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Analysis of glucocorticoid-resistant human leukemic cells by somatic cell hybridization
Abstract:
Glucocorticoid-resistant mutants isolated form the glucocorticoid-sensitive human leukemic cell line CEM-C7 can be divided into three phenotypes: those with almost no glucocorticoid-binding activity (r-); those whose steroid-receptor complexes are unstable during attempted activation but are stabilized by the presence of sodium molybdate (actl:molybdate-sensitive); and those whose steroid-receptor complexes are unstable during attempted activation but are insensitive to the presence of molybdate (actl:molybdate-resistant). To determine if these phenotypes represent different mutations within the glucocorticoid receptor locus itself or reflect alterations in other components modifying receptor function, somatic hybrids were constructed between wild-type cells and all three classes of resistant mutants, as well as between various classes of resistant mutants. Hybrids were analyzed for chromosome content, steroid-induced growth inhibition, induction of the enzyme glutamine synthetase, and glucocorticoid receptor content. Hybrids constructed between wild-type cells and any of the three classes of resistant cells were growth-inhibited in the presence of dexamethasone, displayed normal levels of glutamine synthetase induction, and contained a quantity of glucocorticoid receptor approximately equal to the sum of the glucocorticoid receptor concentrations of the parental cell lines. Hybrids constructed between various classes of resistant cells were not growth-inhibited by dexamethasone, displayed no glutamine synthetase induction, and also contained the sum of the glucocorticoid receptor concentration of the individual parents. Thus, each phenotype is recessive, and there is no complementation between phenotypes. We conclude that the three phenotypes are the result of different mutations within the glucocorticoid receptor locus itself and do not represent the presence of dominant receptor-inactivating factors or the absence of positive regulatory components.
Insights
Three phenotypes of glucocorticoid resistance in human leukemic cells result from distinct mutations within the glucocorticoid receptor gene. These mutations are recessive, indicating alterations directly affect receptor function, not other cellular components.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Glucocorticoids are crucial regulators of cell growth and differentiation.
- Resistance to glucocorticoids in cancer cells poses a significant therapeutic challenge.
- CEM-C7 human leukemic cells exhibit distinct glucocorticoid resistance phenotypes.
Purpose of the Study:
- To elucidate the genetic basis of three distinct glucocorticoid resistance phenotypes in CEM-C7 cells.
- To determine if resistance mutations reside within the glucocorticoid receptor locus or affect other cellular components.
- To analyze the genetic interactions between different resistance phenotypes.
Main Methods:
- Construction and analysis of somatic cell hybrids between wild-type and resistant CEM-C7 cell lines.
- Assessment of phenotypes including steroid-induced growth inhibition and enzyme induction (glutamine synthetase).
- Quantification of glucocorticoid receptor content in parental and hybrid cells.
Main Results:
- Wild-type x resistant cell hybrids showed sensitivity to glucocorticoids, indicating recessive resistance.
- Resistant x resistant cell hybrids displayed no complementation, suggesting mutations are in the same locus.
- All three resistance phenotypes were recessive and mapped to the glucocorticoid receptor locus.
Conclusions:
- The three distinct glucocorticoid resistance phenotypes arise from different mutations within the glucocorticoid receptor gene.
- These mutations directly impact glucocorticoid receptor function, not regulatory components.
- The findings provide insights into the molecular mechanisms of glucocorticoid resistance in leukemia.