Selection of wild-type revertants from methotrexate-resistant cells containing an altered dihydrofolate reductase.
A selection system for wild-type revertants from methotrexate-resistant Chinese hamster ovary cells is described. In the absence of exogenous thymidine, cells use the folate metabolic pathway to generate thymidine 5'-monophosphate from deoxyuridine 5'-monophosphate. Thus, in the presence of methotrexate, the incorporation of labeled deoxyuridine into phenotypic wild-type cells is inhibited whereas resistant cells that are cycling incorporate sufficient radioactivity to be killed. Using several suicide cycles, wild-type revertants have been isolated from methotrexate-resistant cells containing a structurally altered dihydrofolate reductase. These revertants possess a wild-type sensitivity to the cytotoxicity of the drug and contain a reductase with similar properties as wild-type enzyme.
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005260:Female; D005347:Fibroblasts / Q000528:radiation effects; D005821:Genetic Techniques; D008727:Methotrexate / Q000378:metabolism*; D010053:Ovary / Q000166:cytology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014529:Uridine / Q000378:metabolism
D013762:Tetrahydrofolate Dehydrogenase; D014529:Uridine; D008727:Methotrexate