrexate-resistance of two carrot cell lines that overproduce bifunctional dihydrofolate reductase-thymidylate synthase
The results suggest that in carrot cells, as in animal systems, MTX-resistance can be due to different events such as gene amplification and gene modifications ; the latter appear to be responsible for the increased production of a target enzyme with reduced affinity for MTX. Expand
CHAPTER 23 – Development and Application of an Engineered Dihydrofolate Reductase and Cytidine-Deaminase-Based Fusion Genes in Myeloprotection-Based Gene Therapy Strategies
The results demonstrated both the feasibility and efficiency of ex vivo CD34 + stem cell transduction with this retroviral vector and in vivo selection in the presence of MTX. Expand
Assay for expression of methotrexate-resistant dihydrofolate reductase activity in the presence of drug-sensitive enzyme.
The assay was applicable to two quite diverse DHFR variants and may be useful for assaying the expression of other drug-resistant DHFR genes as well after introduction into cells containing drug-sensitive enzyme. Expand
In vitro mutations in dihydrofolate reductase that confer resistance to methotrexate: Potential for clinical application
Introduction of some of these mutants into mammalian cells by retroviral transfer provides substantial protection from toxic effects of the inhibitors, and has promise for the myeloprotection of patients receiving therapy with methotrexate or trimetrexate. Expand
Drosophila dihydrofolate reductase mutations confer antifolate resistance to mammalian cells.
Antifolates, such as methotrexate, are used to inhibit dihydrofolate reductase (DHFR), an enzyme essential for the biosynthesis of thymidylate, purines, and several amino acids that would make excellent candidates for gene therapy and genetic markers in the treatment of certain human disorders. Expand
Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction
A double-copy Moloney leukemia virus-based retroviral construct containing both the NeoR gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 andExpand
Selection for methotrexate resistance in mammalian cells bearing a Drosophila dihydrofolate reductase transgene
Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance. Expand