Bleb survival and characteristics, and intraocular pressure, were evaluated over a 60-day period. Histology of the surgical eyes was performed to evaluate ocular scarring and fibrosis in each group.The IKKβ targeting siRNAs were chemically synthesized and screened in cultured monkey Tenon’s fibroblasts in vitro. Fourteen monkeys underwent trabeculectomy and were randomly allocated to one of three treatment regimens: subconjunctival injection of either CS-g-(PEI-b-mPEG)/IKKβ-siRNA was well tolerated in this model. Both siRNA and MMC significantly prolonged bleb survival compared with the PBS group . Higher blebs were observed in the siRNA group than in the PBS group (p<0.01), while the MMC group showed the highest blebs among three groups (p<0.01). The surgical eyes in both the siRNA and MMC groups had significantly larger bleb area compared with the PBS group (p<0.01), but there was no significant difference between the siRNA and MMC groups (p=0.214). There were no significant differences in IOP readings among the three groups on the designated days after surgery . The histologic examination demonstrated that the eyes treated with siRNA showed a marked reduction in subconjunctival scar tissue compared with the eyes in the PBS group. The conjunctival epithelium appeared healthy without the acellularity that was present in the MMC group.Subconjunctival injection of CS-Subconjunctival injection of cationic nano-copolymers mediated IKKβ targeting siRNA is associated with improved surgical outcome in a monkey model of trabeculectomy. This novel approach may potentially be a more controlled alternative as an anti-scarring agent in glaucoma filtration surgery. The most common reason for glaucoma filtration surgery failures is the excessive subconjunctival scarring at the bleb and sclerostomy sites . The intNuclear factor kappa B (NF-κB) is an evolutionarily conserved family of DNA binding proteins that play a critical role in the regulation of genes involved in a variety of cellular processes . They exg-(PEI-b-mPEG) as the vehicle. The results showed that the expression of IKKβ was downregulated, and the activation of NF-κB in the HTFs was subsequently inhibited. The proliferation of HTFs was effectively suppressed through the blocking of the NF-κB pathway. Furthermore, the non-viral siRNA carrier has indicated good biocompatibility, biodegradability, and transfection efficiency in vitro.RNA interference (RNAi) was originally recognized as an evolutionary conserved defense mechanism in higher eukaryotic cells, and this system can easily and effectively inhibit the expression of one specific gene . Recentlg-(PEI-b-mPEG) mediated IKKβ targeting siRNA on the ocular wound healing process in a non-human primate model of filtration surgery, as well as to assess the safety and tolerance of this CS-g-(PEI-b-mPEG)/IKKβ-siRNA complex in vivo.The purpose of this study was to investigate the effects of CS-IKKβ (IKKβ-siRNA) were chemically synthesized by Ribobio Co. Ltd. . siRNAs were derived from the coding sequence of the Rhesus Monkey (Macaca mulatta) IKKβ gene (GenBank XM_001096913), and were designed using an siRNA Target Finder program. A BLAST search checked all of the duplex sequences and target sequences of these siRNAs to preclude sequences with significant similarity to other genes in the Macaca mulatta genome. The duplex sequences of IKKβ-siRNA1, IKKβ-siRNA2, and IKKβ-siRNA3 were: 5′-GAA CAG AGG UUA AUA CAC AdT dT-3′ and 3′-dTd TCU UGU CUC CAA UUA UGU GU-5′, 5′-GAG CUG UAC AGG AGA CUA AdT dT-3′ and 3′-dTd TCU CGA CAU GUC CUC UGA UU-5′, and 5′-CCA AGA AGA GUG AAG AAC UdT dT-3′ and 3′-dTd TGG UUC UUC UCA CUU CUU GA −5′, respectively. The non-viral siRNA carrier, CS-g-(PEI-b-mPEG), and CS-g-(PEI-b-mPEG)/IKKβ-siRNA complex, were synthesized as previously described [Three pairs of siRNA, which specifically targeted escribed .IKKβ mRNA (data not shown). Therefore, we used IKKβ-siRNA2 as IKKβ-siRNA in the subsequent RNAi procedures.In our primary experiments, we found that IKKβ-siRNA2 (5′-GAG CUG UAC AGG AGA CUA AdT dT-3′ and 3′-dTd TCU CGA CAU GUC CUC UGA UU-5′) was the most effective among the three siRNAs in downregulating the transcription of 2, 95% humidified atmosphere in DMEM supplemented with 10% FBS, 2 mM of L-glutamine, 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 25 μg/ml of amphotericin B. Cells between passages 3 and 6 were used for the following experiments.Tissue explants of the rhesus monkey Tenon’s capsule were obtained from two monkeys . Tenon’s fibroblasts were cultured by a previously reported method , as desc5 cells per well, and incubated for either 12 h or 24 h (reaching 60%–70% confluence). Subsequently, the culture media were replaced with serum- and antibiotic-free DMEM 2 h before transfection. CS-g-(PEI-b-mPEG)/IKKβ-siRNA complexes were prepared, at an N/P ratio of 10, 30 min before transfection (based on our previous results), and cells were incubated with serum- and antibiotic-free DMEM that contained complexes corresponding to the determined final concentrations of IKKβ-siRNA  for 6 h. Then, the cells were maintained in the serum-supplied DMEM for either another 24 h or 48 h before the following assays were performed. Real-time RT–PCR, western blot, and cell proliferation assay were performed using the previously reported method [Tenon’s fibroblasts were plated in six well plates with a density of 6x10A total of 14 rhesus monkeys that weighed between 2 to 3 kg, and ranged in age from 3 to 4 years, were used in this study. A standard ophthalmologic evaluation was performed to exclude eye diseases. Experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.Surgery was performed under general anesthesia with intramuscular injection of ketamine and local anesthesia with 0.5% proparacaine hydrochloride drops. Conventional trabeculectomy was performed in the superior quadrant. Briefly, after a lid speculum was positioned, a superior rectus bridle suture was used to rotate the eye inferiorly. A half-thickness, rectangular, 3x4-mm scleral flap was prepared after a limbus-based conjunctival flap was fashioned. A 1x2-mm sclerostomy was followed by peripheral iridectomy. The scleral flap was closed with two 10–0 nylon sutures at each corner. The conjunctiva was closed with a continuous locking suture. Antibiotics and steroids were applied topically after surgery. The surgery was executed by the same masked individual.g-(PEI-b-mPEG)/IKKβ-siRNA (six eyes) or phosphate buffered saline , or treated with MMC (four eyes). In the IKKβ-siRNA group, 0.1ml of CS-g-(PEI-b-mPEG)/IKKβ-siRNA  was injected subconjunctivally into the bleb area by a 30-gauge needle, after the conjunctiva was closed at the time of surgery. The needle was placed 5 mm behind the limbus, at the nasal margin of the superior rectus muscle. The subconjunctival injection was repeated on day 7 after surgery, under general and topical anesthesia. A similar volume of PBS was administered in the same manner as the negative control. The MMC group was used as the positive control. Eyes were treated at the time of surgery with a solution of 0.2 mg/ml MMC, using a section of soaked microsponge, which was placed between the conjunctiva and sclera over the determined filtration site for 5 min. The area was then thoroughly irrigated with physiologic saline.The animals were randomly allocated to one of three treatment regimens: subconjunctival injection of either CS-The slit-lamp observations were performed twice per week for the first four weeks, and once per week thereafter, until the animals were executed on day 60 to assess the conjunctival hyperemia, corneal edema, either intraocular inflammation or hemorrhage, and anterior chamber depth. The eyes were also stained with fluorescein to look for evidence of either corneal epithelial toxicity or bleb leak. Corneal endothelial cell density was examined preoperatively and 1 month after surgery. Bleb characteristics, including length and width, were measured with calipers, and height was graded semiquantitatively by slit-lamp examinations . Bleb failure was defined as the appearance of a flat, vascularized, scarred bleb in association with a deep anterior chamber. Intraocular pressure (IOP) was measured (average of three consecutive readings) at the same time intervals using an applanation tonometer . All experimental procedures and analyses were conducted by the same masked investigator.Two monkeys in each group were killed on day 60. The eyes were enucleated and prepared for histological examination by conventional optical microscopy. Serial sections were cut through the sclerostomy site, and stained with standard hematoxylin-eosin.t-test. Survival analysis was performed for bleb failure using the Kaplan–Meier log rank test. The area and height of the bleb were analyzed with a generalized linear model  repeated-measures procedure to compare the two groups. IOP measurements were compared among groups using a one-way ANOVA. P-values of less than 0.05 were considered significant.The statistical analyses were conducted using SPSS version 13.0 for Windows . The in vitro results were analyzed by Student's IKKβ in monkey Tenon’s fibroblasts was suppressed in a dose-dependent manner 24 h after 5 to 100 nM of IKKβ-siRNA were transfected , as shown in The RNAi process that targets g-(PEI-b-mPEG)/IKKβ-siRNA were well tolerated in the monkeys. There were no remarkable differences in ocular findings between the control and experimental groups. Superficial punctuate keratitis occurred in 1 eye of the MMC treated group. Not one case of bleb leaks or endophthalmitis was observed.Surgical eyes exhibited slight hyperemia of the conjunctiva, reversible edema of the cornea, and measurable flare and cells in the anterior chamber, which all resolved within the first week post-surgery. The mean endothelial cell density measured 1 month after surgery decreased slightly compared with preoperative values, but this change was not statistically significant in all groups . In the 2=11.548, p<0.01), as shown in the Kaplan–Meier survival curve in IKKβ-siRNA and MMC groups significantly prolonged bleb survival compared with the PBS group in the monkey model  as the vector. The in vitro results have indicated that CS-g-(PEI-b-mPEG) featured good biocompatibility and transfection efficiency. Subconjunctival IKKβ-siRNA/CS-g-(PEI-b-mPEG) application in the animal model appeared clinically safe and well tolerated within the experimental time. Furthermore, it has been proposed that the hydrophobic domains in the CS-g-(PEI-b-mPEG) may be capable of storing hydrophobic drugs and allowing a slow and sustained release [The eye is a relatively isolated compartment, which makes it an ideal target organ for gene therapy . The mai release , which mIn this study no significant differences were detected in IOP among the three groups. There are questions about the usefulness of IOP as a measure of successful filtration surgery and bleb function in the animal model. Several investigations with the normotensive model failed to find the IOP changes after antiproliferative treatments, in spite of effects detected on the bleb morphologic characteristics -31. TherIn summary, the current study demonstrated that inhibition of NF-κB activity by local application of IKKβ-siRNA improved the surgical outcome in a non-human primate model of glaucoma filtration surgery. Further studies are needed to improve the application methods, and to investigate the long-term effects and safety of this novel anti-scarring agent.


Aberrant β-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate β-catenin activity for therapeutic purposes have proven elusive to date.CSNK1E, the gene encoding casein kinase 1 epsilon (CK1ε) as required specifically for the proliferation of breast cancer cells with activated β-catenin and confirm its role as a positive regulator of β-catenin-driven transcription. Furthermore, we demonstrate that breast cancer cells that harbor activated β-catenin activity exhibit enhanced sensitivity to pharmacological blockade of Wnt/β-catenin signaling. We also find that expression of CK1ε is able to promote oncogenic transformation of human cells in a β-catenin-dependent manner.To uncover genetic dependencies in breast cancer cells that harbor active β-catenin signaling, we performed RNAi-based loss-of-function screens in breast cancer cell lines in which we had characterized β-catenin activity. Here we identify These studies identify CK1ε as a critical contributor to activated β-catenin signaling in cancer and suggest it may provide a potential therapeutic target for cancers that harbor active β-catenin. More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors. MYC and CCND1. In addition to ligand-regulated degradation of β-catenin, Wnt signaling is antagonized by extracellular factors that inhibit the ability of Wnt ligands to bind to Fzd and initiate signaling, such as the secreted frizzled-related proteins  and dickkopf (DKK) The Wnt/β-catenin pathway plays a critical role in embryonic development, maintenance of multipotent progenitor cell populations and proliferation of many tissue types APC or AXIN or activating mutations in the gene encoding β-catenin, CTNNB1, lead to aberrant activation of Wnt/β-catenin signaling and have been causally linked to tumorigenesis of the colon, liver and skin WIF1, this downregulation often occurs through methylation-induced epigenetic silencing SFRP1 and DKK have been shown to be transcriptionally repressed by MYC, a well-established breast oncogene Loss-of-function mutations in CSNK1E, a member of the casein kinase family, as an essential regulator of β-catenin activity and proliferation in breast cancer cell lines.Given the potentially important role of Wnt/β-catenin signaling in breast tumorigenesis, we sought to identify potential therapeutic targets, by identifying genes that promote the survival of breast cancer cells with active β-catenin. By using an RNAi-mediated loss-of-function screening approach, we have identified SFRP1 has previously been observed in all three β-catenin-expressing lines We initially characterized the activation status of β-catenin in breast cancer cell lines using an antibody that specifically recognizes the unphosphorylated form of β-catenin, which corresponds to the stable and thus functionally active form CTNNB1, with two distinct short hairpin RNAs (shRNAs) in the three cell lines with active β-catenin, we observed a substantial reduction in cell proliferation  to identify genes specifically required for proliferation of cells that harbor active β-catenin. We chose to focus on kinases as they regulate many key physiological processes and have the potential to rapidly translate to therapeutic targets thanks to the existence of readily available inhibitors. Raw luminescence scores derived from the proliferation/viability assay were normalized to plate medians and corrected for variability due to spatial and batch effects to generate B scores STK6 (AURKA), AURKB, CDK8, CSNK1E, DCK, KDR, FOXO4 (MLLT7), PRKACA, PRKCA, STK16, TK1 and VRK1 that were required for proliferation in the three cell lines that showed active β-catenin but not in the cell line with no evidence of β-catenin activation   , Table 11 (1 uM) , Table 11 (1 uM) . TogetheCSNK1E is required for β-catenin activity, we used the same two CSNK1E-specific shRNA sequences that we tested above in an independent β-catenin reporter assay and observed that suppression of CSNK1E reduces β-catenin transcriptional activity by more than two-fold . From these expression profiles, we used Genepattern CSNK1E-specific shRNA from cells transduced with GFP-specific shRNA to generate a molecular signature of CSNK1E suppression. We then used this signature to query the Connectivity MAP (CMAP) database of expression signatures generated from cells treated with chemical compounds To further investigate CSNK1E-specific RNAi , as well as other cancer cell lines in previously published reports CSNK1E is a cancer-specific essential gene and further postulate that this may be due to its role in regulation of Per1/2 degradation and circadian cycling CSNK1E suppression, and we failed to detect effects on cell viability after Per1 overexpression (data not shown). Given the many functions attributed to CK1ε, it seems likely that CK1ε is an important regulator of several pathways and its function may be dependent on cell context. However, despite the potentially varying mechanisms in different tumor types, these observations further support the view that inhibition of CK1ε may be broadly applicable as a therapeutic strategy for cancer.CSNK1E as a novel cancer-relevant gene, these observations demonstrate the power of integrating complementary unbiased functional genomic approaches to identify genes involved in specific cancer pathways. As high-throughput screening technology becomes increasingly available, systematic integration of the resulting phenotypic annotation datasets will likely prove to be of great use in rapidly pinpointing genes that may provide new avenues for targeting essential cancer pathways.In addition to the identification of Cells were cultured in DMEM , RPMI  or McCoy's (SKBR3) medium with 10% inactivated fetal bovine serum. All established breast cancer cell lines are commercially available from ATCC. Human mammary epithelial cells were cultured in DMEM/F12 with 10 ng/ml EGF, 10 ug/ml insulin and 0.5 ug/ml hydrocortisone www.R-project.org) and BioConductor Cells plated in 384-well microtiter plates were infected with lentiviral shRNA constructs generated by the RNAi Consortium (TRC) as described Cells were fractionated using the Qproteome Cell Compartment kit from Qiagen, according to manufacturer's instructions. For knockdown validation of shRNA constructs by immunoblotting, cells were infected in 6 well plates and lysates were harvested in RIPA buffer containing protease and phosphatase inhibitors three days post-infection.CSNK1E), TRCN0000003843 and TRCN0000003845 (against CTNNB1), TRCP0000008679 (against GFP). For drug response experiments, cells were plated and 24 hours later, treated with the indicated drug concentrations in triplicate for 72 hours. Drug dose response curves were fitted and IC50 values calculated using a non-linear regression model in Graphpad Prism. Retroviral infections, anchorage-independence assays and xenograft assays were performed as described Cells were plated in 96 well microtiter plates and 24 hours later, infected with shRNA lentiviruses in triplicate, followed by Cell Titer Glo assay (Promega) six days post infection. The TRC IDs corresponding to the shRNA sequences used for theses experiments include: TRCN0000009965 and TRCN0000001834 .RNA was harvested from MCF7 cells infected 48 hours prior with lentiviruses harboring shRNAs against either Figure S1(A) Connectivity Map analysis identifies oxaprozin and resveratrol as compounds that cause transcriptional responses similar to CSNK1E suppression in MCF7 cells, with multiple instances scoring highly. The black lines indicate the ranking of the instances for oxaprozin and resveratrol, with the green area indicating samples with positive connectivity and the red area indicating samples with negative connectivity. (B) Effects of resveratrol on relative cell number of MCF7 versus MDA-MB-453 cells.(0.39 MB TIF)Click here for additional data file.Table S1Primary screen B scores for MCF7, MDA-MB-231, MDA-MB-453 AND T47D cell lines. B-scores for all shRNAs tested in the cell viability/proliferation for each of the cell lines screened. All shRNA constructs are listed by their TRC identifier under “clone ID”. Asterisks denote wells that were removed for technical reasons.(0.74 MB XLS)Click here for additional data file.


The entorhinal cortex is commonly perceived as a major input and output structure of the hippocampal formation, entertaining the role of the nodal point of cortico-hippocampal circuits. Superficial layers receive convergent cortical information, which is relayed to structures in the hippocampus, and hippocampal output reaches deep layers of entorhinal cortex, that project back to the cortex. The finding of the grid cells in all layers and reports on interactions between deep and superficial layers indicate that this rather simplistic perception may be at fault. Therefore, an integrative approach on the entorhinal cortex, that takes into account recent additions to our knowledge database on entorhinal connectivity, is timely. We argue that layers in entorhinal cortex show different functional characteristics most likely not on the basis of strikingly different inputs or outputs, but much more likely on the basis of differences in intrinsic organization, combined with very specific sets of inputs. Here, we aim to summarize recent anatomical data supporting the notion that the traditional description of the entorhinal cortex as a layered input-output structure for the hippocampal formation does not give the deserved credit to what this structure might be contributing to the overall functions of cortico-hippocampal networks. Theentorhinal cortex (Brodman area 28) derives its name from the fact that it ispartially enclosed by the rhinal (olfactory) sulcus. This feature isparticularly striking in nonprimate mammalian species, but also in primates atleast the anterior part of the entorhinal cortex is bordered laterally by arhinal sulcus. Interest in the entorhinal cortex arose around the turn of the 20thcentury when Ramon y Cajal, in his seminal studies on the anatomy of thenervous system, described a peculiar part of the posterior temporal cortexwhich is strongly connected to the hippocampus with fibers that merge in theangular bundle and perforate the subiculum. Cajal was so struck by this massiveconnection that he suggested that the physiological significance of thehippocampus had to be related to that of the entorhinal cortex. At that time,he assumed that the entorhinal cortex was part of the olfactory cortex and sowas, therefore, the hippocampus. He even stated that if this part of theposterior temporal cortex, which he called the sphenoidal cortex/angularganglion, would be visual, so would be the hippocampus . How rigOur current insights into the functional relevance of the hippocampal formation,and how its anatomy is related to function, are much more detailed than what weknow about the entorhinal cortex. It therefore seems attractive to turn theargument of Cajal around by stating that in view of the findings that thehippocampus is crucially involved in conscious, declarative memory processes soshould be the entorhinal cortex. This conjecture is apparently supported byavailable functional studies. Although the specific functional contributions ofthe entorhinal cortex to memory remain to be established, they are most likelydifferent from, but complementary to, those of the hippocampus , 16, 17.A cortical area can be defined inmany different ways, using a variety of different criteria, such as location,connectivity, cyto- and chemoarchitectonics. For the entorhinal cortex, all theseapproaches have been applied, resulting in a confusing variety of borders,subdivisions, and description of layers. A good lead, since it has withstoodover a century of arguments, is the definition of the entorhinal cortex on thebasis of its connectivity with the hippocampus as originally suggested by Cajal . In viewThe entorhinal cortex is surrounded by a number of corticalareas. Anteriorly, it meets with olfactory and amygdaloid cortices, such as thepiriform (olfactory) cortex laterally, and medially it is bordered by theperiamygdaloid cortex and the posterior cortical nucleus of the amygdala. Onits medial side, the entorhinal cortex merges with structures that belongeither to the hippocampal formation or the parahippocampal region, such as theamygdalo-hippocampal transition, and the parasubiculum. The lateral andposterior borders are with the other two major constituents of theparahippocampal region, the perirhinal cortex laterally and the parahippocampalcortex posteriorly. The lateral and posterior borders are quite easy to establish onthe basis of a variety of cytoarchitectonic and chemoarchitectonic features.The most prominent features are that the fairly large-sized cells of layer IIin the entorhinal cortex are replaced by much smaller neurons in the perirhinaland postrhinal cortices, the lamina dissecans disappears, and these changescoincide with similarly striking changes in the density of parvalbumin-positiveneuropil, high in entorhinal cortex, virtually absent in perirhinal andparahippocampal areas. The mirror-image pattern appears when staining for heavymetals (Timm stain) or the calcium binding protein calbindin. All additionalcriteria that have been described seem to coincide with these borders. Theanterior and medial borders, in contrast, are somewhat harder to establish.They apparently coincide with a rather striking change in the ease with whichlayers II and III can be differentiated from each other as well as with a lossof differentiation between the deep layers  or even completedisappearance of the deep layers (anterior border). Combined with subtlechanges in chemoarchitectonic features and connectional differences, an overallconsensus has now been reached (for further details see –25).Attempts to subdivide the entorhinal cortex have, likewise, been numerous . Theseareas roughly correspond to the description of Brodmann's areas 28a and b, respectively, 37, 38.A note of caution should be addedhere: the choice for the terms lateral and medial entorhinal cortex is notsimply related to a particular anatomical position of these areas in relationto the hippocampal formation and the rhinal fissure. In general, the lateralarea occupies a more rostrolateral position versus a more caudomedial positionfor the medial area  whereas caudal entorhinal cortex in the monkey and MEC inthe rat project to proximal CA1 (close tothe dentate gyrus) and distal subiculum (far from the dentate gyrus) [Entorhinal connections with the hippocampal formation in the rat have been comprehensivelydescribed and reviewed in a number of recently published papers and reviews towhich the reader is referred for further details , 47–52. e gyrus) , 57, 58.The CA1-subicular projections are topographically organized along the transverse orproximodistal axis as well, such that parts of CA1 and subiculum that receivecomparable inputs that are either from LEC or MEC are connected to each other–61. FinaThemost comprehensive systematic series of studies on entorhinal connectivity inthe rat is from the Burwell lab , 68–71, Althoughwe will address the layered organization of the entorhinal cortex in moredetail below, it is relevant to point out that entorhinal-cortical projectionslargely arise from deep layers, primarily from layer V pyramidal neurons. Possibleexceptions are the entorhinal-infralimbic and entorhinal-olfactory projections,which appear to arise in layers II and III as well , 81. RegStudiesconducted in multiple species indicate extensive subcortical connectivity forthe entorhinal cortex. Although differences exist with respect to the detail ofthe information, it is safe to conclude that the entorhinal cortex hasconnections with the basal forebrain, claustrum, amygdala, basal ganglia,thalamus, hypothalamus, and brainstem (for review see ). The enEntorhinal-amygdala connectivity has beenstudied in rather detail in both monkey and rat. For recent reviews, the readeris referred to McDonald , PitkäneTheentorhinal cortex is connected with thalamic and hypothalamic structures. Majorthalamic input arises in midline nuclei, particularly the reuniens, paratenial,and periventricular nuclei , 98–100.grosso modo, encompassesthree different (groups of) elements, elements receiving inputs, elements thatprovide output, and elements that contribute to the intrinsic architecture ofthe area. This subdivision into three functionally different elements and rolesto be played by different neurons does not necessarily have an exclusivecharacter; it is actually quite likely that all three elements might be anintegral part of one and the same neuron; however, specializations may occur.Our understanding of the entorhinal cortex isstill rather premature, and to a large extent, influenced by our currentfunctional concept for MEC. The generally accepted division of the entorhinalcortex into at least two functionally different domains stresses the need foran answer to the questions whether or not they differ with respect to their intrinsicwiring and neuronal makeup, in addition to their gross differences with respectto cortical and subcortical connectivity summarized above. The entorhinalnetwork, Compared to the details known for thehippocampal formation and some parts of the neocortex, such as the visual orbarrel cortices in rodents, our understanding of the entorhinal cortex israther in its infancy. The first detailed description of the morphology ofentorhinal neurons, based on Golgi impregnated material, was published in 1933by Lorente de Nó . Over thμm in small or 150 μm in the other MPNs, respectively. Own recent dataindicate that the axons of layer I neurons travel towards layer II and III [In layer I throughout the entorhinal cortex, Lorente de Nódescribed two cell types; horizontal cells and short axis cylinder cells,nowadays known as multipolar neurons (MPNs). This latter category constitutesthe majority of cells in layer I, and generally, they are non- or sparsely spiny.MPNs are quite often positive for calretinin (CR) and GABAergic, and two typeshave been described. Small CR positive MPNs are more often located just deep tolayer Ia , whereas and III where th and III , 112.A  and III .μm. Almostspine-free dendrites extend laterally and spread horizontally within layer Iand superficial layer II. The horizontal extent can be up to 700 μm (ownunpublished data). The noncollateralizing axon travels towards the deep layersto the hippocampus [Horizontal cellsare located in the transitional zone between layers I and II , 115 .Tpocampus , 115. Hopocampus .μm) and may cover about one half of themediolateral extend of the MEC [μm) [μm away from the start, the axon gives off very thin collaterals,branching repeatedly and reaching the superficial layers, forming a net thatcolocalizes with the entire dendritic tree, sometimes extending beyond [Layer II is mainly made up of densely packed, large andmedium sized pyramidal and stellate cells. The most abundant cell typethroughout layer II in MEC is the stellate cell, with their preferred locationwithin superficial and middle layer II . The som the MEC –121. AftMEC [μm) . The relMEC [μm) .Up to 4g beyond .Besidesg beyond .Most stg beyond –126 and g beyond .Stellate cells are less common in LEC than in MEC. In LEC, stellate cells are mostlikely replaced by a comparable cell type, called fan cells , 127. Thμm and is thereforesmaller than that of stellate cells. The smooth and thin axons of thepyramidal-like cells originate from the soma, some follow a sinusoidal route withinlayers II and III, giving off collaterals that distribute in layers I–III [Aside from the stellate-like principal neurons, layer II contains a number ofpyramidal-like cells that have medium sized triangular or ovoid shaped somawith a perpendicular elongation with respect to the pial surface. Most arelocated in the deep portion of layer II , 129. Thrs I–III , 128. Surs I–III .Another pyramidalcell type described in LEC has a very thick and sparsely or nonspiny apicaldendrite, which branches in layer II. Thin apical dendritic tufts reach layerI. The apical dendrite is not as frequently tilted as in MEC pyramidal neurons. The neuInterneurons within layer II are described as MPNs, bipolar, basket, and chandelier cells.MPNs have polygonal, fusiform, or round cell bodies with multiple, sparselyspiny dendrites, extending in all directions, reaching layer I and deep intolayer III. It has been described that the axons of MPNs travel to the whitematter but also form local synapses within layer II , 131. MoSparselyspiny horizontal bipolar cells although considered to be local/interneuronsproject to the hippocampus , 131. ThFastspiking basket-like cells have small spherical cell bodies with sparsely spinydendrites that often ramify into layer I. The extensive axonal arbor is mainlyconfined to layer II. They form basket-like complexes mainly around the soma ofother cells, preferably forming symmetric, inhibitory synapses with stellate or pyramidal cells , 132. Baμm wide and300–450 μm high with the main axonal branch dividing into severalcollaterals that form the characteristic vertical aggregations within the upperportion of layer II/III [μm wide and100–200 μm high) thanthat of vertically oriented chandelier cells. Chandelier cells are GABAergic,often PV-positive and form symmetric contacts withinitial axon segments of principal cells [Chandelieror axo-axonic cells are characterized by verticalaggregations of axonal boutons, called candles, which preferably are locatedsuperficial to the cell body. The somata of chandelier cells are medium sizedwith different shapes. The almost nonspiny, poorly ramifying dendritesoriginate from the basal and apical poles of the somata, displaying a bipolaror bitufted arbor that often stays within layer II/III. Vertical chandeliercells that are restricted to MEC issue a vertically oriented axonal tree thatis around 200–300 r II/III . Horizonal cells –138.μm compared to thevertical direction 203 ± 31 μm, allowing for widespread local connectivity [μm and a horizontal extent of 312 ± 37 μm. The main axon projects via the angularbundle to the subiculum [MEC and LEC layer IIIpyramidal neurons have comparable morphological as well as electrophysiologicalcharacteristics , 29, 127ectivity .Apical ubiculum . Some axubiculum , 140.Nonspiny pyramidalcells (NSPCs), also called type 2 cells , have trLayer III alsocontains stellate cells, in particular in the upper part of the layer. The somataof these neurons are elongated, polygonal, or spherical. Cells belonging to thelatter subgroup sometimes have evenly distributed spiny dendrites around thesomata, whereas others have one or two spiny basal dendrites and a variety ofascending dendrites that branch in layer I. The axons reach the white matter,and collaterals are formed in layer III and the lamina dissecans .μm) between each cell body. The cell body of large MPNsis 15–18 μm in diameterwith multiple sparsely spiny dendrites thatelongate in all directions showing moderate branching. The thickest dendritesface towards the superficial layers whereas the thinner ones radiate laterallytowards the deep layers. The axons of MPNs reach the hippocampus via the whitematter with collaterals distributed in the vicinity of the parent cell soma [Also locatedwithin layer III are principal MPN somata. These MPNs are either small and spherical,with laterally extending dendrites, or they are large. The largest MPNs arelocated in the outer half of layer III of the LEC with a conspicuous spatiallateral separation have also been described as Type 3-(Gloveli) or Type 1-(Kumar) cells , 143.PLBipolarcells have been described in layer III of MEC and LEC. They have a spindle-likeperikaryon with one ascending and one descending smooth, thin and sometimeslong dendrite. The ascending dendritic collaterals traverse throughout layerII, reaching layer I. The extent of the descending dendrites has not beendescribed yet. The axon arises from the primary descending dendrite and extendsinto layer III and the lamina dissecans, deep to the parent cell body . AtleasOccasionally, pyramidal-shaped neurons are located inthe lamina dissecans, at the borders to layers III and V. These neurons havethe morphological and physiological properties of either layer III or layer Vpyramidal neurons, respectively (own unpublished data).Furthermore, bipolarcells, whose dendrites grow horizontally instead of vertically to the pialsurface, with axonal collaterals that can travel towards superficial layer IIIand deep layers, have been found in the lamina dissecans (unpublished data). Ithas been shown that bipolar cells might contain VIP, CCK, and CRF .There is no difference between layer V principal neuronsin LEC and MEC , 145. ThA secondprincipal cell type described in layer V is generally referred to as a type of horizontalcell , 145. Soμm. Instead of havinga prominent apical dendrite, these neurons have a multipolar spiny dendriticarborization that extents for long distances in all directions some even intothe subiculum . The basal dendrites and the widely spreading collaterals spread withinlayers VI and V. The axons of pyramidal cells travel towards the angular bundleand subiculum as well as towards superficial layers. Their axon collaterals arelocated within layers V, VI, the angular bundle, and the subiculum .In conclusion, there aredifferences between cell types and the distribution of cell types in LEC andMEC see . In layeμm distance, and inhibitoryconnectivity  of rostrocaudally orientedbands. Connections that link different transverse  regions ofthe entorhinal cortex, thus providing connectivity between these bands, arerather sparse –151. Thesee also . Among lsee also  is likelThe overall organization of the longitudinalintrinsic connections is best considered in relation to the organization of thereciprocal entorhinal connections with the hippocampal formation. Interconnectedportions of the LEC and MEC close to the rhinal fissure, in rats referred to asthe dorsolateral band of entorhinal cortex, are connected to the dorsal(nonprimate) or posterior (primate) part of the hippocampal formation (seeFigures The second organizational level deals withthe local connectivity within and among layers of more restricted portions ofthe entorhinal cortex. As we know from the studies summarized above, neurons indifferent layers have very different inter- and intralaminar connectionalpatterns that include axon collaterals confined to the parent cell layer orspanning several layers. But not only the axonal distribution is of importance,the dendritic trees may also play an essential role in that they either spanseveral layers or are more restricted to the parent cell layer. Althoughdetailed information for quite a few of neuronal types in the entorhinal cortexis still lacking, it is safe to say that the entorhinal network, on the basisof its neuronal composition alone, cannot be properly described in terms ofsuperficial and deep layers as more or less independent layers. All this maynot come as a surprise since comparable concepts have been described withrespect to the organization of the neocortex . This seOne important anatomical observation alreadyreported by Cajal , 28 is tAs illustrated in Whatthen is the functional relevance of inputs from for example the medialprefrontal, cingular, and retrosplenial areas? Afferents from these areaspreferentially, and in some instances even exclusively, terminate in the deeplayers of the entorhinal cortex. Note that in the monkey, however, it hasrecently been reported that projections from the retrosplenial cortex denselyinnervate entorhinal layer I . Do inpuThe functional relevance of theorganization of networks in the brain is often interpreted on the basis of a surprisinglyrestricted point of view. Debates on the functional organization of thehippocampal formation have been strongly influenced by the idea that theprevailing hippocampal circuitry is unidirectional. With regards to the entorhinalcortex, the breakthrough discovery, that deep entorhinal layers receivehippocampal output from CA1 and the subiculum on the one hand, and that thesesame layers are the origin of strong cortical projections, has biased our view towardsthe rather simple concept that the deep layers mediate hippocampal-to-corticalconnectivity, similar to superficial layers providing the way in for corticalinputs to the hippocampal formation. If the entorhinal cortex is such animportant hub, similar to the central station of a large city, and that is whatall data seem to converge on, it is quite likely that it serves yet anotherrole. In addition to serving simply to get into the city or leave the city, thestation also provides the powerful potential for new interactions between andamong incoming and outgoing people. This potential for “new” interactions has beengrossly neglected in case of the entorhinal cortex. The potential of theentorhinal cortex to act as an interactive hub, contributing essentially to thefunctions of the cortico-hippocampal system instead of just transferringinformation, has been underscored not only by the recent finding of the uniquespatial firing properties of grid cells in the entorhinal cortex –9, but aSimilar to the yet unresolvedmystery of the relevance of cortical inputs to deep layers of the entorhinalcortex, it remains to be established what the functional relevance is of LEC.The data summarized above indicate that with the exception of neurons in layerII, it is likely that both LEC and MEC are largely similar with respect totheir intrinsic wiring, both in terms of neuronal elements that comprise the nodalpoints of the network as well as with respect to how these are wired together(see Figures


The Ex-Press Mini Glaucoma Shunt has been available internationally for almost a decade with almost 35,000 implantations world wide. The device shunts aqueous from the anterior chamber to a subconjunctival reservoir in a similar fashion as trabeculectomy, without removal of any sclera or iris tissue. Developments in ophthalmic surgery have been focused on smaller incisions to improve patient outcomes and visual recovery. The Ex-Press is an example of these developments. This article will review the surgical technique for implanting the Ex-Press Mini Glaucoma Shunt and will highlight the clinical experience with the device. The Ex-Press Mini Glaucoma Shunt  was origThe original unguarded technique of implantation under the conjunctiva caused numerous complications including hypotony, erosion, and extrusion of the implant. These complications have been well described in the literature.7To avoid complications associated with placement of the device under the conjunctiva, Dahan and Carmichael proposed implanting the device under a scleral flap.Topical or retrobulbar anesthesia is administered depending on patient selection and surgeon preference.A standard fornix or limbal-based conjunctival incision is performed to gain exposure to the scleral bed adjacent to the limbus. Gentle cautery is performed in this area.A scleral flap is created in a similar manner performed with a standard trabeculectomy. Care is taken to dissect the flap up to clear cornea. Anti-fibrotic agents can be applied either before or after the creation of the scleral flap in the usual manner based on the surgeon's preference.A temporal paracentesis is created through the cornea.The scleral flap is lifted and care is taken to identify the center of the “blue line” adjacent to clear cornea which corresponds to the location of the trabecular meshwork. A 26 gauge needle is inserted into the anterior chamber through the center of the “blue line” at an angle parallel to the iris plane.  The needThe Ex-Press shunt is preloaded on an injector.The shunt is then placed in the anterior chamber through the ostium created with the needle. The angle of entry with the shunt is the same as the angle used to make the ostium . The shuThe scleral flap is then sutured in place using a 10-0 nylon suture with a spatulated needle. One to three sutures are typically required depending on the flow which can be tested by inflating the anterior chamber with balanced salt solution with a 27 or 30 gauge canula through the temporal paracentesis.The conjunctiva is then meticulously closed with the surgeon's suture of choice. I typically use a 9-0 polyglactin suture on a BV or VAS needle in a running or interrupted fashion. A fluorescein strip is used to make certain the wound is water tight.et al.16Thus far, the largest published case series of the Ex-Press Mini Glaucoma Shunt was by Kanner et al. that there is a lower incidence of hypotony with the Ex-Press compared with trabeculectomy.et al. demonstrated a 32% hypotony rate in the trabeculectomy group and 4% in the Ex-Press group.et al., there was 15.6% with hypotony (IOP <5mm Hg) in the first week in the Ex-Press alone group, and 7.9% with hypotony in the first week in the combined group. All of these instances of hypotony during the early postoperative period resolved spontaneously. None of the eyes developed flat anterior chamber with lens-cornea touch.16It has been demonstrated by a previous paper by Maris It is postulated that the lower rate of hypotony is from the resistance to flow that is offered by the 50 micron lumen of the shunt. Whereas, with trabeculectomy, the smallest scleral punch that is manufactured is approximately 750 microns and that is only if one punch alone is used to make the incision. It is clear that scleral sutures offer much of the resistance to flow with either trabeculectomy or the Express shunt; however, with the latter it is likely that both the small lumen of the implant and the suture tensioning offer resistance.Ophthalmic surgery has evolved over the last several decades into sophisticated microsurgery involving continually smaller incisions. This has been well demonstrated by developments in cataract surgery with phacoemulsification through small self-sealing clear cornea incisions and foldable intraocular lenses inserted using injectors. This evolution has also been shown in retina surgery with the advent of 25 gauge vitrectomy. It therefore follows that a similar evolution take place in glaucoma surgery with smaller, more precise wounds with more predictable and reliable results.The Ex-Press shunt is on the forefront of this evolution toward smaller incision glaucoma filtration surgery. Although, there is an added cost to using the Ex-Press rather than trabeculectomy, I believe there are patient safety issues that are provided by the Ex-Press that make the added cost worthwhile. Moreover, it can be argued that fewer return visits to the operating room will occur with the lower rate of hypotony afforded by the Ex-Press. Examples of these benefits include fewer revisions for over-filtering blebs and much less frequent draining of choroidal effusions.For the last decade there has been a philosophic paradigm shift in the surgical treatment for glaucoma with a desire to restore the natural outflow of aqueous humor via Schlemm's canal; however, none of these newer surgeries have been demonstrated to lower IOP as much or as reliably as guarded filtration surgery. Although not a paradigm shift regarding location of outflow, the Ex-Press offers a modification of outflow that is consistent with the general shift in surgery toward smaller incision microsurgery, affording easier utilization of topical anesthesia, less inflammation, greater predictability with less hypotony, and more rapid visual recovery.


The Coxsackie and adenovirus receptor (CAR) has been shown to inhibit cancer cell proliferation, migration, and invasion. The underlying mechanisms, however, are poorly understood.α-catenin was determined by quantitative RT-PCR and western blotting. Proliferation, migration, and invasion after CAR knockdown were assessed by in vitro assays, and cell morphology in a three-dimensional context was evaluated using matrigel.The differential gene expression in the human colon cancer cell line DLD1 on RNAi-mediated functional CAR knockdown was analysed using oligo-array technology. Expression of α-catenin as the strongest downregulated gene after CAR knockdown. Western blotting and quantitative RT-PCR confirmed a reduced α-catenin expression after CAR knockdown in DLD1 cells and in the rat intestinal cell line IEC-6. Functionally, both cell lines showed a marked increase in proliferation, migration, and invasion on CAR knockdown. In matrigel, both cell lines formed amorphous cell clusters in contrast to well-organised three-dimensional structures of CAR-expressing vector controls. Ectopic ‘re’-expression of α-catenin in DLD1 and IEC-6 CAR knockdown cells reversed these functional and morphological effects.Oligo-array technology identified α-catenin mediates the impact of CAR on cell proliferation, migration, invasion, and morphology.These data suggest that an interaction of CAR and  The Coxsackie and adenovirus receptor (CAR) was first characterised as a viral attachment site on the surface of epithelial cells and was later identified as an integral component of the tight junction complex . In seveα-catenin.Aiming to further elucidate genes that facilitate the proposed function of CAR in cancer, we determined differential gene expression after CAR knockdown in the human colon cancer cell line DLD1 and assessed the functional impact of the strongest regulated gene α-catenin was accomplished by the stable transfection of a human full-length α-catenin cDNA  as described previously  and from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH , respectively, and were cultured in recommended growth media. Three-dimensional cultures were set up as described previously  and reverse transcribed with oligo-dT primers and SuperScript II (Invitrogen). cDNAs generated from 200 ng of total RNA were analysed for differential gene expression using HG U133 2.0 plus chips  and processed using GeneSpring software .α-catenin, ZO-1 , or β-Actin , and detected using the enhanced chemiluminescence system .Protein lysates were obtained as previously described (α-catenin gene-specific primers (CCATGCAGGCAACATAAACTTC and AGGGTTGTAACCTGTGTAACAAG) and the qPCR MasterMix for SYBR Green I . Quantification was carried out by the comparative ΔCT method normalising CT values to β-actin (DLD1) or 18S (IEC-6). cDNAs derived from CHO and CHO-CAR cells were used as negative and positive controls, respectively. All experiments were carried out in triplicate and repeated at least twice.Total RNA was isolated using Trizol (Invitrogen) and was reverse transcribed with oligo-dT primers and SuperScript II (Invitrogen). cDNAs generated from 200 ng of total RNA were used for real-time RT-PCR assays on a Stratagene MX3000P cycler using α-catenin (Zymed Laboratories) antibodies served as primary antibodies; a Cy2-conjugated anti-rabbit antibody and a Cy3-conjugated anti-mouse antibody were used as secondary antibodies . Multicolour fluorescence microscopy was carried out using a Zeiss Axiophot microscope .Immunofluorescence staining was carried out as described previously (α-catenin (Zymed Laboratories) antibodies served as antibodies for the initial immunoprecipitation; IgG rabbit (Dianova GmbH) was used as control. Subsequent western blotting was carried out as described above.Co-immunoprecipitations were carried out using the Immunoprecipitation Kit Protein G  according to the manufacturer's instructions. In brief, anti-CAR  and anti-5 cells per well for DLD1 and 2 × 105 cells per well for IEC-6) in recommended media. After 48 h, the cells were detached using trypsin, stained with trypan blue to exclude dead cells, and counted using a haematocytometer counting chamber . All experiments were carried out in triplicate and repeated at least twice.Cells were seeded onto six-well plates -free media were seeded onto the upper part of the chamber, whereas its lower compartment was filled with media containing 10% FCS or serum-free media as control. After 24 h at 37°C, cells that migrated through the filter were fixed and stained with crystal violet containing 10% ethanol. After gentle rinsing with phosphate-buffered saline, non-migrated cells at the upper side of the filter were scraped off. Subsequently, the crystal violet dye retained on the filters was extracted with 10% acetic acid for 5 min at room temperature and was colorimetrically measured at 560 nm. All experiments were carried out in triplicate and repeated at least twice.Assessment of directed cancer cell migration was carried out using an AP48 48 Well Micro Chemotaxis Chamber . Here, 50 000 cells per well in 50 μm pore size PET membranes covered with matrigel matrix. Medium containing 10% FCS was added to the bottom well of the chambers as a chemoattractant, whereas serum-free medium was used as a control. After 24 h at 37°C and 5% CO2, cells that invaded the matrigel-coated membrane, located at the lower membrane surface, were fixed and stained by crystal violet containing 10% ethanol. Cells of three representative areas in each well were counted at a magnification of × 100. Experiments were carried out in triplicate and repeated at least twice.Cells were seeded onto the top of BioCoat Matrigel Invasion Chambers (BD Biosciences) containing 8 t-test, considered significant when P<0.05.Statistical calculations were carried out using GraphPad Prism software  using an unpaired, two-tailed CAR-negative) were compared with those of DLD1control cells (containing the pSuper vector only), using oligo-array technology. Setting a threshold of a 1.5-fold difference between the two cell lines, 1187 genes were found upregulated and 2318 genes downregulated in the DLD1CAR-negative cell line. Among these genes, α-catenin displayed the highest fold change, showing an 18.54-fold lower expression in the DLD1CAR-negative cell line. Western blotting confirmed this finding at the protein level revealing a reduced α-catenin expression in both the DLD1CAR-negative and the IEC-6CAR-negative cell lines. Furthermore, transcriptional dependence of α-catenin by CAR was shown using reverse transcribed (RT)–PCR assays showing a reduced mRNA expression level of α-catenin in DLD1CAR-negative and IEC-6CAR-negative cells  and vector-transfected control cell lines. In contrast, DLD1CAR-negative and IEC-6CAR-negative cells showed a weak intracellular signal only, comparable with that of controls stained by secondary antibodies only (data not shown). After the ectopic expression of α-catenin in DLD1CAR-negative and IEC-6CAR-negative cell lines, a strong intracellular immunopositivity was noted for α-catenin, yet no signal was detected at the plasma membrane . Ectopic ‘re’-expression of α-catenin in DLD1CAR-negative and IEC-6CAR-negative resulted in significantly lower cell numbers  . In contrast, ectopic ‘re’-expression of α-catenin significantly decreased the number of migrating cells for DLD1α-cateninCAR-negative/ and IEC-6α-cateninCAR-negative/ (both: P<0.0001) (CAR-negative (P<0.0001) and IEC-6CAR-negative (P<0.0001) cell lines. Ectopic ‘re’-expression of α-catenin impaired cell invasion significantly , yet was not sufficient to reduce the number of invading cells to the level of DLD1control and IEC-6control cell lines  . Using m<0.0001) . To testll lines .α-catenin expression after downregulation of CAR. On the basis of our data, it may be speculated that this phenomena crucially affects the inhibitory effect of CAR on cell proliferation, migration, and invasion. Moreover, our observations reveal that loss of CAR is potentially accompanied by substantial changes in cellular morphology that may also be explained by an impaired presence of α-catenin.This study marks the first report of an impaired α-catenin mRNA expression represents the most prominent feature after downregulation of CAR. This observation was further validated by our finding of reduced α-catenin protein presence in DLD1 and IEC-6 cell lines upon downregulation of CAR. As DLD1 represents a human cancer cell line and IEC-6 a rat non-transformed small intestinal cell line, it may be speculated that our results account for a more general phenomenon. However, differential protein expression of α-catenin after CAR downregulation differs between cell lines: after a transient transfection of a CAR-specific siRNA, reduced α-catenin protein expression was noted in AGS (gastric cancer) and SW480 (colon cancer), whereas in T84 and HCT116 cells, only a moderate decline was observed and no changes were noted in Caco2 . The underlying mechanisms of these differences remain to be elucidated. Transcriptional regulation of α-catenin expression after CAR downregulation potentially marks the main regulatory measure, as shown by quantitative RT-PCR. However, whether the α-catenin promoter becomes inactivated upon loss of CAR or changes in RNA stability occur, remains to be elucidated.Our findings reveal that a reduced α-catenin, a protein that has been extensively studied for its inhibition of cancer cell growth and motility (α-catenin is frequently described in carcinomas, for example, colon cancer, significantly correlating with depth of invasion, presence of metastasis, and an adverse clinical outcome (α-catenin. Furthermore, our data show that ‘re’-expression of α-catenin is sufficient to markedly reverse these effects. Therefore, it may be speculated that the functional effects on cell growth and motility after loss of CAR are crucially mediated by a reduced expression of α-catenin. However, cell invasion has been reduced to a small extent only on re-expression of α-catenin. Given that our experimental setting sufficiently reflects cell invasion, we found that a CAR–α-catenin interaction seems to be less important in this context compared with cell proliferation and migration.The previously proposed inhibitory effects of CAR on cancer cell growth and motility have been suggested to result from an impaired intercellular adhesion (motility . Moreoveα-catenin and, recently, CAR have been shown to interact with the cytoskeleton (α-catenin on this phenomenon was shown by the ‘re-’expression of α-catenin, which resulted in the formation of organised cell formations as opposed to amorphous clusters after CAR knockdown.Both α-catenin showed a co-localisation of both proteins at the plasma membrane in parental- and vector-transfected control cell lines. However, the ‘re’-expression of α-catenin in CAR-negative cell lines resulted in an intracellular accumulation of α-catenin, yet no ‘re’-distribution to the plasma membrane. Therefore, we hypothesised that CAR may recruit α-catenin to the plasma membrane. Yet, co-immunoprecipitation using either an anti-CAR or an anti-α-catenin antibody failed to proof a binding of these proteins. Therefore, reduced presence of other proteins that link α-catenin to the plasma membrane may account for the intracellular accumulation of α-catenin seen here. Nevertheless, binding of CAR to α-catenin may not be excluded entirely, as it might be weak and/or transient and not be detectable biochemically. Herein, our findings are potentially in line with a recent study showing that α-catenin, rather than forming a stable link to actin, acts as a regulator of actin dynamics and that its binding to the E-cadherin–β-catenin complex is transient and mutually exclusive of its interaction with F-actin (Co-immunofluorescence staining for CAR and α-catenin expression. Underlying regulatory mechanisms remain to be elucidated.In conclusion, it may be speculated that the suggested tumour suppressive function of CAR is at least in part mediated by regulating the


To investigate the potential involvements of E-cadherin and β-catenin in meningioma.Immunohistochemistry staining was performed on samples from patients with meningioma. The results were graded according to the positive ratio and intensity of tissue immunoreactivity. The expression of E-cadherin and β-catenin in meningioma was analyzed by its relationship with WHO2007 grading, invasion, peritumoral edema and postoperative recurrence.The positive rates of E-cadherin in meningioma WHO I, II, III were 92.69%, 33.33% and 0, respectively, (P<0.05); while the positive rates of β-catenin in meningioma WHO I, II, III were 82.93%, 33.33% and 20.00%, respectively, (P<0.05). The positive rate of E-cadherin in meningioma without invasion (94.12%) was higher than that with invasion (46.67%) (P<0.05). The difference in the positive rate of β-catenin between meningioma without invasion (88.24%) and meningioma with invasion  was also statically significant. The positive rates of E-cadherin in meningioma with peritumoral edema 0, 1, 2, 3 were 93.75%, 85.71%, 60.00% and 0 respectively, (P<0.05); the positive rates of β-catenin in meningioma with peritumoral edema 0, 1, 2, 3 were 87.50%, 85.71%, 30.00% and 0 respectively, (P<0.01). The positive rates of E- cadherin in meningioma with postoperative recurrence were 33.33%, and the positive rate with postoperative non-recurrence was 90.00% (P<0.01). The positive rates of β-catenin in meningioma with postoperative recurrence and non-recurrence were 11.11%, 85.00%, respectively (P<0.01).The expression levels of E- cadherin and β-catenin correlated closely to the WHO 2007 grading criteria for meningioma. In atypical or malignant meningioma, the expression levels of E-cadherin and β-catenin were significantly lower. The expression levels of E- cadherin and β-catenin were also closely correlated with the invasion status of meningioma, the size of the peritumoral edema and the recurrent probabilities of the meningioma, all in an inverse correlationship. Taken together, the present study provided novel molecular targets in clinical treatments to meningioma. Meningioma originates from the derivatives of meninges and arachnoid cap cells. Its incidence as primary intracranial tumors is very high (15–20%), ranked just behind the cerebral glioma All specimens involved in this study were collected from 49 meningioma patients with university guidelines carefully followed . Written permissions from patients were obtained. These operative specimens came from the Affiliated Municipal Hospital at Taizhou Medical College and Sir Run Run Shaw Hospital at Zhejiang University. All the patients were diagnosed as meningioma in formal pathological reports between Jan 2003 and Sep 2005.Eighteen cases were male, and 31 cases were female. The age range was 20 to 75 years, and the average age was 56.3±17.1 years. The length of time for which the patients had meningioma varied from 5 years to 2 days. The first symptoms were as follows: increased intracranial pressure (12 cases), visual disturbance (7 cases), disorder of limb activity (20 cases), hearing disorder (4 cases), seizure (6 cases), and other (10 cases). Cranial MRIs revealed the tumor locations as follows: cerebral hemisphere (15 cases), pardsagittal (17 cases), and cerebellopontine (5 cases) Angle, 6 cases in sphenoid ridge, 6 cases in sellar region; The tumor size was as follows: <3 cm (8 cases), 3∼6 cm (25 cases), >6 cm (16 cases). The dural tail sign was not obvious in 20 cases and was obvious in 29 cases. All 17 cases of pardsagittal tumors had MR venous angiography(MRV), and 2 cases underwent DSA. The results showed that no obstructed sagittal sinus or thrombosis was present. In five cases, the tumors were closely related to the cortical draining veins, but it was easy to separate the tumors and veins during the operation, and there were no obstructions or thrombosis with cortical draining veins. In the samples we studied, 22 cases were Simpson resection grade I, and the other 27 cases were Simpson resection grade II. Invasive tumor was defined according to whether the tumor had invaded the pia mater and skull. This was ascertained by surgical findings and pathological examination. In the invasive group, the tumors had invaded the pia mater or the skull and the arachnoidal cleavage plane had disappeared, while in the non-invasive group, the tumors had not invaded the pia mater or the skull and the arachnoidal cleavage plane was well preserved. According to these criteria, 15 cases were classified as invasive. All of them, the tumor >3cm diameter 2 cases, the tumor 3–6cm diameter 8 cases, the tumor >6 cm diameter 5 cases. Goldman's method was used to classify peritumoral edema The expression levels of E-cadherin and β-catenin were measured by immunohistochemical staining and En Vision. Tissues were prepared as paraffin sections. Prior to immunohistochemistry, the sections were deparaffinized with xylene, dehydrated with ethanol, and deoxidised with methanol. The sections were then prepared in a pressure cooker to 121°C for 2 minutes in citrate buffer solution to restore the antigen immunoreactivity. Then the sections were washed in PBS prior to incubation with primary mice monoclonal antibodies of E-cadherin  and β-catenin  overnight at room temperature. Then the sections were processed for DAB visualization. The sections were then mounted with permount medium and observed under a light microscope.The expression of E-cadherin was located in either the membrane or cytoplasm of meningioma cells, more commonly in the former. The expression of β-catenin was located in the membrane, cytoplasm, and perinuclear granules Statistical analyses were carried out using SPSS 11.0 software. The comparison of the expression strength of E-cadherin and β-catenin with the differentiated pathological types was performed by the rank sum test. The comparison of the positive ratio of E-cadherin and β-catenin with the differentiated WHO 2000 grading, incidence of invasion, level of peritumoral edema, and postoperative recurrence was performed by the Chi-square test. P<0.05 was considered as statistically significant.2 = 5.649, 6.274, respectively; P>0.05). This suggested that E-cadherin and β-catenin could be common molecules participated in the development of diverse meningioma.We investigated the expression levels of E-cadherin and β-catenin among different types of meningioma. We found that their expression levels were not statistically different between each other . The positive rates of β-catenin for grades I, II, and III were 82.93%(34/41), 33.33%(1/3), and 20%(1/5), respectively, and these differences were significantly different .To understand if the amounts of E-cadherin and β-catenin could contribute to the servility of meningioma, we graded the samples of meningiomas following WHO2007 standards. We found that the positive rates of E-cadherin were 92.69%(38/41), 33.33%(1/3), and 0 respectively, for grade I, II, and III (χWe further asked if the expression levels of the two proteins could contribute the invasion ability of the tumor. We found significant differences in the expression levels of E-cadherin and β-catenin between invasive or non-invasive meningioma (P<0.05). This strongly suggested that E-cadherin and β-catenin could be potentially negative regulators of the tumor invasion.2 = 11.22, P<0.05). The positive rates of β-catenin for grade 0, 1, 2, and 3 were87.50%(14/16), 85.71% (18/21), 30%(3/10), and 0, respectively .We found that the positive rates of E-cadherin were 93.75% (15/16), 85.71% (18/21), 60%(6/10) and 0, respectively for peritumoral edema graded at 0,1,2,3 .Because the expression levels of E-cadherin and β-catenin could reflect the invasive ability of the tumor cells, therefore they might also be implicative for post-operative recurrence. We therefore investigated the association between expression levels of of E-cadherin and β-catenin in our study. We found that the expression levels of of E-cadherin and β-catenin in postoperative recurrence cases were 33.33% and 11.11%, respectively; and for postoperative non-recurrence cases they were 90% and 85%, respectively. In both cases, the differences were significantly different (χ2+ for its function and the structural rigidity; the extracellular amino-terminus formed the “zipper-like” structure with that would act as a cell tight junction. The intracellular carboxyl-terminus of the cadherin molecule indirectly attached the cytoskeleton via catenin. β-catenin, which was one of four known kinds of catenin, is a multifunctional protein E-cadherin was a calcium-dependent cell-cell adhesion molecule with pivotal roles in epithelial cell behavior, tissue development, and suppression of cancer growth The expression level of E-cadherin could be related to the classification of astrocytomas. For this reason, an assessment of the expression status of E-cadherin in astrocytomas could be one important index in determining the prognosis of patients The results of our research revealed remarkable differences in the involvements of E-cadherin and β-catenin in different pathological grades of meningioma. Moreover, these differences were statically significant. As the pathological grade of meningioma increased, the positive rates of E-cadherin and β-catenin in meningioma decreased. The expression levels of the E-cadherin and β-catenin were actually completely diminished in malignant meningioma. In previous studies, Utsuki et al. tested specimens of 103 meningioma and found that the expression levels of E-cadherin in 5 atypical meningioma were all negative, 3 cases of the expressions of the β-catenin were negative among them Tumor invasiveness and the presence of peritumoral edema were the two major factors that would determine the clinical management of meningioma. There were some biological, physical, and chemical factors that contribute to the peritumoral edema of meningioma The decrease or deletion of the expression of E-cadherin leads to the loss of contact inhibition and unrestricted hyperplasia, the loss of intercellular junction with, stronger invasive ability, enhanced tumor cell diffusion and metastasis, as well as benign tumor malignant transformation in some extreme cases In summary, the present study employed molecular biology and immunohistochemistry tools to understand the potential involvements of two cell-adhesion molecules in development and invasiveness of meningioma, which provided novel targets for pathological analyses as well as therapeutic drugs.


Post-translational stabilization of beta-catenin through mutation of the adenomatous polyposis coli (APC) gene has been proposed as an early step in colorectal carcinogenesis. Beta-catenin may translocate from the cytoplasm to the nucleus, where it might serve as a transcriptional factor to stimulate tumour formation. We investigated intracellular localization of beta-catenin in sporadic colorectal adenomas and cancers as well as familial adenomatous polyposis (FAP). Nuclear over-expression of beta-catenin was observed in 35% (7/20) of intramucosal cancers and 42% (23/55) of invasive cancers but was not seen in any adenomas from sporadic or FAP cases. Cytoplasmic beta-catenin in adenomas was significantly higher than that of normal mucosa in both sporadic and FAP cases. The cytoplasmic intensity index of cancers was significantly higher than that of sporadic adenomas, but the index was not correlated with nuclear expression in cancers. These findings suggest that nuclear translocation of beta-catenin is involved in development of intramucosal cancer rather than adenoma, independent of APC mutations. Cytoplasmic accumulation of beta-catenin may occur in adenomas, but it remains to be determined whether this is a cause or a consequence of colorectal cancer. © 2000 Cancer Research Campaign


Inducible NOS-mediated intracellular killing may represent direct bactericidal activity, because B. mallei showed an exquisite sensitivity to NO generated chemically. Exposure of B. mallei to sublethal concentrations of NO upregulated transcription of [Fe-S] cluster repair genes, while damaging the enzymatic activity of the [Fe-S] protein aconitase. To test whether [Fe-S] clusters are critical targets for RNS-dependent killing of B. mallei, a mutation was constructed in the NO-induced, [Fe-S] cluster repair regulator iscR. Not only was the iscR mutant hypersusceptible to iNOS-mediated killing, but its aconitase pool was readily oxidized by NO donors as compared to wild-type controls. Although killed by authentic H2O2, which also oxidizes [Fe-S] clusters, B. mallei appear to be resilient to NADPH oxidase-mediated cytotoxicity. The poor respiratory burst elicited by this bacterium likely explains why the NADPH oxidase is nonessential to the killing of B. mallei while it is still confined within phagosomes.We show herein that reactive nitrogen species (RNS) generated by inducible nitric oxide (NO) synthase (iNOS) account for the anti-B. mallei. To the best of our knowledge, this is the first instance in which disruption of [Fe-S] clusters is demonstrated as cause of the bactericidal activity of NO congeners.Collectively, these findings have revealed a disparate role for NADPH oxidase and iNOS in the innate macrophage response against the strict aerobe  B. mallei bacillus is the causative agent of glanders, a disease that can be transmitted to humans upon cutaneous, mucosal or aerosol exposure to mucopurulent discharge from the eyes, nose and lips of infected solipeds B. mallei a desirable bioterrorist agent as has been demonstrated empirically in humans and animals in both World War I and II The gram negative, nonmotile 2-dependent antimicrobial mechanisms that are coupled to NADPH oxidase and iNOS enzymatic activities. The NADPH phagocyte oxidase is assembled on endocytic or plasma cell membranes, whereupon it catalyzes the NADPH-dependent univalent reduction of O2 to superoxide (O2•−). This radical serves as precursor to a variety of reactive oxygen species (ROS) endowed with widespread antimicrobial activity 2 and NADPH in the oxidation of L-arginine with the consequent generation of L-citrulline and NO 2, O2•− and metals are endowed with antimicrobial activity against phylogenetically diverse microorganisms Pathogens adapted to an intracellular lifestyle avoid competition with other microbes that colonize host surfaces, while taking advantage of the nutritional resources provided by the cell. The cost of intracellular parasitism is the potential exposure to a variety of host defenses. For example, intracellular pathogens may encounter the OBurkholderia survive within mononuclear phagocytes Burkholderia spp. is made evident by the fact that B. cepacia is the second most lethal infection in chronic granulomatous disease patients carrying mutations in membrane-bound or cytosolic components of the NADPH oxidase B. cepacia has been recreated in p47phox-deficient mice B. pseudomallei arsenal of macrophages Burkholderia activity have been rather controversial. Experimental animal models and macrophage cell cultures have shown that iNOS is dispensable for innate host defense against B. cepacia and B. pseudomalleiBurkholderia growth occurs during maximal NO synthesis B. pseudomalleiB. pseudomalleiB. mallei is susceptible to NO generated by macrophages B. mallei. The major goal of this study was to characterize the contribution of NADPH oxidase and iNOS hemoproteins to the macrophage antimicrobial arsenal against B. mallei.Recent investigations have demonstrated that members of the genus B. mallei strain ATCC 23344 was used in these studies  and the iscR reverse primer containing an SphI restriction site (5′-aagcccgtcgcatgcgcgacgggctccggcttgcgcttg-3′). After double digestion with BamHI and SphI, the PCR product was directionally cloned into the suicide vector pMO90, creating the disruption plasmid pMO126. pMO126 was introduced into B. mallei using triparental mating E. coli DH5α pRK2013 strain E. coli strains. The resulting colonies were re-streaked for isolation on LB plates supplemented with 30 µg/ml kanamycin and 20 µg/ml polymyxin B. Insertion of pMO126 into the B. mallei genome and the disruption of BMA 1709 was confirmed by PCR. The replicative plasmid pMO79 encoding a green fluorescence protein was conjugated into B. mallei strain ATCC 23344 by triparental mating as described above. The presence of pMO79 in B. mallei was confirmed by fluorescence microscopy. studies . Bacteri−/−iNOS−/−−1/100 mg ml−1 of penicillin/streptomycin (Cellgro) (RPMI+ medium). The RPMI+ medium used to culture the THP-1 cell line was supplemented with 50 µM 2-mercaptethanol. The peritoneal exudate cells were seeded in flat-bottom 96-well plates for macrophage killing assays at a density of 2×105 cells per well. The macrophages were selected by adherence after 24 h of culture at 37°C in a 5% CO2 incubator. Murine J774 (clone ATCC TIB-67) and AMJ2-C11 (clone ATCC CRL-2456) and human U937 (clone ATCC CRL-1593.2) and THP1 (clone ATCC TIB-202) macrophage-like cells grown in RPMI+ medium supplemented as described above were used as additional sources of mononuclear phagocytes. Selected groups of macrophages were treated with 200 U/ml of IFNγ  overnight prior to Burkholderia infection. Just prior to infection the macrophages were washed with pre-warmed RPMI without antibiotics.C57BL/6 and congenic SalmonellaB. mallei were grown overnight in LBG broth and sub-cultured to OD600 of 0.6 as described above. The bacteria were spun down for 5 min at 1500 RPM onto the macrophages at an MOI of 200 after opsonization in 10% normal mouse serum in RPMI+ medium. Unless specified, extracellular bacteria were removed from the monolayers after a 2 h incubation by washing with pre-warmed RPMI+ medium containing 6 µg/ml of gentamicin (Sigma-Aldrich). After washing the noninternalized bacteria, the MOI was determined to be 10. Burkholderia-infected macrophages were lysed with 1% Triton X-100 in phosphate buffered saline (PBS) at the indicated times after challenge. The intracellular bacteria recovered at various points after infection were enumerated on LB agar plates. The % survival was calculated as (cfu tn/cfu t0)×100.The macrophage killing capacity was quantified by a gentamicin protection assay following a modified protocol described for 2•− was quantified by the superoxide dismutase–inhibitable reduction of ferricytochrome c Burkholderia resuspended in phenol red–free Earle's balanced salt solution containing 60 µM ferricytochrome c. Because B. mallei is aflagellated, a strain of Salmonella enterica serovar Typhimurium deficient in fliC fljB was used as a positive control. After 1 h incubation in 5% CO2 at 37°C, the OD of the supernatants was determined spectrophotometrically at 550 nm. The concentration of O2•− was calculated by using an ε550 of 2.1×103 M−1 cm−1. All reagents were purchased from Sigma-Aldrich.O2−) produced by Burkholderia-infected macrophages 6 h post-challenge was estimated spectrophotometrically at 550 nm in a Biotek Synergy HT-2 reader after mixing culture supernatants with an equal volume of Griess reagent (0.5% sulfanilamide and 0.05% N-1-naphthylethylenediamide hydrochloride in 2.5% phosphoric acid). The NO2− concentration was calculated by regression analysis using a NaNO2 standard curve.The concentration of nitrite , H2O2 , or spermine  at 37°C. At the end of the incubation, 200 units of catalase  were added to the H2O2-treated samples to consume any remaining H2O2. Serial dilutions were spotted on LBG agar plates and the number of colony forming units counted after 48 h of culture.Overnight cultures of B. mallei was isolated in Triazol reagent from cultures grown to an OD600 of 0.1 in LB medium. Selected cultures were treated with 1 mM DEANO for 30 min before the isolation of the RNA. Synthesized cDNA was labeled as previously described for Mycobacterium tuberculosis2O, 250 µl 20× SSC, 10 µl 10% SDS 10 µl). Samples were heated at 98°C for 2 min, cooled 5 min at 25°C and placed on pre-hybridized Burkholderia array slides (Colorado State University RM-RCE Genomics Core). Hybridization was conducted in standard hybridization chambers overnight in a 42°C water bath and washed with 1× SSC and 0.05% SDS, followed by two washes in 0.06× SSC. The slides were scanned using a Genepix Axon 4000B scanner. Initial data analysis was conducted using Genepix Pro software, followed by analysis using Microsoft Excel as described RNA from B. mallei grown in LBG medium were pelleted by centrifugation and resuspended in an equal volume of LB medium. Selected cultures containing 36 OD were treated with either 500 µM or 1 mM spermine NONOate for 30 min. The cultures were pelleted by centrifugation at 4°C, washed with 50 mM Tris-HCl buffer, pH 7.6 and the cytoplasmic proteins extracted in 400 µL B-PER  after vortexing for 30 sec. The samples were diluted 1∶2 in 50 mM Tris-HCL buffer, pH 7.6. After removing the insoluble fraction by centrifugation, the supernatants were filter-sterilized. Aconitase activity in the soluble fractions was estimated spectrophotometrically at 240 nm by following the formation of cis-aconitate in 50 mM Tris-HCl buffer pH 7.4 containing 30 mM isocitrate. Aconitase activity is expressed as ΔOD240/min/µg protein.3 mL of stationary phase 5 per chamber of a 8-well Permanox Labtek chamber slide system . The macrophages were challenged with B. mallei at an MOI of 10 as described above. Between 2 and 3.5 h after infection, the cells were fixed in 2.5% glutaraldehyde in phosphate buffer, pH 7.4. The specimens were postfixed in 1% osmium tetraoxide, treated with uranyl acetate, dehydrated in ascending ethanol series, and infiltrated with Embed 812. Ultrathin sections were examined in a FEI Technai 62 electron microscope operated at 80 kV.The phagocytes were plated as described above for killing assays at a density of 4×105 cells/well were infected as described above with GFP-expressing B. mallei. The specimens were washed in PBS 3 h after infection and fixed overnight at 4°C in 2% paraformaldehyde  diluted in PBS. After fixation, the coverslips were washed in PBS and the cells were permeablized for 5 min with 0.1% Triton X-100 in PBS. After washing, F-actin was visualized by labeling the infected macrophages with Alexa Fluor 568 phalloidin (Invitrogen). The coverslips were washed with PBS and mounted onto glass slides prepped with Vectashield mounting medium containing DAPI nucleic acid stain . The specimens were examined with a Zeiss Axiophot fluorescence microscope equipped with a charge-coupled-device camera controlled by the SlideBook deconvolution image-processing software .Macrophages plated on 13 mm glass coverslips in a 24-well plate at a density of 2.5×10t-test was used for statistical analysis and the data considered statistically significant when p<0.05.Data are presented as mean ± SEM or in box-and-whiskers plots as median, intraquartile and total ranges. A two-tailed, Student's B. mallei internalized by J774 cells increased as a function of time, reaching approximately 106 CFU/105 macrophages 2 h after infection. Because of this delay phagocytosis, all subsequent Burkholderia infections, unless indicated, were carried out for 2 h before gentamicin was added to the medium. Mice offer a wide variety of genetic reagents that facilitate investigations of O2-dependent host defenses in host–pathogen interactions. Therefore, we deemed it important to compare the intracellular growth of B. mallei in a number of murine  and human  cell lines  the anti-B. mallei activity of primary macrophages isolated from C57BL/6 mice. It should be noted that some of the IFNγ-primed macrophages completely eliminated B. mallei from the cultures, a phenomenon that was seen in all of the IFNγ-primed, gp91phox-deficient macrophages  and the stimulation of TLR4 signaling by LPS purified from B. malleiOB. mallei are selectively killed by RNS generated by IFNγ-activated macrophages. These investigations place B. mallei among the limited number of microorganisms for which NO congeners are microbicidal. Our biochemical and genetic studies indicate that [Fe-S] clusters are critical targets of RNS-mediated killing of B. mallei. The extraordinary hypersusceptibility of B. mallei to RNS raises the intriguing possibility that NO donors could be explored as antibiotics to treat glanders, as shown in the experimental treatment of cutaneous mycosis and leishmaniasis In summary, our studies indicate that despite its susceptibility to ROS and RNS,


Securin is a regulator of chromosome separation and its overexpression has been shown to be involved in different tumour-promoting processes, like transformation, hyperproliferation and angiogenesis, and correlates with tumour cell invasion. However, the molecular mechanism leading to securin overexpression in human colorectal cancer is unknown. Here we show a correlated high expression of β-catenin and securin (hPTTG1) in colorectal adenomas and carcinomas and further demonstrate that securin is a target of β-catenin transcriptional activation. This implies that deregulation of the β-catenin/T-cell factor-signalling pathway leads to overexpression of securin in human colorectal cancer, which subsequently may contribute to tumour progression.Overexpression of the transcriptional activator  The effect is an accumulation of the coactivator protein β-catenin, which translocates to the nucleus and functions as a composite transcription factor together with the DNA-binding proteins of the T-cell factor (TCF/LEF) family  is an oncogene with little expression in normal adult colon tissue . This study comprised well characterised moderate-to-well differentiated colorectal adenocarcinomas and mucosa from six patients. Total RNA was extracted from each tissue sample using the RNeasy kit  including an DNA digest according to the manufacturer's instructions. Linear amplification of RNA was carried out with the MessageAmp aRNA kit . Biotin-labelled cRNA was hybridised to the HG-U133A oligonucleotide microarray . All reactions were performed according to the Affymetrix protocol.Detailed protocols and results will be published elsewhere  and normal mucosa (N). P-values were calculated and corrected in the multiple testing context in order to obtain control over the false discovery rate (FDR), that is, the fraction of false positives in a candidate list of probe sets. Making use of the Benjamini–Hochberg procedure . The expression values were transformed to log-2 scale and normalised by a non-linear procedure based on a scatterplot smoother . All samrocedure  we identβ-catenin/TCF target genes was obtained from the homepage of Joel Nusse (http://www.stanford.edu/~rnusse/wntwindow.html) and expanded by recent reports found in the literature (see for review β-catenin/TCF target genes of which 42 were represented by one or more probe sets on the GeneChip array. We identified a cluster of 13 target genes (including securin) that are (i) significantly regulated in the sense that they are represented by at least one probe set contained in the list of 3018 candidates and (ii) upregulated in tumour tissue compared to normal mucosa.Special attention was paid to targets of the Wnt-signalling pathway. A list of known ATCAAAGGATAGAATTT-3′; securin-mutTBE: 5′-ATAAATCA CTGCCAAAGGAT AGAATTT-3′. Probes were end labelled and incubated with 0.5 μg of bacterially expressed GST-TCF-4 DBD (DNA-binding domain codon 265–496) or GST alone as described previously : securin-TBE: 5′-ATAAATCACTsecurin promoter–reporter plasmid (pGL-Sec. fl.) was constructed by amplification of the primers U1 and D1 described previously .The eviously  using thHuman colon carcinoma cell lines SW480, DLD1 and HeLa (human cervix carcinoma) cells were obtained from the American Type Culture Collection (ATCC). Cells were cultivated under standard conditions in Dulbecco's modified Eagle's medium +10% fetal bovine serum. Transfections and reporter assays were carried out as described previously (β-catenin (CAGUUGU GGUUAAGCUCUUdTdT) or GFP (AAGCUACCUGUUCCAUGGCCAdTdT) as control and Oligofectamine  was used for transient transfection of the colorectal cancer cells according to the instructions of the manufacturer. After 72 h incubation time, cells were harvested, total RNA isolated and real-time reverse transcription—PCR (RT–PCR) was performed as described  specific for securin  in six human colorectal carcinomas compared to the corresponding normal mucosa . Similarly, securin overexpression was found by real-time PCR analysis of seven different colorectal tumours compared to the corresponding mucosa  were identified  and tested for interaction with TCF-4 in electromobility shift assays (EMSA). One TBE  in the β-catenin-overexpressing colorectal carcinoma cell lines SW480 and DLD1, the securin promoter (pGL Sec fl.) exhibits strong transcriptional activity, 74 and 35-fold over background (pGLbasic). Addition of dominant-negative TCF-4 (dnTCF), which lacks the N-terminal β-catenin interaction domain, reduced the securin promoter activity in a dose-dependent manner by up to 51 and 44 % . In and 44 % . As it ill cycle . Unsynchβ-catenin/TCF-signalling pathway, the activity of the securin promoter fragment (−1336 to −625) was enhanced by overexpression of TCF4 and β-catenin in a dose-dependent manner -siRNA; β-catenin expression by β-catenin-specific siRNA transfection was confirmed by Western blot in control experiments , is significantly overexpressed in colorectal cancer compared to normal mucosa using DNA microarray analysis, real-time PCR and immunohistochemistry. Moreover, we found a functional TCF-binding element in the securin promoter sequence identical to the TBE found to be functional in esophageal squamous cell carcinoma, published during the preparation of this manuscript  reduces β-catenin transcriptional activity, but does not completely abolish it, which would be necessary for inducing a G1-phase arrest of the cell cycle  proximal to the transcriptional start site contains additional regulatory elements (β-catenin/TCF in the regulation of the endogenous securin gene by transfection of colorectal carcinoma cells with β-catenin-specific siRNA. Owing to the transient transfection procedure used, no complete abrogation of β-catenin expression was observed and cell cycle progression was not changed. However, a potent inhibition of β-catenin expression was induced, resulting in a significant reduction of the endogenous securin gene expression. Even though we do not exclude the possibility of an indirect effect of β-catenin on securin expression by affecting cell cycle progression, our results show the relevance of β-catenin for the direct regulation of securin gene expression in colorectal tumour cells.Additionally, we show that securin is deregulated in many human tumours and is able to contribute to several main aspects of tumorigenesis. In several tumours, overexpression of securin is associated with tumour metastasis and poor clinical outcome and has been identified as a significant prognostic marker in esophageal carcinoma (securin as a Wnt target gene activated early in the adenoma–carcinoma sequence further supports a decisive role of aberrant overexpression of β-catenin in all phases of colorectal carcinogenesis.The oncogene