Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.
We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.
D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D004351:Drug Resistance; D005784:Gene Amplification; D005786:Gene Expression Regulation; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D010042:Ouabain / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D012333:RNA, Messenger; D010042:Ouabain; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase