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Membrane permeability as a determinant of dexamethasone resistance in murine thymoma cells
Abstract:
The variant MS23 of the murine thymoma cell line W7 , selected for growth at low concentrations of dexamethasone (7.5 nM), is cross-resistant to various unrelated drugs, including colchicine. By stepwise selection in combinations of dexamethasone and colchicine at increasing concentrations, we have isolated a series of variants with increased resistance to dexamethasone and cross-resistance to puromycin, colchicine, daunomycin, gramicidin, and vincristine. Surprisingly, resistance to triamcinolone acetonide, a glucocorticoid structurally related to dexamethasone, did not develop. Assays for specific dexamethasone and triamcinolone acetonide binding sites in variant cell extracts reveal that the glucocorticoid receptors of these variants are unchanged as compared to the W7 parental line. However, whole cell binding assays yielded reduced apparent affinity for dexamethasone in the MS23 variant and drastically reduced dexamethasone binding after selection for increased resistance. We demonstrate that this is due to reduced drug uptake. Procaine, a membrane-active anesthetic, potentiates uptake of puromycin and dexamethasone in the variants. The variants are stable, and karyotypic analysis did not reveal double minute chromosomal structures. These results demonstrate that permeability can be a rate-limiting step in steroid hormone action and is the basis for dexamethasone resistance in these variants.
Insights
Drug resistance in W7 thymoma cells is linked to reduced drug uptake, not altered glucocorticoid receptors. This suggests cell permeability is a key factor in steroid hormone resistance.
Area of Science:
- Cell Biology
- Pharmacology
- Molecular Biology
Background:
- The murine thymoma cell line W7 and its dexamethasone-resistant variant MS23 were used.
- MS23 exhibits cross-resistance to unrelated drugs, indicating a potential multidrug resistance mechanism.
Purpose of the Study:
- To investigate the mechanisms underlying dexamethasone resistance and cross-resistance in W7 cell variants.
- To determine if altered glucocorticoid receptors or changes in drug permeability are responsible for resistance.
Main Methods:
- Stepwise selection of cell variants using increasing concentrations of dexamethasone and colchicine.
- Assays for specific dexamethasone and triamcinolone acetonide binding to glucocorticoid receptors.
- Whole cell binding assays to assess drug affinity and uptake.
- Karyotypic analysis to detect chromosomal abnormalities.
Main Results:
- Selected variants showed increased resistance to dexamethasone and cross-resistance to multiple unrelated drugs, but not to triamcinolone acetonide.
- Glucocorticoid receptor levels and binding affinity remained unchanged in variant cell extracts.
- Whole cell assays revealed significantly reduced dexamethasone uptake in resistant variants.
- Procaine enhanced drug uptake, confirming reduced permeability as the resistance mechanism.
Conclusions:
- Cellular permeability, specifically reduced drug uptake, is a critical factor limiting steroid hormone action and drives dexamethasone resistance in these variants.
- The findings highlight the role of drug transport in modulating cellular responses to steroid hormones.