The amyloid precursor protein (APP) is genetically associated with Alzheimer's disease (AD). Elucidating the function of APP should help understand AD pathogenesis and provide insights into therapeutic designs against this devastating neurodegenerative disease.33, Ser37, and Thr41 (S33/37/T41) residues, which is a prerequisite for β-catenin ubiquitinylation and proteasomal degradation. APP-induced phosphorylation of β-catenin resulted in the reduction of total β-catenin levels, suggesting that APP expression promotes β-catenin degradation. In contrast, treatment of neurons with APP siRNAs increased total β-catenin levels and decreased β-catenin phosphorylation at residues S33/37/T41. Further, β-catenin was dramatically increased in hippocampal CA1 pyramidal cells from APP knockout animals. Acute expression of wild type APP or of familial AD APP mutants in primary neurons downregulated β-catenin in membrane and cytosolic fractions, and did not appear to affect nuclear β-catenin or β-catenin-dependent transcription. Conversely, in APP knockout CA1 pyramidal cells, accumulation of β-catenin was associated with the upregulation of cyclin D1, a downstream target of β-catenin signaling. Together, these data establish that APP downregulates β-catenin and suggest a role for APP in sustaining neuronal function by preventing cell cycle reactivation and maintaining synaptic integrity.We demonstrate that APP expression in primary neurons induces β-catenin phosphorylation at Serin vitro and in vivo. Future studies may investigate whether APP processing is necessary for β-catenin downregulation, and determine if excessive APP expression contributes to AD pathogenesis through abnormal β-catenin downregulation.We have provided strong evidence that APP modulates β-catenin degradation  In the radation -6. SignaSeveral lines of evidence suggest that APP influences β-catenin regulation. APP binds to APP-BP1 which activates the small ubiquitin-like protein Nedd8 ,8. Activin vitro and in vivo. We provide critical evidence that APP downregulates β-catenin in neurons.Both APP and Presenilins are genetically associated with AD. In addition to its role in APP processing, Presenilin-1 (PS1) also interacts with the cadherin/catenin adhesion complex  and affe2 according to the protocol approved by the Institutional Animal Care and Use Committee. The cortices from embryonic brains were gently triturated with a fine-tipped transfer pipette right after dissection. Single cells were counted and immediately plated in Neurobasal medium containing B27 supplements , 0.5 mM glutamine, 1% FBS, 1% equine serum, and 1× penicillin/streptomycin  at a density of 4 × 106 per 60-mm dish or 4 × 105 per well of 24-well plate on poly-D-lysine-coated dishes. The medium was replaced with fresh medium at 2 h after plating. Half of the medium was replaced with fresh medium every three days. The adult rat brain and other organs were dissected and frozen on dry ice before lysis and immunoblot analyses. Primary neurons were used at 7 DIV unless noted otherwise.Pregnant Sprague-Dawley rats  were euthanized after being rendered unconscious with CO695), mycAPP-BP1, and LacZ  in the pHSVprPUC vector [Constructs of full length APP , or at 0.5 or 1 IU per cell . At 14–15 h after infection, cells were washed with cold phosphate-buffered saline (PBS) and sonicated in lysis buffer . Protein concentrations were determined by the bicinchronic acid (BCA) method; equal amount of protein was boiled in SDS sample buffer and resolved on SDS-PAGE gels. The following antibodies were used: rabbit anti-β-catenin and mouse anti-γ-tubulin (both from Sigma); anti-phosphorylated β-catenin antibodies, S33/37/T41 and S45 ; rabbit anti-APP antibody . Chemiluminescence reactions were carried out using ChemiGlow (Alpha Innotech) and the blot digital images were collected by the AlphaEase FC™ Imaging System and the FluorChem™ IS-8800 software (Alpha Innotech). Multiple exposures of each blot were routinely taken, and the exposure that had specific protein bands within the linear range in optical density was used for densitometry.t-Tests (more stringent) for two samples assuming unequal variance using the EXCEL data analysis tool (Microsoft Corporation). A p value of 0.05 or less was considered to be statistically significant.Quantitative western blot analyses were based on three or more independent experiments after densitometry tracing of specific protein bands using the Scion Image software . The optical densities of the protein bands in equal area of space were determined after subtracting the background per individual lane. In order to analyze the results across different experiments, the optical density levels were then normalized to γ-tubulin expressions in the respective western blot. The normalized density units were then analyzed by one-tail or two-tail g for 10 min and resuspended in buffer HD  with protease inhibitors. Membrane fractions were collected from the postnuclear supernatants by centrifugation for 20 min at 14,000 × g and resuspended in buffer HD with protease inhibitors. Proteins in the supernatant (cytosolic fraction) were precipitated with 20% trichloroacetic acid. The precipitated proteins were washed with 95% ethanol. Protein pellets were resuspended in 1% Triton X-100 containing protease inhibitor mix (1:250). Equal amount of proteins from membrane, cytosolic or nuclear fractions were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and analyzed for β-catenin by western blot analyses.The post-nuclear membrane fraction was isolated as described . BrieflyHEK293 cells were transfected with pGL3-OT or pGL-OF   and APP Primary neurons were fixed with 4% paraformaldehyde for 40 m at room temperature. Cells were then washed with PBS, blocked with 3% normal goat serum, and stained with rabbit anti-β-catenin (Sigma) for 2 h at room temperature. Cells were then washed and incubated with Alexa Fluor 488-conjugated goat anti-rabbit for 1 h (Molecular Probe). Counterstaining with DAPI was performed in some cells by incubating for 2 m with DAPI (20 μg/ml). After 4× washes in PBS, cells were mounted in 90% glycerol. Negative controls were without primary antibodies. The specificity of the anti-β-catenin antibody was also demonstrated in western blots. Confocal images were taken under a 63× objective with a Nikon D-Eclipse C1 confocal microscope through z-series at the same settings. Individual z-sections and composite images were further grouped in Photoshop .t-Test assuming unequal variance .APP knockout animals were raised and genotyped as described before  and WT controls  . Brain s695 was expressed in primary neurons using HSV-1 vector-mediated gene transfer. An increase in APP levels resulted in a significant decrease in total β-catenin  [p < 0.02) without significantly altering S45 phosphorylation. Representative blots along with quantitative analyses are presented in Figure Levels of β-catenin are tightly regulated by phosphorylation, ubiquitinylation, and proteasomal degradation. Amino acids S33/37/T41 of β-catenin are co-ordinately phosphorylated by GSK-3β before ubiquitinylation and degradation. Casein kinase 1 additionally phosphorylates β-catenin at Sert-Test revealed that suppressing the endogenous APP by this means resulted in a significant increase in total β-catenin compared to the control (missense shRNA)  targeting rat APP in primary neurons; no human APP was expressed in these experiments. Protein levels were quantified by densitometry, with tracing and normalization to the levels of γ-tubulin on each blot. Statistical analyses by NA) Fig. . APP dowThe function of β-catenin differs depending on its cellular location. We sought to determine if APP expression differentially affects β-catenin levels in crude preparations of membrane, cytosolic or nuclear fractions. Three forms of APP were tested: wild-type, Swedish mutation (Swe), and London mutation (V642I). In primary neurons, wild-type APP and each of the FAD mutants dramatically reduced total β-catenin compared to the control (LacZ) in both membrane and cytosolic fractions Fig. . APP decIn contrast to membrane and cytosolic β-catenin, nuclear β-catenin did not appear to change upon expression of wild-type or FAD APP Fig. . To obtaTo complement the above experiments, confocal microscopy was used to assess β-catenin levels and cellular locations in primary neurons. Primary neurons expressing APP or LacZ (control) were fixed and labelled with the rabbit anti-β-catenin antibody and the Alexa fluor 488 goat anti-rabbit antibody. Representative z-sections of the same cells are presented in Fig. The results presented above show that APP mediates β-catenin degradation in primary neurons. We next sought to determine if β-catenin levels are altered in CA1 pyramidal cells from APP knockout animals. Fig. In this report, we provide strong evidence that APP mediates β-catenin downregulation. We demonstrate that APP downregulates β-catenin levels in primary neurons by facilitating specific phosphorylation of S33/37/T41 residues in β-catenin. APP expression is inversely correlated with the levels of total β-catenin. Corroborating data were obtained using APP siRNAs: suppression of APP by siRNAs increased steady-state β-catenin levels and reduced the levels of S33/37/T41 phosphorylation in neurons. We further show that β-catenin accumulates in hippocampal pyramidal cells in APP knockout animals. Together, these data establish that APP is physiologically important in maintaining β-catenin homeostasis, which is likely critical for neuronal survival and function by preventing aberrant cell cycle reaction.We show that familial AD mutants of APP induce similar β-catenin degradation, suggesting that familial AD mutants retain similar functions. Our method might not be sensitive enough to detect the degree of differences in β-catenin downregulation by these APP mutants upon acute expression in cell cultures. Long-term effects of APP mutants on β-catenin expression could be analyzed in transgenic animals. In primary neurons, APP-induced β-catenin downregulation did not appear to affect nuclear β-catenin or its transcriptional activity. However, β-catenin accumulation in CA1 pyramidal neurons from APP knockout animals does appear to affect transcriptional activity, judging from the increases found in cyclin D1 protein levels in these same neurons. These observations illustrate the limitations of luciferase reporter assays in primary neurons; they also suggest that basal levels of β-catenin are necessary for neuronal function, but that these do not necessarily affect β-catenin-mediated transcription.1–42 in primary neurons [1–42 also appears to downregulate β-catenin (YC unpublished observations). However, it remains to be determined whether APP and Aβ1–42 modulate β-catenin degradation through the same pathways.Several questions remain to be answered, such as whether APP processing products, such as secreted APP, or Aβ, mediate the effect of APP on β-catenin degradation. The effect of APP on β-catenin phosphorylation could also reflect the interaction between APP and APP-BP1. APP-BP1 activates neddylation and increases ubiquitinylation by the Cullin-based E3 ligase. Ubiquitinylation of β-catenin may be facilitated by the interaction between APP and APP-BP1, which is not unidirectional as indicated by the studies in Drosophila . We have neurons . Aβ1–42 The roles of APP and PS1 on β-catenin phosphorylation may be independent of each other. We show that APP facilitates β-catenin phosphorylation at S33/37/T41 residues. Others have shown that PS1 expression can enhance phosphorylation of β-catenin at S45, which may prime the phosphorylation of S33/37/T41  and faciOur new findings not only support a critical role for APP in maintaining neuronal differentiation but also support a role for APP in synaptic dynamics due to the multifaceted functions of β-catenin ,41. Cellin vivo and in vitro. These findings open new avenues for research involving the role of APP processing in β-catenin downregulation. Future studies may also determine whether excessive APP expression contributes to AD pathogenesis involving abnormal β-catenin phosphorylation and degradation in transgenic animals.In summary, this report documents a function of APP in β-catenin downregulation APP, amyloid precursor protein; AD, Alzheimer's disease; Aβ, beta-amyloid; in vitro; DIV, day siRNA, small interfering RNA; shRNA, small hairpin RNA.The author(s) declare that they have no competing interests.YC conceptualized and designed all the experiments and prepared this manuscript. Both YC and AMB collected data described in this manuscript.


Homologous chromosomes separate in meiosis I and sister chromatids separate in meiosis II, generating haploid gametes. To address the question why sister chromatids do not separate in meiosis I, we explored the roles of Shogoshin1 (Sgo1) in chromosome separation during oocyte meiosis.Sgo1 function was evaluated by exogenous overexpression to enhance its roles and RNAi to suppress its roles during two meioses of mouse oocytes. Immunocytochemistry and chromosome spread were used to evaluate phenotypes. The exogenous Sgo1 overexpression kept homologous chromosomes and sister chromatids not to separate in meiosis I and meiosis II, respectively, while the Sgo1 RNAi promoted premature separation of sister chromatids.Our results reveal that prevention of premature separation of sister chromatids in meiosis I requires the retention of centromeric Sgo1, while normal separation of sister chromatids in meiosis II requires loss of centromeric Sgo1. One of the major differences between meiosis and mitosis is that the former consists of two consecutive rounds of chromosome separation with only one round of DNA replication, in which chromosome number is reduced to half to produce haploid gametes. Errors in this process result in aneuploidy The Shugoshin (Sgo) family of proteins has been demonstrated to protect centromeric cohesion during mitosis and meiosis in fission yeast 6-Sgo1 mRNA was injected into oocytes to examine the dynamics of Sgo1 localization at metaphase and anaphase of both meiotic divisions. Then anti-myc-FITC antibody was used for immunofluorescence. The same amount of Myc6 mRNA was injected as control, but no specific signals were found. All 40 chromosomes of mouse oocytes are telocentric, so homologs form bivalents in meiosis I while sister chromatids form univalents in meiosis II To characterize the roles of Sgo1 in chromosome separation during mouse oocyte meiosis, due to the lack of working antibody in mice, low concentration of (≤0.4 mg/ml) MycTo confirm the hypothesis that the loss of Sgo1 is required for the separation of sister chromatids in AII, Sgo1 overexpression experiments were performed in MII oocytes. MII oocytes were injected with high concentrations (≥2.5 mg/ml) of Myc6-Sgo1 mRNA and cultured to AI stage . The resulting images revealed that exogenous overexpressed Myc-Sgo1 along the arms of sister chromatids attached them tightly so that sister chromatids could not separate apart , arrow, 6-Sgo1 mRNA and then arrested at GV stage by 2.5 µM milrinone for 2 hours. After 14 hours of culture in milrinone-free medium, exogenous Sgo1 overexpression significantly decreased extrusion of the first polar body (PBE), from 82.2±3.3% in the control to 55±2.4% in the injection group (P<0.05)  stage were injected with high concentrations (≥2.5 mg/ml) of Myc(P<0.05) . Then we(P<0.05) a. A typi(P<0.05) a, inset.(P<0.05) c, arrow.(P<0.05) , arrow. (P<0.05) . In thisAs exogenous expression of Sgo1 was able to hold homologous chromosomes and sister chromatids tightly together, we were curious about the effects of RNAi. We employed RNAi to delete Sgo1 to analyze separation of homologs and sister chromatids during meiosis. SiRNAs against Sgo1 was injected into oocytes at GV stage. Then the oocytes were arrested at GV by 2.5 µM milrinone for 24 hours before meiosis resumption. We confirmed the long-time GV-arrested oocytes' quality by morphological evaluation under the optical microscope and chromosome spread. The ratio of germinal vesicle breakdown (GVBD) and extrusion of the first polar body (PBE) was not statistically different from normal cultured oocytes without inhibition (data not shown). In addition, the chromosome spread images also did not show misaligned chromosomes. The efficiency of Sgo1 siRNAs was measured by real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. The results indicated that the amount of Sgo1 mRNA was largely reduced . AccordiThe oocytes injected with Sgo1-m siRNA were cultured to MI (8h) or MII (14h) to perform chromosome spreads. The ratio  of PBE in Sgo1-RNAi oocytes was not distinctly different from the control . The chromosome spreads showed that depletion of Sgo1 did not affect the bivalents in MI stage c. After Our RNAi results are like those most recently reported by Lee et al We also injected Sgo1-m siRNA into zygotes and cultured them for 4.5 days to examine the blastocyst rate. The blastocyst ratio  of the Sgo1 RNAi groups was significantly lower than that of the control groups  (P<0.01) . Sgo1 isIn conclusion, prevention of premature separation of sister chromatids in meiosis I requires retention of centromeric Sgo1, while normal separation of sister chromatids in meiosis II requires loss of centromeric Sgo1. Given that Sgo1 is highly conserved among species, mutations in human Sgo1 might increase the frequency of aneuploidy in gametes, which would be a significant cause for genetic disorders.All chemicals and medium were purchased from Sigma Chemical Company  unless stated otherwise.2+/Mg2+-free CZB. For in vivo zygote collection, one female was placed in a cage with one stud male after hCG injection, and the zygotes were collected the next morning. After microinjection, the zygotes were cultured in KSOM for 4.5 days to examine blastocyst rates.ICR mice care and handling were conducted in accordance with policies promulgated by the Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. The oocytes were collected in M2 medium supplemented with 2.5 µM milrinone GGCCGAGATGAATTTCACTATG, R1: GGTCCACGACAGTGCTATTATTC, F2: AGCCCAAGCATAAATCTATGAC, R2: GATCCTCACCCACTTATGTCTTAC. To detect the expressed protein, the Sgo1 cDNA was then NH2-terminally Myc6-tagged. For in vitro transcription reactions, the Myc6-Sgo1 cDNA was subcloned into the modified pRN3p vector , which has a globin 3′ UTR plus a short poly A tail.Total RNA was extracted from 100 mouse GV oocytes using RNeasy micro purification kit (Qiagen), and the first strand cDNA was generated with cDNA synthesis kit (Takara), using poly (dT) primers. The following two nested primers were used to clone the full length of Sgo1 cDNA by PCR. F1: 6-Sgo1-pRN3p plasmid was linearized by SfiI and purified by gel extraction kit (Qiagen). T3 message machine (Ambion) was used for producing capped mRNA which was purified using the RNeasy cleanup kit (Qiagen). The concentration was detected by Beckman DU 530 Analyzer, and diluted into low concentration (0.4 mg/ml) for localization tract or high concentration (2.5 mg/ml) for overexpression of protein.The Myc6-Sgo1 expression, 0.4 mg/ml (or 2.5 mg/ml for overexpression) Myc6-Sgo1 mRNA solution was injected into cytoplasm of GV stage oocytes. Oocytes were arrested at GV stage in 2.5 µM milrinone for 2 hours. The same amount of H2O or Myc6 mRNA  was injected as control. Each experiment consisted of three separate and replicate groups and approximately 100 oocytes were injected in each group. The ratio of GVBD or PBE was counted under an inverted optical microscope. For MII oocyte overexpression experiments, oocyte-cumulus complexes (COC) were cultured in M16 supplemented with 10% FBS for 12 hours and nearly 100% of oocytes extruded first polar bodies. High concentration (≥2.5 mg/ml) of myc6-Sgo1 mRNA was injected into cytoplasm and the oocytes were cultured for 2 hours before they were parthenogenetically activated by 10 mM SrCl2.Microinjections were performed using an Eppendorf microinjector  and completed within 30 minutes. For MycGCUAACUUCCCGACAAAGUtt; Sgo1-2 siRNA, GCAUUGACAAAUACGACCAGtt; Sgo1-3 siRNA, CCAAAUUAGCUUAUGUUCUtt; or Sgo1-m siRNA, the mixed solution of the above three. The same amount of negative control siRNA (Qiagen) was also injected as control. After microinjection of GV oocytes, the oocytes were cultured for 24 hours in M16 supplemented with 10% FBS and 2.5 µM milrinone to prevent meiosis resumption.Small interfering RNAs (siRNAs) of Sgo1 siRNAs (Ambion) were microinjected into cytoplasm to deplete Sgo1. The following 25 µM siRNAs was used. Sgo1-1 siRNA, TGGAGGTATTGGTTCCTGTGATG, reverse, CTGCATTCGAGGTCACTCACTTC. H2afz, forward, ACAGCGCAGCCATCCTGGAGTA, reverse, TTCCCGATCAGCGATTTGTGGA. Real-time PCR was used SYBR Premix Ex Taq™ kit (Takara) in ABI prism 7000 Sequence Detection System. The steps include 95°C 10s, 40 cycles of 95°C 5s and 60°C 31s.Analysis of relative gene expression was measured by real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Immunofluorescence was performed as described previously Data (mean±SE) were from at least three replicates per experiment and analyzed by ANOVA using SPSS software  followed by the student-Newman-Keuls test. Differences at P<0.05 were considered to be statistically significant.


Wnt canonical signaling pathway plays a diverse role in embryonic development and maintenance of organs and tissues in adults. It has been observed that Wnt/β-catenin signaling pathway is involved in the pathogenesis of many carcinomas. Moreover, Wnt/β-catenin pathway has been revealed to be associated with angiogenesis. Wnt canonical pathway signaling has great potential as a therapeutic target. It has been disclosed that some hematological malignancies, such as chronic lymphocytic leukemia, mantle cell lymphoma, may occur partly due to the constitutive activation of Wnt canonical signaling pathway. This review will summarize the latest development in Wnt canonical signaling pathway and its roles in tumorigenesis and angiogenesis. Wnt canonical signaling pathway acts a significant part in embryonic development and in maintenance of organs and tissues in adults. In the past two decades, medical scientists have devoted themselves to understanding the cellular and molecular mechanisms of Wnt signaling. A lot of studies indicate that Wnt canonical pathway involves in the pathogenesis of a range of disease including many kinds of carcinomas. Hematological malignancies are the types of carcinoma that affect blood, bone marrow and lymph nodes. They may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The incidence of hematological malignancies has been increasing steadily in the world for the past years, but their etiology and pathogenesis has not been well understood involving areas of chromosome aberrations, apoptosis inhibition, abnormal activation of signaling pathways, angiogenesis, et al. In this review, we focus on the role of Wnt canonical signaling in carcinomas, especially in hematological malignancies, and then disclose potential therapeutic opportunities of this pathway in hematological malignancies.Wnt signaling pathways are categorized as "canonical "and "non-canonical" Wnt pathways, which are β-catenin-dependent and β-catenin-independent signaling pathways, respectively. Here we will emphatically point out the role of Wnt canonical pathway in hematological malignancies. A simplified model of Wnt canonical pathway is delineated in Fig. Wnt canonical signaling is involved in pathogenesis of several carcinomas and the mechanisms of its over-activation are varied. Dysregulation of Wnt/β-catenin signaling plays a central role in early events in colorectal carcinogenesis. The APC protein which acts as a tumor suppressor protein can down-regulate the transcriptional activation mediated by Wnt/β-catenin. Therefore, inactivation of APC tumor suppressor gene caused by mutation is related to the initiation of colorectal neoplasia and its protein products lose the function of down-regulation of Wnt signaling. Then, colorectal cancer occurs. Furthermore, mutations of β-catenin in the functionally significant phosphorylation sites have been detected in colorectal tumors. In melaSince aberrant activation of Wnt canonical signaling pathway is diversely involved in pathogenesis of carcinomas, there has been great interest in developing therapeutics that circumvent it either by inhibiting Wnt mediated transcription or by inactivating the target genes. In some carcinomas, Wnt canonical signaling pathway has become the potential therapeutic target. The stabilized nuclear β-catenin due to the aberrant activation of Wnt signaling is an attractive therapeutic target for human cancers. Non-steroid anti-inflammatory drugs(NSAIDs) can suppress the activity of β-catenin. These drugs may inhibit Wnt/β-catenin signaling at multiple levels, including induction of β-catenin degradation and disrWnt signaling pathway has been observed to make a difference in vessel development and pathology and in survival and proliferation of primary endothelial cells. Several Wnt ligands have been demonstrated to be expressed in vascular endothelial cells in vitro, including Wnt-7a, Wnt-10b and in vascular smooth muscle cells including Wnt-5a. In vivoAngoigenesis is essential for tumor growth and metastasis. Studies have revealed the close relationship between canonical Wnt signaling pathway and angiogenesis of carcinomas. β-catenin accumulation has been involved in angiogenesis in brain cancer. β-catenin is found in the cytoplasm and nucleus of endothelium in neovessels of rat N-ethyl-N-nitrosurea-induced gliomas and in tIt is believed that Wnt signaling pathway is vital for tumor neovascularization and is a great potential in blocking tumor invasion and metastasis. To further confirm the role of Wnt/β-catenin signaling pathway in tumor angiogenesis and growth, Wnt antagonists WIF1-Fc and sFRP1-Fc were used to treat hepatocellular carcinoma tumors. They revealed that these two fusion proteins could inhibit Wnt signaling and exerted potent antineoplastic activity by increasing apoptosis of tumor cells and by impairing tumor vascularization; including reducing the microvessel density, decreasing expression of vascular endothelial growth factor and stromal cell-derived factor-1.Hematopoiesis is a continuous process in which stem/progenitor cells develop into mature blood cellular components. Wnt/β-catenin signaling pathway has been shown to have an effect on controlling the proliferation, survival and differentiation of hematopoietic cells. The genAberration of Wnt pathway and the related proteins are detected in many hematological patients. ActivatEpstein-Barr Virus (EBV) is consistently detected in the endemic form of Burkitt's lymphoma (BL). An increase in both free and total β-catenin was seen in EBV-infected BL cells compared to EBV-negative cells. The invWnt canonical signaling pathway is not only involved in cell survival, differentiation, apoptosis and maintenance of homeostasis, but also related to the pathogenesis of many carcinomas and hematological malignancies. Moreover, Wnt/β-catenin pathway has been revealed to be associated with angiogenesis of tumors. Its aberration has been detected in leukemia, myeloma and lymphoma. Canonical Wnt signaling may act as a potentially useful therapeutic target for hematological malignancies. Ultimately, further investigation is needed to interfere with Wnt signaling which may lead to new anti-cancer therapies.The authors declare that they have no competing interests.Both authors participated in drafting and editing the manuscript. Both authors read and approved the final manuscript.


P < 0.05 for all). In the survival analysis, reduced α-catenin expression (P = 0.06) and cytoplasmic signal (P = 0.04) were related to unfavourable patient outcome. In the multivariate analysis, including TM-classification and Gleason score, α-catenin expression had independent prognostic value in T1–2 M0 tumors. In the M0 tumours, abnormal α-catenin signal was independently associated with recurrence-free survival as well. The results indicate that down-regulation of α-catenin is related to several malignant cellular features, and it seems to have prognostic significance in the early phases of cancer progression. We suggest that α-catenin expression can provide prognostic information in early prostate cancer. © 1999 Cancer Research CampaignNormally functioning cell–cell adhesion plays an important role in the maintenance of tissue architecture and cell cohesion. E-cadherin is an important adhesion molecule of epithelial cells. In many types of cancer the expression of E-cadherin is reduced leading to increased risk of disease progression. α-Catenin is one of the intracellular elements of the E-cadherin–catenin complex. The abnormalities in the expression of α-catenin seem to associate with malignant cellular features and disease progression in prostate cancer. To further analyse the significance of α-catenin expression, we studied 215 cases of prostate cancer by immunohistochemistry and the results were related to other known prognostic factors and patient survival during a mean follow-up period of 13 years. α-Catenin expression was down-regulated in 19% of the cases and 3% of the tumours were totally α-catenin-negative. The abnormal α-catenin expression and cytoplasmic signal were significantly linked with high T-category, metastatic disease, high Gleason score, perineural growth, high mitotic rate, high S phase fraction and DNA aneuploidy (


In this paper we are tying on this problem introducing a measure called normalized compression distance with each other. Second, it provides the quantification of an error that could have been encountered by comparing samples of finite size from the underlying processes. Hence, the statistic complexity provides a statistical quantification of the statement ‘This complexity measure is different to all other measures in the following senses. First, it is a bivariate measure that compares two objects, corresponding to pattern generating processes, on the basis of the The presented approach, ultimately, transforms the classic problem of assessing the complexity of an object into the realm of statistics. This may open a wider applicability of this complexity measure to diverse application areas. This property of complex systems has fascinated generations of scientists an object is said to be ‘complex’ when it does not match patterns regarded as simple, as López-Ruiz et al. effective measure complexity (EMC) excess entropypredictive informationthermodynamic depthitányi et al. that evaluates the complexity distance among two objects normalized compression distance (NCD) Complex systems is the study of interactions of simple building blocks that result in a collective behavior or properties absent in the elementary components of the system itself. Due to the fact that this problem does not fit into one of the traditional research fields, it is connected to various of these, for instance physics, biology, chemistry or econometrics statistic complexity that is not only different to all other complexity measures introduced so far, but also connects directly to statistics, specifically, to statistical inference normalized compression distance with each other. Second, this measure provides the quantification of an error that could have encountered by comparing samples of finite size from the underlying processes. Hence, the statistic complexity provides a statistical quantification of the statement ‘The purpose of this paper is to introduce a new measure of complexity we call This paper is organized as follows. In the next section we describe the general problem in more detail and introduce our complexity measure. Then we present numerical results and provide a discussion. We finish with conclusions and an outlook.good if: (1) the complexity of simple and random objects is less than the complexity value of complex objects Currently, a commonly acknowledged, rigorous mathematical definition of the complexity of an object is not available. Instead, when complexity measures are suggested they are normally assessed by their behavior with respect to three qualitative patterns, namely simple, random (chaotic) and complex patterns. Qualitatively, a complexity measure is considered i et al. introduced in normalizedinformationdistance (NID) which can be approximated by,normalizedcompressiondistanceWe start by pointing out that criteria (1) provides a relative statement connecting different objects. That means the complexity of an object is always related to the complexity of another object statistic complexity measure we suggest is defined by the following procedure visualized in Criteria (3) of a complexity measure stated above acknowledges the fact that an assessment of an object's complexity cannot be without uncertainty or error in case only finite information about this object is available. That means, for a complexity measure to be applicable to real objects  it has to be statistic in order to deal appropriately with incomplete information. Based on these considerations, the Estimate the empirical distribution function Estimate the empirical distribution function Determine statistic complexityDefine, statistic complexity corresponds to the p-value of the underlying null hypotheses, average and, thus needs to be connected to two ensembles statistic complexity, it is apparent that we are following closely the guiding principle expressed by López-Ruiz et al. This procedure corresponds to a two-sided, two-sample Kolmogorov-Smirnov (KS) test different processes, namely statistic complexity has the very desirable property that the power reaches asymptotically statistic complexity is a statistical test, it borrows part of its strength from the NCD respectively Kolmogorov complexity on which this is based on. Hence, it unites various properties from very different concepts.Regarding the notation and interpretation of the above procedure it is important to note the following. First, the entities In the following we provide different numerical examples for data frequently used when studying complexity measures. This allows a direct comparison of ours with different measures.statistic complexity we study is the influence of the size statistic complexity is capable to differentiate between random and complex objects. For this reason we compare rule The first characteristic of the statistic complexity  are obtained for each value of statistic complexity and the Lyapunov exponent. This property is certainly desirable to hold because it may allows to connect to traditional contributions in the field beyond the logistic map. The possibility of such a connection, despite the seemingly different methods underlying the statistic complexity respectively the Lyapunov exponent, can be attributed to the parametric form of our complexity measure allowing a flexibility that is entirely missing in other measures. More importantly, this flexibility is not imposed into the measure but follows naturally from a consequent interpretation of complexity as a referential measure quantitatively compared.Next, we apply our measure to the logistic map and compare the results with the Lyapunov exponent  objects from a multitude of different application domains. In the future we are planing to investigate the complexity of biological pathways in the context of cancer and other complex diseases goodness-of-fit tests. For example, it would be interesting to study a Cramér-von Mises or an Anderson-Darling test, instead of a Kolmogorov-Smirnov test From an applied point of view, the direct connection of


Here we describe the Genome Variation Format (GVF) and the 10Gen dataset. GVF, an extension of Generic Feature Format version 3 (GFF3), is a simple tab-delimited format for DNA variant files, which uses Sequence Ontology to describe genome variation data. The 10Gen dataset, ten human genomes in GVF format, is freely available for community analysis from the Sequence Ontology website and from an Amazon elastic block storage (EBS) snapshot for use in Amazon's EC2 cloud computing environment. With the advent of personalized genomics we have seen the first examples of fully sequenced individuals -9. Now, GVF  is an ex- an ontology developed by the Gene Ontology Consortium [While there are richer ways of representing genomic features using XML (Extensible Markup Language) and relational database schemas, simple text-based, tab-delimited files have persisted in bioinformatics because they balance human with computer readability. Since its adoption as the basic exchange format, two aspects of GFF3 have emerged as essential for success. First, it must be simple for software to produce and parse; second, its contents need to be typed using terms drawn from an ontology. The first aspect means that humans can easily read and edit files with a text editor and perform simple analyses with command-line software tools. The second aspect not only constrains different database curators to use the same terminologies, but also, because of the formal structure of the ontology, allows automated reasoning on the contents of such a file. It therefore prevents ambiguities and conflicting terminologies. GVF builds upon these strengths of GFF3, adopting GFF3's simple, tab-delimited format; and like GFF3, the contents of GVF files are described using the Sequence Ontology (SO) nsortium  to descrnsortium ,20. Usinnsortium , and proBelow we describe the GVF standard and the various additions we have made to GFF3 and SO to support it. We also briefly describe the conversion of the first ten publicly available personal genomes into GVF format. These GVF files are available for download and for cloud computation. We will refer to these data as the 10Gen dataset. This is provided as a service to the biomedical community as a reference dataset for whole genome comparative analyses and software development. This dataset will hopefully foster the development of new tools for the analyses of personal genome sequences.We have extended both the GFF3 specification and SO to allow the rigorous description of sequence variations with respect to a reference genome. The first eight columns of a GFF3 file specify the type and source of a feature, its location on a reference sequence, and optionally a score, strand and phase. These columns of data are incorporated into GVF unchanged. The GFF3 format additionally provides the option to append attributes to a sequence feature using tag-value pairs in the ninth column and it is here that GVF specifies additional structure to annotate sequence alteration specific data Table . EffectiThe SO has been extended in order to describe both the nature of the observed variants and the effects that the variants might have. SO is part of the Open Biological and Biomedical Ontologies (OBO) Library , and folFigure GVF provides nine new pragmas to describe the reference sequence and the methods used to call variants. These pragmas are described in detail in Table Each of the rows in a GVF file describes a single variant from an individual or population. Each such variant is typed using the SO terms that can describe SNVs, any size of nucleotide insertion or deletion, copy number variations, large structural variations or any of the 38 terms currently related to sequence alterations in SO. In the case of a seemingly complex variation, such as an SNV located within a translocation, each sequence alteration is annotated relative to its location on the reference genome, on a separate line in the file.The most flexible part of a feature description in GFF3 is the ninth column, where attributes of a feature are given as tag-value pairs Table . It is hWhile a great deal of personal genome variation data today comes from next generation sequencing technologies, the GVF standard can also be used to describe variant data from any source creating DNA variation data with nucleotide resolution, including genotyping DNA microarrays, comparative genomic hybridization (CGH) arrays, and others.Because GVF is a fully compliant extension of GFF3, GVF files provide a basis for exploration and analysis of personal genome sequences with the widely used Bioperl , and GMODrosophila melanogaster) [et al. [Gold standards and reference datasets are invaluable for software development, testing and for benchmarking the performance of algorithms and tool sets. Classic examples in genomics include the CASP (Competitive Assessment of Protein fold recognition) workshop and its datasets for protein structure comparisons ,33, the ogaster) , EGASP (ogaster) ,36, and ogaster)  datasets [et al.  gene exp- and impact upon - other features annotated on a genome.To fulfill the promise of personal whole genome sequencing it will be critical to compare individual genomes to the reference genome and to one another. One lesson learned from comparative genomics analyses -34,37 isRigorously grounding GVF upon the GFF3 specification has many other benefits as well. Because both file formats are typed using the SO, GFF3 and GVF files can be used together in a synergistic fashion. Moreover, because GVF is a formal extension of the GFF3 standard, existing parsers, visualization tools and validation software, such as those developed by the GMOD project  to operaIn order to jumpstart such analyses, we have also manufactured a reference dataset of variants from ten personal genomes, the 10Gen dataset. These genomes represent a diverse assortment of ethnicities, and were produced using a variety of sequencing platforms. Our hope is that the 10Gen dataset will be used as a benchmark for personal genomics software development, following in the footsteps of other successful benchmark datasets, such as those used by CASP ,33 for pUsing OBO-Edit  the SO wThe variant files from the ten genomes were downloaded from web sites indicated in the references listed in Table The GVF standard can also be used to describe genotyping DNA microarray-based variant calls. This flexibility means that a single parser can process variant files from both sequencing and DNA genotyping microarray experiments; moreover, because these fields are attributes of the variant, not the file, a single GVF file can contain variants from heterogeneous sets of sequencing and microarray platforms.The 10Gen dataset is available for download . Each vaDrosophila melanogaster; GFF: General Feature Format; GFF3: Generic Feature Format version 3; GMOD: Generic Model Organism Database; GVF: Genome Variation Format; NCBI: National Center for Biotechnology Information; NGASP: Nematode Genome Annotation Assessment Project; OBO: Open Biological and Biomedical Ontologies; SNP: single nucleotide polymorphism; SNV: single nucleotide variation; SO: Sequence Ontology; VCF: Variant Call Format.CASP: Competitive Assessment of Protein fold recognition; EGASP: ENCODE Genome Annotation Assessment Project; GASP: Genome Annotation Assessment in MGR, MY and KE conceived the project. BM, CB, FS, LS, KE developed technical aspects. BM maintains the GVF specification and data repository. All authors contributed intellectually to the development of the project. MGR, MY and KE wrote the manuscript with input from the authors.


Cell adhesion complexes facilitate attachment between cells or the binding of cells to the extracellular matrix. The regulation of cell adhesion is an important step in embryonic development and contributes to tissue homeostasis allowing processes such as differentiation and cell migration. Many mechanisms of cancer progression are reminiscent of embryonic development, for example, epithelial-mesenchymal transition, and involve the disruption of cell adhesion and expression changes in components of cell adhesion structures. Tight junctions, adherens junctions, desmosomes, and focal adhesion besides their roles in cell-cell or cell-matrix interaction also possess cell signaling function. Perturbations of such signaling pathways can lead to cancer. This article gives an overview of the common structures of cell adhesion and summarizes the impact of their loss on cancer development and progression with articles highlighted from the present issue. Tight junctions are regulators of the epithelial microenvironment as they are responsible for the formation of paracellular barriers see , 1, 2]., 2.1, 2]β-catenin and binds to the same region of the cadherin tail [β-catenin and plakoglobin [β-catenin-deficient cell lines [Desmosomes are adhesion complexes tethered to the intermediate filament, (see figure 1), . Desmosorin tail , 13. Whirin tail , 15. Thekoglobin , and thall lines .Desmoplakin connects desmosomes through binding of plakoglobin to the intermediate filament. It is downregulated in oropharyngeal cancer  and a taβ-catenin expression [The desmosomal cadherins, desmoglein 1, and 3, are targets in two autoimmune diseases, Pemphigus foliaceus and Pemphigus vulgaris, respectively . Bindingpression . Mice wipression . Interespression . Effects of desmosomal perturbations on tumorigenesis rarely share the lime light with the well-known consequences of adherens junction loss. However, loss of desmoglein 1 has been associated with poor prognosis in head-and-neck cancer patients . Contrarβ-catenin, are structural components of the desmosomal plaque and regulate the strength and integrity of cell contacts by facilitating the interaction with the intermediate filament [Plakophilins, which are armadillo family members like plakoglobin and filament . Decreasfilament . Furtherfilament . Similarfilament . In addition, other junction types have been identified that use desmosomal components without being desmosomes. A recent review by Pieperhoff et al. , 35 highβ-catenin have shown that adherens junction components are essential for normal development. E-cadherin- and β-catenin-null embryos display lethality due to primary defects in morphogenetic events such as trophectoderm development and ectoderm formation [α-catenin, loss of this gene results in death shortly after birth [Early on, experiments targeting E-cadherin and ormation , 37. Delormation , 38, 39.er birth . Intereser birth , 42. In er birth –45. Alteer birth  to this er birth –49.β-catenin mediates the anchoring of adherens junctions to α-catenin and other actin-binding proteins, and thereby to the cytoskeleton [β-catenin and free β-catenin can tip the balance to induce Wnt signaling [β-catenin is not degraded by the ubiquitin-proteasome pathway, but translocated to the nucleus instead to regulate target gene expression in conjunction with members of the T-cell factor/lymphoid-enhancer factor (TCF/LEF) family of transcription factors [β-catenin degradation and allows its accumulation in the cytosol and translocation to the nucleus leading to the activation of genes such as cyclin D1, c-myc, CD44, and others [β-catenin or loss of adenomatous polyposis coli (APC) function frequently leads to cancer [Cadherins interact through their intracellular domain with cytoplasmic proteins, the catenins see , 50, 5151. β-catskeleton . The relignaling , 53. Thi factors . Activatd others , 56. Cono cancer , as parto cancer .β-catenin, unbound p120ctn can translocate to the nucleus where it binds Kaiso, a zinc finger transcription factor that acts as a transcriptional repressor and tumor suppressor. Once bound to Kaiso, p120ctn relieves the repressor activity of Kaiso by dissociating it from its sequence-specific binding sites [p120ctn binds cadherins at the juxtamembrane domain of the cytoplasmic tail and prevents their internalization and degradation , 58. Simng sites . Wnt signg sites –62. p120ng sites  and has ng sites . In thisng sites . In anotng sites . Furtherng sites . Ablationg sites . Conditing sites .β-catenin or p120ctn, at the cell membrane and thereby negatively control their nuclear translocation.Cadherins can signal in different ways: they can bind to growth factor receptors and modulate their internalization and downstream pathways. They also activate signaling mediators, such as phosphatidylinositol 3-kinase (PI3K) or small GTPases. Alternatively, they can recruit transcriptional cofactors, such as A number of cadherins has been implicated in cell signaling via interaction with receptor tyrosine kinases: both E-cadherin and N-cadherin interact with FGFR-1. To prevent constitutive or prolonged signaling by FGFR-1, it is sequestered by E-cadherin and internalized . Contrarβ-catenin and plakoglobin leading to the disruption of cell adhesion [E-cadherin can also interact with epidermal growth factor receptor (EGFR) . EGFR ovadhesion . At the adhesion , 76. Othadhesion , 78. Simadhesion .β-catenin, p120 and plakoglobin [β receptor-mediated TGFβ signaling. This has been demonstrated through knockdown of VE-cadherin [β-catenin null-endothelial cells are unable to respond to TGFβ stimulation [VE-cadherin is an endothelial specific transmembrane protein concentrated at adherens junctions. Similar to E-cadherin it engages in homophilic cell-cell adhesion. A link to the cytoskeleton is established through the same intercellular partners, koglobin . Upon VEkoglobin . Furthercadherin , but alsmulation .Another interesting aspect is that tumor-inducing viruses alter cell adhesion. In the case of Kaposi-sarcoma-associated herpesvirus, VE-cadherin is targeted inducing endothelial permeability and contributing to the progression and malignancy of this disease . While Kβ-catenin, and Notch) involved in the regulation of this process. However, a central node is the downregulation of E-cadherin [β1 signaling is often increased in tumor cells and induces EMT, thereby leading to tumor cell invasion [in vivo  [β-induced upregulation of transcriptional repressors such as ZEB1, ZEB2, and Snail. This is accompanied by the decreased expression of ZO-1 and keratins [Similar to the cadherin switch, epithelial-mesenchymal transition is an important process of development, but is “hijacked” as a mechanism of malignant transformation resulting in mesenchymal-like high motility cells. The spotlight on EMT is warranted by the many signaling pathways  can also mediate TGFuced EMT . The induced EMT  in this uced EMT , 105. Lyuced EMT  describein vitro , the tum et al. ) and them et al. ).β-catenin and increased invasion as well as increased tumor angiogenesis via VEGF upregulation. miR-145 is an example of a tumor suppressor miRNA, which silences MUC-1, thereby reducing β-catenin and oncogenic cadherin-11 [β1, therefore placing it at the center for the regulation of the epithelial phenotype. Another central regulator of cell invasion and metastasis that is upregulated in the absence of E-cadherinis Twist [β-catenin is released from the E-cadherin cytoplasmic tail.More recently, with the advent of microRNAs, small non-coding RNAs (miRNAs) regulating gene expression, an additional level ofprotein translation regulation has been added. A number of miRNAs that inhibit tumor suppressor genes have been identified as well as miRNAs, which negatively affect the translation of oncogenes. Cadherin scan be targets of miRNAs directly or indirectly through the modulation of transcriptional repressors that target cadherins. Ma et al., identified E-cadherin as a direct target of miR-9  leading herin-11 . The miRis Twist . Twist, is Twist , however397-phosphorylation site, which is responsible for Src recruitment [FAK, focal adhesion kinase, is a crucial mediator of integrin and growth factor signaling. FAK resides within focal adhesion complexes, large integrin clusters that mediate crosstalk between the extracellular matrix and the cytoskeleton, where it regulates outside-in signaling see . High leruitment . Actin rruitment . This isruitment ). Two FAruitment  and possruitment  the suppruitment , N-cadheruitment . The impruitment . These dWhile the main components of cell adhesion structures are well defined, recently new players such as the nectins have been identified . Similar


On immunohistochemistry with an anti-NPBC antibody, the nuclei were positively stained in 22 and 18 of AML and MDS specimens, respectively. Staining of nuclear NPBC was associated with AML subtypes (M6 and M7), low complete remission (CR) rate, and poor prognosis. Nuclear NPBC was also associated with a high score when using the International Prognostic Scoring System (IPSS) for MDS and with −7/−7q and complex karyotypes. These findings suggest that in situ detection of nuclear NPBC by immunohistochemistry could provide new insights into the pathogenesis and prognosis of AML and MDS.Wnt signaling activates the canonical pathway and induces the accumulation of non-phosphorylated beta-catenin (NPBC) in the nucleus. Although this pathway plays an important role in the maintenance of haematopoietic stem cells as well as in oncogenesis, the significance of nuclear NPBC remains unclear in malignant haematopoiesis. This study examined the expression of nuclear NPBC in bone marrow specimens from 54 and 44 patients with  MYC and CCND1  deletion indicated that Ctnnb1 is not indispensable for haematopoiesis (Ctnnb1 compromised haematopoietic stem cell maintenance and blocked differentiation in transgenic mice experiments (In the bone marrow (BM), Wnt proteins activate the beta-catenin pathway and the non-obese severe combined immunodeficient (NOD-SCID)-repopulating capacity of normal haematopoietic stem cells. They lead to increased expression of According to previous studies, the expression of beta-catenin is associated with activation of the Wnt pathway as well as poor prognosis  and myelodysplastic syndrome (MDS) patients using two anti-beta-catenin antibodies: one against C-terminal peptides and another against N-terminal non-phosphorylated peptides. The latter antibody detected nuclear NPBC, and positive staining for nuclear NPBC was associated with particular clinical characteristics of AML and MDS.de novo AML patients consisted of 35 men and 19 women with a median age of 53 years , and FLT3 mutations were detected in seven of 22 patients with AML (31·8%). The MDS patients consisted of 28 men and 16 women with a median age of 57 years . BM mononuclear cells were harvested by standard Ficoll/Paque density gradient centrifugation , and were suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU/ml of penicillin G and 100 μg/ml of streptomycin.For clinical samples, BM clots were obtained during routine diagnostic procedures. Beta-catenin expression was analyzed in BM specimens from patients newly diagnosed at the Nagoya University Hospital between 2000 and 2006 . The de For immunohistochemical and immunoblot studies, two monoclonal antibodies were used; one was against C-terminal peptides , enabling recognition of pan beta-catenin (PBC), and the other was against N-terminal-peptides , enabling recognition of NPBC.FLT3 mutations or karyotypes. We investigated a single case twice for NPBC expression. The entire section was screened to find the region with the highest immunostaining. The score was determined in each case after counting at least 500 nuclei in 3–5 randomly selected regions. When 20% or more of the BM mononuclear cells were positive for nuclear staining of NPBC, they were classified as nuclear NPBC+. The cut-off value of 20% was determined by the median distribution of the percentage of BM mononuclear cells stained by NPBC. As described in the results, some erythroblasts were positive for NPBC but the number was <20% except in the case of M6 patients. On the other hand, almost all blasts in M7 and other cases tested positive. The discrimination of cell types based on the 20% criterion therefore enabled a clear delineation.Samples were fixed with ice-cold 4% paraformaldehyde for 16–24 h, embedded in paraffin, sectioned transversely , and processed for immunohistochemistry to determine the localization of beta-catenin. After removal of paraffin with xylene and dehydration with a series of ethanol solutions, the tissue sections were subjected to microwave irradiation (750 W) for 15 min in 0·01 mol/l citrate buffer (pH 6·0). The sections were then placed in an automated immunostainer  as described . The membrane was initially incubated at room temperature for 1 h with 5% nonfat milk and 0·1% Tween-20 in Tris-buffered saline and then overnight with mouse monoclonal antibodies at a 1:2000 dilution in the same solution. After washing, the membrane was incubated for 1 h with a 1:5000 dilution of horseradish peroxidase-conjugated mouse antibodies to mouse IgG , and immune complexes were then detected with enhanced chemiluminescence (ECL) reagents (Amersham).Cell lysates from AML cells were extracted as previously described . P-values <0·05 were considered significant.The χde novo AML specimens, significant staining was observed only in M6. This antibody also detected BM vessels whose density was increased in AML specimens as previously reported  in AML patients (+ staining was closely associated with AML subtype: it occurred frequently (8/9) in M6 and M7 and rarely (0/7) in M3 .Using an anti-beta-catenin C-terminal peptide antibody, beta-catenin was stained in the membrane and cytoplasm of erythroid cells from normal BM. In reported . On the reported . In M6 sreported . In M7 sreported . In totareported , Fig 1B.patients . However+ was preferentially detected in refractory anaemia with excess blasts in transformation (RAEBT) compared to other MDS subtypes .In MDS specimens, erythroid cells and endothelial cells were stained with the anti-beta-catenin C-terminal peptide antibody . As obseTo confirm whether these two antibodies recognized beta-catenin, a total of 41 samples from AML and MDS patients were subjected to immunoblot analysis. The anti-beta-catenin C-terminal peptide antibody detected bands at a molecular weight of 95 kDa, corresponding to beta-catenin, in most samples except for M3 . The antFLT3 mutation might be associated with the stabilization of beta-catenin. In this study, however, nuclear NPBC was never detected in AML with t or t. In AML/MDS with −7/−7q and a complex karyotype, nuclear NPBC was frequently detected  and t, contributed to the activation of gamma-catenin, or that ion ITD; .+ and NPBC− AML patients. NPBC+ AML patients showed significantly lower hemoglobin levels, lower blast percentages in the BM, and lower CR rates . Accordingly, it was concluded that the nuclear staining with the latter antibody identified nuclear non-phosphorylated beta-catenin. Nuclear NPBC was detected in 22 (40·7%) of 54 AML patients and 18 (40·9%) of 44 MDS patients. Positive staining of nuclear NPBC was associated with particular AML subtypes (M6 and M7), a low complete remission rate, and poor prognosis. The presence of nuclear NPBC was also associated with a high IPSS score of MDS and with −7/−7q and complex karyotypes.ex vivo  is suppressed by deletion and/or methylation. Both alpha-catenin and beta-catenin bind to the inner membrane of hematopoietic cells, and cadherin binds to actin filaments via the catenin complex. If the expression of alpha-catenin is suppressed in the 5q- genotype or for other reasons, beta-catenin may be abnormally located or activated. In this study, abnormalities of chromosome 5 were associated with the presence of NPBC but this was not statistically significant. The reason why NPBC is associated with chromosome 7 abnormalities remains unclear. A gene encoded by chromosome 7 may be associated with the regulation of the Wnt/beta-catenin pathway. Several possible candidate genes including SFRP4, WNT2, and FZD1 and FZD9 are located at human chromosome 7. There are reports that sFRP4 plays a role in tumor suppression via the Wnt pathway (MDS is a clonal hematopoietic stem cell disorder characterized by multi-lineage dysplasia and pancytopenia in which further genetic events may be required for the rapid expansion of leukaemic blasts . Althougin situ detection of nuclear NPBC by immunohistochemistry of paraffin sections from BM specimens could be used to predict the prognosis of AML and MDS. Understanding the mechanisms leading to leukemogenesis in nuclear NPBC+ AML and MDS may lead to new anti-leukemia therapies.In conclusion,


Helicobacter pylori resides in the stomach of about fifty percent of the world's population and represents a risk factor for chronic gastritis, peptic ulcers and, in rare cases, gastric cancer. Alterations of the Wnt/β-catenin signaling pathway have been described in almost every human cancer disease, due to the regulation of target genes being involved in cell cycle control, differentiation, cell migration or stem cell control. Our study aimed to elucidate the role of proximal Wnt signaling components low density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled (Dvl) in the activation of β-catenin early after infection of gastric epithelial cells with H. pylori.The human microbial pathogen H. pylori induces rapid phosphorylation of the Wnt/β-catenin pathway co-receptor LRP6 independent of the cytotoxin-associated gene A (CagA) or vacuolating cytotoxin A (VacA). However, bacteria lacking a functional type 4 secretion system (T4SS) failed to induce LRP6 phosphorylation. Further, we identified proteins of the Dvl family, namely Dvl2 and Dvl3, which are involved in LRP6 phosphorylation. H. pylori-induced nuclear accumulation of β-catenin and its transcriptional activation, and expression of Wnt target genes are strongly reduced in stable knockdown cell lines deficient for LRP6, Dvl2 or Dvl3.Infection of gastric epithelial NCI-N87 cells with H. pylori-induced activation of Wnt-signaling factors and demonstrate for the first time that the canonical Wnt-signaling proteins LRP6 and Dvl2 and Dvl3 are involved in the regulation of β-catenin.We analysed the  H. pylori is a leading cause for the development of gastroduodenal diseases like chronic gastritis, peptic ulcers, gastric adenocarcinoma or mucosa-associated lymphoid tissue (MALT) lymphoma [H. pylori in the regulation of β-catenin remained unclear to date. β-catenin is a ubiquitously expressed protein with a dual role. On the one hand it is important in the establishment and maintenance of adherence junctions and, therefore, mediating cell-cell adhesion by connecting E-cadherin via α-catenin to the actin cytoskeleton [H. pylori suppresses GSK3β activity via the PI3K/Akt pathway, inhibiting proper β-catenin degradation, inducing LEF/TCF transactivation, and upregulation of the β-catenin target gene cyclin D1 in a CagA- and T4SS-independent manner [Persistent infection of the gastric mucosa by the human pathogen lymphoma . Environlymphoma ,3. Additlymphoma ,5 associskeleton . On the skeleton  togetherskeleton -10, whicskeleton . In non-skeleton . This phskeleton . Upon biskeleton ,15, and t manner .H. pylori. We show that 1) infection leads to phosphorylation of LRP6 which is independent of CagA and VacA, but depends on a functional T4SS, 2) members of the Dvl family, namely Dvl2 and Dvl3, are involved in LRP6 phosphorylation 3) H. pylori-induced nuclear translocation of β-catenin, LEF/TCF transactivation and target gene expression are significantly reduced in cells with a stable knockdown of LRP6, Dvl2, or Dvl3.In the presented work we used human gastric epithelial cells NCI-N87 to study LRP6- and Dvl-dependent regulation of β-catenin after infection with H. pylori, cells were infected with H. pylori P1 wild type or the isogenic mutant strains lacking the CagA protein or the virB7 protein (required for the integrity of the T4SS). In addition, a P14 strain lacking the VacA protein was used. Phosphorylation of LRP6 was induced within 30 minutes after infection in a CagA- and VacA-independent manner. However, a bacterial strain lacking a functional T4SS was not capable of inducing LRP6 phosphorylation  or mutated (SuperFOPflash) LEF/TCF binding sites and then infected with H. pylori. After six hours of infection cells showed significant increased levels of luciferase activity in SuperTOPflash transfected cells compared to non-infected cells or SuperFOPflash transfected cells. Treatment of the cells with Wnt3a also increased reporter activity in SuperTOPflash transfected cells, but not in cells with the mutated LEF/TCF binding sites  cells T4SS-independent induction of the epidermal growth factor receptor (EGFR)-PI3K/Akt kinases after H. pylori infection, resulting in downstream GSK3β inhibition and reduced Ser/Thr phosphorylation of β-catenin [et al. showed EGFR phosphorylation and Akt activation after H. pylori infection of gastric AGS and MKN tumor cell lines, which depends on the T4SS and the bacterial outer membrane protein OipA [Germ line or somatic mutations in the Wnt/β-catenin signaling pathway cause several hereditary human diseases and are involved in the initiation or progression of a number of human cancers  mostly c-catenin , but mec-catenin . In contein OipA . Other sein OipA , or CagAein OipA .489 and Tyr654 disrupts binding to E-cadherin, and at Tyr142 to α-catenin [142, switching it from its adhesive to its transcriptional function [H. pylori infection [In contrast to Serine/Threonine phosphorylations of the N-terminus of β-catenin, which are responsible for the degradation of the protein, regulation of the E-cadherin-catenin complex is regulated by phosphorylation of different tyrosine residues. Tyrosine phosphorylation of β-catenin at residues Tyrfunction . Fyn is function . Both prnfection ,27.H. pylori-induced activation of β-catenin. Expression of Dvl1 was weak, therefore, we resigned to establish a knockdown cell line deficient for Dvl1. We used human NCI-N87 cells [In our work we present for the first time an analysis of the role of proximal Wnt signaling pathway components, namely LRP6, Dvl2, and Dvl3, in 87 cells , which e87 cells ,30.H. pylori induced a similar phosphorylation of LRP6 compared to NCI-N87 cells. Concerning the role of LRP6 in H. pylori infected cells we observed that the knockdown of LRP6 tremendously suppresses the amount of β-catenin, the LEF/TCF transactivation and the amounts of Axin 2 expression. Therefore, we suggest that LRP6 exerts an important role in the H. pylori dependent β-catenin regulation. A contributory role of LRP5 could be possible. Studying expression of mRNA we observed no differences between LRP6 and LRP5 in AGS and NCI-N87 cells (data not shown).The Wnt signaling pathway co-receptor LRP6 is crucial for the stabilization of β-catenin  and its Drosophila [H. pylori. Noteworthy, phosphorylation of LRP6 was not fully hindered with either Dvl2 or Dvl3 shRNA. The question remains open, if a double-knockdown of both proteins would completely reverse the observed LRP6 phosphorylation. Dvl proteins have been shown to show at least some redundancy [Overexpression of the cytoplasmic phosphoprotein Dvl has been shown to result in β-catenin signaling in osophila . Moreoveosophila ,35 and Ddundancy , but notdundancy .H. pylori, as previously demonstrated by others [H. pylori to deregulate cell adhesion through disconnection of the E-cadherin-catenin complex from the cytoskeleton [H. pylori-infected cells. Beyond this, proteolytic cleavage of the extracellular domain of E-cadherin in H. pylori-infected epithelial cells contributes in a CagA-independent manner to the disintegration of adherence junctions [Previous studies using different MKN gastric tumor cell lines could show nuclear β-catenin localization after infection with the NCTC11637 strain ,38,39. Wy others . Upregulskeleton . Other rskeleton , or showskeleton  in H. pyunctions .H. pylori. Future studies will have to determine if this difference is related to distinct functions of Dvl2 and Dvl3 in H. pylori-infected cells.Nuclear accumulation of β-catenin, LEF/TCF activation as well as target gene upregulation was clearly affected in cells deficient for LRP6, Dvl2, and Dvl3. Interestingly, a similarly efficient knockdown of Dvl2 when compared to knockdown of Dvl3 is less important for the activation of β-catenin in response to H. pylori-infected epithelial cells and show phosphorylation of LRP6 in a T4SS-dependent manner. Additionally, H. pylori-induced activation of β-catenin involves the Wnt signaling pathway components Dvl2, Dvl3  and fresh, serum- and antibiotic-free medium was added.NCI-N87 cells  and AGS cells were maintained in RPMI 1640  with 10% FCS and 1% penicillin/streptomycin in humid atmosphere at 37°C and 5% COWe used the following antibodies and chemicals: phospho-LRP6 (Ser1490), LRP6, Axin2, Dvl2, HDAC1, anti-mouse-HRP and anti-rabbit-HRP ; Dvl3 and TfR1 (Santa Cruz Biotechnology); β-catenin (BD Transduction Laboratories); GAPDH  Recombinant Wnt3a was purchased from R & D Systems.Lentiviral constructs for the generation of stable knockdown cell lines for LRP6, Dvl2 and Dvl3 as well as a control construct were purchased from Open Biosystems.4 cells per well the day before transfection. 150 ng of total DNA were transfected per well with a ratio between firefly SuperTOPflash or SuperFOPflash  and renilla luciferase (Promega) plasmids of 9:1 using Lipofectamine LTX transfection reagent (Invitrogen). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) at Lumat LB 9507 luminometer (Berthold Technologies). All samples were assayed in triplicates.NCI-N87 cells and the respective knockdown cell lines were seeded into 96-well plates at a density of 102HPO4, 0.5% Nonidet P40, 1 × protease inhibitor cocktail (Roche), 1 mM Na3VO4, 1 mM Na2MoO4, 20 mM NaF, 0,1 mM AEBSF, 20 mM β-glycerol-2-phosphate, 10 mM Na4P2O7). The homogenate was incubated 10 min on ice, drawn through a syringe . After another 10 min of incubation on ice, lysates were cleared by centrifugation for 10 min (12000 × g at 4°C). Aliquots of the respective samples were then boiled with sample buffer  for 5 min at 95°C. Subcellular fractions were prepared using ProteoExtract Kit  following the manufacturer's instructions.Whole cell lysates were prepared using a modified RIPA buffer  membrane (Millipore), blocked with 5% w/v milk or BSA/TBST followed by analysis with antibodies as indicated. Immunoreactivity was detected using the enhanced chemiluminiscence detection kit ECL™ (Amersham Pharmacia Biotech). Blots were semi-quantitatively evaluated by densitometric analysis. Protein bands on X-ray films were scanned using VersaDoc Imaging System (Bio-Rad), and analyzed using Quantity One software (Bio-Rad).Experiments were analysed using ANOVA and unpaired Student's t-test. The values of the experiments are expressed as means ± SEM. Statistical decisions were made with a critical probability of p = 5% without α-adjustment.The authors declare that they have no competing interests.TG performed most of the experiments, contributed to the design of the study and analysis of data, and drafted the manuscript. MF and ML generated the stable knockdown cell lines. UL and MN contributed to the design of the study, analysis of the data and editing of the manuscript. All authors read and approved the final manuscript.


The role of TCF/β-catenin signalling in T cell development is well established, but important roles in mature T cells have only recently come to light.Axin2 and dickkopf is down regulated.Here we have investigated the signalling pathways that are involved in the regulation of β-catenin in primary human T cells. We demonstrate that β-catenin expression is upregulated rapidly after T cell receptor (TCR) stimulation and that this involves protein stabilisation rather than an increase in mRNA levels. Similar to events in Wnt signalling, the increase in β-catenin coincides with an inhibition of GSK3, the kinase that is required for β-catenin degradation. β-catenin stabilisation in T cells can also be induced by the activation of PKC with phorbol esters and is blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PKC). Upon TCR signalling, β-catenin accumulates in the nucleus and, parallel to this, the ratio of TCF1 isoforms is shifted in favour of the longer β-catenin binding isoforms. However, phosphorylated β-catenin, which is believed to be inactive, can also be detected and the expression of Wnt target genes These data show that in mature human T cells, TCR signalling via PI3K and PKC can result in the stabilisation of β-catenin, allowing β-catenin to migrate to the nucleus. They further highlight important differences between β-catenin activities in TCR and Wnt signalling. Wnt/β-catenin signalling is important for cell fate decisions during many developmental programs. The canonical Wnt signalling pathway is initiated upon binding of Wnt to the receptor Frizzled and its co-receptor LRP, which ultimately leads to the stabilisation and accumulation of β-catenin. Stabilised β-catenin translocates to the nucleus and associates with the transcription factors TCF and LEF to drive transcription of Wnt regulated genes reg cells was shown to increase cell survival, resulting in an enhanced protection against inflammatory bowel disease in a mouse model + T cells renders these cells anergic H2 differentiation. TCF1/β-catenin were found to activate the transcription of GATA-3-1b early after TCR activation. Furthermore, in activated effector T cells, β-catenin has been shown to regulate expression of matrix metalloproteinases MMP2 and MMP9 during T cell extravasation, which promotes migration through subendothelial basement membrane + T cells has also been demonstrated Wnt/β-catenin signalling regulates many aspects of T cell development Signalling via the TCR in combination with CD28 is known to inhibit GSK3 To investigate whether β-catenin up-regulation occurs at the mRNA or protein level, we monitored β-catenin mRNA levels in stimulated T cells by qRT-PCR. T cell activation had little impact on β-catenin mRNA levels over a range of time points , despiteIn the absence of a signal, levels of β-catenin are low because of its phosphorylation on residues Ser33/37 and Thr41 2+ and can further enhance GSK3 inhibition by reducing the activity of phosphatases that dephosphorylate Ser21 (GSK3α) and Ser9 (GSK3β) The presence of lithium and anti-CD3/28 stimulation together enhanced β-catenin levels relative to that observed with either anti-CD3/CD28 or lithium alone , lane 10In summary, we conclude that the increase in β-catenin levels upon TCR signalling correlates with GSK3 inhibition, suggesting that it results from a stabilisation of the β-catenin protein. The residual phosphorylation of this stabilised β-catenin may arise from an incomplete inactivation of GSK3.Primary T cells have been shown to express full length and short isoforms of the transcription factor TCF1 in equivalent amounts  [15]. OfTo explore the stabilisation of β-catenin and modulation of TCF1 isoforms further, we next isolated populations of CD4+ and CD8+ T cells and divided these into naïve and memory subsets based on the surface expression of CD45 isoforms . Stimulahttp://www.stanford.edu/~rnusse/wntwindow.html). A robust increase in mRNA levels of CD25 (the IL-2 receptor α-chain) with anti-CD3/CD28 stimulation, demonstrated that efficient T stimulation had indeed occurred  using Lymphoprep (Axis-Shield) and density-gradient centrifugation. T cells were purified on nylon wool (Kisker) columns yielding about 85–90% pure populations. T cells were maintained in RPMI-1640 (Invitrogen), 10% FCS (Helena Biosciences) and the antibiotics Penicillin (100 U/ml)/Streptomycin (0.1 mg/ml streptomycin) (Gibco Ltd.) and used for experiments within 24 hours of isolation. Alternatively, nylon wool purified T cells were immunostained with APC conjugated anti-CD4, PerCP conjugated anti-CD8, FITC conjugated anti-CD45RA and PE conjugated anti-CD45RO (eBiosciences) on ice for 1 hour in PBS 0.2% BSA. Cells were sorted on Becton Dickinson Aria Flow Cytometer and immediately processed.6) were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies  for the indicated times. Where applicable, inhibitors of PI3K , PLC , GSK3  and MEK1/2  were added 1 hour prior to stimulation and remained present during stimulation. PDBu was used at 10 ng/ml and ionomycin at 100 nM. LiCl was added from a 8M stock solution to the indicated final concentrations.T cells (2–5×106 cells with 50 pmoles ON-TARGET plus siRNA targeting human β-catenin (Ctnnb1), or control (Luciferase) siRNA reagents . T cell transfections were performed using the human T cell nucleofector reagent (Amaxa biosystems) and programme U14 of the Amaxa nucleofector. Cells were stimulated and processed 18 hours after transfections.To confirm antibody specificity, T cells were transfected at 5×10Antibody stimulated T cells were removed from plates with PBS containing 5 mM EDTA and lysed in ice cold lysis buffer , 150 mM NaCl, 10 mM NaF, 10 mM disodium pyrophosphate) together with a cocktail of small protease and phosphatase inhibitors (Roche). Equal amounts of post-nuclear supernatants (BCA assay) were subjected to SDS-PAGE and Western blotted with indicated antibodies and visualised with enhanced chemiluminescence (Pierce). To separate nuclear and cytoplasmic fractions, T cells were lysed using the NE-PER Extraction Reagents (Pierce) according to the manufacturer's instructions.The following antibodies were used for immunoblotting: anti-β-catenin, anti-phospho-β-catenin (Ser33/37/Thr41), anti-TCF-1, anti-phospho-GSK3αβ (Ser21/9), anti-phospho-AKT (Ser473) , anti-GSK3αβ (Biosource), anti-lamin B1, anti-ERK1 (Santa Cruz Biotechnology), anti-tubulin (Invitrogen) and rabbit anti-α-catenin  for the indicated time points, washed in cold PBS, pelleted and snap frozen in liquid nitrogen as dry cell pellets. Total RNA was isolated using Trizol (Invitrogen) and equivalent amounts reverse transcribed with the high capacity cDNA reverse transcription kit (Applied Biosystems). PCR was performed in triplicate in TaqMan Universal PCR master mix containing gene specific probes or endogenous control (Applied Biosystems). Ctnna1 (α-catenin) and Ctnnb1 (β-catenin) gene expression were analysed using the ABI PRISM 7900HT instrument and samples normalised to expression of endogenous control. Fold increase was calculated by the ΔΔCt method. Axin2 and DKK-1 mRNA levels were analysed using SYBR green PCR mastermix (Applied Biosystems) and primers (Eurofins MWG Operon) designed using the ProbeFinder software (Roche Applied Science). Abl1 and PPIA were used as reference genes.