The development of lethal, castration resistant prostate cancer is associated with adaptive changes to the androgen receptor (AR), including the emergence of mutant receptors and truncated, constitutively active AR variants. AR relies on the molecular chaperone HSP90 for its function in both normal and malignant prostate cells, but the requirement for HSP90 in environments with aberrant AR expression is largely unknown. Here, we investigated the efficacy of three HSP90 inhibitors, 17-AAG, HSP990 and AUY922, against clinically-relevant AR missense mutants and truncated variants. HSP90 inhibition effectively suppressed the signaling of wild-type AR and all AR missense mutants tested. By contrast, two truncated AR variants, AR-V7 and ARv567es, exhibited marked resistance to HSP90 inhibitors. Supporting this observation, nuclear localization of the truncated AR variants was not affected by HSP90 inhibition and AR variant:HSP90 complexes could not be detected in prostate cancer cells. Interestingly, HSP90 inhibition resulted in accumulation of AR-V7 and ARv567es in both cell lines and human tumor explants. Despite the apparent independence of AR variants from HSP90 and their treatment-associated induction, the growth of cell lines with endogenous or enforced expression of AR-V7 or ARv567es remained highly sensitive to AUY922. This study demonstrates that functional AR variant signaling does not confer resistance to HSP90 inhibition, yields insight into the interaction between AR and HSP90 and provides further impetus for the clinical application of HSP90 inhibitors in advanced prostate cancer. AR gene, the frequency of which increases with tumor stage and in CRPC [The mechanisms underlying the development of castration-resistant prostate cancer (CRPC), characterized by the survival and proliferation of prostate cancer cells following androgen deprivation therapy (ADT), have been the subject of intense scrutiny over the past decade. The totality of research in this field has demonstrated that CRPC is characterized by sustained androgen receptor (AR) signaling within the “castrate” environment  through  in CRPC -8. Funct in CRPC , and the in CRPC .More recently, the isolation of constitutively active, truncated forms of the AR has revealed another mechanism underlying persistent AR signaling in CRPC. These so-called AR variants (ARVs), which arise due to aberrant splicing and/or structural rearrangements of the AR gene , 12, havThe realization that AR signaling is maintained in CRPC has underpinned the clinical development and recent FDA approval of agents that more effectively target androgen biosynthesis (e.g. abiraterone acetate) or the AR LBD . While abiraterone and enzalutamide have improved the clinical outlook of men with CRPC, they are not curative , 21. As www.clinicaltrials.gov).Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone required for the stabilization and correct folding of > 200 proteins . These “Despite the potential of HSP90 inhibitors for the treatment of prostate cancer, the consequence of HSP90 inhibition has not been comprehensively assessed in the context of AR signaling by aberrant forms of the receptor, such as gain-of-function missense mutants and constitutively-active variants lacking the LBD. This is of particular relevance given the emerging realization that HSP90 and other chaperones act to stabilize mutant oncoproteins that are characteristic of many human cancers , 24. In HSP90 inhibitors, 17-AAG, AUY922 and HSP990, were assessed for their ability to decrease wild-type AR (wtAR) transactivation activity by luciferase reporter assay in transfected PC-3 prostate cancer cells. All three HSP90 inhibitors dose-dependently decreased wtAR transactivation activity but AUY922 was the most potent agent, reducing wtAR transactivation activity by >98% at doses of 25nM and above Fig. . We firsWe next assessed the effect of HSP90 inhibition on transcriptional signaling by the two most clinically relevant ARVs, ARv567es and AR-V7. Both of these variants lack the LBD, are active in the absence of androgen, and are resistant to drugs targeting the LBD , 19. As Inhibition of HSP90 function is known to induce proteosomal degradation of HSP90 client proteins, including AR . To deteTo assess the extent to which the constitutively-active AR variants were resistant to HSP90 inhibition, the transactivation assays were repeated at 10-fold higher doses of HSP90 inhibitor (0-1000 nM). No transcriptional activity by wtAR was observed for any AUY922 dose or any HSP990 treatment ≥ 250nM, an observation that mirrored receptor protein levels Fig. . The redTo further investigate the apparent resistance of ARv567es and AR-V7 to these agents, we determined whether HSP90 inhibition affected the subcellular localization of GFP-tagged forms of the variants. We first confirmed that GFP-wtAR, GFP-AR-V7 and GFP-ARv567es were transcriptionally active in PC-3 cells . SubsequTo further examine the relationship between ARVs and HSP90, we tested whether ARV:HSP90 protein complexes exist in prostate cancer cells by co-immunoprecipitation assays. FLAG-tagged forms of wtAR and the ARVs, which exhibited normal transcriptional activity , were usOur results suggest that transiently-expressed ARVs function largely independently of the HSP90 chaperone. To assess whether HSP90 inhibitors affect the expression and/or stability of endogenously-expressed ARVs, protein and RNA was isolated from VCaP cells that had been treated for 24 h with AUY922. VCaP cells were chosen because they express both AR-V7 and ARv567es and exhibit selection of AR-V7 in response to agents targeting the LBD (e.g. MDV3100) . qRT-PCRex vivo in medium containing AUY922. As previously observed [t test, p = 0.0011). ARV expression was also induced by AUY922: ARv567es (detectable in 7/9 tumours) and AR-V7 (detectable in 8/9 tumors) transcript levels increased by an average of 8.8-fold (p = 0.0493) and 1.6-fold (p = 0.1170), respectively  . Two finin vitro and has been linked to treatment resistance and cell survival in prostate cancer [Collectively, the observation that HSP90 inhibitors have a minimal effect on ARV transcriptional activity and nuclear localization and our inability to detect HSP90:ARV protein complexes indicate that AR-V7 and ARv567es function largely independently of HSP90. Whether other proteins “chaperone” ARVs is currently unknown. Classic members of the HSP90 molecular machinery, such as HSP70, p23, FKBP52 and Cyp40, either bind to the AR LBD or indirectly with AR via HSP90 , 43. As e cancer . At lease cancer . ProtectWhile our discussion has focused on the resistance of ARVs to HSP90 inhibition, the observation that high doses of all three agents at least partially suppressed ARV-driven transcription is worth consideration. We envision at least three possible explanations for this finding. First, we cannot rule out the possibility that ARVs retain a residual level of interaction with, and reliance on, HSP90. Indeed, a subset of the variants retain the hinge region, a proposed HSP90 interaction surface. Second, as discussed above, ARVs may utilize alternative chaperones that could be affected by high concentrations of HSP90 inhibitors. Finally, ARV activity is likely to be indirectly affected by HSP90 inhibition through deregulation of alternative client proteins. For example, it was recently found that suppression of the PI3K-AKT pathway inhibits AR-V7 transcriptional activity , and HSPin vitro [Despite the apparent autonomy of ARVs from the HSP90 chaperone system, we observed that the growth/proliferation of cell lines expressing AR-V7 or ARv567es, either endogenously or in an enforced manner from an inducible promoter, is highly sensitive to HSP90 inhibition. Thus, the growth advantage associated with ARV expression is unable to compensate for disruption of other critical cellular pathways by HSP90 inhibitors. It follows that sensitivity to these agents is largely independent from both classical and aberrant AR signaling axes, a concept further supported by the observation that HSP90 inhibitors are highly effective in both AR-positive and AR-negative settings in vitro . CollectAR gene expression. Importantly, we demonstrated that treatment with the potent HSP90 inhibitor AUY922 caused an increased level of transcripts encoding full-length AR (VCaP cells and primary tumors), AR-V7  and ARv567es . One potential explanation for this finding is that liganded AR can negatively regulate its own expression [ex vivo cultured prostate tumors [AR gene. This hypothesis may also explain why AR inhibition (MDV3100 treatment or knockdown with siRNA) leads to accumulation of AR-V7 and emergence of an alternative transcriptome [in vitro experiments, the consequences of such selection during prolonged treatment of men is unknown. Our findings provide a rationale for clinical application of a combinatorial treatment strategy comprising HSP90 inhibitors and agents that target the AR-NTD.This study also provided insight into the relationship between HSP90 inhibition and pression : thus, te tumors , could dcriptome . Alternacriptome . IrrespeAR-V7 and ARv567es were detectable by qRT-PCR in 8/9 and 7/9 primary tumors, respectively. For AR-V7, this proportion is similar to what has been reported in previous studies , 17. By In summary, our study provides key new insights into the clinical potential of HSP90 inhibitors in cells that are characterized by aberrant AR signaling. Despite the independence of constitutively-active AR variants from the HSP90 chaperone system, HSP90 inhibitors are likely to be effective in CRPC by targeting signaling by wild-type AR, missense AR mutants and other critical pathways required for growth and survival of prostate cancer cells.AUY922 and HSP990 were obtained from Novartis and dissolved in dimethyl sulfoxide (DMSO). 17-AAG and MDV3100 were obtained from the National Cancer Institute and Sellicks Chemicals, respectively, and dissolved in DMSO. Primary antibodies used for Western blotting were AR-N20 , AR-V7 , HSP70 , HSP90α/β , GAPDH  and FLAG . A tubulin antibody  antibody was used for immunofluorescence.4.14-probasin-ARR3-tk-luciferase reporter has been reported [in vitro mutagenesis as previously described [The pGLreported . Plasmidescribed . pcDNA-Aescribed . pcDNA-Hescribed . pEGFP-Aescribed . pCMV-3x7 virus particles per 1 × 106 cells then selected with 1 ug/ml puromycin (Invitrogen) for 10 days. Stably transduced LNCaP lines were maintained in RPMI-1640 containing 10% FBS.VCaP, PC-3 and 22Rv1 human prostate carcinoma cells were obtained from the American Type Culture Collection. All cell lines underwent verification by short-tandem repeat profiling in 2010 by CellBank Australia. PC-3 cells were maintained in RPMI-1640 containing 5% FBS or phenol-red free RPMI containing 5% charcoal-stripped serum (DCC-FBS). 22Rv1 cells were maintained in RPMI-1640 containing 10% FBS or phenol-red free RPMI containing 10% charcoal-stripped serum (DCC-FBS). VCaP cells were maintained in Dulbecco's Modified Eagle's Medium containing 10% FBS, 1% sodium pyruvate, 1% MEM non–essential amino acids, and 0.1 nM 5α-dihydrotestosterone . LNCaP lines expressing cumate-inducible 3xFLAG-wtAR, 3xFLAG-ARv567es and 3xFLAG-AR-V7 lentivirus were made using the SparQ cumate switch lentivector system (Systems Biosciences). pCDH-EF1-CymR-T2A-Puro vectors were packaged into lentiviral particles using pPACK packaging systems (System Biosciences). To make stable cell lines, LNCaP cells were infected with 1 × 104 cells/well) and then transfected with 100 ng of pGL4.14-probasin-ARR3-tk-luciferase reporter plasmid and 1.5 ng of the appropriate AR expression construct using Lipofectamine 2000 (Invitrogen). Plasmid quantities were chosen to ensure receptor activity was within the linear range. After 4 h, treatment media containing HSP90 inhibitor (0-1000 nM) and 1 nM DHT (Sigma) was added to the cells. Cells were lysed 24 h later with Passive Lysis Buffer (Promega) and frozen overnight at -80°C. Lysates were assayed for luciferase activity using the Luciferase Assay System (Promega) and a plate reading luminometer . Subsequently, protein from six replicate wells was pooled, centrifuged at 10,000g for 10 min at 4°C, and equal volumes of the supernatants were analyzed by SDS-PAGE and Western blotting.AR transactivation assays were performed essentially as described . Briefly4 cells/well in 8-well Lab-Tek chamber slides (Nunc) and left overnight to adhere. The cells were then transfected with plasmids expressing GFP-tagged AR, AR-V7 and ARv567es using Lipofectamine 2000. After 24 h the cells were treated with AUY922 or DMSO (vehicle control) for 1 h in the absence or presence of 1nM DHT or ethanol (vehicle control). The cells were then fixed using 4% paraformaldehyde, washed with PBS and incubated overnight at 4°C with an antibody specific for tubulin. Alexa-Fluor 594 donkey anti-mouse IgG (Life Technologies) was used to visualize tubulin staining. ProLong Gold Antifade Reagent with DAPI (Life Technologies) was used as mounting media and to detect cell nuclei. Cells were viewed using an Olympus IX71 fluorescent microscope and images were obtained with an Olympus DP70 cooled digital color camera at 20x magnification.PC-3 cells were seeded at a density of 3.0 × 102MoO4, 0.5% NP-40 Alternative, 1 mM EDTA, 1 mM EGTA (pH 8.0), 2 mM DTT and protease inhibitors). The lysates were sonicated twice at low power for 15 sec each  and cleared by centrifugation . Samples were immunoprecipitated for 2 h with rotation at 4°C using anti-FLAG M2 agarose beads (Sigma) and then washed three times with IP buffer. Protein was eluted by boiling in SDS-PAGE loading buffer for 5 min and analyzed by SDS-PAGE and Western blotting.AR:HSP90 complexes were isolated by immunoprecipitation as described previously . Briefly4 cells/well in 24-well plates. The next day, day zero counts were taken and cells were treated with 0.3 ug/ml cumate to induce AR and ARV expression. One day later, cells were treated as appropriate with AUY922 or DMSO (control). Cells were counted after a further 72 hours using trypan blue exclusion assays.Trypan blue exclusion counts were performed on 22Rv1 and VCaP cells treated with AUY922. Growth assays were stopped when DMSO-treated cells reached the stationary phase of growth. For LNCaP lines with inducible AR and ARV expression, cells were seeded out at 2 × 10Fresh prostate cancer specimens were obtained with written informed consent through the Australian Prostate Cancer BioResource from men undergoing robotic radical prostatectomy at the Royal Adelaide Hospital. Tissues were dissected and cultured as described previously  in mediu5 cells/well in 12 well plates and treated for 24 h with AUY922 (40 or 160 nM) or DMSO. RNA was extracted using Trizol using standard techniques. For prostate tumors, samples were homogenized using a Precellys tissue homogenizer (Bertin Technologies) or mortar and pestle and RNA was extracted using Trizol. 200 ng of RNA was reverse transcribed and analyzed by SYBR green qRT-PCR as described previously [For cell line experiments, VCaP cells were seeded at a density of 4 × 10eviously . Primers22 40 or 0 nM or Dt test . All statistical analyses were done using GraphPad Prism Software.Changes in the expression level of AR and AR-V7 in VCaP cells treated with AUY922 were assessed using one-way ANOVA with Tukey's post hoc test. Changes in the expression level of AR and ARVs in tumor explants treated with AUY922 were assessed using a one-sample


Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice.The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of  Flowering time is controlled by endogenous and environmental signals. In Arabidopsis, a long day (LD) plant, extensive genetic analyses have identified four flowering pathways: photoperiod, autonomous, vernalization, and gibberellin induced pathways, as well as a series of signaling molecules ArabidopsisRice, a short day (SD) plant, has a similar molecular mechanism to that of Arabidopsis. Rice heading date 3a (Hd3a) and heading date 1 (Hd1) are the orthologs of FT and CO, respectively Arabidopsis, there are 196 genes encoding RNA recognition motif (RRM)- containing proteins FLK, regulates the autonomous flowering pathway via FLC in Arabidopsis Arabidopsis and shown to be involved in flowering time control and cell fate RNA binding proteins are involved in the synthesis, processing, transportation, translation, and degradation of various types of RNA molecules in the cell Spin1 in transgenic rice causes late flowering under SD and LD conditions Spin1 full-length cDNA as the bait. We identified a novel RRM-containing protein that we have named RNA-binding and SPIN1-interacting 1 (RBS1). RBS1 interacts with SPIN1 in vitro and in vivo and binds RNA in vitro. Overexpression of Rbs1 in transgenic rice leads to late flowering under both SD and LD conditions. These results revealed that RBS1 plays a negative role in rice flowering and is a new component in the SPL11-mediated signaling pathway.We previously demonstrated that Spotted Leaf 11 (SPL11), a functional U-box/ARM E3 ligase, is involved in the regulation of programmed cell death (PCD), defense and flowering time in rice Oryza sativa) seeds of mutant, transgenic and wild-type plants were sterilized and germinated in half-strength Murashige and Skoog medium for 7 d, and then transferred to a pot with sterilized soil in a growth chamber with the same conditions as described by Vega-Sánchez et al. (2008). Rbs1-ox plants were generated in the cv. Nipponbare background, while the knockout mutant was obtained from the Postech collection in Korea (cv. Dongjin). For flowering time measurements, plants were grown either in 10/14 h light/dark for SD or 14/10 h light/dark for LD. Rice flowering time was measured in days from germination until emergence of the first panicle. For diurnal expression analyses, young leaves were harvested from wild-type Nipponbare, Rbs1 overexpression lines, Dongjing and rbs1 plants of 50 day-old (SD) or 60 day-old (LD) plants at 4 h intervals for a total of 24 h.Rice . A rice cDNA library available into the pPC86 vector was used as the prey (22). Putative interacting candidates were identified by sequencing of the inserts in pPC86 at the Plant-Microbe Genomics Facility (PMGF) at the Ohio State University.The ProQuest yeast two-hybrid system (Invitrogen) was used to screen for SPIN1-interacting proteins following the manufacturer’s protocol. A full-length DsRed-Rbs1 translational fusion was obtained by cloning an Rbs1 coding sequence PCR fragment containing the BglII and SalI sites into the pGDR vector to make pGRbs1-Red. The GFP-Spin1 translational fusion was made in the pGDG vector (pGSpin1-GFP) as described previously Agrobacterium tumefasciens strain GV3101 and used to agroinfiltrate 4 week-old N. benthamiana plants as described previously The Hd1, Hd3a, Ubq, Rbs1, Spin1 and Spl11 are listed in Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer’s protocol. RNA was treated with DNAse1 (Invitrogen) and 1.5 µg was used for first strand cDNA synthesis with the reverse transcription system from Promega, following the kit’s instructions. Real-time quantitative RT-PCR (qPCR) was performed in a final volume of 50 µL, including 25 µL iQ SYBR Green Supermix (Biorad), 2 µL of the diluted first-strand cDNA as templates and 0.2 µM of each primer. The reactions were carried out with the Biorad IQ5 system in the following program: 94°C for 4 min, 40 cycles of 95°C for 5 s, and 58°C for 20 s, 72°C for 30 s. Every experiment was repeated at least three times. The primers of Rbs1 coding sequence was cloned by PCR into the HindIII and XhoI sites of pET28a to obtain pET-Rbs1. As a negative control, unrelated protein Avr-Pita was cloned into BamHI and SalI site of the same vector and designated as pET-AvrPita. The6XHIS-RBS1 or 6XHIS-Avr-Pita protein was induced in E. coli and purified using HIS-Select® Nickel Affinity Gel (Sigma) according to manufacturer’s instruction. Either purified 6XHIS-RBS1 or 6XHIS-Avr-Pita was incubated with beads containing polyuridylic, and polyguanylic ribohomopolymers as well as calf thymus single- and double stranded DNA purchased from Sigma. Incubation was done in 500 µl of buffer KHN  for 10 min under rotation. Beads were then washed in KHN buffer 5 times and proteins retained in the beads were identified by Immunoblotting using anti-HIS antibody (Thermo Scientific).The Spin1 or Rbs1 cDNA template, followed by ligation into a desired vector. For the BiFC assay, the Rbs1 and Spin1 constructs in the pA7-NYFP and pA7-CYFP vectors were constructed following the procedure by Chen et al. (2006). The Rbs1 overexpression construct was made into the Gateway (Invitrogen)-compatible vectors Ubix.nc1300.ntap.gck, following the Gateway cloning protocols. Two artificial miRNA constructs were made that targeted the 21 bp of Rbs1’s 3'UTR , which was obtained from the WMD3 website (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) In general, constructs were generated by PCR amplification of the target gene using primers containing appropriate restriction enzyme sites and Fluorescence microscopy analyses were done by transfection of rice protoplasts with various constructs as described in vivo Co-IP assay, Agrobacterium strain GV3101 carrying expression vectors of RFP:RBS1and SPIN1:HAwere agroinfiltrated N. benthamiana leaves to express the proteins in vivo. Two days after agroinfiltration, N. benthamiana leaf tissues were harvested, and total proteins were extracted with a native buffer including 50mM K+-HEPES (pH 7.4), 110mM KOAc, 2mM MgCl2, 0.1%Tween-20, 0.2% Triton, and plant protease inhibitor. A 15-µl volume of anti-HA agarose suspension (Sigma) was added to the protein samples loaded in the column, and the mixtures were kept at 4°C with head-to-tail shakingovernight. The samples were washed five times using 1X IP buffer following the manufacturer’s instruction (Sigma). After 50 µl of 1X SDS sample loading buffer was added to each column, samples were heated to 95°C for 5 min. Twenty microliters of each sample was loaded to the protein gel for immunoblot analysis using anti-HA (Sigma) and anti-RFP  antibodies.For The protein extraction and protein gel blot assay were done as described Spin1 cDNA as the bait and a rice cDNA library as the prey. Approximately half a million-yeast colonies were screened and 140 putative positive clones were selected for validation after the first round of screening. Only clones #69-2 and 72 were able to reproduce the interaction based on yeast growth on different selection media and appearance of blue colonies in an X-GAL assay  Arabidopsis, grape (Vitis vinifera) and the moss Physcomitrella patens subsp. patens  technique to test the interaction between SPIN1 and RBS1 in rice protoplasts in silico data, we performed an in vitro RNA binding assay. 6xHis-RBS1 protein was purified from E. coli and incubated with ribohomopolymer beads containing poly U and poly C RNA molecules, as well as calf thymus single and double-stranded DNA. Detection of proteins bound to nucleic acids was performed by western blot analysis using an anti-His antibody after SDS-PAGE and blotting. The assay showed that 6xHis-RBS1 bound to both RNA and DNA molecules in vitro .To analyze the function of  primers . Real-ti primers . However Dongjin . The RbsRbs1 overexpression vector in which the Rbs1 full-length cDNA was fused with an HA tag under the control of the maize ubiquitin (Ubi) promoter . However, like the Spin1 overexpression plants Rbs1 overexpression plants show late flowering under SD and LD conditions. Expression analysis showed that Rbs1 overexpression leads to the suppression of both Hd3a, the rice florigen We identified RBS1 as a novel RNA binding protein of the hnRNP-R type in rice and showed that it interacts with SPIN1 in both yeast and rice protoplasts. To assess the functional relationship between Arabidopsis and 221 proteins in rice Os10g06130 also encodes an RRM-containing protein, which has 88% protein sequence similarity with RBS1. The expression of this gene is not affected in the Rbs1 overexpression and artificial microRNA plants (data not shown). This gene may have redundantfunction with Rbs1 in rice flowering, which might explain our observationthat the rbs1 knockout mutant and artificial microRNA plants did not show any significant flowering time change under SD and LD conditions.Genome analysis showed that there are 196 RRM-containing proteins in Rbs1 overexpression plants, the expression of Spin1 is upregulated, which correlates with enhanced SPIN1 protein accumulation. We have previously shown that the Spin1 overexpression plants flower later than the wild type Nipponbare plants under SD and LD conditions Hd1 is reduced in the Spin1 overexpression plants, indicating that SPIN1 influences flowering time via the Hd1 pathway in flowering-promoting conditions. However, in LD conditions, the Hd1 expression, is the same in Nipponbare and Spin1 overexpression plants Hd3a under LD conditions. By contrast, in this study we found that the expression of Hd1 is similar between the Rbs1 overexpression and wild type Nipponbare plants under both LD and SD conditions . It is plausible that RBS1 is one of the components in the SPL11 protein complex and the interaction between RBS1 and SPL11 requires the presence of another protein that interacts with both proteins, for example SPIN1.Our previous study revealed the negative relationship between SPIN1 and SPL11 in vitro, it might be involved in RNA metabolism to regulate flowering time. In mammals, it has been recently shown that hnRNP-R binds to the 3′UTR of serotonin N-acetyltranferase and to the AU rich element of c-Fos mRNA to promote their degradation Rbs1 is involved in mRNA turnover in rice needs to be determined in future studies. It is interesting to note that the putative ortholog of RBS1 in Arabidopsis, LIF2, is also involved in flowering time control Arabidopsis lif2 loss-of-function mutants with the rice Rbs1 gene should provide clues as to whether these pathways are conserved or not between the two species.Because RBS1 is a putative RNA binding protein and has RNA binding activity Figure S1Identification of Rbs1 by yeast two-hybrid screen in a rice cDNA library using the full length Spin1 as the bait. Numbers 72 and 69-2 are independent yeast clones containing the pDBleu-Spin1 and pPC86-Rbs1 constructs. Positive clones were identified by an X-GAL assay.(TIFF)Click here for additional data file.Figure S2Molecular analysis and flowering time of the rbs1 mutant. A. Structure of the T-DNA insertion. T-DNA was inserted into the fourth intron of Rbs1. Arrows indicate the primers used for analyzing the insertion site. LB and RB represent the left and right borders of T-DNA; B. PCR genotyping of the rbs1 mutant. DJ denotes Dongjin, 1 is a segregated wild type, 2 and 3 are homozygous plants and 4 is a heterozygous plant; C. Expression of Rbs1 for the plants in S3B; D. Flowering time of the rbs1 mutant under LD and SD conditions.(TIFF)Click here for additional data file.Figure S3Western blot analysis of the protein level of RBS1 in the Rbs1 overexpression and wild type plants.(TIFF)Click here for additional data file.Figure S4Expression analysis of Rbs1 in the Spin1 overexpression plants, spl11 mutant plants and wild type Nipponbare (NPB) plants. A. RT-PCR analysis of Rbs1 and Spin1 in the Spin1 overexpression and RNAi transgenic plants; B. RT-PCR of Rbs1 in the leaves of the wild type IR68 and spl11 mutant plants at different developmental stages. For spl11, young (Y) without lesions, fully expanded (FE) with few lesions and old leaves (O) with many lesions were taken for RNA extraction; C. RT-PCR of Rbs1 in leaves from 55 day-old plants of the wild type IR64 and spl11 mutant. D. Western blot analysis of RBS1 in the spl11 mutant and Spl11 overexpression lines.(TIFF)Click here for additional data file.Table S1(DOCX)Click here for additional data file.


C-terminally truncated AR lacking the AR-ligand binding domain (LBD). Due to the absence of a LBD, these receptors, termed ARΔLBD, are unable to respond to any form of anti-hormonal therapies. In this study we demonstrate that the multikinase inhibitor sorafenib inhibits AR as well as ARΔLBD-signalling in CRPCa cells. This inhibition was paralleled by proteasomal degradation of the AR- and ARΔLBD-molecules. In line with these observations, maximal antiproliferative effects of sorafenib were achieved in AR and ARΔLBD-positive PCa cells. The present findings warrant further investigations on sorafenib as an option for the treatment of advanced AR-positive PCa.Recent evidence suggests that the development of castration resistant prostate cancer (CRPCa) is commonly associated with an aberrant, ligand-independent activation of the androgen receptor (AR). A putative mechanism allowing prostate cancer (PCa) cells to grow under low levels of androgens, is the expression of constitutively active,  Prostate cancer (PCa) is the most common neoplasm and the third leading cause of cancer-related deaths in elderly men of the western world . LocalizC-terminally truncated AR-variants has emerged as an important mechanism of CRPCa cells to grow and survive under subphysiological levels of circulating androgens [C-terminus, these AR-variants are referred to as ARΔLBD. Several molecular mechanisms enabling CRPCa cells to synthesize ARΔLBD have been identified: Most ARΔLBD found in cell lines and tissue specimens of metastatic PCa are products of alternative splicing (AR-V) [Q640X) [N-terminal domain and the DNA-binding domain (NTD/DBD core) is sufficient for AR-variants to access the nucleus and to activate AR-target genes [Continued androgen receptor (AR)-signalling remains the dominant growth pathway in prostate cancer progressing under low levels of circulating androgens . Variousndrogens –7. As thg (AR-V) ,8,9. How [Q640X)  have als [Q640X) ,6. Althoet genes . Due to et genes . Thus, tet al. reported that the multikinase inhibitor sorafenib  was able to decrease proliferation of PCa cells via inhibition of the canonical AR-signalling pathway. Inhibition of AR-signalling was paralleled by a downregulation of AR protein-levels [N-terminus [Q640X as well as the AR/AR-V expressing PCa cell line 22Rv1 as experimental models.AR-function as well as AR-stability largely depend on post-translational modifications like phosphorylation on serine/threonine or tyrosine residues ,14. Recen-levels . As mostterminus , a regioversus DHT-treated controls: 23% ± 11%, p = 0.034), reaching its maximum at 10 μM,  reaching its maximum at 10 μM  . As LNCaet al. it is tempting to speculate that the multikinase-inhibitor sorafenib targets the AR phosphorylation via blockade of a yet undefined kinase [N-terminus of the receptor molecule, we hypothesized that sorafenib might also affect phosphorylation of the ARΔLBD-N-terminus.Posttranslational modifications like phosphorylation on serine, threonine or tyrosine residues are involved in a large variety of steroid receptor functions ,14. Based kinase . As the Q640X, the product of a nonsense mutation leading to a stop codon in the hinge region adjacent to the LBD of the AR [Q640X was shown to be very strong on artificial androgen-responsive promoters (ARE(2x)) but was very weak on the PSA promoter [C-terminally truncated ARQ640X in a dose-dependent manner. Inhibition was significant at a concentration of 5 μM , reaching its maximum at 10 μM . The data suggest that sorafenib affects wild type AR and ARΔLBD signalling in a similar way.As an experimental model we transfected PC-3 cells with the AR-mutant ARpromoter ,18. In cpromoter –19. As sQ640X is largely sufficient to perform reportergene assays , predominant nuclear localization in the absence of androgens and constitutive activity as shown by activation of PSAP1 luciferase reporter plasmid [There is experimental evidence that kinase inhibitors directed against p42/p44 MAPK, GSK-3β or CDK1 are able to trigger AR-signalling by modulating AR-protein levels –22. Recee assays , the expnt AR-V7 . Althoug plasmid .Interestingly, sorafenib was able to diminish both full length AR as well as AR-V in 22Rv1 cells. Downregulation of AR and AR-V protein levels following sorafenib treatment could be rescued by the proteasome inhibitor MG132, the latter suggesting that sorafenib induces a proteasomal degradation of AR- and ARΔLBD molecules in PCa cells .Q640X-fusion proteins. As seen in Q640X.Besides its effects on AR-stability, various kinase inhibitors were shown to modulate the intracellular localization of the AR ,24–26. Iversus PC-3: 82% ± 3%, p = 0.002 and p = 0.036, respectively).Based on the observation that sorafenib is able to inhibit AR as well as ARΔLBD-signalling we further investigated the antiproliferative effects of the compound on the androgen sensitive LNCaP (AR+) cells, the castration resistant 22Rv1  cells as well as the androgen insensitive PC-3 (AR−) and DU-145 (AR−) cells using a MTT cell viability assay . As depiC-terminally truncated ARQ640X (aa 1–640) were provided by and Dr. J. Céraline . The PSA reporter plasmid pPSA-61luc under control of a 6kb-fragment of the human PSA-promoter was a generous gift of Dr. J. Trapmann . The artificial ARE(2x) reporter plasmid pLC0548 (pARE(2x)-luc) under control of a synthetic ARE-promoter was created by H. Lebedur and provided by Dr. A. Allera . Renilla reniformis luciferase reporter plasmid (pRL-TK) was purchased from Promega . Dihydrotestosterone (DHT) and the proteasome inhibitor MG132 were purchased from Sigma-Aldrich GmbH . Sorafenib was a product of LKT Laboratories Inc. . All other chemicals, if not specified, were products of Sigma-Aldrich GmbH .pSG5-AR encoding a wild-type full-length AR (919 amino acids) was supplied by Dr. H. Klocker . pAR-t1EosFP coding for a green fluorescent Eos-AR-fusion protein was a generous gift from Dr. F. Oswald . pCruz-ARQ640X and pEGFP-ARQ640X coding for the PC-3, DU-145, 22Rv1 and LNCaP cells were purchased from the American Type Culture Collection . Cell culture was performed as recently described .PC-3 cells were transiently cotransfected in 24-well plates with AR-expression plasmids  and different reporter gene constructs -luc) using the transfection reagent Polyfect . pRL-TK was cotransfected as an internal control for transfection efficiency. Subsequently, cells were treated with/without 1 nM DHT. After 24 h, reporter gene activity was measured using the Dual-Luciferase Reporter Assay . In this experimental set-up, the PSA- and ARE-reporters are correlated with the effects of the specific experimental conditions, while the activity of the co-transfected constitutive pRL-TK reporter provides an internal control that serves as the baseline response. Normalizing the activity of the experimental PSA- and ARE-reporters to the activity of the internal control minimizes experimental variability caused by differences in cell viability, transfection efficiency, general effects on transcription, translation or protein stability. All experiments were performed as recently described .PC-3-cells were seeded in 24-well plates and grown in the absence of DHT for 24 h. Subsequently, cells were transfected with pAR-t1EosFP and pEGFP-ARQ640X. After 24 h, cells were treated with ethanol (solvent control), 10 nM DHT, ethanol + 5 μM Sorafenib or 10 nM DHT + 5 μM Sorafenib for 2 h. The fluorescent cells were subsequently counted using a fluorescent microscope as recently described ,28.Cell viability was determined by means of a colorimetric MTT assay. This assay is based on the the reduction of tetrazolium salts to formazan derivatives by functional mitochondria. The assay was performed as described by Mosmann .v/v) (TBS-T). Beta-actin served as a loading control using a mouse monoclonal antibody directed against beta-actin . Immunoreactive bands were detected using a 1:20.000 dilution of peroxidase-coupled secondary goat anti-mouse IgG  in TBS-T. Signals were visualized by the ECL Plus Chemiluminescent Substrate from Pierce .Total proteins were extracted from cells using RIPA buffer. 40 μg of lysate were separated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page). Subsequently proteins were transferred onto a PVDF membrane  by semi-dry blotting. AR and ARΔLBD were detected using the monoclonal antibody AR441  at a dilution of 1:2.000 in Tris buffered saline, 0.1% Tween20 . One-way ANOVA results were confirmed with Bonferroni’s multiple comparison tests. All analyses were performed with the use of SPSS Statistics .Only recently, the efficacy of various tyrosine kinase inhibitors has been evaluated in the treatment of advanced PCa –33. So fIn vitro studies analyzing the precise molecular effects of the multikinase inhibitor sorafenib on CRPCa cells are sparse [et al. showed that sorafenib affects AR-signalling in PCa cell lines grown in presence of the synthetic androgen R1881. Inhibition of the canonical AR-signalling pathway after sorafenib treatment was due to a downregulation of AR-levels by an unknown mechanism [C-terminally truncated, constitutively active AR variants (ARΔLBD), the latter are thought to be key players in the development of CRPCa. Interestingly, inhibition of AR- as well as ARΔLBD-signalling was paralleled by a sorafenib-induced proteasomal degradation of both receptor types. Consistently, the antiproliferative effects of sorafenib in our experimental model were the most pronounced in AR- or AR/ARΔLBD-positive PCa cells as compared to their AR-negative counterparts (proliferation rate: 22Rv1/LNCaP < PC-3/DU-145). Our finding of a dose-dependent decrease in PCa cell growth after sorafenib treatment is in accordance with previous findings by Oh et al. [et al. At least partially, this may be due to the varying experimental settings used in both studies. Oh and colleagues determined the antiproliferative effect of sorafenib treatment by measuring 3H-thymidine incorporation after 48 h in PCa cells that were grown and treated in serum free HITES medium . By contrast, in our experiments PCa cells were grown in RPMI-1640 under standard conditions , cell viability following sorafenib treatment was determined after 72 h using an MTT-assay which reflects mitochondrial activity by measuring the reduction of tetrazolium salts to formazan derivatives. Therefore it is conceivable that the concentration dependencies may differ in both studies.e sparse ,39. Receechanism . To our h et al. . Howeveret al. presented encouraging results using a combination therapy of sorafenib with the nonsteroidal anti-androgen bicalutamide in 39 chemotherapy naïve CRPCa patients. PSA declines or stable disease (≥6 months) were observed in 47% of patients including 26% of patients previously progressing on bicalutamide monotherapy [In line with our observations, Beardsley otherapy .In summary, the present results suggest that a subset of advanced CRPCa patients, especially those who are expressing AR and/or ARΔLBD, might benefit from a sorafenib treatment. Moreover, strategies to combine multi-targeted kinase inhibitors like sorafenib with hormonal therapies warrant further experimental studies in CRPCa.


Intra-tumoral steroidogenesis and constitutive androgen receptor (AR) activity have been associated with castration-resistant prostate cancer (CRPC). This study aimed to examine if CRPC bone metastases expressed higher levels of steroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation to expression of constitutively active AR variants (AR-Vs) and to clinical and pathological variables.Untreated, hormone-naïve  and CRPC bone metastases samples (n = 45) were obtained from 54 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry and immunoblotting. No differences in steroidogenic enzyme levels were detected between CRPC and HN bone metastases. Significantly higher levels of SRD5A1, AKR1C2, AKR1C3, and HSD17B10 mRNA were however found in bone metastases than in non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNA levels in metastases were significantly lower. A sub-group of metastases expressed very high levels of AKR1C3, which was not due to gene amplification as examined by copy number variation assay. No association was found between AKR1C3 expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only one exception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individual bone metastases.nd line androgen-deprivation therapy.Induced capacity of converting adrenal-gland derived steroids into more potent androgens was indicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage of metastasis. Sub-groups of bone metastases could be identified according to their expression levels of AKR1C3 and AR-Vs, which might be of relevance for patient response to 2 The first-line treatment for patients with advanced prostate cancer (PC) is androgen deprivation therapy (ADT). This therapy is effective in most patients, but after a period of initial remission tumors generally relapse, predominantly within the bone, and are then termed castration-resistant PC (CRPC).nd line ADT and this might be due to intra-tumoral steroidogenesis and production of testosterone, dihydrotestosterone (DHT), or other androgens at levels high enough for AR activation, as reported by others In spite of low circulating levels of androgens in castrated men the majority of CRPC tumors show androgen receptor (AR) activity and expression of AR regulated genes The study was approved by the local ethic review board of Umeå University and participants gave written or verbal consent. Due to the acute situation when bone metastasis surgery is performed in order to relief spine symptoms and paresis, logistics do not always allow written consent and the local ethic review board therefore specifically approved also verbal consent. Verbal consent is documented by the physician in the patient journal.Bone metastasis tissue was obtained from series of fresh-frozen and formalin-fixed biopsies collected from patients with PC operated for metastatic spinal cord compression or pathologic fractures at Umeå University Hospital (2003–2011). Clinical characteristics and therapies are summarized in Representative areas of bone metastases, primary PC and non-malignant prostate tissue were cryo-sectioned into extraction tubes and RNA was isolated using the Trizol protocol  or AllPrep DNA/RNA/Protein Mini Kit . For cases were DNA and protein were analyzed in parallel to RNA, nucleic acids and protein were isolated from the same tissue sections using the AllPrep method. The percentages of tumor cells in the samples were above 50% in the majority of cases. The RNA and DNA concentrations were quantified by absorbance measurements using a spectrophotometer . Protein concentration was determined by the BCA Protein assay . The RNA quality was analyzed with the 2100 Bioanalyzer  and verified to have a RNA integrity number ≥6.RNA was reversed transcribed with Superscript II reverse transcriptase (Invitrogen) in a total volume of 10 µl. The subsequent PCR amplification was performed using the Biorad iQ5 iCycler  or the ABI PRISM 7900HT Instrument  in a total volume of 20 µl using the IQ SYBR Green Supermix (Bio-Rad laboratories) or the TaqMan gene expression mastermix  according to manufacturers' protocols. Primer sequences are shown in Samples were fixed in buffered formalin, decalcified in formic acid, and embedded in paraffin The copy number of the AKR1C3 gene in PC bone metastases was examined using the Hs03060693_cn and the Hs02574521_cn TaqMan copy number assays , targeting AKR1C3 exon 2 and exon 7, respectively. The assays were run according to the manufacturer's description, with RNaseP as reference gene, and analyzed using the Copy Caller software (Applied Biosystems).22Rv1 cells were maintained according to manufacturer's instructions (ATCC) and protein was extracted as above. Samples (10 µg protein) were separated by 7.5% SDS-PAGE under reducing conditions and subsequently transferred to PVDF membranes . Membranes were blocked in 5% milk followed by primary antibody incubation in 4°C over night  in order to detect the full length AR and approximately 80 kDa truncated AR-Vs with intact N-terminals. The secondary anti-rabbit IgG  antibody (diluted 1∶20 000 in 2.5% milk) was applied after washing in PBST and incubated for 1 h in RT. Protein expression was visualized after extensive washing using the ECL Advanced detection kit . Filters were then stripped, according to standard procedures, and analyzed for actin protein levels using the rabbit anti-actin antibody from SIGMA (Saint Louis. Missouri), diluted 1∶8000, in order to ensure equal sample loading. ECL signals were quantified using a ChemiDoc scanner and the Quantity One 4 software (Bio-Rad Laboratories). The intensity of the 80 kDa band in each patient sample was analyzed and expressed in relation to the corresponding full length AR band intensity. The 22Rv1 cell extract was run as a positive control sample on each gel.Correlations between variables were analyzed using Spearman rank test. Groups were compared using the Mann-Whitney U test for continuous variables and the Chi-square test for categorical variables. Kaplan-Meier survival analysis was performed with death of PC as event and follow-up time as time between metastasis surgery and the latest follow-up examination (December 2012). Statistical analyses were performed using the Statistical Package for the Social Sciences, SPSS 20.0 software . A P-value less or equal to 0.05 was considered statistically significant.Gene expression levels of key enzymes involved in the formation of testosterone and DHT from cholesterol  were assNone of the analyzed enzymes in the steroidogenesis pathway  and 2 shTranscript levels of enzymes in the early steps of steroidogenesis were reduced in HN and CRPC bone metastases as compared to non-malignant prostate tissue ; median Transcript levels of many enzymes involved in later steps of androgen synthesis were increased in metastases compared to non-malignant prostate and primary tumor tissue . The medde novo synthesis of androgens from cholesterol is less likely.Taken together those results indicate that PC bone metastases in patients may have induced capacity to convert androgens with low AR affinity into more potent androgens, while Although none of the examined steroidogenic enzymes showed significantly increased expression levels in CRPC compared to HN bone metastases, individual CRPC metastases were noted to express very high transcript levels . One enzTo determine whether the AKR1C3 mRNA levels in the metastases were reflected in the corresponding protein levels, we assessed the AKR1C3 protein expression by immunohistochemistry and by using a scoring system that took both staining intensity and distribution into account . CytoplaIn the CRPC bone metastases, the AKR1C3 total staining score was correlated to the UGT2B15 mRNA levels , which might indicate DHT production in metastases with high AKR1C3 protein expression. It was however obvious that high AKR1C3 protein expression was not directly linked to high AR activity in CRPC metastases; 14 out of 18 (78%) samples with high AKR1C3 staining scores, defined as an AKR1C3 immunostaining score above median (6), and 15/25 (60%) samples with low AKR1C3 staining scores (≤6) showed high nuclear AR staining scores .The AR protein levels were studied by immunoblot analysis of 29 CRPC bone metastases selected based on AR immunohistochemistry results to include AR low to AR high score metastases, and where adequate tissue remained to allow immunoblot analysis. The full length AR of approximate 110 kDa was detected in most cases while truncated AR-Vs of approximately 80 kDa, hypothesized to represent AR splice variants lacking the LBD C-terminal domain, were detected in some cases . The intnd line ADT and clinical progression Interestingly, only one out of 13 (7.8%) CRPC metastases with high AKR1C3 expression (immunostaining score above median) also showed high AR-V protein levels. This was a significantly lower fraction than found among metastases with low AKR1C3 levels . We further hypothesized that tumor cell expression of AKR1C3 and constitutively active AR variants could be of relevance for individual tumor response to 2Notably, none of the metastases with homogeneous AKR1C3 staining, i.e fraction of positively stained cells above 75%, showed high AR-V levels compared to 7/15 metastases with <75% positively stained cells (P = 0.004). This observation deserves to be further examined, as it might indicate that high AR-V levels are induced preferable in tumors cells without endogenous steroidogenesis. Unfortunately, we were not able to study this hypothesized heterogeneity among tumor cells as antibodies for immunohistochemical evaluation of AR-Vs were not available.nd line therapies.Intra-tumoral synthesis of testosterone and DHT has been suggested to contribute to CRPC growth. In this study we compared expression levels of steroidogenic enzymes in CRPC bone metastases with levels in metastases obtained from un-treated patients and, surprisingly, found no induction of the examined enzymes in CRPC compared to HN metastases. We however found high expression levels of certain steroidogenic enzymes; SRD5A1, AKR1C2, AKR1C3, and HSD17B10, in bone metastases when compared to non-malignant prostate and primary prostate tumor tissue. Furthermore, we were able to show distinct differences in expression levels of AKR1C3 and levels of AR splice variants in individual cases of CRPC bone metastases, which we hypothesize could be of relevance for patient response to 2de novo synthesis from cholesterol de novo synthesis of androgens from cholesterol within metastases is less likely, except maybe in individual cases with correlated expression of CYP11A1, CYP17A1, and HSD3B2.Tissue androgens might be derived by We found high expression levels of AKR1C3 in a sub-group of PC bone metastases, which confirm results previously reported in castration-resistant primary prostate tumors In conclusion, we want to emphasize that increased tumor expression of steroidogenic enzymes in individual patients is not clearly associated with CRPC but merely associated with advanced tumor stage, as it can be seen not only in CRPC tissue of different origin nd line therapies of CRPC needs to be tested upfront in clinical studies.A limitation to this work is the restricted number of bone metastases, and furthermore the single metastasis sample examined from each patients, which obviously make conclusions regarding this heterogeneous disease and overall patient status uncertain. We are also aware that expression levels not necessarily reflect biological activities and our hypothesis that tumor expression pattern of steroidogenic AKR1C3 and constitutively active AR variants would predict response to 2Table S1Primer sequences used for real-time RT-PCR analysis.(DOC)Click here for additional data file.


In the well-mixed prisoner’s dilemma game, individuals are typically assumed to have no choice about whether to interact with other individuals in the population. In this paper, we instead consider reputation-based conditional interaction and its consequences for the evolution of cooperation. Each individual has a tolerance range, and only interacts with other individuals whose reputation lies within its tolerance range in a chosen sample of the population. Reputation contains information about the number of interaction partners an individual has just cooperated with. We find that the introduction of conditional interaction promotes cooperation in well-mixed populations, and there exist moderate tolerance ranges for which this effect is maximized. For a given tolerance range, there is a critical cost-to-benefit ratio below which cooperation can be promoted. Interestingly, we find that if cooperation evolves, different cooperators’ interaction clusters are typically maintained in the population, each around a different reputation level. We further investigate some properties of these cooperators’ clusters. Moreover, we examine the effects of the sample number on the evolution of cooperation. Our results highlight the importance of the detailed consideration of modes of interaction for the evolution of cooperation in well-mixed populations. How to understand the emergence of cooperation is a central problem in evolutionary biology c that leads to the other individual receiving a benefit b, while a defector pays no cost and provides no benefit. In the evolutionary PDG, cost and benefit are measured in terms of payoffs, which are interpreted as reproductive success. In general, the strategy of someone who receives a higher payoff is more likely to be successfully reproduced/propagated In the PDG framework, a cooperator is an individual who pays a cost In a well-mixed PDG, all individuals are equally likely to interact with each other (no assortment), defectors have a higher average payoff than unconditional cooperators. Therefore, the relative abundance of defectors increases via natural selection, and drives cooperators to extinction In natural systems, individuals do not always deterministically interact with others in a population. Nor do they interact purely stochastically  or defect (D). The strategy of player x is denoted by Consider the evolutionary PDG in a well-mixed population with size k , and h is the tolerance range of player x. In this study, we assume that each player has the same tolerance range. Similarly, player y will be willing to interact with player x if x can interact with player y, and obtain the payoff from the game.Then, player x chooses its interaction partners from the sample of the population, and collects its payoff x’s interaction partners, According to the above interaction rule, player x’s reputation level is adjusted, and its updated reputation level, t. Formally,k), and is a measure of how much player x cooperated with others. In this study, the reputation level for N agents is initially random and can vary between 0 and 1, and correspondingly the tolerance rang is set to Following the interactions with the sample of the population, player x selects another player y randomly from the entire population for strategy updating. Whenever y’s strategy with probability given byM ensures the proper normalization and is given by the maximum possible difference between the payoffs of x and y. It is important to point out that the selection under such strategy update rule is strong and can dismiss some stochastic effects, so that we can focus more forcefully on the effects of the reputation-based conditional interactions.Following the reassignment of reputation levels, player h on the evolution of cooperation. Moreover, we have checked that the main results remain unaffected qualitatively when changing α within realistic limits.In this study, we set We study this model by Monte Carlo (MC) simulations, which are carried out in the well-mixed population with size


Cardiac rehabilitation (CR), a multidisciplinary program consisting of exercise, risk factor modification and psychosocial intervention, forms an integral part of managing patients after myocardial infarction (MI), revascularization surgery and percutaneous coronary interventions, as well as patients with heart failure (HF). This systematic review seeks to examine the cost-effectiveness of CR for patients with MI or HF and inform policy makers in Singapore on published cost-effectiveness studies on CR.Electronic databases  were searched from inception to May 2010 for published economic studies. Additional references were identified through searching bibliographies of included studies. Two independent reviewers selected eligible publications based on the inclusion/exclusion criteria. Quality assessment of economic evaluations was undertaken using Drummond’s checklist.A total of 22 articles were selected for review. However five articles were further excluded because they were cost-minimization analyses, whilst one included patients with stroke. Of the final 16 articles, one article addressed both centre-based cardiac rehabilitation versus no rehabilitation, as well as home-based cardiac rehabilitation versus no rehabilitation. Therefore, nine studies compared cost-effectiveness between centre-based supervised CR and no CR; three studies examined that between centre- and home based CR; one between inpatient and outpatient CR; and four between home-based CR and no CR. These studies were characterized by differences in the study perspectives, economic study designs and time frames, as well as variability in clinical data and assumptions made on costs. Overall, the studies suggested that: (1) supervised centre-based CR was highly cost-effective and the dominant strategy when compared to no CR; (2) home-based CR was no different from centre-based CR; (3) no difference existed between inpatient and outpatient CR; and (4) home-based programs were generally cost-saving compared to no CR.Overall, all the studies supported the implementation of CR for MI and HF. However, comparison across studies highlighted wide variability of CR program design and delivery. Policy makers need to exercise caution when generalizing these findings to the Singapore context. Cardiovascular diseases are a major cause of mortality and morbidity, contributing to about 30% of all-cause mortality and 10% of the total disability-adjusted life-years globally . Risk faBesides risk factor management , the other core components of cardiac rehabilitation include nutritional and physical activity counseling, psychosocial interventions and exercise training . In partSeveral systematic reviews over the past three decades have consistently demonstrated cardio-protective effects of exercise-based cardiac rehabilitation programs -9. ExercEconomic evaluation of cardiac rehabilitation has been reported since the 1980s. A systematic review of economic evaluation studies on cardiac rehabilitation, which identified 15 studies, was reported in 2005. Based on studies published between 1985 to 2004, supervised cardiac rehabilitation, compared to usual care, resulted in USD2,193 to USD28,193 per life year gained, and USD668 to USD16,118 per quality-adjusted life years . Most ofThe overall objective of the current systematic review was to describe and summarize published economic evaluations of cardiac rehabilitation for comparing the cost-effectiveness of different modes of delivery of cardiac rehabilitation. The specific aims were to compare the following modes of delivery:(a) supervised cardiac rehabilitation versus no cardiac rehabilitation,(b) supervised versus home-based cardiac rehabilitation,(c) inpatient  versus outpatient cardiac rehabilitation, and(d) home-based cardiac rehabilitation versus no cardiac rehabilitation.In this review, cardiac rehabilitation is considered as consisting of at least exercise training sessions, as this is usually the component studied as well as being the main cost driver of cardiac rehabilitation programs.Prior to developing the search strategy, “PICO” statements were used to address the specific aims of the systematic review Table . The eleFor the purpose of this review, the inclusion criteria were as follows:Economic evaluation study design that was either prospective alongside a clinical trial or based on modeling;Adult patients diagnosed with acute myocardial infarction (including post-infarction and after revascularization surgery or percutaneous coronary intervention for infarct), or chronic heart failure;Intervention that included exercise-based cardiac rehabilitation;Study with at least one of four comparators listed in Table Outcomes included either cost-effectiveness, cost-utility or cost-benefit analysis. Cost-minimization analysis was not included.Articles were excluded if one of the elements of PICO Table  was not The 10-item Drummond checklist was used to assess the methodological quality of the economic evaluation studies . If the Two independent reviewers (JF and WPW) selected eligible publications initially based on titles and abstracts. Potentially relevant articles were abstracted using standardized data abstraction form. This form was also used for data synthesis. Any disagreement between the reviewers was resolved by consultation with a third reviewer (KHP).A total of 896 citations were retrieved based on the search strategy. Of the 62 articles retrieved for more detailed evaluation, only 20 were included. An additional two references from hand searches yielded a total of 22 -33 articThe majority of the articles clearly stated that they included patients after acute myocardial infarction, following revascularization surgery or percutaneous coronary interventions, or who had a diagnosis of chronic heart failure, and therefore satisfied the selection criterion on diagnosis. Two articles employed the same sample for analyses, involving patients with coronary artery disease, defined as acute myocardial infarction or angina pectoris ,27. One The quality of these articles varied. Articles fulfilled two to nine items on the Drummond checklist, with none of them meeting all the items  ,16-18, wAmong 10 studies, six yielded no significant differences in the clinical outcome measures, and were therefore technically considered cost-minimization analyses ,24,28,33The only study that evaluated cost-effectiveness between inpatient and outpatient cardiac rehabilitation demonstrated no significant difference  Table 44.Four studies considered the cost-effectiveness of home-based program compared to no cardiac rehabilitation program. Home-based program was considered affordable  and moreThis systematic review summarizes the cost-effectiveness of cardiac rehabilitation compared to no cardiac rehabilitation, for patients after myocardial infarction, revascularization surgery or percutaneous coronary interventions, as well as those with chronic heart failure. Pooling of results is not possible given the heterogeneity in perspectives, health systems, study designs, details of cardiac rehabilitation interventions and types of patients that exist among the studies included in this review. However, we contend that these studies provide sufficient evidence for policy development concerning cardiac rehabilitation.Inclusion of a supervised outpatient cardiac rehabilitation program is clearly more cost-effective than not including cardiac rehabilitation program  into the overall management of patients after myocardial infarction or those with chronic heart failure. The centre-based programs consisted of exercise-based sessions, three times a week, over a period of 8 to 12 weeks. In addition, risk factor management and other multidisciplinary input were included in half of these studies.Four of the nine studies were economic evaluations alongside prospective randomized controlled trials ,16-18. TComparisons between home-based and centre-based programs were predominantly cost-minimization studies ,24,28,33Although all 13 studies  on home-based cardiac rehabilitation, compared to either supervised centre-based programs or no cardiac rehabilitation, have demonstrated home-based model to be cost-effective or cost-saving, the contents of the home-based programs varied widely. The contents of home-based program ranged from actual exercise sessions at home ,22,33, fversus home-based programs [versus centre-based programs in terms of cost-effectiveness.Early studies (before 2005) tended to demonstrate that home-based programs were more cost-effective and cost-saving than centre-based ones ,21-24,33programs ,26,28. Iprograms . The “hoprograms . Thus, tNone of the 16 articles met all of Drummond’s 10-item checklist Table . ArticleIn conclusion, evidence exists that supports the inclusion of supervised outpatient centre-based or home-based cardiac rehabilitation compared to no cardiac rehabilitation in patients after myocardial infarction, revascularization surgery or percutaneous coronary interventions, as well as those with chronic heart failure. Based on the reviewed articles, it would appear that the costs and outcomes of home- versus supervised centre-based cardiac rehabilitation were no different. Therefore the choice of the mode of delivery (home- versus centre-based) should be left to purchasers and patients. To the policy decision makers, there could be possible economic advantage of home-based program over centre-based ones. However, the details of what constitute “home-based program” are important.The authors declare that they have no competing interests.WPW and JF performed article search and screened the abstracts. KHP resolved any disagreements arising from the primary reviewers’ interpretations of the articles and provided input to the manuscript. JL provided input to the interpretation of the review and discussion. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/12/243/prepubAppendix 1. Quality assessment with the 10-item Drummond checklist [.hecklist .Click here for fileAppendix 1. Summary of Studies on Economic Evaluations of Cardiac Rehabilitation (CR).Click here for file


After anti-androgen treatment, AR expression was remarkably down-regulated within both the cytoplasm and the nucleus; however, when CPA had an agonist activity, the AR associated with the nuclear matrix (NM) increased approximately 2.5 times. This increase was synchronous with a higher PSA expression, indicating that the NM-associated AR represents the active complex. After BIC treatment, hnRNP K expression was significantly lower in the NM, the protein was hypophosphorylated and the co-localization of AR and hnRNP K decreased. In contrast, CPA as an agonist caused hnRNP K hyperphosphorylation and an increase in the co-localization of two proteins. These findings demonstrate that, in vitro, there is a strong relationship between NM-associated AR and both cell viability and PSA levels, indicating that AR transcriptional activity is critically dependent on its subnuclear localization. Moreover, the agonistic/antagonistic activity of anti-androgens is associated with modifications in hnRNP K phosphorylation, indicating an involvement of this protein in the AR transcriptional activity and likely in the onset of the androgen-independent phenotype.The androgen receptor (AR) plays a central role in the development and progression of prostate cancer (PCa) and anti-androgen therapy is a standard treatment. Unfortunately, after a few years, the majority of patients progress, developing androgen-independent PCa. AR-driven gene transcription recruits a large number of co-activator/co-repressor complexes; among these, the heterogeneous nuclear ribonucleoprotein K (hnRNP K) directly interacts with and regulates the AR translational apparatus. Here we examined AR and hnRNP K expression in response to the treatment of LNCaP cells with anti-androgen cyproterone acetate (CPA) or bicalutamide (BIC). AR and hnRNP K modulation and compartmentalization were studied by Western blot and confocal microscopy. Phosphate-affinity gel electrophoresis was employed to examine how anti-androgens modified hnRNP K phosphorylation. 10 Prostate cancer (PCa) is currently a leading cause of morbidity in the western male population AR-mediated gene transcription involves the recruitment of a large number of co-activator/co-repressor complexes, and it has recently been demonstrated that the heterogeneous nuclear ribonucleoprotein K (hnRNP K) directly interacts with and regulates the AR translational apparatus It is known that the unbound AR resides predominantly in the cytoplasm in a complex containing heat-shock proteins; the presence of androgen initiates a cascade of events that leads to receptor dimerization and translocation into the nucleus. Interaction of the AR with anti-androgens has been intensely investigated; however, the precise molecular mechanisms of their action remain unclear. Little is known regarding the way by which these drugs influence AR subnuclear localization and the dynamics of coactivator recruitment. Therefore, in this study, we examined the distribution of AR and its regulator hnRNP K in the cytoplasm, nucleus and NM in response to treatment of androgen-sensitive LNCaP cells with the steroidal anti-androgen cyproterone acetate (CPA) and the non-steroidal BIC. The elucidation of the mechanisms underlying AR suppression may provide a basis to understand the pathways involved in PCa progression and in the development of androgen escape.2. Subsequently, the medium was changed, and the cells were incubated for 48 or 72 h with BIC (SIGMA) or CPA (SIGMA) in the presence of 0.1 nM DHT. To elucidate the phosphorylation status of hnRNP K, LNCaP cells were also treated with 200 nM Wortmannin (SIGMA) for 5 h. For each treatment, viable cell numbers were determined using an MTT assay according to the methods recommended by the manufacturer (SIGMA). Absorbance was recorded at 595 nm, and the data are presented as the percentages of the absorbance of BIC- or CPA-treated cells with respect to control cells.LNCaP cells were obtained from ATCC  and maintained in RPMI-1640 medium without phenol red  containing heat-inactivated 10% fetal bovine serum , 100 U/ml penicillin G, 34 µM streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 25 mM glucose (Celbio). The passage numbers at which these cells were placed on maintenance medium ranged from 24 to 34. Control cells were cultured in monolayer in the presence of 0.1 nM 5-α-dihydrosterone  for 72 h at 37°C in 5% CO2S2O5, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 20 µg/ml leupeptin, 10 µg/ml pepstatin A, 25 µg/ml aprotinin and 1 mM dithiothreitol. All steps were performed at 4°C in the presence of 1 mM Na3VO4 to inhibit phosphatase activity. About 1×107 cells were mechanically harvested with a sterile plastic disposable cell scraper and recovered by centrifugation at 3000×g for 15 min. Then, the pellet was washed twice in 30 ml of PBS; cytoplasmic and nuclear extracts were prepared as reported by Kim and Tucker 2, 2 mM (CH3COO)2Mg, 0.1 mM EDTA, 100 mM Tris-HCl, pH 8.0 and 0.5% (v/v) NP-40) and centrifuged at 900×g for 2 min. The pellet contained the nuclei; the supernatant was centrifuged again at 17,000×g for 15 min and the resulting supernatant was the cytoplasmic fraction. The nuclear pellet was washed with 1 ml of sucrose buffer lacking NP-40, centrifuged at 200×g for 2 min, suspended in 200–400 µl of 10% SDS, 3% DTT and sonicated for 30 sec. The NM was isolated according to Barboro et al.To minimize proteolysis, all buffers were supplemented with 5 mM NaProtein concentrations were determined using the Bio-Rad  protein microassay with bovine serum albumin as a standard.in situ as described by Zeng et al. 2, 0.5% (v/v) Triton X-100) for 15 min each. The chromatin fraction was removed by digestion in CSK buffer with 50 mM NaCl (digestion buffer) containing 100 µg/ml DNase I  for 30 min, followed by extraction in digestion buffer containing 0.25 M (NH4)2SO4 for 10 min, then, the coverslips were fixed in 4% formaldehyde in PBS.For confocal microscopy, the NM was extracted 2+-Phos-tag™ ligand , according to Kimura et alAll samples were delipidated, cleaned and solubilized for 1D or 2D-PAGE as previously described t-test within the OriginPro 7.5 software, exporting the single values of each film.The proteins separated by 1D or 2D-PAGE were transferred to a Hybond-P membrane , and immunodetection was performed using mouse monoclonal antibodies anti-hnRNP K/J , anti-AR , anti-PSA (1 µg/ml sc-69664 Santa Cruz), anti-α-Tubulin , anti-PARP-1/2  and anti-lamin B . All the concentration quoted are value in the final reaction mixture. In the compartmentalization experiments, it was not possible to use an internal control to ensure equal gel loading because the major components of the NM undergo appreciable changes, as we have previously demonstrated Detection and alignment of spots present in all 2D-WBs were carried out digitizing the autoradiography films with a GS-800 densitometer (BioRad) under the same scanning conditions; then, the images were matched using the software package PDQuest . The relative amount of each spot was obtained by normalizing the spot volume to the total density of all spots.−5 M BIC or 10−6 M CPA) for 24 h. RNA was reverse transcribed with oligo(dT) primers and PSA mRNA expression was analyzed by semi-quantitative real-time PCR by using the following primer: sense 5′-ACGGATGCTGTGAAGGTCAT and antisense 5′-CTTTGGGGTCAAGAACTCCT. The relative expression of PSA gene was assessed in comparison with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplified with the following primers: sense 5′-GAAGGTGAAGGTCGGAGT and antisense 5′-CATGGGTGGAATCATATTGGAA. cDNAs were amplified for 50 cycles using iQ Supermix  containing the intercalating agent SYBR Green in a two-step amplification scheme . Fluorescence was measured during the annealing step on a Mastercycler Eppendorf realplex. Blank controls, that did not contain cDNA, were run in parallel. Each treatment was repeated two times and all samples were run in triplicate. Following amplification, melting curves with 80 steps of 15 seconds and a 0.5°C temperature increase per step were done to control for amplicon identity. Relative expression values with SEs and statistical comparison (unpaired two tailed t-test) were obtained using Qgene software Total RNAs were obtained from treated or non-treated LNCaP cells grown with 0.1 nM DHT and incubated with anti-androgens (10http://www.uhnresearch.ca/facilities/wcif/imagej/) that provides both the Pearson’s correlation coefficient (R) and the co-occurrence Manders’ coefficients (M1 and M2). Three-dimensional measurements of proteins cluster sizes were carried out on image stacks as already reported Analysis of the spatial relationship between AR and hnRNP K in the NM was performed on both treated and untreated LNCaP cells using triple–color confocal microscopy. The following antibodies were used: rabbit anti-hnRNP K , mouse anti-AR at a final concentration of 33 mg/ml , CF633-conjugated donkey anti-rabbit IgG  and CF555-conjugated donkey anti-mouse IgG (Biotium), both at a final concentration of 40 µg/ml. Goat anti-lamin B at the concentration of 8 µg/ml (sc-6216 Santa Cruz) and Alexa 488-conjugated donkey anti-goat at the concentration of 40 µg/ml  were employed to detect effective NM purification. All confocal images were collected according to the Nyquist criterion as 3D data sets (z-stacks) with a step size of 250 nm using an Olympus FV-500 laser scanning confocal microscope equipped with a 60×/1.4 NA oil-immersion objective. Image processing and co-localization analyses were performed as previously described 50, the concentration required for a 50% effect, were 1.53×10−4 and 26.38×10−4 for BIC and CPA, respectively. These results are consistent with previous investigations demonstrating that BIC and CPA cause a decrease in LNCaP cell viability in androgen-containing medium −6 M CPA showed a very small, but significant (P<0.01), agonist effect. After treatment for 72 h with 10−5 M BIC or 10−4 M CPA, approximately 75% of cells were viable, indicating that at these concentrations, the two drugs had the same antagonistic effect. Thus, subsequent experiments were performed on cells treated for 72 h with 10−5 M BIC or 10−4 M CPA. The agonistic effects of 10−6 M CPA were also studied.The data reported in −5 M BIC or 10−6 M CPA caused comparable effects (approximately 50% reduction with respect to control), and at the higher concentration of CPA used the AR expression plummeted to 10% of control. In the previous section, we reported that LNCaP viability was equally affected by 10−5 M BIC and 10−4 M CPA; therefore, these results indicate that in our experimental conditions, cellular growth was independent of AR expression. Interestingly, when the cells were supplemented with 10−6 M CPA, an approximately 2.5-fold increase was observed in the AR associated with the NM, and this increase was synchronous with higher intracellular PSA expression levels  was observed, indicating a higher hnRNP K fraction overlapping the AR. Moreover, the AR was distributed in hyperspeckled sites, as after DHT treatment.Because the principal effects on AR and hnRNP K appear to implicate the NM, we performed confocal microscopy analysis in this subnuclear compartment to define the changes in AR/hnRNP K co-localization after anti-androgen treatment, utilizing BIC and CPA concentrations that did not induce NM protein degradation. Representative optical sections are shown in 3, corresponding to a sphere of 0.1 µm in diameter, a value very close to the dimension of nuclear speckles The number and size of hyperspeckled sites, determined on image stacks, confirmed these findings. The size frequency distribution curves reported in −5 M BIC, some acidic species decreased, while they increased in the cells treated with 10−6 M CPA, suggesting that the two drugs induced an opposite effect on hnRNP K phosphorylation , as already evident in 1D-WB. Therefore, we examined the effects of BIC and the lower CPA concentration on different hnRNP K isoforms using phosphate-affinity electrophoresis, a technique able to detect phosphoproteins based on a shift in their electrophoretic mobility. As expected, multiple hnRNP K forms were observed  disappeared, and spots 5 and 6 (isoform 1) decreased, demonstrating hnRNP K hypophosphorylation. A parallel increase in spot 18 (isoform 4) and the appearance of the spot 14 (isoform 3) were also evident. In the presence of 10−6 M CPA, a strong increase in spots 5, 6 and 8 (isoform 1), 4, 9, 10 (isoform 2) and 14 (isoform 3) occurred, demonstrating that this condition is associated with a global hyperphosphorylation of the protein. The spots that after anti-androgen treatment undergo major changes are expected to be phosphorylated at serine 116 , serine 284 (spots 8) and serine 353  Because it is known that hnRNP K possesses multiple sites that can be phosphorylated in response to extracellular signals rylation . In the observed . BecauseAs Akt kinase activity is elevated in LNCaP cells These results support the hypothesis that hnRNP K, and above all its phosphorylation, plays an important role in the response to anti-androgen treatments.The current study shows that there is a strong relationship between the level of AR localized in the NM and both cell viability and PSA expression, indicating that AR transcriptional activity is critically dependent on its subnuclear compartmentalization. Moreover, the agonistic/antagonistic activity of anti-androgens is associated with modifications in the phosphorylation status of hnRNP K, a protein that directly interacts with the AR.−4 M CPA, the level of AR in the cytoplasm and the nucleus was approximately 4 times lower than that present after treatment with 10−5 M BIC, whereas there was no significant difference in the AR associated with the NM. Therefore, this AR fraction could be sufficient to determine LNCaP proliferation. These results are in agreement with the notion that NM, in particular the internal NM, contains acceptor sites for AR et al.It is well recognized that LNCaP cell proliferation is especially stimulated by 0.1 nM DHT, and in the presence of anti-androgen treatment, cell proliferation decreases. Here, we report that, under the same conditions of viability, CPA is more potent than BIC in inhibiting AR transactivation function. This effect is associated with a strong degradation of hnRNP K. A peculiar characteristic of LNCaP cells is the presence of a point mutation in the ligand-binding domain in the AR gene at codon 877 (ACT-GCT), which results in the transition of a threonine into an alanine −6 M CPA, as opposed to BIC exposure, which showed a diffuse and weak fluorescence. These results suggest that the hyperspeckled distribution of AR may be considered a consequence of interactions with the NM et al. In addition, the agonist activity displayed by CPA appears to be strongly bound to the amount of AR associated with the NM; in fact, an elevated PSA expression, found by WB analysis and real time PCR, is synchronous with a significant increase in the proportion of AR in the NM as detected by WB analysis and confocal microscopy. Hyperspeckled sites in the internal NM are visible after treatment with 10Heterogeneous nuclear ribonucleoproteins (hnRNP), including hnRNP K, are among the most abundant components of the NM and share in the stabilization of the internal NM network We have shown that a phosphorylated isoform of hnRNP K is overexpressed in the NM extracted from prostate carcinoma tissues In conclusion, our data provide evidence that the transcriptionally active form of AR is localized into the NM, and the bond between AR and hnRNP K depends on the phosphorylation of the latter. The bond between hnRNP K and AR facilities the stabilization of the latter into the NM and/or its interaction with MAR sequences and promoted transcriptional activity. Treatments with agonist/antagonist drugs, modifying the level of hnRNP K phosphorylation, increased/decreased its interaction with AR and consequently its activity.Work is in progress in our laboratory, using specific inhibitors of the kinase activities, to define the potential therapeutic and/or chemopreventive role of hnRNP K hypophosphorylation in PCa progression and in androgen resistance.Figure S1Cell fractionation effectively separates cellular compartments. Eight µg of proteins extracted from whole cells (W), cytoplasm (Cyto), nucleus (Nu) and nuclear matrix (NM) were immunoblotted for α-Tubulin, PARP or lamin B as indicated. α-Tubulin was found only in the cytoplasmic fraction, the nuclear marker PARP was present in the nucleus and in small quantity in the NM as reported by Kaufmann et al (TIF)Click here for additional data file.Figure S2Effects of BIC or CPA exposure on PSA mRNA expression. LNCaP cells grown in presence of 0.1 nM DHT were treated for 24 h with 10−5 M BIC or 10−6 M CPA and real time semi-quantitative PCR carried out as reported in Materials and Methods. Mean normalized expression values were calculated by comparison with housekeeping gene GAPDH amplified in parallel. Two treatments were performed and all amplifications were done in triplicate. Error bars correspond to SE.(TIF)Click here for additional data file.


Polymorphism of the interleukin-23 receptor gene corresponds with susceptibility to several immune-related diseases. For the terminal differentiation of IL-17-producing effector T-helper cells in vivo, the interleukin-23 receptor gene is of vital importance. As shown recently, Th17 cells probably have a great influence on the pathogenesis of allergic airway diseases. Our intention was to establish an association between polymorphisms in the IL-23R gene and allergic rhinitis (AR) in the Chinese Han population.We included 358 AR patients and 407 control Chinese subjects in a case-control comparison. The study involved obtaining blood samples for DNA extraction genotyping and determination of 4 selected single-nucleotide polymorphisms in IL-23R by performing PCR restriction fragment length polymorphism analysis (PCR-RFLP).A substantially growing prevalence of the homozygous rs7517847 GG genotype and G allele appeared in the AR patients unlike that observed in the control individuals (P<0.001). In addition, substantially high frequencies of the GGCA and GGCG haplotypes were observed in the AR patients, unlike that observed in the control individuals (P<0.05). The results suggest that the AGTG haplotype may provide protection against AR (P<0.001).To the best of our knowledge, this is the first study to demonstrate an important association between polymorphisms in IL-23R and AR in the Chinese Han population. A strong association between rs7517847 in a SNP of IL-23R, and AR was identified. In the past few decades, the prevalence of allergic diseases, including asthma, rhinitis, eczema, and food allergies, has risen sharply As shown recently, Th17 cells might have a great influence on the pathogenesis of allergic airway diseases. The IL-17 levels in the in nasal lavage, induced sputum, and serum of patients with AR showed a substantial increase Therefore, in this study, we aimed to determine the association between IL-23R polymorphisms and susceptibility to AR. In accordance with our aim, we analyzed 4 SNPs, including rs17375018, rs7517847, rs1343151, and rs11209032.From April to August 2012,358 patients aged 4 to 70 y were selected and evaluated on the basis of the ARIA criteria Meanwhile, 407 unrelated healthy subjects from the same region were enrolled as the control individuals during the same time. To evaluate the impact of occupation on the association of the 4 polymorphisms, we divided the samples into 2 groups according to the place of work (indoor versus outdoor work). All the control individuals were of the Han ethnicity of the general population and had undergone a comprehensive medical screening. They did not show clinical features or family history of allergies and had not experienced an upper respiratory tract infection in the 4 weeks prior to the study. Individuals diagnosed with AR and those with eczema, asthma, or a tumor in the nasal cavity were excluded from the study. The case-control ratio of our population (0.88∶1) was considered to be one of the parameters of analysis. The clinical characteristics of the AR patients were assessed at the time of diagnosis and are summarized in The local ethics authorities  provided permission, and helped to obtain informed consent from all the participants. Written informed consent was obtained from all the participants. Informed consent was obtained from the next of kin, caretakers, or guardians of the minors and children participating in this study.The SNPs were selected according to the results of the earlier studies on the association of IL-23R polymorphisms with other autoimmune diseases All the blood samples were collected in ethylenediaminetetraacetate tubes and stored at −70°C until use. Using the QIAamp DNA Blood Mini Kit, genomic DNA was extracted and the target region was amplified by performing PCR. PCR restriction fragment length polymorphism analysis was applied to genotype these SNPs. The obtained digestion products were visualized on a 4% agarose gel and stained with Gold View . In order to validate the method used in this study, Invitrogen Biotechnology Company  was assigned to execute direct sequencing by using randomly selected subjects .2 test. The χ2 test was applied to compare the allele and genotype frequencies between the patients and control individuals.Estimation of genotype frequencies was performed by direct counting. The online software platform SHEsis  was used to analyze the haplotype and probabilities. We used non-risk alleles as the reference, and tested all the other haplotypes. Logistic regression analysis was used to analyze the genotype allele, controlling for age, gender, and occupation as the covariables. All the statistical analyses were performed using the SPSS software (version 13.0).Call rates were compared between AR and control by the χversus outdoor). The groups were comparable with respect to mean age, gender, and workplace ratio. Pearson Chi-Square analysis of the ratios of male: female and indoor: outdoor work between the control and the study groups was performed (P>0.05). The mean age values for the control individuals and the AR cohorts were 28.51y (ranging from 4–70 y), and 34.09y (ranging from 7–75 y), respectively, which were not significantly different . Further investigation was performed to determine whether any association existed between the IL-23R SNPs and certain allergens of AR, such as seasonal grass pollens, house dust mites, dog and cat fur, molds, and cockroach. Any association of these parameters with the tested IL-23R SNPs has not yet been determined. The 4 SNPs analyzed in the IL-23R gene were in Hardy–Weinberg equilibrium in both the AR cohort and the control cohort. The 4 SNP call rates in the patients and the control individuals and their HWE p-values are shown in Clinical and demographic characteristics of the patients and the control individuals are summarized in Logistic regression analysis was used to analyze the genotype allele controlling for age, gender and occupation as the covariables. The results of genotypic and allelic frequency analysis are shown in The Haploview V3.32 program and the online software platform SHEsis were used to analyze the haplotype data and probabilities in We further investigate whether the IL-23R SNPs were associated with certain allergen category of AR, such as house dust mite, pollens and other mixed allergens. The analysis failed to find any association of these parameters with the tested IL-23R SNPs (P>0.05).This study determined an association between IL-23R polymorphisms and AR susceptibility in a Chinese Han population and confirmed a novel SNP, rs7517847, in IL-23R that has a relationship with AR. The haplotype AGTG was found to protect against AR, whereas other haplotypes, GGCA and GGCG, were associated with susceptibility to AR.In order to establish a correlation with immune diseases, we have investigated many SNPs. The most common IL-23R SNP related to immune diseases are rs7517847, rs11209032, and rs1343151, although rs17375018 was not shown to be associated with other diseases, it was found to be associated with Behcet’s in our laboratory of Ophthalmology; therefore, these 4 were analyzed in our study. Considering that results on the association of gene polymorphisms with disease can be influenced by many factors, we aimed to ensure the correctness of our results. First, we selected AR patients strictly according to the criteria of ARIA and excluded the ones who did not fit the criteria. Second, we chose unrelated healthy individuals from the same geographic region as that of the AR patients’; all the study participants were similar with respect to their age, sex, occupation, and ethnicity. Finally, to verify the results of genotyping by PCR-RFLP, we repeated the sequencing of 20% of the samples, for which we obtained the exact same results as those obtained for the first genotyping. These approaches were taken to ensure the accuracy of the results of this study.In previous reports, the strategy of selecting SNPs has been noted in association studies of certain genes with disease. As far as we know, we have for the first time investigated the association of 4 SNPs with allergic disease, and found that only rs7517847 was associated with AR. As stated in our study, the frequency of presence of the GG genotype and G allele of rs7517847 in AR patients  4.248, 95% CI 2.60 to 6.95; Pc<0.001, OR 1.62, 95% CI 1.32 to 1.98, ) presented a significant increase, suggesting an important susceptibility factor to this disease. These results conform to those of the studies on Crohn’s disease and ulcerative colitis, in which rs7517847 had the strongest association Further studies are needed to investigate how rs7517847 is directly involved in susceptibility to AR. The intronic polymorphism rs7517847 might exert its influence by regulating the specific splicing of the gene The association of AR with 3 other SNPs of the IL-23R gene  has been under investigation. The results show that there are small differences between the AR patients and the control individuals concerning the frequencies of rs17375018 and rs1343151. It is interesting to note that the frequency of the rs17375018 G allele was as high as 70.1% and 68.1% in the AR patients and control individuals, respectively, and the frequency of the rs1343151 T allele was as low as 2.0% and 3.7% in the AR patients and control individuals, respectively. Further studies on the influence of rs17375018 and rs1343151 IL-23R variants on AR will require genetic analysis of a very large cohort. The results of a new UK case–control study and meta-analysis of published series show a significant association between rs11209032 and both AS and IBD Haplotype analysis revealed that the haplotype AGTG conferred a reduced risk of AR, whereas 2 other haplotypes GGCA and GGCG were associated with susceptibility to AR. By analyzing the results presented in As with other studies regarding the association of gene polymorphisms in AR, our study also has limitations. As AR is a heterogenetic disease, the environment is critical for the disease; further studies including gene-gene and gene-environment interactions in AR are needed to clarify the impact of IL-23R on allergic disease. From another point of view, this study only investigated AR patients from a Chinese Han population. However, by performing the test on multiethnic populations, the association of IL-23R with AR found in this study could be clarified. In addition, GWAS coverage was insufficient for many asthma candidate genes, imputation based on these data is reliable but incomplete In conclusion, a strong association between rs7517847 in the IL-23R gene and AR was identified in our study. The GG genotype and G allele of rs7517847 were confirmed as incentive factors to AR. In addition, the biological function of rs7517847, a strongly associated SNP expressed in our study, needs to be further researched. Moreover, we also believe that further studies are required to determine whether rs17375018, rs1343151and haplotype AGTG influence AR. Validation and understanding of this IL-23R relationship and the influence of IL-23R variants on the IL-17A/IL-23 pathway will be important to increase the understanding about the pathogenesis of AR.