Absence of gene amplification in human cell mutants resistant to cardiac glycosides.

In HeLa cells, four different types of mutants resistant to cardiac glycosides viz. ouabain and SC4453, which differ from each other in cross resistance pattern, have been isolated after single-step selections [J Biol Chem 260 (1985) 6843-6850; J Biol Chem 261 (1986) 2034-2040]. Using cloned cDNA probes specific for the alpha and beta subunits of Na+/K+ ATPase, these mutants have been investigated for amplification and/or increased transcription of Na+/K+ ATPase genes. Results from dot blots, Southern and Northern hybridizations provide evidence that these mutants do not involve any amplification or increased transcription or gross structural alterations in Na+/K+ ATPase genes of their transcripts. Similar results were obtained with the mutant cells grown either in the absence or presence of cardiac glycosides, the latter conditions of which cause 3-4-fold increase in the resistant form of the enzyme within the mutant cells. These results are consistent with the inference that resistance to cardiac glycosides in these mutants may be due to specific point mutations within the structural gene(s) of Na+/K+ ATPase leading to an altered enzyme that is resistant to inhibition by different cardiac glycosides.