MENA  as a novel transcriptional target of the Wnt/β-catenin signalling pathway. We show that the expression of MENA is upregulated upon overexpression of degradation-resistant β-catenin. Promoters of all mammalian MENA homologues contain putative binding sites for Tcf4 transcription factor – the primary effector of the Wnt/β-catenin pathway and we demonstrate functionality of these Tcf4-binding sites using luciferase reporter assays and overexpression of β-catenin, Tcf4 and dominant-negative Tcf4. In addition, lithium chloride-mediated inhibition of GSK3β also resulted in increase in MENA mRNA levels. Chromatin immunoprecipitation showed direct interaction between β-catenin and MENA promoter in Huh7 and HEK293 cells and also in mouse brain and liver tissues. Moreover, overexpression of Wnt1 and Wnt3a ligands increased MENA mRNA levels. Additionally, knock-down of MENA ortholog in D. melanogaster eyeful and sensitized eye cancer fly models resulted in increased tumor and metastasis formations. In summary, our study identifies MENA as novel nexus for the Wnt/β-catenin and the Notch signalling cascades.Wnt/β-catenin signalling pathway plays important roles in embryonic development and carcinogenesis. Overactivation of the pathway is one of the most common driving forces in major cancers such as colorectal and breast cancers. The downstream effectors of the pathway and its regulation of carcinogenesis and metastasis are still not very well understood. In this study, which was based on two genome-wide transcriptomics screens, we identify  Wnt/β-catenin signalling pathway is critical for early and late embryonic development Wnt/β-catenin pathway is strongly implicated in breast carcinogenesis, in addition to many other cancer types. Transgenic mice expressing degradation-resistant β-catenin in mammary gland tissue develop breast tumors MENA is an actin-regulatory protein that belongs to ENA/VASP protein family et al.MENA (hMENA) is overexpressed in ∼75% of primary breast cancers.Many actin-associated proteins play important roles in carcinogenesis of various types of cancers MENA to be differentially expressed and in this study we show that MENA is a transcriptional target of the Wnt/β-catenin pathway.In our previous study MENA in tumorigenesis, we tested whether knock-down of the Drosophila homolog of MENA (Ena) can affect tumor formation in the D. melanogaster eye cancer fly models “eyeful” and “sensitized”. Eyeful flies have a metastatic eye tumor phenotype induced by activated Notch signalling due to overexpression of the Notch ligand Delta and overexpression of polycomb genes lola and pipsqueak. Sensitized flies overexpress only Delta and have an increased eye size phenotype, but no metastatic tumors. Interestingly, we found that Ena knock-down increases tumor formation and metastasis in both fly models, indicating that Ena has a tumor suppressor role at a crosstalk between the Wnt/β-catenin and the Notch signalling cascades.Wnt and Notch pathways have essential roles in development with well-studied crosstalks, yet their interplay in cancer is not well understood. In order to investigate the role of MENA expression was upregulated 5.5-fold upon overexpression of the S33Y-β-catenin MENA mRNA levels.This study stems from our previous work with Huh7 cells as a hepatocellular carcinoma model, in which SAGE and genome-wide microarray analysis were used to identify novel cancer markers related to Wnt/β-catenin pathway. This pathway is known to be relatively silent in Huh7 cells Homo sapiens, Macaca mulatta, Mus musculus and Rattus norvegicus MENA ortholog genes using Transcriptional Regulatory Element Database of Zhang Lab at CSHL (http://rulai.cshl.edu) and found two putative TBEs in all four of the promoters  differed by only 1 nucleotide and the second TBEs, which were more distal to the TSS were identical   . In HeLaΔN-Tcf4) . Importah7 cells . In orde tissues . PCR anaMENA mRNA levels. In Huh7 cells, we performed a time-course of the LiCl treatment (MENA mRNA levels was observed after 24 hrs of LiCl-treatment. Huh7 cells entered senescence (data not shown) after 72 hrs of LiCl-treatment, which can be the reason for the drop in the β-catenin protein and MENA mRNA levels compared to the 48 hrs timepoint. Treatment of U373, Sk-Mel-103 and Sk-Mel-19 cells with LiCl for 24 hrs also lead to accumulation of endogenous β-catenin and increase in MENA mRNA levels  of eyes showing tumor formation and increased frequency (14%) of flies showing metastasis, compared to eyeful flies expressing a negative control RNAi (white gene) that showed only 42% of eyes with tumor formation and 8% of flies with metastases  which did not result in any tumor or metastasis formation  is promoting metastasis, whereas high levels of growth signalling-related function of Mena (in human cancers) result in increased proliferation and thus, increased neoplasia. Therefore, our findings put MENA at a critical point of crosstalk between Wnt/β-catenin and Notch pathways in regulating actin cytoskeleton and provide a novel example of a Wnt target that represses Notch signalling. Further studies are necessary to determine the role of MENA at the crossroads of Wnt and Notch signalling pathways, in the context of tumorigenesis and potentially brain development.The Wnt and Notch signalling pathways play important roles in development and tumorigenesis and have reported crosstalks in these processes, which are not well understood. These two pathways have opposing effects during development. However, in tumorigenesis, there are many examples where Wnt and Notch pathways must be both activated to induce neoplasia, whereas there are only a few examples where these pathways contradict. For example, in epidermis, Notch1 is a tumour suppressor gene that represses the Wnt/β-catenin pathway http://www.atcc.org) and maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37°C in 5% CO2. Passages 6–15 were used for the experiments.All cell lines were obtained from American Tissue Culture Collection (MENA gene were amplified from human genomic DNA using the following primers: “Forward”: [TAA GCT AGC GAA GTC ATC CCT ATA CCT AGT G] and “Reverse”: AAA AAG CTT CAT GGT GCC GGC GGC]. The PCR fragments were cloned into a Firefly luciferase reporter vector pGL3-basic (Promega) via NheI and HindIII sites which were designed into the forward and the reverse primers, respectively. To characterize β-catenin/TCF4 activity of various cell lines, pGL3-OT and pGL3-OF Firefly luciferase reporter plasmids with wild-type and mutant TCF4 binding sequences, respectively, were used 5′-upstream fragments of h0.5 µg (per well of 6-well plate) of either pcDNA3 or pcDNA3-S33Y were transfected into Huh7 cells by using Lipofectamine 2000 reagent (Invitrogen), following the manufacturer's instructions. . Cells were harvested at indicated time points into 1X Triton-HEPES Lysis Buffer  and frozen at −80°C. Luciferase activities were measured using Dual Glo™ Luciferase Assay System (Promega), as per the procedure provided by the manufacturer.Cells were harvested into 350 µL of Buffer RLT and frozen at −80°C at indicated time points. Total RNA was isolated using the RNeasy® Mini Kit (Qiagen) according to the manufacturer's protocol. RNA was reverse transcribed using ImProm-II™ Reverse Transcription System (Promega) as per manufacturer's instructions RNA extraction was performed from 30 mg head tissue of F1 generation flies using the Rneasy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA samples were reverse transcribed using the First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's instructions and used as template in RT-PCRs. For Drosophila experiments, total RNA was isolated from head tissues of Drosophila fly lines. RT-PCR was performed in a total volume of 25 µl containing 50 ng of genomic DNA and 0.25 mM primers in the presence of 1.5 mM MgCl2, 0.25 mM of each dNTP and 0.5 U Taq DNA polymerase (Fermentase). The following PCR conditions were used: 5 min at 94°C; 25 cycles of 30 s at 94°C, 30 s at 57°C and 30 s at 72°C, followed by a final extension of 5 min at 72°C. The amplified PCR products were analyzed on a 1,5% agarose gel containing ethidium bromide. qRT-PCR was performed as described previously 4, 4 mM EGTA, 1 mM DTT]. The samples were passed through syringe needle (23G) and analyzed by Bradford assay for total protein levels. Equal protein amounts (100 µg) were resolved on 10% SDS-PAGE and analyzed by Western Blotting using monoclonal anti-β-catenin antibody 9E10  and anti-α-actin polyclonal antibody .Cells were seeded into 6-well plates and at ∼80% confluence, lithium acetate was added to a final concentration of 25 mM. Cells were harvested at indicated timepoints by adding 350 µL of RLT lysis buffer RNeasy® Mini Kit (Qiagen) and total RNA was isolated using the RNeasy® Mini Kit (Qiagen) according to the manufacturer's protocol. For Western Blotting analysis, the above procedure was repeated, but the cells were harvested at indicated timepoints by adding 700 µL of 1X Triton-HEPES Lysis Buffer , 70°C 5 min; Hot-start at 92°C.The procedure was adapted from a previously published protocol white gene was knocked-down and as a positive control, Axin was knocked-down. To analyze the tumor prevalence, 180 flies were examined and each eye was scored separately. Eyes were counted as tumorous when the eye showed at least one fold, and metastasis could be seen as masses of amorphous red-pigmented cells outside of the eye field.Fly strains used were “ey-GAL4, GS88A8, UAS-Dl/Cyo” (eyeful flies), “ey-GAL4, UAS-Dl/Cyo” (sensitized flies), “UAS-whiteRNAi” (kind gifts from Bassem Hassan), “UAS-AxinRNAi”, “UAS-EnaRNAi” (obtained from Vienna Drosophila RNAi Center). All flies were raised at 18°C and all crosses were stocked at 25°C on standard fly food. Eye specific promoter eyeless was used to drive Ena RNAi expression. The eyeful flies with the downregulated Ena expression were selected from F1 generation using specific balancer markers in order to analyze the tumor and metastasis prevalence. As a negative control, C57BL/6 mice were used in this study, which was approved by the Bogazici University Ethics Committee. Mice were culled by cervical dislocation and tissues were frozen in liquid nitrogen until further use. For ChIP experiments, procedure described in the “Chromatin ImmunoPrecipitation” section was applied to the brain and liver tissues.http://rulai.cshl.edu/cgi-bin/TRED/tred.cgi?process=home) and TFexplorer (http://www.tfexplorer.org) P-values less than 0.05 were considered significant (one stars) and p-values less than 0.005 were considered very significant (two stars).Promoter sequences were analyzed using Transcriptional Regulatory Element Database located at Cold Spring Harbor Laboratory website (


The Wnt signaling pathway plays crucial roles during embryonic development, whose aberration is implicated in a variety of human cancers. Axin, a key component of canonical Wnt pathway, plays dual roles in modulating Wnt signaling: on one hand, Axin scaffolds the “β-catenin destruction complex” to promote β-catenin degradation and therefore inhibits the Wnt signal transduction; on the other hand, Axin interacts with LRP5/6 and facilitates the recruitment of GSK3 to the plasma membrane to promote LRP5/6 phosphorylation and Wnt signaling. The differential assemblies of Axin with these two distinct complexes have to be tightly controlled for appropriate transduction of the “on” or “off” Wnt signal. So far, there are multiple mechanisms revealed in the regulation of Axin activity, such as post-transcriptional modulation, homo/hetero-polymerization and auto-inhibition. These mechanisms may work cooperatively to modulate the function of Axin, thereby playing an important role in controlling the canonical Wnt signaling. In this review, we will focus on the recent progresses regarding the regulation of Axin function in canonical Wnt signaling. Wnt signaling is one of the most conserved pathways during evolution, playing essential roles in multiple biological processes like embryonic development and adult tissue homeostasis. Dysregulation of the Wnt pathway underlies the pathogenesis of a variety of human cancers. So far, two types of Wnt signaling pathways have been characterized: canonical Wnt pathway  and noncanonical Wnt pathway . The Wnt/β-catenin pathway is distinguished from the others in the way of its dependency on β-catenin, which is nevertheless not required for the two noncanonical Wnt pathways. As a central player in canonical Wnt signaling, β-catenin dictates the signaling intensity through its cellular levels and subcellular localization. In the absence of Wnt signaling, β-catenin is sequestered in the cytoplasm by the “destruction complex” composed of Axin, glycogen synthase kinase 3 (GSK3) and adenomatous polyposis coli (APC), etc. β-catenin is phosphorylated by GSK3 and subsequently subjected to degradation through the ubiquitin-proteasome pathway. Upon Wnt stimulation, low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) is co-clustered with Frizzled (Fz) and Dishevelled (Dvl) and recruits the active Axin and GSK3 complex; these proteins thus form a high-molecular-weight assemblage close to the plasma membrane that is referred to as “LRP signalosome”  is constitutively expressed, while Axin2  is subjected to the regulation of Wnt signaling  for ATP-dependent activation of ubiquitin (Ub), ubiquitin-conjugating enzyme (E2) for delivery of Ub, and ubiquitin ligase (E3) for ligation of Ub to the substrates. In this process, the E3 ligases are responsible for substrate selection and the specificity of Ub-linkage. There are seven lysines  in Ub molecule. Generally, proteins marked by K48-linked poly-Ub chains are destined to proteasome-dependent degradation, while other types of Ub chains could regulate a variety of cellular processes by both proteolytic and non-proteolytic functions  plays an inhibitory role in Axin’s binding with its partners  also targets and represses the translation of Axin2. lethal-7 (let-7) is one of the first two known miRNAs (the other one is lin-4) . However, it is unclear how reduced Axin2 expression conferred by miR-205, rather than facilitates tumorigenesis as it usually does, contributes to the suppression of KB oral cancer cells.MicroRNA (miRNA) is a small noncoding RNA molecule normally containing 19–25 nucleotides in length, which regulates gene expression both at transcriptional and post-transcriptional level. So far, thousands of transcripts have been reported to subject to microRNA regulation, playing extensive and important roles in eukaryotic development and physiology. Moreover, increasing evidences have implicated the role of miRNA deregulation in human disease including cancer. Recently, there are several researches addressing potential roles of miRNAs in Axin regulation. In a screen for miRNAs affecting Wnt pathway, Silver et al. found that ectopic miR-315 directly inhibits Axin as well as Notum , and forced expression of Axin2 results in rapid cell death in an MSI+ CRC cell line, indicating a role of epigenetic silencing of Axin2 in carcinogenesis of MSI+ CRC  and Axin2 expression is correspondingly downregulated, which may contributes to the pathogenesis and growth of NETs (Kim et al., Axin2 promoter (Chen et al., In additional to the post-transcriptional regulations mentioned above in affecting Axin levels, epigenetic modulations could also affect the expression of Axin and the change thereof may relate to tumor development. It is well accepted that genetic changes in components of Wnt signaling are closely related with human cancer. One leading example is colorectal cancer: alterations in Wnt-related genes, mainly APC, have been observed in ~90% of colorectal cancer (Miyaki et al., Besides those post-transcriptional modulations mentioned above, another modification—methylation—is also observed for Axin (Cha et al.,


Activation of the IKK-NFκB pathway increases the resistance of cancer cells to ionizing radiation (IR). This effect has been largely attributed to the induction of anti-apoptotic proteins by NFκB. Since efficient repair of DNA double strand breaks (DSBs) is required for the clonogenic survival of irradiated cells, we investigated if activation of the IKK-NFκB pathway also regulates DSB repair to promote cell survival after IR. We found that inhibition of the IKK-NFκB pathway with a specific IKKβ inhibitor significantly reduced the repair of IR-induced DSBs in MCF-7 cells. The repair of DSBs was also significantly inhibited by silencing IKKβ expression with IKKβ shRNA. However, down-regulation of IKKα expression with IKKα shRNA had no significant effect on the repair of IR-induced DSBs. Similar findings were also observed in IKKα and/or IKKβ knockout mouse embryonic fibroblasts (MEFs). More importantly, inhibition of IKKβ with an inhibitor or down-regulation of IKKβ with IKKβ shRNA sensitized MCF-7 cells to IR-induced clonogenic cell death. DSB repair function and resistance to IR were completely restored by IKKβ reconstitution in IKKβ-knockdown MCF-7 cells. These findings demonstrate that IKKβ can regulate the repair of DSBs, a previously undescribed and important IKKβ kinase function; and inhibition of DSB repair may contribute to cance cell radiosensitization induced by IKKβ inhibition. As such, specific inhibition of IKKβ may represents a more effective approach to sensitize cancer cells to radiotherapy. The IκB kinase (IKK)-nuclear factor κB (NFκB) pathway is one of the most important cellular signal transduction pathways In addition, an increasing body of evidence suggests that activation of the IKK-NFκB pathway also play a pivotal role in the development of cancer resistance to ionizing radiation (IR) and chemotherapy It has been well established that IR kills cancer cells primarily by induction of DSBs and efficient repair of DSBs is required for the clonogenic survival of irradiated cells Activation of NFκB by IR depends on IKKβ NU7026 (NU) and KU55933 (KU) are well characterized DNA-PK and ATM inhibitors, respectively in vitro kinase assays. As shown in Although BMS is a selective inhibitor of IKKβ, it is not know whether it inhibits DNA-PK and ATM To further explore the requirement of IKK in efficient DSB repair, we generated stable IKKα and/or IKKβ knockdown cell lines, e.g. IKKα(-), IKKβ(-), and IKKα/β(-) cells, by transfection of MCF-7 cells with lentiviral short hairpin RNAs (shRNAs) that specifically target IKKα and/or IKKβ mRNA. As shown in To further validate the essential role of IKKβ in DSB repair and determine whether the kinase activity of IKKβ is required for the regulation, we reconstituted MCF-7/IKKβ(-) cells with wild-type IKKβ (WT- IKKβ), kinase-dead IKKβ (K44M-IKKβ), and constitutively active IKKβ (SSEE-IKKβ) Since efficient repair of IR-induced DSBs is required for the clonogenic survival of irradiated cells IR is one of the most widely used therapeutic modalities for cancer. Unfortunately, many tumor cells are inherently more resistant to IR or can acquire radioresistance shortly after radiotherapy, which inevitably leads to treatment failure and relapse of the disease Among various inhibitors of the IKK-NFκB pathway, IKKβ inhibitors have emerged as the most promising anti-tumor agents and novel tumor sensitizers for IR and chemotherapy in vivoIt has been well established that IR kills cancer cells primarily by induction of DSBs. DSBs are considered the most detrimental DNA lesions and a single unrepaired DSB is sufficient to kill a cell. Therefore, targeted inhibition of the DSB repair pathways has been actively pursued as a way to sensitize tumor cells to IR and other chemotherapeutic agents The mechanisms by which IKKβ regulates DSB repair have yet to be elucidated. Our preliminary data showed that selective inhibition of the NFκB transcriptional activity by ectopical expression of a mutant IκBα or down-regulation of RelA by RNAi had no effect on the repair of IR-induced DSBs , indicatIn conclusion, for the first time, we demonstrate that IKKβ regulates the repair of IR-induced DSBs. Moreover, IKKβ, but not IKKα, is primarily responsible for promoting survival of certain tumor cells after IR at least in part by facilitating DSB repair. Surprisingly, NFκB-RelA is dispensable for IKKβ-dependent repair of DSBs. Therefore, IKKβ inhibition or critical processes involved in the IKKβ-dependent DSB repair pathway may be exploited as a novel therapeutic strategy to increase the sensitivity of tumor cells to IR.TPCA-1, SC-514 and NU-7026 (NU) were purchased from Calbiochem . KU-55933 (KU), BMS-345541 (BMS), Hoechst-33342 (Hoe) and Propidium iodide (PI) were purchased from Sigma-Aldrich .2) at 37°C.MCF-7, H1299 and H1648 cell lines were originally obtained from ATCC . They were selected for our study because IR is a common therapeutic modality for breast and lung cancer and these cell lines have been extensively used in radiation research. MCF-7 cells stably transfected with the dominant-negative mutant IκBα (mIκBα or IκBα A32/36) were kindly provided by Dr. Jian Jian Li . Immortalized wide type, IKKα, IKKβ, and IKKα/β double knockout mouse embryonic fibroblasts were kindly provided by Dr. Shigeki Miyamoto  with the permission of Dr. Inder Verma . All these cells were maintained in Dulbecco's modified Eagle's minimum (DMEM) supplemented with 10% fetal bovine serum , penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified incubator  was digested with EcoRI and then ligated with synthetic oligonucleotides containing PmeI, HpaI and BstBI sites to generate PLVUT-1 vector. The PCR fragment containing PCMV IE-GFP and BstBI sites was generated from pEGFP-C1 vector  by PCR using Phusion® Hot Start High-Fidelity DNA Polymerase . It was inserted into BstBI site of PLVUT-1 vector to generate PLVUT-GFP vector. According to the sequences of IKKβ/WT, IKKβ/K44M and IKKβ/SSEE vectors (Addgene), IKKβ/WT, IKKβ/K44M and IKKβ/SSEE fragments containing PmeI and HpaI sites were generated by PCR using Phusion® Hot Start High-Fidelity DNA Polymerase. PLVUT-GFP-IKKβ/WT, PLVUT-GFP-IKKβ/K44M, PLVUT-GFP-IKKβ/SSEE lentiviral vectors were constructed after insertion of these fragments into PmeI and HpaI sites of PLVUT-GFP vector. The resulting vectors were confirmed by DNA sequence.Lentivirus was produced after transient infection of human embryonic kidney (HEK) 293T cells with individual lentiviral vectors along with the packaging plasmids pCMV-VSV-G and psPAX2 (Addgene) using FuGEN6-HD  as the infection reagent according to Roche's protocol. The supernatants containing viral particles were collected 48 h after the infection and filtered through 0.22 µm filter. The viral particles were concentrated using a kit (PEG-itTM Virus Precipitation Solution) from System Biosciences  according to the manufacturer's instructions.137Cesium -irradiator  at a dose rate of 2.4 Gy/min. Cells were irradiated on a rotating platform.Cells were exposed to various doses of IR in a JL Shepherd Model 143 Cells grown on a 4-chamber CultureSlide  after various treatments were fixed, permeabilized, and stained as previously described 32P)ATP in the presence or absence of BMS (5 or 10 µM) or NU (5 µM) for 5 min at 30°C according to the manufacturer's instructions. The biotinylated substrate was captured on a streptavidin membrane, washed and quantified by a Storm 860 Phosphorimager .The kinase activity of DNA-PK was measured using a SignaTECT DNA-Dependent Protein Kinase Assay System . The biotinylated peptide substrate was incubated with 50 units purified DNA-PK  and (γ-32P)ATP in the presence or absence of BMS (5 or 10 µM) or KU (5 µM) at 30°C for 10 min. After SDS-polyacrylamide gel electrophoresis, γ-32P-H2AX was visualized by autoradiography.ATM was purified from irradiated MCF-7 cells by immunoprecipitation with anti-ATM antibody  as previously described g) at 35°C for 30 min. Stably transduced cells were selected with puromycin (2 µg/ml). IKKα and/or IKKβ knockdown in MCF-7/IKKα(-), MCF-7/IKKβ(-) and MCF-7/IKKα/β(-) cells, respectively, was confirmed by Western blot.Control lentiviral pLKO.1 vector and pLKO.1 vectors containing shRNAs for human IKKα (RHS4533-NM_001278) and IKKβ (RHS4533-NM_001556) were obtained from Open Biosystems . Viral particles were produced as described above. To establish stable IKKα and/or IKKβ knockdown MCF-7 cell lines, MCF-7 cells were infected twice with the viral particles under centrifugation (900×MCF-7/IKKβ(-) cells were infected twice with the viral particles containing PLVUT-GFP vector or the vector encoding IKKβ/WT, IKKβ/K44M and IKKβ/SSEE as described above. The cells expressing a moderate level of GFP were enriched twice by cell sorting to generate stable MCF-7/IKKβ(-)/vector, MCF-7/IKKβ(-)/WT, MCF-7/IKKβ(-)/K44M, and MCF-7/IKKβ(-)/SSEE cell lines. The expression of IKKβ in these cell lines was confirmed using Western blot.Total cell lysates were separated on 10% SDS–PAGE. The proteins were transferred onto a PVDF membrane, blocked with 5% nonfat milk for 1 h at room temperature, and probed with a primary antibody (1∶1000) overnight at 4°C. The primary antibodies used include anti-IKKα (Cat#2682), IKKβ (Cat# 2678) and RelA (Cat# 4764) from Cell Signaling Technology ; and anti-phosphorylated IκBα (Cat# 2859), anti-IκBα (Cat# 4814) and Actin (Cat# SC-1616) from Santa Cruz Biotechnology . The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody at 1∶10,000 dilutions  for 1 h. Protein bands were visualized using Amersham ECL Western Blotting Detection Reagents  and exposed to ECL Plus film .TM NFκB-RelA kit (Active Motif) using 5 µg of nuclear extract proteins according to the manufacturer's instructions.Nuclear proteins were extracted from cells using the Nuclear Extract Kit  per the manufacturer's protocol and were not contaminated by cytoplasmic elongation factor 2 (EF2) based on the result of Western blot using an antibody against EF2. NFκB-RelA DNA-binding activity was determined by a TransAM4 MCF-7 cells were seeded into a well of six-well plates. After overnight incubation, the medium was removed and then replaced with transfection media containing control  or RelA  siRNA  along with DharmaFECT4 transfection reagent (Dharmacon) according to the manufacturer's protocol. After 24 h incubation, the transfection medium was removed and replaced with cell culture medium. The cells were allowed to grow for an additional 48 h to achieve maximal knockdown of RelA as shown by real-time RT-PCR and Western blot analyses.To down-regulate the expression of RelA with siRNA, 5×105′-CCTCAATGTCCTCTTTCTGC-3′; and GAPDH, forward 5′-CCC CAC ACA CAT GCA CTT ACC-3′ and reverse 5′-CCT ACT CCC AGG GCT TTG ATT-3′. The threshold cycle (Ct) value for RelA was normalized to the Ct value of GAPDH. The relative RelA mRNA expression was calculated using the comparative CT (2-ΔΔCt) method as previously described Real-time PCR was done as previously described using the following primers: RelA, forward 5′-CCTTCCTCATCCCATCTT TG- 3′ and reverse 4 cells/well. After overnight incubation, they were exposed to various doses  of IR with or without pretreatment as indicated in individual experiments. The cells were allowed to grow for additional 12 days to form colonies before stained with 0.1% crystal purple. Colonies with more than 50 cells were counted. Survival fraction was calculated according to the plating efficiency of control cultures.MCF-7 cells were seeded into wells of 12-well plates at 1×10t test. Differences were considered significant at P<0.05. All of these analyses were done using GraphPad Prism from GraphPad Software .The data were analyzed by analysis of variance (ANOVA). In the event that ANOVA justified post hoc comparisons between group means, these were conducted using the Student-Newman-Keuls test for multiple comparisons. For experiments in which only single experimental and control groups were used, group differences were examined by unpaired Student Figure S1IKKβ but not IKKα knockout inhibits the repair of IR-induced DSBs in mouse embryonic fibroblasts. Mouse embryonic fibroblasts (MEF) from wild-type (WT), IKKα, IKKβ, and IKKα/β knockout mice were exposed to 2 Gy IR. DSBs were analyzed by γH2AX and 53BP1 immunofluorescent staining at 1 h and 6 h after IR. Un-irradiated cells were included as controls (CTL). Representative photomicrographs (100× magnifications) of γH2AX (red) and 53BP1 (green) immunofluorescent staining and nucleic counterstaining with Hoechst-33342 (blue) are shown in (A) and the average numbers of γH2AX and 53BP1 foci/cell from three independent experiments are presented (B) and (C) as mean ± SE. * p<0.05, ** p<0.01, and *** p<0.001, vs. WT MEFs.(TIF)Click here for additional data file.Figure S2IKKβ regulates DSB repair in a NFκB-RelA independent manner.(A) and (B) Ectopic expression of mIκBα inhibits IR-induced phosphorylation of IκBα and NFκB activation in MCF-7 cells. The levels of phosphorylated IκBα (p-IκBα) and total IκBα in the lysates from vector- or mIκBα-transfected MCF7 cells before (CTL) or 30 min after IR (2 Gy) were analyzed by Western blots. NFκB activation was analyzed by quantification of the levels of RelA in the nuclear extracts from vector- or mIκBα-transfected MCF7 cells before (CTL) or 30 min after IR (2 Gy) by an ELISA assay. The data presented in (B) are mean ± SE (n = 3). *** p<0.001, vs. vehicle. (C) Ectopic expression of mIκBα has no effect on the repair of IR-induced DSBs in MCF-7 cells. DSBs were analyzed by γH2AX immunofluorescent staining at 1 and 6 h after vector- or mIκBα-transfected MCF7 cells were exposed to 2 Gy IR. Un-irradiated cells were included as a control (CTL). The average numbers of γH2AX foci/cell from three independent experiments are presented as mean ± SE. (D) Down-regulation of RelA mRNA expression by siRNA was confirmed by real-time PCR. The expression of RelA and GAPDH mRNA in RelA siRNA-treated cells was expressed as a percentage of that in control siRNA-treated cells. The data are presented as mean ± SE (n = 3). *** p<0.001, vs. control siRNA treatment. (E) Down-regulation of RelA expression by siRNA was confirmed by Western blot in MCF-7 cells transfected with control (CTL) or RelA siRNA. Un-transfected MCF-7 cells (Control) were included as a control. (F) Down-regulation of RelA expression by siRNA has no effect on the repair of IR-induced DSBs in MCF-7 cells. DSBs were analyzed by γH2AX immunofluorescent staining at 1 and 6 h after control (CTL siRNA) or RelA siRNA-transfected MCF7 cells were exposed to 2 Gy IR. Un-irradiated cells were included as controls (CTL). The average numbers of γH2AX foci/cell from three independent experiments are presented as mean ± SE.(TIF)Click here for additional data file.


Initially, male Sprague Dawley rats were divided into 5 groups, with 6 rats in each group. The rats received two intraperitoneal (i.p.) injections of AOM in saline (15 mg/kg body weight) over a 2-week period to induce CRC. IP6 was given in three concentrations, 0.2% (w/v), 0.5% (w/v), and 1.0% (w/v), via drinking water for 16 weeks. The deregulation of the Wnt/β-catenin signaling pathway and the expression of cyclooxygenase (COX)-2 have been implicated in colorectal tumorigenesis. β-Catenin and COX-2 expressions were analysed using the quantitative RT-PCR and Western blotting. Herein, we reported that the administration of IP6 markedly suppressed the incidence of tumors when compared to the control. Interestingly, the administration of IP6 had also markedly decreased β-catenin and COX-2 in colon tumors. Thus, the downregulation of β-catenin and COX-2 could play a role in inhibiting the CRC development induced by IP6 and thereby act as a potent anticancer agent.Nutritional or dietary factors have drawn attention due to their potential as an effective chemopreventive agent, which is considered a more rational strategy in cancer treatment. This study was designed to evaluate the effect of IP These concentrations were achievable physiologically in rat tumor models considering the doses used which give rise to inhibition of tumor incidence [Rats were acclimatized for seven days and randomized into experimental and control groups. The animals were injected intraperitoneally with azoxymethane (AOM), a specific carcinogen that was diluted in saline, once per week (15 mg/kg body weight), over a 2-week period to induce colonic tumors , 25. Ratncidence , 26. AllμM) were stained with hematoxylin and eosin (H & E).The colons were resected and flushed with 10% neutralized formalin to remove residual bowel contents, cut open longitudinally and submerged overnight in 10% (v/v) neutralized formalin. The tissue samples were dehydrated in an ascending series of alcohol dehydration, cleared with xylene, and wax impregnated with paraffin wax for 14 hours in an automatic tissue processor machine. Sections  in a final reaction volume of 20 μL, according to the manufacturer's protocol. The reverse transcription reaction was carried out at 42°C for 30 minutes in an authorized thermal cycler , followed by a 10-minute step at 99°C to denature the enzyme; then, it was cooled to 4°C. This cDNA was then used as a template for amplification in real-time PCR reactions. Quantitative real-time PCR was performed in a reaction volume of 20 μL, according to the manufacturer's instructions for the KAPA SYBR Green FAST qPCR kits . Nucleotides primer sequences of rat origin were used , and 1 μL of template cDNA (20 ng) were added to each well. After a brief centrifugation, the PCR plate was subjected to 40 cycles of the following conditions: (i) PCR activation at 95°C for 20 seconds, (ii) denaturation at 95°C for 3 seconds, and (iii) annealing/extension at 60°C for 20 seconds. All samples and controls were run in triplicate on a Mastercycler realplex system . The quantitative RT PCR data were analyzed using a comparative threshold (Ct) method, and the fold inductions of the samples were compared with the untreated samples. β-actin was used as an internal reference gene to normalize the expression of the target genes.The extraction of total RNA from fresh whole colon tumor tissue was performed using TRI Reagent . Tissue disruption and homogenization were performed such that the colon tumor tissue was weighed out (20–30 mg), immediately placed in liquid nitrogen, and homogenized using a tissue grinder. The tissue powder and liquid nitrogen were decanted into an RNase-free, 2 mL microcentrifuge tube, and the liquid nitrogen was allowed to evaporate. Then, the tissue samples were homogenized in 1 mL of TRI Reagent per 50–100 mg of tissue for 10 minutes. RNA extraction was then performed according to the manufacturer's instructions, and 1 ere used . Brieflyμg) was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a piece of PVDF membrane using transfer buffer  methanol, pH 8.3). After transfer, the PVDF membrane was blocked at room temperature with blocking solution   for 30 minutes. The membrane was then incubated overnight with primary antibodies at a 1 : 1000 dilution , followed by a 1-hour incubation with alkaline phosphatase-labeled goat anti-mouse secondary antibody  at a 1 : 10000 dilution in Tris-buffered saline (TBS) and 0.5% Tween 20. Immunodetection was performed by the addition of a developing solution. The developing solution for alkaline phosphatase conjugated antibodies consisted of 10 mL alkaline phosphatase buffer , 33 μL BCIP  in 10 mL of 100% (v/v) dimethylformamide (DMF)), and 66 μL NBT  in 10 mL of 70% (v/v) DMF). The reaction was stopped when the desired protein band appeared. Densitometric analysis of band intensities obtained from western blotting experiments were carried out using ImageJ software , USA).The fresh colon tumor tissue disruption and homogenization steps for extracting proteins were similar to those described for total RNA extraction. The samples were homogenized in Buffer RLT using a QIAshredder homogenizer. Protein extraction was performed using the AllPrep DNA/RNA/Protein Mini Kit, according to the manufacturer's protocol . Next, the protein concentration was determined by the Bradford assay, according to the manufacturer's protocol . The protein (50 P < 0.05.Data were expressed as the mean ± standard deviation (SD) and statistically analyzed using SPSS version 10, with a one-way ANOVA with Tukey's test and a significance level of 6 was analyzed by reversed-phase high-performance liquid chromatography (HPLC). HPLC chromatograms were compared with those of standard phytic acid. As we reported earlier [IP earlier , 23, bas6, AOM + 0.5% (w/v) of IP6, and AOM + 1% (w/v) of IP6). The plotted graph in 6-fed groups (AOM + 0.2% (w/v) of IP6, AOM + 0.5% (w/v) of IP6, and AOM + 1% (w/v) of IP6) showed no significant differences in body weight, even after 16 weeks of IP6 treatment. However, there was a significant (P < 0.05) difference between the body weight of the positive control group (AOM only) and the body weight of the rats in the IP6-fed and normal groups. In addition, the body weight of the AOM-only group was significantly lower than that of the IP6-fed and normal groups; these results support the expectation that exposure to the carcinogen AOM would cause weight loss. Additionally, administration of IP6 during the early treatment may boost growth  (0.2% (w/v), 0.5% (w/v), and 1% (w/v)) on the reduction of ACF development was further demonstrated. As shown in 6 significantly reduced the total number of ACF (P < 0.05) in all IP6-fed groups in comparison with the untreated rats and AOM-only group, thereby leading to the inhibition of ACF formation which may or may not develop into the invasive carcinoma. 6).ACF is a preneoplastic lesion which can be characterized by clusters of mucosal cells with an enlarged and thicker layer of epithelia compared to the surrounding normal crypts. ACF may progress into polyps, later develop into adenomas, and eventually become invasive carcinomas . Althoug6).An adenoma is defined as a benign tumor of glandular origin. Colonic adenoma consists of proliferation on colonic glands lined by neoplastic colonic epithelium, while an adenocarcinoma is a malignant tumor originating in glandular epithelium. Colonic adenocarcinoma is composed of invasive glands lined by pleomorphism and hyperchromatic epithelium. 6 ) significantly reduced the incidence of tumors in a dose-dependent manner when compared with the results of the AOM-only group (no IP6 treatment) (P < 0.05). None of the rats in the normal group developed colon tumor. Additionally, not all rats consist of both adenoma and adenocarcinoma, namely, total tumors  in different study groups is summarized in l tumors , and in 6 treatment is accompanied by the downregulation of β-catenin. 6-treated rats expressed less β-catenin at the mRNA level, especially for rats treated with the highest concentration of IP6, 1% (w/v). After treatment with 0.2%, 0.5%, and 1% (w/v) of IP6, β-catenin was significantly downregulated by 0.93-, 0.514-, and 0.385-fold, respectively, demonstrating a dose-dependent relationship (P < 0.05). Our study successfully demonstrates that inhibition of tumor growth in AOM-induced rat CRC using IP6 also significantly inhibited the expression of COX-2 in AOM-induced rat colon carcinogenesis (P < 0.05). As shown in 6 ), COX-2 mRNA expression was significantly downregulated by 0.783-, 0.661-, and 0.426-fold, showing a dose-dependent relationship (P < 0.05).Moreover, based on the fold changes relative to the control, IPβ-catenin and COX-2 proteins for the control and experimental group of rats are presented in β-catenin and COX-2 were both overexpressed in the AOM-induced rats. As shown in β-catenin was detected as a 92-kDa band in AOM-induced rat colon carcinogenesis. Significant reduction in β-catenin protein expression was observed after treatment with IP6  by 0.272-, 0.189-, and 0.045-fold relative to the control in a dose-dependent manner (P < 0.05). The expression levels of 6 at the mRNA level, it was also downregulated at the protein level. COX-2 protein expression in AOM-induced rat colon carcinogenesis was significantly decreased by 0.38-, 0.102-, and 0.031-fold after treatment with 0.2%, 0.5%, and 1% (w/v) of IP6, respectively, compared with the control group, thus showing a dose-dependent relationship (P < 0.05). All together, these results showed that the regulation or stability of β-catenin and COX-2 protein levels should be affected by IP6.Furthermore, as shown in 6 administration during the 16-week study period, were assessed to evaluate potential adverse health effects of the treatment. As mentioned earlier, all of the IP6-fed groups experienced body weight gain that was similar to the normal group and, therefore, did not show any significant differences. These results were also observed by Norazalina et al. [6 from rice bran. Similar findings were observed in the suppression of prostate cancer growth for IP6-fed mice [The primary parameters used in cancer chemoprevention studies to assess the toxicity of a test compound include body weight gain profiles. Accordingly, changes in animal body weight, as a result of IPa et al.  who prevfed mice . 6-fed groups were reliable because, according to Alfin-Slater and Kritchevsky [6-fed groups, even though all of the rats were initially treated with the carcinogen (AOM), their body weights showed no significant difference compared with that of the normal rats after 16 weeks of continuous IP6 treatment. Thus, the administration of IP6 appears to have an inverse effect on the reduction in body weight seen with AOM alone. However, the mechanism behind this effect is unknown.A significant reduction in the body weight of AOM-induced group was observed when compared with the other groups; the body weights observed for the normal and IPtchevsky , the wei6 in drinking water suppressed the number of ACF and thereby leading to the inhibition of tumor incidence in the colons of the IP6-fed groups as compared with the AOM-induced group. IP6 has been shown to reduce the number and size of colon adenomas, which are the precursor lesions of CRC. Our study suggests that IP6 extracted from rice bran might reduce the formation of tumors in the colon. The tumor inhibition action of IP6 has been supported by previous studies, which showed that the addition of IP6 to drinking water had a chemopreventive effect on CRC [6 from rice bran on CRC and hepatic cancer in vitro [The current data shows that the administration of IPt on CRC , 30, 31.in vitro , 33.APC or β-catenin gene are regarded as early critical events during CRC and are therefore considered to play a gate keeper role in the development of CRC in both humans and preclinical models [APC or β-catenin gene were proved to repress the degradation of the protein and generate β-catenin accumulations in the cytosol [β-catenin functions as a transcriptional activator when complexed with members of the T-cell factor (TCF) family of DNA binding proteins [β-catenin signaling pathway, such as c-myc and cyclin D1, are growth-promoting genes, suggesting that this pathway is potentially an oncogenic pathway [β-catenin was elevated in AOM-induced experimental CRC [β-catenin during colon tumorigenesis [β-catenin which was increased while induction with AOM [6 might be involved in regulation or stability of β-catenin level in CRC as a previous study has also reported the role of inositol polyphosphates in Wnt signaling pathway, by which IP5 accumulates in response to Wnt3a, thereby playing an essential component of signaling of the canonical pathway to the level of β-catenin accumulation [Alterations in the l models –36. Muta cytosol , 38. Theproteins , 40. Fur pathway , 42. Expntal CRC , also contal CRC . Other nigenesis –45. Our with AOM . We assumulation .6 may be mediated through inactivation of COX-2 gene. The results showed a dose-dependent reduction in the level of COX-2 in IP6-treated rats with AOM-induced CRC. Although the mechanism underlying how IP6 modulates the expression of the COX-2 is still unknown, it is likely that the downregulation of COX-2 might be the cause of repressed tumor growth and subsequent tumor cell death. The current data indicate that oral administration of IP6 was associated with a decreased incidence of CRC through reductions in the number and size of adenomas and invasive carcinomas. These observations suggest the protective effects of IP6 against AOM-induced colon cancer in animal models. Interestingly, we demonstrated that the inhibition of CRC by IP6 occurs via a reduction in tumor incidence, which subsequently leads to tumor suppression and the depletion of COX-2 and β-catenin expressions, whereby all of these are implicated in AOM-induced colon carcinogenesis. These data further support previous studies suggesting that IP6 acts as an anticancer agent by reversing the proliferative effects of carcinogens [Our data demonstrate that the anticancer action of IPβ-catenin signaling pathway [6 may downmodulate Wnt signaling via the inhibition of β-catenin, thus reducing levels of COX-2. This effect was shown by a marked inhibition of COX-2 expression after treatment with IP6. Real-time PCR determination of the COX-2 mRNA level in IP6-treated rats with AOM-induced CRC indicates that COX-2 is significantly downregulated; this effect correlates with the observed protein levels as well. Therefore, COX-2 expression may be suppressed via inhibition of Wnt/β-catenin signaling. The pharmacological inhibition of COX-2 may therefore provide an effective chemoprotective method against CRC. To further support this finding and previous studies that correlate β-catenin with COX-2, we suggest that the underlying inhibitory mechanism of IP6 occurs via the suppression of β-catenin expression through COX-2 regulation or vice versa. This correlation at the mRNA and protein levels strongly supports the reduction of intestinal neoplasia by β-catenin and COX-2 inhibitions, suggesting that IP6 may play a therapeutic role in CRC and may contribute to new strategies in the prevention and treatment of this disease.Interestingly, several studies have shown that COX-2 may be regulated by the Wnt/ pathway –50. It s6 at the level of cell proliferation, by inhibiting β-catenin and COX-2 during AOM-induced CRC, have not been noted previously and suggest that the intake of rice bran IP6 may be used in future clinical chemopreventive trials to monitor the responsiveness of the CRC development to the chemopreventive agents. The observed modulatory influences of rice bran IP


The RNAi-mediated targeting of DKK1 gene expression in Ishikawa EC cells resulted in increased tumor cell invasion and migration. DKK1 was identified as an inhibitor of EC cell invasion and migration via its novel role in the Wnt signaling pathway. Targeting DKK1 may therefore represent an effective anti-invasion and -migration strategy for the treatment of EC.The Wnt signaling pathway plays an essential role in tumor invasion and migration. DKK1 functions as an important inhibitor of the pathway and represents a promising target for cancer therapy. The aim of the present study was to determine the role of DKK1 in endometrial carcinoma (EC) cell invasion and migration using RNA interference (RNAi) technology. Ishikawa EC cells were transfected at high efficiency with specific DKK1 siRNA. RT-PCR and western blot analysis were used to determine the mRNA and protein levels of DKK1, β-catenin and metalloproteinase 14 (MMP14) in siRNA-treated and -untreated cells. In addition, the invasion and migration of the EC cells were detected by invasion and migration assays. Transient transfection of DKK1 siRNA significantly inhibited the mRNA and protein levels of DKK1. Markedly increased cell invasion and migration was observed following treatment with DKK1 siRNA when compared with the negative control siRNA-treated and siRNA-untreated cells. The knockdown of DKK1 also elevated the mRNA and protein levels of β-catenin and MMP14 involved in the Wnt signaling pathway, indicating that targeting this gene may promote intracellular Wnt signal transduction and thus, accelerate EC cell invasion and migration  Endometrial carcinoma (EC) is a common malignant tumor of the female genital tract, which has notably increased in incidence over recent years . Tumor i2 at 37°C. Routine testing confirmed that the cells were free of mycoplasma and viral contaminants. The cells were subcultured every 2 days at a ratio of 1:2.Ishikawa EC cell lines were obtained from the American Type Culture Collection . The cells were maintained in DMEM/F12 medium  supplemented with 10% fetal bovine serum , 100 μg/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere containing 5% CODKK1 were used: i) (RNA)-AUA GCG UUG GAA UUG AGA ACC GAG U; ii) (RNA)-ACU CGG UUC UCA AUU CCA ACG CUA U; and iii) (RNA)-AAU CCU GAG GCA CAG UCU GAU GAC C. Stealth™ RNAi Negative Control Med GC was used as a negative control for the siRNA (siRNA sequences were obtained from Invitrogen Life Technologies).The following primer sequences for siRNAs targeting human 6 cells/well prior to transfection with DKK1 siRNA or negative control siRNA using Lipofectamine 2000 reagent, according to the manufacturer's instructions. Lipofectamine 2000 (5 μl) diluted in 250 μl Opti-MEM was prepared. In addition, 10 μl DKK1 siRNA (20 μM) and 10 μl negative control siRNA (20 μM) were diluted with 250 μl Opti-MEM and incubated for 20 min. The 500 μl complexes of Lipofectamine 2000 and siRNA plus 1,500 μl DMEM/F12 were introduced to 35-mm culture dishes and incubated in a humidified atmosphere containing 5% CO2 at 37°C. After 5–6 h, the medium was replaced with 10% serum-supplemented DMEM/F12 and the cells were incubated for 24–96 h for further use in various procedures .The EC cells were transfected with DKK1 siRNA or negative control siRNA or untransfected . The EC cells were then seeded in 35-mm culture dishes at 1×10BLOCK iT™ fluorescent oligos (Invitrogen Life Technologies) were transfected into the cells of the DKK1 RNAi and control groups to ensure the successful transfection of siRNA into the cells., since it was identified as the most effective for inhibiting DKK1 expression.The silencing efficiency was determined by RT-PCR and western blot analysis using DKK1-specific primers and antibodies. Subsequent experiments focused on the primer previously described as primer ii in the cell transfection methods for siRNAs targeting human DKK1® reagent (Invitrogen Life Technologies). The reverse transcription reaction was set up using RT reaction mix  and the resultant cDNA was used for PCR. The following primers for DKK1, β-catenin, MMP14 and GAPDH were used: i) DKK1 sense, 5′-CTGCATGCGTCACGCTATGT-3′ and antisense, 5′-TCCTCGGAAATGATTTTGATCA-3′; ii) β-catenin sense, 5′-CGGGATGTTCACAACCGAAT-3′ and antisense, 5′-TTGGATGTTTTCAATGGGAGAA-3′; iii) MMP14 sense, 5′-CAGGGTCTCAAATGGCAACA-3′ and antisense, 5′-TTGCGAATGGCCTCGTATG-3′; and iv) GAPDH sense, 5′-CAGTCAGCCGCATCTTCTTTT-3′ and antisense, 5′-GTGACCAGGCGCCCAATAC-3′. Experiments were performed in triplicate.mRNA levels of DKK1, β-catenin, MMP14 and GAPDH  were determined by RT-PCR. Following cell incubation, total RNA was extracted from the cells using TRIzolProtein levels of intracellular DKK1, active-β-catenin, MMP14 and β-actin  were detected by western blot analysis. When the cells reached 80–90% confluence, they were lysed in lysis buffer with protease inhibitors at 4°C. Cell lysates were also collected and protein concentrations were determined using the Bradford method. The lysates were cleared by centrifugation and quantified using the DC protein assay . The protein samples (50 μg) were boiled for 5 min prior to being loaded onto 10% SDS-PAGE. Following electrophoresis, proteins were transferred onto a nitrocellulose membrane . The membranes were blocked with 5% skimmed milk in PBS and probed with primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies  were used and the membranes were developed following an enhanced chemiluminescence detection protocol . The following primary antibodies and dilutions with blocking solution were used: DKK1 ; active-β-catenin ; MMP14  and β-actin . All experiments were performed in triplicate.5 cells/well with 300 μl serum-free DMEM/F12, whilst 500 μl serum-supplemented DMEM/F12 was filled into the lower wells. Plates were incubated at 37°C for 48 h, then non-invaded cells on top of the transwell were scraped off with a cotton swab. The cells in the chambers were maintained in an incubator at 37°C and allowed to migrate for 48 h. After 48 h, the non-migrated cells in the upper compartments were scraped carefully with cotton swabs. Migrated cells adhering to the lower surface of the membranes were fixed with methanol and stained with hematoxylin and eosin. The membranes were excised from the insert and mounted onto glass slides for light microscopic analysis, and migrated cells (penetrating Matrigel matrix and pores) were counted  from ten randomly selected fields. Each sample was assayed in triplicate.An invasion assay was performed using the Transwell chamber assay according to previous studies –11, withA migration assay was performed following the method for the invasion assay, with the exception of the exclusion of the Matrigel matrix and the use of polycarbonate membranes only.Data are presented as the mean ± SD. Statistical analyses were performed using SAS Version 9 . P<0.05 was considered to indicate a statistically significant difference. Quantitative analyses were performed using the Student's t-test for RT-PCR, western blot analysis and invasion and migration assay results among the three groups.The EC cells were transfected successfully with DKK1 and negative control siRNAs with high efficiency. DKK1 mRNA levels in total cell extracts were measured by semi-quantitative RT-PCR. Negligible changes in DKK1 mRNA levels were identified in the blank and control groups, however, in the DKK1 RNAi group, the DKK1 mRNA levels showed a decrease between 24, 48 and 72 h . In the DKK1 RNAi group, DKK1 gene expression was significantly inhibited between 24 and 48 h, however, there was no marked difference after 72 h. mRNA levels of GAPDH, as a loading control, were also analyzed, however, no changes were observed .The levels of DKK1 protein in DKK1 siRNA-treated cells were identified to significantly decrease between 48, 72 and 96 h  when compared with the untreated and control-treated cells. Inhibition was identified as statistically significant between 48 and 72 h, however, no significant difference was identified after 96 h. No change in the protein levels of β-actin, which served as a loading control, were identified. In addition, no significant differences were identified in protein levels in the blank and control groups .Each field was observed under a light microscope  and a significant difference was identified between the number of migrated cells in the DKK1 RNAi group (149.80) when compared with the blank group (122.50), and the knockdown of DKK1 was identified to result in accelerated EC cell invasion .Cell migration numbers for DKK1 siRNA-treated and -untreated cells were 173.90 and 135.80, respectively. The results indicated that the cells treated with DKK1 RNAi had significantly accelerated EC cell migration when compared with the blank group .The mRNA levels of β-catenin and MMP14 were increased in the DKK1 siRNA-treated cells when compared with the untreated and control-treated cells after 72 h. In addition, the mRNA levels of β-catenin and MMP14 were significantly elevated following the knockdown of DKK1, however, no significant differences were identified in the blank and control groups  and IV.Western blot analyses of cell lysates were performed to analyze whether protein expression correlated with mRNA expression following the knockdown of DKK1. The protein levels of β-catenin and MMP14 in the DKK1 RNAi group were significantly elevated after 96 h when compared with that of the additional two groups  and VI, EC is the most common malignant tumor in females and has a high incidence worldwide . The majPrevious studies have identified that abnormalities in the Wnt signaling transduction pathway contribute to tumorigenesis involved in cell migration, invasion and metastasis –8. A numβ-catenin functions as a significant component in the Wnt signaling pathway, and interactions with frizzled and LRP5/6 receptors result in the dephosphorylation of β-catenin (non-phosphorylated active β-catenin). The dephosphorylated form of β-catenin has been shown to have significant effects on the Wnt signaling pathway ,19. SubsRNAi is a sequence-specific, post-transcriptional gene-silencing method initiated by double-stranded RNA and homologous to the gene being suppressed. RNAi is now routinely used for the transient knockdown of gene expression in a wide range of organisms for the analysis of gene function . In the In the present study, the transfection of DKK1 siRNA downregulated the mRNA and protein levels of DKK1 in the DKK1 RNAi group. DKK1 siRNA was identified to significantly inhibit DKK1 mRNA levels after 48 h, and protein levels were downregulated after 72 h. However, no marked changes in the levels of mRNA and protein were identified in the blank and control groups. These results demonstrated that the mRNA and protein levels of DKK1 were successfully downregulated by transfection of DKK1 siRNA, however, no significant decrease in the levels of mRNA and protein were identified in the DKK1 siRNA-treated cells after 72 and 96 h, respectively. This is hypothesized to be due to the transient nature of the DKK1-knockdown by DKK1 siRNA.The knockdown of DKK1 has been identified to accelerate EC cell invasion and migration. In the present study, EC cells transfected with DKK1 siRNA showed increased tumor cell invasion and migration when compared with the untreated and control-treated cells, indicating an upregulation of β-catenin and MMP14. A significant increase in the mRNA and protein levels of β-catenin and MMP14 and cell invasion and migration were identified in the DKK1 RNAi group when compared with the additional two groups. The results of the current study are consistent to that of previous studies reporting the involvement of the knockdown of DKK1 in tumor cell invasion and migration –18. In aUpregulation of β-catenin and MMP14 by the knockdown of DKK1 interferes with the Wnt signaling pathway and its downstream signaling events. Intracellular signaling transduction pathways are often exploited during tumor invasion and migration and the involvement of β-catenin and MMP14 in the Wnt signaling transduction pathway has been identified to accelerate tumor invasion and migration. Therefore, the hypothesized effects of the DKK1 RNAi-mediated upregulation of β-catenin and MMP14 on EC cell migration and invasion were investigated in the present study. A significant increase in the levels of β-catenin mRNA and protein in the DKK1 RNAi group was identified. The knockdown of DKK1 increased the levels of β-catenin dephosphorylation into active-β-catenin, which resulted in elevated activation of the Wnt signaling pathway. Accumulated active-β-catenin in the cytoplasm was translocated into the nucleus where it interacted with lymphocyte enhancers and T-cell transcription factors to activate downstream target genes, including MMP14. Marked increases in the MMP14 mRNA and protein levels in the DKK1 RNAi group indicated significant MMP14 activation. The results indicated that the upregulation of β-catenin and MMP14 the knockdown of DKK1 may result in the elevated activation of the Wnt signaling pathway and downstream signaling events involved in tumor migration and invasion.In summary, the results of the present study indicate a novel biological function for DKK1 in the inhibition of EC cell invasion and migration. In addition, targeting DKK1 may represent an effective anti-invasion and -migration strategy for the treatment of EC, as DKK1 may contribute directly or indirectly to anti-tumorigenesis.


ABCD1 gene that encodes the peroxisomal ATP-binding cassette (ABC) transporter subfamily D member 1 protein (ABCD1), which is referred to as the adrenoleukodystrophy protein (ALDP). Induction of the ABCD2 gene, the closest homolog of ABCD1, has been mentioned as a possible therapeutic option for the defective ABCD1 protein in X-ALD. However, little is known about the transcriptional regulation of ABCD2 gene expression. Here, through in silico analysis, we found two putative TCF-4 binding elements between nucleotide positions −360 and −260 of the promoter region of the ABCD2 gene. The transcriptional activity of the ABCD2 promoter was strongly increased by ectopic expression of β-catenin and TCF-4. In addition, mutation of either or both TCF-4 binding elements by site-directed mutagenesis decreased promoter activity. This was further validated by the finding that β-catenin and the promoter of the ABCD2 gene were pulled down with a β-catenin antibody in a chromatin immunoprecipitation assay. Moreover, real-time PCR analysis revealed that β-catenin and TCF-4 increased mRNA levels of ABCD2 in both a hepatocellular carcinoma cell line and primary fibroblasts from an X-ALD patient. Interestingly, we found that the levels of very long chain fatty acids were decreased by ectopic expression of ABCD2-GFP as well as β-catenin and TCF-4. Taken together, our results demonstrate for the first time the direct regulation of ABCD2 by β-catenin and TCF-4.X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the  ABCD1 gene located in Xq28, which encodes the peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D member 1 (ABCD1), also known as adrenoleukodystrophy protein (ALDP) ABCD1 gene by a lentiviral vector has been shown to provide clinical benefit in X-ALD patients β-oxidation. Recently, it was demonstrated that human ABCD1 was able to transport VLCFA-CoA into the peroxisome in a yeast system β-oxidation of VLCFAs in peroxisomes X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is caused by mutations or large deletions of one or more exons in the ABCD2 encodes the ALDP-related protein (ALDRP or ABCD2) that share high homology with ABCD1 implying their functional redundancy or functional overlap β-oxidation and prevent accumulation of VLCFAs in cultured human fibroblast cells obtained from an X-ALD patient Abcd1 knockout mice model ABCD2 gene β-catenin is not only a key protein involved in cell-cell adhesion complexes with E-cadherin, but also an important component of the Wnt signaling pathway, which is involved in diverse cellular processes including cell growth, migration, differentiation, gene transcription, development of the nervous system, and self-renewal of stem cells β-catenin translocates into the nucleus et al., via promoter studies, demonstrated that sterol regulatory element-binding proteins (SREBPs), a family of transcription factors that control the metabolism of cholesterol and fatty acids, are involved in transcription of the ABCD2 gene ABCD2 gene are not known. In this study, we investigated putative transcription factors affecting the expression of ABCD2 gene through in silico analysis, and found two putative LEF-1/TCF binding elements between nucleotide positions −360 and −260 of the promoter of the ABCD2 gene. To our knowledge, this is the first report that β-catenin and TCF-4 directly regulate the expression of ABCD2; this direct regulation may provide a new drug discovery strategy for X-ALD.Weinhofer HepG2 cells (human hepatocellular carcinoma cell line) were cultured in RPMI1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen). Huh7 cells (human hepatoma cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% antibiotics. Human X-ALD fibroblasts from a single patient with X-ALD  were cultured in Eagle’s minimumessential medium  supplemented with 15% FBS and 1% antibiotics. Human dermal fibroblast (HDF) cells were purchased from Invitrogen (#C-004-5C) and were cultured as recommended by the supplier.β-catenin , Myc-tagged wild-type TCF-4, and dominant negative TCF-4  were obtained from Addgene  ABCD2 promoter fragments  were amplified by PCR using i-MAX™ II DNA polymerase  from HDF genomic DNA as a template, and subcloned into the pGL3-basic (Promega) vector. The primer sequences containing restriction sites are listed in ABCD2 promoter sequence (starting from −1300 bp) was aligned with the sequence of human chromosome 12 . Mutations in the LEF-1/TCF binding sites were introduced using the 800-luc reporter plasmid by overlap extension PCR as described previously Expression vectors for Flag-tagged constitutively active CCTGCTCCTCTCCTCAGATGTAATTCG -3 andmTBE1-5′primer, 5- GGGAGCACGAATTACATCTGAGGAGAG -3;mTBE1-3′ primer, 5- AGAAGTTGGAAGCTGCGAGGTTCAAGG -3 andmTBE2-5′ primer, 5- CTAACTTCCTTGAACCTCGCAGCTTCC -3. Putative LEF-1/TCF binding sites are shown in bold, and mutated sequences are underlined. After two rounds of PCR, each full-length construct with the desired mutation (800 bp DNA fragment) was amplified with primers for 800-luc . The total amount of transfected DNA was kept constant by adding an empty vector. Fibroblasts isolated from an X-ALD patient were electroporated with 0.5 µg reporter plasmids and 0.2 µg of pRL-SV40 plasmid using a microporator transfection system . Cells were pulsed twice with a voltage of 1,100 (V) for 30 ms. After the two pulses, cells were seeded in a 12-well culture plate. At 36 h after transfection (or electroporation), cells were harvested and luciferase activities were determined by measuring luminescence activity. Data were normalized by Renilla luciferase activity. For overexpression of β-catenin and TCF-4, cells were transfected (or electroporated) with each plasmid using Fugene6 (for HepG2 and Huh7) or a microporator (for X-ALD fibroblasts). Forty-eight hours after transfection, cells were harvested for luciferase activity measurement, western blot analysis, and immunofluorescence staining.For reporter assays, HepG2 cells were seeded in a 24-well culture plate and transiently transfected with 0.3 µg reporter plasmids and 50 ng of pRL-SV40 plasmid (Promega) containing the β-catenin and ABCD2 siRNA were purchased from Genolution Pharmaceuticals (Korea). HepG2 or X-ALD fibroblasts were transfected with scrambled siRNA (5-ACGUGACACGUUCGGAGAA-3), β-catenin siRNA (5-CUGGGACCUUGCAUAACCU-3) or ABCD2 siRNA (5-GAUUAUGUCUUCAUACAAA-3 and 5-GCAUGAUAAAGGUUAUACA-3) using Lipofectamine™ RNAiMAX (Invitrogen). Three to four days after transfection, cells were harvested to measure mRNA or protein levels, luciferase activities, and VLCFA levels.Scrambled, β-catenin antibodies overnight at 4°C, then for another 2 h at 4°C with protein G-agarose/salmon sperm beads. Following washes, the protein/DNA cross-links were reversed by heating at 65°C overnight, and the DNA fragments eluted. Eluted DNA fragments were used for PCR analysis using the following human ABCD2 promoter-specific primers:ChIP assay was performed as described previously 5-GTTTTGTTCGCCAGCAGATGGCCTGAT-3;forward, 5-CCGCTGCATCTACCGGGAATGATTCTC-3. Amplified DNA fragments were visualized by agarose gel electrophoresis.reverse, β-catenin, ABCD2, and GAPDH was determined by real-time PCR using SYBR® Premix Ex Taq (Takara) on a CFX Connect™ real-time PCR system (Bio-Rad) according to the manufacturer’s instructions. Real-time PCR was performed using the following specific primer sequences: β-catenin-f, 5-ATGTCCAGCGTTTGGCTGAA-3 and.Total RNAs were isolated from cells using TRIzol reagent (Invitrogen). The cDNAs were synthesized usingthe iScript™ cDNA synthesis kit (Bio-Rad) from 1 µg of total RNA. Expression of β-catenin -r, 5-TGGTCCTCGTCATTTAGCAGTT-3;ABCD1-f, 5-GGAGCTGGTGGCAGAGGA-3 andABCD1-r, 5-ACAGCCACCATGAGCAGG-3;ABCD2-f, 5-GATAACTGGTCCCAATGGTTG-3 andABCD2-r, 5-TCCCGAAGACTTCCAAGAGA-3;GAPDH -f, 5-CCCCTCAAGGGCATCCTGGGCTA-3 andGAPDH -r, 5-GAGGTCCACCACCCTGTTGCTGTA-3.Total cells lysates were prepared using cell lysis buffer containing protease inhibitor cocktail (Roche) and sodium orthovanadate (Sigma) for 20 min on ice as described previously X-ALD fibroblasts were electroporated with ABCD2-GFP expression plasmid, and cells were seeded on cover glass. Two days after electroporation, cells were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde and 0.1% Triton X-100 for 20 min at RT. Cells were further incubated in 5% bovine serum albumin (BSA) for 20 min at RT, and then incubated with anti-PMP70 polyclonal antibody (Millipore) for 2 h at 37°C. After three washes in PBS, cells were incubated with Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Invitrogen) for 1 h at 37°C. After two washes with 0.1% Triton X-100 in PBS and three washes in PBS, cells were analyzed using a confocal microscopy system (Olympus).5 cells) was dissolved in PBS. VLCFA analysis was performed by Seoul Clinical Laboratories (Korea) as described previously X-ALD fibroblasts were harvested by trypsinization, and the cell pellet  of the ABCD2 promoter subcloned in this study was identical to that of reported sequence on human chromosome 12  (data not shown). By in silico analysis with Genomatix software (MatInspector™), we identified the two putative LEF-1/TCF binding elements (TBEs) located at nucleotide positions −324 to −318 (TBE1) and −299 to −293 (TBE2) within the upstream sequence of the ABCD2 promoter  showed full transcriptional activity in HepG2 cells , wild-type TCF-4, dominant negative (DN)-TCF-4 expression plasmids. As shown in β-catenin mutant protein resulted in only 2.6-fold activation of the ABCD2 promoter (lane 2) due to the presence of an endogenous active mutant form of β-catenin in HepG2 cells ABCD2 gene was 15-fold greater in cells expressing wild type TCF-4 protein than cells expressing empty vector (lane 3). In contrast, promoter activity was slightly decreased in cells that expressed DN-TCF-4 protein (lane 5). These results strongly suggest that β-catenin and TCF-4 interact to activate transcription of the ABCD2 gene. Consistent with these results, we found that cotransfection of β-catenin and wild-type TCF-4 expression plasmids resulted in a 21.1-fold induction in promoter activity (lane 4), whereas cotransfection of β-catenin and DN-TCF-4 plasmids resulted in only a 2.5-fold induction in promoter activity (lane 6).To further investigate transcriptional regulation of promoter , lane 2 y vector , lane 3. protein , lane 5.activity , lane 4,activity , lane 6.β-catenin in ABCD2 promoter transactivation in HepG2 cells, endogenous β-catenin expression was knocked-down using specific siRNAs. Real-time PCR analysis revealed that the expressions of both wild-type (WT) and active mutant forms (Mut) of β-catenin in HepG2 cells were efficiently reduced by siRNA transfection  assay. We used either normal rabbit IgG or β-catenin antibodies for ChIP, and amplified the DNA using specific primers for the ABCD2 promoter containing the two TBEs. As shown in ABCD2 promoter was immunoprecipitated by the β-catenin antibody, but was not immunoprecipitated by the normal IgG antibody. These data indicate that a transcription factor complex containing β-catenin binds directly to the ABCD2 promoter.To determine if the ABCD2 gene was increased by ectopic expression of active mutant β-catenin (4A) and wild-type TCF-4 in HepG2 cells. mRNA expression of ABCD2 was compared between control vector-transfected or β-catenin and TCF-4-transfected cells after total RNA extraction. As shown in ABCD2 expression was elevated by ectopic expression of β-catenin and TCF-4. In contrast, ABCD1 expression was unaffected by these proteins  by the ABCD2 protein (ALDRP) has been shown to reduce VLCFA accumulation in fibroblast cells isolated from X-ALD patients Since C26∶0/C22∶0 ratio is being used widely for diagnosing X-ALD β-catenin and TCF-4-induced ABCD2 induction in X-ALD fibroblasts. X-ALD fibroblasts were electroporated with either GFP or active β-catenin and TCF-4 expression vectors. We showed that mRNA level of the ABCD2 gene was increased by overexpression of β-catenin and TCF-4 using real-time PCR analysis  in the ABCD2 promoter has been reported ABCD2 gene expression.Induction of ABCD2 in addition to the GC and CCAAT boxes reported in earlier studies ABCD2 gene transcription, we performed luciferase reporter assays using various lengths of the ABCD2 promoter. Because the promoter of the ABCD2 gene has putative TBEs, we chose HepG2 cells that contain active β-catenin protein to investigate transcriptional activation. In both HepG2 cells and fibroblasts from an X-ALD patient, the −260 promoter fragment showed minimal activity, while the activity of the −360 fragment differed according to cellular context , we found two putative LEF-1/TCF binding elements (TBEs)  in the p context . We furtis study . Interesis study , indicatABCD2 was elevated by ectopic expression of β-catenin and TCF-4 (lane 4). We confirmed this finding in Huh7 cells that do not express active β-catenin ABCD2 promoter are functionally involved in ABCD2 transcriptional regulation -liganded thyroid hormone receptor α (TRα)/SREBP-1 cis-retinoic acid ABCD2 gene. However, we found that β-catenin and TCF-4 not only increased transcription of the ABCD2 gene, but also decreased levels of VLCFAs according to the level of ABCD2 induction. We therefore speculate that induction of ABCD2 by β-catenin and TCF-4 may decrease levels of VLCFAs.Transcription factors such as SREBPs ABCD2 gene expression by β-catenin and TCF-4, which are members of the canonical Wnt signaling pathway. These findings may provide a new strategy for developing drugs to treat X-ALD.In this study, we demonstrated direct regulation of Figure S1Mutation of TBE sites within the ABCD2 promoter decreased β-catenin/TCF-4 mediated promoter activity in Huh7 cells. Huh7 cells were cotransfected with the indicated expression plasmids along with either wild-type (WT) 800-Luc reporter plasmid containing wild-type (WT) TBE sequences or 800-Luc reporter plasmids containing the mutation of one or both TBE sites of the ABCD2 promoter . Mutated sequences are underlined (lower panel). At 36 h after transfection, cells were harvested, and luciferase activities were determined. The amount of DNA in each transfection was kept constant by adding an appropriate amount of pcDNA3 empty vector. Data represent the mean (± SEM) of triplicate experiments. *p<0.001.(TIF)Click here for additional data file.Figure S2Contribution of the two TBE sites within the ABCD2 promoter to β-catenin/TCF-4-mediated transcriptional activation in fibroblast cells from an X-ALD patient. Primary fibroblasts isolated from an X-ALD patient were electroporated with the indicated expression plasmids along with either wild-type (WT) 800-Luc reporter plasmid containing the wild-type (WT) sequences or the double mutant (D-Mut) 800-Luc reporter plasmid containing mutations in the two TBE sites of the ABCD2 promoter. The mutated sequences are underlined (lower panel). At 36 h after electroporation, cells were harvested, and luciferase activities were determined. The amount of DNA in each transfection was kept constant by adding an appropriate amount of pcDNA3 empty vector. Data represent the mean (± SEM) of triplicate experiments. *p<0.001.(TIF)Click here for additional data file.Figure S3β-catenin and TCF-4 increase the transcriptional activity of ABCD2 in Huh7 cells. Huh7 cells were cotransfected with ABCD2 promoter reporter (800-Luc) plasmids together with the indicated expression plasmids. At 36 h after transfection, cells were harvested, and luciferase activities were determined. The amount of DNA in each transfection was kept constant by adding an appropriate amount of pcDNA3 empty vector. Data represent the mean (± SEM) of triplicate experiments.(TIF)Click here for additional data file.


In 1993, the European Society for Traumatic Stress (ESTSS) was founded by a group of men from all over Europe. At the time, 20 years ago, Europe was shaken by a war in southeast Europe. The Berlin Wall had fallen, the eastern countries of the continent were recovering from the consequences of problematic regimes, and the European Union was smaller than it is today. The continent of Europe was in some areas very rich and well developed; in other regions, it was poor and suffering from ongoing conflicts. This thematic cluster of papers has contributions from almost all past presidents of ESTSS, since the start of ESTSS in 1993 up to its 20th anniversary in 2013. Erica van der Schrieck-de Loos has written a memorial contribution given the sad loss of her father Walter de Loos. Bas Schreuder was unable to contribute but sent his congratulations and has explained his connectedness with ESTSS.Erica van der Schrieck-de Loos cites an opening speech of her father, Wolter de Loos (the first president of ESTSS), in which he reflected about the victims, the victor, and the perpetrator , our journal.Most of the past presidents met in Bergen in 1993, the founding place of ESTSS. This was the case for Dean Ajduković and Ulrich Schnyder. Dean Ajduković, from the University of Zagreb, came from a country involved in a horrible civil war. In his contribution, he reflects on the introduction of the notion of social context of collective trauma, an immediate influence of the war situation in former Yugoslavia (Ajduković, Finally, I describe some professional steps in my own career, including a variety of ones related to traumatic stress. I use my own experience to reflect on the further development of ESTSS as a multilingual and culturally sensitive society for all matters concerning psychotraumatology. I consider that the integrative capacity of ESTSS, with the diverse European background in history, culture, scientific approaches, and languages will enrich our future activities and bring its services closer to individuals in need. This richness will be the contribution of ESTSS to the Global project that ISTSS has launched.ESTSS has developed into a large society for professionals. Trauma, PTSD, and psychotraumatology have moved closer to the center of mental health. Leaders of the global field have a European background. Psychotraumatology has become a field of interest that attracts many people. ESTSS and its past presidents have worked hard to achieve the current status. I am deeply grateful for their pioneering work.As the current president of ESTSS, it was my wish to celebrate the 20th birthday of ESTSS. Twenty years is an age when the first biographical notes can be put down. I, therefore, invited our past presidents to contribute to an ESTSS birthday edition of EJPT. Their answers came immediately, positive and enthusiastic.The papers of all past presidents stress one recurrent theme: ESTSS is a professional home, built on knowledge, the sharing of experiences, a very active engagement in the field of psychotraumatology and friendship. Dealing with trauma is sometimes hard, frightening, and stressful. Having a professional home that includes high quality friendship is fantastic, especially when one is overwhelmed or in need of advice. I hope that ESTSS will retain its special qualities, for at least the next 20 years.Brigitte Lueger-SchusterESTSS president, 2011–2013Guest Editor


ATP8B1 gene were identified as a cause of low γ-glutamyltranspeptidase cholestasis with variable phenotype, ranging from Progressive Familial Intrahepatic Cholestasis to Benign Recurrent Intrahepatic Cholestasis. However, only the coding region of ATP8B1 has been described. The aim of this research was to explore the regulatory regions, promoter and 5′untranslated region, of the ATP8B1 gene.Mutations in ATP8B1 transcripts were determined by quantitative reverse-transcription PCR and compared with the non-variable part of ATP8B1. Three putative promoters were examined in vitro using a reporter gene assay and the main promoter was stimulated with chenodeoxycholic acid. Four novel untranslated exons located up to 71 kb upstream of the previously published exon 1 and twelve different splicing variants were found both in the liver and the intestine. Multiple transcription start sites were identified within exon −3 and the proximal promoter upstream of this transcription start site cluster was proven to be an essential regulatory element responsible for 70% of total ATP8B1 transcriptional activity. In vitro analysis demonstrated that the main promoter drives constitutive ATP8B1 gene expression independent of bile acids.5′Rapid Amplification of cDNA Ends using human liver and intestinal tissue was performed to identify the presence of 5′ untranslated exons. Expression levels of ATP8B1 gene is complex and the previously published transcription start site is not significant. The basal expression of ATP8B1 is driven by a housekeeping-like promoter located 71 kb upstream of the first protein coding exon.The structure of the  ATP8B1 (18q21-q22) cause variable cholestatic phenotypes ranging from progressive to benign recurrent forms  Mutations in In vivo experiments using “Byler“ Atp8b1G308V/G308V mice or ATP8B1 deficient hepatocytes demonstrated defective membrane order due to the impaired flippase activity of ATP8B1 The ATP8B1/FIC1  protein, a member of the P4-type ATPases subfamily, is widely expressed in epithelial tissues ATP8B1 mutations were detected in fewer than 50% of the families screened, irrespective of severity of their disease. It may be partly because only the coding region of the gene was analyzed. Even though the ATP8B1 gene is important biologically and clinically, our knowledge of its regulatory regions remains limited. Our aim was to characterise the transcriptional control of the ATP8B1 gene by identifying its promoter and 5′untranslated (5′UTR) regions, and to search for putative regulatory sites in any newly discovered parts of the gene.In a large study by Klomp et al. ATP8B1 mRNA isoforms . These new exons have been designated exons −1 to −4 (Ex −1 to Ex −4) and their lengths and positions are summarized in isoforms .Inr) Using commercially available First Choice® RACE-Ready cDNA from human liver and intestinal tissue, several putative transcription start sites were identified: One each at the beginning of the novel UTR exons depicted in ATP8B1 transcripts we used 7 normal human livers and 3 normal human intestinal samples in qRT-PCR experiments with individually-designed probe/primer sets covering all identified alternative exon-exon boundaries found by 5′RACE (Tab. S1). The results are presented in ATP8B1 expression, whilst the alternative splicing event, Ex −3/Ex −1 that in fact comprises two mRNA isoforms: Ex−3/Ex −1/Ex +1 and Ex−3/Ex −1/CAG/Ex +1, comprises less than 10% of the total transcripts. Splicing variants Ex −1/Ex +1 and Ex −1/CAG/Ex +1 account for almost 26% but splicing events including Ex −2 account for less than 4% of the total transcripts. The expression level of Ex −4/Ex +1  revealed one ATP8B1 transcript with Ex −3 spliced to Ex +1 without the CAG triplet . This transcript does not include the protein coding Ex +2, and thus Ex +1 must be spliced directly to Ex +3. The resultant predicted protein sequence would have a premature stop codon (TAA) at position 62, which would not give a viable ATP8B1 protein. We searched for the existence of Ex +1/Ex +3 splicing event by PCR using cDNA templates prepared from liver and intestinal RNA. qRT-PCR using the specifically-designed probe for the Ex +1/Ex +3 boundary demonstrated 20-fold lower expression of this transcript in normal human liver and intestinal tissue compared to the Ex +1/Ex +2 splicing variant (data not shown). The biological significance of the low abundance transcript therefore remains unclear.Searching of the NCBI EST database , showed only basal transcription activity which was less than 10% that of the Prom 1 construct upstream of Ex −3  and aligned with nucleotide sequences of newly identified human UTR exons using the ClustalW  program. A high degree of conservation was found in the region corresponding to Ex −3 and Ex −4: 83% and 82%, respectively, for a human-mouse alignment and 66% and 69%, respectively, for a human-rat alignment  were identified in that DNA sequence. No liver- or intestine-specific transcription factor binding sites were found in the vicinity of the TSS cluster of Ex −3 , proving HepG2 as a suitable model to assess the response of the ATP8B1 promoter constructs to bile acids.To investigate the effect of bile acids on P3 promoter activity, HepG2 cells were transfected with the Prom 3, Prom 4, and Prom 6 ATP8B1 under CDCA stimulation, none of the luciferase constructs tested demonstrated a significant change in promoter activity in HepG2 cells after CDCA  ATP8B1 is driven by three newly identified promoters  versus the promoter upstream of Ex −3 (P3). However, this difference was not replicated at the level of abundance of the mRNA transcripts associated with these promoters: Whereas the reporter gene activity mediated by promoter P1 was 15-fold lower than that of the principle promoter P3   which can affect gene expression by inhibition of mRNA stability and translational repression G (uAUG) . WhereasATP8B1 deficiency is associated with cholestatic liver disease, most studies have focused on the role of the gene in cholestasis. Zollner and colleagues ATP8B1 mRNA level in cholestatic patients. In our experiments, stimulation of HepG2 cells with CDCA or TCA respectively showed no change in ATP8B1 mRNA levels and CDCA or TCA treatment of HepG2 or rNTCP-HepG2 cells expressing various ATP8B1 promoter constructs did not significantly alter the luciferase activity compared with untreated cells. This observation is in agreement with the gene’s ubiquitous expression and suggests that bile acid independent mechanisms regulate ATP8B1 expression across different cell types.Since ATP8B1 gene regulation. Newly identified ATP8B1 mRNA isoforms differ in their 5′UTRs and both transcriptional and post-transcriptional efficiency. The basal expression of ATP8B1 gene in the liver and the intestine is driven by a promoter with house-keeping like properties. Regulatory parts of ATP8B1 characterised in this study represent a feasible region for mutational search in patients with features suggestive of ATP8B1 deficiency, in whom no mutations have been identified within the coding region.In conclusion our study provides fundamental data about the complexity of ATP8B1 gene were mapped using the 5′/3′ RACE kit, 2nd Generation  according to manufacturer’s instructions. Total RNA was isolated using RNA-Bee  from 50 mg of human liver or intestinal tissue, or 5×106 of HepG2 cells. All procedures were conducted with written informed consent under an institutional-review-board approved protocol or using anonymised bank samples, previously collected with consent for research. To confirm any newly identified transcriptional origins, liver and intestinal RACE-ready cDNA  was used. ATP8B1 gene-specific primers for RACE were designed to span the junctions of exons 3/4, 2/3 and 1/2. Resultant PCR products were cloned into the pDrive Cloning Vector  and sequenced in both directions using ABI Big Dye (Version 3.1) on a 3100 automated DNA Sequencer  using vector specific primers.The 5′ends of the ATP8B1 transcripts on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Expression levels of the studied transcripts and the overall expression of the ATP8B1 coding sequence represented by Ex +1/Ex +2 boundary, were corrected to the level of 18S rRNA (delta Ct) . A PCR of non-reverse transcribed RNA was performed as a negative control to check for any genomic DNA contamination. Delta delta Ct values were calculated using ABI SDS software with RQ study application  and the data analysed using Microsoft Excel.Twelve sets of individually designed TaqMan® MGB probes labelled with Fam and non-fluorescent quencher and primers were generated using Primer Express® Software Version 2.0 , to cover all variants of alternative splicing of the untranslated exons , No.1-12http://genome.ucsc.edu/, http://www.genomatix.de, http://bimas.dcrt.nih.gov/molbio/proscan/) were used to analyse the 5′UTR of the ATP8B1 gene. The University of California Santa Cruz Genome Bioinformatics server was also used to predict the gene’s first exon. The data obtained were compared with the EST database and the 5′RACE experimental results.Three independent algorithms for promoter prediction  and specific primers containing XhoI restriction sites. PCR products were cloned (Invitrogen Zero blunt kit or Qiagen Cloning kit), sequenced, digested with XhoI, and ligated into XhoI-digested luciferase reporter gene vector pGL3-Basic  using Quick Ligation Kit . Resultant constructs were checked for the correct sequence with various restriction enzymes and by direct sequencing prior to transfection. Stocks were prepared using an Endotoxin-free Maxiprep kit . Human retionid X receptor α (hRXRα) and farnesoid X receptor (hFXR) cDNAs were PCR amplified, cloned into Invitrogen’s TOPO TA- cloning kit, sequenced and then sub-cloned to the mammalian expression vector pCI (Promega).Twelve fragments of the 5′UTR . For some stimulation experiments, 50 ng of pCI_hFXR and 50 ng of pCI_hRXR contructs were co-transfected with the luciferase constructs. Twenty-four hours after transfection, cells were treated with 0 to 100 µM dimethyl sulfoxide, chenodeoxycholic acid, CDCA or taurocholic acid, TCA (Sigma Aldrich), respectively.HepG2 cell lines were purchased from the ATCC . Cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 5% or 10% fetal calf serum (PAA) and glutamine. rNTCP-HepG2 cells, kindly provided by Ulrich Beuers  and Christopher Rust , were maintained in Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum and 1 mg/ml G418 (Invitrogen). For transfection, cells were seeded in 24-well plates (TPP) in medium containing 5% fetal calf serum or 5% charcoal-stripped bovine calf serum (GibcoBRL). For transient transfection, 1.5 µl of FuGene HD (Roche) and 500 ng of plasmid DNA were used per well. Plasmid DNA comprised 450 ng of the appropriate LucFirefly and Renilla luciferase activities in cell lysates were determined using the Dual luciferase system (Promega) on a Glomax luminometer (Promega). All reporter gene assays were performed in triplicate and results are presented as the average value from at least three independent experiments corrected for the transfection efficiency using Renilla luciferase activities. The data from individual experiments were related to the activity of the control expression plasmid pGL3 Basic (Promega).Cells were harvested 48 hours after transfection (24 hours after stimulation with bile acids) and then Figure S1In silico analysis of first exon and promoter region for ATB8B1 gene compared to the 5′RACE results. Four putative first exons were predicted by “First EF” computer prediction software  (TIF)Click here for additional data file.Figure S2Putative secondary structures of ATP8B1 5′UTR isoforms depicted in  of the main text. Drawings of the minimum free energy (MFE) structures for each splicing variant of ATP8B1 5′UTR suggest their possible different role in regulation of gene expression. One of the regulatory mechanism, formation of stable secondary structures, was shown to impede the progress of the scanning ribosome (TIF)Click here for additional data file.Table S1Individually designed TaqMan® MGB probes labelled with Fam and non-fluorescent quencher and primers were generated using Primer Express® Software Version 2.0 (Applied Biosytems), to cover all variants of alternative splicing of the untranslated exons. The abundance of each splicing variant was compared relatively to a non-variable coding region of ATP8B1 represented by Ex+1/+2 boundary. Probe/primer set for Ex+1/+3 boundary (No.14) was used to test the biological significance of transcript excluding protein coding Ex +2, found in EST database (GenBank accession: DR005588.1). All probe sets were designed across exon/exon boundaries to eliminate the possibility of genomic contamination. Primers used in various amplifications are indicated by upper index . The amplification efficiency was tested for each probe/primer set on control templates (obtained by cloning the appropriate cDNA region) using different concentrations of positive and negative controls. As each probe set worked with a slightly different efficiency, the concentration of probes was adjusted for each positive control to reach a cycle threshold (Ct) value difference not greater than 1.(DOC)Click here for additional data file.Text S1Supporting references.(DOC)Click here for additional data file.