Ouabain-resistant mutants of mouse and hamster cells in culture

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 (OUA R) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUA R 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 OUA R 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 OUA R clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUA R 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 OUA R 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.