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  • Title: Methotrexate-induced overexpression of functional glucocorticoid receptors in Chinese hamster ovary cells.
    Author: Bellingham DL, Sar M, Cidlowski JA.
    Journal: Mol Cell Endocrinol; 1992 Feb; 83(2-3):153-71. PubMed ID: 1547909.
    Abstract:
    We have used a modified cotransfection and selection strategy to create a series of mammalian cell lines that stably express high levels of intact glucocorticoid receptors. These cell lines were produced by subjecting Chinese hamster ovary (CHO) cells, which had been previously cotransfected with a glucocorticoid-responsive dihydrofolate reductase (DHFR) gene and the human glucocorticoid receptor gene, to growth in increasing concentrations of methotrexate (MTX). By linking the MTX selection process to glucocorticoid receptor function via the DHFR gene, stable cell lines resistant to a range of MTX concentrations (50 nM to 3 microM) were isolated that were strictly dependent upon glucocorticoids for growth. Quantitation of steroid binding capacity in MTX-resistant cells revealed a progressive increase in the number of glucocorticoid receptors as a function of increasing MTX concentration. This increase in receptor content was maximal at the highest level of MTX resistance examined (3 microM MTX) and represented a 25-fold elevation in glucocorticoid receptor number relative to CHO cells expressing only endogenous hamster receptor. The increases in steroid binding obtained after MTX selection were reflected by similar increases in the level of glucocorticoid receptor protein as determined by immunoblot analysis. Examination of glucocorticoid receptor structure by sucrose density gradient centrifugation revealed that oligomeric (9 S) steroid receptor complexes were formed at all levels of receptor expression. Subcellular localization of the glucocorticoid receptor protein by immunocytochemical staining revealed effective nuclear translocation of the overexpressed receptors in MTX-resistant cells. Functional transfection studies using a glucocorticoid-responsive reporter gene indicated that the additional glucocorticoid receptors in CHO cells were competent to activate transcription. To determine the molecular basis for the MTX-induced increases in functional glucocorticoid receptors, steady-state levels of glucocorticoid receptor mRNA were examined. MTX selection produced a 5- to 7-fold increase in transfected glucocorticoid receptor gene expression relative to untreated cells. MTX-resistant cells also expressed increased levels of a putative hamster glucocorticoid receptor mRNA species. Interestingly, the observed increases in receptor gene expression in these cells could not be accounted for by amplification of either the human or the hamster glucocorticoid receptor genes.
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