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Comparison of the Protection of Cells from Antifolates by Transduced Human Dihydrofolate Reductase Mutants
- Source :
- Human Gene Therapy. 8:2069-2077
- Publication Year :
- 1997
- Publisher :
- Mary Ann Liebert Inc, 1997.
-
Abstract
- Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (or =7.5-fold increased resistance), piritrexim (or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.
- Subjects :
- Cell Survival
Mutant
Gene Expression
Transfection
Dihydrofolate reductase
Genetics
Animals
Humans
Cytotoxic T cell
Molecular Biology
biology
fungi
Genetic Variation
food and beverages
Growth Inhibitors
Aminopterin
Kinetics
Tetrahydrofolate Dehydrogenase
Methotrexate
Pyrimidines
Biochemistry
Drug Resistance, Neoplasm
Trimetrexate
biology.protein
Folic Acid Antagonists
Molecular Medicine
Rabbits
Retroviral transduction
Thymidine
Subjects
Details
- ISSN :
- 15577422 and 10430342
- Volume :
- 8
- Database :
- OpenAIRE
- Journal :
- Human Gene Therapy
- Accession number :
- edsair.doi.dedup.....c1ef28c5edd80b0feac18214d2c98d81