Sprague Dawley rats (6 weeks of age) were randomly divided into 5 groups: (G1) positive control , (G2) fed with 2.5 g/kg of GBR (GBR (g)/weight of rat (kg)), (G3) fed with 5 g/kg of GBR, (G4) fed with 10 g/kg of GBR and (G5) negative control . GBR was administered orally once daily via gavage after injection of 15 mg/kg of body weight of azoxymethane (AOM) once a week for two weeks, intraperitonially. After 8 weeks of treatment, animals were sacrificed and colons were removed. Colonic aberrant crypt foci (ACF) were evaluated histopathologically. Total number of ACF and AC, and multicrypt of ACF, and the expression of β-catenin and COX-2 reduced significantly (p < 0.05) in all the groups treated with GBR  compared to the control group (G1). Spearman rank correlation test showed significant positive linear relationship between total β-catenin and COX-2 score . It is demonstrated that GBR inhibits the development of total number of ACF and AC, and multicrypt of ACF, reduces the expression of β-catenin and COX-2, and thus can be a promising dietary supplement in prevention of colon cancer.Chemoprevention has become an important area in cancer research due to the failure of current therapeutic modalities. Epidemiological and preclinical studies have demonstrated that nutrition plays a vital role in the etiology of cancer. This study was conducted to determine the chemopreventive effects of germinated brown rice (GBR) in rats induced with colon cancer. GBR is brown rice that has been claimed to be richer in nutrients compared to the common white rice. The male  Colon cancer develops through a multistage process that could be recognized at a histopathological level by progression from mucosa to invasive carcinoma. Colorectal cancer is becoming increasingly common in Asian countries while remains the second leading cause of cancer death in the United States. Approximately 110,000 new cases are diagnosed each year .GBR is brown rice, which has been soaked in water for up to a day and has a germ of approximately 1 mm long. During germination, nutrients in the brown rice change drastically. Nutrients that increase in content include γ-amirobutyric acid (GABA), dietary fiber, inositols, ferulic acid, phytic acid, tocotrienols, magnesium, potassium, zinc, γ-oryzanol, and prolylendopeptidase inhibitor . AccordiAdenomatous polyposis coli (APC) or β-catenin genes, are associated with majority of human and rat colon tumors [et al. [β-catenin gene mutations and accumulations of the protein are involved in the initial stage of colon carcinogenesis induced in rats by AOM.β-catenin, which was originally a cadherin-binding protein, has recently been proven to function as a transcriptional activator when it formed a complex with members of the T cell factor (Tcf) family of DNA binding protein ,11. Actin tumors ,14 and wn tumors , which ln tumors . Yamada  [et al.  had demoAPC gene and markedly elevated in carcinogen-induced rat colonic tumours [Cyclooxygenase (COX) catalyses the rate limiting step in the conversion of arachidonic acid to prostaglandin H2 (PGH2), which is further metabolized to form PGD2, PGE2, PGF2α, PGI2, thromboxanes A2 and leukotrienes -19. COX  tumours ,24.et al. has also demonstrated that COX-2 overexpression enhances neovascularization thus conferring survival advantage of colon cancer cells [Studies have led to the recognition of the importance of COX-2 in colorectal tumorigenesis and in the development of intestinal neoplasms in genetically manipulated animal models, in which overexpression of COX-2 in colon cancer as well as its established cell lines is frequently demonstrated . Tsujii er cells .Continuous intake of germinated brown rice has been predicted to prevent cancer of colon, but there is no scientific proof on that, as yet . The goaSprague Dawley rats were purchased from the Faculty of Veterinary Medicine, UPM. Animal Care and Use Ethic was obtained from the Animal Care and Use Committee (ACUC), Faculty of Medicine and Health Sciences, Universiti Putra Malaysia . AOM was purchased from Sigma Chemical Company, St. Louis USA. The rats were housed in plastic cages (2 rats per cage) with woodchip bedding in a well ventilated room at room temperature, 29-32°C; 50-60% relative humidity; 12 hours light and darkness cycle. Hygienic conditions were maintained by weekly changes of the woodchip beds. GBR was kindly supplied by the Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia. Briefly, GBR was soaked at 32°C for 24 hours until germinated. Subsequently, GBR was ground using Rotary type grinding and pulverizing machines (RT-34) to produce powder. The GBR powder was dissolved in distilled water according to the dosage/treatment.Five-week-old male Sprague Dawley rats were divided into five groups (n = 10), which were (G1) positive control , (G2) fed with 2.5 g/kg of GBR (GBR (g)/weight of rat (kg), (G3) fed with 5 g/kg of GBR, (G4) fed with 10 g/kg of GBR and (G5) negative control . At 6 weeks of age, the animals in groups 1-4 were injected with AOM intraperitonially (i.p) (15 mg/kg body weight) once weekly for two weeks. At 9 weeks of age, rats in group 2-4 were treated with GBR at 2.5 g/kg, 5 g/kg and 10 g/kg (weight of GBR (g)/weight of rat (kg)) by oral administration via gavage once daily for 8 weeks. The animals were fed with commercial basal diet in the form of pellet throughout the experimental period. The GBR free animals were given distilled water (that has been used to dissolve GBR prior to treatment) via gavage throughout the experimental period. Body weight of the rats was recorded weekly until the termination of the study. Upon termination, all animal were sacrificed by decapitation under chloroform anesthesia. Colon was removed and cut longitudinally. The colon was washed with phosphate-buffered saline, fixed in 10% of formalin and stained with hematoxylin and eosin. The total number of ACF and AC per colon was recorded.Following a one week of acclimatization, the male ®+ Kit, DAKO, USA) was used as the secondary antibody. The peroxidase activity was developed by incubating the slides in the substrate 3,3'-diaminobenzidine (DAB) . A semi-quantitative scoring system adopted from Rajnakova et al. [The paraffin embedded sections were heated at 60°C for 60 min, deparaffinized in xylene, and rehydrated through graded alcohol at room temperature. Four-μm-thick section obtained from whole part of the colon of each rat was treated with 3% of bovine serum albumin (BSA) for one hour at room temperature to block the non-specific antigen sites, and subsequently incubated with primary antibody for another one hour. The expression of β-catenin was determined using a monoclonal antibody raised against a peptide corresponding to the C-terminus of mouse β-catenin  (1:200). COX-2 protein expression was detected by tissue culture supernatant of rabbit IgG  raised against a synthetic peptide from C-terminus of rat COX-2 (1:100). All the antibodies were diluted in antibody diluent . Streptavidin-biotin complex peroxidase kit  of five field, randomly selected from each section of colon/rat. The data were presented as mean ± SEM.p < 0.05) in the body weight between the groups treated with GBR and the control (G1) (data not shown). The effect of GBR on the incidence of ACF is shown in Table p < 0.05) in the total number of ACF and AC, and multicrypt of ACF per colon in all the groups treated with GBR  compared to the control group (G1). Reduction in the total number of ACF and AC, and multicrypt of ACF with increase in the percentage of GBR was observed but yet insignificant.There was a significant difference (control G (data nocontrol G (data nop = 0.0001) in the expression of β-catenin.All tissue samples from G1 showed intense and heterogenous β-catenin immunoreactivity throughout the colon epithelia Figure . In sampp = 0.0001) in the expression of COX-2 compared to the treatment groups . Weak, diffused and heterogenous expression of COX-2 was observed in the tissue samples from G5  in the expression of COX-2.The colon epithelia from G1 showed more intense immunoreactivity of COX-2 indicating significant increase .Spearman rank correlation test showed significant positive linear relationship between total β-catenin and COX-2 score /weight of rat (kg)) significantly suppressed the total number of ACF, AC and multicrypt of ACF per colon, yet was insignificant among the GBR-treated groups. It is postulated that the chemopreventive effects of GBR was dose-independent. The ACF was found to consist of 1, 2, 3, 4 or more crypts. It is believed that the number of crypts increases with time due to crypt multiplication or branching . The cryet al. [Sprague Dawley rats and the other samples (human and other breeds of rats).All tissue samples from G5, which represent the normal colon tissue did not show β-catenin immunoreactivity Figure . There wet al.  also shoIn the tissues of positive control group G1), where colon carcinogenesis was initiated by injection of AOM but no treatment with GBR was given, intense β-catenin stain  was observed, representing much higher level of β-catenin Figure . The int, where cAccording to Goss and Groden , β-caten-catenin, APC, axin and GSK-3β may result in the inability of the complex to phosphorylate β-catenin, resulting in failure of ubiquitination; whereas E-cadherin mutation can cause dissociation of β-catenin from the complex, releasing free β-catenin into the cytoplasm. This explains loss or reduced β-catenin protein at the cell-to-cell borders but an over-expression in the cytoplasm of the G1 tissues.Mutations in β In the treatment groups , heterogenous expression of β-catenin was observed in the cell membrane and cytoplasm compartment. Nevertheless, all the three treatment groups showed significant decrease in β-catenin expression compared to G1. This indicates that GBR has the potential of reducing the expression of β-catenin. It is postulated that GBR is a natural chemopreventive agent which acts through pathway involving β-catenin.et al. [et al. [Tissue samples from G5, which represent the normal colon tissue showed some degree of COX-2 immunoreactivity, where the expression of COX-2 was found to be weak, diffused and heterogenous Figure . Lack ofet al.  and Taka [et al.  have alset al. [et al. [In the tissues of G1, the colon epithelia showed much greater COX-2 immunoreactivity compared to all other groups Figure . The staet al.  and Taka [et al.  have repp = 0.0001) between G2 and G3, and between G2 and G4. This indicates that the expression of COX-2 decreased with increase in the concentration of GBR. APC, β-catenin, axin, and GSK-3β mutations can cause increased expression of β-catenin in the cytoplasm and nucleus of colorectal carcinoma, which leads to Wnt pathway activation. Since COX-2 may be a downstream target of Wnt signaling pathway, COX-2 gene is upregulated and COX-2 expression is increased in tumour cells [As for the comparison among the treatment groups, there were significant differences , ferulic acid, inositol and dietary fiber . It has in vitro . In addiin vitro . Ferulicin vitro .As functional food component, Shamsuddin  has repoThis findings show that germinated brown rice has chemopreventive properties in rats induced with colon cancer. The administration of 2.5 g/kg, 5 g/kg and 10 g/kg of GBR (weight of GBR (g)/weight of rat (kg)) significantly reduced the total number of ACF, AC, and multicrypt of ACF, and the expression of β-catenin and COX-2 compared to the positive control (without GBR). It is suggested that GBR can be a promising dietary intake for prevention of human colon cancer.The authors declare that they have no competing interests.SYL was the chief investigator for this project in planning the study design and contributed to manuscript preparation. NA, HTT and YA took part in planning the study design, scoring of ACF and AC, immunohistochemistry analysis of β-catenin and Cox-2, statistical analysis and manuscript preparation. AHN processed the GBR. SN and IH assisted in scoring of ACF and AC, and immunohistochemistry analysis of β-catenin and Cox-2. MS performed the statistical analysis. IM was responsible for funding, study design and GBR supply. All authors read and approved the final manuscript.


Because of their high sequence identity with mammalian IL-8, fish IL-8-like CXCs have been named as piscine 'IL-8' and included in the ELR+ subgroup, even though there is no reliable functional or evolutionary evidence to support this classification.The ELRAcanthopagrus schlegeli), called BS CXC, has been cloned and analyzed. The results revealed that BS CXC has a high gene similarity and tertiary structure similarity with piscine and mammalian CXC chemokines, both ELR-CXC and ELR+CXC, although it has a lower identity with ELR-CXC, compared with ELR+CXC chemokines. Like other piscine IL-8, BS CXC has only an incomplete ELR motif, which is essential for the mammalian ELR+CXC ability to attract granulocytes. Bioactivity assay demonstrated that the BS rCXC produced in E. coli significantly stimulated migration of fish neutrophils and macrophages, but had no effect on rat neutrophils and macrophages, whereas hrIL-8 induced strong chemotaxis of fish neutrophils but did not affect fish macrophages. BS CXC seems show some structural and functional properties of the intermediate between ELR-CXC and ELR+CXC.In this investigation, a homologue of piscine 'IL-8' from black seabream (+CXC chemokine from a modern fish, BS CXC provides some clues on the evolution from ancient ELR-CXC to ELR+CXC by retaining some properties of the intermediate stage in evolution, and it may be more appropriate to call this molecule 'piscine CXC with an incomplete ELR', instead of terming it fish 'IL-8'.As an incomplete ELR Chemokines are a group of small peptide chemotactic cytokines, which are multifunctional mediators that can trigger inflammatory cell chemotaxis toward a site of infection and injury by binding to a G-protein-coupled cell surface receptor ,2. Chemo+ subgroup and the ELR- subgroup -x- [LIVMFY]-x- [RKSEQ] -x- [LIVM]-x(2)- [LIVM]-x(5)- [STAG]-x(2)-C-x(3)- [EQ]- [LIVM](2)-x-C-L- [DN]  was idenC X-rays -24  and ELR-CXC(49-36%), and has higher identity with ELR+CXC (60.5-25.9) but a lower identity with ELR-CXC(28.1-16.1%)  and mammalian ELR+CXC (25.9–40%). But the Agnatha (jawless fishes) lamprey only has a lower identity (33.6-18.6%) with all the CXC chemokines [Alignment of amino acid sequences of the BS CXC with the other CXC chemokines indicates that the BS CXC has a higher similarity with all the CXC chemokines (36–72%), both ELR1%) Fig. . The BS emokines .+CXC of mammalian CXC and incomplete ELR+CXC of piscine CXC, which are orthologous genes that represent independent lineages and have diverged from ELR-CXC, share sister-taxon relationships on the evolutionary tree. All the piscine incomplete ELR+CXC chemokines formed one clade. In this clade, lamprey CXC of the Agnatha (jawless fishes) is in an independent branch that evolved first from ancestral CXC and later from cartilaginous fish CXC and other bony fish CXC. In the mammalian ELR+CXC clade, the orthologous genes of IL-8 and chicken K60 ligands share parallel evolutionary relationships, with the orthologous genes of CXC 5-7 and CXC 1–3 forming sister branches. Piscine incomplete ELR+CXC chemokines originated almost contemporaneously through a series of gene duplication events. There is no solid evidence to confirm that fish incomplete CXC were clearly orthologous with mammalian IL-8. The results indicate that the ELR+CXC chemokine ligands may have evolved from ancestral ELR-CXC and diverged further during evolution to perform a special function. Fish incomplete ELR+CXC retain some properties of the intermediate stage during the evolutionary development of highly specific CXC chemokines.A phylogenetic tree was constructed based on the mature peptide of BS CXC and part of the known CXC ligands by using the neighbour-joining algorithm Fig. . The ELRAcanthopagrus schlegeli). The results showed basal expression of BS CXC in various organs, including the heart, gill, spleen, kidney and HK, and the basal expression was higher in the blood than in other tissues . The construct was transformed into +CXC and ELR-CXC. A migratory assay of leukocytes was performed to test our hypothesis. The neutrophils were collected from peripheral blood and macrophages were isolated from the lungs of rats and the head kidneys of black seabream and common carp. Dose-dependent chemotactic responses induced by BS CXC were observed in the migratory assays of BS neutrophils and macrophages  was used to do a migratory assay of fish neutrophils. As shown in Fig. The first reported fish chemokine gene from a jawless fish lamprey belonged to the CXC family; the gene encodes an IL-8-like peptide and was called lamprey 'IL-8'. Following these discoveries, similar molecules were identified in different fishes, including shark, trout, flounder and carp . Because+CXC and ELR-CXC in different species. Mammalian IL-8 belongs to the ELR+CXC subgroup of CXC chemokines containing the ELR motif (ELR+). This group includes CXCL1 to 8, except CXCL4, and these chemokines have implications in the specificity for granulocytes. In contrast, the ELR- subgroup includes CXCL4 and CXCL9 to 14, and these chemokines attract lymphocytes and monocytes, with poor chemotactic ability for granulocytes [+ subgroup but the classification seems unsuitable [CXC chemokine ligands are grouped into two subgroups, ELRulocytes ,14,27,28suitable , not onl-CXC chemokine [+CXC and piscine incomplete ELR+CXC are derived from ancestral ELR-CXC. Piscine incomplete ELR+CXC represents an early evolutionary stage of ELR+CXC. Among the fishes, lamprey 'IL-8' (jawless fishes), which occupies an independent branch, may be the first that evolved from the ELR-CXC ancestor, followed by shark 'IL-8' (cartilaginous fish), and finally leading to common carp 'IL-8', rainbow trout 'IL-8', flounder 'IL-8' and BS CXC, etc (bony fishes). Similar to the CXC protein molecules, there is also an evolutionary trend for the fish ELR-like motif, from GGR (jawless fishes) to SLR/SLQ (cartilaginous fish), then to SLH/DLR/ELH (bony fishes), and finally to ELR . The entire process has probably occurred through multi-time mutations of amino acids that have led to the formation of an ELR motif–a special binding site for the CXC receptor in mammals.In this study, we constructed a phylogenetic tree using the neighbour-joining algorithm Fig.  and a sihemokine ,14, and In mammalian species, all CXC chemokines have a similar tertiary structure which contains three β strands in the N terminal and an α helix in the C terminal, and all the functional CXC chemokines act by binding to G-protein-coupled cell surface receptors via a seven-transmembrane-domain, and signaling through a heterotrimeric GTP-binding protein ,30. Ther-CXC chemokine. Unlike fish 'IL-8', the mammalian IL-8 evolved a more specialized chemotactic function, inducing only granulocytes, but lost its attraction for macrophages. It is generally acknowledged that the ELR motif is an essential structure for the chemotaxis of granulocytes [The differences in molecular structures hint at differences in functions between mammalian IL-8 and fish 'IL-8'. Before this investigation, almost no functional investigations about fish 'IL-8' had been performed, so it was unclear whether fish 'IL-8' and mammalian IL-8 have the same function or not. In this study, we used BS CXC as a model of fish 'IL-8' to investigate the chemotactic functions of fish 'IL-8' and found that BS CXC developed a chemotactic ability for granulocytes, but still kept the chemotactic property of inducing macrophages and neutrophils, compared with its ancestors of the ELRulocytes , howeverulocytes . These d-CXC chemokine. It seems that fish incomplete ELR+CXC still retain some properties of the intermediate stage during the evolution from ancient ELR-CXC to ELR+CXC chemokines. Therefore, several papers have noted that fish IL-8 is an inappropriate name for piscine chemokines , and it may be more appropriate to call this molecule 'piscine CXC with an incomplete ELR', instead of terming it fish 'IL-8'.Taken together, although fish 'IL-8' with an incomplete ELR motif already has granulocyte-attracting ability similar to mammalian IL-8, it still retains a strong chemotactic ability for monocytes, similar to its ancestor, the ELR+CXC and ELR-CXC, although it has a lower identity with ELR-CXC, compared with ELR+CXC chemokines. Like other piscine IL-8, BS CXC has only an incomplete ELR motif, which is essential for the mammalian ELR+CXC ability to attract granulocytes. Bioactivity assay demonstrated that the BS rCXC significantly stimulated migration of fish neutrophils and macrophages. As an incomplete ELR+CXC chemokine from a modern fish, BS CXC provides some clues on the evolution from ancient ELR-CXC to ELR+CXC by retaining some properties of the intermediate stage in evolution.A homologue of piscine 'IL-8' from black seabream, called BS CXC, has been cloned and analyzed. The results revealed that BS CXC has a high gene similarity and tertiary structure similarity with piscine and mammalian CXC chemokines, both ELRAcanthopagrus schlegeli), weighing about 100 g, were collected from a local fish farm in Shenzhen, Guangdong province, and acclimated for 10 days in a 200 L fiberglass tank containing clean seawater at 25 ± 3°C. Each day 25% of the water was replaced by fresh seawater. Fish were fed commercial food at a daily rate of 1.5% of their estimated body weight. All the fishes were handled according to the regulations of "U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training".Black seabream (Vibrio anguillarum (about 2 × 108 cells per fish). Then 24 hours after stimulation, the head kidney HK was isolated and homogenized for RNA isolation. Total RNA was extracted by Trizol reagent  following the user's manual. The cDNA was synthesized from total RNA by MMLV reverse transcriptase  with oligo dT or adapter dT primer.Healthy black seabream were challenged with an attenuated marine fish bacterial pathogen of E. coli competent cells. Plasmid DNA was isolated using a GenElute plasmid miniprep Kit (Sigma). The insert in the plasmid DNA was sequenced using the ABI377a Automated Sequencer (Applied Biosystems).PCR (polymerase chain reaction) was performed using the cDNA template prepared above and a pair of degenerated primers dCXC-F/dCXC-R  approaches , using gBased on the candidate BS CXC sequence, a pair of primer gF(5' -ATG ATG AGC AGC AGA GTC- 3')/gR (5'-CAG ATT GTC AGA GCC AGA-3'), which was located in 5'UTR and 3'UTR of the BS CXC sequence, respectively, was designed. RT-PCR was carried out with these primers to get the full length transcript of BS CXC.The nucleotide sequences were analyzed by the BLAST  program -1), 3 μL cDNA and 5 μL DEPC-treated water. The thermal program for real-time PCR was 95°C for 5 min followed by 35 cycles at 95°C for 30 s and 61°C for 1 min. Dissociation curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. After the PCR program, fluorescent real-time PCR data from three replicate samples were analyzed with 7300 System SDS Software v.1.4.0 . The comparative CT method was used to analyze the BS CXC expression level. All analyses were based on the CT values of the PCR products. The CT was defined as the PCR cycle at which the fluorescence signal crossed a threshold line that was placed in the exponential phase of the amplification curve. The CT for target amplification of BS CXC and that for the internal control β-actin were determined for each sample. The difference between the CT values of the target and internal control, termed dCT, was calculated to normalize the differences in the amount of total nucleic acid added to each reaction and the efficiency of the RT-PCR. One of the samples was chosen as the reference sample and was called the calibrator. The dCT for each sample was subtracted from the dCT of the calibrator, and the difference gave the ddCT value. The BS CXC expression level could be calculated by 2-ddCT; the value represented an n-fold difference relative to the calibrator. The data obtained from real-time PCR were subjected to the Student's t-test to determine differences in the mean values between the control and treated groups. P values less than 0.01 were regarded as significant.Expression of the BS CXC transcript was studied by fluorescence real-time RT-PCR. Healthy black seabream were challenged with LPS (Sigma) or poly(I:C) (Sigma) at a dose of 3 mg/kg body weight, and the PBS-challenged fish were used as the negative control. Twenty-four hours after stimulation, total RNA was extracted from different organs, including the gill, heart, head kidney (HK), kidney, spleen and blood. Single-strand cDNA was synthesized with MMLV reverse transcriptase (Promega) and the oligo dT primer (Promega) using DNase I (Promega)-treated total RNA as the template. The gene-specific primer F85 and GR primers  containing a 6 His tag at the N-terminus for purification purposes and cloned into E. coli competent cells M15. The positive clones were screened by PCR with primers pQE30 F/rCXC R and the plasmids of positive clones were purified and sequenced using pQE30-R primer  under denaturing conditions, following the user manual. The protein was dialyzed in refolding buffer . Expression and purified protein were checked by 4–18% SDS PAGE gel. Western blot analysis was performed to confirm the identity of the BS rCXC; the monoclonal antibody of 6His  was used as the primary antibody for the detection of the Western blot.Head kidney (HK) macrophages and blood neutrophils were isolated according to the method of Solem J (1995)  and refiRat blood neutrophils were isolated like fish blood neutrophils isolation, and Rat macrophages were isolated from rat lungs by the perfusion method. Briefly, the lungs and trachea were picked out carefully, HBSS was added from the trachea to wash and perfuse the lung macrophages. The perfusion liquid containing macrophages was sedimented at 400 × g for 10 min at 4°C. The pellet was washed and resuspended with RPMI 1640 medium  supplemented with 5% fetal calf serum. The viable cells were determined by 0.5% trypan blue exclusion. All the rats were handled according to the principles of "Public Health Service Policy on Humane Care and Use of Laboratory Animals".6 cell/ml of the neutrophils or macrophages in fresh medium were added to the Transwell inserts; each dose had three replicates. After 2 h incubation at 25°C or 37°C in a humid chamber, the polycarbonate membranes were removed from the insert, and the upper layer cells of the membrane were rinsed with HBSS to remove the cell residues. Then the membranes were fixed in methanol and stained by Giemsa stain and the migration cells passing through the polycarbonate membranes were counted in twenty randomly selected fields under the microscope.The migration of the macrophages and the blood neutrophil cells induced by different concentrations of BS rCXC or hrIl8 was investigated using Transwell permeablThe means and standard errors were calculated from the replicates. The mean values were compared using the Origin 6.0 program . A one-way ANOVA test was performed when significant differences occurred at the 5% level. Differences were considered significant at p-values of < 0.05.CZ Conceived and designed the study and drafted the manuscript. GC, ZY and X K Performed the experiments and analyzed the data. ZYO participated in design of the study, helped to draft the manuscript and revised the manuscript.Table for cxc primerClick here for file


Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1). Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, β-catenin, Glycogen Synthase Kinase 3ß (GSK3β), axin and α-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and β-catenin interaction and did not induce transcriptional activation of β-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.Previous studies have indicated that Epstein-Barr virus (EBV) can modulate the  The oncogenic potential of Epstein-Barr virus (EBV) is well recognized, and the virus is associated with a number of human malignancies, including Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC) Wnt pathway Wnt cascade. Here we show that transient or stable expression of LMP1 sequences from normal B cells and NPC does not impair the expression of E-Cadherin and other mediators of the Wnt pathway. Furthermore, we also demonstrate that LMP1 expression in human cells had minimal effect on the interaction of E-cadherin and β-catenin thus no evidence of β-catenin-mediated transcriptional activation was observed.Over the last few years, there has been increasing evidence to suggest EBV is capable of modulating the To explore the effect of LMP1 on other mediators of the Wnt pathway, we transiently transfected HaCaT and MDCK cells with expression vectors encoding LMP1-GFP or the control EGFP vector. These LMP1 sequences were either derived from the prototype B95.8 isolate, spontaneous LCLs  or NPC (NPC9 and CAO). After transfection, these cells were examined using confocal microscopy for the expression of E-cadherin, β-catenin or actin. Representative data from a series of experiments is presented in To further confirm these observations, we resolved the protein samples  from these transfected cells on SDS-PAGE followed by immunoblotting. Data presented in Wnt cascade and there is no evidence of accumulation of β-catenin either in the cytoplasm or nucleus following expression of LMP1.To ensure that these observations were not influenced by the transient expression of LMP1, we established stable transfectants of the HaCaT cell line expressing B95.8-LMP1 and EGFP. These cells were processed for E-cadherin, β-catenin and actin expression using confocal microscopy. Similar to data obtained with transient transfection, HaCaT cells stably expressing LMP1 showed minimal change in the expression of E-cadherin and β-catenin when compared to the cells expressing EGFP alone , panel AAlthough the data presented above clearly indicated that LMP1 does not influence the expression of individual components of the Wnt pathway, it is possible that LMP1 may disrupt the interaction of E-cadherin and β-catenin. A series of experiments were designed to immunoprecipitate E-cadherin and β-catenin complexes from LMP1 and EGFP expressing cells. In the first set of experiments, E-Cadherin was immunoprecipitated from the whole cell extract and then resolved on SDS-PAGE followed by immunoblotting. The immunoblots were probed with anti-E-cadherin, anti-β-catenin and anti- α-catenin antibodies. Data presented in Previous studies have suggested that EBV-mediated activation of β-catenin involves stabilization of this protein which leads to β-catenin-mediated increased transcriptional activity. To explore the possibility that this effect may be mediated by LMP1, stable transfectants (LMP1 and EGFP-positive) were pretreated with cycloheximide to block fresh protein synthesis and protein samples collected at different time intervals. These samples were then resolved on SDS-PAGE followed by immunoblotting with the β-catenin-specific antibody. Data presented in Another possible approach to test the stabilization of β-catenin is to assess downstream transcriptional activity mediated by this protein. It is now well established that stabilized β-catenin forms a complex with Tcf/lymphoid enhancer factor transcriptional factors and that this complex transactivates various cellular oncogenes (e.g. c-myc and cyclin D1) which play crucial role in cell transformation and tumour development These experiments were undertaken to reassess the role of LMP1 in modulating the canonical Wnt pathway. We have used human epithelial and keratinocyte cell lines to express different sequence variants of LMP1 and studied its effect on the regulation of E-cadherin and β-catenin interaction/function. The effect of LMP1 expression was assessed using both a transient and stable expression system. Confocal microscopic studies showed none of the LMP1 sequences  had any dramatic effect on the expression of E-Cadherin and β-catenin. Furthermore, we also found no effect of LMP1 on the interaction of E-Cadherin and β-catenin and the downstream β-catenin-mediated transcriptional activity. Based on this extensive and in depth analysis, we propose that it is unlikely that LMP1 plays any significant role in the modulation of the Wnt pathway. It is very difficult to precisely identify the reason for difference in the results described here and those described previously by other groups It is important to stress here that our studies do not refute a potential role of EBV in activating β-catenin and its transcriptional activity. It is possible that another EBV protein(s) play a crucial role in regulating β-catenin activity in virus-infected normal and malignant cells. Indeed, recent studies by Morrison and colleagues have shown that EBV-encoded Latent membrane protein 2A (LMP2A) activates β-catenin in epithelial cells through the PI3/AKt pathway LMP1 sequences were amplified from prototype B95.8 isolate (referred to as B95.8-LMP1), NPC  or spontaneous lymphoblastoid cell lines  using sequence-specific primers and PCR and cloned in frame into the pEGFP-N1 vector . Amplified LMP1 sequences were ligated into the EcoR1 and BamHI sites of pEGFP-N1 in frame so as to express a fusion protein with the Green Fluorescent Protein (GFP) at the C-terminus. Human epithelial or keratinocyte cell lines  or Madin-Darby canine kidney cell line  were transfected with either pEGFP-N1 or LMP1-GFP expression vectors using lipofectamine 2000 . In some experiments, an expression vector (pcDNA3.1) encoding full-length LMP1 without GFP fusion protein was also used These cells were cultured for 36–48 h in RPMI-1640 supplemented with 10% FCS (growth medium) and the transfection efficiency was assessed by the expression of EGFP using FACScalibur . In some cases transfected cells were cultured in growth medium supplemented with Geneticin  for 3 weeks and stable transfectants isolated by FACSort .For immunofluorescence studies, LMP1 or EGFP expressing cells were seeded onto coverslips and cultured overnight in growth medium. After incubation, these cells were washed in PBS plus Calcium and Magnesium (PBSCM) and then fixed in 3% paraformaldehyde and permeabilised with 0.1% Saponin  in 5% FCS/PBSCM. After fixation, cells were incubated with monoclonal or polyclonal antibodies specific for LMP1 , β-Catenin  and E-Cadherin , and TRITC conjugated anti-Phalloidin antibody  in 5% FCS/PBSCM and incubated on the cells for 1 hour at room temperature. After washing in 0.1% Saponin/PBSCM, cells were incubated with anti-mouse Cy3 . Finally the cells were washed and examined by confocal microscopy .Monolayers of HaCaT cells stably expressing LMP1 or EGFP were washed with cold PBS (pH 8.0) and labelled with 2 mM EZ-Link Sulfo-NHS-Biotin Reagent . The reaction was quenched with PBS and 100 mM Glycine prior to solubilisation with modified radioimmunoprecipitation (RIPA) buffer . These cell extracts were initially incubated with E-Cadherin-specific antibody for 2.5 hours followed by an overnight incubation with Protein G Sepharose beads . After incubation, beads were extensively washed with RIPA buffer and protein samples resolved using standard SDS-PAGE, transferred to nitrocellulose membrane and incubated with HRP-conjugated streptavidin , and antibodies specific for E-Cadherin, β-catenin or α-catenin ,. Protein bands were detected using Chemiluminescence Reagent Plus  and their intensity compared by densitometric analysis using Imagequant software . In some experiments protein samples from EGFP and LMP1-GFP expressing cells were directly resolved on the SDS-PAGE, transferred to nitrocellulose membrane and incubated with HRP-conjugated streptavidin, and antibodies specific for LMP1, E-Cadherin, β-catenin, α-catenin or GSK3β , GAPDH  and Axin .6 cells and equal aliquots of cells were removed at time points 0 min, 45 min, 90 min, 135 min and 180 min; and lysed in RIPA buffer, resolved on SDS-polyacrylamide gel, transferred to nitrocellulose membrane and incubated with antibodies specific β-catenin. Protein bands were detected using Chemiluminescence Reagent Plus densitometricaly analysis using Imagequant software.The human keratinocyte cell line HaCaT was transiently transfected with expression constructs encoding LMP1-GFP or EGFP as described above. At 30 h posttransfection, cyclohexamide (50 µg/ml) was added to 1×106 HaCaT cells were co-transfected with TOPFlash or FOPFlash . in combination with EGFP or LMP1-GFP expression plasmids  at a ratio of 1∶2. After incubation for 24 h, a small aliquot of these cells was analysed for GFP expression by FACSCalibur and cell numbers were accordingly standardised based on GFP positive cells. These cells were used in luciferase reporter assays according to the manufacturer's instructions . The TOPFlash luciferase activity produced by each sample is shown relative to the matching FOPFlash activity produced.Approximately, 106 cells, and 0.8 µg total DNA was added to 2×105 cells when using Effectene™ (Qiagen) for the NF-κB co-transfections. After growth for 24 hours in RPMI medium supplemented with antibiotics and 10% FCS, cells were harvested and resuspended in PBS supplemented with 2% FCS. To measure successful transfection a small aliquot was taken for FACScan (Becton Dickson) analysis of GFP expression, and the volume remaining was subsequently standardized to the sample of lowest efficiency. The transfection efficiencies were generally between 40–50%. To report the transcription factor activity, the cells were pelleted and lysed with 120 µl of Cell Culture Lysis Reagent (Promega) and the luciferase activity measured on 20 µl triplicates by addition of Luciferase Assay Reagent (Promega). The luciferase activity produced by each sample was calculated relative to the activity produced by the prototype B95-8-LMP1.HaCaT cells were grown to late-log phase of cell growth prior to co-transfection. On the day of transfection, luciferase reporter plasmids for either NF-κB (3.Enh-Luc) or STAT (pGRR5-Luc)  were combined with LMP1-GFP expression plasmids  and pEGFP-N1 expression vector in a 1∶2 ratio. Electroporation was used for the STAT co-transfections where 9 µg of total DNA was used for 5×10


The Human papillomavirus plays an important role in the initiation and progression of cervical cancer. However, it is a necessary but not sufficient cause to develop invasive carcinoma; hence, other factors are required in the pathogenesis of this malignancy. In this review we explore the hypothesis of the deregulation of wnt/β-catenin signaling pathway as a "second hit" required to develop cervical cancer. Cervical carcinoma (CC) is one of the most common cancers and a leading cause of death among women worldwide. . The numOver 30 years ago, some authors pointed out that CC had sexually transmitted disease behavior ,5. But iAt present, more than 100 types of HPV have been identified , 40 of wOnce HPV has infected basal cells, the viral genome is replicated actively as episome and early genes E1–E7) are expressed. E1 and E2 are important proteins for viral genome replication and viral cycle completion . E1 play–E7 are eMYC, NR4A2, hTERT, APM-1, FANCC, and TNFAIP2, etc., are found . 2.. 2.39].  pathway . The canFollowing the trimerization of Wnt/Fzd/LRPs, the LRP co-receptor is phosphorylated leading to the binding and phosphorylation of Disheveled (Dvl), which transduces the Wnt signal into the cell through direct binding with FZD ,43. Dvl An important hallmark in the canonical Wnt pathway is the stabilization of β-catenin in cytoplasm and its accumulation into the nucleus. This is possible due to the detachment of β-catenin from the degradation complex, a multi-protein assembly activated in the absence of wnt signaling, whose main task is to add ubiquitins to β-catenin resulting in its inactivation by means of the ubiquitin-proteasome pathway . In an uAfter the FZ/LRP activation by its ligand, the degradation complex kinase activity is inhibited. Consequently, non-phosphorylated β-catenin is stabilized, and increasing levels tend to accumulate in the cytoplasm. β-catenin accumulation in cytoplasm leads to its nuclear accumulation, where it is associated with lymphoid enhancer-binding factor 1/Tcell-specific transcription factor (LEF/TCF) and transcriptional activator Pygopus (Pygo). Pygo contains a domain (PHD), which is shared by many nuclear proteins with a role in chromatin remodeling and transcriptional co-activation . MutatioIn the absence of wnt signaling, TCF is bound to Groucho, forming a repressor complex of Wnt target genes. Groucho can repress gene transcription by inhibition of basal transcriptional machinery and recruitment of histone deacetylases (HDACs) ,52. AlthWhen β-catenin enters the nucleus, it converts the repressor LEF/TCF/Groucho into a transcriptional activator complex. This is possible due to Groucho displacement by interaction between β-catenin and LEF/TCF, thereby activating wnt target genes . SeveralTo this point we have reviewed how the wnt/β-catenin pathway turns on complex signaling events leading to the activation of target genes. Mutations in several components of this pathway have been studied and identified in nearly all human cancers. However, in cervical cancer only few studies have shown the involvement of certain wnt/β-catenin genes in their pathogenesis. In this regard, it has been suggested that HPV-immortalized human keratinocyte transformation requires a second hit and could be achieved thanks to the activation of the canonical wnt pathway . This hyAnother mechanism involved in the upstream activation of the wnt/β-catenin pathway is over-expression of pathway activators such as wnt ligands, frizzled receptors, and disheveled. There is evidence showing over-expression of WNT10B, -14, FZD10, and DVL-1 in cervical cell lines -65; noneWe recently conducted a genome-wide expression analysis in HPV16 CC compared against normal cervix epithelia . We wereInterestingly, gene components of the planar cell polarity (PCP) pathway were actively expressed in normal cervices, showing that this branch of wnt signaling is downregulated in CC Figure . In vertEven though the extensive use of the Papanicolau smear and colposcopy examination have significantly decreased the mortality rates, CC remains as the second cause of death in women worldwide. HPV sequences have been found in more than 99% of CC and HPV infection is the most important etiologic factor in cervical carcinogenesis. Besides, HPV infection is very common among the young sexually active population, but only a small fraction of infected individuals will develop cervical carcinoma later in life. Thus, HPV can be considered as an initial hit in the multistep carcinogenesis that leads to the development of CC. The molecular pathways involved in the progression of HPV-infected cells to CC have not yet been accurately identified. Here, we reviewed the role of Wnt/β-catenin pathway activation and the inactivation of planar cell polarity pathway in CC cells as a second hit to develop CC.In CC mutations on CTNNB1 gene are uncommon; thus the activation on wnt signalling pathway it seems to be activated upstream of/β-catenin. This fact could be achieved by means of negative regulators inactivation or over-expression of pathway activators.Other branch in wnt signaling pathway that could be relevant during CC pathogenesis is planar cell polarity (PCP) pathway, which is involved in differentiation processes. PCP is a key differentiation and morphogenetic process involved in development of epithelia. In normal cervical epithelia, cells are polarized and migrate from basal to the luminal space as they differentiate. Interestingly, PCP component genes are down-expressed in CC, indicating that this mechanism could be abated prior the establishment of the neoplasia. From the diagnostic point of view this fact could be important, because if we were able to show as an early process, we could account with potential molecular markers for early detection.The authors declare that they have no competing interests.CPP conceived and wrote the manuscript, ADG and BAT participated in the discussion and analysis of the content.


The expression pattern of runt-related transcription factor 2 (Runx2) was similar to that of β-catenin. Immunofluorescence staining indicated that Runx2 was also expressed in β-catenin–positive odontoblast-like cells and DPCs during reparative dentin formation. Knockdown of β-catenin disrupted odontoblastic differentiation, which was accompanied by a reduction in β-catenin binding to the Runx2 promoter and diminished expression of Runx2. In contrast, lithium chloride (LiCl) induced accumulation of β-catenin produced the opposite effect to that caused by β-catenin knockdown. In conclusion, it was reported in this study for the first time that β-catenin can enhance the odontoblastic differentiation of DPCs through activation of Runx2, which might be the mechanism involved in odontoblastic differentiation during reparative dentin formation.An intense stimulus can cause death of odontoblasts and initiate odontoblastic differentiation of stem/progenitor cell populations of dental pulp cells (DPCs), which is followed by reparative dentin formation. However, the mechanism of odontoblastic differentiation during reparative dentin formation remains unclear. This study was to determine the role of β-catenin, a key player in tooth development, in reparative dentin formation, especially in odontoblastic differentiation. We found that β-catenin was expressed in odontoblast-like cells and DPCs beneath the perforation site during reparative dentin formation after direct pulp capping. The expression of β-catenin was also significantly upregulated during odontoblastic differentiation of  Dental pulps have regenerative capacity to form reparative dentin in cases of tooth injury A number of molecular mechanisms are involved in odontoblastic differentiation of DPCs β-catenin regulates a number of genes in various biological processes All animal experimental procedures were approved by the Institutional Animal Care Committee of Wuhan University. The study was approved by the Ethics Committee of Wuhan University School of Stomatology. Informed written consent was obtained from the parents/guardians of the children. Written informed assent was additionally obtained from the children who participated in the study.Direct pulp capping was prepared as previously described by us After deparaffinization and rehydration, the sections were treated with pepsin  for 10 min. For immunohistochemical staining, endogenous peroxidase was blocked with 3% hydrogen peroxide followed by blocking with goat serum for 30 minutes. The sections were incubated with rabbit anti-β-catenin  and mouse anti-Runx2  primary antibodies at 4°C overnight. The sections were washed and stained using the SP kit  according to the manufacturer's instructions. Then stainings were visualized by diaminobenzidine solution . For double immunofluorescence staining, the sections were incubated with primary antibodies mixtures of rabbit anti-β-catenin and mouse anti-Runx2 antibodies at 4°C overnight, followed by incubation with a mixture of goat anti-rabbit-Cy3 and goat anti-mouse-Dylight 488 antibodies for 1 h at room temperature. Sections were counterstained with DAPI to reveal nuclei. Photos were obtained by a fluorescent microscope with a camera . Excitation and emission wavelengths were 405 and 425–460 nm (blue) for DAPI, 473 and 485–545 nm (green) for DyLight 488, 559 and 575–620 nm (red) for Cy3. Negative controls were obtained by substitution of primary antibodies with phosphate-buffered saline (PBS).2). These explants were transferred to T-25 flasks containing alpha-modified Eagle's medium supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. After 2 weeks, the explants were discarded and DPCs were serially passaged. Cells at passage 3 were used in the study. The culture medium was changed at 3-day intervals.Healthy premolars were collected from 13-to 15-year old patients undergoing premolar extractions for orthodontic reasons. Immediately after extraction, the teeth were washed with PBS and then fractured mechanically. The pulp tissue was removed with forceps and minced into small fragments (1–2 mmOdontoblastic differentiation induction was performed as previously described 5-AAGUCCUGUAUGAGUGGGAAC-3) 5-TTCTCCGAACGTGTCACGT-3)  was constructed by GenePharma as previously described. 9 pfu/ml. DPCs were seeded at a density of 1.5×104 cells/cm2. The next day, the cells were infected for 24 h with supernatants containing viral particles (150 multiplicity of infection) in culture medium containing 5 µg/ml polybrene. The cells were then washed with PBS and treated with odontoblastic differentiation medium as mentioned above β-catenin knockdown by shRNA was performed in the DPCs. A self-inactivating lentivirus containing a CMV-driven, enhanced green fluorescent protein (EGFP) reporter and a U6-driven shRNA against β-catenin  solution for 20 min and washed twice in PBS.Alkaline phosphatase (ALP) activity assay was performed using a commercial ALP activity assay kit  according to the manufacturer's instructions. All results were normalized to total protein content. ALP activity was calculated as moles of p-nitrophenol/mg protein.−ΔΔCT method, and the values were normalized to the levels of GAPDH. Each experiment was performed in triplicate. Specific primers used were β-catenin,5-GTGCTATCTGTCTGCTCTAGTA-3(Forward),5-CTTCCTGTTTAGTTGCAGCATC-3(Reverse);Runx2,5-GAACCACAAGTGCGGTGCAA-3(Forward),5-ACTGCTTGCAGCCTTAAATGACTCT-3(Reverse); BSP,5-CAAGCATGCCTACTTTTATCCTC-3(Forward),5-CTTCTTGGGAAGCTGGATTG-3(Reverse);OCN,5-GGTGCAGCCTTTGTGTCCAA-3(Forward),5-CCTGAAAGCCGATGTGGTCA-3(Reverse);DSPP,5-CCATTCCCACTAGGACTCCCA-3(Forward),5-TGGCGATGCAGGTCACAAT-3(Reverse);DMP-1,5-AGGAAGTCTCGCATCTCAGAG-3(Forward),5-TGGAGTTGCTGTTTTCTGTAGAG-3(Reverse);ALP,5-AACATCAGGGACATTGACGTG-3(Forward),5-GTATCTCGGTTTGAAGCTCTTCC-3(Reverse);and GAPDH, 5-GCACCGTCAAGGCTGAGAAC-3(Forward),5-ATGGTGGTGAAGACGCCAGT-3(Reverse).Total RNA was extracted from the cells with Trizol reagent . Reverse transcription was carried out using a PrimeScript RT reagent kit  according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) was performed using SuperReal Premix  on the ABI 7500 Real-Time PCR System . Relative gene expression was calculated by the 2Cultured DPCs were lysed in radioimmunoprecipitation (RIPA) buffer. Total proteins were measured using the BCA Protein Assay kit . Equal amount of protein was separated in 10% SDS-polyacrylamide gels and electrophoretically transferred onto a polyvinylidene difluoride transfer membrane. After blocking with 5% nonfat milk, the membranes were incubated overnight with specific primary antibodies against β-catenin , Runx2 , DSP , BSP , or GAPDH , followed by incubation with horseradish peroxidase(HRP)-conjugated secondary antibody . The blots were then visualized by enhanced chemiluminescence (ECL).5-CGTAGTAGTACACAACGCCG-3(Forward) and 5-GTTTCGTGTCTGT CTTCCCC-3(Reverse). qPCR reactions were carried out in the ABI 7500 Real-Time PCR System and enrichment of the immunoprecipitated DNA was normalized with the 1/10 inputs.The changes in binding of β-catenin to the promoter of Runx2 were analyzed with ChIP assays as previously described t test (comparing two groups) using software Sigma Plot 10.0 , P<0.05 was considered to be statistically significant.Statistical significance was evaluated by the Student-Newman-Keuls multiple comparison test after a one-way ANOVA or Student's To examine the temporospatial expression of β-catenin during reparative dentin formation, H&E and immunohistochemistry staining assays were performed on dental pulps at different stages of the reparative process. Immediately after tooth preparation (day 0), the odontoblast layer with weak β-catenin expression was detached from the dentin surface and the cells beneath the perforation site were irregularly arranged . At 7 daFirst, we confirmed odontoblastic differentiation of human DPCs by demonstrating mineralization nodules formation  and incrTo further determine whether the upregulation of β-catenin was necessary for odontoblastic differentiation, we used lentivirus that express specific β-catenin shRNA to knock down β-catenin in DPCs and found that more than 90% of the infected cells were EGFP positive on day 14 after odontoblastic induction . qPCR anTo further investigate the effect of β-catenin on odontoblastic differentiation of DPCs, we treated the cells with LiCl that was known to cause β-catenin accumulation We found that Runx2 expression was undetectable in normal healthy dental pulps . However2+, that is released from MTA, reacts with phosphates to yield precipitates similar to hydroxyapatite Reparative dentin formation is a hallmark of dental pulp healing process following direct pulp capping with MTA, calcium hydroxide, and other materials Previous studies suggest that there is a hierarchy of stem/progenitor cells in the adult dental pulp, including a small population of stem cells and relatively more committed progenitor cells in vitro odontoblastic differentiation of DPCs. These data indicate that β-catenin participates in reparative dentin formation and might also be necessary for odontoblastic differentiation of DPCs. We found that knockdown of β-catenin reduced the calcium nodule formation and the expression of DSPP, DMP-1, ALP, and BSP. In contrast, LiCl induced accumulation of β-catenin promoted calcium nodule formation accompanied by upregulated expression of DSPP, DMP-1, ALP, BSP and OCN. These findings suggest that odontoblastic differentiation of DPCs can be enhanced by β-catenin. These findings are consistent with a previous study showing that activation of β-catenin enhances odontoblastic differentiation of stem cells from the apical papilla et al. reported that stable expression of β-catenin inhibits differentiation and mineralization of dental pulp stem cells et al. found that activation of β-catenin signaling inhibited the differentiation of bone mesenchymal stem cells. However in cells that are committed to the osteoblast lineage, β-catenin enhanced osteoblast differentiation β-catenin has been reported to play fundamental roles in various biological processes et al. demonstrated that β-catenin/CBP complex plays a critical role in the maintenance of mES cell self-renewal capacity et al. have shown that the β-catenin/TCF1 complex occupies the proximal promoter to control physiological levels of Runx2 In vitro study of the odontoblastic differentiation of DPCs showed that expression of Runx2 was upregulated as well. Furthermore, we found that knockdown of β-catenin significantly diminished enhanced expression of Runx2 in differentiated DPCs, while LiCl induced β-catenin accumulation upregulated Runx2 expression. These findings indicate that Runx2 is a downstream mediator of β-catenin to promote odontoblastic differentiation of DPCs during reparative dentin formation. ChIP assays have consistently demonstrated that odontoblastic differentiation resulted in enhancement of β-catenin binding to the promoter region of the Runx2 gene. Additionally, knockdown of β-catenin caused a disassociation of β-catenin from the examined ChIP region, and accumulation of β-catenin by LiCl treatment enhanced the binding of β-catenin.Previous studies showed that β-catenin interacts with several transcriptional co-factors to form DNA binding complex that regulates transcription of target genes. Miyabayashi in vitro. Additionally, odontoblastic differentiation of DPCs was accompanied by a remarkable increase of Runx2, which was controlled by β-catenin. Moreover, coexpression of Runx2 and β-catenin was observed in the odontoblast-like cells and DPCs beneath the perforation site during reparative dentin formation. Therefore, the β-catenin/Runx2 pathway may enhance odontoblastic differentiation of DPCs during reparative dentin formation. It has been reported that Runx2 can regulate the transcription of the DSPP gene Collectively, our study showed that β-catenin was expressed in the DPCs and odontoblast-like cells beneath the perforation during reparative dentin formation. β-catenin enhanced odontoblastic differentiation of the DPCs


MicroRNAs of the miR-302 cluster are involved in early embryonic development and somatic cell reprogramming. Expression of the miR-302 gene is regulated by the binding of the pluripotency factors Oct4, Sox2 and Nanog to the miR-302 promoter. The specific expression pattern of the miR-302 gene suggested that additional transcription factors might be involved in its regulation. Here, we show that the miR-302 promoter is a direct target of the Wnt/β-catenin signaling pathway. We found that the miR-302 promoter contains three different functional Tcf/Lef binding sites. Two of the three sites were located within the cluster of Oct4/Sox2/Nanog binding sites and were essential for Wnt/β-catenin-mediated regulation of the miR-302 gene. Tcf3, the only Tcf/Lef factor that bound to the miR-302 promoter, acted as a repressor of miR-302 transcription. Interestingly, mutations in the two Tcf/Lef binding sites and the Oct4/Nanog binding sites abolished miR-302 promoter responsiveness to Wnt signaling, suggesting that the Tcf/Lef and the Oct4/Nanog sites interact genetically. Xenopus laevis embryos, the miR-302 ortholog miR-427 promotes mesendoderm formation by targeting the Lefties, inhibitors of the Nodal pathway , centrifuged through Qia Shredder Columns  and boiled for 5 min at 95°C. Thirty micrograms of total protein from each sample was resolved on 10% SDS-Page gels and blotted onto PVDF membranes. The antibodies used in this study were anti-β-catenin (BD Transduction), anti-GAPDH  and anti-Oct-3/4 .6 cells (P19 or ESCs) were seeded in 15 cm dishes coated with gelatin one day prior to starting the procedure. Cells were washed once with PBS and subsequently fixed by addition of 1% formaldehyde solution in PBS. Plates were placed on a rocking platform at room temperature for 10 min. After rinsing twice with ice-cold PBS, cells were scraped off and collected by centrifugation . The pellet was lysed by adding 500 µl of lysis buffer (containing a mix of protease inhibitors) per 15-cm dish and incubating on ice for 15 min. Sonication with the Covaris S2 sonication system was performed to promote chromatin fragmentation. The lysate was cleared from cellular debris by centrifugation . DNA concentration was measured using a NanoDrop 1000 spectrophotometer. For chromatin immunoprecipitation, 250 µg of chromatin was diluted with dilution buffer  to a final volume of 1 ml. An aliquot of the chromatin sample (input control) was removed and stored at -20°C. After pre-clearing with protein G dynabeads (Invitrogen), 5 µg of the respective antibody was added, and the samples were incubated on a rotating wheel over night at 4°C. The following day, an equal volume of protein G dynabeads was added to each sample and incubated on a rotating wheel for 1 hour at 4°C. Beads were collected using a magnetic rack and subjected to sequential washings: 2x low salt wash buffer, 2x high salt wash buffer, 1x LiCl wash buffer and 3x TE buffer. Each washing step was performed at 4°C on a rotating wheel for 10 min. Antibody-protein complexes were eluted by adding 100 µl of freshly prepared elution buffer to the beads and constantly shaking at 1250 rpm at room temperature for 1 hour. Eluates as well as the input controls were subjected to reverse crosslinking by incubation at 65°C with constant shaking (950 rpm) over night. DNA was purified using the MinElute PCR Purification Kit (Qiagen). DNA was stored at -20°C until used for qPCR analysis.ChIP experiments were performed as described previously . In brieThe antibodies used for the ChIP experiments were anti-Tcf1 (Santa Cruz), anti-Tcf3 (Santa Cruz), anti-Tcf4 (Santa Cruz) or anti-Lef1 (Santa Cruz). Primer sequences are given in Table S3 in File S1.4 pH 7.8; 10% glycerol; 0,1% NP-40) on a rocking platform for 30 min at 4°C. The lysates were collected and cleared from cellular debris by centrifugation  immediately or prior to measurement. The lysates were stored at -20°C until use. Firefly and Renilla luciferase activities were separately measured in duplicate in 96-well microtiter plates using a Centro LB 960 luminometer (Berthold Technologies). The values obtained for the firefly luciferase activities were normalized to the corresponding Renilla luciferase activities.For the luciferase assays, transfected cells were incubated with NP40 lysis buffer  correlated with active β-catenin in ESCs, embryonic carcinoma cells and the developing mouse embryo  and analTo rule out a cell line-specific effect, we also knocked down β-catenin in the embryonic carcinoma cell line P19 . This ceTo determine whether the expression of the miR-302 gene is regulated by Wnt signaling, we stimulated the Wnt pathway by treating mouse ESCs with Wnt3a. Upon treatment, expression of both the pri-miR-302 transcript and the mature miRNAs increased up to three-fold . In contflox/-: CreERT2 ESCs were derived from the β-cateninflox/- ESCs. The β-cateninflox/-: CreERT2 ESCs constitutively express fusion proteins composed of Cre recombinase fused to the ERT2 mutant ligand-binding domain of the human estrogen receptor. The recombinase activity of these fusion proteins is blocked by their binding to heat shock proteins. The synthetic ligand 4-hydroxy-tamoxifen (Tam) releases the heat shock proteins and induces recombinase activity [flox/-: CreERT2 ESCs with Tam for 3 days led to an efficient depletion of β-catenin from these cells, as evidenced by Western blot analysis  were used. The β-cateninactivity . Treatmeanalysis . As a coanscript . In agreanscript . Furtherwith Tam . Expresswith Tam . These rA fragment of the miR-302 promoter containing the binding sites for OCT4, SOX2 and NANOG is sufficient to drive OCT4-dependent activation of the miR-302-367 cluster in human ESCs and P19 cells [To determine which of the three identified binding sites were the effectors of Wnt/β-catenin signaling, we performed two different sets of experiments. First, we knocked down β-catenin in mESCs. Second, we treated mESCs and P19 cells with Wnt3a prior to performing luciferase assays. In agreement with our previous results, β-catenin knockdown repressed the wildtype miR-302 promoter, whereas Wnt3a treatment activated this promoter . MoreoveFour Tcf/Lef proteins are expressed in embryonic stem cells: Tcf1, Tcf3, Tcf4 and Lef1. It is thought that Tcf1, Tcf4 and Lef1 fulfill activating functions, whereas Tcf3 mainly acts as a transcriptional repressor. To determine which Tcf/Lef factors bind to the miR-302 promoter, we carried out chromatin immunoprecipitation (ChIP) experiments in mESCs and P19 cells . We coulHere, we identify the miR-302 gene as a new transcriptional target of the Wnt/β-catenin pathway in mESCs and P19 cells. Wnt/β-catenin mediated transcription of the miR-302 gene through two non-canonical Tcf/Lef binding sites bs 5 and bs 6 located in the miR-302 promoter. As the only member of the Tcf/Lef family that demonstrated binding to the miR-302 promoter, Tcf3 bound to the bs 5 and bs 6 positions of the miR-302 promoter and repressed miR-302 transcription through these sites. All other members of the Tcf/Lef family are considered to be transcriptional activators, whereas Tcf3 is a transcriptional repressor –34. It wIt was originally thought that upon activation of the Wnt/β-catenin pathway, β-catenin shuttles from the cytoplasm to the nucleus and activates target gene transcription by replacing members of the family of groucho co-repressors from the Tcf/Lef factors. Subsequent studies revealed that additional mechanisms of action may exist. In addition to its interaction with a number of transcription factors other than the Tcf/Lef factors ,25,38,39in vitro expression pattern, the expression of miR-290 cluster miRNAs is already upregulated in four-cell stage mouse embryos and reaches the highest level in the blastocyst, whereas miRNAs of the miR-302 cluster begin expression later at E6.5 and reach their expression peak at E7.5 [in vivo in the blastocyst, whereas miR-302 cluster miRNAs may be important at later stages. Although it was clearly demonstrated that Oct4, Sox2 and Nanog regulate the expression of the miR-302 gene, the expression pattern of these pluripotency factors and the miR-302 gene only partially overlap [Bs 5 and bs 6 are located within a cluster of binding sites for the pluripotency factors Oct4, Sox2 and Nanog in the miR-302 promoter. Because of their close proximity to each other, these binding sites might genetically interact. This idea is supported by the fact that the responsiveness of the miR-302 promoter to β-catenin knockdown and the Wnt3a treatment was abolished not only when Tcf/Lef binding site bs 5 and bs 6 were mutated but also when the Oct4/Nanog binding sites were mutated. Moreover, the derepressive effect observed upon depletion of Tcf3 from mESCs was abrogated not only by the simultaneous mutation of bs 5 and bs 6 but also by the mutation of the Oct4/Nanog sites. An interdependence of the Tcf/Lef sites and the Oct4/Nanog sites is further supported by the specific expression pattern of the miR-302 cluster in the mouse embryo. In mESCs derived from the E3.5 blastocyst, the miR-290 cluster miRNAs are expressed at much higher levels than the miRNAs of the miR-302 cluster . In cont at E7.5 ,43. Thes overlap . Althoug overlap ,16The activation of the Wnt pathway perfectly mirrors the increase in miR-302 gene expression, and thus, active Wnt/β-catenin signaling could be another missing factor that drives miR-302 expression in the mouse embryo. This idea is further supported by the fact that the miR-302 cluster functionally overlaps with the Wnt/β-catenin pathway. The miRNAs from the miR-302 cluster, in addition to Wnt/β-catenin signaling, promote the formation of mesendoderm in the gastrulating embryo, whereas the loss of Tcf3 results in the expansion of axial mesoderm in the mouse ,15,19.Several of the miR-302 and miR-290 cluster miRNAs share similar seed sequences, and it has been suggested that miRNAs of the two clusters act redundantly . TherefoFile S1Table S1, siRNA sequences. Table S2, Primer sequences and numbers of universal probes used in the qPCR experiments. Table S3, Sequences of the primers used in ChIP experiments.(DOCX)Click here for additional data file.


Perturbations in cell adhesion molecules are linked to alterations in cadherin-catenin complexes and likely play major roles in invasion and metastasis; their impact on early precancerous stages remains yet unknown. We showed ALCAM overexpression in early oral lesions and its cytoplasmic accumulation in oral squamous cell carcinoma (OSCC) to be a predictor of disease progression and poor prognosis. This study tested the hypothesis that alterations in E-cadherin and β -catenin expressions are early events in oral tumorigenesis, associated with disease prognosis, and correlate with perturbations in ALCAM expression.Expressions of E-cadherin and β-catenin were analyzed in the same cohort of 105 OSCCs, 76 oral lesions and 30 normal oral tissues by immunohistochemistry and correlated with clinicopathological parameters and prognosis. The effect of siRNA mediated ALCAM knockdown on E-cadherin and β -catenin was determined using western blot, confocal microscopy and RT-PCR analysis in oral cancer cells.trend <0.001); and correlated with cytoplasmic ALCAM accumulation in OSCCs (p  = 0.006). Multivariate analysis revealed β-catenin membrane loss and ALCAM/β-cateninnuclear/cytoplasmic accumulation to be significant predictors for late clinical stage  and nodal metastasis . Cox’s regression showed E-cadherin membrane loss/ALCAM cytoplasmic expression  to be independent adverse prognosticators in OSCCs. siRNA mediated silencing of ALCAM resulted in concurrent increase in E-cadherin and β-catenin both at the transcript and protein levels.Significant loss of membranous E-cadherin and β-catenin expression was observed from normal, hyperplasia, dysplasia to OSCCs (pin vitro and in in vivo suggests that these dynamic changes in cell adhesion system may play pivotal role in oral cancer.Losses of E-cadherin and β-catenin expressions are early events in oral tumorigenesis; their associations with aggressive tumor behavior and disease recurrence underscore their potential as prognostic markers. Correlation of loss of E-cadherin and β-catenin with cytoplasmic ALCAM accumulation both  Perturbations in orchestrated modulation of cell adhesion cause defects in tissue architecture that play critical roles in cancer development The development of oral squamous cell carcinoma (OSCC) is a multistep process involving interactions between several factors such as tobacco-associated intra-oral carcinogens, areca nut, betel quid and alcohol consumption, and/or viral infections Dynamic changes in cell adhesion manifested by dissociation of membranous E-cadherin-β-catenin complex are implicated in loss of epithelial cohesion as an important event in invasion and metastasis. Activated leukocyte cell adhesion molecule (ALCAM)/MEMD/CD166) is a transmembrane glycoprotein of immunoglobulin superfamily that mediates cell-cell adhesion through both homophilic (ALCAM-ALCAM) and heterophilic (ALCAM-CD6) interactions This study was approved by Human Ethics Committee of All India Institute of Medical Sciences, New Delhi, India. For immunohistochemical analysis surgically resected tissues or biopsy specimens from OSCC, dysplasia, hyperplasia and histologically normal oral tissues were obtained from Surgical Oncology Unit of Dr. B.R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India, with prior informed written consent of the patients.2 and stored at −80°C till further use and one piece was collected in 10% formalin and embedded in paraffin for histopathological and immunohistochemical analyses.One hundred and five primary OSCC patients  undergoing curative oral cancer surgery, at the Surgical Oncology Unit of Dr. B.R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India were enrolled in this study after obtaining prior written consent of the patients. The clinical and pathological data including clinical TNM staging , site of the lesion, histopathological differentiation, age, and gender were recorded in a pre-designed performa as described previously et al. Paraffin embedded sections (5 µm thickness) of human oral tissue specimens were stained with hematoxylin and eosin for histopathological analysis, and immunostaining was done on serial sections as described previously by Sawhney The immunostaining was evaluated in randomly selected five non-overlapping areas of the tissue sections with more than 80% epithelial cells. For E-cadherin, specific staining in the membrane was defined as positive staining. The slides were scored as follows: ≥50% tumor cells showing immunoreactivity were graded as positive for E-cadherin expression, while those showing immunostaining in <50% tumor cells were graded as negative 1 and T2 tumors were treated with radical radiotherapy or surgery alone, whereas majority of patients with T3 and T4 disease were treated using a combination of radical surgery followed by postoperative radical radiotherapy as described Seventy-two of the 105 OSCC patients, who underwent treatment of primary OSCC from 2002–2005, could be followed regularly in the follow up clinic, while 33 patients were lost to follow up. Survival status of patients was verified and regularly updated from Tumor Registry records, Institute of Rotary Cancer Hospital, as of December 2010. As per our protocol, OSCC patients with TThe immunohistochemical data were subjected to statistical analysis using SPSS 17.0 software (Chicago IL). The relationships between E-cadherin, β-catenin and ALCAM expression and clinicopathological parameters was tested in univariate analysis by Chi-Square test, Chi-Square test for trend, Fisher’s exact test and logistic regression analysis. To determine independent predictors for tumorigenesis, logistic regression analysis was carried out in stepwise manner for the individual variables, clinicopatholgical parameters and the proteins E-cadherin, β-catenin and ALCAM. Different combinations of variables were generated to assess the association between these combinations and patient prognosis. Follow-up studies were analyzed by Kaplan-Meier, and Cox’s Proportional Hazards test. Only disease free survival was evaluated in the present study, as the number of deaths due to disease progression did not allow a reliable statistical analysis. The association between patient outcome and variables was assessed by log–rank test. Two sided p values were calculated and p<0.05 was considered to be significant.2. SCC-4 cells were transiently transfected using Hiperfect reagent (Qiagen) and a blunt-ended duplex of the RNA oligonucleotides, 5′- AAG CCC GAU GGC UCC CCA GUA UU-3′and 5′-AAU ACU GGG GAG CCA UCG GGC UU-3′. SCC-4 cells were treated with varying concentrations of siRNA (1–15 nM) and with varying concentrations of Hiperfect reagent (1–4.5 µL). ALCAM siRNA (15 nM) with 4.5 µL of Hiperfect reagent for 48 h was found to be the best concentration with maximum transfection efficiency (95%) and maximum silencing of ALCAM gene expression (data not shown). Forty-eight hours post-transfection, siRNA-treated cells were used in subsequent experiments.Human head and neck squamous carcinoma cell line, SCC-4 cells were maintained in DMEM containing 10% FBS, 100 µg/ml streptomycin, 100U/ml penicillin at 37°C in humidified atmosphere of 5% COCTG GCC ATA TCC ACC AGA GT -3′ were used. β-actin was used as an internal control to ensure that equal amount of RNA was used in control and siRNA transfected cells. For β-actin cDNA, forward primer 5′- CAG CCA TGT ACG TTG CTA TCC AG -3′ and reverse primer 5′- GTT TCG TGG ATG CCA CAG GAC -3′ were used. PCR products were separated on 1.5% agarose gel, stained with ethidium bromide, and visualised with AlphaEase FC Software (version 3.1.1) The intensities of PCR bands were quantified with Chemi Imager IS-4400  as described earlier Total RNA was isolated from SCC-4 cells (control and siRNA transfectants) as described earlier ALCAM siRNA transfected SCC-4 cells for 48 h and untreated control cells were used for the preparation of cell extracts by boiling the cell pellet in SDS-lysis buffer. Briefly, whole cell extract (100 µg protein/lane) were resolved by SDS-PAGE and transferred onto nitrocellulose membrane SCC-4 cells grown on coverslips were treated with ALCAM siRNA for 48 h and processed for confocal laser microscopy as described previously trend <0.001). Loss of E-cadherin membranous expression was observed in 61/105 (58%) OSCCs  of the histologically normal oral tissues with occasional cytoplasmic expression . Signifi%) OSCCs  and was Seventy-two OSCC patients could be followed up for a maximum period of 91 months (median 24 months). Kaplan–Meier survival analysis and Cox’s proportional hazard model revealed that patients with E-cadherin positive tumors had increased median disease-free survival (DFS) of 74 months as compared to those with loss of membranous immunopositivity (median DFS = 18 months)  .trend <0.001 and ptrend = 0.003 respectively).β-catenin was mainly present in the membrane of epithelial cells in 27/30 (90%) of histologically normal oral tissues; occasionally cytoplasmic/nuclear staining was also observed . SignifiLoss of β-catenin membranous expression was observed in 65 of 105 (62%) OSCCs , Table 1Loss of membranous β-catenin immunopositivity was associated with reduced disease free-survival (median DFS = 15 months) in comparison with patients with β-catenin membrane positive tumors (median DFS = 78 months)  .−/β-cat M-) correlated significantly with advanced tumor stage , nodal metastasis  and late clinical stage . Chi-Square trend analysis showed significant loss of membranous E-cadherin and β-catenin (E-cad−/β-cat M-) in different stages of oral carcinogenesis . Furthermore, loss of E-cad−/β-cat M- immunopositivity was significantly associated with reduced disease free-survival [median DFS = 14 months as compared to β-catenin and E-cadherin membrane positive tumors with median disease-free survival of 71 months ] ] .To explore the significance of ALCAM in biological development of invasive cancer and metastasis, it is important to determine its correlation with perturbations in expression of adherens junction components. Associations among protein expressions of E-cadherin, β-catenin and ALCAM were determined  as ALCAM−/β-catM-) correlated significantly with overall ALCAM immunopositivity in hyperplasia. Among the dysplasias analyzed (n = 20), loss of β-catenin membranous expression was found to be significantly associated with ALCAM cytoplasmic immunopositivity .Among the hyperplasias analyzed (n = 56) in this study, loss of membranous E-cadherin was found to be significantly associated with loss of β-catenin expression . Loss of β-catenin membranous expression was found to be significantly associated with overall ALCAM expression . Loss of membrane E-cadherin and β-catenin taken together as one phenotype (E-cad−/β-cat M-) correlated significantly with overall and cytoplasmic ALCAM immunopositivity in OSCC  and β-catenin nuclear/cytoplasmic accumulation  were the most significant predictors for increased tumor burden. β-catenin membrane loss  and overall ALCAM expression/β-catenin nuclear/cytoplasmic accumulation  were the most significant predictors for nodal metastasis. Combined markers, ALCAM expression/β-catenin nuclear/cytoplasmic accumulation  and β-catenin membrane loss  were the most significant predictors for late clinical stage  = 2.5; 95% C.I. = 1.1–5.6].+/E-cad− ] or excluding cancer recurrence  in OSCC patients was measured by the ratios: PPVrelapse/OSCC (76 months | ALCAM C+/E-cad−)/PPVrelapse/OSCC (76 months) = 100/61.6; NPVrelapse/OSCC (76 months| ALCAM C+/E-cad−)/NPVrelapse/OSCC (76 months) = 63.5/46.0  were transiently transfected with siRNA against ALCAM. ALCAM-depleted transfectants (15 nM siRNA) for 48 h showed 3-folds decreased levels of ALCAM transcripts , lane T ALCAM-depleted transfectants (15 nM siRNA for 48 h) showed 7-folds decreased level of ALCAM protein compared to the untreated control cells as determined by western blot analysis , lane T.These results were further corroborated using confocal microscopic analysis; 95% down-regulation was observed in expression of ALCAM protein upon siRNA transfection for 48 h as compared to the control cells . IncreasThe results of our study are summarized in −/β-cat M−) correlated significantly with overall ALCAM, and cytoplasmic ALCAM immunopositivity in OSCCs; (iii) a panel of markers, ALCAM overexpression, β-catenin nuclear/cytoplasmic accumulation and β-catenin membrane loss were the most significant predictors for late clinical stage; (iv) Cox regression (multivariate) analysis revealed increased cytoplasmic ALCAM/E-cadherin loss to be the most significant adverse prognosticators for OSCC patients; (v) siRNA mediated ALCAM knockdown resulted in increase in E-cadherin and β-catenin, both at the transcript and protein levels, suggesting the link between these proteins and supporting our clinical findings.The salient findings of the study are:(i) loss of E-cadherin and β-catenin membranous expression, and cytoplasmic/nuclear accumulation of β-catenin are early events in oral tumorigenesis, occurring in pre-neoplastic stages (dysplasia) and sustained in frank malignancy; (ii) loss of E-cadherin/loss of membranous β-catenin . Multivariate logistic regression analyses revealed that loss of membranous E-cadherin expression was the most significant phenotype associated with malignancy. Multivariate analysis showed that concomitant loss of membranous E-cadherin and increased cytoplasmic ALCAM expressions was the most significant adverse prognosticator in OSCC patients. Our study underscores the clinical relevance of aberrant expression of cell adhesion molecules in oral cancer. This is particularly important because differentiation is often a consequence of reduced or altered intercellular adhesion and maintenance of a differentiated phenotype of epithelial cells is critically dependent on the presence of functional adhesion complexes. However, the exact mechanism underlying these changes in oral carcinogenesis remains to be unraveled. In this context, siRNA mediated ALCAM knock down demonstrated increased cytoplasmic accumulation of β-catenin and E-cadherin, suggesting that there is an orchestrated modulation of these adhesion molecules which are dynamically controlled, supporting our clinical findings. The molecular mechanisms implicated are under investigation. We speculate that disturbances in this equilibrium might cause the cells to progress down the tumorigenic pathway. Notably, loss of E-cadherin and membranous β-catenin are hallmarks of epithelial- mesenchymal transition (EMT), defined as the loss of epithelial differentiation and gain of mesenchymal phenotype, that is associated with acquisition of aggressive traits by cancer cells, invasion and metastasis in-vitro studies support the clinical findings and suggest these dynamic changes in the cells adhesion system are likely to play pivotal roles in oral tumorigenesis.In conclusion, our study underscores the clinical significance of loss of E-cadherin and β-catenin membrane expression in relation to ALCAM expression in early precancerous stage (dysplasia), their sustained deregulation in OSCCs and correlation with aggressive tumor behavior and poor prognosis, underscoring their potential as candidate biomarkers for disease prognosis. Further, our


The Wnt/β-catenin pathway is involved in the regulation of a multitude of physiological processes by controlling the differential expression of target genes. In certain tissues such as the adult liver, the Wnt/β-catenin pathway can attain different levels of activity due to gradients of Wnt ligands and/or intracellular pathway components like APC. How graded pathway activity is converted into regionally distinct patterns of Wnt/β-catenin target gene expression is largely unknown. Here, we apply a mathematical modeling approach to investigate the impact of different regulatory mechanisms on target gene expression within Wnt or APC concentration gradients. We develop a minimal model of Wnt/β-catenin signal transduction and combine it with various mechanisms of target gene regulation. In particular, the effects of activation, inhibition, and an incoherent feedforward loop (iFFL) are compared. To specify activation kinetics, we analyze experimental data that quantify the response of β-catenin/TCF reporter constructs to different Wnt concentrations, and demonstrate that the induction of these constructs occurs in a cooperative manner with Hill coefficients between 2 and 5. In summary, our study shows that the combination of specific gene regulatory mechanisms with a time-independent gradient of Wnt or APC is sufficient to generate distinct target gene expression patterns as have been experimentally observed in liver. We find that cooperative gene activation in combination with a TCF feedback can establish sharp borders of target gene expression in Wnt or APC gradients. In contrast, the iFFL renders gene expression independent of gradients of the upstream signaling components. Our subsequent analysis of carcinogenic pathway mutations reveals that their impact on gene expression is determined by the gene regulatory mechanism and the APC concentration of the cell in which the mutation occurs. Wnt ligands are secreted signaling molecules that can generate concentration gradients across tissues . However, detailed regulatory mechanisms of their expression have been suggested for only a few. Experiments have shown that the expression of E-cadherin is directly inhibited by the β-catenin/TCF complex  an activation mechanism, (ii) an inhibition mechanism, and (iii) an iFFL are investigated. Moreover, we consider the impact of an additional possible feedback via TCF. By simulating the effects of pathway mutations, we furthermore compare target gene mRNA concentrations under wild type and carcinogenic conditions. Our study demonstrates that the combination of specific gene regulatory mechanisms and a gradient of either Wnt or APC is sufficient to generate the distinct target gene expression patterns that have been observed in the liver.Various aspects of the Wnt/β-catenin pathway have been investigated in theoretical studies . β-Catenin is constantly produced (reaction 1) and degraded in an APC-independent as well as an APC-dependent manner . β-Catenin can reversibly form complexes with APC (reaction 4) and TCF (reaction 5). TCF is produced and degraded  while the total concentration of APC is assumed to remain constant within the considered time period. The APC-dependent degradation of β-catenin is inhibited by activated Dishevelled. Dishevelled is activated by a Wnt stimulus (reaction 8) and can become deactivated again (reaction 9). Like APC, Dsh obeys a conservation relation.The Wnt/β-catenin signal transduction module of our minimal model consists of β-catenin, TCF, APC, the β-catenin/TCF complex, the β-catenin/APC complex as well as active and inactive Dishevelled  are adopted from the detailed model. They are given in Tables Overall, the signal transduction module consists of five ordinary differential equations (ODEs) and two conservation relations . Taken together, the minimal model very well reproduces the dynamical properties of β-catenin that are relevant for our analysis.First, we compare the dynamics of β-catenin between the minimal model  and the detailed model  and Glt1 (glutamate transporter 1)  given by the Eq. n = 3 as derived from the analysis of the reporter data is used  is described by Eq. KM value is , KM additionally influences the width of the APC concentration range in which the mRNA levels change. Under these conditions, KM also reduces the possible relative mRNA concentration range.Next we compared the effects of the cooperative target gene activation with Hill coefficients e is Eq. , as showE-cadherin is a well-known example of negatively regulated Wnt target genes  van Figure , and (iin Figure . In the n Figure .Ki in target gene repression or KM in target gene activation. Different parameter values shift the APC concentration range in which changes of gene expression occurs (Figures Different target genes regulated by the same mechanism may still be expressed in different regions along the periportal-pericentral axis in the liver. Examples are the two negatively regulated target genes Gls2 and Arg1. While Gls2 is only expressed in the proximal periportal region, Arg1 is expressed in the proximal and distal periportal area (Benhamouche et al.,  Figures . Thus, dWhile the activation and inhibition mechanisms discussed above cause changes in the mRNA level along a Wnt or an APC gradient, the iFFL motif renders the steady state of the target gene independent of the gradient Figure A, line 6Signal transduction in the Wnt/β-catenin pathway is perturbed in several types of cancer (Giles et al., In our study we simplified the mutation mechanisms and only considered their potential to modify the APC-dependent degradation of β-catenin. It might be speculated that a mutation in β-catenin not only affects its stability but also its transcriptional activity. In an experimental study, the expression of target genes in hepatocellular carcinoma harboring a mutation in either Axin or β-catenin has been measured (Zucman-Rossi et al., In our modeling approach, we consider individual cells independently of their neighbors. However, some Wnt/β-catenin target genes encode for proteins which affect intercellular signaling, the gradient and/or its effectiveness. For instance, DKK1 is a Wnt/β-catenin target gene and its product influences Wnt/β-catenin signaling of the surrounding cells by competing with Wnt ligands for receptor binding (Mao et al., Our minimal signaling model reproduces the β-catenin steady states and dynamics upon constant Wnt stimulation of the detailed model and in fact extends the detailed model with respect to the representation of β-catenin/TCF complex concentration ranges and the inclusion of gene regulatory mechanisms. The parameters and total concentrations of the minimal model are predominantly derived from the detailed model (Lee et al., The changes of the concentrations of the system’s components are described by a set of ODEs and algebraic conservation equations. The particular equations and parameters are listed in Section Published experimental data of β-catenin reporter constructs (Biechele and Moon, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.