Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts.We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR).ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA , the rate-limiting enzyme in heme catabolism.In arsenite-exposed cells, 186 probe set–identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set–identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.http://dx.doi.org/10.1289/ehp.1408626Qiu LQ, Abey S, Harris S, Shah R, Gerrish KE, Blackshear PJ. 2015. Global analysis of posttranscriptional gene expression in response to sodium arsenite. Environ Health Perspect 123:324–330;  Inorganic arsenic compounds are potent environmental toxicants. Humans are exposed to various forms of arsenic mainly through oral consumption of contaminated water, food, or drugs as well as via inhalation of arsenic-containing dust or smoke in agricultural and industrial settings . ChronicSP1, JUN, and nuclear factor-kappa B (NFKB)  . Genome-B (NFKB) . These eB (NFKB) .STAT1  mRNA, apparently mediated through the RNA binding protein nucleolin  bodies, both thought to be involved in localizing mRNA decay . Posttraucleolin . More reucleolin .The purpose of our study was to perform a genome-wide analysis of sodium arsenite–induced changes in gene expression in human diploid fibroblasts and to determine whether these changes could be due, at least in part, to changes in mRNA stability. We chose diploid human foreskin fibroblasts as the test cells because skin is sensitive to the effects of chronic arsenic exposure and it is where the first manifestations of exposure often appear . We alsoCell culture and stimulation. BJ human diploid foreskin fibroblasts  were obtained from ATCC. Cells were maintained in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were plated into 100-mm dishes until they reached 80% confluence, and then various concentrations of sodium arsenite  or vehicle (water) were added to the medium for 24 hr. We determined cytotoxicity of various concentrations of sodium arsenite after 24 hr using propidium iodide staining followed by flow cytometric analysis  according to the manufacturer’s instructions. Residual genomic DNA was removed by on-column digestion with RNase-free DNase I supplied with the kit. Quality and integrity of RNA samples was checked on denaturing formaldehyde/agarose gels stained with acridine orange. After fibroblasts were treated with sodium arsenite or water (vehicle) for 24 hr, actinomycin D was added to the culture medium at a final concentration of 5 μg/mL; treated cells were then harvested at 0, 1, 2, 3, and 4 hr for RNA extraction. This concentration of actinomycin D has been validated in other cell types to be both effective and noncytotoxic for these relatively short times . This entire human genome array contains > 54,000 probe sets, reflecting > 38,500 genes and approximately 47,000 transcripts  was then fragmented, and 10 μg was hybridized onto each GeneChip 3´ expression array for 16 hr at 45°C in a rotating hybridization oven, using the Affymetrix Eukaryotic Target Hybridization controls and protocol. Array slides were stained with streptavidin/phycoerythrin using a double-antibody staining procedure; slides were then washed using the EukGE-WS2v5 protocol of the Affymetrix Fluidics Station FS450 for antibody amplification. Arrays were scanned in an Affymetrix Scanner 3000, and data were obtained using GeneChip Command Console software . Data processing, normalization, and error modeling were performed using the Rosetta Resolver system . Pathway and functional analyses of the differentially expressed transcripts were performed using Ingenuity Pathway Analysis  and Partek Genomics Suite (Partek Inc.).nscripts . For ourAlthough using microarrays for the analysis of mRNA levels has various disadvantages, as reviewed recently , we haveMicroarray data accession number. The microarray analysis results have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) Profiles database (accession no. 57051).Real-time reverse transcription polymerase chain reaction (RT-PCR). Selected transcripts were analyzed by real-time RT–PCR. For each sample, 1 μg of total cellular RNA was reverse transcribed using oligo(dT)12–18 primers and SuperScript III Reverse Transcriptase (Invitrogen) following the manufacturer’s protocol. All cDNAs were diluted and subjected to real-time PCR using SYBR Green master mix and the ABI Prism 7900 Sequence Detection System (both from Applied Biosystems). Transcript-specific primers were designed and validated for their amplification efficiency prior to being used in the study, and are listed in Supplemental Material, Table S1. Relative transcript abundance was determined by normalizing values to the β-actin (ACTB) transcript as an internal control, which was then used to calculate the fold changes relative to vehicle control, or the relative abundance of each transcript compared with the pre-actinomycin D values, according to the ΔΔCt method . For the real-time PCR results, significant differences in mRNA levels were determined by two-tailed unpaired Student’s t tests. For the decay rate comparisons, significant differences were determined using EDGE, version 1.1.291 (http://www.genomine.org/edge/) (http://www.niehs.nih.gov/research/resources/software/biostatistics/oriogen/) (g/edge/)  and ORIOriogen/)  softwareCytotoxicity of sodium arsenite in BJ cells. To determine a noncytotoxic concentration of sodium arsenite in these cells, we incubated BJ cells (at ~ 80% confluence) in normal growth medium for 24 hr and then added sodium arsenite at concentrations ranging from 0.001 to 1,000 μM; cell viability was determined by propidium iodide staining and flow cytometric analysis . After 24 hr of arsenite exposure, the percentages of viable cells were 98.7 ± 0.3% (mean ± SD of 4–5 samples), 98.5 ± 0.8%, and 95.0 ± 2.3% for arsenite concentrations at 0.1, 1, and 10 μM, respectively, with rapidly increasing cytotoxicity at higher concentrations. We therefore chose 1 μM as the highest clearly noncytotoxic concentration. Similar concentrations have been used in the literature, for example, in human keratinocytes and fibroblasts , 54 (29%) of which were increased more than 2-fold after arsenic stimulation  demonstrated steady-state probe set–identified transcript levels that were significantly altered after 24 hr of treatment with sodium arsenite. Analysis of these data by IPA is summarized in Supplemental Material, Tables S2–S5; these results highlight effects on many pathways, including NRF2-mediated oxidative stress response, the pentose phosphate pathway, vitamin C transport, heme degradation, methylglyoxal degradation, heme biosynthesis, and others.Effect of sodium arsenite on mRNA decay rates. To determine whether the observed changes in probe set–identified transcript levels were due, at least in part, to changes in their decay rates, we performed microarray analysis on RNA samples isolated from control or arsenite-treated cells at hourly intervals for 4 hr after the addition of actinomycin D; little or no actinomycin D–induced cytotoxicity is thought to occur before 4 hr, but longer exposures can complicate interpretation of results  decayed by ≥ 25% after 4 hr in the control cells, equating to approximate half-lives of ≤ 8 hr. The slower decay rates of the remaining 90% meant that we were unlikely to be able to detect decay rate differences for them using this method of analysis. We therefore focused the rest of our analysis on the 4,992 probe set–identified transcripts that decayed by ≥ 25% in 4 hr.ACTB and GAPDH mRNAs  for the differences between the control and arsenite decay curves. For these 5, arsenite increased stability in every case. The mRNAs identified by these probe sets were those encoding ALAS1, GATA3 (twice), MAP3K8, and KRTAP1  was in the same direction as the steady-state increase of 2.68-fold caused by arsenite after 24 hr  had significant EDGE and ORIOGEN d KRTAP1 . Of the er 24 hr ; for GAT.48-fold .ALAS1 and GATA3 mRNAs, we performed real-time RT-PCR on the same mRNA samples, using primers designed specifically for these transcripts [see “Real-time reverse transcription polymerase chain reaction (RT-PCR)”]. The real-time RT-PCR analysis confirmed the highly significant differences between the decay curves for ALAS1 mRNA observed in the microarray data  differences in both EDGE and ORIOGEN p-values for the decay curves. After removing the transcripts discussed above that had significant steady-state changes, uncharacterized genes, and duplicates, 162 probe set–identified transcripts remained, of which 35 were significantly destabilized by arsenite; the remaining 127 were stabilized. Of these 162, 49 had EDGE and ORIOGEN p-values that were significant at the p < 0.01 level for both tests. Supplemental Material, Table S6, provides a list of these 49 probe set–identified transcripts along with their average percentage decreases at 4 hr compared with time 0 and the ORIOGEN and EDGE p-values for the decay curves. Examples of probe set–identified transcripts stabilized by arsenite are shown in Supplemental Material, Figure S2, and examples of those destabilized by arsenite are shown in Supplemental Material, Figure S3. It should be noted that the differences between the decay curves for these transcripts were deemed highly significant (p < 0.01) by both tests, resulting in a fairly stringent selection. Of the 4,992 probe set–identified transcripts that decayed by > 25% in 4 hr in the control cells, 466 were considered significant by ORIOGEN analysis (p < 0.05) but not by EDGE analysis. Similarly, 98 of these probe set–identified transcripts were deemed significant by EDGE analysis (p < 0.05) but not by ORIOGEN analysis. These individual tests provide less stringency than the two together, but they may provide clues in some instances to biologically significant changes in mRNA stability.We then investigated the remaining probe set–identified transcripts that decayed > 25% in the control cells at 4 hr but did not exhibit significant differences in steady-state mRNA levels after 24 hr of control or arsenite treatment. Of the original set of 4,992 probe set–identified transcripts that decayed > 25% after 4 hr of actinomycin D treatment, 340 exhibited significant , and the decay curves showed highly significant transcript stabilization after arsenite treatment. This significant stability change identified by microarray was confirmed by real-time RT-PCR. These data suggest that, at least for transcripts with more rapid decay rates in fibroblasts, changes in mRNA decay rates in response to arsenite accounted for very few of the steady-state changes observed after 24 hr of treatment.The major goal of this study was to determine whether changes in mRNA stability contribute to the changes in steady-state mRNA levels seen in human diploid fibroblasts treated with sodium arsenite. In this genome-wide microarray analysis, we identified 353 probe set–identified transcripts  whose steady-state levels were significantly altered after 24-hr exposure to a noncytotoxic concentration of arsenite. Of these 353, only 70 decayed rapidly enough after actinomycin D treatment  to make comparisons between the decay rates feasible by these techniques. Of these 70, only 4 transcripts exhibited differences in decay rates between control and arsenite-treated cells, and only 1 probe set, corresponding to the transcript of This analysis was limited by the apparent stability of most mRNAs under these experimental conditions, in which approximately 90% of probe set–identified transcripts were apparently too stable to make decay-rate comparisons feasible using these methods. This is one of the inherent difficulties with the actinomycin D method because cytotoxicity prevents longer-term experiments that would allow measurements on slower-decaying transcripts. Using recently developed techniques such as ribonucleoside labeling , it may In addition to the few changes in mRNA stability that could account, at least in part, for changes in steady-state mRNA levels after arsenite treatment, 49 probe set–identified transcripts exhibited highly significant arsenite-induced changes in stability that were not reflected in significant changes in steady-state levels after 24 hr, and only 8 of these were destabilized in the arsenite-treated cells. Many more transcripts were identified whose decay rates differed at lower rates of significance, or in only one of the two tests of significance. The fact that these transcripts did not exhibit changes in steady-state levels suggests the possibility of compensatory changes in transcription rates in these cases.ALAS1 mRNA observed in both microarray and real-time RT-PCR presumably contributed to its steady-state increase after arsenite treatment. ALAS1 encodes aminolevulinate, delta-, synthase 1, a mitochondrial protein that is the rate-limiting step in heme biosynthesis. Heme has long been known to affect ALAS1 expression at the levels of transcription, mRNA decay, mitochondrial import and export, and protein stability , a plausible mechanism for the effect of arsenite to increase ALAS1 mRNA steady-state levels and mRNA stability would be as follows: Arsenite treatment would cause massive increases in HMOX1 expression, resulting in increases in cellular heme catabolism and decreases in heme levels; the decreased heme levels would then lead to increases in ALAS1 transcription and mRNA stability, resulting in increased heme biosynthesis. It seems possible that this effect on heme biosynthesis could lead to known effects of arsenic exposure on exacerbations of porphyria  was not accompanied by significant changes in HMOX1 mRNA decay rates, at least on a percentage basis. It should be noted that many previous studies have demonstrated induction of HMOX1 after treatment of cells with various forms of arsenic  PDFClick here for additional data file.


The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling.2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided.Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χP = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts.CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa. Prostate cancer (PCa) is a major cause of cancer-related deaths worldwide . The andThese diverse resistance mechanisms highlight the reliance of PCa on the maintenance of AR signaling, which controls a number of cellular pathways including metabolic fuelling of tumor growth , progres2.Unless stated otherwise, all cell lines were verified by genetic profiling of polymorphic short tandem repeat (STR) loci as per ATCC standards. We used either AmpFISTR test or GenePrint10 test  and analyzed all data using GeneMapper v4.0 software. LNCaP, C4-2, VCaP, PC3, PNT1a, RWPE-1, DUCaP, 22Rυ1, and DU145 cells were obtained from commercial suppliers and grown in RPMI cell culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified incubator at 37 ºC with 5% COR1-AD1 was a subline derived from the CWR-R1 cell line. The identity of R1-AD1 was authenticated by positivity for the H874Y point mutation in the AR LBD as determined by polymerase chain reaction (PCR) and Sanger sequencing, and negativity for copy number imbalances along the length of the AR gene was determined by multiple ligation-dependent probe amplification (MLPA) assay. The identity of R1-D567 was authenticated by PCR and Sanger sequencing of the signature break fusion junction generated by transcription activator-like effector endonuclease (TALEN)–based genome engineering.Prostate tissues were obtained from 359 patients with a median age of 64 years (range = 46–74 years) who underwent radical prostatectomy between 1993 and 2003. Samples and clinico-pathological data were retrieved from the files of the Department of Pathology, Centro Hospitalar do Porto, Portugal, and tissue microarray blocks (TMAs) were prepared. Prior to TMA construction, haematoxylin and eosin (H&E)–stained tumor sections of each radical prostatectomy specimen were re-assessed using the modified Gleason and 2010 pTNM classification . RepreseAll experiments were carried out in compliance with the UK Animals (Scientific Procedures) Act 1986 under a project licence with the approval of the Cancer Research UK Cambridge Institute Animal Welfare and Ethical Review body. Xenografts were initiated in NOD scid gamma mice (n = 6 per group) by subcutaneous injection of 1 million C4-2b-luciferase (C42b-Sh#1 and nontargeting shLuc C4-2b) cells in 100 µL . Mice were given doxycycline in drinking water (0.5mg/mL in 2% sucrose solution) while control mice received 2% sucrose solution. Tumor size was measured with callipers weekly and calculated using the formula volume = (π/6)/abc or (π/6)/abb (if only 2 diameters are available), and a, b, c are the orthogonal axis of the tumor. Tumors were also monitored weekly by luminescence imaging after intraperitoneal injection of 150mg/kg d-luciferin  using Xenogen Imaging Analysis software Living Image 3.0 , and luminescence was plotted as photons per second for graphic analysis of growth kinetics. Turbo red fluorescent protein (tRFP) emission was recorded using Xenogen camera once after three weeks while luciferase expression was the measured weekly. Mice were culled at the completion of experiment or when tumors reached 10% of body weight.Chi square tests in R were used to look at the association of clinical variables with CHKA, all dichotomized. Survival analysis of the patient data was carried out using the survival package . Cox regP values of less than .05 . Further details are given in the Supplementary Methods (available online). All statistical tests were two-sided.Recursive partitioning (RP) using a conditional inference framework was used to find a significant cutoff for each input gene for predicting recurrence-free survival using a prostate cancer gene expression dataset with accompanying survival data from 79 patients . This waSupplementary Table 4 (available online) contains all the genomic data used in the manuscript including GEO accessions or web links. The probe set used in each data is also specified. The RNA-seq data generated during this work has been submitted to Gene Expression Omnibus and is available for viewing at the following link http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token = ytazouoixxedjal&acc=GSE63700.Supplementary information includes experimental procedures; Supplementary Table 1, available online). By integrating gene expression with our chromatin immuno-precipitation sequencing (ChIP-seq) datasets . Clinical gene expression data revealed that many androgen-upregulated kinases were also upregulated in primary and metastatic PCa . Importantly, we confirmed the in vivo androgen regulation of a subset of these genes in tumor tissues from chemically castrated PCa patients (n = 15) in contrast to untreated tumors (n = 19) from age/tumor grade-matched patients, finding that 18 of the 25 in vitro androgen-upregulated kinases were statistically downregulated in response to castration in men with PCa (t test P < .05) .To identify a therapeutically relevant subset of AR targets, we defined the AR-regulated kinome by performing a cross-platform analysis of an androgen-stimulated gene expression time course  and a hidatasets , we founSupplementary Figure 1F, available online). These datasets indicated that the CHKA gene could be a clinically relevant AR target gene. Supporting this idea, the CHKA has strong intragenic AR binding sites in PCa cell lines and in PCa tissue . CHKA mRNA was statistically significantly overexpressed in localized (primary) and metastatic PCa . Of greater biological relevance, CHKA expression was decreased in tissues from patients treated with the LHRH analogue degarelix, both at the transcript  and protein level compared with control patients without androgen deprivation (P = .006)   and showed some evidence of association with Gleason grade (≤ vs 7) and tumor stage (pT2 vs pT3). There was no evidence of an association of CHKA expression with age, PSA, or biochemical recurrence . However, CHKA was an independent predictor of biochemical recurrence-free survival when other clinical variables were included , a finding supported by Kaplan-Meier analysis of a separate cohort of clinical samples   . TogetheSupplementary Figure 2C, available online). Evidence of an interaction between the AR and CHKA was also observed in a reciprocal co-immunoprecipitation (CoIP) assay (pulling down CHKA and western blotting for the AR) . Fluorescence microscopy imaging of m-Cherry tagged CHKA (red) and green fluorescent protein tagged AR  revealed evidence of colocalization between CHKA and the AR both in the cytosol and nucleus . Surprisingly, AR protein levels were reduced not only by AR knockdown but also by CHKA knockdown using either siRNA   and with subsequent incubation ± DHT with a recombinant CHKA2. In the presence of both DHT and CHKA protein, there was reduced cleavage of the ARDBD-LBD polypeptide at both low and high concentrations of trypsin  and this could be reversed by the addition of both DHT and CHKA, although not by DHT or CHKA individually. Together these data suggest that CHKA exerts a chaperone function on the AR.To determine whether the interaction between the AR and CHKA was direct or indirect, we screened several AR domain constructs . Using aLBD) . NotablyLBD) . We therLBD using R1-D567 cells that exclusively express an AR variant (ARv567es) lacking the LBD  in R1881-induced AR transcriptional activity following CHKA knockdown using siCHKA (P < .005) , a structural analogue of choline that binds to CHKA and inhibits conversion of choline into phosphocholine  in C4-2 cells . Using a bipartite N-C AR reporter assay . CHKAi also decreased androgen-stimulated nuclear AR levels .To further investigate the functional consequences of CHKA on AR signaling, we used a reporter assay to assess AR transactivation capacity in C4-2 PCa cells and observed a 2.5-fold decrease (g siCHKA . CHKA kn < .005)  but fail < .005)  lacking  < .005) , indicatocholine , a metab-2 cells . The lever assay , we founer assay . In lineP < .001) (P < .05) (KLK3 (PSA), TMPRSS2, and SLC45A3 were downregulated by CHKA knockdown . These analyses highlight a functional link between AR and CHKA signaling, consistent with our biochemical and reporter gene studies, indicating a cooperative function of CHKA in driving the AR-regulated transcriptome by stabilizing AR protein levels.Transcriptome sequencing from CHKA knocked down C4-2 cells highlighted the global impact of CHKA on the AR transcriptome. We found a statistically significant bidirectional overlap between siCHKA and androgen-regulated genes  , F and G < .001) , which i < .001) . Most noP < .05) , J and Knockdown  and thatSupplementary Figure 4A, available online). CHKA knockdown in LNCaP cells led to a dose-dependent reduction in androgen-stimulated growth in these AR-expressing cells  . Similarly, CHKAi had profound growth inhibitory effects on AR-positive C4-2 and LNCaP-LN3 cells, which were partially reversed by the synthetic androgen R1881. CHKAi only modestly inhibited growth of PC3, DU145, and PNT1a cells . These data are collectively consistent with a greater effect of CHKA on the growth of AR-expressing PCa cells, likely resulting from dual inhibition of AR signaling and the Kennedy pathway in these cells. In clonogenic survival assay with LNCaP cells, AR antagonists bicalutamide and enzalutamide decreased colony number by 1.5- (P < .001) and 2.3-fold (P < .001), respectively, while CHKAi reduced the clonogenic survival of androgen-dependent LNCaP by more than 50-fold (P < .001) . In androgen-independent VCaP cells, bicalutamide was not effective at clonal repression while enzalutamide was marginally effective ; however, CHKAi treatment led to a 7-fold decrease in clonogenic potential (P < .01) . The growth-promoting role of CHKA does not depend on the catalytic activity of CHKA because the addition of exogenous phosphocholine or phosphatidylcholine did not rescue growth inhibition by CHKAi . In an ex vivo culture assay, treatment of hormone-naïve primary PCa tissue with bicalutamide and CHKAi decreased AR expression in tumor epithelia while both anti-androgens and CHKAi increased levels of cleaved caspase-3, a marker of apoptosis . Collectively, these experiments indicate that CHKA inhibition opposes androgen action, thus triggering apoptosis in PCa cells and tissue.Because of its vital role in the Kennedy pathway, all investigated PCa cell lines express CHKA , providictively) . In a pa < .001) . SimilarP < .01) . ResultsP < .05) (P < .05) . Further analysis of the excised tumor xenografts confirmed a tendency towards decreased CHKA levels and clearly decreased AR protein expression in CHKA sh#2 xenografts from mice treated with doxycycline . CHKA activity can be considered to have conferred an invasive phenotype on PCa cells because both CHKA and AR depletion delayed “wound healing” in a scratch wound assay to a comparable extent (1.6-fold decrease as compared with siNT) (P < .001) . These observations are consistent with PCa cells requiring CHKA and the AR for motility, as suggested by transcriptome analysis enrichment of pathways regulating GTPase activity  in a doxycycline-inducible manner. In the presence of doxycycline, sh#1 did not robustly decrease CHKA levels, but the two other CHKA shRNAs (sh#2 & sh#3) were effective at decreasing CHKA levels . These sP < .05)  and showP < .05) . The doxth siNT)  and simi < .001)  and a Boactivity .The data presented here demonstrate the importance of CHKA inhibition in decreasing AR levels and repressing PCa growth and invasion, highlighting the potential future benefits of inhibitors of this kinase in the clinical management of PCa.By employing genome-wide approaches, we characterize the AR-regulated kinome in PCa and provide direct evidence that CHKA is a chaperone for AR, promoting its stability and function. Indeed, to our knowledge this is the first report indicating that kinases can act as chaperones. Decreasing CHKA protein levels, both by siRNA knockdown and CHKA inhibitor, antagonized AR signaling and constrained development of aggressive phenotypes in models of PCa. We propose that CHKA promotes AR signaling and that therapeutic targeting of CHKA has the potential to improve management of advanced PCa.Initial profiling of the AR-regulated kinome in PCa revealed functionally and clinically important signaling events downstream of the AR in PCa. Within the AR-regulated kinome, we characterized CHKA as a chaperone that regulates AR signaling, elucidating a feed-forward AR-CHKA signaling loop that reinforces AR activity and allows CHKA to maintain its expression in PCa. We show that the chaperone function of CHKA confers a growth advantage on PCa by stabilizing the AR in addition to its well-known function in fuelling membrane production through the Kennedy pathway  Figure , suggestIn advanced PCa models, AR knockdown is very effective in repressing tumor growth ,40, but The discovery that CHKA acts as a chaperone provides a potential explanation for an earlier study in breast cancer, which reported promotion of cancer cell growth by both wild-type and kinase-dead CHKA mutants, suggesting that the CHKA protein level per se, and not its catalytic function, is required for tumor cell growth . AnotherLBD and would be predicted to not interact with CHKA. Appropriate patient selection can avoid unnecessary overtreatment and toxicity.A number of studies have documented the overexpression of CHKA protein in multiple cancer types including breast, colorectal, endometrial, lung, ovarian, and prostate ,52. ConsCHKA contains multiple “NR” box or LxxLL motifs often found in coregulatory proteins of nuclear receptors. Because the LBD is highly conserved among members of the nuclear receptor superfamily, it is likely that CHKA may function as a chaperone for other nuclear receptors. In view of the approximately 76% sequence homology between the LBD of the glucocorticoid receptor (GR) and AR , and theThis study also had some limitations. Firstly, the CHKAi employed in this study is a generic inhibitor and some of its effects on the cell growth may have been more pronounced as compared with the siRNA studies owing to the inhibition of other related kinases, although many experiments with the inhibitor and siRNA produced concordant results. This underscores the need for developing highly specific CHKA inhibitors. Secondly, as CHKA protects AR by binding to its LBD, PCa patients whose tumors are driven by ARVs may still maintain AR signaling despite therapeutic targeting of CHKA. Moreover, the results we reported are underpinned by appropriate statistics, although in some of the functional studies the outcomes should be interpreted against the number of experiments and variance we reported.The discovery of a chaperone function of CHKA for AR signaling might also have wider implications in the treatment of other cancers and should accelerate the screening of potential CHKA inhibitors that will not only inhibit the catalytic function but also decrease the CHKA protein level. We believe our work will direct efforts in the treatment of PCa in particular, but also for other cancers, by stimulating a systematic re-evaluation of CHKA as a therapeutic target, especially in tumors where it is overexpressed and may exhibit chaperone-analogous effects on other oncogenic proteins.Cancer Research UK program grant (to DEN) and also by the US Department of Defense , PCFA/Cancer Australia/Movember (grant IDs 1012337 and 1043482 to WDT and LAS), Cancer Australia (grant ID 1043497 to WDT and JC), and The Ray and Shirl Norman Cancer Research Trust (to WDT and LAS). The Dame Roma Mitchell Cancer Research Laboratories were supported by an establishment grant from the Prostate Cancer Foundation Australia (ID 2011/0452). FO was supported by a PhD project grant from Prostate Cancer UK (S10-10). LAS is supported by a Young Investigator Award from the Prostate Cancer Foundation (the Foundation 14 award).This work was supported by a


Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to prevent cell-matrix disruption, and is less likely to impede cancer cell invasion. AR signaling in prostate cancer stroma appears therefore to alter patient outcome by maintaining an ECM microenvironment inhibitory to cancer cell invasion. This paper provides comprehensive insight into AR signaling in the non-epithelial prostate microenvironment, and a resource from which the prognostic and therapeutic implications of stromal AR levels can be further explored. Prostate cancer causes more than 28,000 deaths each year in the United States . CriticaProstate development and homeostasis requires bidirectional signaling between epithelial cells and stromal constituents, including fibroblast and smooth muscle cells, vasculature, soluble factors and extracellular matrix (ECM) proteins. This signaling is disrupted in cancer -10, wherIn the adult prostate, activation of epithelial androgen receptor (AR) by testosterone (T) and 5α-dihydrotestosterone (DHT) is necessary for cell survival and regulation of seminal fluid proteins including prostate specific antigen (PSA) , which iIn this study, we compared AR levels in epithelial and stromal compartments of patient-matched benign and malignant prostate tissue, and demonstrate an association between low stromal AR levels and death from prostate cancer at one, three and five years post diagnosis. This is the first time that stromal AR changes have been shown to be specific to the immediate cancer microenvironment and not due to differences between patients, and are related to adjacent malignant but not benign regions of the same prostate. We further show that androgen signaling in human prostatic myofibroblasts induces a microenvironment inhibitory to the movement and invasion of tumor cells, primarily by altering ECM composition. This protective AR-mediated phenotype in prostate cancer-associated stroma has implications for understanding the early stages of cancer progression, and for the use of androgen withdrawal in the absence of surgical management.−8, Fig. The relationship between prostate cancer outcome and AR levels in stroma and epithelium was investigated by AR immunohistochemistry on 64 patient-matched BPH and prostate cancer samples in patients of median age 87 years Fig. . Similarin vivo tissue recombination [−/CK8.18+, was similar in grafts from the four patients with PShTert-AR  and PShTert-ctrl  lines. As expected, a significantly lower proportion of stroma in the grafts containing PShTert-ctrl myofibroblasts expressed AR .In order to more precisely define the transcriptional role for AR in PShTert-AR cells, we performed expression microarray analysis, identifying 2615 DHT regulated genes in PShTert-AR myofibroblasts and 1000 in C4-2B epithelial cells (>0.5 log2 fold change). Importantly, only 254 of those regulated genes were common between the two cell lines, and half of those (127/254) were regulated in the opposite direction Fig. . RT-qPCRF-box protein 32 (FBXO32) gene product. FBXO32 is a member of the family of DNA-licensing proteins that regulates progression from G1 phase by inhibiting cyclin D1 [One mediator of the anti-proliferative effect of androgen in myofibroblasts may be the fibroblast-specific androgen regulated yclin D1 . To deteWe next considered whether AR activity in myofibroblasts could affect epithelial growth. Conditioned media was collected from PShTert-AR and PShTert-ctrl myofibroblasts treated with or without DHT. Compared to vehicle, media from DHT treated AR positive myofibroblasts increased C4-2B and PC-3 proliferation by 1.64 and 2.72 fold respectively , a predominantly fibroblast-specific AR coregulator and a component of the focal adhesion (FA) complex, plays an important role in AR-mediated activity in myofibroblasts [We recently reported that hydrogen peroxide-inducible gene 5 , and inhibition of ECM degrading enzymes  squeezing of cells through the ECM. The amount and arrangement of ECM fibers, enzymes, and ECM pore size are capable of altering each type of migration, and have been implicated in malignant disease -47, and in vivo knockdown of stromal AR was found to be more effective at inhibiting tumor growth in early stages of progression rather than at later stages [It appears from our results and those of others that stromal AR may also promote prostate cancer proliferation, as suggested here by the production of an unidentified soluble mediator, and/or ECM-bound growth factor -52. On tr stages , 56. In In conclusion, this manuscript is the first to show that unique androgen/AR transcriptional responses in prostate myofibroblasts play an important role in stromal-mediated alterations to the ECM and microenvironment. Clinically, it will be important to determine the key factors affected by a loss of stromal AR that may influence patient outcome and could be exploited by targeted therapies. The precise composition of the ECM may be one such key mediator of epithelial cancer cell invasiveness and thus indicative of patient outcome, tumor aggressiveness and treatment response.http://www.sa-pccoc.com/) tracks men diagnosed with prostate cancer in the South Australian public health system. Using the SA-PCCOC database, we identified 66 sequential patients whom underwent TURP for symptomatic relief of BPH urinary obstruction at the Repatriation General Hospital  between 2000 and 2007, in which there was (i) a first diagnosis of prostate cancer on histological Gleason grading, (ii) cancer comprising >5% of the specimen, and (iii) sufficient areas of BPH and cancer in each sample from which multiple cores could be obtained. Areas of BPH and cancer were identified by H&E staining and mapped onto paraffin embedded material by a pathologist. Duplicate five mm cores of BPH and cancer from each individual were then used to generate tissue microarrays. Clinical data relating to each patient was acquired from the SA-PCCOC database. Sample and data acquisition was performed according to protocols approved by the Flinders Medical Centre and RGH Ethics Committees (Protocol #042/10).The South Australia Prostate Cancer Clinical Outcomes Collaborative  and, detected using the LSAB+ System-HRP kit . Staining was scored additively by two researchers in three independent fields from 0 (no staining) to 3 (very intense staining), yielding sample scores of 0-9 in epithelial and stromal compartments of both cancer and BPH. No stromal compartment achieved very intense staining. The mean sample score from the two researchers yielded the AR staining intensity score. Differences in staining intensity, Gleason Score, serum PSA and percent prostate cancer were assessed using two-tailed Mann-Whitney U tests. In samples dichotomized by median AR level, differences in prostate cancer-specific death were assessed using Barnard's Exact test. Significance was set at p<0.05.in vitro between passages 3-6. The integrity of primary fibroblast cultures was confirmed in vitro by growth properties, immunological markers and RNA expression, and their tumorigenic potential in vivo using tissue recombination with BPH-1 cells.Human tissue was obtained from consented patients in accordance with Human Ethics Research Approvals 34306 at Epworth Hospital, 03-14-04-08 at Cabrini Hospital and RMO 2006/61082004000145 at Monash University, and processed as previously published . Brieflyin vitro experiments C4-2B [For ts C4-2B  and PC-3ts C4-2B , and WMPts C4-2B  were use™  or LTX-plus (Life Technologies) for transfection of luciferase constructs. Following transfection, cells were treated with 0.1-1000 nM of steroids or equivalent vehicle (ethanol) control for 22 h. Results are presented as mean (± SEM) of six independently transfected wells.Transactivation studies were performed as described previously  using LiChIP was performed as described previously , using sELISA was used to measure collagen 1 levels in media collected from confluent PShTert-AR myofibroblasts treated with 50 μg/ml ascorbic acid  and either DHT, vehicle control and or BIC. Media collected from six independent treated confluent cells was plated into 96-well Maxisorp  plates and incubated overnight at 4ÐC. Plates were washed in PBS supplemented with 0.1% Tween (PBST), blocked in 2.5% BSA and washed in PBST, plates were probed with rabbit anti-collagen type 1 antibody  for 3 h and detected via a europium-tagged anti-rabbit secondary antibody. The concentration of collagen was subsequently fluorescently detected using 340 nm excitation/615 nm emission spectra.5) were combined with 2 mm X 2 mm X 1 mm pieces of patient-derived primary human prostate cancer tissue in 30 μl of collagen/RPMI 1640 + 5% FBS with 0.1% penicillin-streptomycin for 24 h, and grafted under the renal capsule of NOD-SCID mice for 8 weeks. Mice were castrated, and grafts allowed to grow for an additional 3 days before being removed, paraffin-embedded and sectioned. Immunohistochemistry for Ki-67 (Sigma-Aldrich), caspase-3 (Sigma-Aldrich), and AR (Sigma-Aldrich) was performed.Renal capsule tissue recombination grafting of PShTert-AR or PShTert-ctrl cells with pieces of patient-derived primary human prostate cancer tissue into NOD-SCID mice was performed and analyzed as previously described , 35, 60.http://david.abcc.ncifcrf.gov/home.jsp .RNA extracted from cells treated with either DHT or vehicle using the RNeasy Kit , was analyzed using Affymetrix 1.0st Gene Arrays. Data was Bioinformatically analyzed using either in R using Gene Ontology categories, or in R or using DAVID Bioinformatics Resources GAPDH, PPIA, and mRPL19 as per GeNorm (http://medgen.ugent.be/~jvdesomp/genorm/#introduction).cDNA created from sample RNA was analyzed via RT-qPCR as previously described , using SProtein lysates in RIPA buffer were prepared as previously described  and immu4 PShTert-AR, PShTert-ctrl, or C4-2B cells were added to 24-well plates containing treatment media and left to adhere for 15-240 min at 37ÐC. Media was removed and cells were washed with PBS before manual counting. Cellular attachment (trypsinization resistance) was measured using a crystal violet assay adapted from a previous study [4 cells/well in 96 well plates) overnight and treated with 1-100 nM DHT ± 10 μM BIC or equivalent vehicle control for 16 h. Cells were washed with PBS and incubated with trypsin for 2.5 - 15 min. Cells were washed, ethanol fixed and stained with 1% crystal violet solution. Dye was eluted from cells with 10% glacial acetic acid and the concentration measured at an absorbance of 595 nm. Motility and invasion was tested described previously [Proliferative response of PShTert-AR or C4-2B cells to DHT and or BIC was measured in quadruplicate via Trypan blue exclusion. Adhesion of PShTert-AR, PShTert-ctrl, or C4-2B cells was measured using an adhesion assay as described previously . Brieflyus study . Brieflyeviously , using cMatrices produced from confluent fibroblasts treated with 50 μg/ml ascorbic acid and 10 nM DHT or vehicle or 10μM BIC, were decellularized with EDTA and used in adhesion assays (above) and trypsinization assays adapted from previous descriptions .3D-matrices were produced from DHT or vehicle treated fibroblasts seeded into gelatin coated wells as previously described . After dWhen the cells had grown to 100% confluence, media was replaced with stripped media supplemented with 50 μg/ml ascorbic acid and 10 nM DHT or equivalent vehicle control. Treatment was repeated every 48 h. After 8 days, myofibroblasts were removed via an extraction buffer containing PBS, 0.28% ammonium hydroxide (Sigma-Aldrich), and 0.5% Triton-X (Sigma-Aldrich). Remaining 3D matrix was gently washed in PBS prior to adherence, proliferation and invasion assays.4 cells per chamber) in stripped medium were aliquoted. Following 16h by incubation, Ibidi inserts were removed, leaving a 500μm cell-free gap. Migration of PC3 cells across the gap was monitored for 0, 3, 7, 11, and 15 h time-points, using a Zeiss Axio Observer.Z1 with HBO 100 microscope illuminating system . Migration was measuring as cell-free gap-closure using AxioVision Rel 4.8 software, and analysed with the MRI Wound Healing Tool .For cancer cell gap closure assays, into each well, sterile silicon culture-inserts (Ibidi 80209) were positioned into wells containing 3-D matrices, and PC3 cells (3.5 × 10


Despite developments in mental healthcare over recent decades, there remains a group of people with very complex needs who require lengthy admissions and high levels of support in the community on discharge. This is the group that mental health rehabilitation services focus on. In the context of contemporary mental health services that minimise in-patient lengths of stay, the needs of this group must not be overlooked. Providing a local, ‘whole system, integrated rehabilitation care pathway’ requires intelligent commissioning in order to avoid the social exclusion of this group to the ‘virtual asylum’ of out-of-area placements. In the UK, mental health rehabilitation services focus on this group. Simply put, these are the people who have not recovered adequately to be discharged home. The majority have a diagnosis of psychosis complicated by treatment refractory symptoms, severe negative symptoms and cognitive impairments (particularly dysexecutive problems). Many have coexisting mental health problems  and most have physical health problems as a result of a combination of poor diet, lack of exercise, smoking and the side-effects of psychotropic medication. As a consequence of these multiple issues, everyday functioning tends to be severely impaired and challenging behaviours and poor engagement are common.2 However, they usually require lengthy in-patient admissions and more intensively supported accommodation on discharge, which results in them absorbing 25–50% of the mental health and social care budget.3 They can therefore be considered a ‘low volume, high needs’ group.This group represent a relatively small proportion of people in receipt of mental healthcare; it is estimated that around 14% of people newly diagnosed with psychosis will develop the kinds of complex problems that will require rehabilitation services.4 which may suggest that acute services are keen to exhaust therapeutic possibilities before referral or, perhaps more likely, that there is often a long wait for a rehabilitation bed because of underprovision. The remaining 20% of referrals come from forensic/secure services,4 illustrating the importance of local rehabilitation services in the pathway out of secure services.Most people are referred to in-patient rehabilitation services when the admitting service has exhausted treatment options . However, the mean length of admission prior to transfer to rehabilitation services is around 10 months,5 This pathway includes different types of in-patient rehabilitation units and a range of supported accommodation services for people to be discharged to. Almost all NHS mental health trusts in England provide in-patient rehabilitation services.4 These include: ‘high dependency units’ that are usually sited within a hospital setting, with an expected length of stay of up to 2 years and where most patients are involuntarily detained; ‘community rehabilitation units’, the majority of which can also take detained patients and have an expected length of stay of up to 3 years; and ‘complex care units’ for those who require a longer period of in-patient care that are usually hospital based, take detained patients and have an expected length of stay of over 5 years. The private sector also provides in-patient rehabilitation units, usually with a similar specification to high dependency units or with a higher level of security and staffing. These were previously referred to as ‘low secure rehabilitation units’ but now tend to be called ‘locked rehabilitation’ to differentiate them from forensic services (as a result of new commissioning arrangements for the latter). Some offer specialist facilities for people with especially challenging behaviours and less common combinations of complex problems. Supported accommodation services are usually provided by the independent or voluntary sector and include 24 h staffed residential/nursing care homes, individual or shared staffed tenancies and floating outreach services providing support to those living in independent tenancies. Over half the NHS mental health trusts in England have a community rehabilitation team4 that provides clinical input to local supported accommodation services to enable patients in their ongoing recovery and progress through the care pathway.6 For those without a community rehabilitation team, clinical input is provided by other community mental health services such as community mental health teams, recovery teams or assertive outreach teams.Rehabilitation services operate as a ‘whole system, integrated care pathway’ across in-patient and community settings provided by statutory and non-statutory health and social care sectors.7 there has been relatively little research into its effectiveness. We previously surveyed 140 patients of mental health rehabilitation services in one inner London trust and found that those in complex care units had much higher needs and poorer social functioning than those in high dependency and community-based rehabilitation units, and those in high dependency and complex care units had more severe challenging behaviours than those in community units.8 This cohort was followed up after 5 years, by which time two-thirds had achieved and sustained successful community discharge and 9% had achieved fully independent living. The only factor found to be associated with positive progress along the pathway was medication adherence.9 In Ireland, a prospective case–control study compared outcomes over 18 months for people with complex needs receiving mental health rehabilitation services and those awaiting them. Those receiving rehabilitation services were eight times more likely to achieve and sustain community discharge and there was a greater improvement in social functioning. Those with higher levels of unmet need, substance misuse and challenging behaviours were less likely to achieve successful community living.10Although the mental health rehabilitation care pathway has been well described,The Psychiatric Bulletin, Meaden and colleagues describe a survey of over 100 patients of ten NHS mental health rehabilitation units in the West Midlands .11 Their results illustrate the high level of challenging behaviours among these patients and the difficulties of engaging them in treatment. Their findings concur with those of previous studies; there were higher levels of aggressive challenging behaviours among those in the high dependency units and more socially unacceptable challenging behaviours among those in the complex care units, whereas those in the community rehabilitation units were better engaged with their rehabilitation and presented with fewer challenging behaviours. The data are cross-sectional and causality cannot be inferred, but they suggest that these different components of the pathway are necessary to support people at different stages of their recovery.In this issue of 12 Holding therapeutic optimism is no easy task. Staff need to receive training to develop an understanding of the process of individual recovery and how to support this.14 They need to hold in mind the long-term view for the gradual improvement of symptoms and everyday functioning that does come about for the majority of those who receive rehabilitation, that enables them to leave hospital and move to the community successfully without further relapse, readmission or placement breakdown. Working with people who improve at a relatively slow rate requires patience and commitment. Most staff work in only one component of the rehabilitation pathway and it can be difficult to hold on to that longer-term view. In addition, the metric of success in contemporary in-patient settings is an average length of stay that is as short as possible. Understanding the need for longer-term treatment of the complex needs group is vital and making the case for local rehabilitation services is therefore a key skill for rehabilitation practitioners.15The results also highlight the importance of, and difficulty in, maintaining therapeutic optimism when working with people with such complex needs. Meaden et al acknowledge the need to build on the rapport between patients and their primary nurse to enable the person to engage in rehabilitative interventions that involve other members of the team and they suggest approaches to facilitate this. Staff working in these settings need to hold hope for patients who have often been considered ‘untreatable’ in other parts of the mental health system and who may have lost a sense of their own identity, purpose and connections with family and friends through the course of their illness.16 This phenomena has been widely reported in deinstitutionalised countries17 and heavily criticised.20 In 2011, the Royal College of Psychiatrists published a helpful guide for commissioners in England and Wales on best practice in the delivery of services for people with complex needs to address this problem that called for ongoing investment in services that make up the local rehabilitation care pathway.21 However, with recent changes in commissioning and commissioners in most areas of England, this guidance has probably been at best forgotten and at worst ignored. With the imminent introduction of a tariff-based system of mental healthcare, it is imperative that commissioners and senior managers understand the need for appropriate investment in local specialist mental health rehabilitation services and supported accommodation for those with complex needs if we are to avoid a repeat of the shameful exportation of this group into out-of-area placements that lack a rehabilitative ethos and compound the social dislocation of this vulnerable group.20We already know what happens if we do not invest in local mental health rehabilitation services; those with complex needs become stuck on acute in-patient units and are eventually relocated to out-of-area placements in private hospitals, nursing and residential care homes where they often remain out of sight and out of mind in a ‘virtual asylum’.22 The evidence base for mental health rehabilitation services is growing. We now know that the majority of people receiving rehabilitation are able to achieve and sustain successful community living and that improving the quality of care in in-patient rehabilitation units promotes patient autonomy, the main aim of rehabilitation.4 Studies like that of Meaden and colleagues11 are important in furthering our understanding of the needs of this group. In addition, two national programmes of research are underway across England, both funded by the National Institute of Health Research, to investigate the aspects of care delivered in different components of the rehabilitation pathway that are most beneficial (the Rehabilitation Effectiveness and Activities for Life (REAL) study and the Quality and Effectiveness of Supported Tenancies (QuEST) study). We hope that these programmes will help to guide future practice and ensure appropriate investment in the treatment, care and support for those with the most complex mental health needs.Two years ago, the UK Schizophrenia Commission called for investment in high-quality services to deliver evidence-based treatments for people with long-term psychosis.


Allergic Rhinitis (AR) is a worldwide spread illness having an important impact on social life, sleep quality, school, and work productivity and huge costs. Patient preference is becoming an important aspect in medical care. ISMAR was designed as the first-over global survey to identify differences in attitudes and preference in patients and physicians about AR. ISMAR 2 is the second phase.ISMAR is an international, multicenter, non-interventional, cross-sectional study in adults and children (≥ 6 years) with physician-diagnosed AR of at least 1 year of duration conducted in Algeria, Egypt, United Arab Emirates, Qatar, Kuwait, India, Islamic Republic of Iran, Pakistan, and Saudi Arabia.Physicians  working in public and private sectors or both were selected from master lists of physicians attending AR patients and invited to participate in the study. They answered to the Investigator’s Questionnaire  and recruited consecutive patients to whom the ISMAR questionnaire was administered. Data collection was performed during a single visit. A patient's questionnaire and a Case Record Form were also filled in. Statistical analysis was descriptive. Herein we are showing the physician's view.1 antihistamines (Oa-H1: 68.5%) and intranasal corticosteroids (ICS: 64.0 %) [5 in a 0–5 scale].One hundred and seventy eight physicians participated. They were aware about ARIA (82.6%) and GINA (76.4%) and followed them to classify patients severity (83.7%) and for choosing the treatment accordingly (82.0%). Key symptoms to diagnose AR were congestion (85.4%), sneezing (93.3%), and anterior watery rhinorrhea (89.3%). SQ and AR severity were assessed mainly by clinical history (93.8% and 98.7 %). The most commonly tests were serum total IgE (52.8%), eosinophilia (50.0%), x-rays (48.9%), CT scan (44.4%), and allergen skin tests (39.3%). The main reasons to prescribe medication were symptom severity/frequency (98.3 %), drug efficacy (80.3% %) and safety (78.7 %). Other less relevant reasons were personal experience (60.7 %), route of administration (55.1%), cost (52.8%), and frequency of doses (48.9%). The preferred medications were oral anti-H1 and ICS were the most widely recommended treatment for AR and were considered effective and safe.Guidelines are well known and useful to physicians. Clinical history was the main instrument to evaluate patient's SQ, classification and severity of AR, and choice of treatment. Allergen skin tests were not commonly performed. Oa-H


Objective: To describe endometrial abnormalities on transvaginal ultrasonography and histopathology in women after quinacrine sterilization.Methods: It was an analytical cross sectional study conducted during February 2012 to April 2013. The sample size calculated at 95% confidence level was 540. Sampling technique used was simple random sampling. The medical history, examination, transvaginal ultrasonography and biopsy of suspected lesion was performed in quinacrine sterilized women.Results: The calculation of statistics showed the mean age at quinacrine sterilization was 38.5 years, standard deviation 6.517, and standard error 0.461. The endometrium was regular and smooth with homogenous images in 86% (n= 466), irregular endometrium with heterogeneous images on transvaginal ultrasound in 9.4% (n =51) and endometrial growth with high level echoes in 4.2% women (n= 23). The histological findings included hyperplasia and well differentiated adenocarcinoma in two patients respectively.Conclusion: The irregular endometrium, adhesions, and growths were found after quinacrine sterilization. The risk of endometrial growth was more after 10 years duration of quinacrine sterilization. More than 11,000 women underwent quinacrine sterilization in Faisalabad Pakistan.6Quinacrine is a very old drug that is used for many years by humans for many medical conditions including treatment of malaria, arthritis, giardiasis6Quinacrine insertion was performed by inserting quinacrine pellets in the uterus by intra uterine device inserter under aseptic conditions and all pellets were released in a line near fundus of the uterus during proliferative stage. Same procedure was repeated after one month duration for double insertion. 90% women opted for single insertion of quinacrine. Only 10% had double insertion of quinacrine in Faisalabad.7 The maternal morbidity and mortality was improved after quinacrine sterilization in developing countries.8 The data collected in follow-up of the participants identified a cluster of cancer cases among women who had received quinacrine but it was statistically insignificant.9,10The official statistical maternal mortality in Pakistan was 286 deaths per 100,000 live registered births.11World Health Organization (WHO) had suggested that there should be continued surveillance of women who have received quinacrine sterilization in the past for risk of health complications, such as endometrial adhesion-related morbidity, or adverse maternal and fetal outcomes.Nevertheless, many peer reviewed research studies had suggested that quinacrine sterilization was relatively safer than surgical sterilization but scanty literature was available about transvaginal ultrasonography and endometrial histopathology after quinacrine sterilization. Our aim was to identify endometrial abnormalities like adhesions and growths after intrauterine insertion of quinacrine for female sterilization on transvaginal ultrasonography and histopathology.This population based cross sectional study was conducted in Faisalabad, Pakistan. The ethical issues were considered, informed consent was obtained and all information was kept confidential. The ethical permission was obtained from Advanced study and Research Board King Edward Medical University. 6 The expected frequency of endometrial adhesions in quinacrine sterilization was 6.4%.12 The worst acceptable result was 8.45% used for calculations. Sample was calculated at 95% confidence level and 80% power of the test. According to this formula, the sample used in the study was 540 subjects who were selected randomly from the study population. To find the optimal sample from the study population, the sample size calculator Epi info2000 software was used. The study population of quinacrine sterilization was 11000.The women with history of hormonal treatment, pelvic inflammatory diseases, polycystic ovary syndrome were excluded to control bias. Endometrial thickness was measured from echogenic border to echogenic border across the endometrial cavity in a sagittal midline plane by transvaginal ultrasound. The double layer of endometrium less than 5mm thick and no focal thickening was defined as thinning of endometrium. Homogenous, regular, smooth endometrium measuring 5-7mm was considered as normal endometrium. If endometrial thickness was 8mm or greater it was described as thickened endometrium. Endometrial adhesions were defined as thick fibrotic bands that had distorted the uterine cavity. On transvaginal scan they were reported as heterogeneous, hyperechoic, punctated shadows and irregularities. Hyperplasia was visualized as diffuse or focal hyperechoic endometrial thickening or asymmetrical heterogenous images and irregularity. Endometrial abnormalities and irregular bleeding per vagina were advised biopsy under ultrasound guidance by a gynecologist. The biopsy was obtained under aseptic conditions. The histopathology of the specimen was done in histopathology section of Pathology Department in King Edward Medical University Lahore by three histopathologists. Each histopathologist gave independent report to control bias.Data was analyzed using Statistical Package for Social Scientists (SPSS) soft ware version 16. The data were stratified according to women age at quinacrine sterilization and duration of quinacrine sterilization to control confounders. All variables in the study were expressed as frequencies and percentages. The continuous variables like age were reported as mean, standard deviation and standard error. The risk estimation was performed by constructing 2×2 tables, calculating crude odds ratio and 95% confidence intervals. P values were described to show significance. The p value less than 0.05 was considered significant. The missing data were excluded because it was less than 5% for all variables. The chi-square-test was used to test for differences in the distribution of quinacrine sterilization failure as well as endometrial growth stratified by age and duration of quinacrine sterilization.The multivariate analysis was performed to calculate adjusted odds ratio. The confounders identified were age, parity, obesity, socioeconomic status, and general heath status. Age was coded as more than 35 years, equal or less than 35 years, parity was coded as less than 5 children, more than 5 children. Obesity was coded as Body mass index more than 30, less than 30. Socioeconomic status was derived from social status score developed by the National Institute for Social Research based on income, employment, and level of education. These scores were divided into low socioeconomic status (below 25th centile), medium (between 25th and 75th centile), and high socioeconomic status (above 75th centile). The general health status was coded as good if no chronic illness was present and worse if chronic illness (disease more than 3 months) present.The random sample of 540 quinacrine sterilized females was selected. The calculation of statistics showed the mean age at quinacrine sterilization was 38.5 years, standard deviation 6.517, standard error 0.461. The sterilization failure rate 10.74% was present in the study.Demographic features of study participants: The demographic features showed that the most of the females were more than 35 years old when sterilized with quinacrine (80%), living in rural areas (67%), poor (51.85%), unemployed (89.7%) and uneducated (65%). (d (65%). Endometrium on transvaginal ultrasonography: The transvaginal ultrasound showed endometrial adhesions in 15% and endometrial thickness less than 5mm in 13% females after receiving quinacrine sterilization.The endometrial patterns found in present study were regular and smooth endometrium with homogenous images in 86% (n= 466) women, irregular endometrium with heterogeneous images on transvaginal ultrasound in 9.4% (n =51) and endometrial growth with high level echoes in 4.2% women (n= 23) after quinacrine sterilization.  The histological findings included adenocarcinoma in two and hyperplasia in two women. One woman had simple hyperplasia and other had complex hyperplasia. In simple hyperplasia, there was increased number of proliferative glands. The glands were large in number, crowded and separated by stroma. In complex hyperplasia with atypia the glands were present ‘back to back’. There was marked cytological atypia, pleomorphism, hyperchromatism and abnormal chromatin patterns. The adenocarcinoma was seen with irregular glands lined by malignant columnar epithelial cells.Univariate Analysis: The univariate analysis had shown that the age equal or less than 35 yrs at quinacrine sterilization was 9.63 times more at risk of sterilization failure than the age more than 35 yrs at quinacrine sterilization. It was found statistically significant. . It was found statistically significant. Multivariate Analysis: The logistic regression analysis was employed to control the confounders. The multivariate analysis had shown that the 35 years or younger age when received quinacrine sterilization was 8.08 times more at risk of sterilization failure than the age more than 35 yrs. . The quinacrine sterilization duration more than ten years was 9.39 times more at risk of endometrial growth than duration of quinacrine sterilization of ten years or less. . It was found statistically significant. 13 Contrary to this, the latest studies had documented the anti cancerous role of quinacrine by reversing multidrug resistance in cancer cells.14 The expert panel of WHO had recommended surveillance regarding adverse maternal outcomes and endometrial abnormalities in women after quinacrine sterilization.The quest for a secure and effective contraceptive has puzzled the pubic for centuries. Quinacrine sterilization was hypothesized to cause mutagenesis and carcinogenesis in women after intrauterine insertion. Theoretically, quinacrine was described to bind with deoxyribonucleic acid (DNA). Therefore the risk of genetic mutations and carcinogenesis was increased.15 The risk of sterilization failure was found more in 35 years or younger age in recent study.In the present study mean age at quinacrine sterilization was 38.5 years, standard deviation 6.517, standard error 0.461. Suhadia and Sokal had described follow up findings of quinacrine sterilization in females having mean age 33.2 years, standard deviation 9.75 and mean age 36.4 years, standard deviation 6.61 respectively.16,17 In Vietnam, the double insertions of quinacrine pellets were used to sterilize women while in Faisalabad single insertion of quinacrine pellets was the preferred method. The training of health care workers had shown to decrease sterilization failure rates from 5.4%(SE2.3) to 0.9%(SE0.4).6The sterilization failure rate in quinacrine sterilization was found 10.74% in Faisalabad. In Vietnam after 5 years follow up of different sterilization methods, quinacrine sterilization had the highest failure rate 13.3%, tubectomy had the lowest failure rate 1.0% and followed by 4.1% with vasectomy.12The endometrial patterns found in present study were regular and smooth endometrium with homogenous images in most of the women, irregular endometrium with heterogeneous images on transvaginal ultrasound in 10% and endometrial growth with high level echoes in 4.2% women after quinacrine sterilization. Ferriera had described the endometrial patterns on transvaginal ultrasound after quinacrine sterilization as irregular high-level echoes, or variable echogenicities showing irregular endometrium. The irregular endometrial lining was produced by intrauterine adhesions in 6.4%. The decrease in endometrial thickness as measured from one echogenic border to another border across the cavity of the uterus was observed in 11% women.18 In this study there was endometrial hyperplasia in two (0.37%) and endometrial carcinoma in 2(0.37%) after quinacrine sterilization. In an international study the women were investigated for endometrial abnormalities through transvaginal ultrasound and subsequent histopathology for confirmation and it was found that the chances of histopathological evidence of endometrial abnormalities were increased if history of irregular bleeding was present along with abnormal endometrium on ultrasound.19 In present study the biopsy was performed in women after quinacrine sterilization that had irregular, non homogenous, suspicious images on transvaginal ultrasound and suffering from irregular uterine bleeding to increase the yield on histopathology. The insufficient family planning is the leading cause of maternal mortality in developing countries.20 In a study it was found that measurement of endometrial thickness was important in diagnosing abnormal endometrium with irregular uterine bleeding.21 Similarly in the present study the cut off value of 5mm for endometrial thickness was used.Another research showed the difference in thickness of endometrium in cancer and hyperplasia were found statistically significant.22In international studies conflicting evidence was presented about endometrial tumors and growths after quinacrine sterilization. Many experimental studies were conducted on animals to provide evidence in this regard. In present study the quinacrine sterilization duration more than ten years was 7.59 times more at risk of endometrial growth than duration of quinacrine sterilization less or equal to ten years. However, research studies in Chile showed no significant evidence of increased risk of reproductive tract cancers for this method.23 Hypertension and adverse maternal outcomes were found after quinacrine sterilization.24,25Sokal and his colleagues, in a follow-up study of quinacrine female sterilization in Vietnam, had shown no association between carcinogenesis and quinacrine sterilization. They also demonstrated that the number of hysterectomies for endometrial abnormalities was much higher (8%) in females surgically sterilized than in quinacrine sterilization i.e.0.5%.The strength of present study was that the population based study was conducted and endometrial abnormalities of quinacrine sterilization were investigated by transvaginal ultrasonography and histopathology. Further research studies should be designed for better policy making regarding sterilization and reproductive health.Most of the women had normal, smooth, regular and homogenous endometrium after 6-17 years of quinacrine sterilization. The cases of irregular endometrium, endometrial adhesions, growths and sterilization failure were also found after quinacrine sterilization. The sterilization failure rates were more in women younger than 35 years during quinacrine sterilization. The risk of endometrial growth was more after 10 years duration of quinacrine sterilization. Thus, quinacrine sterilized women should be followed for long term side effects.SA conceived, designed the research, did data collection, statistical analysis, writing and editing of manuscript.MHB designed, did histopathology, research writing, interpretation of results, critical analysis, review, and final approval of manuscript.SA takes the responsibility and is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.