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High Level Expression of Wild Type and Variant Mouse Glucocorticoid Receptors in Chinese Hamster Ovary Cells

Institute of Cancer and Developmental Biology, Syntex Research Palo Alto, California 94304
Howard Hughes Medical Institute, Beckman Center, Stanford University School of Medicine Stanford, California 94305

Address requests for reprints to: Dr. Gordon M. Ringold, Syntex Research, Institute of Cancer and Developmental Biology, 3401 Hillview Avenue, Palo Alto, California 94304.

Abstract

We have isolated Chinese hamster ovary (CHO) cell lines expressing elevated levels of wild-type (W) and mutant forms of the glucocorticoid receptor (GR) using the technique of coamplification with a selectable dihydrofolate reductase (dhfr) cDNA. A prominent doublet at 90/92 kilodaltons was observed by Western blotting or labeling with [³H]-dexamethasone mesylate in extracts from cells transfected with W, the hormone binding mutant (NA), and the DNA binding mutant (NB). Quantification of receptor number by [³H]dexamethasone binding revealed the presence of approximately 10⁶ receptors per cell in the W and NB-producing lines. This represents a 25- to 50-fold increase in receptor density over control CHO cells which were not transfected with GR. Comparative quantitation by Western blotting of extracts from cells expressing GR showed that cells producing NA contain a level approximately 500-fold over control CHO cells.

Function of the amptified receptors was examined by transient transfection with the glucocorticoid-responsive reporter plasmid pMMTV-chloramphenicol acetyl transferase (CAT). Our results indicate that inducible CAT activity increases with the abundance of W receptor and no evidence of saturability was observed even at the highest levels of receptor. This supports previous suggestions that the concentration of the hormone-regulated transcription factor is definitely limiting with regard to maximal transcription efficiency. Interestingly, cells expressing even highly amplified levels of NA-GR or NB-GR showed no inducible response above that seen with control CHO cells. Thus these mutations are exceedingly nonleaky and are not dominant over the low endog-enous activity of the CHO GR. The overexpression of various forms of the GR in CHO cells serves as a paradigm for detailed biochemical analysis of this and other hormone-regulated transcription factors.

FOOTNOTES

This work was supported by NJEH Postdoctoral Fellowship ES05410 (to M.A.H.) and NIH Grant GM-25821 (to G.M.R.)

Preliminary data regarding this work was previously reported at a satellite symposium of the International Endocrinology Meetings (1988) and published in Cancer Research Supplement. Danielsen, M., Hinck, L, and Ringold, G.M. (1989) Mutational analysis of the mouse glucocorticoid receptor. Cancer Res. [Suppl] 49:2286s-2291s

^* Present address: Department of Biochemistry and Molecular Biology, Georgetown University Medical School, 3900 Reservoir Road, Northwest, Washington, DC 20007.

Received for publication August 11, 1989. Revision received October 2, 1989. Accepted for publication October 3, 1989.

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