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Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Published on: February 24, 2014
Molecular analysis of ouabain-resistant mutants of the mouse lymphoma cell line L5178Y
W J Muriel1, J Cole, A R Lehmann
1MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, UK.
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.
Insights
Mouse lymphoma cells resistant to ouabain likely have single base changes in the Na,K-ATPase alpha subunit gene. This molecular alteration, not gene amplification, confers ouabain resistance.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Ouabain is a cardiac glycoside that inhibits the Na,K-ATPase pump.
- Understanding mechanisms of drug resistance is crucial for therapeutic development.
- Mouse lymphoma cell line L5178Y is a model for studying drug resistance mutations.
Purpose of the Study:
- To investigate the molecular basis of ouabain resistance in mouse lymphoma cells.
- To differentiate between gene alteration and gene expression changes contributing to resistance.
Main Methods:
- Molecular and biochemical analyses of spontaneous and mutagen-induced ouabain-resistant mutants.
- Analysis of gene structure, copy number, and expression of Na,K-ATPase.
- Measurement of 86Rb+ uptake and Na,K-ATPase activity in the presence of ouabain.
Main Results:
- Ouabain-resistant mutants exhibited significantly higher resistance to ouabain toxicity compared to wild-type cells.
- Gene structure, copy number, and Na,K-ATPase gene expression remained unaltered in mutants.
- Na,K-ATPase activity in mutant cell extracts showed increased resistance to 10(-3) M ouabain.
- A subset of mutants displayed increased sensitivity to 10(-5) M ouabain, suggesting complex effects.
Conclusions:
- The primary mechanism of ouabain resistance in these mutants is likely due to single base-changes within the Na,K-ATPase alpha subunit gene.
- Gene amplification or overexpression is not the cause of resistance in this specific cell line model.
- Findings contrast with previously reported mechanisms of ouabain resistance in other cell lines.
