FUNCTION OF COFACTORS MODIFYING THYROID HORMONE RECEPTOR FUNCTION

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1. ANALYSIS OP MUTATIONS IN p53 AND ACTH RECEPTOR (ACTH-R) GENES IN CORTISOL-PRODUCING ADENOMAS (CPA)Possible mutations in p53 and ACTH-R genes were screened in benign adrenocortical adenomas producing cortisol. In eleven CPA samples obtained at surgery, no mutation was detected either in p53 and ACTH-R genes. It was thus suggested that mutations in these genes are rare cause of CPA.2. ANALYSIS OF ACTH RECEPTOR (ACTH-R) EXPRESSION IN CORTISOL-PRODUCING ADENOMAS (CPA)We and others demonstrated that expression of ACTH-R gene is upregulated by its own ligand ACTH.If the same regulation applies to CPA, expression of ACTH-R should be suppressed because of the lack of endogenous ACTH secretion. However, clinical evidence that ACTH administration to the patients with Cushing's syndrome resulted in further increase in cortisol production, suggesting the expression of functional ACTH-R.Northem blot analysis demonstrated that CPA expresses ACTH-R mRNA more abundantly than the adjacent non-neopla … More stic adrenal gland. It was thus suggested that regulation of ACTH-R gene expression is different from that of normal adrenal gland. To gain more insight how ACTH-R expression is regulated, we cloned the promoter region of ACTH-R gene. To assess the function of the promoter, reporter genes were constructed by placing the promoter region or its deletion mutants to the upstream of the firefly luciferase gene. By transfecting the reporter gene constructs, it was demonstrated that SF-1 is responsible for adrenal specific expression. Furthermore, both AP-1 and SF-1 are shown to be involved in ACTH-dependent upregulation of its receptor gene.3. SEARCH FOR GENES SPECIFICALLY EXPRESSED IN CORTISOL-PRODUCING ADENOMAS (CPA)To gain insight for the pathogenesis of CPA, mRNAs were separated from 10 CPA samples and adjacent atrophic normal tissues. After the synthesis of corresponding double stranded cDNA, they were restricted with DpnII and adaptors were ligated, resulting in the construction of each amplicon representing CPA or normal cDNAs. PCR-based subtraction revealed that several clones were identified to be preferentially expressed in CPA.Among them, glutathione-S-transferase A-1 (GST-A1) was marked upregulated in all the CPA samples when the expression was compared with that in corresponding normal tissues. Inhibition of this enzyme by ethacrinic acid caused severe reduction of the proliferation of the cell line derived from adrenocortical cancer. It was thus suggested GST-A1 plays an important role for the tumorigenesis of CPA. Less