In vitro mutations in dihydrofolate reductase that confer resistance to methotrexate: potential for clinical application.
Mammalian cells cultured in the presence of the chemotherapeutic agent, methotrexate, develop resistance to this drug. Sometimes this is due to mutations in the gene for dihydrofolate reductase, the primary target of methotrexate. However, it has not been possible to link such polymorphism to resistance of neoplastic disease to therapy with methotrexate. Nevertheless, interest in this possibility lead to the introduction of many mutations into the cDNA for human DHFR by mutagenesis. Most of the corresponding enzyme variants have been expressed in Escherichia coli and characterized. Many mutations in codons for hydrophobic residues at the active site greatly decrease inhibition by methotrexate, and by the related substrate analogue, trimetrexate, while allowing the retention of considerable catalytic efficiency. 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. Another potential use is in therapy for inherited disorders of hematopoiesis, where genetic modification of enough cells is a perennial problem. After transplantation of bone marrow that has been transduced with a bicistronic vector encoding both the mutant DHFR and a therapeutic gene, subsequent administration of methotrexate or trimetrexate should permit selection and enrichment of genetically modified hematopoietic cells.
Blakley
R L
RL
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Sorrentino
B P
BP
D016026:Bone Marrow Transplantation; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015316:Genetic Therapy; D014644:Genetic Variation; D006410:Hematopoiesis / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate