Up-regulation of the levels of androgen receptor and its mRNA by androgens in smooth-muscle cells from rat penis

Smooth-muscle cells cultured from the penis of sexually immature (I-PSMC) and adult (A-PSMC) rats express similar high levels of the androgen receptor (AR) mRNA. This contrasts with the marked in vivo decline of both AR mRNA and androgen binding in the penile smooth muscle of adult rats, which appears to be responsible for the cessation of androgen-dependent penile growth upon sexual maturation. PSMC is therefore a good model to study down-regulators of AR expression as a function of cell proliferation in the smooth muscle of androgen-reputative sponsive vascular tissue. In order to determine whether AR protein levels in PSMC correlate with AR mRNA levels, the immunocytochemical detection of ARs and their androgen binding capacity were compared between I- and A-PSMC. The number of ARs and their protein half-lives suggested similar levels of translation of the AR mRNA in both cell lines. The effect of the synthetic analog methyltrienolone (R-1881) on androgen binding was studied in contact-inhibited androgen-deprived PSMC. In contrast to the postulated role of androgens as down-regulators of AR expression in rat penis, ARs were up-regulated in A-PSMC by R-1881. Contact inhibition of A-PSMC combined with serum depletion and androgen deprivation down-regulated AR mRNA levels, and dihydrotestosterone (DHT) counteracted this effect. These results suggest that the loss in A-PSMC of the age-dependent down-regulation of ARs observed in vivo in adult corpora cavernosa smooth muscle is related to the in vitro resumption of cell proliferation and that DHT acts directly on the penile smooth muscle as a positive modulator of AR levels.     

雄激素和雄激素受体对肝癌细胞株PEG10表达的作用

目的 探讨雄激素和雄激素受体(AR)对肝癌细胞株PEG10表达的调控作用.方法 设计合成针对人ARsiRNA,并转染HepG2和7404肝癌细胞株.用双氢睾丸酮(DHT)干预HepG2细胞.Western Blot检测AR和PEG10表达水平.结果 从3对AR siRNA中筛选到1对siRNA(AR siRNA-3),它在2种肝癌细胞株中均可有效抑制AR的表达,其抑制作用呈剂量依赖关系.2种肝癌细胞株中,浓度为240 nmol/L的AR siRNA-3在转染后24 h,对AR抑制效率可达80%以上,且抑制效果可持续至72 h.AR siRNA-3转染24h后PEG10表达水平降低,转染48 h后,PEG10表达水平降低非常明显,72 h后PEG10表达有所上升.DHT可促进HepG2细胞PEG10的表达,呈剂量依赖关系.DHT对AR表达未见明显作用.结论 雄激素和AR参与了肝癌细胞株PEG10表达的调控.这可能是男性肝细胞癌发病率较高的原因之一.     

雄激素受体基因新突变致雄激素不敏感综合征

目的 分析2例雄激素不敏感综合征患者及其家系的临床及分子遗传学.方法 收集2例雄激素小敏感综合征患者的临床资料,从患者及其家系成员的外周血单个核细胞抽提基因组DNA,应用PCR扩增雄激素受体基因并直接测序,明确患者及其父母基因有无突变.结果 患者1表现为女性外生殖器、单侧乳房发育、原发性闭经、阴毛腋毛缺如.患者2表现为男性化不全,体毛稀少、双侧乳房发育、尿道下裂.基因检测证实患者1雄激素受体基因第2号外显子第579位密码子点突变(S579N),并证实为一新突变.患者2第5号外显子第747位密码子点突变(V747M).结论 该2例雄激素受体不敏感综合征系分别由雄激素受体基因S579N及V747M所致,其中S579N突变尚未见文献报道.     

Synthesis and post-translational modification of the androgen receptor in LNCaP cells

Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting with a specific polyclonal antibody showed that the androgen receptor migrated as a closely spaced 110–112 kDa doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Most of the receptor protein is present in the higher molecular mass form. Pulse labelling experiments with [³⁵S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Alkaline phosphatase treatment of cytosols from [³⁵S]methionine pulse labelled cells caused a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform. This indicates that the observed 110 to 112 kDa upshift of the newly synthesized androgen receptor reflects receptor phosphorylation. Both isoforms can bind hormone and can undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.     

再生障碍性贫血雄激素受体与T细胞亚群的变化

目的:检测慢性再生障碍性贫血(CAA)患者骨髓单个核细胞(MNC)雄激素受体(AR)以及骨髓中T细胞亚群水平,探讨AR在CAA免疫病理机制中的作用。方法:①免疫细胞化学SP法检测CAA患者骨髓MNC内AR的表达水平;②流式细胞术检测CAA骨髓中T细胞亚群(CD3 CD4 细胞、CD3 CD8 细胞)的含量。结果:①CAA患者骨髓MNC中AR阳性水平[(35．18±8．78)个／200个MNC]显著低于对照组[(48．46±9．82)个／200个MNC];不同性别间AR的含量差异无统计学意义。②CAA患者骨髓中的CD3 CD8 细胞含量为(28．54±7．57)％,显著高于对照组。③CAA患者骨髓中的AR阳性水平与骨髓中的CD3 CD8 细胞呈负相关(r=-0．576,P<0．01);而与CD3 CD4 细胞含量未见明显的直线相关关系。结论:CAA患者骨髓AR的表达减少,从而使雄激素刺激造血的作用减弱。患者AR的表达与CD3 CD8 细胞含量呈明显负相关,表明AR的异常可能在一定程度上参与了CAA发病机制中的细胞免疫。     

Tandem CAG repeats of the androgen receptor gene and prostate cancer risk in black and white men

The most common malignancy in men worldwide is cancer of the prostate. Androgens play a direct role in normal and malignant growth of prostate cells via the androgen receptor (AR). This study analyzed the polymorphic CAG repeat sequence in exon 1 of the AR gene to determine if the number of repeats might be an indicator of prostate cancer risk or aggressive disease. DNA was extracted from blood samples of 20 black and 20 white men with well-documented prostate cancer and 40 healthy, controls (20 blacks and 20 whites). PCR amplifications was followed by gel electrophoresis and DNA sequencing. This region normally contains between 9 and 29 repeats. Patients and controls both had minor variations in the number of repeats, which ranged from 13 to 27 with 21 being the most frequent allele. Black controls and patients both had a mean of 20±3, repeats; in whites the mean was significantly lower in patients than controls (21±2 versus 23±2; p=0.004). Combined black and white patients also had a lower number than the combined group of controls (20±3 versus 22±3; p=0.02). Similarly, black and white patients with aggressive disease has a lower number than patients whose disease was more slowly progressive (19±2 versus 22±3; p=0.02). We conclude that the small differences in the number of CAG repeats in both black and white patients do not appear to be a strong indicator of risk or aggressive disease but that this size polymorphism may be one of many genetic and environmental risk factors involved in prostate cancer.     

The androgen receptor of the human endometrium

Available androgen binding to soluble proteins from the cytosol of human endometrium was studied using the dextran coated charcoal adsorption method and sucrose density centrifugation analysis. Specific binding of [3H]-5 alpha-dihydrotestosterone ([3H]-DHT) was observed with both methods. The apparent dissociation constant (Kd), for DHT binding is 1.3 +/- 0.2 (SEM) nM and the binding capacity 177 +/- 42 (SEM) fmol/mg protein. Sucrose density ultracentrifugation identifies specific [3H]-DHT binding that sediments at 4S and 8S. The stability of the androgen receptor in human endometrium is increased by the addition of 10% glycerol to the homogenization buffer. The addition of trypsin or pronase and heating at 60 degrees C reduces specific binding which demonstrates that the specific [3H]-DHT binder is a protein. The uptake of [3H] DHT in endometrial tissue minces indicated that 20% of the bound radioactivity was nuclear. Steroid specificity suggests that the binding protein from the uterus is specific for androgens. These observations indicate that androgen binding protein in the human uterus has the characteristics of the androgen receptor.     

Interaction of androgen and glucocorticoid receptor DNA-binding domains with their response elements

Fusion proteins containing the glucocorticoid and the androgen receptor DNA-binding domain (ARF1 and GRF1) were produced in Escherichia coli. DNAse I footprinting was used to compare the interaction of these proteins with responsive elements (REs) in a typically glucocorticoid-responsive gene (mouse mammary tumour virus (MMTV)) and in an androgen-responsive gene (the C3(l) gene of rat prostatic binding protein). It is demonstrated that response elements which most closely resemble the consensus sequence show identical footprinting patterns for ARF1 and GRF1. The protected regions suggest that these sequences are occupied by two DNA-binding domains (DBDs) forming a dimer. Regions that constitute imperfect RE sequences, however, are apparently recognized by only one DBD, which mainly protects the TGTTCT motif. At these REs, the protection patterns produced by ARF1 and GRF1 are not identical. In the long terminal repeat (LTR) of MMTV but not in C3(1), a mechanism other than classical dimer formation seems to increase the affinity of ARF1 and GFR1 for these imperfect REs.     

Infertility with defective spermatogenesis and steroidogenesis in male mice lacking androgen receptor in Leydig cells

Androgen and the androgen receptor (AR) have been shown to play critical roles in male fertility. Our previous data demonstrated that mice lacking AR (AR^−/y) revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in Leydig cells remain largely unknown. Using a Cre-LoxP conditional knockout strategy, we generated a tissue-specific knockout mouse (L-AR^−/y) with the AR gene deleted by the anti-Müllerian hormone receptor-2 (Amhr2) promoter driven Cre expressed in Leydig cells. Phenotype analyses show that the outside appearance of L-AR^−/y mice was indistinguishable from wild type mice (AR^+/y), but with atrophied testes and epididymis. L-AR^−/y mice were infertile, with spermatogenic arrest predominately at the round spermatid stage and no sperm could be detected in the epididymis. L-AR^−/y mice also have lower serum testosterone concentrations and higher serum leuteinizing hormone and follicle-stimulating hormone concentrations than AR^+/y mice. Further mechanistic studies demonstrated that hypotestosteronemia in L-AR^−/y mice is not caused by reducing numbers of Leydig cells, but instead by the alterations of several key steroidogenic enzymes, including 17β-HSD3, 3β-HSD6, and P450c17. Together, L-AR^−/y mice provide in vivo evidence that functional AR in Leydig cells is essential to maintain normal spermatogenesis, testosterone production, and required for normal male fertility. Qingquan Xu, Hung-Yun Lin, and Shauh-Der Yeh contributed equally to this study.     

Combined profile of the tandem repeats CAG, TA and CA of the androgen and estrogen receptor genes in breast cancer

Purpose Case–control studies have reported inconsistent results concerning the association between polymorphisms in the androgen and estrogen receptor (ER) genes and breast cancer. While several studies investigated the association between the androgen receptor (AR) gene, CAG repeat and breast cancer, for the CA and TA repeats in the ER genes there are considerably fewer studies (one for CA and none for TA).Methods We have investigated the potential link between three tandem repeats (CAG, TA, and CA) in the AR, ERs α and β genes, respectively, and breast cancer. DNA was isolated from 153 invasive breast tumors and 318 controls, and the three tandem repeats were sized by polyacrylamide electrophoresis. Number of repeats in each allele and the total repeats of both alleles were taken as variables for classification into dichotomous groups using the median of each variable in the control group as cut-off point. Relationship between polymorphic tandem repeats and breast cancer was assessed by multivariate logistic regression models.Results Three variables combined, longer CAGsum (≥28), shorter TA (<23) and CA (<23) repeats could constitute a possible genetic profile associated with breast cancer.Conclusions Our results confirm previous reports regarding an association between longer CAG repeats and breast cancer. In addition to that, we found that the combination of long CAG, short TA and CA repeats are strongly associated with breast cancer.This paper was initially submitted with an additional co-author who withdrew the co-authorship during the review process. All listed authors have now signed that they agree with the content of the current paper.     

人类表皮生长因子受体2蛋白及雄激素受体在前列腺癌中的表达及其意义

目的 检测Her-2/neu蛋白和雄激素受体(androgen receptor,AR)在前列腺癌组织中的表达情况,探讨其在前列腺癌发生发展中的意义.方法 构建前列腺病变的组织芯片,其中包括前列腺癌107例(Gleason评分6分29例,7分20例,8分46例,9分12例),良性前列腺组织42例;采用EnVsion两步法进行Her-2/neu蛋白和AR的免疫组织化学染色,分析其在前列腺癌及良性前列腺组织中的差异;从免疫组织化学染色强度及阳性细胞数两个方面评价蛋白表达情况,分析其与前列腺癌Gleason评分的关系.结果 Her-2/neu蛋白在前列腺癌组织中的阳性表达率为43.9%,高于在良性前列腺组织中的阳性表达率(14.3%)(x2=11.562,P=0.009),其阳性表达强度前列腺癌高于良性前列腺组织(x2=11.764,P=0.008).在不同Gleason评分组中,Her-2/neu蛋白的阳性表达强度差异有统计学意义(x2=20.512,P=0.015),且与Gleason评分呈正相关(r=0.269,P=0.005).前列腺癌组织AR阳性表达率(67%)高于良性前列腺组织(50%)(x2=3.843,P=0.050),但其阳性表达强度在前列腺癌及良性前列腺组织中的差异无统计学意义(x2=4.318,P=0.229).在不同Gleason评分组中AR的阳性表达强度差异无统计学意义(x2=13.385,P=0.146),与Gleason评分无相关性(r=-0.065,P=0.505).前列腺癌组织中Her-2/neu蛋白和AR的阳性表达强度无相关性(r=-0.115,P=0.237).结论Her-2/neu蛋白在前列腺癌中的高表达提示其可能在前列腺癌发生中起一定作用.Her-2/neu蛋白的阳性表达强度与Gleason评分呈正相关,提示Her-2/neu蛋白与前列腺癌的预后有一定的相关性.

Abstract：

Objective To observe the expression of Her-2/neu protein and androgen receptor (AR) in human prostate cancer and to evaluate their significances in the progression of prostate cancer. Methods The Her-2/neu protein and AR immunohistochemical stain were carried out in human prostate tissue microarray that consisted of prostate cancer (107 cases) and benign prostate tissue (42 cases). The prostate cancer cases were divided into 4 groups: group one (Gleason score 6),group two (Gleasonscore 7), group three (Gleasonscore 8) and group four (Gleasonscore 9) according to the Gleason score. The immunostains immunohistochemical stain were interpreted in two aspects of the staining intensity and the percentage of positive cells. The significance and relationships between the expression of Her-2/neu protein and AR in prostate cancer and benign prostate tissue (BPT) and the grouping of different Gleason scores of prostate cancer were then evaluated. Results The positive expression rate of Her-2/neu protein was significantly higher in prostate cancer tissue than in BPT [43.9%(47/107) vs. 14.3%(6/42), x2=11.562, P=0.009], and the positive expression intensity of Her-2/neu immunoreactivity was also higher (x2= 11.764, P=0.008). There were significant differences in positive expression intensity of Her-2/neu immunoreactivity among the different Gleason scores groups (x2 = 20. 512, P = 0. 015), and the expression intensity was significantly positively correlated with Gleason scores ( r= 0. 269, P = 0. 005). There was significant difference in AR immunoreactivity between in prostate cancer (67 %, 72/107) and in BPT (50 %, 21/42, x2 =3. 843, P=0. 050). Among prostate cancer cases, the positive expression intensity of AR was not significantly different among groups 1 through 4 (x2 = 4. 318, P = 0. 229), and was not significantly correlated with Gleason scores ( r = - 0. 065, P = 0. 505 ). Moreover, the positive expression intensity of Her-2/neu protein was not significantly correlated with that of AR (r = -0. 115, P=0. 237). Conclusions Overexpression of Her-2/neu protein in human prostate cancer tissue suggests that Her-2/neu may have some role in prostate tumorigenesis. Her-2/neu protein expression is positively correlated with Gleason score in prostate cancer, which suggests that Her-2/neu may be a potential prognostic predictor of prostate cancer.     

Correlation study between the polymorphism of repetitive sequence in gene CAG of androgen receptor and the occurrence and progression of prostate cancer

Objective:To explore the relation between the polymorphism of repetitive sequence in gene CAG of androgen receptor(AR)and the susceptibility and clinical stages as well as pathological grading of prostate cancer among Han population.Method:Sixty-eight cases with prostate cancer hospitalized in Urinary Surgery Department from Feb.2010 to Feb.2012 and 60 healthy cases were chosen as research subjects.Methods of PCR and direct sequencing were adopted to detect DNA sequence of AR gene and the length of repetitive sequence in CAG.Results:The lengths of repetitive sequence in CAG of patients with prostate cancer and healthy people were(22.3±4.6)and(23.0±4.9),respectively showing no statistical significance.Comparing length(repetitive sequence of CAG)22,those with that22 suffer a remarkably higher risk of prostate cancer(P0.05).The number of repetitive sequence in CAG of patients at clinical stage C-D was less than that of patients at stage B,and the number of repetitive sequence in CAG of patients with poorly differentiated prostate cancer was also less than that of patients with moderately and highly differentiated prostate cancer.But there was no statistical significance int the difference(P0.05);the proportion of patients with length22 at clinical stage C-D was much larger than that of patients at clinical stage B(P0.05),and as the aggravation of pathological grading,the proportion of patients with the length22 was also remarkably increased and there was significant difference between patients with highly differentiated prostate cancer and those with poorly differentiated prostate cancer(P0.05).Conclusions:There is correlation between the occurrence and development of prostate cancer in Han population and the polymorphism of repetitive sequence in gene CAG of androgen receptor.The less the number of repetitive sequence in CAG is,the higher the risk of prostate cancer will be and the more severe the clinical stage and pathological grading will be.     

Changes in androgen receptor mRNA expression in the forebrain and oviduct during the reproductive cycle of female leopard geckos,Eublepharis macularius

Successful reproduction requires the coordination of reproductive physiology with behavior. The neural correlates of reproductive behavior have been elucidated in a variety of amphibians, mammals, and birds but relatively few studies have examined reptiles. Here we investigate differences in androgen receptor (AR) mRNA expression in the forebrain and oviduct between previtellogenic and late vitellogenic female leopard geckos, Eublepharis macularius. Plasma concentrations of testosterone (T) are low when females are previtellogenic and sexually unreceptive but increase dramatically during late vitellogenesis when females are receptive. In addition, receptivity can be induced by treatment with exogenous T. The relative abundance of AR-mRNA across various nuclei was greater in late vitellogenic than in previtellogenic females. This general pattern was observed in the medial preoptic area, anterior hypothalamus, external nucleus of the amygdala, dorsolateral aspect of the ventromedial hypothalamus, lateral septum, and periventricular hypothalamus. There were also clear differences in AR-mRNA expression among these nuclei. The pattern of gene expression observed in the brain was reversed within stromal cells of the oviduct where expression of AR-mRNA decreased from the previtellogenic stage to the late vitellogenic stage. Overall, these data demonstrate that T concentration in the plasma, abundance of AR-mRNA in the brain and oviduct, and sexual behavior change coordinately during the reproductive cycle of female leopard geckos. Although the function of AR in the female leopard gecko is not yet clear, our results are in accord with growing evidence that androgens regulate numerous aspects of female physiology and behavior in vertebrates.