Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Androgens down-regulate myosin light chain kinase in human prostate cancer cells

Androgens play a major role in the growth and survival of primary prostate tumors. The molecular mechanisms involved in prostate cancer progression are not fully understood but genes that are regulated by androgens clearly influence this process. We searched for new androgen-regulated genes using the Affymetrix GeneChip Human Genome U95 Set in the androgen-sensitive LNCaP prostate cancer cell line. Analysis of gene expression profiles revealed that myosin light chain kinase (MLCK) mRNA levels were markedly down-regulated by the synthetic androgen R1881. The microarray data were confirmed by ribonuclease protection assays. RNA and protein analyses revealed that LNCaP cells express both long (non-muscle) and short (smooth muscle) isoforms, and that both isoforms are down-regulated by androgens. Taken together, these data identify MLCK as a novel downstream target of the androgen signalling pathway in prostate cells.     

Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.     

A novel anti-proliferative property of clusterin in prostate cancer cells

Clusterin is a ubiquitous secretory glycoprotein that is known to suppress certain forms of apoptosis. Since apoptosis and proliferation are two opposing cellular events, it remains unclear if clusterin has any effect on cellular proliferation. The objective of the present study was to examine the effects of clusterin on proliferation in a prostate cancer cell line, LNCaP. We found that clusterin inhibited EGF-mediated proliferation in these cells, as measured by (3)H-thymidine incorporation and by cell counting. Clusterin did not bind with EGF nor did it block phosphorylation of the EGF receptor. Treatment of LNCaP cells with EGF resulted in a transient increase in the expression of both c-Fos and c-Jun. Addition of clusterin to these cultures significantly down-regulated the protein level of c-Fos, but not c-Jun. These results demonstrated a novel biological role for clusterin. Clusterin is not only anti-apoptotic but also anti-proliferative. The anti-proliferative event maybe associated with a down-regulation of c-Fos.     

Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter

Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.     

A differential ligand-mediated response of green fluorescent protein-tagged androgen receptor in living prostate cancer and non-prostate cancer cell lines.

Androgen has been shown to promote the proliferation of prostate cancer through the action of the androgen receptor (AR). Mutation (T877A) of the AR gene found in an androgen-sensitive prostate cancer cell line, LNCaP, has been postulated to be involved in hypersensitivity and loss of specificity for androgen. In the present study, trafficking of AR and AR (T877A) in living prostate and non-prostate cancer cell lines under high and low concentrations of androgen and antiandrogen was investigated by tagging green fluorescent protein (GFP) to the receptors. In the presence of a high concentration of androgen, AR-GFP localized in the nucleus by forming discrete clusters in all cell lines. AR (T877A)-GFP was also translocated to the nucleus in LNCaP and COS-1 cells by the addition of a high concentration of androgen. In contrast, in the presence of a low concentration of androgen, the translocation of AR-GFP and AR (T877A)-GFP was observed in LNCaP cells, but not in COS-1 cells. Upon the addition of antiandrogen, AR-GFP was translocated to the nucleus but did not form subnuclear foci in both COS-1 and LNCaP cells, whereas AR (T877A)-GFP in both cells was translocated to the nucleus with subnuclear foci. The present study demonstrates the differential response of nuclear trafficking of AR and its mutant in prostate cancer cell lines and COS cells, and the subcellular and subnuclear compartmentalization provide important information on the sensitivity of the AR mutation.     

The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines

The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT coVon encoding a threonine residue to GCT, encoding alanine.     

Androgens as therapy for androgen receptor-positive castration-resistant prostate cancer

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in Western countries. While surgery is often successful for organ-confined prostate cancer, androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. Shortening the period of androgen ablation therapy may benefit prostate cancer patients. Intermittent Androgen Deprivation therapy improves quality of life, reduces toxicity and medical costs, and delays disease progression in some patients. Cell culture and xenograft studies using androgen receptor (AR)-positive castration-resistant human prostate cancers cells (LNCaP, ARCaP, and PC-3 cells over-expressing AR) suggest that androgens may suppress the growth of AR-rich prostate cancer cells. Androgens cause growth inhibition and G1 cell cycle arrest in these cells by regulating c-Myc, Skp2, and p27^Kip via AR. Higher dosages of testosterone cause greater growth inhibition of relapsed tumors. Manipulating androgen/AR signaling may therefore be a potential therapy for AR-positive advanced prostate cancer.     

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.     

Lack of causal relationship between clusterin expression and photoreceptor apoptosis in light-induced retinal degeneration

Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.     

MicroRNAs are mediators of androgen action in prostate and muscle

Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3' UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues.