During natural vision the entire retina is stimulated. Likewise, during natural tactile behaviors, spatially extensive regions of the somatosensory surface are co-activated. The large spatial extent of naturalistic stimulation means that surround suppression, a phenomenon whose neural mechanisms remain a matter of debate, must arise during natural behavior. To identify common neural motifs that might instantiate surround suppression across modalities, we review models of surround suppression and compare the evidence supporting the competing ideas that surround suppression has either cortical or sub-cortical origins in visual and barrel cortex. In the visual system there is general agreement lateral inhibitory mechanisms contribute to surround suppression, but little direct experimental evidence that intracortical inhibition plays a major role. Two intracellular recording studies of V1, one using naturalistic stimuli (Haider et al.,  In retinal ganglion cells, the response to a small stimulus of the appropriate luminance polarity (light or dark) positioned in the center portion of the receptive field is larger than the response evoked by a larger stimulus of the same polarity extending beyond the center. Similarly, in the rodent whisker system, single whisker stimulation typically evokes larger responses than stimulation of two or more whiskers. At first glance, results like these seem puzzling because they suggest that spatially extensive and therefore more naturalistic stimuli that engage the entire eye or multiple whiskers are somehow less effective at driving sensory responses than spatially restricted stimuli.phenomenon of suppression can be driven by a number of different mechanisms including inhibition, but also withdrawal of excitation or even changes in intrinsic cell properties , presumably by presynaptic GABA release. This distinction is critical because the Note that increased response sparseness is often associated with a reduction in spiking activity, either driven or spontaneous, although this is not necessarily the case  stimuli that elicit surround suppression should increase inhibition, i.e., engaging the surround should lead to an increase in the amplitude or frequency of inhibitory post-synaptic potentials, (2) inhibitory interneurons should show surround facilitation, not surround suppression, (3) inhibitory interneurons should have larger spatial receptive fields than their target pyramidal neurons, and (4) excitatory pyramidal cells with receptive field centers near the edges of the stimuli, that would provide the excitatory drive to local interneurons, should exhibit elevated firing rates.Haider and colleagues  measuredOzeki and colleagues  also meaOne potentially important difference between these two studies is the spectrotemporal content of the stimuli. Haider and colleagues  used broBoth of these studies, however, support the idea that surround suppression in V1 is not a purely thalamocortical mechanism, but rather one that can depend on local intracortical inhibition. Both studies provide evidence that when spatially extended stimuli engage the surround region, there is either a sustained or transient increase in intracortical inhibition that ultimately leads to a reduction in firing rate and, in the case of naturalistic stimulation, an increase in response sparseness. The specific origin of the long-range excitatory signals that drive inhibitory interneurons during surround stimulation has yet to be nailed down. These signals could originate within V1, perhaps even from specific laminae and cell types, or they could originate from extrastriate areas. Additional experiments are required to resolve these questions.The rodent somatosensory vibrissal system is very different from the visual system. Moreover, the manner in which these two systems have been studied to date is also very different. First, in the vibrissal system, the representation of the sensory space is discrete and organized around individual whiskers, from the periphery through primary somatosensory cortex. The visual system, with its dramatically higher receptor count, instantiates a more continuous representation of the visual environment at the periphery but discrete sampling and columnar organization does not arise until V1. Second, in the vibrissal system, there is no established notion of classical and non-classical or surround receptive fields. The nCRF, where stimuli that evoke no increase or decrease in firing rate but interact with stimuli delivered to the center receptive field, is rarely studied. The most commonly investigated surround interactions in the vibrissal system are responses to a center whisker and the adjacent surround whiskers, where stimulation of either the center or a surround whisker can evoke increases in firing rate. In contrast to visual cortex, where the CRF is defined as the retinal area where stimuli of the right orientation, size, and luminance can elicit or suppress spikes, in barrel cortex the center receptive field is defined by the single whisker that elicits the shortest latency, largest magnitude response. The surround receptive field includes all the whiskers that evoke longer latency, weaker responses. Aside from the trigeminal ganglion cells, which are directly connected to a single whisker follicle, virtually all neurons in the vibrissal system, from brainstem to barrel cortex, respond to individual stimulation of more than one whisker.Another major difference between the visual and whisker system is that we have a reasonably well-established intuitive, idea of what constitutes a natural visual stimulus, while the same cannot be said for whisker stimulation. The natural, and therefore ethologically relevant, stimulus set could be single whiskers making discrete contacts during object localization, multiple whiskers making synchronous or asynchronous contact, high frequency slip-stick synchronous or asynchronous movements, or active or passive movement of single or multiple whiskers . These neurons primarily target layer IV barrel cells  shrink to a single whisker after lesions of the spinal trigeminal nucleus, indicating that surround receptive fields are constructed in the brainstem and relayed to thalamus  whiskers. Though a few studies show that paired whisker stimulation can evoke facilitation  all contribute to surround suppression. There are several potential differences between experiments that support intracortical inhibition . Biological systems must balance the potential gains obtained from sparse coding against the cost of building more neurons.A general rule for both, or even all, sensory systems may be that the spatial and temporal dynamics of natural sensory stimulation invariably activate both the center and surround components of the receptive field in most sensory neurons. The lateral interactions, at many synaptic levels, engaged by spatially extensive stimuli are likely to increase the sparseness of the neural code and increase selectivity in sensory cortex.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite several studies in recombinant systems and insightfrom genetically modified mice, our understanding of the physiological relevance and specificity of the α1-AR subtypes is still limited. Constitutive activity and receptor oligomerization have emerged as potential features regulating receptor function. Another recent paradigm is that βarrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. The aim of this review is to summarize our current knowledge on some recently identified functional paradigms and signaling networks that might help to elucidate the functional diversity of the α1-AR subtypes in various organs.The α The AR family includes nine different gene products, three β . Activation of the three α1-AR subtypes causes polyphosphoinositide hydrolysis catalyzed by phospholipase C (PLC) via pertussis toxin-insensitive G proteins in most tissues where this effect has been examined  was a novel receptor subtype not clearly identified by previous pharmacological studies  ligand-binding; (ii) receptor activation and coupling to G protein; and (iii) desensitization. These findings have been reviewed elsewhere  and of A271(6.34) in the cytosolic extension of helix 6 in the α1b-AR and α1a-AR, respectively ((3.49) and D123(3.49) of the E/DRY motif in the α1b-AR and α1a-AR, respectively ectively .1a and α1b-AR in recombinant systems measuring the inositol phosphate response. The agonist-independent activity of both the wild type α1b-AR and its constitutively active mutants was significantly higher than that of the wild type α1a-AR or its mutant. In contrast, the epinephrine-induced inositol phosphate accumulation above basal at the α1a-AR was significantly higher than that at the α1b-AR or its constitutively active mutants expressed at comparable levels  or bioluminescence resonance energy transfer (BRET) between GPCRs carrying the appropriate pair of fluorescent/bioluminescent labels suggested oligomerization of a variety of GPCRs. Each technique employed has its own shortcomings: whereas co-immunoprecipitation cannot rule out indirect interaction, energy transfer techniques can only certify that the two partners are in close proximity, not necessarily in immediate contact. However, convergent results obtained through independent methods eventually led to the widespread acknowledgment of the existence of GPCR oligomers .1-AR subtypes in recom-binant systems   upreagulation of contractile proteins (myosin light chain-2) and reactivation of embryonic genes , β-myosin heavy chain, skeletal α-actin).Most of the work investigating α studies ,25 demon1-AR-induced hypertrophic response in cardiomyocytes. A recent study supports these earlier findings suggesting that α1-AR-induced cardiac hypertrophy is mediated by three parallel pathways: G12/13-Rho-JNK, Gq-JNK (Rho-independent) and Gβγ (JNK independent)  . Recent ertrophy .1-AR subtypes expressed in heart, the α1a and α1b-AR, does not emerge clearly from the in vitro studies published so far probably because of the limited selectivity of the pharmacological tools available. In one study on rat neonatal cardiomyocytes, a constitutively active form of the α1a-AR activated gene expression of the ANF, whereas the analogous constitutively active mutant of the α1b-AR stimulated gene expression of c-fos, but not of ANF (1b-AR in transgenic mice resulted in a marked increase in ANF (see below). In the future, it would be interesting to carry on a systematic investigation of different signaling pathways comparing the α1-AR subtypes expressed in the same cellular systems and to correlate these findings with the growing information provided by in vivo studies on genetically modified mice (see below).The respective role in stimulating cardiac hypertrophy of the two αt of ANF . However1-AR subtypes display quite divergent regulatory properties. In fact, the α1b-AR in recombinant systems undergoes rapid phospohorylation, desensitization and endocytosis upon exposure to the agonist  as a potential α1a-AR interacting protein , a zinc-finger matrix metal-loprotease of the astacin family, interacted with αd screen . Overexp1a-AR  and the third intracellular loop of the α1a-AR . RGS proeceptors  and it i1b-AR: the μ2 (or AP50) subunit of the clathrin adaptor complex AP2  as well as in anchoring of protein kinase A. Therefore, it would be interesting to test whether ezrin is also involved in ρ-signaling mediated by the α1b-AR.In addition to AP50, the same yeast two-hybrid screen identified ezrin as a potentially direct binding partner of the α1b-AR . Ezrin b1b and the α1d-AR , was reported to interact with the same arginine-rich sequence in the αe α1d-AR . gC1qR i1b-AR is spinophilin which interacts with other GPCRs, including the α2-AR, as well as with the N-terminal domain of RGS proteins  which participates in GPCR recognition  mouse was the first model to be created (1bKO mice displayed: (i) decreased blood pressure response to phenylephrine with normal resting pressure  hypressure ; and  (1d-AR in large conducting arteries (aorta and carotid)   and the 1-AR in cardiac function and hypertrophy. As mentioned above, the α1a and α1b-AR subtypes are both expressed in cardiomyo-cytes with the α1a predominating in humans and the α1b in rodents. Transgenic mice overexpressing a constitutively active α1b-AR mutant specifically in the heart display cardiac hypertrophy without any change in blood pressure  all three α1-AR subtypes contribute to the regulation of blood pressure with a prominent role for the α1a and α1d; (b) both the α1a and α1b-AR play a role in cardiac pathological hypertrophy (independent from pressure overload) or physiological hypertrophy associated with postnatal growth; and (c) the α1-ARs maintain normal heart function as demonstrated by the fact that the double deletion of the α1a and α1b-AR results in some features of failing heart.In conclusion, as summarized in 1-AR subtypes have emerged from studies on the genetically modified mice including their effects on heart contractile function, cardiac rhythm and protection from ischemic injury (Other interesting features of the αc injury . Additio1-AR subtypes both from in vitro and in vivo studies.In the past years, we have gained significant information of some molecular properties and functional implications of the αin vitro and in vivo, to better assess differences and similarities among the three α1-AR subtypes.Several studies focused on individual receptor subtypes whereas only few others attempted to compare the behavior of different receptors in similar experimental conditions. This latter approach should be implemented in future studies, both 1-AR subtypes (1-AR subtypes have been performed in recombinant systems. A big challenge in the future will be to explore the functional implications of a variety of interactions in different tissues and physiological conditions. The α1-AR subtypes are important regulators of several physiological parameters as highlighted by studies in genetically modified mice (The elucidation of receptor-mediated signaling events in time and space will depend on a much deeper understanding of the interactions among receptors and signaling molecules which has recently emerged as an important paradigm in the GPCR field. Beyond receptor oligomerization , a numbesubtypes , but foried mice , and fur


Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Abnormal accumulation and aggregation of alpha-synuclein (a-syn) within neurons, and mutations in the a-syn and UCH-L1 genes have been shown to play a role in the pathogenesis of PD. In light of recent reports suggesting an interaction between a-synuclein and UCH-L1, we investigated the effects of UCH-L1 inhibition on a-syn distribution and expression levels in primary neurons and hippocampal tissues derived from non transgenic (non tg) and a-syn over expressing tg mice. We show that suppression of UCH-L1 activity increased a-syn levels in control, non tg neurons, and resulted in a concomitant accumulation of presynaptic a-syn in these neurons. In contrast, blocking UCH-L1 activity in a-syn over expressing neurons decreased a-syn levels, and enhanced its synaptic clearance. In vitro studies verified the LDN-induced inhibition of UCH-L1 had minimal effect on LC3 (a marker of autophagy) in control cells, in cells over expressing a-syn UCH-L1 inhibition resulted in increased LC3 activity. These findings suggest a possible differential role of UCH-L1 function under normal and pathological conditions. Furthermore, in the context of a-syn-induced pathology, modulation of UCH-L1 activity could serve as a therapeutic tool to enhance the autophagy pathway and induce clearance of the observed accumulated/aggregated a-syn species in the PD brain. Furthermore, transgenic mice that over express the I93M mutant, show behavioral and pathological deficits characteristic of Parkinsonism In vitro, UCH-L1 was found to possess a dimerization-dependent ligase activity responsible for K63-linked polyubiquitination of a-syn The implied involvement of UCH-L1 in PD is based on the discovery of a I93M mutation in the We have previously shown that inhibition of UCH-L1 activity leads to dysregulation of monomeric ubiquitin homeostasis and dramatic alterations in synapse structure UCH-L1 (LDN-57444 (LDN)) inhibitor was purchased from Calbiochem . The HA-tagged Ubiquitin probe  was synthesized as described previously The following antibodies were used in this study: mouse anti-PSD-95 ; rabbit anta-synapsin I ; chicken anti-Map2 ; rabbit anti-UCH-L1 ; rabbit anti-Ubiquitin ; mouse anti-HA ; mouse anti-a-syn ; rabbit anti-a-syn ; mouse anti-a-syn (Syn211) ; rabbit anti-P62 , and rabbit anti-cleaved LC3 .2) on coverslips (12 mm in diameter) coated with poly-D-lysine. Cultures were maintained in B27 supplemented Neurobasal media (Invitrogen) until 19–21 days in vitro (DIV).Hippocampal neuron cultures were prepared from P1 CD-1 mice or transgenic mice over expressing human a-syn tagged to GFP at its C-terminus (h-a-syn-GFP tg) ACA GGA AGT TAG CCC TAA A (#2) corresponding to nucleotides 385–403 or GCA GCT TTA GCA CTT AGA A (#4) corresponding to nucleotides 909–927 of mouse UCHL1 mRNA. The shRNA sequences were cloned into the pSIH1-copGFP vector (SBI Biosystems) to generate LV-siUCHL1#2 and LV-siUCHL1#4 vectors. A control siRNA vector was generated by cloning the sequence CGT GCG TTG TTA GTA CTA ATC CTA TTT designed against the sequence of luciferase (SBI Biosystems) into the same vector to generate pLV-siLuc. Lentiviruses expressing siUCHL1#2 or #4, a-synuclein (a-syn) or empty vector (LV-Control) were prepared by transient transfection in 293T cells The siUCHL1 lentivirus vectors were constructed with the following sequence To determine the effects of down-regulation of UCHL1 on accumulation of a-syn, B103 cells were plated on poly-lysine coated glass coverslips and infected with either LV-Control or LV-a-syn and then co-infected with either LV-siUCHL1#2 or LV-siUCHL1#4 for 72 hours. Cells were then fixed in 4% PFA and processed for immunoflouresence.For the purpose of this study, 6 months old mice over expressing human (h) a-syn under the control of the murine (m) Thy-1 promoter were used in vitro, cultured neurons were treated with 10 µM of UCH-L1 inhibitor (LDN) for 24 hr. This dose was selected based on previous IC50 studies that have shown a specific for LDN to UCH-L1 at this concentration in vivo, wild type and a-syn transgenic mice were injected with LDN (0.5 mg/Kg) intraperitoneally at time zero. Mice were injected again with the same dosage of LDN four hours after the first injection. Mice were then sacrificed four hours after the second injection. This dose of LDN was chosen for in vivo injections based on similar doses reported previously For immunofluorescence staining experiments To investigate levels of UCH-L1 activity we measured the activity of deubiquitinating enzymes (DUB). The DUB activity assay was done as previously described The rat neuroblastoma cell line B103 was used for in vitro experiments At the end of each experiment, hippocampal neurons plated on coverslips were rinsed briefly in PBS and fixed with 4% paraformaldehyde (PFA) and 4% sucrose in PBS-MC  for 10 min at room temperature. Neurons were then rinsed 3× with PBS-MC and subsequently blocked and permeabilized with blocking buffer containing  for 20 min. After rinsing neurons 3× with PBS-MC, primary antibodies were added in blocking buffer (without 0.2% Triton* X-100) and cultures were incubated overnight at 4°C. The following antibodies and dilutions were used for immuno-fluorescence staining: mouse anti-PSD-95 (1∶1000), rabbit anti-synapsin I (1∶5000), rabbit anti-UCH-L1 (1∶2500), chicken anti-Map2 (1∶5000), and mouse anti-a-syn (1∶2500) and rabbit-a-syn (1∶2500). After three washes with PBS-MC, neurons were incubated in goat anti-rabbit, -mouse or -chicken secondary antibodies conjugated to Alexa 488, Alexa 568, or Alexa 678  at room temperature for 1 hr. Neurons were washed 3× with PBS-MC and mounted on slides with Aqua Poly/Mount .Confocal images were acquired using a Leica  DMI6000 inverted microscope outfitted with a Yokogawa  Spinning disk confocal head, a Orca ER High Resolution B&W Cooled CCD camera (6.45 µm/pixel at 1X) , Plan Apochromat 40×/1.25 na and a Melles Griot  Argon/Krypton 100 mW air-cooled laser for 488/568/647 nm excitations. Exposure times were held constant during acquisition of all images for each experiment. Pyramidal-like cells were chosen in a random fashion. Confocal z-stacks were taken at 0.4–0.5 µm depth intervals in all experiments. For image analysis, the NIH ImageJ software was utilized. Max Z-projected images were thresholded equally 1.5–2 times above background. Dendrites from individual neurons were then straightened and used for analysis. Fluorescence intensity associated with pre- and postsynaptic protein puncta was measured to determine the size and number of puncta  in control and LDN-treated neurons. Statistical significance was determined by unpaired two-tailed Student's t test. All imaging and analysis were performed in a blinded fashion.At the end of each experiment, mouse brains were removed and divided sagittaly. One hemibrain from each brain was snap-frozen and stored at −70°C for protein and RNA analysis, while the other hemibrain was fixed in PBS/4% PFA (pH 7.4) at 4°C. 40 µm thick free-floating Vibratome sections were immunolabeled with the mouse anti-a-syn (1∶2500) and/or rabbit anti-UCH-L1 antibodies. After overnight incubation with the primary antibodies, sections were incubated with the Thyramide Signal Amplification-Direct (Red) System (NEN Life Sciences) and/or FITC-conjugated secondary antibody (Vector laboratories). Sections were then transferred to SuperFrost slides , and mounted under glass coverslips with anti-fading media (Vector Laboratories). All sections were processed under the same conditions. Sections were imaged with a Zeiss objective on an Axiovert 35 microscope  with an attached MRC1024 laser scanning confocal microscope (LSCM) system . Fluorescence intensity associated with a-syn immunoreactive structures, was measured using NIH ImageJ software. A total of six mice per group and three sections per mouse were analyzed. Four fields in the hippocampus, per section, were examined. Statistical significance was determined by unpaired two-tailed Student's t test. All imaging and analysis were performed in a blinded fashion.Neurons in the CA1 region of the hippocampus were visualized with Nissl staining. Neuron density measurements were performed using an image-based analysis system. Seven sections per condition were analyzed. The region of interest in each section was selected with a 4× objective on an Olympus BX51 microscope . Each region was then divided into randomly selected squares by the Stereo Investigator software (MBF bioscience). An average of 20 squares per selected area in each section was used to count the total number of nucleoli-containing neurons using a 100× oil objective. The total number of neurons was calculated according to the optical fractionator Mouse hippocampi were homogenized in RIPA buffer in teflon-glass homogenizers. Hippocampal homogenates were centrifuged at 18,000 g and supernatants were removed and protein concentration was determined with the BCA™ Protein Assay Kit  using bovine serum albumin as a standard. Protein samples were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were then blocked for 1 hr in TBST blocking buffer  at room temperature and then incubated with primary antibodies in blocking buffer overnight at 4°C. The antibodies used were at the following dilutions: rabbit anti-ubiquitin (1∶1000), rabbit anti-P62 (1∶1000), rabbit anti-cleaved LC3 (1∶1000), mouse anti-a-syn  and mouse anti-a-syn (1∶2500). Blots were then washed 3× in TBST washing buffer  and incubated with goat anti-rabbit or -mouse IgG conjugated to horseradish peroxidase (1∶5000). Protein bands were visualized by Chemiluminescence plus reagent (PerkinElmer) and were digitized and quantified using NIH ImageJ software. For statistical analysis unpaired Student t-test was performed between any two conditions.5′-TGG CAG AAG CAG CAG GAA AG, 3′-AGC CAC TGT TGC CAC ACC AT) and human a-syn . For statistical analysis one-way ANOVA and post hoc Bonferroni was performed between all conditions.Hippocampi from non tg and a-syn tg control and LDN-treated mouse brains were isolated. Total RNA from each hippocampus was then extracted with the RNeasy Lipid Tissue kit (Qiagen). cDNA from extracted RNA was generated with the qScript cDNA synthesis kit according to the manufacturor's protocol. cDNA was then quantified using the 2× SYBR Green SuperMix for iQ (Quanta Biosciences) with specific primers for murine a-syn  for endogenous a-syn, the presynaptic marker Synapsin I  and UCH-in vitroOur previous work determined that UCH-L1 activity is required for maintaining synapse structure Next, to confirm whether down regulation of UCH-L1 activity affects PSD-95 and a-syn by an independent method, primary neuronal cultures were infected with a lenti-viral vector expressing a si-control (LV-siLuc) or si-UCH-L1 (LV-siUCH-L1) and then analyzed by immunocytochemistry and confocal analysis. As expected, the LV-siUCH-L1 led to a robust reduction in the expression of UCH-L1 . Consistin vitro, we set out to determine whether inhibiting UCH-L1 activity plays a role in modulating a-syn distribution in vivo. To test this possibility, we assessed the effects of UCH-L1 inhibition under normal and pathological conditions.Since our data demonstrate that blocking UCH-L1 activity affects a-syn distribution We first examined the immunohistochemical distribution and expression levels of a-syn in hippocampal tissue sections from control and LDN-treated mice. Immunolabeling of hippocampal tissues with a-syn in non tg mice treated with LDN, showed a significant increase in a-syn expression compared to those in control, untreated mice  and CA3 We then determined UCH-L1 activity levels in hippocampal lysates obtained from non tg and a-syn tg mice that received injections of either vehicle (DMSO) or LDN. We found that in the hippocampi of LDN-treated non tg mice, UCH-L1 activity levels were reduced by 37% .In order to investigate the relationship between a-syn and UCH-L1 in vivo, detailed confocal imaging was performed on double-labeled sections. High magnification images of double-immunolabeled hippocampal sections with antibodies against a-syn and UCH-L1 demonstrate that in both non tg  and a-syTo assess whether the observed alterations in a-syn levels in LDN-treated mice was not due to changes in neuronal cell density, we performed stereological analysis of the hippocampus using Nissl staining . We did The immunohistochemical data demonstrate that inhibition of UCH-L1 activity has differential effects on a-syn distribution in the hippocampi of non tg vs. a-syn tg animals. To complement the immunohistochemical data, we compared a-syn protein expression levels in hippocampal homogenates obtained from non tg and a-syn tg mice that received injections of either vehicle (DMSO) or LDN. We first assessed whether suppression of UCH-L1 activity had an effect on mono-ubiquitin levels in the hippocampi of LDN-treated mice. We have previously shown that UCH-L1 dependent mono-ubiquitination is a good marker of activity in vivo studies show that the levels of a-syn are modulated differentially in non tg and a-syn tg mice with suppressed UCH-L1 activity. In order to validate our in vivo results from a-syn tg mice, we used an independent in vitro model to test the effects of UCH-L1 suppression in a-syn over expressing neurons treated with LDN. We used cultured primary hippocampal neurons from h-a-syn-GFP tg mice for this purpose as transgenic mice are readily detected and distinguished from non tg mice using a GFP flashlight. In this mouse model, h-a-syn expression is driven by platelet-derived growth factor (PDGF) promoter which restricts h-a-syn expression largely to the limbic and cortical areas In vitro, average h-a-syn expression levels in mature synaptic boutons was ∼2.5 times higher than that of wild type littermates, levels that reasonably approximate those observed in a-syn related human diseases in vivo results, exposure of h-a-syn-GFP neurons to LDN led to significant reduction in the intensity and size of h-a-syn-immunoreactive puncta  by which UCH-L1 leads to modulation of a-syn protein expression levels, we set out to examine components of the autophagy pathway. Recent studies have demonstrated that UCH-L1 interacts with the components of the chaperone-mediated autophagy (CMA), a pathway also known to be involved in degradation of a-syn Assessment of cleaved LC3 levels in hippocampal homogenates showed that compared to control non tg mice, the levels of LC3 II decreased by 26% in a-syn tg mice  reduces a-syn protein levels, and this could potentially be beneficial for neuronal survival. Many lines of evidence have suggested that dysfunction in the autophagy pathway is common in many neurodegenerative disorders including in PD and DLB M), and C-terminal farnesylation of UCH-L1 promotes its association with cellular membranes M expressed in cells and correlates with accumulation of a-syn and neurotoxicity M/UCH-L1S (soluble UCH-L1) ratio and disease, in this case, in cortical tissues from PD patients was not detected in vivo.A recent study by Liu et al. demonstrated that a population of UCH-L1 is membrane-bound (UCH-L1In conclusion, we show that suppression of UCH-L1 activity has differential effects on a-syn in neurons that express normal or excessive levels of a-syn. Under normal physiological conditions, perturbation of UCH-L1 activity has a dramatic effect on distribution and protein levels of presynaptic a-syn, which in turn could have detrimental effects on normal neuronal function. Under pathological conditions, however, loss of UCH-L1 function proves to be beneficial as it not only enhances a-syn degradation but also relieves the a-syn-mediated block of the autophagy pathway. Further studies are needed to evaluate the potential value of blocking UCH-L1 as a therapeutic target in a-synucleinopathies.


Prostate cancer is the most common cancer in men in the United States, and it is the second leading cause of cancer-related death in American men. The androgen receptor (AR), a receptor of nuclear family and a transcription factor, is the most important target in this disease. While most efforts in the clinic are currently directed at lowering levels of androgens that activate AR, resistance to androgen deprivation eventually develops. Most prostate cancer deaths are attributable to this castration-resistant form of prostate cancer (CRPC). Recent work has shed light on the importance of epigenetic events including facilitation of AR signaling by histone-modifying enzymes, posttranslational modifications of AR such as sumoylation. Herein, we provide an overview of the structure of human AR and its key structural domains that can be used as targets to develop novel antiandrogens. We also summarize recent findings about the antiandrogens and the epigenetic factors that modulate the action of AR. Prostate cancer (PC) is the second most prevalent cause of death in men in the USA and Europe. The dependence of PC on androgens has been recognized for more than 7 decades. Medical and surgical androgen deprivation therapy (ADT) has been a standard palliative therapy for metastatic PC. However, an estimated 217,730 new cases and 32,050 PC-related deaths in the USA alone in 2010 despite ADT . Th. Th13]. The role of the AR CTD is of particular importance for PC, because the current androgen ablation therapies target this domain of the AR. This prevents both AR nuclear translocation and the exposure of AF-2. Antiandrogens such as bicalutamide bind the LBD, block the activity of AF-2 , and cauThe transcriptional activity of most steroid hormone receptors is predominantly through the activation function AF-2 region in the LBD. However, in the AR, it is the AF-1 region in the NTD that contributes most to the transcriptional activity . AR LBD The AR NTD is highly flexible and displays intrinsic disorder in solution, which has hampered elucidation of its three-dimensional structure , 24. In The AR DBD/hinge region plays important roles in mediating AR nuclear localization, receptor dimerization, and DNA binding. All the hormone receptor DBDs are highly conserved. It consists of two zinc-fingers and a loosely structured carboxy-terminal extension (CTE) . It is rLigand binding induces a conformational change in AR inducing phosphorylation , nuclearDimerization mediated through the AR-DBD is an absolute requirement for AR signaling. By assembling on DNA targets, the AR homodimer specifically binds DNA to regulate the expression of AR target genes. Whereas the N/C (NTD/CTD) interaction provides a potential mechanism for regulation of AR activity, further studies are necessary to determine the precise role and contribution of each model of AR N/C interaction to AR signaling. Based on evidence that AR-LBD dimerization can occur, it is likely that this interaction also contributes to AR signaling. Centenera et al. showed that interaction of multiple AR domains is required for optimal AR-mediated signaling .Currently, all conventional therapy has been focused on androgen-dependent activation of the AR through its C-terminal LBD. A schematic of their mechanism of action is shown in . Below is a summary of the currently used therapies targeting androgen signaling.Surgical castration completely eliminates testosterone production by the testes, whereas administration of an LHRH agonist  generates castrate levels of serum testosterone  by having a negative hormonal feedback on the hypothalamus  R1881, VN/124-1 was equipotent to bicalutamide in LNCaP cells. Transcriptional activation assays showed VN/124-1 to be a pure AR antagonist of the wild-type AR and the T877A mutation found in LNCaP cells , 8988, 8On a general note, HDAC inhibitors cause histone hyperacetylation, activating many genes, which leads to growth inhibition, differentiation, or apoptosis . ClinicaSome of the HDAC inhibitors serve the dual functions of disrupting AR signaling and reducing AR protein levels in the cell. Multiple reports have shown that HDAC inhibitors suppress AR expression , 90–92. Kang et al.  showed tThe SUMO-1 modification (sumoylation) pathway resembles that of ubiquitin conjugation, but the enzymes involved in the two processes are distinct . SUMO-1 N-CoR and SMRT are well characterized corepressors. Given the evidence that SMRT and NCoR form complexes with HDAC3 , 102, thHistone methylation was originally thought to be a stable, irreversible mark as only histone methyltransferases had been identified with no known data about demethylases. However, the discovery of the first histone demethylase LSD1 (lysine-specific demethylase 1) confirmed that histone methylation is reversible , 106. ThAndrogen deprivation has been the major therapy for prostate cancer for 7 decades. After a few months of ADT, PC progresses despite castrate serum levels of testosterone. We now have a greater understanding of the mechanisms of sustained AR signaling in these CRPCs that have progressed despite androgen deprivation. Cofactors such as HDACs, SUMO-1, N-CoR, and SMRT provide additional level of control of AR signaling. Upregulation of cofactors such as HDACs that facilitate AR target gene activation is an important event in the development of CRPC. Whether targeting histone deacetylases and other AR coactivators or AR protein stability will be safe and efficacious remains unknown, but their preclinical data holds a great promise. Most of the currently developed therapies targeting AR signaling aim at the LBD of AR. However, with the knowledge that AR NTD can function independent of AR LBD, newer therapies such as EPI-001 and decoy AR NTD are being developed to target AR NTD. All of these antiandrogens and HDAC inhibitors may show synergistic/additive activities when used in combination. There is a great interest to combine these agents to effectively treat CRPC.


Diabetes is a chronic metabolic disorder, characterized by hyperglycemia resulting from insulin deficiency and/or insulin resistance. Recent evidence suggests that high levels of reactive oxygen species (ROS) and subsequent oxidative stress are key contributors in the development of diabetic complications. The FOXO family of forkhead transcription factors including FOXO1, FOXO3, FOXO4, and FOXO6 play important roles in the regulation of many cellular and biological processes and are critical regulators of cellular oxidative stress response pathways. FOXO1 transcription factors can affect a number of different tissues including liver, retina, bone, and cell types ranging from hepatocytes to microvascular endothelial cells and pericytes to osteoblasts. They are induced by oxidative stress and contribute to ROS-induced cell damage and apoptosis. In this paper, we discuss the role of FOXO transcription factors in mediating oxidative stress-induced cellular response. Diabetes mellitus is a chronic disease characterized by elevated blood sugar levels resulting from either lack of insulin production or resistance to insulin. In 2010, there were nearly 230 million individuals with diabetes worldwide which is estimated to reach 430 million by 2030 . Accumul2−) and hydroxyl radicals (HO•), and hydrogen peroxide (H2O2), are highly reactive and can cause damage to biological macromolecules such as DNA, proteins, and lipids [Reactive oxygen species (ROS) are oxygen free radicals that are generated as by-products of mitochondrial metabolism and function as signaling molecules in various intracellular processes including cell proliferation, migration, and apoptosis . ROS prod lipids . Major sd lipids , 10. In d lipids . Other mROS leads to the generation of intracellular signals that stimulate inflammation and cell death. They include protein kinase C (PKC), c-Jun-N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) –15. In mFOXO transcription factors are critical mediators of oxidative stress and are activated by various kinds of cellular stress stimulus. Oxidative stress regulates FOXO activity through various posttranslational modifications including phosphorylation, acetylation, and ubiquitination, which in turn regulate the subcellular localization of FOXOs, protein-protein interactions, and transcriptional activity of FOXO proteins. While some of these modifications promote FOXO transcriptional activity, others are inhibitory. For example, stress-activated kinase JNK directly phosphorylates FOXO4 at residues Thr447 and Thr451, which leads to its nuclear translocation and induces FOXO4 transcriptional activity . Anotherin vitro and in vivo. SIRT1 deacetylation of FOXO3 increases expression of its target genes involved in cell cycle arrest and DNA repair such as p27 and GADD45. In contrast, SIRT1 deacetylation reduces expression of FOXO3 proapoptotic target genes such Bim and Fas ligand. These results indicate that deacetylation can both enhance and reduce FOXO3-induced activity depending upon the target gene.FOXO transcriptional activity is also regulated by acetylation. The effects of oxidative stress-induced acetylation on FOXO function vary based upon the experimental conditions. Sirtuins (SIRTs), mammalian homologs of the yeast silent information regulator 2 (sir2) deacetylase, are critical regulators of FOXO transcriptional activity and are induced by oxidative stress , 23. It Besides phosphorylation and acetylation, FOXO proteins are further regulated by ubiquitination during oxidative stress. In response to insulin or growth factor signaling, FOXO transcription factors are phosphorylated, polyubiquitinated, and degraded . It has 2O2). Enzymes catalase and glutathione peroxidase further breakdown H2O2 into water and oxygen [FOXO proteins play an important role in protection of cells against oxidative stress. Oxidative stress is caused by overproduction of ROS or inefficient breakdown of ROS. Efficient detoxification of ROS by cellular detoxification systems protects cells against oxidative damage. The levels and enzymatic activities of various antioxidant enzymes such as manganese superoxide dismutase (MnSOD), catalase, and glutathione peroxidase are decreased during hyperglycemia-induced oxidative stress . It is nd oxygen , 31. Thed oxygen . Consistd oxygen , 34. Deld oxygen , 34. It d oxygen . In the d oxygen . In thisd oxygen . Thus, tα (GADD45α) [kip1 [α [FOXO transcription factors play a role in cell proliferation and survival by regulating the expression of genes involved in a number of cellular processes including cell cycle arrest, DNA repair, and apoptosis. In response to certain levels of oxidative stress, FOXO factors induce expression of target genes that control cell cycle progression and DNA repair, including p27Kip1, retinoblastoma-like protein p130, and cyclin D1/2, growth arrest, and DNA damage-inducible gene 45GADD45α) –41. For α) [kip1  and by rα) [kip1 . BesidesL [FOXO transcription factors typically induce either cell death by regulating proapoptotic genes but depending upon the context can enhance survival. In response to certain ROS levels, FOXO transcription factors switch from prosurvival to proapoptotic signaling leading to cell death. However, the exact molecular mechanisms by which FOXO switches from prosurvival to prodeath signaling remain unknown. In diabetes, chronic hyperglycemia-induced mitochondrial ROS stimulate various signaling pathways leading to activation of FOXO, which in turn activates several proapoptotic factors. FOXO1 activation is elevated in diabetic connective tissue and mediates advanced glycation endproduct and TNF-alpha-induced apoptosis both of which are elevated in diabetic connective tissue –44. It hL . It was L . High glL . MoreoveL , 47. In α, interlukin-1β (IL-1β), and interlukin-6 (IL-6), ultimately leading to decreased insulin sensitivity and diabetic complications. Hyperglycemia-induced FOXO plays an important role in the induction of proinflammatory cytokines. It was shown that FOXO1 directly binds to IL-1β promoter and increases its expression in macrophages [in vivo by diabetes-enhanced TNF-α and also induces expression of TNF-α levels in these cells [β and TNF-α production has been implicated in pathogenesis of obesity and diabetes. Hyperglycemia in diabetes also stimulates toll-like receptor (TLR) signaling, which results in prolonged inflammation and tissue damage. Recent studies show that FOXO1 promotes inflammation during diabetes by enhancing TLR4-mediated signaling, suggesting FOXO1 as a key mediator of inflammatory responses during obesity and diabetes [α promoter. Moreover, diabetes-enhanced TNF-α activates FOXO1 in chondrocytes in vivo by enhancing its nuclear localization [Inflammation has long been considered as a major risk factor in diabetes and associated with development and progression of diabetic complications. Hyperglycemia-induced oxidative stress promotes inflammation through increased endothelial cell damage, microvascular permeability, and increased release of proinflammatory cytokines, including TNF-rophages . FOXO1 ise cells . Increasdiabetes . In diabdiabetes , 51. It lization .κB [κB pathway has been implicated in the development of diabetic complications, including retinopathy, and has been shown to regulate expression of various proinflammatory cytokines, including TNF-α and IL-1β [κB and FOXO leading to increased inflammation and cellular damage. In most cell types, NF-κB is directly antiapoptotic, while FOXO1 is directly proapoptotic. Thus, in inflammatory conditions when both NF-κB and FOXO1 are activated, their relative balance may determine whether a cell ultimately survives or undergoes apoptosis [Another transcription factor that plays an important role in stimulating inflammation during hyperglycemia and oxidative stress is NF-κB . Activatnd IL-1β . Chronicpoptosis , 47.2 leading to formation of O2− and the generation of various reactive oxygen species in the mitochondria. Furthermore, changes caused by diabetes alter the redox balance and affect redox-sensitive proteins such as protein kinase C-epsilon, which can result in enhanced mitochondrial ROS production. Advanced glycation end products (AGEs) generated under conditions of hyperglycemia stimulate NADPH oxidase that in turn can induce production of ROS. In a surprising development, increased Wnt signaling stimulates mitochondrial biogenesis that can lead to enhanced ROS levels in mitochondria and greater oxidative damage [κB by TNF-α and IL-1 [A mechanism through which diabetes can increase oxidative stress involves electron transport in mitochondria. It has been proposed that high intracellular glucose levels increase the follow of electrons through the electron transport chain in mitochondria during oxidative respiration . This cae damage . The ince damage . When thand IL-1 .ROS may affect insulin signalling. Insulin signalling is reduced under conditions of oxidative stress, which may contribute to insulin resistance. This may occur through several mechanisms. In one scenario, ROS induces serine phosphorylation of insulin receptor substrate, decreasing tyrosine phosphorylation thereby interfering with insulin signaling . Similar Insulin signalling inactivates FOXO1, which is mediated by insulin receptor substrates-1 and -2 through AKT. A characteristic feature of insulin resistance is the elevated production of glucose that contributes to hyperglycemia. FOXO1 regulates glucose production in the liver through the expression of genes that promote gluconeogenesis . Thus, aHyperglycemia-induced ROS and subsequent oxidative stress are major contributors to the development and progression of diabetes and related complications. However, effective therapeutic strategies to prevent the generation of these free radicals remain limited. It is now well established that FOXO transcription factors are the critical regulators of cell fate and play a major role in diabetes-induced oxidative stress resistance and in diabetes-enhanced apoptosis. It seems that FOXO transcription factors might function as molecular switches that determine cell fate in response to various levels of oxidative stress by either promoting antioxidants  responses or alternatively enhancing proapoptotic gene expression and cell death. However, the precise mechanism by which FOXO mediates prosurvival/proapoptotic response remains unclear and elucidation of molecular mechanisms involved may provide new targets for therapy. Furthermore, multiple signaling pathways, including JNK and MAPK, regulate the activity of FOXO transcription factors in response to hyperglycemia in diabetes. Because our understanding of how these diverse signaling pathways coordinate their effects to regulate FOXO activity during diabetes remains limited, detailed understanding of these pathways may provide insights into development of new therapeutic strategies for treatment of diabetes.


Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa).Using 2 antibodies against different regions of AR protein (N- or C-terminus), we successfully showed the existence of AR variant in the LuCaP 86.2 xenograft. To evaluate the prevalence of AR variants in human PCa tissue, we used this method on tissue microarrays including 50 primary PCa and 162 metastatic CRPC tissues. RT-PCR was used to confirm AR variants. We observed a significant decrease in nuclear C-terminal AR staining in CRPC but no difference between N- and C-terminal AR nuclear staining in primary PCa. The expression of the AR regulated proteins PSA and PSMA were marginally affected by the decrease in C-terminal staining in CRPC samples. These data suggest that there is an increase in the prevalence of AR variants in CRPC based on our ability to differentiate nuclear AR expression using N- and C-terminal AR antibodies. These findings were validated using RT-PCR. Importantly, the loss of C-terminal immunoreactivity and the identification of AR variants were different depending on the site of metastasis in the same patient.We successfully developed a novel immunohistochemical approach which was used to ascertain the prevalence of AR variants in a large number of primary PCa and metastatic CRPC. Our results showed a snapshot of overall high frequency of C-terminal truncated AR splice variants and site specific AR loss in CRPC, which could have utility in stratifying patients for AR targeted therapeutics. Although circulating levels of androgens are depleted in CRPC, tumor progression is often concomitant with elevated levels of the androgen receptor (AR), activation of the AR, and the expression of AR-regulated genes. However, an increase in AR expression by itself is generally not sufficient to engage the AR transcriptional program v567es encodes a protein comprised of exons 1–4, and because of a frame-shift due to loss of exons 5–7, exon 8 has a stop codon generated after the first 10 amino acids resulting in a shortened exon8. In addition to mechanisms leading to AR activation in CRPC that require ligand, recent evidence points to the existence of alternatively spliced forms of AR mRNAs that encode receptors devoid of the LBD, but retaining the ability to engage transcriptional machinery and promote the regulation of known---and potentially new---sets of transcriptional targets FL). We hypothesized that this feature of AR variants afforded an opportunity to identify AR protein variants in formalin-fixed tissues by comparing the differential staining of antibodies recognizing either N- or C-terminus of the AR. Therefore, in the present study, we used antibodies against the N- or C-terminus of the AR protein to interrogate a large number of benign prostate tissue, primary hormone naïve PCa and a series of metastatic CRPC to ascertain the prevalence of C-terminal truncated AR variants.Several studies evaluating the expression of AR splice forms in a small number of prostate cancers suggest that AR variants are more readily detected in CRPC compared to hormone-naïve cancers, and may emerge due to the selective pressure of AR targeted therapy The antibodies used in this study and the working conditions are listed in v567es that is constitutively active. The LuCaP 35 xenograft only expresses wide type AR Human primary and metastatic PCa tissues were obtained as part of the PCa research program and University of Washington Medical Center Prostate Cancer Donor Rapid Autopsy Program, which is approved by the University of Washington Institutional Review Board. The Institutional Review Board of the University of Washington Medical Center approved all procedures involving human subjects, and all subjects signed written informed consent. Human tissue microarrays (TMAs) consists of 42 patients from Prostate Cancer Donor Rapid Autopsy Program (including 65 soft tissue metastases and 120 bone metastases) Total tissue RNA was isolated from minced fresh tissue using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Two micrograms of total RNA was digested with DNase I, and reverse transcribed using Superscript First-Strand Synthesis System (Invitrogen). PCR was performed using AmpliTaq Gold® PCR Master Mix (Applied Biosystems). PCR products were run on 2% agarose gel and image pictures were taken by using AlphaDigiDoc Pro imaging system from Alpha Innotech . qRT-PCR reactions were done using an Applied Biosystems 7900 sequence detector with 5 ng of cDNA, 200 nM of each primer pair and Power SYBR Green PCR Master Mix or TaqMan Universal PCR Master Mix from Applied Biosystems.Gene expression levels were measured by relative quantification between RNA samples, and fold expression changes were determined by the 2–ΔΔCT method The primer sequences are listed in FL and ARv567es variant were grown to 80% confluence in 30 mm plates in RPMI 1640 medium with 5% serum, and then switched to RPMI-1640 medium with 5% charcoal stripped serum for 24 h. Dihydrotestosterone (DHT) 10−9 M, MDV-3100 50 nM, or MDV-3100 plus DHT were added to the cultures. After 24 h, total RNA was collected from duplicate wells for qRT-PCR to detecting ARFL, ARv567es and AR-V7 transcripts. The experiment was repeated 6 times with triplicates each time, and the qRT-PCR results were normalized to DHT treatment group.VCAP cells that expressed both AR2, 1 mM EGTA, 1% Triton X-100) containing Halt™ Phosphatase Inhibitor Cocktail and protease inhibitors (Thermoscientific). Complete lysates were separated on SDS-PAGE, transferred onto a nitrocellulose membrane, blocked in 5% milk-PBS-Tween and probed with respective overnight at 4°C. Membranes were incubated with a horseradish peroxidase-conjugated secondary antibody , and developed with ECL (Pharmacia Biotech). The membranes were stripped for 30 min in Stripping Buffer (Thermoscientific) and re-probed with anti-β-actin antibody as a loading control (Sigma-Aldrich). Independent experiments validated that this stripping procedure did not lead to loss of signal.Fresh tissue was homogenized and lysed with cold lysis buffer  were deparaffinized and rehydrated. Antigen retrieval was performed with 10 mM citrate buffer (pH 6.0) in a pressure cooker (20 psi for 10 min). Endogenous peroxide and biotin/avidin was blocked for 15 min with respective agents (Vector Laboratories). After incubating with 5% normal goat-horse-chicken serum at room temperature for 1 h, sections were incubated with primary antibodies  at 4°C oA few unusable cores were found in the TMAs due to tissue core missing, cancer necrosis, or insufficient cancer cells. These cores were excluded from the results.Immunostaining was assessed using a quasi-continuous nuclear AR score, created by multiplying each intensity level  by the corresponding percentage of positive cells, and then summing the results.2 in each tissue site. Total cell number and Ki67 positive cell number were counted, and the final Ki-67 index was calculated using positive cell number divided by total cell number.Ki-67 staining was measured by randomly choosing up to 4 fields of 250 µmAR antibodies used in this study targeted three distinct regions of the human AR protein. The immunogens of AR F39.4.1 (against aa301–320) and AR441 (against aa299–315) were located in the N-terminal of AR while AR C-19 (against aa900–919) in the C-terminal. Therefore, we describe AR F39.4.1 and AR441 as N-terminal AR antibodies and AR C-19 as a C-terminal AR antibody. Specific patterns of AR nuclear immunostaining were assessed as: N+C+ ; N+C↓  and N-C- . The tissues in N↓C+ group were not included because they were rare, and not relative to C-terminal truncated AR variants.p values ≤0.05 were chosen as statistical significant.Statistical analyses of the results were performed using Prism software (Prism Graphpad), where we used the Mann-Whitney test, and v567es. The LuCaP 35 xenograft was derived from a PCa lymph node metastasis and expresses full-length AR (ARFL)-encoding mRNA comprised of 8 exons and a protein of appropriate size for this transcript Several AR transcript variants have been described that encode AR polypeptides devoid of the C-terminal LBD due to alternative exon splicing FL protein. In the LuCaP 35 tumor, both AR antibodies detected a similar 110 KDa AR polypeptide that corresponded to the size of ARFL. In the LuCaP 86.2 tumor, beside a weak 110 KDa band, N-terminal AR antibody also identified an 80 KDa AR isoform that corresponded to the predicted size of the C-terminal truncated AR splice variant-ARv567es while C-terminal AR antibody did not  or C-terminus (AR C-19) of AR did not .FL by Western analysis, did not show any difference between N- and C-terminal AR staining by IHC  of LuCaP 86.2 showed very weak nuclear staining using the C-terminal AR (AR c-19) antibody, but intense nuclear staining in the serial section with AR antibody against N-terminal (AR F39.4.1). The LuCaP 35 tumor, shown to express only ARg by IHC . These dv567es has been shown to be constitutively active ARp = 0.5756), normal prostate and PCa samples (p = 0.7428), and hyperplastic prostate and PCa samples (p = 0.7508)   a and b. 0.7508) .To determine the frequency of variant AR expression in metastatic PCa, we scored IHC staining of metastatic sites from 42 patients who died of advanced CRPC.v567es transcripts p = 0.38, data not show), but the frequency of decreased nuclear C-terminal AR expression in metastatic CRPC was significantly higher than in primary PCa (p = 0.0027). The comparison of nuclear AR expression between primary PCa and metastatic CRPC is shown in Among 162 metastatic sites, 103 (63.6%) sites showed consistent nuclear AR expression with both antibodies (N+C+). However, 39 (24.1%) sites had a nuclear C-terminal AR score (N+C↓) that was at least 50% less than the corresponding N-terminal AR score, and 20 (12.3%) sites of metastasis had no nuclear AR expression (N-C-) . These Ip = 0.0505). PSA expression in N-C- metastatic sites was significantly lower than both N+C+ and N+C↓ metastatic sites (p<0.001) (p = 0.0097), but PSMA in N-C- metastatic sites was significantly lower than both N+C+ and N+C↓ metastatic sites (p<0.0001) (p = 0.045). TMPRSS2 in N-C- metastatic sites was significantly lower than both N+C+ and N+C↓ metastatic sites (p<0.01)  . Additio<0.0001) . Finally(p<0.01) . The losWe next examined the genes that have been identified as having their expression increased in response to AR C-terminus truncated variants v567es) and unknown AR splice variants. To determine if the N- and C-terminal immunoreactivity was associated with AR transcript variants, we performed RT-PCR on a subset of metastatic CRPC tissues and compared the RT-PCR results to IHC results . Of 8 metastatic samples classified as N+C↓ by IHC analysis, all expressed the ARFL and 6 (75%) also expressed at least one known AR variant (AR-V7/AR3 and/or ARv567es) by RT-PCR. These N+C↓ metastatic samples also exhibited lesser levels of PSA and PSMA immunoreactivity. None of the four N-C- metastatic samples expressed the ARFL examined by RT-PCR, although one showed very low level of ARv567es expression.We determined that loss of C-terminal AR immunoreactivity occurred frequently in metastatic CRPC. This loss of C-terminal immunoreactivity might be due to the expression of a number of known , and then treated with Dihydrotestosterone (DHT), MDV-3100 or MDV-3100 with DHT, separately. qRT-PCR showed that AR DHT+MDV . The varMDV-3100 . AR-V7 m3100 , N+C↓ (n = 39), and N-C- (n = 18). The Ki67 index for the N+C+ sites was 18%. This was not significantly different from the N+C↓ sites (20.8%) (p = 0.4225) or the N-C- sites (17.8%) (p = 0.95).Traditional concepts of AR translocation to the nucleus that involve ligand binding, dissociation from chaperones and nuclear translocation imply that without androgen ligand, AR would be found primarily in the cytoplasm and CRPC tumor progression would be driven by mechanisms other than those involving AR. However, this is not the case excepting most neuroendocrine tumors. CRPC usually possess a transcriptionally active AR that modulates the expression of AR regulated messenger RNAs Multiple mechanisms have been proposed to explain AR nuclear localization and some or all may be responsible for that seen in CRPC v567es splice variants, at least 20 AR splice variants have been reported to date. With specific antibodies available for only 2 of the variants, the use of specific antibody staining on tissue to detect castration-induced C-terminal truncated AR variants is not feasible Although antibodies have been developed for the AR-V7/AR3 and ARThe data reported here correlating the expression of AR protein by IHC with 2 antibodies and validation of the expression of AR splice variants by RT-PCR, strongly suggest that the variation between AR N- and C-terminus immunoreactivity results from the expression of alternative AR mRNAs. However, alternative explanations should be entertained. The primary tumors and soft tissue metastases were formalin fixed and paraffin embedded while the metastatic lesions of bone were formalin fixed and decalcified with 10% formic acid before embedding in paraffin. We observed no significant difference in AR staining in soft tissue versus bone metastasis . Furthermore, we have not seen differences with other antibodies between bone and soft tissue preparation methods using the same tissues and methods in vitro. We have also shown here that IHC on the LuCaP 86.2 xenograft that predominantly expresses one of the AR C-terminal truncated splice variants had a similar staining pattern to 24% of the metastatic CRPC tissues  and occur relatively rapidly if ADT results in a significant decrease in circulating and intratumoral androgens  tissues . Since tOne of our limitations was the inability of the assay to separate constitutively active from inactive C-terminal deleted AR proteins. To address this, we examined the expression of known AR regulated proteins. We observed a decrease in PSA and PSMA expression in the CRPC sites with the loss of C-terminal AR staining. However the decrease in PSA and PSMA expression was limited, suggesting constitutively active AR variants constitute a significant portion of the C-terminal truncated AR variants in CRPC.v567es regardless of whether the host is castrated or not v467esIn order to further explore the activity of AR variants among the tissues, we selected a group of genes that are purported to be AR variant regulated in human tissue We observed considerable heterogeneity in AR staining within and between patients with CRPC. This suggests that the truncation and/or loss of the AR are not necessarily early clonal events in the development of PCa, rather late stage events occurring independently of one another in CRPC. As proposed by us and others, C-terminal AR loss, may be associated with mutations, chromosomal rearrangements, or splicing events that occur after androgen withdrawal FL and enhance ARFL transactivation by ligand FL for activity and thus respond to MDV-3100 v567es is increased after androgen withdrawal and androgen receptor inhibitor MVD-3100 treatment, which correlates with our findings herein of an increase in AR variants with a decrease in C-terminal IHC reactivity in CRPC. However, as shown herein, there is heterogeneity among the metastatic sites with regard to variant status and this confounds potential clinical management decisions. Further studies are required to determine whether assessments of AR splice variants will be useful in stratifying patients for AR pathway-targeted therapies.An important question for a patient in whom there is a conversion to decreased C-terminal AR is whether or not it should effect treatment decisions, especially since two new treatments for PCa that has recurred following “traditional” ADT, i.e. abiraterone and MDV-3100, require the LBD of the AR to be effective v567es) in CRPC v567es are the three variants identified in human tissue so far, emerging studies have suggested the existence of other AR variants which should also be considered in regard to evaluation of total AR functions. Our study showed a relatively complete profile of C-terminal truncated AR variants in CRPC patients, and found AR regulated gene products are associated with the distribution of AR variants. Secondly, we investigated multiple metastatic sites for each CRPC patient, whereas only single metastatic site per patient was assessed in Hornberg's study. Third, in our study, we did not find any correlation between AR variants and time from diagnosis to death or starting androgen ablation treatment to death. This would not be unexpected since in Hornberg's study, the AR variants were detected at a specific event prior to death, i.e. pathologic fracture; whereas our tissues were collected at the time of death and thus the time from appearance of variants to death is unknown. Furthermore, we have recently shown that recurrence of human PCa xenografts following castration and inhibition of steroid synthesis with the CYP17 inhibitor abiraterone may be associated with either AR splice variants expression or intracrine steroidogenesis Hornberg et al published an independent study showing increased mRNA levels of 3 specific AR splice variants (AR-V1, AR-V7 and AR


Niabella soli Weon et al. 2008 is a member of the Chitinophagaceae, a family within the class Sphingobacteriia that is poorly characterized at the genome level, thus far. N. soli strain JS13-8T is of interest for its ability to produce a variety of glycosyl hydrolases. The genome of N. soli strain JS13-8T is only the second genome sequence of a type strain from the family Chitinophagaceae to be published, and the first one from the genus Niabella. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,697,343 bp long chromosome with its 3,931 protein-coding and 49 RNA genes is a part of the  Genomic Encyclopedia ofBacteria andArchaea project. T (= DSM 19437 = KACC 12604) is the type strain of the species Niabella soli [Niabella [soli, of soil [T was found to assimilate several mono- and disaccharides and to produce numerous glycosyl hydrolases [Niabella are recorded. Here we present a summary classification and a set of features for N. soli JS13-8T, together with the description of the genomic sequencing and annotation.Strain JS13-8N. soli JS13-8T was compared using NCBI BLAST [Niabella (34.8%), Terrimonas (21.0%), Flavobacterium (14.9%), 'Niablella'  and Niastella (8.2%) . Regarding the single hit to sequences from members of the species, the average identity within HSPs was 99.7%, whereas the average coverage by HSPs was 96.8%. Among all other species, the one yielding the highest score was 'Niablella koreensis'  were deN. soli in a 16S rRNA based tree. The sequences of the two 16S rRNA gene copies in the genome differ from each other by one nucleotide, and differ by up to one nucleotide from the previously published 16S rRNA sequence (EF592608), which contains three ambiguous base calls.Balneola and Gracilimonas, listed as belonging to Chitinophagaceae by [Bacteroidetes was conducted, either unconstrained or constrained for the monophyly of all families [Balneola and Gracilimonas are misplaced as members of Chitinophagaceae . Chitinophagaceae should thus be regarded to only contain the genera listed by [Flavitalea [In a preliminary phylogenetic analysis of the 16S rRNA sequences from the family, we observed that two genera, aceae by ,28,29, ffamilies . The alifamilies  , iso-C15:1 G (18.4%), iso-C17:0 3-OH (11.8%), and summed feature 3 (11.1%), which is generally reported to include iso-C15:0 2-OH and/or C16:1 ω7c, although careful examination of the MIDI fatty acid reports generally allow a more precise identification [anteiso-C15 : 0 (1.2%), iso-C15:0 3-OH (2.2%), C16:0 (6.8%), C16:0 2-OH (1.3%), C16:0 3-OH (2.2%), C18:0 (3.8%), C18:1 ω7c (1.5%), C18:1 ω9c (1.0%), Summed feature 5  and an unknown peak with an equivalent chain length of 13.565 (1.1%) were also detected. The presence of major amounts of branched chain saturated and unsaturated fatty acids, together with significant amounts of 3-OH and 2-OH fatty acids is characteristic of members of this evolutionary group and also points to the presence of characteristic lipids, for which data is missing from this strain.The major respiratory quinone found in  Genomic Encyclopedia ofBacteria andArchaea project [This organism was selected for sequencing on the basis of its phylogenetic position , and is  project . The gen project  and the N. soli strain JS13-8T, DSM 19437, was grown in DSMZ medium 830 (R2A medium) [et al. 2009 [ medium)  at 37°C.al. 2009 . DNA is al. 2009 .The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website . PyroseqGenes were identified using Prodigal  as part The genome consists of one circular chromosome of 4,697,343 bp length with a 45.2% G+C content . Of the Chitinophagaceae – Chitinophaga pinensis [N. koreensis (unpublished) – and the permanent draft genome of Sediminibacterium sp. OR43 is available from the IMG/GEBA website [N. soli is most closely related to N. koreensis  but larger than that of Sediminibacterium sp. OR43 (3.8 Mbp). Using the genome-to-genome distance calculator [N. soli and C. pinensis, which corresponds to a DNA-DNA hybridization value of 26.60±2.42%. For N. koreensis, these values were 78.29% and 20.20±2.31%, respectively.Two other complete genomes are available in GenBank from the family pinensis  and N. k website . Of thesoreensis . The genlculator ,50 versiN. koreensis has 228 glycosyl hydrolases, while C. pinensis has 187 [N. soli and strain OR43 and found that they encode 164 and 86 glycosyl hydrolases, respectively. When viewed as a percentage of the total protein-coding sequences, glycosyl hydrolases constitute 4.2% of the N. soli genome and 3.1% of the N. koreensis genome. In the C. pinensis and OR43 genomes, glycosyl hydrolases account for 2.6% of the protein-coding genes. Thus N. soli has the highest density of glycosyl hydrolases in this family examined to date. In addition N. koreensis has 28 polysaccharide lyases while C. pinensis has only six [N. soli has 15 polysaccharide lyases and OR43 has only two. Thus N. soli also has a substantial number of polysaccharide lyases in addition to glycosyl hydrolases.A major feature of the previously sequenced genomes from this family is the presence of large numbers of glycosyl hydrolases.  has 187 . We analonly six . We founN. soli, some are also prevalent in N. koreensis and C. pinensis, for example families GH2, GH28, GH29, GH43, and GH78. However, there are GH families in which N. soli has a greater number of members than the genomes from other Chitinophagaceae – GH20 and GH106. N. soli also has enzymes from GH116 and GH123, which are not found in the other three genomes. There is also one GH family (GH92) for which N. soli has only two members, while N. koreensis and C. pinensis have 10 and 9, respectively.Of the glycosyl hydrolase families with many members in Bacteroides thetaiotaomicron, the SusC and SusD outer membrane proteins are required for starch utilization [B. thetaiotaomicron genome contains many proteins related to SusC and SusD [Chitinophagaceae also contain large numbers of these proteins. N. soli has 60 SusC family and 50 SusD family proteins, which is about half as many as in the larger N. koreensis and C. pinensis genomes.In lization  and the and SusD . The genChitinophagaceae appear to rely mainly on symporters for sugar transport. Only two sugar ABC transporters were found in N. soli, one in N. koreensis, and none in the other two genomes. The phosphotransferase system is not found in any of the four genomes. In contrast N. soli has 23 sugar symporters, N. koreensis has 27, C. pinensis has 14, and OR43 has 12. The sugar symporters belong to several families, with the most prevalent being the Major Facilitator Superfamily (TC 2.A.1) and the Solute:Sodium Symporter Family (TC 2.A.21).The


First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor.The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence  Transgenic mice overexpressing wild-type AR in the prostate epithelium fail to develop PCa in vitro and in vivo cause cellular senescence, which can be attenuated by AR antagonist, anti-androgen flutamide. Cellular senescence is one of the events underlying tumor suppression. Senescence can be triggered by telomere shortening or stress, including inappropriate expression of growth-promoting genes, GTPases or reactive oxygen species (ROS) Here we demonstrate that continuous AR activation promyelocytic leukemia) tumor suppressor In contrast with p53, a p53-related protein p63 opposes cellular senescence. Transcriptionally active p63 isoform, TAp63, can induce apoptosis by activating p53 targets genes; however its inactive, short isoform ΔNp63 can block p53 and TAp63 function in a dominant-negative fashion In our model, AR-induced senescence occurred independently of DNA damage and p53. Instead, it involved increased p21 levels, decreased phospho-Rb and p63. Additionally, we observed increased numbers of PML nuclear bodies due to AR-dependent p63 depletion. P21 expression was directly regulated by AR, as was shown by chromatin immunoprecipitation (ChIP). Paradoxically, p21 had no effect on Rb phosphorylation: the decrease of the phospho-Rb was due to AR-dependent ROS increase. Elevated P21, on the other hand, caused depletion of the ΔNp63. AR-dependent senescence was blocked by p21 or Rb silencing, as well as by ROS quenching and vice versa, mimicked by p63 knockdown. Thus we identified a novel AR function, the induction of senescence, previously not ascribed to any of the nuclear hormone receptors, and delineated underlying signaling pathways.To avoid the loss of androgen sensitivity due to persistent AR expression/activity we generated PC-3 PCa cells expressing tetracycline-inducible wild-type AR (PC3-AR) In AR-positive LNCaP PCa cells persistent AR activation caused similar phenotype . Other sWe then proceeded to verify the possibility of AR-induced senescence in vivo. One possible in vivo experiment, the treatment of male animals with ectopic testosterone, involves testosterone concentrations exceeding physiological levels. The detection of senescence in normal prostate on ambient testosterone background would not unequivocally link senescence to AR and testosterone, since in the only possible control (castration or testosterone depletion) causes apoptosis and rapid involution of the gland, which preclude the detection of senescence. As was reported previously, inducible (tet-on) AR expression inhibits the growth of PC3-AR tumors on ambient testosterone background in male mice We observed no decrease in telomerase activity or telomere following DHT treatment –B, thus We examined AR effect on known mediators of growth arrest and senescence. PC-3 cells are p53 and p16 null ATCC and ; RWPE-1 Previous studies demonstrated that AR increased p21 promoter activity and identified putative ARE AR activation can increase reactive oxygen species (ROS) in LNCaP cells P63, a member of p53 family, is a marker of AR-negative prostate stem cells and transiently amplifying basal prostate epithelium; it is also expressed by less differentiated, more aggressive PCa One possible p63 target involved in senescence is PML tumor suppressor whose link to senescence is well documented Growth arrest may be resolved via quiescence or senescence and the choice is dictated by mTOR activity. In the absence of p53, low mTOR activity allows cells to remain quiescent while high mTOR activity promotes senescence Stress-induced senescence can cause cytokine release reciprocated by the host inflammatory response, which has tumor promoting effect We have shown that persistent AR activity promotes cellular senescence in prostate cancer and in normal prostate epithelial cells. Senescence is recognized as a tumor-suppressive event whereas the proportion of senescent cells is high in pre-cancerous lesions and falls in the course of malignant progression Unaltered telomerase activity suggested that AR activation caused either DNA damage- (DDR) or stress-induced (SIS), rather than replicative senescence. The involvement of Ras in AR-dependent cellular senescence was unlikely, since Ras expression and activity are constitutively high in PaCa cells regardless of AR status DDR and SIS can cross-communicate via p53 downstream target, p21, an inhibitor of Cdks, or via MDM2 downregulation downstream of Rb, which increases p53 stability G1 arrest after 3-day DHT exposure was followed by a significant increase in senescence at day 5 and thereafter and senescence increased after the passage of the surviving cell population or the transfer to androgen-free environment, further supporting senescence and not temporary growth arrest (quiescence). At the same time, PC3-AR cells remained resistant to stress-induced apoptosis, despite the decrease in pro-survival Bcl-2, likely due to lacking p53. Seeking molecular mechanisms of AR-dependent growth arrest, we identified p21 as direct AR transcriptional target. On the other hand, we detected striking reduction of phospho-Rb downstream of AR, which, to our surprise, was not obliterated by p21 knock-down. Moreover, the pattern of Cyclin D1 decrease over time bore no direct correlation with p21 or Rb. On the other hand, AR activation enhanced ROS accumulation, which appeared critical for Rb dephosphorylation as was shown by ROS quenching. Increased ROS levels may be caused by the elevated PKCδ expression downstream of AR, which was documented previously Arf (p14Arf in humans) to the nucleoplasm and subsequent p53 activation TA-p63 and ΔNp63 were expressed in PC3-AR and RWPE-1 cells and ΔNp63 significantly downregulated upon AR activation. A member of p53 family p63 has been linked to senescence, although its mechanisms are not completely understood. The role of p63 PCa is complex; while in most cases PCa tissues stain negative for p63 The fact that AR induces senescence at a much lower rate in RWPE-1 cells lacking both p53 and Rb, compared to Rb-positive PC-3 cells suggest that AR activation triggers parallel pathways, which both contribute to senescence and may be engaged in a cross-talk . In one In cells that like PC-3 are p53-deficient, senescence mTOR promotes senescence AR induction of cellular senescence may be interpreted as a tumor suppressive action, or as inappropriate oncogene activation, similar to Ras and Myc In conclusion, our study clearly demonstrates that there is more than one side to AR activation: the canonical pro-proliferative, oncogenic AR activity is counterbalanced by its less studied activity where it promotes terminal differentiation and senescence. Our observations may explain why initial robust clinical response to androgen ablation is frequently followed by emergence of a more aggressive hormone-independent PCa.PC3 cells inducibly expressing wild-type AR (PC3-AR) have been established previously nu/nu, National Cancer Institute, 4–6 weeks), 106 cells/site, 5 animals/group, 2 sites/animal. To induce AR expression, Dox (1 mg/ml) was given in drinking water. The tumors were measured every 3 days; the volumes were calculated as length×width2×0.52. Flutamide  was given in drinking water. LNCaP cells were inoculated into the flanks of the same numbers of female athymic mice; the mice simultaneously received s.c implants of DHT or mock control pellets . Absorbance was measured in the microplate reader .3, 1% FBS) and fixed overnight . Fixed cells were rinsed in wash buffer with 0.25% Triton-X100, re-suspended in wash buffer with FITC-conjugated Cyclin B1 monoclonal antibody  and incubated 1 hr (RT). The cells were washed, re-suspended in 0.5 ml propidium iodide solution  and analyzed by flow cytometry (ModFit software). FITC-conjugated mouse IgG  served as a negative control.Cells were collected by trypsinization, washed 2 times in wash buffer  and rinsed several times in PBS. The coverslips were incubated overnight at 37°C in staining solution 2, 1∶20000 and 1∶10000 dilution, respectively) were added at RT for 45 min in 5% milk/TBS-T. Bands were visualized with chemiluminescent substrate . Tubulin or GAPDH served as loading controls for the total and cytoplasmic extracts; TATA binding protein (TBP) was used for the nuclear extracts.Total cell lysates were prepared in RIPA buffer ; cytoplasmic and nuclear extracts were prepared using CNM compartment protein extraction kit . Protein extracts (20–50 µg/sample) were separated on 4–20% gradient PAAG, transferred to PVDF membranes and probed with appropriate primary antibodies  followin5/well) 24 hrs before transfection in antibiotic-free growth media. siRNA were diluted in 200 µl serum-free media to final 100 nM. DharmaFECT reagent was diluted 1∶50 in serum-free medium, mixed 1∶1 with diluted siRNA, incubated for 20 min at RT and added to the cells (400 µl per well) for 24 hrs.siRNA  and scrambled siRNA controls were from Dharmacon, . The cells were seeded in six-well plates (3×105′-GCTCGAGAGGATGGAAGTGCAGTTAGG and 5′-CGGATCCGCTTCACTGGGTGTGGAAATAGATG primers) ligated into Xho I/Bam H I sites of a bicistronic lentiviral vector pLVX-IRES-ZsGreen1 .Human AR cDNA  was PCR-amplified  with lentiviral vector plasmid (pLVX-AR or pLVX control) and packaging plasmids pPAX2 and pMD2.G. After overnight exposure, the transfection media was replaced with complete growth medium and the incubation continued for 48 h. Viral supernatants were harvested and concentrated using Lenti-X concentrator . To determine titers, concentrated supernatant was serially diluted and transfected into HEK 293T cells in a 24-well cell plate and efficiency determined by ZsGreen fluorescence. RWPE-1 cells were transduced with pLVX-AR or pLVX lentiviral particles at MOI 10. Transduced cells were visualized by epi-fluorescent microscopy and enriched by FACS. AR expression was verified by Western blotting.Total RNA was extracted using GeneElute extraction kit  following the manufacturer's protocol. RNA (0.5 µg) was converted to cDNA with Superscript III enzyme  and Q-PCR performed with iQ SYBRGreen Supermix  using primers in 4/ml) were seeded on glass coverslips, allowed to attach, AR was induced and activated. At indicated time points the cells were fixed 10 min in methanol (−20 C). All subsequent steps were done at room temperature (RT). The coverslips were washed with PBS, incubated 30 min in blocking solution  and 1 hr in blocking solution with primary antibodies. The slides were washed in PBS and incubated 30 min with secondary antibodies conjugated with respective fluorescent marker (Cells (2×10t marker  and washROS levels were assessed by Dihydroethidium (DHE) fluorescence . PCa cells were grown on coverslips. DHE was added for the last 30 min of treatment to 10 µM final concentration. The cells were washed in PBS, fixed in 1.6% PFA and fluorescence was evaluated by epi-fluorescent microscope (Nikon Diaphot 2000).7/condition) were cross-linked with formaldehyde , harvested and lysed. The lysates were sonicated on wet ice to obtain ∼1 kb fragments , sheared DNA precipitated with AR antibody . For each sample pull-down with IgG was run as a negative control. DNA-protein complexes were isolated on Protein G agarose beads, cross-linking reversed, protein digested with proteinase K. DNA was purified on spin columns and real-time PCR performed with p21 promoter primers ((−ΔΔCT [exp-con]) .We used EZ-ChIP kit  following manufacturer's protocol. The cells (∼10 primers . Input gChemokine detection was performed in conditioned media (CM) after 3 and 5 days of treatment with Dox, alone or in combination with DHT and Flutamide. CM was collected, cleared of debris by cenrifugation. Samples were tested in triplicates using Q-Plex™ Human Cytokine/Chemokine array . The results were normalized per mg of protein in respective cell lysates, to account for different proliferation rates.Figure S1AR causes cellular senescence. (A) AR activation did not cause increased apoptosis. PC3-AR cells were cultured in Dox 2, 5 and 7 days with (▪), or without (□) DHT. The cells were harvested, and cell cycle distribution analyzed by flow cytometry of propidium iodide stained cells. The percentage of cells with decreased DNA content (Sub-G0 population) is reported. (B) AR activation caused morphology consistent with senescence or autophagy: PC3-AR cells were cultured on coverslips in Dox, with or without DHT. Note flat, vacuolated morphology upon DHT treatment. (C) AR activation failed to increase autophagy marker, Beclin (BCN-1). PC3-AR cells cultured in Dox, with DHT or without DHT for 1, 3 and 5 days and BCN-1 expression evaluated by Western blot. * indicates non-specific band. (D–E) LNCaP cells mount senescence response to DHT. (D) Representative images of LNCaP cells cultured for 3 days with DHT or control EtOH. Senescence was measured using SA-βGal assay. Note increased βGal positivity upon AR activation. (E) Senescent cells were counted on the digital images of 5 random fields using Image Tool 3.00 software (UTHSCSA); means of three independent experiments with S.D.M. are shown. (F) AR-positive senescent cells fail to recover. PC3-AR cells were grown in testosterone-free maintenance media , or treated with Dox/DHT . After 5 days the cells were treated with Dox/DHT for additional 3 and 6 days  or transferred in testosterone-free maintenance media for the same time period . Senescence was measured as % of β-Gal positive cells.(PDF)Click here for additional data file.Figure S2AR activation halts tumor growth.  Male nude mice were injected as in experiment shown in 6 PC3-V , or PC3-AR cells (B). Dox was administered in drinking water; Flutamide (Fl) was given in subcutaneous pellets, as above. Sham pellets were used as control. The arrows indicate the beginning of Dox treatment. Note decreased volume of the PC3-AR tumors in Dox/DHT-treated group and reversal by Fl.  To test the effect of testosterone on AR-positive LNCaP tumors castrated male mice are traditionally used. The use of ectopic DHT precluded chemical castration. Surgical castration of 20–30 male mice is a technically challenging and lengthy procedure. We therefore used female nude mice, which lack testes and therefore the bulk of circulating and tissue androgens. LNCaP cells in Matrigel (2×106 cells/0.1 ml/site) were injected subcutaneously in the flanks (10 mice per treatment group). Mice were given subcutaneous pellets containing DHT or sham pellets (control). Tumors were measured every 3 days. The arrow indicates the beginning of DHT treatment (pellet implantation). Note the lower tumor volume in the DHT-treated group (P<0.002). (E) Senescence was measured in snap-frozen tumor sections using SA-βGal assay.(PDF)Click here for additional data file.Figure S3AR-induced senescence did not involve telomerase or DNA damage response. (A) Telomerase in the cells treated with Dox, or Dox/DHT for the indicated time periods. In telomerase-positive samples 6-base ladders are seen, starting at 50 bp, with a 36 bp internal control band (indicated with arrowheads). Extra band between internal control and the ladder indicates higher telomerase activity, but does not affect overall detection. M50, M25, size control ladders. P, control for primer dimers. +veC, positive control ; Q-veC, quantitative control. (B) Average telomere length of the telomeres in the samples: PC3 wt AR : 2.6 kbp; PC3: 3.2 kbp; Control DNA: 8.6 kbp. Lane1-PC3 wtAR dox, lane2-PC3 wtAR dox/dht, lane3- PC3 wtAR dox/dht/flu, lane4- PC3 wtAR untreated, lane5-PC3, lane6-Control DNA (C–D) DNA damage assessment. RWPE-AR and PC3-AR cells were grown on coverslips and treated 24 hours as indicated, fixed and stained for phosphorylated histone γH2-AX (C), to assess DNA damage. The extent of DNA damage was quantified as the average number of γH2-AX positive foci per nucleus in at least 300 cells (D). While PC3-AR tumor cells predictably showed higher extent of DNA damage compared to the normal RWPE-1 cells, the moderate differences between DHT-treated and control cells were not statistically significant .(PDF)Click here for additional data file.Figure S4Senescence-associated proteins in DHT-treated PCa cells. (A) p16 levels in PCa cells were found below detection (measured by Western blot). PC3-AR cells were treated as indicated and western blot of total cell lysates probed with p16 antibodies.  LNCaP cells were treated with DHT (D) or DHT/Flutamide (DF) for 3 days. C indicates untreated control (0.1% EtOH). Total (T) or nuclear (N) cell extracts were isolated. Nuclear extracts were probed for AR and TATA binding protein (TBP), to assess loading; total extracts were probed for p53 and tubulin. (D) PC3-AR cells were treated with Dox and DOX/DHT, where indicated. Phospho-Rb and Cyclin D1 were assessed by Western blot. Note the difference between proteins in the regulation patterns. (D) PC3-AR cells were treated for up to 5days with Dox, DHT and flutamide at indicated combinations and Bcl-2 was measured by Western blot. Note decreased Bcl-2 expression in the presence of DHT and the reversal of the effect by flutamide.(PDF)Click here for additional data file.Figure S5Regulation of cytokine secretion by AR. PC3-AR cells were treated with Dox (D) in the presence of DHT (DD) or DHT and Flutamide (DDF) and conditioned media collected after 3 and 5 days. Cytokines were measured in triplicate using multiplex ELISA assay. Only the factors whose expression is changed in the presence of DHT are shown. Statistical significance was calculated by one-way analysis of variance (ANOVA). * indicates P<0.03 and ** P<0.003, respectively.(PDF)Click here for additional data file.Table S1Antibodies used in the study.(PDF)Click here for additional data file.Table S2PCR primers/conditions used in this study.(PDF)Click here for additional data file.