【作者】 陈光椿；

【导师】 卢建；

【作者基本信息】 第二军医大学 ， 病理学与病理生理学， 2002， 博士

【摘要】 前列腺癌(PC)是雄激素依赖的肿瘤，雄激素可促进PC的增殖，而内分泌治疗即去势和应用雄激素拮抗剂可抑制肿瘤生长并诱导癌细胞凋亡，因此，该疗法一直是PC的重要治疗手段之一。但在临床工作中早已发现，一部分患者在内分泌治疗前或治疗一段时间后其肿瘤细胞出现了对内分泌治疗的不敏感或抵抗，其机制迄今尚未完全阐明。由于雄激素与PC的发生、发展和临床治疗关系密切，而雄激素受体(AR)是介导雄激素作用的关键大分子，因此，PC与AR关系的研究一直是前列腺癌研究的热点之一。一般认为，PC细胞为适应内分泌治疗后体内出现的极低的雄激素环境，可通过多种机制，使AR的信号转导增强(1)AR基因扩增，蛋白表达增加；(2)AR基因突变，造成AR转录激活功能增强，与配体结合的特异性改变等；(3)辅助因子表达的异常；(4)配体非依赖性激活。 本文对我们以前应用PCR-SSCP分析，从50余例前列腺癌组织标本中，发现的4例AR基因突变(G142V、D221H、E872Q和M881I)进行了体外功能分析。 首先，我们应用pALTER体外致突变系统成功地对这些突变位点进行了人工致突变，构建了这些突变的表达载体，然后应用Western blot分析，对转染入无内源性AR表达的COS-7细胞中的表达载体的表达进行了分析，结果表明，表达载体能有效表达AR，其大小与野生型AR一致。而应用完整细胞的放射配体结合分析法，对转染入COS-7细胞中的AR进行了Scatchard分析，结果表明，与野生型AR相比，这些 博士学位论文 陈腑 突变与人N合成的雄激素R1881结合的亲和力及最大结合容量均无明显。 改变。在此基础上，我们对受体的转录激活功能进行了研究。 以无内源性AR表达的＊＊l细胞；为模型，我们首先应用＊＊T对受－体的转录激活功能进行了研究。结果表明，与野生型AR相比，DHT 在 Zxlo－“mol／L时，即对位于 TAD的两突变体＊ 和 D22lH即具有 明显的车录激活增强作用（P＜O．05），Zx ic“’moL几时则有显著的激活作 用（P＜o刀1＊＊＊T对另外两个突变体的转录激活作用无明显的异常。 应用雄激素桔抗剂CPA、Flutamide及雌激素、孕激素、RU486、Dex 对AR突变体进行转录激活功能研究表明，与野生型AR相比，CPA、 雌激素和孕激素对E872Q的转录激活功能异常增强（P＜o刀1）。而其余 三种突变体则无明显异常。这表明E872Q与2：PA、雌激素和孕激素结 合发主了特异性改变。而RU486和I儿x对四种突变体的转录功能均无 明显异常。 作为配体依赖性的转录调节因于，AR发挥其正常的转录调节作用， 有赖于辅助因子的参与。我什1应用共激活因子 1IF上、CBP及 P300和 共抑制因子NCOR、SMRTMRT及p53对AR突变体转录激活功能的影响进 行了研究。与野生型AR相比，TD上和CBP对突变发生于LBD的两＿突变体＊872和＊8861具有明显异常的转录激活·增强作用（P＜0刀1），而 对发兰于TAD的两个突变体的转录则无明显的异常作用。而P300对四 种突变体的作用，则与野生型AR基本相似，无明显的异常。 对共抑制因子所进行的研究则表明，SMRT和 P53能完全进行 DHT 以及CPA对野生型AR和AR突变体的转录激活作用。而NCOR作用 存在一些差异：能完全阻断 CPA对所有受体的转录激活作用；仅能部＿分抑制＊＊T对野生型AR及＊142、IR21H的转录激活作用，完全阻断 DHT对E872和M8861的转录激活作用。以上研究提示，AIt的转录激 3 博士学位论文 陈光椿 活作用的强弱，不仅与共激活因子表达强弱有关，也与共抑制因子表达 强弱有关；二者表达比例的高低，对受体的转示：活性尤为重要。这也可 能正是受体的转录激活存在细胞特异性的机制之一。 AR的配体非依赖性转录激活功能在前列腺癌的发展过程中，也具－有重要的作用。尽管机制尚不清楚。我们应用PKC激动剂PMA对AR 进行的研究表明，＊M A对野生型AR和AR突变体均有基本相同的配 体依赖性转录激活作用，并且还与］n｛T一起，对野生型AR及AR突 变体的转录激活起协同的作用。尽管 c＊un是I。MA信号转导途径中的 下游蛋白，我们对c＊un在AR季录激活中的作用研究 表明，c－Jun育完 全阻断 AR的转录激活作用，表明 c＊un并不参与 l。MA对 Aft的的配体 非依赖性激活作用。 G CHO为模型，研究了 AR7含 pZI启动子的报告基因 pZI－LUC 的转录激活作用，结果表明，与野生型＊R相比，＊R突变体对报告基更多还原

【Abstract】 Prostatic carcinoma (PC) is the most commonly diagnosed malignancy in men in the United States and is second only to lung cancer in cancer-related deaths. However, in recent years, with the change of life style, there is also obvious augment of morbidity in our country.Androgens are essential for the development, growth, and maintenance of the prostate. They exert their effects via the intracellular androgen receptor (AP.), which is a ligand-dependent transcription activator. As is the case with normal prostate development, primary prostatic cancers are largely dependent on androgens for growth and survival. Most patients respond favorably to androgen ablation and antiandrogen therapy, which has become a standard treatment of metastatic disease. However, virtually all patients will relapse with clinically defined androgen-independent cancer. This phenomenon raises the question of how cancer cells survive and grow in the low androgen environment? Two of the routes cells can take to adapt are (1) bypassing and (2) sensitizing the AR pathway. The vast numbers of AR abnormalities observed in prostate tumors from patientstreated with hormonal therapy suggest that many cells sensitize or change the AR pathway. To continue to activate this pathway in a low androgen environment, cells can (I) mutate the AR to become promiscuously activated by different steroids, (2) amplify the AR, (3) activate the AR in a ligand-independent manner by growth factors and cytokines, or (4) amplify coactivators. Alternatively, prostate cancer cells that have lost AR expression must have bypassed the AR pathway. Activation of oncogenes and autocrine growth factor stimulation are two mechanisms that likely contribute to becoming completely androgen-independent. From all the studies on AR function in prostate cancer, it is clear that the AR plays an important role in cancer development and progression. Moreover, the AR pathv/ay remains important in most cells from patients with clinically defined androgen-independent prostate cancer.AR like other nuclear receptors displays a modular structure with three main conserved regions: an amino-terminal region harboring an autonomous activation function (AF-1), a centrally located E’NA-binding domain (DBD), and a carboxyl-termirial region containing the ligand-binding domain (LBD) The AR is normally associated with heat shock proteins and in this state cannot bind to androgen-responsive elements). Upon ligand binding, the receptor acquires a new conformational state. Androgen binding induces dissociation from heat shock proteins, hyperphosphorylation, conformational changes, and dimerization of the receptor. This allows binding of the AR to specific DNA sequences called androgen-responsive elements located within the promoters of androgen-responsive genes. In conjunction with cofactor proteins and other transcriptional factors, the AR is then able to up- or down-regulate the transcription of genes, such as C(3), Sip, probasin, PSA, p21 and hKLK2, et.al.The first AR mutation in prostate cancer was described in LNCaP cells several years ago. This mutant receptor binds estrogenic and progestagenic steroids and antiandrogens hydroxyflutamide and nilutamide with high affinity. These substances activate the mutant AR more efficiently than the wild-type one. All mutant ARs detected inprostate cancer that are functionally characterized are "promiscuous" receptors; they can be activated by other steroid hormones arid by nonsteroidal antiandrogens. Subsequently, amino acid substitutions were reported in organ-confined as well as rnetastatic tumors Of the several AR mutants in prostate cancer that have been characterized thus far, the majority are functional and can mediate androgen-induced transactivation, in contrast to loss of function germ-line AR mutations that cause androgen insensitivity.Previous years, in our laboratory, we have identified four missense mutations (G142V, D221H, E872Q, and M886I) in AR from nearly fifty PC samples by PCR-SSCP analysis. However, the effect of these mutati更多还原