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Myocyte Androgen Receptor Modulates Body Composition and Metabolic ParametersFernando, Shannon M. 31 December 2010 (has links)
Androgens (such as testosterone) have been shown to increase lean body mass and reduce fat body mass in men through activation of androgen receptors (AR). While this suggests a potential clinical use for androgens, attempts at utilization of this class of hormones as a therapeutic are limited by side effects due to indiscriminate AR activation in various tissues. Thus, a greater understanding of the tissues and cells involved in promoting these changes would be beneficial. Here we show that selective overexpression of AR in muscle cells of transgenic (HSA-AR) rodents both increases lean muscle mass and significantly reduces fat mass in males. Similar effects can be induced in HSA-AR females treated with testosterone. Metabolic analyses of HSA-AR males show that these animals demonstrate increased O2 consumption and hypermetabolism. Thus, targeted activation of AR in muscle regulates body composition and metabolism, suggesting a novel target for drug development.
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Myocyte Androgen Receptor Modulates Body Composition and Metabolic ParametersFernando, Shannon M. 31 December 2010 (has links)
Androgens (such as testosterone) have been shown to increase lean body mass and reduce fat body mass in men through activation of androgen receptors (AR). While this suggests a potential clinical use for androgens, attempts at utilization of this class of hormones as a therapeutic are limited by side effects due to indiscriminate AR activation in various tissues. Thus, a greater understanding of the tissues and cells involved in promoting these changes would be beneficial. Here we show that selective overexpression of AR in muscle cells of transgenic (HSA-AR) rodents both increases lean muscle mass and significantly reduces fat mass in males. Similar effects can be induced in HSA-AR females treated with testosterone. Metabolic analyses of HSA-AR males show that these animals demonstrate increased O2 consumption and hypermetabolism. Thus, targeted activation of AR in muscle regulates body composition and metabolism, suggesting a novel target for drug development.
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Testosterone acts at the cell surface to induce granulosa/theca cell death via an apoptotic pathway in Atlantic croaker (Micropogonias undulatus)Zhang, Chenan 08 April 2014 (has links)
The teleost ovarian follicle undergoes extensive remodeling and regression during the reproductive cycle—a process involving apoptosis and cell death. However, the hormonal regulation of these processes remains unclear. In the current study the role of testosterone in regulating regression of Atlantic croaker (Micropogonias undulatus) ovarian follicles was investigated in co-cultured granulosa/theca (G/T) cells. Testosterone (T) treatment enhanced serum starvation-induced cell death and apoptosis of G/T cells during the mature stage of oocyte maturation. This effect was mimicked by a cell-impermeable T conjugate, T-bovine serum albumin, indicating that this androgen action is initiated at the cell surface. Mibolerone, a nuclear androgen receptor agonist, was ineffective in promoting apoptosis and cell death, which suggests that T actions are independent from the nuclear receptor. Together, the data suggests that T-induction of apoptosis and cell death are through a novel membrane androgen receptor in the croaker ovary. T treatment also increased expression of a pro-apoptotic member of the Bcl-2 gene family, Bax, and two Bax upstream regulators, JNK and p53. These results suggest that T induces cell death of G/T cells in croaker through the apoptotic pathway involving JNK, p53 and Bax. An opposite response of cell death protection by T was also observed in G/T cells cultured from late-stage ovaries. This response was accompanied by a rapid increased ERK-1/-2 phosphorylation not seen in Mibolerone treatment. By examining the role of T in croaker follicle cell death and elucidating the corresponding basic mechanisms of androgen action, we are learning more about the regulatory components involved in the breakdown and remodeling stages of the teleost reproductive cycle. / text
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Androgen receptors in the bonnethead shark, Sphyrna tiburo: CDNA cloning and tissue-specific expression in the male reproductive tractTyminski, John P 01 June 2007 (has links)
Androgens and the androgen receptor (AR) play important roles in virilization, spermatogenesis, and sexual behavior in vertebrates. An understanding of the distribution and levels of expression of the ARs on the cellular and tissue level demonstrates the pattern of responsiveness to the androgenic hormones in a given organism. In this study, a fragment of the AR gene was cloned and sequenced from the bonnethead shark, Sphyrna tiburo, an elasmobranch species with a well-defined annual reproductive cycle. Acquiring this gene sequence facilitated the construction of species-specific AR polymerase chain reaction (PCR) primers and species-specific AR mRNA probes that were used to screen reproductive tissues for evidence of AR gene expression using reverse transcription (RT)-PCR and in situ hybridization (ISH), respectively. The RT-PCR screens demonstrated AR gene expression in the testes, epididymides, seminal vesicles, and claspers of male sharks.
The use of relative PCR revealed that these organs have variable levels of AR gene expression that significantly differ with the stage of the shark's seasonal reproductive cycle. ISH results localized the AR RNA in the interstitial cells, Sertoli cells, and developing sperm of the testes, and mature spermatozoa within the seminal vesicles and the epididymides. Immunocytochemical methods used to detect the AR protein using a rabbit polyclonal antibody, PG-21, produced comparable results in the shark testes but did not yield positive results in the seminal vesicles or the epididymides. However, the Leydig gland, whose secretions contribute to the seminal fluid, demonstrated consistent AR immunoreactivity. Results of ICC in male and female embryos of S. tiburo revealed AR protein in the developing kidney but not in the embryonic reproductive structures.
By characterizing AR distribution in the reproductive tract of male S. tiburo, this study provides the basis for future research on the direct and indirect effects of androgenic hormones in this species.
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New insights into targeting the androgen receptor for cancer therapy: from selective delivery of gold nanoparticles and histone deacetylase inhibitors, to potent antagonists and inverse agonistsGryder, Berkley Eric 12 January 2015 (has links)
Cancer is the second leading cause of death in the United States (more than half a million people each year), and even with billions of dollars in medical effort patients are rarely cured. This dissertation research is devoted to meeting this medical need by providing new cancer therapeutics that are more potent and safer than current chemotherapies. This is achieved by using two state of the art anticancer “warheads”: 1) gold nanoparticle (AuNP) technology and 2) a new class of epigenetic anticancer small molecules, histone deacetylase inhibitors (HDACi). These warheads are then selectively delivered to cancer cells via “homing devices” targeted to receptors that are overexpressed in the cancers.
This work primarily focuses on the androgen receptor (AR) to target prostate cancer.
The 1st chapter sets the stage, providing scientific rationale and background for the central hypothesis: small molecules that engage the AR can, upon conjugation to a therapeutic agent, enable selective delivery of that agent to prostate cancer cells.
Chapter 2 delves into the structural molecular biology of the androgen receptor. There is a survey of the crystallographic data for all nuclear receptors, providing structural information which is used to build AR homology models for antagonist and inverse agonist modes of ligand binding. These models are used to design AR targeting ligands (Chapters 3, 5, 6 and 7).
The application of the targeting technology is illustrated by attaching them to AuNPs for selective delivery to prostate cancer cells (Chapter 3). Next, in order to appreciate the importance of using targeting agents in HDACi cancer therapeutics, we reviewed this recently emerged field in Chapter 4. In this chapter we argue that the failure of HDACi in solid tumors, despite more than 500 clinical trials in the last decade, is primarily due to an inability of these small molecules to accumulate at effective concentrations in the cancer. We provide an analysis of the paradigms being pursued to overcome this barrier, including HDAC isoform selectivity, localized administration, and targeting cap groups to achieve selective tissue and cell type distribution. In Chapter 5, this last approach (targeting cap groups, or a “homing device”) is illustrated with HDACi targeted to prostate cancer via antiandrogens that bind the AR. The second generation of improved “homing devices” is disclosed in Chapter 6 (for both AuNPs and HDACi), in addition to preliminary ADMET data and safety studies in mice.
Excitingly, our three dimensional understanding of binding to the AR allowed design and structure-activity-relationship studies that lead to the first reported examples of AR inverse agonists (Chapter 7)
Several points of significance:
• AuNP targeted to AR
∙ have the strongest binding affinity ever reported (IC50 ~14 picomolar)
∙ are actively recruited to prostate cancer cells
∙ overcome treatment resistance in advanced prostate cancer cells
∙ exhibit nanomolar anticancer potency
∙ resolved the identity of the “membrane AR” as the GPRC6A
• HDACi targeted to AR
∙ have HDACi activity and AR binding affinity superior to their clinical precursors
∙ exhibit potent AR antagonist activity
∙ induce AR translocation to the nucleus in a HDACi dependent fashion
∙ selectively and potently kill prostate cancer cells that express AR
∙ are safer than Tylenol®, as tested in small animals
• Pure AR binding ligand studies
∙ resulted in the discovery of the first examples of AR inverse agonists, which are vastly more potent that clinically available antiandrogens for prostate cancer
∙ work via a never-before-seen mechanism of action, by localizing to the nucleus and recruiting corepressors to actively shut off AR genes
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FUNCTION OF ANDROGEN RECEPTOR IN PROSTATE CANCER EPITHELIAL MESENCHYMAL TRANSITION AND MICROTUBULE TARGETINGZhu, Menglei 01 January 2010 (has links)
Prostate cancer is the most frequently diagnosed non-skin cancer and the third leading cause of cancer mortality among men in the US. Androgens are functionally required for the normal growth of the prostate gland and play a critical role in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development, and cancer cell metastasis.
This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells and evaluate the effect of taxol chemotherapy on androgen signaling in prostate cancer cells in prostate cancer. The EMT pattern was evaluated on the basis of expression of the epithelial markers as well as cytoskeleton reorganization in respond to DHT (1nM) and/or TGFβ (5ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. The AR transcriptional activity was determined on the basis of prostate specific antigen (PSA) mRNA expression and the androgen-response element (ARE) luciferase reporter assay. The interaction of AR and tubulin was investigated using immunoprecipitation, immunofluorescence as well as introduction of a truncated AR in human prostate cancer cells.
Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and led to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. Our study also reveals that treatment of prostate cancer cells with Paclitaxel or Nocodaxol inhibits androgen-dependent, as well as androgen-independent AR nuclear translocation and activation potentially via targeting the interaction of AR and microtubule cytoskeletal structures. Our findings on multiple aspects of AR function in prostate cancer development and progression may enhance the understanding of AR targeting therapy being a double-sided sword in the context of tumor microenvironment. These studies provide new insights into the mechanism of action of chemotherapy agents and the development of therapeutic resistance within tubulin/microtubule repertoire in prostate cancer cells.
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A Characterization of the Role of Post-translational Modification in Transcriptional Regulation by the Histone Variant H2A.ZDraker, Ryan 11 December 2012 (has links)
H2A.Z is an essential histone variant that has multiple chromosomal functions. One such role is transcriptional regulation. However, its role in this process is complex since it has been reported to function both as a repressor and activator. Earlier work in our lab showed that H2A.Z can be post-translationally modified with monoubiquitin (H2A.Zub1) and this form of H2A.Z is linked to transcriptional silencing. We further predicted that changes in the H2A.Z ubiquitylation status directly modulated its function in transcription. Furthermore, H2A.Z-containing nucleosomes possess a unique set of post-translational modifications (PTMs), compared to H2A nucleosomes, many of which are linked to transcriptional activation. The central aim of this thesis was to characterize the role of PTMs on H2A.Z nucleosomes in transcriptional regulation. To this end, I have provided the first evidence linking H2A.Z deubiquitylation to transcriptional activation. I demonstrated that ubiquitin specific protease 10 (USP10) is a deubiquitylase that targets H2A.Z in vitro and in vivo. Moreover, I found that both H2A.Z and USP10 are required for activation of androgen-receptor (AR)-regulated genes, and that USP10 regulates the levels of H2A.Zub1 at these genes. To understand how H2A.Z engages downstream effector proteins, in the nucleosome context, we used a mass spectrometry approach to identify H2A.Z-nucleosome-interacting proteins. Many of the identified proteins contained conserved structural motifs that bind post-translationally modified histones. For example, we found that Brd2 contains tandem bromodomains that engage H2A.Z nucleosomes through acetylated H4 residues. To investigate the biological relevance of this interaction, I present evidence that Brd2 is recruited to AR-regulated genes in a manner dependent on H2A.Z and the bromodomains of Brd2. Consistent with this observation, chemical inhibition of Brd2 recruitment greatly inhibited AR-regulated gene expression. Collectively, these studies have defined how H2A.Z mediates transcriptional regulation through multiple mechanisms and pathways.
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Development of androgen receptor messenger RNA targeted molecular beacons for use in the study of prostate cancer progressionGlick, Cindy Jennifer 31 July 2008 (has links)
Messenger RNA (mRNA) posttranscriptional regulation has been implicated in the development and/or progression of several diseases including many types of cancer, rheumatoid arthritis, vascular disease, and Alzheimer's disease. Differential regulation of Androgen Receptor (AR) mRNA has been associated specifically with prostate cancer progression. In this thesis, molecular beacons were developed to allow for the detection of the expression and localization of AR mRNA in live prostate cancer cells. These beacons were then applied as a tool for studying how AR mRNA regulation is involved in prostate cancer growth and advancement. Two AR mRNA targeted beacons were designed and tested in solution and in live cells to determine their functionality. The beacon-based approach for AR mRNA detection was then optimized through the use of the two beacons in tandem and alteration of their backbone chemistry. A series of validation tests were performed on these beacons, including testing their abilities to: 1) produce a feasible localization pattern, 2) discriminate between AR positive (AR+) and AR negative (AR-) prostate cancer cell lines and 3) follow stimulus-induced changes in AR mRNA expression. Based on these results, a dual chimeric beacon approach was selected to determine the role of AR mRNA regulation in two systems that represent important stages in prostate cancer growth and progression: 1) hormone stimulation of androgen-dependent prostate cancer cells and 2) progression of androgen-dependent prostate cancer cells to the androgen-independent state. Our results suggest that changes in AR mRNA expression, organization, and localization may be indicative of molecular mechanisms involved in these critical transitions associated with prostate cancer progression. Taken together, this work provides a feasibility study for visualizing changes in AR mRNA state as a diagnostic measure for evaluating the aggressiveness of the disease and demonstrates the possible utility of therapeutically targeting AR mRNA regulation in order to prevent prostate cancer advancement.
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Androgen signalling in normal and malignant breast epithelial cells.Peters, Amelia Alice January 2008 (has links)
The growth and survival of normal breast epithelial cells and breast cancer cells is promoted by estrogens. In contrast, androgens inhibit the proliferation of normal and malignant breast epithelial cells. While this effect of androgens on breast cells appears to be androgen receptor (AR) dependent, the precise mechanism of inhibition and its functional significance are unknown. The aims of this thesis were to investigate the effect of androgen signalling on growth of normal and malignant breast epithelial cells, and to assess the interactions between androgen and estrogen signalling in the breast. To investigate the role of androgen signalling in the growth and development of the normal mammary gland, female mice were treated with either the native androgen 5α- dihydrotestosterone (DHT) or the antiandrogen, flutamide. Analysis of the mammary glands at the end of the treatment period demonstrated that DHT reduced ductal branching and mammary epithelial cell proliferation when treatment commenced mid-puberty. Conversely, flutamide treatment that commenced post-puberty significantly increased ductal branching and proliferation of mammary epithelial cells. This data demonstrates that androgen signalling inhibits proliferation in the normal mammary gland, and may therefore oppose to the growth stimulatory effects of estrogen signalling to regulate breast growth and development. The antiproliferative effects of androgens on breast epithelial cells may be due in part to direct AR-mediated activation of androgen regulated genes, or alternatively, androgens could act indirectly through AR to inhibit estrogen receptor alpha (ERα) activity. Expression of fulllength AR or a truncated, constitutively active AR (AR-T707) significantly inhibited the activity of ectopically expressed ERα in MDA-MB-231 breast cancer cells (ERα- and ARnegative), in a dose-dependent manner. The functional consequences of inhibition of estrogen signalling by overexpressing AR were investigated in the T-47D breast cancer cell line (ERα- and AR-positive). Expression of AR-T707 in T-47D cells resulted in inhibition of both basal and estradiol-induced cell proliferation and a marked reduction in the steady-state protein levels of the estrogen regulated gene, PR. The final chapter investigated the mechanism by which AR inhibits ERα activity. A coimmunoprecipitation assay demonstrated an interaction between ectopically expressed AR and ERα in COS-1 cells, but not endogenous AR and ERα in a breast cancer cell line. To delineate the regions of AR required for inhibition of ERα signalling, various functional domains of the AR were mutated or deleted. Reporter gene assays showed that the inhibitory effects of AR were abrogated by deletion or mutation of the DNA binding domain (DBD). Furthermore, overexpression of the AR-DBD alone was sufficient to inhibit ERα activity. Consistent with a requirement for the DBD of AR to inhibit ERα activity, mobility shift assays demonstrated binding of AR to the Xenopus vitellogenin A2 consensus estrogen response element (cERE); however AR/ERα heterodimers were not detected on a cERE. Consistent with these findings, molecular modelling demonstrated that it is feasible for the DBD of AR to bind to a cERE and that it is unlikely that AR/ERα heterodimers could bind. Chromatin immunoprecipitation demonstrated recruitment of AR to the promoters of endogenous estrogen regulated genes. The findings suggest that the inhibitory effect of AR on ERα activity may occur either via formation of non-functional AR/ERα heterodimers that are unable to bind to EREs, or AR homodimers competing effectively for binding to EREs, in ERα target genes. The results in this thesis demonstrate an inhibitory effect of androgen signalling on growth of normal and malignant breast epithelial cells. Additionally, the inhibition of breast epithelial cell proliferation by androgen signalling can be attributed, at least in part, to inhibition of ERα activity. These studies have provided insight into androgen action in the breast, and support a model whereby androgens balance the stimulatory effects of estrogen signalling in normal and malignant breast epithelial cells. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008
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Over-Expression of Aryl Hydrocarbon Receptor (AhR) Enhances Src Kinase Activity to Functionally Induce AR Signaling and Promote Prostate Cancer ProgressionGhotbaddini, Maryam 21 May 2018 (has links)
The aryl hydrocarbon receptor (AhR) has been reported to interact with multiple signaling pathways during prostate development including the androgen receptor. AhR was overexpressed in LNCaP using PLNCX2 retrovirus vector containing AhR cDNA to determine if ectopic overexpression induces castrate resistant phenotype. The highly overexpressed AhR clone illustrated further increase in transcriptional and promotor activity for AhR and AR compared to the moderately overexpressed AhR clone and control. Western blot analysis showed more AhR, AR, cSrc, and pSrc protein expression in clones. AhR overexpression was found to induce several biological properties such as migration, invasion, proliferation, and promotion of G1 to S phase during the cell cycle. Bicalutamide treatment had no effect on AR transcriptional activity in either clone, proving resistance to anti-androgen therapy. Our results confirm that overexpression of AhR induces constitutive activity and stimulates androgen receptor signaling. This suggests a role for AhR in the development of CRPC.
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