FUNCTION OF COFACTORS MODIFYING THYROID HORMONE RECEPTOR FUNCTION

1. 分析在产生皮质醇的良性肾上腺皮质腺瘤（CPA）中筛选p53和ACTH受体（ACTH- r）基因可能突变的p53和ACTH- r基因。在手术中获得的11份CPA样本中，p53和ACTH-R基因均未检测到突变。因此，这些基因的突变是导致cpa的罕见原因。促肾上腺皮质激素分泌腺瘤（CPA）中促肾上腺皮质激素受体（ACTH- r）的表达分析我们和其他人证明了ACTH- r基因的表达可被其自身的配体ACTH上调。如果同样的调控也适用于CPA，则ACTH- r的表达应受到抑制，因为内源性ACTH分泌不足。然而，临床证据表明，库欣综合征患者给予ACTH可导致皮质醇分泌进一步增加，提示功能性ACTH- r的表达。northern blot分析表明，与邻近的非肿瘤肾上腺相比，CPA更丰富地表达ACTH-R mRNA。提示ACTH-R基因表达调控与正常肾上腺不同。为了进一步了解ACTH-R的表达是如何调控的，我们克隆了ACTH-R基因的启动子区域。为了评估启动子的功能，通过将启动子区域或其缺失突变体置于萤火虫荧光素酶基因的上游来构建报告基因。通过转染报告基因构建，证明SF-1负责肾上腺特异性表达。此外，AP-1和SF-1均参与acth依赖性受体基因的上调。为了深入了解促肾上腺皮质激素分泌腺瘤（CPA）的发病机制，我们从10个CPA样本和邻近的萎缩正常组织中分离了mrna。在合成相应的双链cDNA后，用DpnII限制它们并连接接头，从而构建代表CPA或正常cDNA的每个扩增子。基于pcr的减法分析显示，有几个克隆在CPA中优先表达。其中谷胱甘肽- s -转移酶A-1 （GST-A1）在所有CPA样品中与相应的正常组织相比表达均显著上调。乙二酸对这种酶的抑制导致肾上腺皮质癌细胞系的增殖严重减少。提示GST-A1在CPA的肿瘤发生中起重要作用。少

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