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Absence of gene amplification in human cell mutants resistant to cardiac glycosides
1Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Abstract:
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.
Insights
Four HeLa cell mutants resistant to cardiac glycosides were studied. Resistance is likely due to point mutations in Na+/K+ ATPase genes, not gene amplification or increased transcription.
Area of Science:
- Cell biology
- Molecular genetics
- Biochemistry
Background:
- Cardiac glycosides like ouabain are potent inhibitors of the Na+/K+ ATPase.
- Drug resistance in cell lines often involves alterations in the target enzyme or its gene expression.
- Understanding mechanisms of drug resistance is crucial for developing new therapies.
Purpose of the Study:
- To investigate the molecular basis of cardiac glycoside resistance in four distinct HeLa cell mutants.
- To determine if gene amplification, increased transcription, or structural alterations of Na+/K+ ATPase subunits are responsible for resistance.
Main Methods:
- Isolation of cardiac glycoside-resistant HeLa cell mutants.
- Utilizing cloned cDNA probes for alpha and beta subunits of Na+/K+ ATPase.
- Employing dot blot, Southern, and Northern hybridization techniques to analyze gene copy number and transcript levels.
Main Results:
- No evidence of Na+/K+ ATPase gene amplification or increased transcription was found in the resistant mutants.
- Southern and Northern blots showed no gross structural alterations in the Na+/K+ ATPase genes or their transcripts.
- Cardiac glycoside treatment increased the resistant enzyme form but did not alter gene expression patterns.
Conclusions:
- Cardiac glycoside resistance in these mutants is not due to gene amplification or transcriptional changes.
- The findings strongly suggest that specific point mutations within the Na+/K+ ATPase structural genes confer resistance.
- These mutations likely result in an altered enzyme with reduced sensitivity to cardiac glycoside inhibition.