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Isolation of ouabain-resistant human diploid fibroblasts
Authors:R Mankovitz  M Buchwald  R M Baker
Institution:Department of Pathology Queen''s University Kingston, Ontario, Canada;Research Institute The Hospital for Sick Children, and Departments of Medical Genetics and Medical Biophysics University of Toronto Toronto, Ontario, Canada
Abstract:Seventeen clones resistant to the cytotoxic action of ouabain were isolated in culture by direct selection from 5 independent strains of diploid human fibroblasts. Resistant clones were recovered at frequencies on the order of 10−7 per wild type cell selected from populations treated with the mutagen EMS, but no resistant cells were detected among 108 unmutagenized cells. Most selected clones remained ouabain-resistant following further propagation in the absence of drug. The growth of wild type cells was inhibited by 50% at ouabain concentrations of 2–5 × 10−8 M, while resistant clones required 15–180 fold higher drug concentrations to cause equivalent inhibition. Ouabain-resistant clones showed increased resistance of K+ transport function to ouabain inhibition that paralleled their increased resistance to growth inhibition. Initial experiments suggest that under selective conditions the resistant diploid fibroblasts differ significantly from wild type in binding of 3H-ouabain per unit surface area. The ouabain-resistant cells were similar to wild type in transport properties unrelated to ouabain inhibition. Resistant cells had normal karyotypes and senesced with a lifespan similar to control clones. The ouabain-resistant phenotypes of these diploid human fibroblast isolates apparently reflect point mutations that specifically affect the Na+/K+ transport ATPase with respect to ouabain-binding and/or response to bound ouabain.
Keywords:Address reprint requests to: Dr  M  Buchwald  Genetics Department  The Hospital for Sick Children  555 University Avenue  Toronto  Ontario M5G 1X8  Canada  
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