Molecular analysis of ouabain-resistant mutants of the mouse lymphoma cell line L5178Y

Abstract

We have carried out molecular and biochemical analyses on several spontaneous and mutagen-induced ouabain-resistant mutants from the mouse lymphoma cell line L5178Y. Mutant cells were much more resistant than wild-type cells to the toxic effects of ouabain. The gross structures and copy numbers of two ouabain-resistant genes were unaltered in the mutants, as was the expression of the Na,K-ATPase gene. Uptake of 86Rb+ was more resistant to ouabain in the mutants than in wild-type cells. There was no evidence for an inducible uptake mechanism. The Na,K-ATPase activity in extracts of the mutants was, in all cases examined, more resistant than wild-type cells to the inhibitory action of 10(-3) M ouabain. At 10(-5) M ouabain, the Na,K-ATPase activity in three out of eleven mutants was actually more sensitive to ouabain than in wild-type cells. These data suggest that the ouabain-resistance of the mutants probably results from single base-changes in the alpha subunit of the Na,K-ATPase gene, rather than from amplification or overexpression of ouabain-resistance genes. The latter types of alterations have been described for ouabain-resistant lines derived in other laboratories.