Analysis of co-factor function in a glucocorticoid-resistant small cell carcinoma cell line.
Human small cell lung carcinoma (SCLC) tumours exhibit neuroendocrine differentiation, secreting hormones such as ACTH and related peptides. While glucocorticoids inhibit ACTH secretion from the pituitary, this does not occur in SCLC tumours and SCLC cell lines. Failure of glucocorticoids to suppress ACTH peptides is accompanied by a global lack of glucocorticoid action in a number of SCLC cell lines. In the human SCLC cell line, COR L103, activation of a human tyrosine aminotransferase (TAT3)-luciferase reporter gene is resistant to glucocorticoids despite similar glucocorticoid receptor (GR) expression to the glucocorticoid-sensitive A549 human lung cancer cell line; moreover, the GR is free of deleterious mutations. Over-expression of a wild-type GR restores glucocorticoid regulation of TAT3-luciferase, and this is enhanced when the activation function (AF)-2 domain is deleted but much reduced when the AF-1 domain is deleted. This suggests aberrant AF-2 activation domain function. We identified defective steroid receptor co-activator 1 (SRC1) recruitment to the GR AF-2 in COR L103 cells, although SRC1 was successfully recruited to the steroid X receptor with rifampicin, suggesting a defect in the GR. Analysis of other GR C-terminal co-factors identified increased expression of nuclear co-repressor (NCoR) in COR L103 cells. To determine the impact of this, NCoR was over-expressed in A549 cells, where it reduced GR transactivation by 55%. In summary, glucocorticoid resistance is associated with altered SRC protein recruitment and increased expression of NCoR in these SCLC cells, suggesting that glucocorticoid sensitivity may be modified by subtle changes in co-factor recruitment.
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Endocrine Sciences Research Group, 3.810 Stopford Building, Schools of Biological Sciences and Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Stevens
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D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D018288:Carcinoma, Small Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D018274:Electroporation / Q000379:methods; D004789:Enzyme Activation; D017930:Genes, Reporter; D015756:Genes, tat; D005938:Glucocorticoids / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008175:Lung Neoplasms / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D000324:Adrenocorticotropic Hormone; D008156:Luciferases