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Glucocorticoid receptor structure and function in an adrenocorticotropin-secreting small cell lung cancer
D Gaitan1, C R DeBold, M K Turney
1Division of Endocrinology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Abstract:
ACTH secretion by tumors of nonpituitary origin is characteristically resistant to negative feedback regulation by glucocorticoids. One possible mechanism for the phenomenon could be a structural defect in the intracellular glucocorticoid receptor (GR). We studied the GR in DMS-79 cells derived from a human ACTH-secreting small cell lung cancer. Compared with control cells, DMS-79 cells were found to have greatly diminished GR ligand-binding activity and immunoreactive 94-kilodalton (kDa) GR content. Northern blot analysis revealed expression of GR transcripts that appeared to be slightly larger than those in control cells. A DMS-79 cell GR cDNA was cloned by reverse transcription/polymerase chain reaction amplification of mRNA using primers specific for full-length normal GR. The derived sequence of this full-length GR differed from the reported sequence by a single altered codon (G to A; Asn to Ser at codon 363) outside the steroid-binding domain. This N363S DMS-79 GR functioned normally to activate a target gene [mouse mammary tumor virus-chloramphenicol acetyl transferase (MMTV-CAT)] in transient transfection experiments in COS cells. Evidence for expression of a second type of GR mRNA was obtained by screening a DMS-79 cell cDNA library. This GR cDNA contained normal GR sequence up to nucleotide 2155, corresponding exactly to the end of exon 7 in the normal GR gene. The sequence appended to the GR sequences was not matched by any known sequence in DNA databases and included an in-frame termination codon after only 6 bases. The predicted truncated GR protein product (GR delta) has a mol wt of 73,740 and lacks most of the ligand-binding domain. Transient transfection of the GR delta form into COS cells did not reveal any dominant negative effect on the function of a cotransfected normal GR.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Tumors secreting ACTH (adrenocorticotropic hormone) often resist feedback regulation due to glucocorticoid receptor (GR) defects. This study identified a novel truncated GR variant in lung cancer cells, potentially explaining resistance.
Area of Science:
- Endocrinology
- Molecular Biology
- Oncology
Background:
- Adrenocorticotropic hormone (ACTH) secretion by nonpituitary tumors typically shows resistance to glucocorticoid negative feedback.
- A potential cause for this resistance is a structural defect in the intracellular glucocorticoid receptor (GR).
Purpose of the Study:
- To investigate the glucocorticoid receptor (GR) in DMS-79 cells from a human ACTH-secreting small cell lung cancer.
- To identify molecular mechanisms underlying glucocorticoid resistance in these tumor cells.
Main Methods:
- Studied GR in DMS-79 cells, comparing ligand-binding activity and immunoreactive GR content to control cells.
- Utilized Northern blot analysis to examine GR transcript expression.
- Cloned and sequenced a full-length GR cDNA from DMS-79 cells and screened a cDNA library for other GR variants.
Main Results:
- DMS-79 cells exhibited significantly reduced GR ligand-binding activity and 94-kDa GR content.
- A single codon alteration (N363S) was found in the cloned full-length GR, outside the steroid-binding domain; this variant retained normal function in gene activation assays.
- A second, novel GR mRNA variant (GR delta) was identified, predicting a truncated protein lacking the ligand-binding domain, which did not exhibit dominant-negative effects.
Conclusions:
- The identified N363S GR variant in DMS-79 cells is functional and does not explain glucocorticoid resistance.
- A novel truncated GR delta variant, lacking the ligand-binding domain, is expressed in these cells and may contribute to ACTH-secreting tumors' resistance to glucocorticoid feedback.