Ouabain-resistant mutants of mouse and hamster cells in culture

R. M. Baker, D. M. Brunette, R. Mankovitz, L. H. Thompson, G. F. Whitmore, L. Siminovitch, J. E. Till

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Abstract

Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5-6 mM K+, or ≥ 0.1 mM with 0.5 mM K+, inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUAR) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUAR L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUAR cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUAR clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUAR cells in a wild-type population can be substantially increased, to approximately 10-4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5-6 × 10-8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUAR and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.

Original languageEnglish (US)
Pages (from-to)9-21
Number of pages13
JournalCell
Volume1
Issue number1
DOIs
StatePublished - 1974
Externally publishedYes

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Medicine(all)

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    Baker, R. M., Brunette, D. M., Mankovitz, R., Thompson, L. H., Whitmore, G. F., Siminovitch, L., & Till, J. E. (1974). Ouabain-resistant mutants of mouse and hamster cells in culture. Cell, 1(1), 9-21. https://doi.org/10.1016/0092-8674(74)90149-4