雄激素应答元件假冒DNA对PSA基因启动子的抑制作用

研究雄激素应答元件假冒DNA(AREdecoy)对前列腺特异抗原 (PSA)基因启动子的抑制作用 .联合运用报告基因和假冒DNA策略 ,构建了含PSA基因 5′侧启动子区 6 40bpDNA的萤光素酶表达载体pGL3 PSA ,与人工合成的双链硫代ARE假冒DNA共转染前列腺癌细胞株PC3 M并作用不同的时间 (2 4h、4 8h、72h) .应用双萤光素酶测定系统 ,检测萤光素酶的表达活性 .结果显示 :AREdecoyDNA显著抑制报告基因萤光素酶的表达 ,抑制率可达 95 % ,而对照decoyDNA无此作用 .作用不同的时间对萤光素酶活性的抑制无显著性差异     

雄激素通过雄激素受体途径调控垂体肿瘤转化基因(PTTG1)的表达

雄激素能上调大鼠前列腺中垂体肿瘤转化基因1(PTTG1)的表达,在前列腺癌标本中,发现PTTG1的表达明显高于正常组织.因此,用雄激素依赖的前列腺癌细胞LNCaP来研究雄激素调控PTTG1表达的分子机制.在LNCaP细胞中,通过雄激素刺激后,PTTG1的表达明显升高.根据序列分析以及5-缺失体实验(deletion)和突变实验(mutation),发现PTTG1的启动子上游-950到-933的序列上有1个雄激素受体反应元件(ARE),染色体免疫共沉淀实验(CHIP)也证实了雄激素能促进雄激素受体与PTTG1启动子上ARE结合,从而在转录水平调控PTTG1的表达.     

Identification of two novel cis-elements in the promoter of the prostate-specific antigen gene that are required to enhance androgen receptor-mediated transactivation.

A monomeric androgen responsive element (ARE) is not sufficient to mediate significant androgen induction of the prostate-specific antigen (PSA) gene. Co-transfection experiments using a series of 5'deletion fragments of the proximal promoter region of the PSA gene linked to bacterial chloramphenicol acetyltransferase (CAT) as a reporter have identified two motif sequences which are indispensable for androgen receptor (AR)-mediated transactivation of the PSA promoter and have been designated as motifs A and B respectively. Of note, motif B alone has very little independent enhancer activity regardless of the presence or absence of androgen, whereas multi-copies of motif A exert androgenic inducibility for a heterologous promoter independent of the presence of ARE. Nucleotide substitutions in either motif significantly decrease the androgen inducibility and the nuclear protein binding ability. Furthermore, gel band shift experiments consistently demonstrate that nuclear proteins can bind these motifs, and they are non-receptor factors. Our data indicate that these two DNA motifs are novel cis -regulatory elements and exhibit different mechanisms in cooperation with ARE for AR-mediated transactivation.     

Convergent evolution of crystallin gene regulation in squid and chicken: The AP-1/ARE connection

Previous experiments have shown that the minimal promoters required for function of the squid SL20-1 and SL11 crystallin genes in transfected rabbit lens epithelial cells contain an overlapping AP-1/antioxidant responsive element (ARE) upstream of the TATA box. This region resembles the PL-1 and PL-2 elements of the chicken B 1-cry stallin promoter which are essential for promoter function in transfected primary chicken lens epithelial cells. Here we demonstrate by site-directed mutagenesis that the AP-1/ARE sequence is essential for activity of the squid SL20-1 and SL11 promoters in transfected embryonic chicken lens cells and fibroblasts. Promoter activity was higher in transfected lens cells than in fibroblasts. Electrophoretic mobility shift and DNase protection experiments demonstrated the formation of numerous complexes between nuclear proteins of the embryonic chicken lens and the AP-1/ARE sequences of the squid SL20-1 and SL11 crystallin promoters. One of these complexes comigrated and cross-competed with that formed with the PL-1 element of the chicken B1-crystallin promoter. This complex formed with nuclear extracts from the lens, heart, brain, and skeletal muscle of embryonic chickens and was eliminated by competition with a consensus AP-1 sequence. The nonfunctional mutant AP-1/ ARE sequences did not compete for complex formation. These data raise the intriguing possibility that entirely different, nonhomologous crystallin genes of the chicken and squid have convergently evolved a similar cis-acting regulatory element (AP-1/ARE) for high expression in the lens. Correspondence to: S. I. Tomarev     

Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein

We demonstrate that the 204 bp intronic gene fragment of C3(1), which has a specific in vitro affinity for the androgen receptor, is able to confer androgen responsiveness to a heterologous promoter. This characteristic is completely destroyed by a single G----T substitution, affecting a 5'-TGTTCT-3' element that closely resembles the consensus sequence of the glucocorticoid and progesterone response elements (GRE/PRE). In fact we could show that this androgen response element (ARE) also acts as a similarly weak GRE or PRE in T-47D cells.     

Down-regulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer

Human DAB2IP (hDAB2IP), a novel GTPase-activating protein modulating the Ras-mediated signaling and tumor necrosis factor-mediated apoptosis, is a potent growth inhibitor in human prostate cancer (PCa). Loss of hDAB2IP expression in PCa is due to altered epigenetic regulation (i.e. DNA methylation and histone modification) of its promoter region. The elevated polycomb Ezh2, a histone methyltransferase, has been associated with PCa progression. In this study, we have demonstrated that an increased Ezh2 expression in normal prostatic epithelial cells can suppress hDAB2IP gene expression. In contrast, knocking down the endogenous Ezh2 levels in PCa by a specific small interfering RNA can increase hDAB2IP expression. The association of Ezh2 complex (including Eed and Suz12) with hDAB2IP gene promoter is also detected in PCa cells but not in normal prostatic epithelial cells. Increased Ezh2 expression in normal prostatic epithelial cells by cDNA transfection facilitates the recruitment of other components of Ezh2 complex to the hDAB2IP promoter region accompanied with the increased levels of methyl histone H3 (H3) and histone deacetylase (HDAC1). Consistently, data from PCa cells transfected with Ezh2 small interfering RNA demonstrated that reduced Ezh2 levels resulted in the dissociation of Ezh2 complex accompanied with decreased levels of both methyl H3 and HDAC1 from hDAB2IP gene promoter. We further unveiled that the methylation status of Lys-27 but not Lys-9 of H3 in hDAB2IP promoter region is consistent with the hDAB2IP levels in both normal prostatic epithelial cells and PCa cells. Together, we conclude that hDAB2IP gene is a target gene of Ezh2 in prostatic epithelium, which provides an underlying mechanism of the down-regulation of hDAB2IP gene in PCa.     

Inhibition of monoamine oxidase A promotes secretory differentiation in basal prostatic epithelial cells

Abstract Monoamine oxidase A (MAO-A) expression is associated with high-grade prostate cancer. Immunohistochemistry showed that MAO-A is also expressed in the basal epithelial cells of normal prostate glands. Using cultured primary prostatic epithelial cells as a model, we showed that MAO-A prevents basal epithelial cells from differentiating into secretory cells. Under differentiation-promoting conditions, clorgyline, an irreversible MAO-A inhibitor, induced secretory cell-like morphology and repressed expression of cytokeratin 14, a basal cell marker. More importantly, clorgyline induced mRNA and protein expression of androgen receptor (AR), a hallmark of secretory epithelial cells. In clorgyline-treated cells, androgen induced luciferase activity controlled by the promoter of prostate-specific antigen, an AR target gene, in a dose-dependent manner. This activity was blocked by the AR antagonist Casodex, showing that AR is functional. In turn, androgen decreased MAO-A expression in clorgyline-treated, secretory-like cells. Our results demonstrated that cultured basal epithelial cells have the potential to differentiate into secretory cells, and that inhibition of MAO-A is a key factor in promoting this process. Increased expression of MAO-A in high-grade prostate cancer may be an important contributor to its de-differentiated phenotype, raising the possibility that MAO-A inhibition may restore differentiation and reverse the aggressive behavior of high-grade cancer.     

Nuclear factor Nrf2 and antioxidant response element regulate NRH:quinone oxidoreductase 2 (NQO2) gene expression and antioxidant induction

Human NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic protein that catalyzes the metabolic reduction of quinones and provides protection against myelogenous hyperplasia and chemical carcinogenesis. NQO2 gene expression is induced in response to antioxidant tert-butylhydroquinone (tBHQ). Sequence analysis revealed six putative antioxidant response elements (ARE1 through 6) in the human NQO2 gene promoter. Deletion mutagenesis and transfection studies suggested that the ARE region between nucleotides -1433 and -1424 is essential for basal expression and antioxidant induction of NQO2 gene expression. Mutation of this ARE from 3.8 kb NQO2 gene promoter significantly repressed expression and abrogated the induction in response to antioxidant in transfected cells. Band shift, supershift, and chromatin immunoprecipitation (ChIP) assays demonstrated binding of nuclear factors Nrf2 and JunD with human NQO2 gene ARE. Coimmunoprecipitation experiments revealed an association between Nrf2 and JunD. Overexpression of Nrf2 upregulated and overexpression of Nrf2 dominant-negative mutant downregulated ARE-mediated NQO2 gene expression. The treatment of Hep-G2 cells with Nrf2-specific RNAi significantly reduced Nrf2 and NQO2 gene expression and tBHQ induction. The results combined demonstrated that Nrf2 associates with JunD, binds to ARE at nucleotide -1433, and regulates human NQO2 gene expression and induction in response to antioxidants.