This study investigates the impact of activated, gain-of-function β-catenin on the chemoresistant phenotype.Colorectal cancers are often chemoresistant toward antitumour drugs that are substrates for ABCB1-mediated multidrug resistance (MDR). Activation of the Wnt/β-catenin on ABCB1 expression and promoter activity was examined using HCT116 human colon cancer cells and isogenic sublines harbouring gain-of-function or wild-type β-catenin, and patients’ tumours. Chemosensitivity towards 24 anticancer drugs was determined by high throughput screening.The effect of mutant (mut) β-catenin showed high ABCB1 promoter activity and expression. Transfection and siRNA studies demonstrated a dominant role for the mutant allele in activating ABCB1 expression. Patients’ primary colon cancer tumours shown to express the same mut β-catenin allele also expressed high ABCB1 levels. However, cell line chemosensitivities towards 24 MDR-related and non-related antitumour drugs did not differ despite different β-catenin genotypes.Cell lines with mut β-catenin, this did not lead to drug resistance in the isogenic cell line model studied. In patient samples, the same β-catenin mutation was detected. The functional significance of the mutation for predicting patients’ therapy response or for individualisation of chemotherapy regimens remains to be established.Although ABCB1 is dominantly regulated by mut  The generation of the MDR phenotype was shown directly by ABCB1 cDNA transfection (The phenomenon of multidrug resistance (MDR) was identified as one of the most frequent causes for therapy resistance in cancer and represents a major cause of failure of cancer chemotherapy . The MDRHigh ABCB1 levels have been detected in normal tissues with excretory or secretory function, which include colorectal epithelium, and in tumours originating from these organs  β-catenin on in vitro drug sensitivity.Reports of T-cell factor 4 (TCF4)-binding sites in the promoter  suggest β-catenin Δ45, exon 3; resulting in the loss of the serine phosphorylation site, S45), the β-catenin knockout cell lines HAB-18mut and HAB-68mut , and HAB-85wt and HAB-92wt (express only one wild-type (wt) β-catenin allele) were kindly obtained from Todd Waldman, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC (β-Catenin genotypes were previously confirmed by sequencing exon 3 and by reverse transcription (RT)–PCR-based restriction fragment length polymorphism  or scrambled control siRNA  were transfected using oligofectamine. For each transfection experiment, at least three independent transfected clones of each cell line were analysed; representative clones are shown. Sulindac treatment was performed with 100 μM sulindac sulphide  were obtained with written consent of the patients . These patients with adenocarcinomas of UICC stages I–III were previously untreated, did not have a history of familial colon cancer, did not suffer from a second tumour of the same or a different entity, underwent surgical R0 resection, and had not developed distant metastases at time of surgery. Tumour specimens were snap-frozen in liquid nitrogen and blinded for analysis. Three of these patients were identified to harbour the heterozygous in-frame deletion mutation of Δ45 in exon 3 of the β-catenin/TCF4-mediated gene expression was analysed in pTOP-CAT- and pFOP-CAT-  transfected cells. pTOP-CAT utilises the multimerised TCF4 consensus DNA-binding sequence to drive the CAT-reporter expression  was determined in parallel from the same RT reaction by using gene-specific hybridisation probes, each done in duplicate per sample. For ABCB1: forward primer 5′-TTGAAATGAAAATGTTGTCTGG-3′, reverse primer 5′-CAAAGAAACAACGGTTCGG-3′, FITC probe 5′-CACTGAAAGATAAGAAAGAACTAGAAGGTGCT-3′, LCRed640 probe 5′-GGAAGATCGCTACTGAAGCAATAGAAAACT-3′ for ABCC1: forward primer 5′-TGCCGAAGGAGAGATCATCATC-3′, reverse primer 5′-CGGAGGGAACCCGAAAACA-3′ FITC probe 5′-GCCTGCACGACCTCCGCTTCAAGA-3′, LCRed640 probe 5′-CACCATCATCCCCCAGGACCCTGTT-3′ for ABCG2: forward primer 5′-AGCAGGGACGAACAATCATC-3′, reverse primer 5′-AGGCCCGTGGAACATAAGTC-3′ FITC probe 5′-TCATCAGCCTCGATATTCCATCTTCAAGTT-3′, LCRed640 probe 5′-TTTGATAGCCTCACCTTATTGGCCTCAG-3′ for MVP: forward primer 5′-CGCATCCCCCCATACCACTA-3′, reverse primer 5′-GGCAAACAGTACCCTCTCATTGTCC-3′ FITC probe 5′-CAGAACAGCAACGTGTCCCGTGTGGA-3′, LCRed640 probe 5′-GTCGGGCCAAAGACCTACATCCGGC-3′ . The calibrator cDNA was employed in serial dilutions simultaneously in each run, derived from the ABCB1-overexpressing cell line KBV-1 and the ABCC1-overexpressing cell line MCF-7/VP16 , from the ABCG2-overexpressing cell line SW1573/2R120 and from the MVP-overexpressing cell line GLC-4/ADR .RT reaction was performed with 100 ng of total RNA . Quantitative real-time PCR  was performed using the LightCycler  as previously described . For immunocytochemistry, cells were cultured on eight-well chamber slides; cells without primary antibody served as controls. For immunohistochemistry, consecutive cryosections were incubated on the same slide; tissue sections without primary antibodies served as controls. Cells or tissue sections were fixed, endogenous peroxidase was inactivated and cell membranes were permeabilised. After blocking, cells or tissue sections were incubated with the anti-ABCB1 or the anti-β-catenin antibody for 2 h. Detections were performed with the StreptABComplex/HRP Duet system . Nuclei were counterstained with hemalum . Microphotographs were taken with a Zeiss Axioplan 2 microscope and an Axiocam HRc camera  using the Axiovision 4.2 software (Zeiss).We used a monoclonal anti-ABCB1 antibody  and a monoclonal anti-4 cells was measured with a FACSCalibur  and expressed as mean fluorescence. After titration of the antibody, at least two independent experiments, each performed in duplicate, were carried out.We used a monoclonal anti-ABCB1 antibody , a monoclonal anti-ABCC1 antibody , a monoclonal anti-ABCG2 antibody , a monoclonal anti-MVP antibody . Goat anti-mouse IgG1 and IgG2a antibodies were from Life Technologies . Cells were prepared and were incubated as previously described (M Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, supplemented with complete protease inhibitor tablets; Roche Diagnostics) for 30 min on ice. The nuclear, cytoplasmic and membrane protein fractions were isolated with the Qproteome Cell Compartment Kit . Western blotting was carried out as described previously  served as loading control.Total protein extractions of the cells were performed with RIPA buffer , and were then kept in rhodamine 123-free medium for another 60 min at 37 °C. Fluorescence intensity of 1 × 104 cells per group was measured in duplicate per sample by using the FACSCalibur .HCT116 cells and the M glutamine). Aliquots of diluted compounds were then transferred to 384-well plates containing tumour cells and the plates were incubated for 4 days in a 37 °C, humidified incubator with an atmosphere of 5% CO2. Viable cell numbers were then evaluated using an Alamar Blue assay. Briefly, cells were treated with Alamar Blue dissolved in serum-free RPMI-1640 and incubated for 4 h. Plates were then read on a Wallac Victor reader (PerkinElmer) at an excitation wavelength of 530 nm and emission wavelength of 590 nm. Values for triplicate wells at each concentration were then averaged and expressed as percent of vehicle (dimethyl sulfoxide) control. Concentration-response curves were generated and IC50 values extracted from the curves by linear interpolation. Response of cell lines across the drug library was compared in terms of concentration-response curves, derived IC50 values and patterns of sensitivity in relation to genotype.A library of 24 prototype drugs  includint-test or the non-parametric two-sided Mann–Whitney rank sum test depending on whether the data passed or failed a normal distribution test.Levels of statistical significance were evaluated by using the β-catenin. We compared ABCB1 gene expression levels in the parental human colon carcinoma cell line HCT116, that is heterozygous for an in-frame Δ45 deletion in exon 3 of the β-catenin gene, in the HAB-18mut and HAB-68mut as well as in the HAB-85wt and HAB-92wt knockout strains of HCT116 cells, in which either the wt or the mut β-catenin allele was ablated by homologous recombination , seven TCF4-binding sites were identified at −275 to −261, −419 to 405, −580 to −566, −964 to −950, −1017 to −1003, −1652 to 1638 and −1814 to −1800 . Using a previously developed RT–PCR-based restriction fragment length polymorphism analysis, we characterised these specimens for their nin gene . Consistmutation .mut and HAB-92wt cells. Expression of each of these chemoresistance-associated genes remained unchanged in the knockout sublines compared with HCT116, and was not dependent on the β-catenin genotype as demonstrated at the mRNA level by quantitative RT–PCR (We also analysed the expression of ABCC1, ABCG2 and MVP in HCT116, HAB-68e RT–PCR , and at e RT–PCR  as well e RT–PCR .β-catenin genotype and ABCB1 expression, we performed accumulation assays for rhodamine 123. However, rhodamine 123 accumulation was comparable in all the three cell lines, either after 10 min or 15 min of rhodamine 123 uptake (β-catenin genotype and ABCB1 levels.To analyse whether drug accumulation is affected in cells with different 3 uptake , despiteβ-catenin knockout cell lines to a mechanistically diverse library of anticancer drugs and prototype compounds did not reveal evidence of drug resistance mediated by β-catenin. A focus on known MDR substrates in this library does not indicate a consistent pattern of relative resistance based on the presence of oncogenic β-catenin. Indeed, the response of HCT116 and the derived knockout cell lines to the 24 compounds in this library were remarkably similar.As shown in β-catenin pathway activation and intrinsic response to chemotherapy, as activation of this pathway is observed in almost all colorectal cancers. We investigated this hypothesis using a frequently occuring mutation of β-catenin in the context of its MDR-associated target gene ABCB1. Here we report that mut β-catenin signalling regulates the expression of the ABCB1 gene in a dominant fashion. We investigated the relevance of mut β-catenin with respect to ABCB1 expression levels to clinical cancer with surgical samples of primary colon carcinomas. We demonstrate that tumours heterozygous for mut β-catenin showed nuclear β-catenin together with overexpression of ABCB1. However, chemosensitivities towards MDR-related as well as non-related antitumour compounds measured by high throughput screening in tumour cell lines harbouring gain-of-function or wt β-catenin did not differ despite their differences in β-catenin genotype and ABCB1 expression levels.In this report we addressed the potential interplay between Wnt/β-catenin (β-catenin, knockout strains harbouring either the mut or the wt β-catenin allele exclusively, and cell clones with reconstituted heterozygous β-catenin genotype were analysed (β-catenin, either intrinsically or by transfection, show higher ABCB1 levels than cells with wt β-catenin. Moreover, consistent with the β-catenin mutation status, we found higher ABCB1 promoter activity in cells harbouring gain-of-function β-catenin. These findings underline in particular that gain-of-function β-catenin acts in a dominant manner to control ABCB1 transcription. Thus, the dependence of ABCB1 expression on β-catenin genotype was clearly shown using this cell line model. Furthermore, intervening in β-catenin expression using siRNA acting on β-catenin led to ABCB1 expression knockdown. Treatment with sulindac reduced ABCB1 expression independent of the β-catenin genotype. Sulindac inhibits expression and nuclear accumulation of β-catenin in colorectal cancer cell lines, and induces proteasome-dependent degradation of β-catenin. Consequently, β-catenin target genes like Met, c-myc, cyclinD1 and S100A4 are downregulated following sulindac treatment (β-catenin transcriptional target.We began our studies with HCT116 cells that are heterozygous for gain-of-function β-catenin and ABCB1 was also shown in side-population colon cancer cells, however, ABCG2 expression was also found to be dependent on β-catenin levels (β-catenin signalling was also reported for breast cancer cells (Our findings are in line with observations of β-catenin targets, including c-myc and cyclin D1, which are implicated in the development of colorectal cancer (β-catenin has also been shown for β-catenin targets such as matrilysin, BMP4 and S100A4, thereby underlining the impact of activated β-catenin on tumourigenesis and metastasis (β-catenin/TCF4-mediated ABCB1 expression represents only one mechanism within the complex transcriptional regulation of the ABCB1 gene, our findings on the dominant action of gain-of-function β-catenin adds to the current knowledge on the role of ABCB1 in the biology of colorectal cancer.A large number of genes have been identified as in vitro analyses to archival samples of human primary cancer. We identified tumours that are heterozygous for this in-frame deletion mutation of Δ45 in exon 3 of the β-catenin gene. These tumours showed concomitant nuclear β-catenin and high ABCB1 expression. This finding, exploiting the defined Δ45 mutation as an example for gain-of-function β-catenin, provides a new link between high ABCB1 levels in human colon tumours and an activated Wnt pathway. Furthermore, increased ABCB1 expression also correlated with β-catenin in adenomatous polyps from patients with familial adenomatous polyposis (We extended our β-catenin on ABCB1 and drug resistance and potential response to therapy in colon cancer cells we screened a mechanistically diverse library of anticancer drugs and prototype compounds. Using a high throughput screening approach designed to minimise the impact of drug sample handling on measured sensitivity, we found no evidence for dependence of cell response on β-catenin genotype. This was rather unexpected, particularly for MDR substrate drugs, in view of the clear evidence for upregulation of ABCB1 by oncogenic β-catenin. On the other hand, these results are consistent with the rhodamine efflux studies, which showed no difference between the cell lines. It may be that the extent of upregulation in these cell lines is not sufficient to confer functional drug resistance. It is also possible that expression of other genes in this HCT116-based cell line model, that is, ABCC1, ABCG2 and MVP, contribute to the measured drug sensitivity phenotype. The simultaneous expression of MDR genes has already been described for HCT116 cells (β-catenin genotype in HCT116 cells and the knockout sublines thereof. The simultaneous, unmodulated expression of the MDR-associated proteins ABCC1, ABCG2 and MVP may well contribute to the comparable chemosensitivities towards the 24 drugs tested.To assess the impact of gain-of-function 16 cells . We repoβ-catenin silencing by siRNA (β-catenin by siRNA resulted in increased chemosensitivity towards doxorubicin and etoposide (β-catenin and ABCB1 expression was found with respect to neoadjuvant chemotherapy (In SW480 colon cancer cells, however, increased chemosensitivities towards paclitaxel and irinotecan were reported following β-catenin on intrinsic ABCB1 expression and in vitro chemotherapy response. We clearly demonstrate that the mutation status of β-catenin determines ABCB1 expression in a defined cell line model and in colon cancer specimens; however, no correlation of this finding with chemosensitivity towards 24 MDR-related and non-related antitumour compounds was detected. Further work is needed to evaluate the role of gain-of-function β-catenin with respect to additional anticancer compounds of potential use in treatment of colorectal cancer. Further studies will also be required to reveal the potential importance of an activated Wnt/β-catenin pathway for intrinsic ABCB1-mediated resistance in other tumour types. Likewise, additional insight is needed into factors important for expression of other ABC transporters, which may mediate drug resistance. In summary, although ABCB1 is validated as β-catenin target gene in a cell line model with defined β-catenin genotypes, the facile detection of β-catenin mutations using the diagnostic PCR procedure reported here is not sufficient for predicting therapy response or for individualisation of chemotherapy regimens for patients.For advanced and metastatic tumours, chemotherapy is frequently the only feasible treatment. However, the success of chemotherapy differs from patient to patient. Some patients show complete responses, others respond partially and/or transiently. Here we report the impact of gain-of-function


Although the prognostic roles of β-catenin expression in non-small cell lung cancer (NSCLC) have been reported in several immunohistochemical (IHC) studies, the results were not consistent because some studies lack sufficient number of the positive cases or did not evaluate the subcellular localization features of the protein.In this study, we have evaluated the expression levels and subcellular localization of β-catenin and Nanog proteins IHC staining in tissue specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome.p=0.064) than those with positive expression. In contrast to previous studies, we found that increased expression of either cytoplasmic or nuclear β-catenin was strongly associated with poor prognosis and was an independent prognosticator for overall survival (p <0.01). We further found that NSCLC cells frequently exhibited an abundance of nuclear Nanog protein which was significantly correlated with nuclear β-catenin expression (p <0.01) and poor prognosis (p <0.01). Interestingly, immunofluorescent staining results revealed that increased expression of Nanog and nuclear translocation of β-catenin occurred concomitantly in response to epidermal growth factor receptor(EGFR) signaling in A549 and H23 cells. Furthermore, western blot analysis show that nuclear β-catenin rather than cytoplasmic β-catenin expression in the A549 and H23 cells can be enhanced by adding EGF, Nanog expression in the A549 and H23 cells with knockdown of β-catenin can not be obviously enhanced by adding EGF.We showed that patients with negative expression of membranous beta-catenin had a trend towards shorter survival (We propose that evaluation of subcellular localization of β-catenin and Nanog expression is of clinical significance for patients with NSCLC. Lung cancer is the most common cancer worldwide, and has the highest incidence and mortality levels of any cancer . NSCLC iβ-catenin is a key component of the canonical Wnt pathway that plays pivotal roles in pattern formation during embryogenesis and in malignant transformation . At the Nanog is a core transcription factor required for maintenance of the pluripotency and self-renewal of embryonic stem cells. Nanog protein has been identified as one of four factors essential for reprogramming adult cells into induced pluripotent stem (iPS) cells . Recent Nanog is critically involved in regulation of cancer stem cells in several types of tumors -12 and hIn this study, we have evaluated the expression levels and subcellular localization of β-catenin and Nanog proteins in primary tumor specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome.Primary tumor specimens from 309 NSCLC patients who underwent curative surgical resection were evaluated in this study. None of these patients had undergone chemotherapy or radiotherapy prior to the surgery. Of these patients, 203 were collected from 2001 to 2005 at Hunan Tumor Hospital and the rest of 96 were from 2002 to 2006 at the Affiliated Hospital of Guilin Medicine University. Patients in the study were examined and treated according to provincial guidelines. This study was approved by the Research Ethics Boards of the Southern Medical University, China.Cores from formalin-fixed paraffin embedded tumor tissues of NSCLC were arrayed in triplicate onto a tissue microarray (TMA) and each TMA block consisted of up to 40 tissue cores, before the area of cores were chosen, the preliminary experiment of β-catenin were made to ensure the typical cores. TMAs were constructed using an automated tissue arrayer (Beecher Instruments Inc. USA) by Auragene Bioscience Corporation, China. Tissues core with a diameter of 3 mm was transferred to a recipient paraffin block (array margin of 15mm×20 mm). The block was sectioned at a series of 4-μm-thick slices. Once the TMA slices were made, they were heat 60°C for 1 h to aid cutting.Relevant clinical pathologic features Table  were obtSerial tumor sections and IHC staining were used to evaluate both β-catenin and Nanog proteins. β-catenin immunoactivity was examined using a mouse polyclonal antibody . Nanog protein was detected with a rabbit polyclonal antibody . The TMA blocks containing tissue sections (4 μm) were de-waxed in xylene, rehydrated through a graded series of ethanol solutions, rinsed in distilled water for 5 min, and then immersed in boiling citrate buffer in methanol for 1.5 min for antigen retrieval. After deparaffinization and rehydration, blocks were incubated in 3% hydrogen peroxide to block endogenous peroxidase activity. Then the sections were subject to immunohistochemistry staining using the primary antibodies overnight at 4°C in a humidity chamber. The avidin-biotin technique was applied using DAB for visualization and hematoxylin for nuclear counterstaining. Negative controls were prepared by omitting the primary antibody. Histological and IHC evaluation were independently performed by two pathologists without knowledge of the clinicopathological outcomes of the patients. We classified the IHC staining results into three categories according to subcellular localization of β-catenin, e.g. membranous, cytoplasmic and nuclear. Briefly, each slide was examined in its entirety under a light microscope, and an initial score was assigned which represented the estimated proportion of positive tumor cells . The score 0 was defined as low and 1, 2, 3 as high. Slides with indeterminate evaluation were re-evaluated, and a consensus was reached.For immunofluorescent staining, A549  or H23  cells grCytoplasmic and nuclear proteins of A549 and H23 cells were extracted by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime), then equal amount of protein were subjected to electrophoresis on a SDS-PAGE gel. The separated proteins were transferred to PVDF membranes (Millipore) and probed with appropriate primary antibodies. Protein bands were detected by enhanced chemiluminescence reagents (Pierce Biotechnology).Both pLKO.1 lentiviral shRNA vector and control shRNA targeting against GFP were from Aldrich -Sigma. β-catenin targeting sequences are GCTTGGAATGAGACTGCTGAT, which was previously described in our earlier study . The senp-value < 0.05 was considered statistically significant.Statistical analysis was performed using the SPSS 13.0 software package for Windows. Associations between clinicopathological features and IHC β-catenin and Nanog expression were analyzed using the chi-squared test. Multivariate survival analyses were performed with the Cox regression model. Overall survival (OS) was measured from the onset of treatment to the date of death or the survival status at the last date of follow-up. OS probabilities were estimated by the Kaplan-Meier method and the significance of differences was assessed by the log-rank test. Correlations between β-catenin and Nanog expression with clinicopathological factors were analyzed using Fisher’s exact probability test or the chi-squared test. A p <0.01, Table Tumor specimen from 309 NSCLC patients consisting of 134 males and 175 female were included in this study. The median age was 52 years (range: 37–82 years). Of these patients, 167 (32.9%) were diagnosed as adenocarcinoma and 142 (33.1%) diagnosed as squamous carcinoma. Clinical stages, histological grades, and studied proteins were correlated as shown in Table Representative images of β-catenin immunohistochemical staining in NSCLC tissues are shown in Figure Nanog is critically involved in regulation of cancer stem cells in several types of tumors and has been reported to be transcriptionally regulated by β-catenin. Thus we further examined Nanog expression in NSCLC specimen. We found that 30.4% (94/309) tumor tissues displayed Nanog immunoactivity that was located in the nucleus in most cases Figure D.p <0.01, Figure p <0.01, Figure p=0.064, Figure p <0.01) . The 1-, 3- and 5-year overall survival (OS) rates were 82.4%, 52.5%, 30.6%, respectively. The survival rates of 309 NSCLC patients according to status of β-catenin and Nanog were shown in Table Univariate analyses showed no significant association between OS and age (>52 yr vs. ≤52 yr), sex , histological subtype (adenocarcinoma vs. squamous carcinoma), T stage (T3-T4 vs. T1-T2), clinical stage (stage III vs. I-II) (Table p < 0.001). On the contrary, only 55 (26.6%) of the 207 specimen with membranous β-catenin expression showed Nanog immunoreactions (Table p = 0.036). Likewise, cytoplasmic β-catenin expression is not correlated with Nanog protein (Table p = 0.166) even though cytoplasmic β-catenin is associated with poor prognosis. However, expression of cytoplasmic and nuclear β-catenin is correlated, among the 133 specimen with cytoplasmic β-catenin expression, 105 (78.9%) showed nuclear β-catenin immunoreactions  than those with positive expression. This result is in agreement with the findings reported by others previously.The prognostic roles of β-catenin expression in NSCLC have been extensively studied in the past years ,25. In aIn addition to the similarity, there are some striking differences between our results and those of earlier studies. We found that increased expression of nuclear β-catenin was strongly associated with poor prognosis of patients with NSCLCs. In addition, multivariate analysis revealed that the expression of nuclear β-catenin was an independent prognosticator for OS. These data are consistent with recent observations that increased Wnt/β-catenin pathway activity is associated with metastasis and relapse in primary lung adenocarcinoma . ImportaIn contrast to our investigation, some earlier studies evaluated only the overall intensity of IHC staining of β-catenin in tumor cells without stratification of the data based on subcellular localization of the protein. Thus the distinct impact of β-catenin at different cellular compartments on patient prognosis and survival was ignored. Some of the studies ,29 evaluNanog protein is elevated in many human tumors including NSCLC and might positively regulate tumor metastasis through enhancing EMT in lung adenocarcinoma . We founTaken together, we showed that negative expression of membranous β-catenin correlated with a shorter survival time than the normal expression level of the protein. However, high expression of nuclear β-catenin was strongly associated with poor prognosis and was an independent prognosticator for OS. We further found that NSCLC cells frequently exhibited nuclear Nanog protein abundance that is significantly correlated with nuclear β-catenin expression and poor prognosis. Furthermore, results from IHC staining with established lung cancer cell lines revealed that increased expression of Nanog and nuclear translocation of β-catenin occurred concomitantly in response to EGFR signaling. In conclusion, we propose that evaluation of subcellular localization of β-catenin and Nanog expression is of clinical significance for patients with NSCLC.The authors declare no conflict of interest.XQL carried out the cases collection, immunohistochemical staining and western blot analysis. XLY and GZ conducted immunofluorescent staining. SPW and XBD performed the statistical analysis. SJX constructed the tissue microarrays. QZL participated in the design of the study. GHX and KTY designed and conceived of the study, XQL helped to draft the manuscript. GHX wrote the manuscript. All authors have read and approved the final manuscript.


Kindlin 2, as an integrin-associated protein, is required for myocyte elongation and fusion. However, the association of Kindlin 2 with muscle differentiation-related signaling pathways is unknown. Here, we identified a mechanism that Kindlin 2 regulates myogenic regulatory factors myogenin via a canonical Wnt/β-catenin signaling. We found that knockdown of Kindlin 2 leads to the abolishment of β-catenin/TCF4-mediated transcription in C2C12 cells, followed by the impairment of myogenic differentiation. Mechanistically, nuclear translocation of both Kindlin 2 and β-catenin is induced during myogenic differentiation. In particular, Kindlin 2 forms a tripartite complex with active β-catenin and TCF4, and hence co-occupied on the promoter of myogenin to enhance its expression. Functionally, depletion of Kindlin 2 impairs myogenic differentiation via downregulation of myogenin. Taken together, our data reveal that Kindlin 2 is required for Wnt signaling-regulated myogenic differentiation, providing a mechanistic insight into the role of Kindlin-2 in muscle development. Skeletal myogenesis is a complex and highly regulated process that mediates the formation of multinucleated myotubes by fusion of mononucleated myoblasts. During myogenesis, Wnt signaling plays an essential role Integrins and proteins that bind to integrins are required for muscle development and muscle function Our recent study indicated that Kindlin 2 is a novel regulator of Wnt signaling. The Kindlin 2 in the cytoplasm is mainly responsible for stabilizing β-catenin, whereas the Kindlin 2 in the nucleus significantly activates the target genes of Wnt signaling 2 humidified incubator. To induce myogenic differentiation, the growth medium (GM) containing 15% FBS was substituted with differentiation medium (DM) contained 2% horse serum. The differentiation medium was exchanged every day.C2C12 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 15% fetal bovine serum , and an appropriate amount of penicillin/streptomycin in a 37°C, 5% COBoth Western blotting and Co-IP assay were performed as described previously 5′- AGTGGAATGTCAACTGGGAGATC -3′ and reverse primer, 5′- GGACAACCGGACCTCATCTG-3′; MyHC forward primer, 5′- AGAAGGAGGAGGCAACTTCTG-3′ and reverse primer, 5′- ACATACTCATT GCCGACCTTG-3′; GAPDH forward primer, 5′- TGTGTCCGTCGTGGATCTGA -3′ and reverse primer, 5′- CCTGCTTCACCACCTTCTTGA -3′.Total RNA was extracted using Trizol reagent (Invitrogen). cDNA was synthesized using the SuperScript kit (Invitrogen). The primer sequences were as followed: Kindlin 2 forward primer, C2C12 cells were seeded into 24-well plates the day before transfection. Control or Kindlin 2 siRNA was transfected into C2C12 cells with Lipofectamine RNAiMAX (Invitrogen). At 24 h post transfection, 100 ng of super 8x TOPFlash/FOPFlash plasmid with 1 ng of pRL were transfected per well using Lipofectamine 2000. After 24 h, C2C12 cells were induced myogenic differentiation for two days. The reporter activity was measured using a Dual-lusiferase Reporter Assay System (Promega).A Co-IP assay was performed as described previously Sequences of RNA interference (RNAi) oligonucleotides were as follows: Control siRNA, UUCUCCGAACGUGUCACGU; Kindlin 2 siRNA, AAGUUGG UGGAAAAACUCGAU.After the cells were fixed with 4% paraformaldehyde solution at RT for 15 min, they were treated with 0.5% Triton X-100 at 37°C for 5 min and blocked with 5% BSA at room temperature for 1 h. The cells were then incubated with 1∶100 dilution of anti-Kindlin 2, anti-β-catenin, anti-active β-catenin or anti-TCF4 antibodies overnight at 4°C, and then with a 1∶100 dilution of Alexa Fluor 488 or 568-conjugated IgG (Invitrogen) for 1 h at room temperature. The images were captured with a TCS SP5 confocal microscope .3VO4, and protease inhibitor mixture) for 5 min on ice. After the cells were scraped and centrifuged, the supernatant was the cytoplasmic fraction. Then the pellet was lysed in buffer B . After centrifuging, the supernatant was the nuclear fraction.Cells were rinsed twice in cold PBS, and then incubated with buffer A  was used to perform chromatin immunoprecipitation (ChIP) assay. The antibodies used in ChIP assay are anti-β-catenin (BD), anti-Kindlin 2 (Millipore) and anti-TCF4 (Upstate). The primers for quantitative ChIP (q-ChIP) PCR were as follows: myogenin-F, in vitro, C2C12 murine myoblasts are used as a model of skeletal muscle differentiation. In C2C12 cell culture model system, C2C12 cell differentiation is triggered by serum removal. Proliferating C2C12 cells are cultured in growth medium and regarded as undifferentiated cells (GM stage). Upon serum removal, the proliferation of C2C12 cells is arrested, and the elongation and fusion of C2C12 cells are induced (DM stage). Eventually, multinucleated myotubes are formed. To evaluate the role of Kindlin 2 during skeletal muscle differentiation, we detected the levels of Kindlin 2 at different stages of cell differentiation, including proliferating C2C12 cells with 50% confluence (GM), proliferating C2C12 cells with 100% confluence (DM day 0) and the first five days after serum removal (DM day 1–5). As determined by Western blot analysis, the level of Kindlin 2 was low in undifferentiated cells (GM) (To mimic the process of muscle development lls (GM) . Upon C2lls (GM) . Myosin lls (GM) , reflectlls (GM) . TogetheFurthermore, to confirm whether Kindlin 2 is required for muscle cell differentiation, small interfering RNA (siRNA) to murine Kindlin 2 gene was designed and synthesized. The efficacy of siRNA was determined by Western blot in Given the critical role of Wnt signaling in muscle development, SuperTop/Fopflash reporter assay was performed. SuperTopflash is a luciferase reporter of β-catenin/TCF4-mediated transcriptional activation, which contains seven TCF/LEF binding sites. SuperFopflash is an appropriate control plasmid, which has some mutant TCF/LEF binding sites. As shown in To scrutinize how Kindlin 2 regulates Wnt signaling during myogenic differentiation, we investigated the regulation of Kindlin 2 on β-catenin. Firstly, the expression of β-catenin was detected in undifferentiated and differentiated C2C12 cell. As determined by Western blot analysis, β-catenin expression was obviously induced during C2C12 cells differentiation . The levTo determine the subcellular localization of Kindlin 2 or β-catenin, immunofluorescence staining was performed using anti-Kindlin 2 or anti-β-catenin antibodies. In undifferentiated C2C12 cells, either Kindlin 2 or β-catenin mainly resided in the cytoplasm –a. Upon Furthermore, distribution patterns of Kindlin 2 and β-catenin were determined by Western blot analysis of subcellular fraction. Results showed that, in undifferentiated cells, Kindlin 2, β-catenin or active β-catenin was only detected in the cytoplasmic fraction . Upon myin vivo, the interaction of Kindlin 2 and β-catenin was identified by co-immunoprecipitation (Co-IP) assay. As shown in To investigate the physical association between Kindlin 2 and β-catenin Nuclear β-catenin has been shown to interact with members of the TCF/LEF family to activate Wnt target gene expression. To uncover the relationship of Kindlin 2 and TCF/LEF family, we first explored the effect of Kindlin 2 on TCF4 expression. As determined by Western blot analysis, TCF4 was induced expression at day 1of C2C12 cell differentiation. When Kindlin 2 was knocked down, the induction of TCF4 expression was obviously delayed, suggesting that Kindlin 2 was involved in the regulation of TCF4 expression . To deepMyogenic regulatory factors function to induce the expression of some muscle specific proteins, such as, alpha-actin, myosin heavy and light chains, tropomyosin, troponin-C and troponin-I, which are critical for muscle development. Myogenin, a typical myogenic regulatory factor, was obviously activated during muscle cell differentiation . FurtherMost studies of Kindlin 2 focused on its binding to integrin at focal contacts Although it is well established that the nuclear translocation of β-catenin is a key factor of Wnt signaling activation, the detail mechanisms remain elusive. Smad3 is reported to enhance the stability of β-catenin and facilitate the nuclear translocation of β-catenin in chondrocytes Kindlin 2– β-catenin complex was originally confirmed in breast cancer cells, in which Kindlin 2 was recruited by β-catenin to activate Wnt target gene AXIN2, and, in turn, promote the EMT process


The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation. Primary cilia are expressed by most mammalian cell types and exist as a singular cytosolic compartment, often projected into the extracellular environment. They consist of a tubulin scaffold wrapped in a specialized section of the plasma membrane. Increasingly, the cilium has been implicated in various aspects of cell biology including regulation of the cell cycle, migration and the cellular response to external stimuli such as hedgehog, wnt, growth factors, inflammatory cytokines and mechanics123456789101112141617181920et al., recently identified that the primary cilium accomplishes negative regulation of canonical wnt signalling by sequestering β-catenin away from the nucleus by means of the protein Jouberin, also the mediator of β-catenin nuclear entry. In doing so, the cilia compartment brings about a reduction in the cells responsiveness to wnt3a2313252627et al., demonstrated a role for the primary cilium in mediating a mechanotransduction response to oscillatory fluid flow resulting in an increase in osteogenic differentiation of MSCsIn vitro increases in cilia length have been attributed to various factors including a reduction in intracellular calcium, alterations in cyclic AMP and the signaling pathways activated on reception of growth factors and inflammatory cytokines781718The primary cilium is well established as the location for the transduction of ligand-induced hedgehog pathway activationSurface topography-mediated changes in intracellular tension has been widely documented to regulate both the differentiation and self-renewal of MSCs333436373839414243et al., observed that MSCs under increased cellular contractility and intracellular tension are more susceptible to soluble factors. We propose that in the case of wnt signaling this is due to an alteration in cilia prevalence and length, both functions of cilia trafficking, and which is also regulated by actin organization and contractility. Therefore this study tests the hypothesis that grooved topographic surfaces induce a cell shape mediated decrease in intracellular actin-myosin tension, which leads to an increase in primary cilia length and prevalence and an associated alteration in wnt signaling.Previous studies by Kilian We show that primary cilia respond to reduced actin cytoskeletal organization induced by surface topography with subtle, but statistically significant, elongation. Using a ROCK inhibitor to prevent actin stress fiber formation on the flat surface, primary cilia length was found to increase to lengths comparable to those measured on untreated grooved surfaces. Reductions in cilia length with siRNA to IFT88 increased nuclear β-catenin localization, a marker for canonical wnt signaling response. Similarly nuclear β-catenin localization was lower in MSCs on flat surfaces with shorter cilia compared with cells on grooved topography. The response to grooves was prevented when cilia structure was disrupted using siRNA-IFT88. In addition grooved topography abolished downstream axin-2 induction and proliferation in response to wnt3A. This study not only adds to the growing body of evidence for the importance of the cilium in stem cell biology, but highlights how subtle alterations in ciliogenesis, in this case induced by topography regulate wnt signalling, a role particularly crucial within the stem cell niche.To investigate the effect of microtopography on the structure of the primary cilium, MSCs were cultured on microgrooves  and a flMSCs cultured on the grooved surfaces and subjected to 24 hrs serum starvation, were found to have increased primary cilia length when compared with those on flat surfaces, the difference being statistically significant . The majority of MSCs on the grooves had primary cilia with lengths greater than 3 μm, with an overall increase in average length of 26% with respect to flat . The angWe next examined the actin cytoskeleton in MSCs cultured on grooved and flat topographies as studies have identified actin organisation as a regulator of primary cilia length. Micro-contact printed surfaces have been shown to increase cytoskeletal tension and inhibit primary cilia extension whilst surfaces that lead to a reduced cytoskeletal tension result in longer ciliain vitro differentiation protocols, MSC's are normally grown in the presence of serum, a prominent regulator of cell cycle re-entry. Therefore it is important to examine the role of the cilium under conditions without serum starvation. In the presence of serum, cilia length was not assessed due to the more dynamic nature of cilia assembly and disassembly in these cultures. Furthermore, cilia lengths were generally found to be < 0.5 μm making accurate measurement difficult. However, the presence of the primary cilium  was assessed as both length and prevalence are ultimately governed by ciliary trafficking.Having shown that topography modulates primary cilia length, presumably through the control of IFT and associated cilia trafficking, we wished next to examine whether this influences MSC responses to potential differentiating stimuli. For Under serum conditions, many MSCs cultured on both flat and grooved surfaces exhibited ki67 positive cells as exemplified in To investigate the influence of primary cilia structure in regulating wnt signaling we assessed wnt signaling following disruption of intraflagellar transport mediated ciliary assembly by means of IFT88 (anterograde IFT particle) directed siRNA transfection. Previously, mutations of retrograde intraflagellar associated motors, such as retrograde dynchc2, showed ability to modulate wnt signaling including at the level of β-catenin translocation to the nucleusWe finally sought to establish the effect of the grooved topography on canonical wnt signaling and whether this required the increase in primary cilia assembly shown to occur through reduced cytoskeletal tension. This was initially indicated by the intensity of active β-catenin localization in the nucleus but also supplemented by assessment of axin-2 gene expression, another canonical wnt marker. The topographical microenvironment affected the response to wnt3a such that active β-catenin levels in the nucleus were statistically significantly lower on grooves with a 12% reduction in mean staining intensity compared with that on flat surfaces  supplemented with 10% FBS and Penicillin-Streptomycin followed by 24 hours serum starvation to induce ciliogenesis. For activation of canonical wnt signaling, wnt3a  was added to serum-free media for 3 hours following serum starvation. A ROCK inhibitor  was added to the serum-free media 24 hours after cell seeding to biochemically inhibit actin stress fibre formation.Surfaces were first sterilised under UV light for 20 min, then coated in fibronectin  to increase cell adhesion followed by two sequential washes in PBS. Human bone marrow-derived mesenchymal stem cells (MSCs)  were seeded onto the grooved and planar surfaces at 1.25 × 10Cells were fixed in 4% paraformaldehyde for 10 min at 37°C and permeabilized in 0.5% triton X in phosphate buffered saline  for 5 min at room temperature. Non-specific binding sites were blocked by incubating cells for 30 min in 5% goat serum in PBS at room temperature. The samples were then washed in 0.1% bovine serum albumin in PBS before incubation with the appropriate primary antibody. Samples were washed a further three times in 0.1% PBS/BSA and incubated for 2 hr at room temperature in 0.1% PBS/BSA with the secondary antibody, either anti-mouse 488/555 or anti-rabbit 488/555 . The following primary antibodies were used: mouse anti-acetylated α-tubulin , mouse anti-active β-catenin , mouse anti-Ki67  and rabbit anti-pericentrin . In addition F-actin was labeled with alexa 555-conjugated phalloidin  in 0.1% PBS/BSA overnight at 4°C. All samples were finally washed three times in 1% PBS/BSA and mounted onto glass slides using Prolong Gold mountant containing DAPI.Samples were imaged using a confocal microscope (Leica SP2) with a x63/1.05 NA objective and a pixel size of 0.1 × 0.1 μm. Confocal z-series were made throughout the entire cell monolayer with a z step size of 0.5 μm. Primary cilia lengths and x/y orientation were measured from reconstructed maximum projection images using Image J software as in previous studiesSamples were imaged using a fluorescent microscope (Leica) and nuclear β-catenin intensity quantified using Image J software. DAPI staining was used to identify nuclei and to create a mask that was overlaid onto the β-catenin image enabling the mean β-catenin intensity to be calculated for each nucleus. For each group, at least 60 nuclei were assessed from multiple coverslip preparations.4 cells/cm2 in antibiotic-free medium. 24 hrs post seeding cells were transfected as per manufacturers protocol with 25 μM human IFT88 siRNA , a non-targeting siRNA  or with water as a control using Dharmafect 1 transfection reagent (4 μL/mL) . Transfection media was replaced after 24 hours and serum free media added for induction of ciliogenesis. After 24 hours serum starvation, 150 ng/mL wnt3a was added for a further 3 hours.MSC's were seeded onto surfaces at 1.25 × 10Total RNA was extracted and isolated following the standard protocol associated with Qiagen RNeasy mini kit, quantity and integrity were checked by Nanodrop ND-1000 spectrophotometer  and gel electrophoresis analysis. Reverse transcription reactions (including genomic DNA clean up) were performed using Quantitect RT kit (Qiagen). For real time PCR, cDNA was diluted in nuclease free water and triplicate 10 μL reactions were performed using KAPA SYBR® FAST Universal 2X qPCR Master Mix (KAPA Biosystems) containing SYBER-green dye, 0.2 μM ROX reference dye. Optimised primer pairs were as follows axin-2, FOR _GCCAAGTGTCTCTACCTCAT, REV _ACAGGTGATCGTCCAGTATC; GAPDH, FOR _GACAAAATGGTGAAGGTCGG, REV_TCCACGACATACTCAGCACC. IFT88, FOR _ GCAATCCTACGAAACAGTGCC, REV _ CACTGACCACCTGCATTAGC.An annealing temperature of 60°C was used for PCR reactions and fluorescence data was collected using the MX3000P QPCR instrument (Stratagene). Data was analyzed using the relative standard curve method, target genes were normalised to GAPDH expression.Unless otherwise stated in the text, parametric, non-parametric and contingency statistical tests were used were appropriate and outlined here. For primary cilia length and orientation comparisons non-parametric Mann-Whitney U tests were used as many data sets failed normality testing . Primary cilia length data is depicted for the purposes of figures as mean and S.E.M. Prevalence is identically presented but statistical significance was assessed by means of fisher's exact tests using frequency of incidence data for each field. ki67 data is presented as means and S.E.M and also used fisher's exact testing from positive or negative staining in immunofluorescence preparations. Nuclear β-catenin expression is shown relative to the mean of the respective control with figures showing mean and S.E.M values and significant differences assessed using two-tailed unpaired student's t-tests. For gene expression studies two-tailed paired student's t-tests with Bonferoni adjustments to avoid type 1 errors. In all cases statistically significant differences are indicated at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Where important, non-significant differences are labeled ‘ns' on figures. All data analysis and presentation was done using GraphPad prism 5. See text for n numbers.R.J.M., A.K.T.W., J.T.C. and M.M.K. conceived and designed experiments. R.J.M., A.K.T.W. and C.L.T. performed experiments and analysed data. R.J.M., A.K.T.W., C.L.T., J.T.C. and M.M.K. prepared the manuscript. All authors reviewed the manuscript.Supplementary material


Splitting describes fragmentation of conscious experience that may occur in various psychiatric disorders. A purpose of this study is to examine relationships between psychological process of splitting and disturbed cognitive and affective functions in schizophrenia and borderline personality disorder (BPD).In the clinical study, we have assessed 30 patients with schizophrenia and 35 patients with BPD. The symptoms of splitting were measured using self-reported Splitting Index (SI). As a measure of semantic memory disorganization we have used verbal fluency test. Other psychopathological symptoms were assessed using Health of the Nation Outcome Scale (HoNOS).Main results show that SI is significantly higher in BPD group than in schizophrenia, and on the other hand, verbal fluency is significantly lower in schizophrenia group. Psychopathological symptoms measured by HoNOS are significantly higher in the BPD group than in schizophrenia. Significant relationship was found between verbal fluency and the SI “factor of others”  in schizophrenia patients.Processes of splitting are different in schizophrenia and BPD. In BPD patients splitting results to mental instability, whereas in schizophrenia the mental fragmentation leads to splitting of associations observed as lower scores of verbal fluency, which in principle is in agreement with Bleuler’s historical concept of splitting in schizophrenia. Splitting reflects shifts of mind related to a consciously experienced conflict of opposing mental forces. In principle it describes fragmentation of conscious experience that is typically related to long-term or acute stress that significantly disturbs self-concept, identity, memory and perception of the external world In schizophrenia the term splitting was developed by Bleuler These alterations on mental level consequently may be linked to great and abrupt changes in patterns of neural activity that may dissociate, or split off, certain external and internal stimuli and information out of awareness, which may lead to distinct states of divided consciousness With respect to recent findings a purpose of this study is to examine relationships between psychological process of splitting and disturbed cognitive and affective functions in schizophrenia and BPD.The participants were recruited from regular daily outpatients treatment programs for schizophrenic and BPD patients at the Psychotherapeutic and Psychosomatic Clinic ESET in Prague. All participants signed informed consent and the study was approved by Charles University ethical committee. In the study were included only patients who had not compromised capacity and ability to consent. This ability was confirmed by clinical data about the patients and specific written statement regarding each participant by his/her psychiatrist. Each included participant was able to consider his/her participation and no one was included in the study based on agreement of legally authorized representative consented on the behalf of a participant.The participants had diagnosis of schizophrenia or borderline personality disorder. Exclusion criteria were organic illnesses involving the central nervous system, substance, and/or alcohol abuse and mental retardation (IQ Raven lower than 90) With respect to current theoretical concepts and empirical data we have tested relationship between splitting based on Splitting Index score The symptoms of splitting were measured using self-reported Splitting index (SI) Other psychopathological manifestations in both groups of patients were measured using Health of the Nation Outcome Scales (HoNOS) As a measure of semantic memory disorganization, which is very close to Bleuler’s concept of mental fragmentation, we have used verbal fluency test Statistical evaluation of the results of SI and other psychometric measures included descriptive statistics, Mann-Whitney test for independent samples and Spearman correlation coefficients. The non-parametric analyses were preferred because SI data have not normal distribution. All the methods of statistical evaluation were performed using the software package Statistica version 6. To prevent Type II error which would disable to reject null hypothesis that the measure of splitting is not linked to verbal fluency and psychopathological symptoms we performed Power Analysis and assessed the effect sizes characterizing differences between means and correlation coefficients.Results show significant differences in scores of splitting, verbal fluency and psychopathological symptoms measured by HoNOS between BPD and schizophrenia groups that were compared using Man-Whitney test . Mean scOn the other hand score of verbal fluency was significantly lower in schizophrenia group. In both assessments of HoNOS, for external evaluators and for self evaluation, the BPD group scored significantly higher in means of total scores. In the power analysis we have tested significant differences which show that all differences between means have strong effect size (r = 0.5 or higher; Results also show significant Spearman correlation coefficients characterizing relationships between splitting, verbal fluency and psychopathological symptoms measured by HoNOS in both samples . Very siMain results of this study indicate significant differences in splitting, verbal fluency and psychopathological symptoms between schizophrenia and BPD patients. These findings show significantly higher level of splitting measured by SI in BPD patients compared to schizophrenia. On the other hand schizophrenia patients show significantly lower scores of verbal fluency most likely as a consequence of cognitive disorganization which in principle is in agreement with Bleuler’s historical concept of splitting in schizophrenia Recent findings also show that impaired verbal fluency is associated with psychomotor slowness These typical neurophysiological changes might reflect typical differences related to splitting in schizophrenia and BPD based on psychological mechanisms of defense against unacceptable affective impulses In summary, the results show that the process of splitting has different forms in schizophrenia and BPD. In BPD patients splitting results to mental instability manifested as shifts in emotional perception of objects, other persons and the self, which are linked to increased mental tension and excessive prefrontal and amygdala activation. This specific form of splitting that occur in BPD is not typically present in schizophrenic patients, which is in agreement with the results indicating that SI score as a measure of borderline splitting is higher in BPD than in schizophrenia patients. On the other hand in schizophrenia the mental fragmentation leads to splitting of associations observed as lower scores of verbal fluency which in principle is in agreement with Bleuler’s historical concept of splitting in schizophrenia


Beta-catenin is a multifunctional oncogenic protein that contributes fundamentally to cell development and biology. Elevation in expression and activity of β-catenin has been implicated in many cancers and associated with poor prognosis. Beta-catenin is degraded in the cytoplasm by glycogen synthase kinase 3 beta (GSK-3β) through phosphorylation. Cell growth and proliferation is associated with β-catenin translocation from the cytoplasm into the nucleus.This laboratory was the first to demonstrate that selenium-containing compounds can enhance the efficacy and cytotoxicity of anticancer drugs in several preclinical xenograft models. These data provided the basis to identify mechanism of selenium action focusing on β-catenin as a target. This study was designed to: (1) determine whether pharmacological doses of methylseleninic acid (MSeA) have inhibitory effects on the level and the oncogenic activity of β-catenin, (2) investigate the kinetics and the mechanism of β-catenin inhibition, and (3) confirm that inhibition of β-catenin would lead to enhanced cytotoxicity of standard chemotherapeutic drugs.In six human cancer cell lines, the inhibition of total and nuclear expression of β-catenin by MSeA was dose and time dependent. The involvement of GSK-3β in the degradation of β-catenin was cell type dependent . However, the pronounced inhibition of β-catenin by MSeA was independent of various drug treatments and was not reversed after combination therapy.Knockout of β-catenin by ShRNA and its inhibition by MSeA yielded similar enhancement of cytotoxicity of anticancer drugs.Collectively, the generated data demonstrate that β-catenin is a target of MSeA and its inhibition resulted in enhanced cytotoxicity of chemotherapeutic drugs.This study demonstrates that β-catenin, a molecule associated with drug resistance, is a target of selenium and its inhibition is associated with increased multiple drugs cytotoxicity in various human cancers. Further, degradation of β-catenin by GSK-3β is not a general mechanism but is cell type dependent. Beta-catenin protein is a vital component of the canonical Wnt/β-catenin signaling pathway, which is described as an oncogenic cause in many human cancers . In headOver 90% of colorectal cancers (CRC) demonstrate a deregulated Wnt/β-catenin signaling pathway . PublishIn addition to its role in cell growth and adhesion, activated canonical Wnt/β-catenin signaling pathway is linked to cancer stem cells ,27 that The response rates of combination regimen of irinotecan and 5-fluorouracil/leucovorin (5-FU/LV) is 39% in metastatic CRC . TreatmeIn our preclinical models, selenium-containing compounds enhanced the efficacy of multiple chemotherapeutic agents (CPT-11 or docetaxel) against various types of cancers  ,33. In mThis study is designed mainly to determine if β-catenin is a target of MSeA in CRC, HNSCC and CaP cancers; and to evaluate the role of GSK-3β in the degradation of β-catenin and if such an effect is associated with enhanced cytotoxicity of anticancer drugs.To evaluate the effect of MSeA on the expression of β-catenin, various tumor cell types were treated with multiple time and doses of MSeA. In all treated cells, MSeA decreased the expression of β-catenin in dose and time dependent manners Figure . The datTo determine whether MSeA down regulates the activity of β-catenin, nuclear and cytoplasmic extracts of colorectal cancer cells were tested for the level of β-catenin before and after MSeA treatment. The data in Figure To determine whether the observed down regulation of β-catenin by MSeA results from inhibition of its synthesis or from increased degradation, cells were treated with MSeA and cycloheximide (an inhibitor of de novo protein synthesis ) alone aTo determine the mechanism of β-catenin degradation by MSeA, the role of GSK-3β was evaluated in HT-29 and HCT-8 Figure . TreatmeTo evaluate further the role of GSK-3β, cells were treated with lithium chloride (LiCl) alone (GSK-3β inhibitor ) and in To determine whether MSeA in combination with a chemotherapeutic agent would affect β-catenin expression and activity, cells were treated with MSeA ± chemotherapeutic agent and analyzed for total and nuclear β-catenin expression. The data in Figure To determine whether inhibition of β-catenin can be correlated with enhanced efficacy of chemotherapy, β-catenin in tumor cells was knockdown by specific ShRNA. Two β-catenin ShRNA tranfectant clones (HCT-8RH7 and HCT-8RF4) that demonstrated inhibition of β-catenin expression when compared with scrambled control (HCT-8SC) and wild type (HCT-8WT) were selected for further testing Figure .To evaluate the effect of silencing β-catenin on cell growth, HCT-8WT, HCT-8SC and HCT-8R (HCT-8RH7 and HCT-8RF4) transfectants were treated with various classes of chemotherapeutic agents. Treatments with 0.5 μM of SN-38 were more effective (p < 0.05) against HCT-8R (~50% cell growth inhibition) when compared with all other groups  and various chemotherapeutic agents (SN-38 or docetaxel) were proven synergistic against various cancers including HNSCC, CRC and CaP ,33. StudTo determine that inhibition of β-catenin by MSeA will translate into enhanced drug-cytotoxicity, cells knocked down β-catenin by ShRNA were more sensitive to growth inhibition by SN-38 than wild type. Collectively, this study indicates that the decreased level of β-catenin is associated with enhancement of drug induced inhibition of cell growth. table .Further, the data in Figure These results support the hypothesis that β-catenin is a target of MSeA and its inhibition results in enhanced drug-cytotoxicity in multiple cancers. Degradation of β-catenin by GSK-3β is not a general mechanism but it is cell type dependent.Although Selenium is a multi-target agent ,33,40, i3SeO2H) was purchased from PharmaSe Inc. . Toptecan was obtained from GlaxoSmithKline . Puromycin dihydrochloride, plasmid transfection medium and transfection reagents were purchased from Santa Cruz biotechnology Inc. .Human cancer cell lines of colorectal (HCT-8 and HT-29), head and neck (FaDu and A253) and prostate (PC3 and C42) were purchased from American type cell culture  and maintained in RPMI 1640 with 10% fetal bovine serum (FBS). The cell lines were tested regularly using Stratogene's mycoplasma plus PCR Primer set  and they were free from Mycoplasma. SN-38, docetaxel, 5-FU, paclitaxel, oxaliplatin, lithium chloride (LiCl) and cycloheximide (CHX) were purchased from Sigma Aldrich . MSeA  for various times . MSeA and SN-38 or docetaxel were given alone or in sequential combination. In sequential combination, 2 h treatments with SN-38  or docetaxel (2 nM against PC3 and C42) started 22 h after treatment with MSeA (1 or 5 μM). HCT-8 parental and β-catenin knockout cells were treated with SN-38 (2 h), docetaxel (2 h), paclitaxel (24 h), oxaliplatin (24 h), 5-FU (24 h) and topotecan (2 h) using various doses. The doses were for SN-38 , for docetaxel , for paclitaxel , for oxaliplatin (1 and 5 μM), for 5-FU  and for topotecan (0.5 and 1 μM). LiCl was applied for 24 h in multiple doses  alone or in combination with MSeA (5 μM).Cycloheximide (CHX) was applied for 5, 10, 20, 30 minutes and 24 hours at a nontoxic concentration of 100 μM alone or in combination with MSeA (5 μM). Puromycin dihydrochloride was used at concentration of 20 μM for clones' selection.2, 25 mM CaCl2, 0.15 M sucrose, 2 mM PMSF, 8 mM dithiothreitol). After overnight storage at -80°C, cells were passed through a 28-gauge needle, centrifuged and the supernatant collected to represent the cytoplasm extract. The remaining pellet was re-suspended in lysis buffer, sonicated, centrifuged and the supernatant collected to represent the nuclear extract.Cytoplasm and nuclear extracts were prepared as previously described . Briefly5 cells/well (6-well plate) one day before the transfection. A well with 70-80% cells confluence was transfected with control shRNA Plasmid-A (a negative control) that encodes a scrambled shRNA sequence that does not inhibit β-catenin to generate HCT-8 scrambled control (HCT-8SC). Another well with the same cells confluence, was transfected with β-catenin shRNA plasmid DNA, a β-catenin-specific lentiviral vector plasmid to knock down expression and generate HCT-8 recombinant clones (HCT-8R). Clones of stable transfectants were selected using 20 μM of puromycin dihydrochloride. After selection, 10 individual clones were evaluated using western blots and the 2 clones (HCT-8RH7 and HCT-8RF4) that demonstrated the most effect of survivin suppression were selected for further studies.HCT-8 cells were utilized to generate a stable transfection using small hairpin β-catenin RNA (β-catenin ShRNA) purchased from Santa Cruz Biotechnology Inc. . Transfection was carried out following the manufacture's instructions. Briefly, HCT-8 cells were plated 5 × 10Western blots were performed as described previously  to deterCell growth was evaluated using sulforhodlamine B (SRB) assay as previously described and performed . BrieflyEach experiment has been repeated at least 3 times. Values were presented as the mean plus or minus standard deviation. Statistical analyses were performed comparing all treatments groups using unpaired t-student test. Significant difference between groups was noted when the p value was less than 0.05.The authors declare that they have no competing interests.MSS performed and designed experiments, prepared and wrote the manuscript. DRR performed cytotoxicity experiments. YMR participated in study design, data interpretation and preparation of the manuscript. RGA designed the research strategy, supervised the project, assisted in data generation, results interpretation and correction of the manuscript. All authors read and approved the final manuscript.


Our results suggest that the function of the network is to buffer the transcriptional activity of Oct4, which appears to be the main determinant to exit pluripotency. The protein network explains the mechanisms underlying the gain and loss of function in different mutants, and brings us closer to a full understanding of the molecular basis of pluripotency.Pluripotency in embryonic stem cells is maintained through the activity of a small set of transcription factors centred around Oct4 and Nanog, which control the expression of ‘self-renewal’ and ‘differentiation’ genes. Here, we combine single-cell quantitative immunofluorescence microscopy and gene expression analysis, together with theoretical modelling, to investigate how the activity of those factors is regulated. We uncover a key role for post-translational regulation in the maintenance of pluripotency, which complements the well-established transcriptional regulatory layer. Specifically, we find that the activity of a network of protein complexes involving Nanog, Oct4, Tcf3, and  Embryonic stem (ES) cells are clonal cell populations derived from mouse pre-implantation embryos that, upon differentiation, give rise to derivatives of all the lineages of an organism and, under certain culture conditions, propagate this capacity, that is, they are pluripotent . GeneticAnalysis of the expression of Oct4 and Nanog in standard self-renewal conditions, for example, Serum and LIF (S+L), reveals a high degree of heterogeneity in gene expression and protein levels  of single mES cells shows that pluripotency is associated with specific ratios, rather than absolute levels, of Oct4 and Nanog protein expression . These rNanog-Oct4-Complex), we assume that Oct4 and Nanog exist in one of the two forms: either free or bound together in a complex. We do not exclude the possibility that the free forms of Nanog and Oct4 interact with other proteins to exert additional functions (see below). This model aims to describe the stochastic dynamics of Oct4 and Nanog expression and translation without relying on any specific transcriptional regulation  an increase in the levels of β-catenin as a consequence of Chiron and (b) an increase in the levels of Nanog expression as a consequence of PD03 .The model considers the existence of three binary complexes: Oct4 with Nanog O:N, ; β-catenThere is a hierarchy in the half-lives of the elements of the network, and also in the stabilities of the complexes, that can be arranged from less to more stable as: Nanog<Oct4<O:N<T, β:T<β:O:N, β:O, and β. This hierarchy of degradation rates is compatible with our experimental observations of the stability of the different protein species of the network .The total amount of β-catenin in the system is unlimited, but only a fraction of it is available to the pluripotent protein network. The available amount of β-catenin varies from cell to cell, as we see experimentally  and alloUnder standard growth conditions, there is no clear correlation between the levels of β-catenin and Nanog (β/N), and the correlation between β-catenin and Oct4 (β/O) is weak  Figures, and useIn the model, the activity of the individual factors is altered when they become part of a complex; for instance, Tcf3 is unable to repress Nanog expression when in complex with β-catenin (β:T) . StochasWe surmise that the main function of the complexes is to stabilize ground-state pluripotency through the attenuation of the activity of Oct4, represented by free Oct4, which has been shown to lead to differentiation when in excess . A bioinThe results and model presented here create a framework to analyze mutants, gain some insights into the molecular basis for their phenotypes, and thereby on the mechanisms underlying the maintenance of pluripotency in wild-type conditions. For example, Nanog is a key element of the ES cell regulatory network but, surprisingly, while it is absolutely required for the establishment of pluripotency , its rem+/− cells; Our model is also able to reproduce and provide a molecular understanding of the phenotype of β-catenin mutant ES cells, which can be unstably maintained in 2i+LIF . AlthougThe ability of the model to reproduce and explain mutants with weakened pluripotency is important, but there are also mutants, in particular Tcf3 mutants, that have been suggested to exhibit increased pluripotency . It is tFinally, we examined the response of the TBON circuit to perturbations in Oct4. Loss of Oct4 results in the loss of pluripotency , 2002 anWe have shown that a competitive network of protein complexes involving Oct4, Nanog, Tcf3, and β-catenin can account for the major characteristics of pluripotency in mouse ES cells. At the centre of this network is the transcriptional activity of Oct4, whose levels are crucial for the maintenance of pluripotency and differentiation . We suggGenetic studies have revealed other proteins involved in the regulation of pluripotency, such as Sox2, Tbx3, Klf4, and Essrb . Within in vivo and establishing the impact that these have on the half lives and dynamics of the different components. As our model indicates that these are important variables in the state of the system, they will have to be addressed experimentally in addition to the more conventional measurements of the transcriptional activity of gene regulatory networks.A plethora of recent studies is making increasingly clear that pluripotency does not rely on a single linear program of transcriptional activity, but rather on the convergence of several molecular activities that balance homeostatically self-renewal and differentiation. Here, we have shown that post-transcriptional interactions are an important component of the mechanism that maintains, and perhaps establishes pluripotency. In particular, our results suggest that a noise-driven competitive protein interaction network involving Oct4, Nanog, Tcf3, and β-catenin provide a key element of the regulatory system that maintains pluripotency. In this network, the levels of β-catenin control the stability and dynamics of the different complexes. The impact that protein complexes appear to have on the maintenance of pluripotency places the focus on the need to characterize their biochemical and functional properties. While this work see also  is a ste−/−  and the parental line (CK044) were cultured in SL or 2i for two passages before lysis in RIPA buffer and concanavilin A fractionation (see To determine the half-lives of proteins, E14Tg2A cells were plated on gelatine in serum+LIF and the next day the medium was changed to serum+LIF+40 μM cycloheximide to inhibit translation. Cells were lysed using RIPA buffer after 0, 2, 4, and 8 h in cycloheximide. Quantitative western blots were performed by loading equivalent number of cells in each lane for normalization. Specific antibodies to Nanog, Oct4, TCF3, and total β-catenin were used and the half-life was obtained assuming a first-order degradation.


Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1) expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes  were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G) or minimal media plus casamino acids (M9CG) facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG) did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc. Burkholderia mallei, the etiologic agent of glanders, is a Gram-negative bacillus that primarily infects horses, mules and donkeys, and occasionally humans. In nature, chronically infected horses are believed to be the only reservoir of this host-adapted pathogen B. mallei. Due to the high risk of aerosol infection and the historical use of this organism as a biological weapon, B. mallei is currently listed as a Tier 1 Select Agent by the Centers for Disease Control and Prevention (CDC) B. mallei is a facultative intracellular pathogen that can survive and replicate within the cytosol of eukaryotic cells. Several studies have shown that the organism persists within murine macrophage cell lines in vitro and can be observed within phagocytic cells in animal models of glanders B. mallei rapidly escapes from endocytic vacuoles, enters into the cytosol and uses actin-based motility to promote intra- and inter-cellular spread B. mallei also possesses the unique ability to induce host cell fusion resulting in multinucleated giant cell (MNGC) formation; a phenomenon that is observed both in vitro and in vivoB. mallei in animals have recently been shown to influence key processes during the interactions of the organism with eukaryotic cells. Included among these are an animal pathogen-like Type III secretion system (T3SSAP), the cluster 1 Type VI secretion system (T6SS-1) and the VirAG two-component regulatory system AP is essential for early vacuolar escape, T6SS-1 is necessary for optimal intracellular growth, robust actin-based motility, and MNGC formation in RAW 264.7 murine macrophages virG is required for actin tail formation by B. mallei in J774.2 cells B. mallei T3SSAP, T6SS-1 and VirAG mutants are unclear.Vibrio cholerae VasX protein, T6SS-secreted, non-VgrG effectors that act directly on eukaryotic targets remain largely unidentified Type VI secretion systems (T6SSs) are contact-dependent protein translocation machines expressed by many Gram-negative bacteria in vivo, but are repressed during routine growth in bacteriological culture media B. mallei T6SS-1 genes are transcribed poorly during the routine culture in rich medium, but are expressed following uptake by murine macrophages prior to vacuolar escape in vitro growth of B. mallei, Hcp1 becomes the major protein detected in culture supernatants T6SS expression is precisely regulated at the genetic level and typically involves two-component regulatory systems, transcriptional activator proteins, posttranscriptional modulators or posttranslational activation mechanisms in vitro while still supporting robust growth of B. mallei. We describe the use of minimal media formulations that facilitate Hcp1 production and demonstrate that virG is required for T6SS-1 gene expression during growth in this media. In addition, we show that divalent metals influence T6SS-1 gene expression and Hcp1 production. Since two closely related species, B. pseudomallei (etiologic agent of melioidosis) and B. thailandensis , also harbor homologous T6SS-1 gene clusters in their genomes In the present study, we sought to identify defined growth conditions that would induce T6SS-1 gene expression Escherichia coli, B. pseudomallei and B. thailandensis strains were routinely cultured at 37°C on Luria Bertani (LB) agar  or in LB broth . B. mallei strains were routinely cultured at 37°C on LB agar or in LB broth supplemented with 4% glycerol (LB4G). For growth of the Burkholderia strains in minimal media, the following formulations were used: Difco M9 Minimal Salts supplemented with 0.4% glucose (M9G); M9G supplemented with 0.5% (w/v) Bacto Casamino Acids (M9CG); or M9G supplemented with 0.5% Bacto Tryptone (M9TG). Metal-deplete M9TG media (M9TG-C) was prepared by supplementing M9G media with 0.5% Bacto Tryptone that had been chelated two times with 2.5% (w/v) Chelex 100. When appropriate, antibiotics were added at the following concentrations: 25 µg/ml kanamycin (Km), 50 µg/ml zeocin (Zeo), 15 µg/ml polymyxin B (Pm), and 100 µg/ml ampicillin (Ap) for E. coli; 5 µg/ml Km, 5 µg/ml Zeo for B. mallei; and 100 µg/ml Km for B. thailandensis. Bacterial stocks were maintained at −80°C as 20% glycerol suspensions. All studies utilizing viable B. mallei and B. pseudomallei were conducted in a CDC select agent-certified biosafety level 3 containment facility.The bacterial strains used in this study are shown in Unless stated otherwise, chemicals were purchased from Sigma-Aldrich. Zeocin was purchased from Invitrogen. Puratronic metals were obtained from Alfa Aesar as follows: Cu as Copper (II) sulfate, 99.999%; Fe as Iron (II) sulfate, 99.999%; Mg as Magnesium sulfate, 99.997%; Mn as Manganese (II) sulfate, 99.999%; Ni as Nickel (II) sulfate, 99.9985%; Zinc sulfate, 99.999%.E. coli TOP10 cells were transformed as per manufacturer’s instructions (Invitrogen). Oligonucleotide primers and dual-labeled probes were obtained from Integrated DNA Technologies or Sigma-Aldrich. DNA sequencing was performed by ACGT Inc. The plasmids and primers used in this study are shown in DNA manipulations were performed using standard methods. Restriction enzymes and T4 DNA Ligase (New England Biolabs) were used according to manufacturer’s instructions. PCR was performed using an Expand High Fidelity PCR System (Roche Applied Science) or GoTaq DNA Polymerase (Promega); 1 M Betaine was included in all polymerase chain reaction (PCR) procedures. PCR was performed using the following conditions: 97°C for 5 min; 30 cycles, each consisting of 97°C for 45 s, 55°C for 45 s, and 72°C for 3 min; a final extension step of 72°C for 10 min was included. PCR and restriction digested products were purified using a QIAquick Gel Extraction Kit (Qiagen). Plasmids were purified using a QIAprep Spin Miniprep Kit (Qiagen). Genomic DNA was purified using a Wizard Genomic DNA Purification kit (Promega). Chemically competent B. mallei SR1A were performed with the sacB-based allelic exchange vectors pEX18Zeo and pMo130 virG gene  from pGRV2-ΔA0746 HindIII and KpnI and cloned into pEX18Zeo digested with the same enzymes resulting in plasmid pEXZΔvirG. E. coli S17-1 was used to mobilize pEXZΔvirG or pMo130-ΔBPSS1498 into B. mallei SR1A via conjugative mating essentially as previously described 4, spotted onto LB4G agar plates and incubated for 8 h at 37°C. To select for transconjugants, mating mixtures were plated onto LB4G-Pm-Zeo or LB-Pm-Km agar and incubated at 37°C for 48 h. To select for sucrose resistant colonies, transconjugants were inoculated into LB4G broth without antibiotics and 10-fold dilutions of the overnight cultures were spread onto M9G plates containing 5% sucrose. Following incubation at 37°C for 48–72 h, sucrose resistant colonies were screened for loss of the Zeo or Km resistance markers by replica plating individual colonies onto LB4G and LB4G-Zeo or LB4G-Km. The resolved co-integrates were screened for the presence of the mutant alleles (ΔvirG or Δhcp1) by PCR using the appropriate primer pairs (virG-FH/virG-RKp or BPSS1498-1/BPSS1498-4).Gene replacement experiments with virAG expression experiments, pBHR2 and pvirAG were mobilized into B. mallei SR1A and its derivatives via conjugative mating as described above and transconjugants were selected for on LB4G-Pm-Km agar. Plasmid pBtvirAG was constructed by PCR amplifying virAG from B. thailandensis E264 genomic DNA using the BtvirAG-FKp/BtvirAG2-RXb primer pair and then cloning the resulting PCR product into the KpnI and XbaI sites of pBHR2. pBtvirAG was mobilized into B. thailandensis DW503 via conjugative mating essentially as described for B. mallei.For B. mallei Hcp1 with a C-terminal 6xHis-Tag (BmHcp1-6xHis), the hcp1 ORF (BMAA0742) was PCR amplified from B. mallei ATCC 23344 genomic DNA using the Bmhcp1-HisF1 and Bmhcp1-HisR1 primer pair. For expression of recombinant B. thailandensis Hcp1 with a C-terminal 6xHis-Tag (BtHcp1-6xHis), the hcp1 ORF (BTH_II0868) was PCR amplified from B. thailandensis E264 genomic DNA using the Bthcp1-HisF1 and Bthcp1-HisR1 primer pair. The resulting DNA fragments were digested with BsaI and cloned into pBAD/HisA digested with NcoI/HindIII resulting in pBmhcp1-His and pBthcp1-His, respectively.For expression of recombinant E. coli TOP10 (pBmhcp1-His) or TOP10 (pBthcp1-His) were grown in 500 ml of LB broth plus Ap at 37°C with aeration (200 rpm). When the cultures reached an OD600 of 0.8, protein expression was induced using 0.02% L-arabinose for 5 h. Bacteria were harvested by centrifugation , resuspended in B-PER (Pierce) plus 1 µl/ml Benzonase (Novagen) and incubated for 10 min at room temperature (RT). The insoluble material was pelleted by centrifugation  and resuspended in B-PER and incubated for 10 min at RT. The insoluble material was again pelleted, then resuspended in Solubilization Buffer (50 mM Tris (pH 8.0), 50 mM NaCl, 10 mM Imidazole and 0.2% Sarkosyl) and gently agitated for 60 min at RT. The remaining insoluble material was removed by centrifugation, the supernatant was filter sterilized and loaded onto a gravity fed Ni-NTA agarose (Qiagen) column. The flow through was collected and applied to the column a second time followed by washing of the column with Wash Buffer (50 mM Tris (pH 8.0), 300 mM NaCl and 40 mM Imidazole). Protein was eluted with Elution Buffer . Fractions were analyzed by SDS-PAGE and those containing BmHcp1-6xHis or BtHcp1-6xHis were pooled, concentrated, dialyzed against saline and stored at −20°C. Protein concentrations were determined using a BCA protein assay kit (Pierce).For purification of BmHcp1-6xHis or BtHcp1-6xHis, Purified BmHcp1-6xHis was used to raise BmHcp1-specific polyclonal antiserum in rats, and purified BtHcp1-6xHis was used to raise BtHcp1-specific polyclonal antiserum in rabbits, at Cocalico Biologicals, Inc.  using a standard protocol.All procedures involving animals were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were approved by the Cocalico Biologicals, Inc. Animal Care and Use Committee.B. mallei, B. pseudomallei or B. thailandensis strains grown in various media  overnight (16–18 h) at 37°C with shaking (200 rpm). Briefly, 0.5–1 ml of bacterial culture was pelleted by centrifugation, the resulting pellets were resuspended in 0.2 ml of 1X Novex Tris-glycine SDS Sample Buffer (Invitrogen) with β-mercaptoethanol and then boiled for 10 min. For immunoblot analyses, the whole cell lysates (10 µl/lane) were separated on 12% Novex Tris-glycine gels (Invitrogen) and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with StartingBlock T-20 Blocking Buffer (Thermo Scientific) for 20 min, incubated with a polyclonal rat anti-BmHcp1 or rabbit anti-BtHcp1 serum  for 1 h, and then incubated with either goat anti-rat or anti-rabbit IgG horse radish peroxidase conjugate antibodies (1/5000 dilution in TBS-T) for 1 h. Membranes were washed three times with TBS-T following each incubation step. Blots were visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific) and a ChemiDoc XRS imaging system (BioRad).Whole cell lysates were prepared from B. mallei or B. pseudomallei strains grown in various media  for 8 h at 37°C with shaking (200 rpm). RNA extracted from bacterial pellets using TRIzol Reagent (Invitrogen) was treated with RNase-free DNase I and purified using an RNeasy Miniprep Kit (Qiagen) according to the manufacturer’s instructions. SuperScript III (Invitrogen) was used to synthesize first-strand cDNA from 1 µg of RNA following manufacturer’s instructions. qRT-PCR primers and probes specific for target genes were designed using Primer Express 3.0 and are shown in rpoA. Fold changes were calculated using the ΔΔCT method. The housekeeping gene dnaK was used as a control for comparative purposes since expression of this gene has been shown to remain similar in various growth conditions For quantitation of transcript levels, RNA was purified from B. mallei T6SS-1 gene cluster (BMAA0744-0730) is part of the VirAG regulon and is co-regulated with the Burkholderia intracellular motility (bim) locus and the clan CA cysteine protease encoding gene, tssM  in order to monitor T6SS-1 expression during growth of the organism in various media. Utilizing whole cell lysates prepared from B. mallei SR1A (pvirAG) and BM0742 (pvirAG), Western immunoblot analysis confirmed the specificity of the polyclonal antiserum for Hcp1  or pvirAG (expresses virAG) grown in M9 minimal media formulations. Consistent with previous findings, Western immunoblot analysis confirmed that SR1A (pBHR2) grown in LB4G medium did not produce Hcp1  or tryptone (M9TG). When Hcp1 levels were assessed under these growth conditions, results demonstrated robust Hcp1 production during growth in M9CG, but much lower Hcp1 production during growth in M9TG  that induced Hcp1 production by B. mallei during in vitro growth.To confirm that non-plasmid bearing strains of  in M9TG . As expehcp1 transcript levels increase significantly in both B. mallei and B. pseudomallei when virAG is over-expressed in vitroB. mallei in minimal media conditions, we harvested RNA from SR1A grown in M9G, M9CG, M9TG and LB4G, and used qRT-PCR to determine hcp1 and virG transcript levels. As shown in hcp1 transcript levels. Consistent with the observations in hcp1 transcript when compared to growth in LB4G  in comparison those grown in LB4G  and assessed Hcp1 production. As shown in hcp1 and tssA mRNA levels in BM0746 in comparison to SR1A during growth in M9CG media using qRT-PCR. In agreement with the immunoblot analysis, results indicated that in the absence of VirG, hcp1 and tssA transcripts were undetectable  used in this study. While both are digests/hydrolysates of casein, casamino acids preparations are largely devoid of iron in comparison to tryptone. To determine if the presence of iron or other divalent metals in tryptone suppressed Hcp1 production, B. mallei was grown in minimal medium supplemented with Chelex-treated tryptone (M9TG-C) and Hcp1 production was assessed. Results of Western immunoblots showed that, although not as robust as in M9CG grown B. mallei, Hcp1 was detectable in M9TG-C grown cultures , or if the iron concentration was increased 2-fold (20 µM), Hcp1 was virtually undetectable  during growth of B. mallei in M9CG containing both iron and zinc (bimA transcript levels decreased (∼5-fold), but virG, tssM and dnaK transcript levels remained relatively unchanged in the presence of iron and zinc. Taken together, these findings indicated that divalent metals, particularly iron and zinc, play a role in negatively regulating T6SS-1 and bimA expression in B. mallei.The observation that cultures . Extendioduction . When B.tectable . In suppand zinc . In addiB. pseudomallei and B. thailandensis are closely related to B. mallei, and harbor homologous T6SS-1 gene clusters  virAG and bimBCADE as shown in B. mallei, B. pseudomallei T6SS-1 expression is induced following uptake by host cells and requires VirG B. mallei . To determine if Hcp1 production in B. pseudomallei was influenced by divalent metals, strains K96243 and 1026b were grown in M9CG alone or supplemented with various cations and Hcp1 levels were assessed. Results demonstrated that either iron or zinc negatively regulated Hcp1 production in both strains  and used it to monitor Hcp1 production during growth of the organism in LB, M9G, M9CG and M9TG. In contrast to the pathogenic species, B. thailandensis did not produce Hcp1 under any of the media conditions tested  and VirAG virAG results in transcription of the T6SS-1 gene cluster as well as the production and export of Hcp1 virG is expressed during growth in M9CG media and is required for T6SS-1, bimA and tssM expression. Our results showed that virG transcript levels increased (∼32-fold) during growth of B. mallei in M9CG in comparison to LB4G. Analysis of a B. mallei ΔvirG mutant confirmed a critical role for VirG in the expression of T6SS-1 genes (tssA and hcp1) and tssM transcription, and to a lesser extent bimA transcription. These findings are comparable to those from a previous study reporting that the activation of T6SS-1 in B. pseudomallei was completely dependent on VirAG within the intracellular environment virAG in B. pseudomallei had no effect on T6SS-1 gene expression when bacteria were grown in RPMI medium. This discrepancy may be due to differences in the levels of T6SS-1 gene expression in RPMI in comparison to M9CG. It is conceivable that VirAG regulated genes may be transcribed at much higher levels during growth of the organisms in M9CG than in RPMI. Further studies will be necessary to address this issue. Taken together, however, these findings suggest that growth of B. mallei and B. pseudomallei in M9CG provides a signal(s) sensed by VirAG which induces its expression in vitro.Although the regulation of T6SSs varies widely from species to species, some common mechanisms that govern T6SS gene expression include quorum sensing, changes in temperature and pH, and two-component regulatory systems Edwardsiella species, T3SS and T6SS genes have been shown to be activated under conditions that mimic those of a phagosome  and repressed in the presence of high iron Pseudomonas aeruginosa and enteroaggregative Escherichia coli, expression of the H2-T6SS and sci1 T6SS gene clusters, respectively, is repressed in the presence of iron B. mallei, we made the observation that when M9G media was supplemented with tryptone (iron-replete) rather than casamino acids (iron-deplete), Hcp1 production was repressed. Extending upon this finding, we showed that iron and zinc negatively regulate Hcp1 production in both B. mallei and B. pseudomallei. Analysis of mRNA levels revealed that transcription of tssA, hcp1 and bimA was down-regulated in the presence of iron/zinc, but that tssM transcript was unchanged, indicating that not all VirAG regulated genes are metal regulated. While virG transcript levels remained relatively constant in both organisms in the presence of iron/zinc, virG transcript decreased ∼5-fold B. pseudomallei in the presence of iron alone. The reason for this result is currently unclear. Additional experiments will be necessary to investigate this phenomenon as well as to determine the levels and activation state of VirG under these growth conditions.Several recent studies have identified a variety of environmental cues that influence T6SS gene expression including the extracellular concentrations of iron, phosphate and magnesium B. mallei T6SS-1 is expressed within phagolysosomal compartments and with the findings in the current study demonstrating that iron and zinc limiting conditions induce expression of genes encoding this critical virulence factor Based on the results obtained herein, it appears that while VirAG positively regulates T6SS-1 expression, iron and zinc negatively regulate the expression of this important secretion system. Since vertebrate hosts sequester iron as a protective mechanism against bacterial infections, and pathogens often sense this iron depletion as a signal to express virulence genes, our findings are in keeping with previous studies demonstrating that T6SS-1 is expressed within host environments sci1 T6SS in enteroaggregative E. coli, the H2-T6SS in P. aeruginosa and, a T6SS in Edwardsiella tardaE. coli and P. aeruginosa, Fur negatively regulates T6SS expression in the presence of iron by binding to Fur boxes in the promoter regions of the gene clusters encoding these systems. The situation in E. tarda appears to be more complex, and it has recently been reported that in addition to directly suppressing T6SS, Fur also acts indirectly through unidentified downstream transcriptional regulators that interact with components of the PhoB regulon B. mallei ATCC 23344 and B. pseudomallei K96243 B. mallei/B. pseudomallei Fur proteins play a role in regulating T6SS-1 gene expression.The ferric uptake regulator protein Fur has been shown to be involved in the regulation of expression of the B. thailandensis produces a number of homologs of known virulence determinants that are expressed by B. mallei and B. pseudomallei and is commonly used as a model system to study specific aspects related to the pathogenesis of glanders and melioidosis B. thailandensis can invade and replicate within host cells and exhibits a similar intracellular lifestyle as the pathogens B. thailandensis T6SS-1 mutants are similar those displayed by B. mallei and B. pseudomallei T6SS-1 mutants, i.e. these strains do not stimulate MNGC formation and are attenuated in a mouse model of acute pneumonic B. thailandensis infection B. thailandensis activates T6SS-1 expression and results in Hcp1 production. Curiously, however, in contrast to B. mallei and B. pseudomallei, growth of B. thailandensis in M9G and M9CG media did not induce Hcp1 production. These findings suggest that the regulatory mechanisms controlling T6SS-1 expression in B. thailandensis may differ from those used by the pathogens. Although VirAG plays a role in regulating T6SS-1, it appears that under the conditions tested, divalent metals do not. Whether B. thailandensis is an appropriate surrogate for studying the regulation of this virulence-associated T6SS requires additional investigation. It is tempting to speculate that differences in the regulatory mechanisms governing virulence factor expression in these three Burkholderia species may account, in part, for the differences in their pathogenicity.in vitro growth conditions that induce Hcp1 production in B. mallei and B. pseudomallei. We demonstrated that virG is expressed under these conditions and is required for T6SS-1 gene expression. In addition, we showed that iron and zinc down-regulate tssA, hcp1 and bimA expression. Other genes  under the control of VirAG did not appear to be as tightly regulated by these metals. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc. The regulation of T6SS-1 appears to be complex and may involve VirG-independent mechanisms that have not yet been identified. Nevertheless, the media conditions described in this study will facilitate additional investigations aimed at characterizing the functions of these important intracellular virulence factors at a molecular level and will be useful for identifying in vivo expressed antigens for use in the development of glanders and melioidosis diagnostics and vaccines.In summary, we have identified