However, δ-catenin is upregulated in human prostatic adenocarcinomas, although the effects of δ-catenin overexpression in prostate cancer are unclear. We hypothesized that δ-catenin plays a direct role in prostate cancer progression by altering gene profiles of cell cycle regulation and cell survival.2 Profiler™ PCR Arrays demonstrated gene alterations involved in cell cycle and survival regulation. δ-Catenin overexpression upregulated cyclin D1 and cdc34, increased phosphorylated histone-H3, and promoted the entry of mitosis. In addition, δ-catenin overexpression resulted in increased expression of cell survival genes Bcl-2 and survivin while reducing the cell cycle inhibitor p21Cip1.We employed gene transfection and small interfering RNA to demonstrate that increased δ-catenin expression promoted, whereas its knockdown suppressed prostate cancer cell viability. δ-Catenin promoted prostate cancer cell colony formation in soft agar as well as tumor xenograft growth in nude mice. Deletion of either the amino-terminal or carboxyl-terminal sequences outside the armadillo domains abolished the tumor promoting effects of δ-catenin. Quantitative RTTaken together, our studies suggest that at least one consequence of an increased expression of δ-catenin in human prostate cancer is the alteration of cell cycle and survival gene profiles, thereby promoting tumor progression. Tumor progression is the result of loss of balance in cellular functions including cell growth, adhesion, division and apoptosis. Among the many oncogenes and tumor suppressors, cell-cell junction associated proteins, such as β-catenin and adenomatous polyposis coli (APC), contribute to cancer development by disrupting the E-cadherin based cell-cell junction, as well as interfering with cell proliferation, altering karyotype, and reducing apoptosis ,2.neural plakophilin related armadillo protein)/Neurojungin, is an adhesive junction associated protein [ctn subgroup in the armadillo/β-catenin superfamily [ctn are ubiquitously expressed in the body, δ-catenin distribution is principally restricted to the brain in healthy individuals. However, it has become increasingly clear that δ-catenin is expressed in a variety of cancers of peripheral tissues, including breast, prostate, and esophageal tumors [ctn in primary prostatic adenocarcinomas, and the forced overexpression of δ-catenin in cultured prostate cancer cells can induce the redistribution of E-cadherin and p120ctn [ctn, are involved in cell adhesion and motility as well as cancer cell growth [δ-Catenin, or NPRAP  and carboxyl- (COOH) terminal sequences flanking the armadillo repeat domains. In addition, quantitative RT2 Profiler™ PCR arrays revealed a wide range of gene alterations involved in cell cycle and survival regulation. These findings support the notion that at least one consequence of an increased δ-catenin expression in prostate cancer development is the alteration of cell cycle and survival gene profiles, thereby promoting tumor progression.In this study, we tested the hypothesis that δ-catenin plays a direct role in prostate cancer cell growth by altering gene profiles of cell cycle regulation and cell survival. We demonstrated, for the first time, that δ-catenin overexpression promotes anchorage-independent prostate cancer cell growth and tumor xenografts in nude mice. We determined that the ability of δ-catenin overexpression to promote prostate tumor xenograft growth is dependent on the amino-  or COOH-terminal (ΔC207) deletions . Similarly, ΔN280 and ΔC207 did not show tumor promoting effects in nude mice  ions Fig , arrow wions Fig , arrow. ions Fig . In factmice Fig , althougTo investigate if the effect of δ-catenin overexpression on the increase in viable cell number was to increase mitosis, we conducted experiments to compare the mitotic index of δ-catenin expressing cells with that of vector transfected cells. Cells entering mitosis show intense chromosomal staining with Hoechst 33258 dye highlighting different phases of cell division Fig , insert.To independently confirm the increased mitotic index in δ-catenin overepressing cells, we analyzed phosphorylated histone-H3 immunolocalization Fig . Histone2 Profiler™ PCR Array, we selected cyclin D1, Bcl2L1, and HK2 genes to further study their changes in expression by real-time PCR  and cdc34 (CDC34), activation of which promotes G1 to S transition , showed  PCR Fig . Cyclin ults Fig .Cip1 is a downstream effector of p53 mediated cell cycle interruption [Cip1 expression in δ-catenin overexpressing cells was reduced when compared to vector transfected cells , however, is a unique member of the family because it is primarily expressed in the central nervous system of normal individuals. However, δ-catenin is now well established as being overexpressed in prostate cancer [β-Catenin/armadillo superfamily proteins are expressed in cells of all major tissue types including epithelial cells. They play important roles in cancer with some of its members as oncogenes while others act as tumor suppressors ,2. δ-Cate cancer ,26.Catenin gene amplification was observed in cervical cancer [catenin was observed in prostate cancer patients [The frequently increased expression of the neuronal protein δ-catenin in peripheral prostate cancer tissue raised important questions such as what mechanisms are in place in cancer to result in a high level of δ-catenin expression. δ-l cancer  and bladl cancer . Transcrl cancer . Recentll cancer . Furtherpatients , furtherRegarding cell proliferation and colonization, earlier studies evaluating MDCK cells transfected with δ-catenin showed that there were no significant differences between MDCK cells expressing δ-catenin and control cells . Interes2- or COOH-terminal sequences on their own do not localize to cell-cell junctions [2-terminal 280 amino acids also remarkably altered the effects of δ-catenin on 3T3 cell morphology [2-terminal 280 amino acids or COOH-terminal 207 amino acids lost its ability to promote tumor development. These studies underscore the importance of these sequences outside the armadillo domain to the functions of δ-catenin in cancer.The armadillo repeating units reveal the most significant homology among β-catenin superfamily proteins, while the sequences flanking the armadillo domain are quite variable ,7. This unctions . In addiunctions ,33,34. Rrphology . In our Gene profiling can provide initial indications of what may be the potential molecular pathways that δ-catenin employs to contribute to tumor progression. We identified a number of genes that displayed changes in expression when δ-catenin is overexpressed in prostate cancer cells. Several of these genes, such as cyclin D1, cdc34, Bcl2L1, and HK2, are especially interesting. Both cyclin D1 and cdc34 are involved in G1 to S transition, and Bcl2L1 is a long isoform of Bcl2 which protects cells from undergoing apoptosis . HK2 phoThis study is the first to demonstrate the effects of δ-catenin overexpression in prostate cancer cells. We show that these effects rely on the δ-catenin domains outside armadillo repeating sequences, providing future direction for investigating the molecular basis of δ-catenin mediated prostate cancer development. Our studies suggest that an increased expression of δ-catenin in human prostate cancer permits the alteration of cell cycle and survival gene profiles, which may advance tumor progression.Cip1 were from BD Biosciences . Rabbit anti-cyclin D1 were from Santa Cruz Biotech . Mouse anti-actin, histone-H3, and rabbit anti-Erk1/2 and survivin were from Cell Signaling . Rabbit anti-phosphorylated histone-H3 (Ser 10) was obtained from Upstate Biotech . Unless otherwise indicated all chemicals were from Sigma .Mouse anti-δ-catenin and p212 until they were used for further experimental analyses.CWR22Rv-1 and PC-3, as well as NCI-H1299, were obtained from ATCC and cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum (FBS). All cultures were maintained at 37°C with 5% CO2-terminal deleted (ΔN280), or COOH-terminal deleted (ΔC207) δ-catenin cDNA with or without pEGFP fusion [pEGFP  were used as a vector control. Stable cell lines transfected with untagged δ-catenin were established by selection using 250 μg/ml G418. For selection of pEGFP-δ-catenin transfected cells, cells were first selected in G418 containing medium. Then, pEGFP-δ-catenin trasnfected cells were further selected by GFP-based cell sorting using a FACS Vantage (BD Biosciences). The stable cell lines were maintained in RPMI 1640 medium containing G418.To produce stable δ-catenin overexpressing cell lines, full length, NHP fusion  was tranCatenin gene specific shRNA constructs (gene sequence accession number NM 001332) used in this study were from Origene Technologies, Inc . They were as follows: shRNA1, 5'-ggatggagtaggacctcttccagactgtg-3', shRNA2, 5'-ctcacgctttgtttactctcttcatccgt-3', and a negative control shRNA pRS-GFP plasmid. CWR22Rv-1 cells were transfected with shRNA specific to δ-catenin gene using Lipofectamine 2000 (Invitrogen) and co-transfected with pGFP. Following transfection, the medium was replaced by RPMI 1640 with G418 for selection. The G418 resistant cells were further sorted using GFP as a marker to enrich for shRNA expressing cells.δ-To determine δ-catenin knockdown efficiency, cultured cells were lysed in 10 mM HEPES, pH 7.3, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.2% SDS with protease inhibitor cocktails . Insoluble materials were removed by centrifugation. The lysates were mixed with sample buffer and equal protein amounts were loaded onto gels for SDS-PAGE and Western blot analysis. After proteins were transferred to nitrocellulose membranes , anti-δ-catenin and anti-actin immunoreactivities were revealed by antibody immunoblotting using enhanced chemiluminescence  for detection.t-tests or one-way ANOVA were conducted and p-values were assigned. The significance level was set at 0.05.Stably transfected cells were seeded and counted after 6 days in culture using a hemacytometer in at least three independent experiments. Trypan blue exclusion was used to determine viable cells for counting. To determine mitotic index, cells were either stained with Hoechst 33258 or immunostained with anti-phospho-histone H3. Following the incubation with Cy3™-conjugated affinity purified secondary antibody , the coverslips were mounted using Antifade. At least 300~500 cells in 10 randomly selected fields were counted to determine mitotic figures or cells intensely labeled with anti-phosphorylated histone H3. Statistical analysis was performed using MS Excel and SigmaPlot . Student 2 in air at 37°C. Colonies were stained with 0.04% Crystal violet and counted in 20 randomly selected fields. The number of colonies was plotted as the mean ± SD.A soft agar colony formation assay was performed using six-well plates. Each well contained 1% agar in 2× RPMI 1640 medium as the bottom layer and 0.6% agar in 2× RPMI 1640 medium as the top layer. 5,000 cells were plated into the top layer. The plates were incubated in a humidified incubator with 5% CO6 CWR22Rv-1 cells in RPMI 1640 medium plus cold Matrigel (BD Biosciences) or PC-3 cells in RPMI 1640 medium without Matrigel into the dorsal flank of nude mice. Once xenografts became established, their size was measured with a caliper once a week. The tumor volume was calculated by the formula: length × width × height × 0.5236 [Male, athymic nude (nu/nu) mice  were obtained and used at 4–8 weeks of age. Animals were kept under pathogen free conditions according to the guidelines of East Carolina University Animal Use Protocol. Animals were given subcutaneous injections of 4 ×10× 0.5236 . At the Five-micrometer sections were prepared from the tumors in nude mice and placed onto charged glass slides. They were stained with Hematoxylin. For visualization of EGFP or EGFP-δ-catenin expressing cells in nude mice, 5 μm sections were prepared and fixed in 4% paraformaldehyde. They were analyzed under the Zeiss Axiovert inverted fluorescent light microscope equipped with MetaMorph imaging software.Cip1, anti-survivin (1:1000), anti-histone H3 (1:1000), and anti-phosphorylated histone H3 (1:1000). Anti-actin (1:5000) was used as a loading control. Following appropriate secondary antibody incubations, the blots were developed using enhanced chemiluminescence.Cells were lysed in RIPA buffer with protease and phosphatase inhibitor cocktails, and subjected to protein solubilization and SDS-PAGE analysis. Proteins were western blotted using anti-Erk1/2 (1:1000), anti-p212 first strand kit (SABioscience). Real-Time PCR was performed according to the User Manual of RT2 Profiler PCR array system (SABioscience) using SYBR Green PCR Master Mix in an iCycler iQ Multicolor Detection System (Bio-Rad). Three selected PCR arrays, including pathways of apoptosis  and cell cycle  were repeated three times and the data were analyzed using Excel-based PCR Array Data Analysis Templates (SABioscience). Additional real-time PCR analyses were performed to compare RNA expression of cyclin D1, Bcl2L1 and HK2 between control vector transfected cells and that of δ-catenin overexpressing cells. For these experiments, the primer set for cyclin D1 was 5'-AGAAGCTGTGCATCTACACCGACA-3' (forward) and 5'-TGGAGGGCGGATTGGAAATGAACT-3' (reverse), the primer set for BcL2L1 was 5'-GTCGCATTGTGGCCTTTTTCTCC-3' (forward) and 5'-AGCTGCGATCCGACTCACCAATAC-3' (reverse), while the primer set for HK2 was 5'-GCCTTCGGGGACAATGGATGC-3' (forward) and 5'-TCTGCTTGCCGGGGTTGAGTG-3' (reverse).CWR22Rv-1 cells with or without δ-catenin overexpression were re-plated in RPMI 1640 media supplemented with 10% FBS and 0.25% G418 at 37°C in a 5% CO2 atmosphere. After 2 days, the total RNA was isolated using RNeasy Mini kit (Qiagen). The single strand cDNA from 2–3 μg total RNA was synthesized using RTThe authors declare that they have no competing interests.YZ performed most of the experiments and contributed to the data analyses. AA contributed to the cell viability measurements while JL performed PCR arrays. TW contributed to the mouse transplantation experiments. KK designed and subcloned δ-catenin truncation constructs. YHC and DMT contributed to the design of the experiments, and QL contributed to the design of the entire study and the editing of the manuscript.


Drosophila development. Limited functional information is available for its two mammalian relatives, BCL9 and B9L/BCL9-2: like Legless, these proteins bind to β-catenin, and RNAi-mediated depletion of B9L/BCL9-2 has revealed that this protein is required for efficient β-catenin-mediated transcription in mammalian cell lines. No loss-of-function data are available for BCL9.Most cases of colorectal cancer are initiated by hyperactivation of the Wnt/β-catenin pathway due to mutations in the APC tumour suppressor, or in β-catenin itself. A recently discovered component of this pathway is Legless, which is essential for Wnt-induced transcription during We have used overexpression of dominant-negative forms of BCL9, and RNAi-mediated depletion, to study its function in human cell lines with elevated Wnt pathway activity, including colorectal cancer cells.APC mutation. Dominant-negative mutants of BCL9 indicated that its function depends not only on its β-catenin ligand, but also on an unknown ligand of its C-terminus. Finally, we show that BCL9 and B9L are both Wnt-inducible genes, hyperexpressed in colorectal cancer cell lines, indicating that they are part of a positive feedback loop.We found that BCL9 is required for efficient β-catenin-mediated transcription in Wnt-stimulated HEK 293 cells, and in the SW480 colorectal cancer cell line whose Wnt pathway is active due to BCL9 is required for efficient β-catenin-mediated transcription in human cell lines whose Wnt pathway is active, including colorectal cancer cells, indicating its potential as a drug target in colorectal cancer. A keyoplasias -19.Drosophila, called Pygopus (Pygo) and Legless (Lgs), which are required for the transcriptional activity of Armadillo (the Drosophila β-catenin) [Recently, two new nuclear components of the canonical Wnt pathway have been discovered in catenin) -23. Eachcatenin) ,24,25. Lcatenin) -29. NothLgs/BCL9 proteins are adaptors between Armadillo/β-catenin and Pygo proteins ,30, and Given their activity in colorectal cancer cells ,24,25, PA pCDNA3.1 vector encoding FLAG-tagged human BCL9 was kindly provided by H. Clevers. pCDNA3.1 encoding FLAG-tagged mouse BCL9-2 , B9L or 35S-labeled proteins were generated by in vitro transcription/translation using T7 polymerase (Promega). For GST-fusion pull-down assays, glutathione beads containing 10 μg of GST or GST-ARD (GST fused to amino acids 134–671 of β-catenin [in vitro translated 35S-labeled wild-type (wt) or mutant HD2 domains in PBS containing 0.1% Tween-20 and 1% BSA (PBS-TB) for 4 hours at 5°C. Beads with bound proteins were washed five times in PBS-TB, once in PBS and subsequently resolved by SDS-PAGE prior to autoradiography.-catenin ) were inHEK 293, HEK 293T and HCT116 cells were grown in Dulbecco's modified Eagles medium (DMEM) while SW480 cells were grown in Leibowitz L-15 medium. All media was supplemented with 10% fetal calf serum. Wnt pathway activity was induced by treating cells with 20 mM LiCl  or conditioned media obtained from L-cells expressing Wnt3A for 6 hours. Cells were transfected with the Lipofectamine 2000 transfection reagent according to Manufacturer's protocol (Invitrogen). siRNAs against human BCL9, BCL9-2 and β-catenin were obtained from Dharmacon; 20 or 80 nmol of 4 pooled siRNAs were transfected per well . Co-immunoprecipitation or quantitative RT-PCR (RT-qPCR) experiments were carried using cells grown in 6-well plates and transfected with 1 μg per well of each plasmid or 80 pmols of siRNAs. For co-immuno-precipitations, HEK 293 cells were harvested and lysed 24 hours after transfection; mouse α-FLAG monoclonal antibody M2 (Sigma Aldrich) was used for immunoprecipitation. Cells for RT-qPCR were harvested 24- and 48-hours post transfection (with plasmids or siRNAs), and RNA was isolated using the TRIZOL reagent (Invitrogen) according to the manufacturer's protocol.For immunofluorescence, 250 ng of each plasmid was used to transfect cells grown on 13 mm coverslips in 24-well plates. SW480 and HCT116 cells were fixed and stained with rabbit α-FLAG (Sigma Aldrich) or mouse monoclonal α-β-catenin C19220 (BD Transduction Laboratories) antibodies 24 hours after transfection as described . For TOPB9L (Hs00699441), HPRT1 (Hs99999909), AXIN2 (Hs00610344), CD44 (Hs00153304), and β-Actin (Hs99999903). Additional gene target primer and probe sets were designed with the GenScript qPCR primer design software, and used as follows : c-myc, FAM-AAGACAGCGGCAGCCCGAAC-TAMRA, TCAGAGAAGCTGGCCTCCTA and AGGTACAAGCTGGAGGTGGA; TATA-box binding protein (TBP), FAM-TACCGCAGCAAACCGCTTGG-TAMRA, AAAGACCATTGCACTTC GTG and GGTTCGTGGCTCTCTTATCC; BCL9, FAM-CGATTATTCCATCTGAGAAGC CCA-TAMRA, CCACCGGAATACCTGAGTTT and AGGGAAATATTGCAGCGTCT. Primers and probes were used at a final concentration of 0.2 μM and 0.5 μM respectively. Values for gene expression in each case were calculated relative to a standard curve of TBP expression whose consistency from different samples was confirmed in each case using HPRT1 and β-Actin expression. Comparative qPCR revealed that the levels of BCL9 and B9L transcripts in transfected HEK 293 cells were 45× and 90×, respectively, above those of endogenous genes.cDNA was synthesized from total RNA (2.5 μg) primed with random hexamers and Superscript III (Invitrogen). RT-qPCR reactions were carried out with the ABI7900 Taqman thermocycler (Applied Biosystems). Reaction mixtures for amplification (10 μl) contained 0.5 μl of each gene expression assay (AB Biotech), 5 μl of 2 × Master Mix (AB Biotech) 1 μl of cDNA (~50 ng) and water to 10 μl. The following gene expression assays (from Applied Biosystems) were used: 2 and a mixture of protease inhibitors. Total protein content was determined with Coomassie Plus Protein Assay Reagent (Pierce). Lysates corresponding to 100 μg of total protein were analysed by PAGE with 3–8% gradient Tris-acetate gels (Invitrogen). Proteins were transferred to a PVDF membrane in transfer buffer (Invitrogen) at 25 V for 2 hours. The following primary antibodies were used: mouse α-FLAG monoclonal antibody M2 (Sigma Aldrich), rat α-HA 3F10 (Roche Applied Science), mouse α-β-catenin monoclonal antibody C19220 (BD Transduction Laboratories). For detection of proteins, secondary antibodies coupled to HRP (Sigma Aldrich) and ECL Plus Western Blotting Detection Reagents (Amersham Biosciences) were used.Cell lysates were prepared in lysis buffer consisting of 50 mM Tris-HCl pH 7.4, 1% NP-40, 150 mM NaCl and 1 mM MgClin vitro and in vivo [Drosophila lgs  of BCL9 proteins mediates their binding to Armadillo/β-catenin  in vivo ,24,25,32in vivo, we introduced them into full-length FLAG-tagged BCL9, and also generated the corresponding mutants in BCL9-2 . We optimized this assay, minimizing the Wnt exposure time as much as possible, which allowed us to focus on the primary transcriptional Wnt response and to avoid secondary knock-on effects. We thus found that culturing 293 cells in the presence of Wnt3A for 6 hours resulted in a 2× increase in TOPFLASH activity Fig. . Like inAs in SW480 cells, overexpression of BCL9-2 in Wnt3A-stimulated HEK 293 cells resulted in a 2× increase of TOPFLASH activity Fig. . Its ΔHDc-myc and AXIN2 [HPRT. As expected, we observed a 2–2.5× upregulation of c-myc and AXIN2 transcripts in response to both Wnt3A and LiCl stimulation  after Wnt stimulation  although the converse was not true, possibly because this gene is only mildly Wnt-inducible, and is less hyperactive than B9L in colorectal cancer cell lines  are located at the interface between the two domains and are engaged in direct contacts with β-catenin  [in vitro also eliminated association of BCL9 and B9L with β-catenin in vivo, so HD2 is the only domain through which these proteins can bind to β-catenin in cells. Consistent with this, the corresponding L>K mutation in Drosophila  results in Lgs loss-of-function, and loss of Armadillo binding [in vitro, and not detectably in vivo  . Importa binding . Somewhaelopment .Lgs counterpart in Drosophila [The functional link between BCL9 and β-catenin indicated a role for BCL9 in Wnt signaling. Indeed, our loss-of-function analysis by RNAi in Wnt-stimulated HEK 293 and SW480 cells provided strong evidence for this, revealing that BCL9 is required for efficient TCF-mediated reporter gene transcription and for Wnt-inducibility of endogenous TCF target genes. Therefore, BCL9 is a positive regulator of the Wnt pathway in human cells, similarly to its relative B9L ,25, and osophila .Drosophila, Pygo is associated with dTCF target genes even in the absence of Wingless signaling [B9L in human cells and its overexpression in colorectal cancer cells, it is conceivable that the constitutively nuclear B9L/BCL9-2 proteins serve to maintain, or boost, Wnt-mediated transcription in cells whose Wnt pathway is persistently active (see below).We confirmed earlier findings that BCL9 exhibits a subcellular distribution distinct from that of B9L/BCL9-2 ,25: BCL9ignaling . Pygo prThe distinct subcellular locations of BCL9 and B9L may explain why only B9L, but not BCL9, is able to potentiate Wnt-induced transcription when overexpressed in HEK 293 and SW480 cells Fig. ; Fig. 5.in vivo associations with β-catenin were comparable . Secondly, a C-terminal truncation of BCL9 also behaved as a DN in Wnt-stimulated mammalian cells, like the equivalent truncation of B9L [lgs mutants in Drosophila [lgs mutations results in a similar C-terminal truncation [Drosophila.Two pieces of evidence indicated an additional unknown ligand of the BCL9 C-terminus that is critical for the function of BCL9 in Wnt-mediated transcription. Firstly, a BCL9 double-mutant whose binding to β-catenin and Pygo was eliminated behaved as a DN in Wnt signaling assays. This DN effect is unlikely due to direct interference with endogenous BCL9 (and/or B9L) via dimerization, since we were neither able to detect self-association of differently tagged BCL9 proteins, nor heterodimerization between BCL9 and B9L, in transfected HEK 293 cells; lack of interaction between the two proteins was also apparent in sensitive n of B9L . This waosophila . Howeveruncation , indicatEvidence for an additional ligand of BCL9 proteins with a function in Wnt signaling was also provided by the double-mutants whose binding to Pygo and β-catenin was eliminated: these behaved as DNs, possibly by sequestering a functionally important ligand. If so, and if this third ligand bound to the C-terminus of BCL9 proteins, then the DN effects of the double-mutants should be eliminated in triple-mutants that also lack the C-terminus, which was indeed the case. It thus appears that the triple-mutants are no longer able to sequester any functional ligand, supporting the notion that BCL9 proteins have three rather than two functionally important ligands. Our evidence for a third C-terminal ligand of BCL9 proteins argues against the simple adaptor hypothesis as initially proposed, which envisaged that the sole function of BCL9 proteins was to link Pygo and Armadillo/β-catenin .During the final stages of manuscript preparations, a study was published that uncovered a cell-type specific function of BCL9 in TCF-responsive transcription in B cell lines . This fuBCL9 and B9L are both Wnt-inducible genes, and that they are hyperexpressed in colorectal cancer cells whose Wnt pathway is constitutively active. Therefore, both genes are likely to be TCF target genes, much like c-myc and AXIN2 [B9L expression was highly responsive to changes in Wnt pathway activity, and its hyperexpression in colorectal cancer cells was pronounced. Notably, high levels of B9L transcripts have also been found in 6 of 14 of colorectal tumour samples with elevated Wnt pathway activity [B9L overexpression correlated with the progression from benign colorectal adenomas to invasive carcinomas [In the course of our analysis, we discovered that nd AXIN2 ,16,17. Iactivity , and thercinomas .Our findings suggest that BCL9 and B9L are part of a positive feedback mechanism. In particular, B9L may function to maintain, or boost, TCF-mediated transcription once initiated by the stimulation of cells by extracellular Wnt ligand. Indeed, overexpression of B9L and BCL9 may contribute to, or even confer, persistent pathway activity that is independent of Wnt ligand, for example in colorectal tumours whose progression may thus be promoted by these proteins.Although the Wnt/β-catenin pathway is an important cancer pathway, there are no well-established, validated small molecule inhibitors of this pathway to date. The likely reason for this is that the cancer-promoting Wnt pathway mutations typically occur at the level of, or immediately above, β-catenin ,48. Howein vitro and in vivo (Fig. Our evidence for the requirement of BCL9 in the TCF-dependent transcription of colorectal cancer cells, and its hyperexpression in these cells, has revealed its potential as a target for inhibitory drugs. Furthermore, the potential for disruption of the binding between β-catenin and BCL9 is indicated by our result that single point mutations in HD2 disrupt the association of β-catenin and BCL9, or B9L, ivo Fig. , consistBCL9 and B9L are Wnt-inducible genes that are hyperexpressed in colorectal cancer cell lines, indicating that they are part of a positive feedback loop, reinforcing Wnt pathway activity. The functional importance and hyperexpression of BCL9 in colorectal cancer cells indicates the potential of this protein as a cancer drug target.Our loss-of-function analysis of BCL9 in human cell lines has led to the following three conclusions. Firstly, BCL9 is required for efficient TCF-mediated transcription in Wnt-stimulated and colorectal cancer cells. Secondly, this function of BCL9 depends on β-catenin, but also on its C-terminus, implying that this conserved part of the protein binds to an unknown ligand that is important for its Wnt-related function. Thirdly, both The authors declare that they have no competing interests.MdlR conducted most of the experiments in this study. JW initiated the study, and contributed Fig. The pre-publication history for this paper can be accessed here:


Activation of the transcription factor NF-κB by cytokines is rapid, mediated through the activation of the IKK complex with subsequent phosphorylation and degradation of the inhibitory IκB proteins. The IKK complex is comprised of two catalytic subunits, IKKα and IKKβ, and a regulatory protein known as NEMO. Using cells from mice that are genetically deficient in IKKβ or IKKα, or using a kinase inactive mutant of IKKβ, it has been proposed that IKKβ is critical for TNF-induced IκB phosphorylation/degradation through the canonical pathway while IKKα has been shown to be involved in the non-canonical pathway for NF-κB activation. These conclusions have led to a focus on development of IKKβ inhibitors for potential use in inflammatory disorders and cancer.Analysis of NF-κB activation in response to TNF in MEFs reveals that IKKβ is essential for efficient phosphorylation and subsequent degradation of IκBα, yet IKKα contributes to the NF-κB activation response in these cells as measured via DNA binding assays. In HeLa cells, both IKKα and IKKβ contribute to IκBα phosphorylation and NF-κB activation. A kinase inactive mutant of IKKβ, which has been used as evidence for the critical importance of IKKβ in TNF-induced signaling, blocks activation of NF-κB induced by IKKα, even in cells that are deficient in IKKβ.These results demonstrate the importance of IKKα in canonical NF-κB activation, downstream of cytokine treatment of cells. The experiments suggest that IKKα will be a therapeutic target in inflammatory disorders. The transcription factor nuclear factor-kappaB (NF-κB) plays critical roles in inflammation, control of cell death pathways and cell proliferation which are hallmarks of many human diseases IκB phosphorylation by the high molecular weight IκB kinase (IKK) complex (approximately 700 kDa) is a critical regulatory step in the NF-κB activation pathway Insight into the physiological roles of the two catalytic IKK subunits comes from gene targeting studies. IKKβ knockout mice display a phenotype similar or identical to knockout of RelA, namely embryonic lethal with severe liver apoptosis Mouse embryonic fibroblast (MEF) and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM), complemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin. SKBr3 cells were cultured in McCoy's 5A medium complemented with 10% fetal calf serum (FCS), 100 units/ml penicillin and 100 µg/ml streptomycin. Wild type, IKKα, IKKβ single and IKKα/β double knockout cells (DKO) were the kind gift from Dr. Inder Verma. Antibodies to phospho-specific NF-κB p65 (Ser-536) and IκBα (Ser 32/36) were obtained from Cell Signaling. Antibodies to β-tubulin and to IκBα were obtained from Santa Cruz. Antibodies to IKKα and IKKβ were obtained from Upstate Biotechnology Inc. RhTNF-α (Promega) was used at a final concentration of 10 ng/ml.g. The amount of total protein was measured and equal amounts (20 µg) were fractionated by NuPAGE Novex 4–12% Bis Tris gels (Invitrogen) and electro-transferred to polyvinylidene difluoride membranes. Membranes were blotted with the indicated antibodies, and proteins were detected using an enhanced chemiluminescence detection system . Where indicated, membranes were stripped and re-probed with the indicated antibody.After stimulation, cultured cells were lysed on ice for 5 min in RIPA lysis buffer with freshly added protease and phosphatase inhibitor cocktails. Lysates were cleared by centrifugation at 4 °C for 15 min at 13,000 EMSAs were performed as previously described IKKα and IKKβ mRNA were knocked down with siRNA obtained from Dharmacon. Dharmafect 1 (Dharmacon Company) transfection reagent was used for all si-RNA transfection as described in the manufacturer's protocol. SiRNA was transfected for 72 hrs, and lysate preparation and westerns were performed as described SKBr3 cells stably expressing the 3x-κB plasmid were plated in equal number in triplicate in 24-well plates and transfected with siRNA for 72 hours. Cells were lysed in MPER and luciferase activity was measured with Promega Luciferase Assay System (Promega). Luciferase levels were normalized by protein concentration using a Bradford assay. MEF cells were seeded in 24-well plates at 30–50% density and transfected the next day with the indicated expression vectors and 3x-κB Luciferase reporter gene for 48 h using Effectene (Qiagen) transfection reagent according to the manufacturer's instruction. β-galactosidase reporter gene was used as an internal control. The total amount of transfected DNA (500 ng of DNA) in each well was adjusted by adding empty plasmid vector (pcDNA3.1). Where indicated, 100 ng and 200 ng of IKKβ KM vector has been used. Luciferase activity of whole cell lysates was measured by using a luciferase assay kit (Promega). β-galactosidase activity was measured by liquid -galactosidase assay with chlorophenolred-β-D-galactopyranoside substrate. Relative luciferase activity was calculated by normalizing the assay results to β-galactosidase expression values. Luciferase-fold induction was calculated by normalizing the results to control treatment, which was assumed as 1-fold induction. HeLa cells, seeded in 24-well plates were transiently transfected with the indicated siRNAs for 48 hr. Media was then replaced and cells were further transfected with the NF-κB response 3X-κB luciferase reporter and a control Renilla luciferase construct. 24 hr later, cells were lysed and dual luciferase assays were performed. Luciferase readings in untreated and control vector transfected cells were normalized to 1.in vitro activity, directed to recombinant IκBα, is not diminished in IKK1 (IKKα) null MEFs, yet is significantly reduced in IKK2/IKKβ null cells.Experiments were initiated to assay roles of IKKα and IKKβ in inducing IκBα phosphorylation and activation of NF-κB in response to a well-studied NF-κB inducer, TNFα. For this purpose, mouse embryonic fibroblast cells (MEFs) that are deficient for IKKα or for IKKβ singly as well as IKKα/β double knock-out cells have been utilized. As shown in In addition to IκBα degradation and p65 phosphorylation, we have studied whether IKKα and IKKβ differentially affect NF-κB DNA binding activity in response to TNF as measured by EMSA/gel shift assay. NF-κB DNA binding activity was investigated in WT, IKKα −/−, and IKKβ −/− cells. As shown in Most studies regarding the roles of IKKα and IKKβ have been performed in MEFs null for either subunit. To expand these studies, we have analyzed the differential roles of IKKα and IKKβ in response to TNF in Hela cells . For thiTo further analyze the roles of individual IKK kinases on NF-κB activity and to analyze another cell type, we utilized siRNA knockdown of IKKα, IKKβ, and IKKα/β in SKBR3 breast cancer cells . These cThe data presented so far indicate that IKKα as well as IKKβ have significant roles in canonical NF-κB activation. Previous results derived from expression of an IKKβ kinase inactive mutant have suggested that IKKβ activity is highly dominant in canonical NF-κB activation. In order to further examine this hypothesis, we have utilized WT and IKKβ KO MEF cells for transfection studies. WT and IKKβ −/− MEF cells were transfected with empty vector or with an expression vector encoding IKKα along with an NF-κB luciferase reporter plasmid. Results shown in Based on the phenotypes of IKKα and IKKβ animals, and on results utilizing IKKα −/− and IKKβ −/− MEFs, it has been concluded that IKKβ is the more important IKK catalytic subunit relative to the control of NF-κB activation in the canonical pathway Original data using knockout MEFs indicated that IKKβ is the critical kinase downstream of TNF in inducing IκBα phosphorylation and degradation Based on the results obtained in MEFs, we extended our studies to HeLa cells. Using siRNA knockdown of IKKα or IKKβ in these cells, we demonstrate that loss of either IKK subunit suppresses IκBα phosphorylation, and delays IκB degradation see . These rWe analyzed the effect of expression of a kinase-inactive form of IKKβ on NF-κB-driven reporter gene expression see . PreviouWhy MEFs and HeLa cells appear to utilize IKKα and IKKβ differently regarding effects on IκBα phosphorylation is unclear. This observation may indicate species differences or that different cells/tissues utilize IKKα and IKKβ differently, a concept that should be considered in potential approaches to disease therapy. This latter point may relate to different levels of key upstream regulators of IKK. In this regard, it was reported that knockout of IKKβ in adult hepatocytes did not significantly suppress the ability of TNF to activate NF-κB in these cells, with activity presumably derived from IKKα IKKβ inhibitors have been developed and have shown therapeutic responses in different animal models of diseases and are in early clinical trials


Breast cancer is a common malignant disease, which may be caused by a number of genes deregulated by genomic or epigenomic events. Deregulated WNT/β-catenin signaling with accumulation of β-catenin is common in breast tumors, but mutations in WNT signaling pathway components have been rare. An aberrantly spliced internally truncated LRP5 receptor  was shown recently to be resistant to DKK1 inhibition, and was required for β-catenin accumulation in hyperparathyroid tumors and parathyroid tumor growth.in situ and metastatic carcinoma. LRP5Δ was required in MCF7 breast cancer cells for the non-phosphorylated active β-catenin level, transcription activity of β-catenin, cell growth in vitro, and breast tumor growth in a xenograft SCID mouse model. WNT3 ligand, but not WNT1 and WNT3A augmented the endogenous β-catenin activity of MCF7 cells in a DKK1-insensitive manner. Furthermore, an anti-LRP5 antibody attenuated β-catenin activity, inhibited cell growth, and induced apoptosis in LRP5Δ-positive MCF7 and T-47D breast cancer cells, but not in control cells.Here we show, by reverse transcription PCR and Western blot analysis, that LRP5Δ is frequently expressed in breast tumors of different cancer stage (58–100%), including carcinoma Our results suggest that the LRP5Δ receptor is strongly implicated in mammary gland tumorigenesis and that its aberrant expression present an early event during disease progression. LRP5 antibody therapy may have a significant role in the treatment of breast cancer. Deregulated Wnt signaling with accumulation of β-catenin in the cytoplasm/nucleus plays an important role in a variety of human cancers. The binding of WNT ligand to frizzled and LRP5/6 cell surface receptors normally leads to inhibition of a “destruction complex” consisting of APC/Axin/GSK-3β/Ck1/Dvl and other factors, with subsequent accumulation of dephosphorylated stabilized β-catenin, and regulation of its target genes Aberrant activation of Wnt signaling by cytoplasmic/nuclear β-catenin has been reported in over 60% of human breast cancers in vitro, and parathyroid tumor growth in vivo in SCID mice. Furthermore, LRP5Δ and stabilizing mutation of CTNNB1 was found to be mutually exclusive in those tumors A novel mechanism for aberrant activation of Wnt signaling was recently disclosed by us in parathyroid tumors We have now extended our analysis of aberrant Wnt signaling to breast carcinoma, and demonstrate a fundamental role for the internally truncated LRP5 receptor in deregulated β-catenin signaling and tumor growth.3 1α-hydroxylase To investigate whether the internally truncated LRP5Δ666–809 receptor (LRP5Δ) was expressed in breast carcinoma we initially analyzed nineteen breast cancer specimens (T1–T19) that had been randomly selected in a previous study regarding 25-hydroxyvitamin DIn order to relate accumulation of β-catenin to expression of LRP5Δ, Western blotting analysis was done on cryosections of the tumor material, including two normal breast tissue specimens. The sixteen tumors with LRP5Δ showed increased accumulation of non-phosphorylated active β-catenin when compared to the normal breast tissues, and the tumors expressing LRP5wt only  showed similar relative amounts . Thus, cin situ and metastases. Thus, expression of LRP5Δ was very common in carcinoma of the breast and may be an early event during cancer progression.Encouraged by the above results we then screened a commercially available cDNA panel of 96 breast tissue specimens that covered eight cancer stages. LRP5wt and LRP5Δ was observed in 79 out of 95 (83%) samples . The remIn order to study the consequences of LRP5Δ receptor expression we employed the widely used MCF7 mammary adenocarcinoma cell line which expressed the LRP5Δ receptor but not LRP5wt , and hasControl siRNAs and three highly specific siRNAs directed against LRP5 mRNA were transfected to MCF7 cells. The specificity and silencing potential of the LRP5 siRNAs were ascertained at the mRNA and protein level in MCF7 cells , as we hNext we employed the natural β-catenin responsive DKK1 promoter 5 cells/35 mm dish, 104 cells/96-well microplates) as compared to for example 80% confluency In an attempt to address possible explanations for conflicting results regarding detection of β-catenin activity by the TOPFLASH and pTOPGlow assays In summary, the results showed that maintained expression of the internally truncated LRP5 receptor in MCF7 cells appeared necessary for accumulation of transcriptionally active β-catenin.We reported previously that WNT3 ligand conditioned medium (CM), but not WNT1 and WNT3A, further activated endogenous β-catenin driven TOPFLASH reporter transcription in sHPT-1 parathyroid tumor cells. WNT1, WNT3 and WNT3A are expressed in MCF7 cells in vitro, and this may contribute to the total aberrant signaling level in LRP5Δ-positive breast tumors. DKK1 was found to be expressed in all (n = 16) analyzed breast tumors, as determined by immunohistochemistry (not shown).The LRP5 antagonist DKK1 requires several amino acid residues included in the LRP5Δ truncation, and indeed the DKK1 ligand could not inhibit endogenous LRP5Δ-induced transcriptional activity in parathyroid tumor cells or in LRP5Δ-transfected HEK 293T cells The data presented so far provide compelling evidence for a major role of the internally truncated LRP5 receptor in sustained aberrant β-catenin signaling in breast cancer.Control siRNA, siLRP5wt, siLRP5Δ, and siLRP5tot were transfected to MCF7 cells and the cell viability was determined 24 hrs and 48 hrs later. Growth inhibition and induction of cell death were seen only with siLRP5Δ, and siLRP5tot at both time points . ReducedTumor growth was then evaluated in a xenograft SCID mouse model. Tumor growth was significantly reduced in transplants of MCF7 cells pretransfected with siLRP5Δ and siLRP5tot, but not with control siRNA when compared to non-transfected cells . Thus, LIf breast tumor cell growth is dependent on LRP5Δ, as strongly suggested by the above experiments, an appropriate anti-LRP5 antibody may reduce cell viability as well as β-catenin activity. The anti-LRP5 goat polyclonal antibody, that immunoprecipitated both LRP5wt and LRP5Δ , signifiThus, breast tumor cell growth was dependent on maintained expression of LRP5Δ and continued β-catenin signaling by the receptor. The latter result is in line with the observation that WNT antagonist SFRPs were shown to suppress MCF7 and T-47D cell colony formation Although the mechanism by which the LRP5Δ receptor is made and how β-catenin signaling is activated remain to be understood, the results presented here strongly support an important role of the truncated LRP5 receptor in mammary gland tumorigenesis, and its expression may present an early event during disease progression. Our results furthermore suggest that antibody therapy directed against LRP5Δ, possibly in combination with chemotherapy Cryosections of nineteen breast cancer specimens, including seventeen invasive ductal carcinoma and two invasive lobular carcinoma C-3′5′-TCCACAGGATCTCCCTCGAGACCAATAACAACGAC; and reverse (position 2543), 5′-TTGGACGACTCGATCATGTTGGTGTCCAGGTCGGTC-3′ . The underlined C residue in the forward primer is unique to the Δ666–809 truncation point 2 and 0.25 U Platinum Taq DNA polymerase . Denaturation at 95°C for 60 s, followed by 40 cycles of denaturation for 10 s, annealing at 60°C for 20 s and extension at 72°C for 10 s and a final extension at 72°C for 7 min. The LRP5Δ-specific PCR is more sensitive since no competition between LRP5wt and LRP5Δ fragments occurs during the PCR. Additional positive tumors of the cDNA panel was detected by this protocol. A total of sixty-two LRP5 truncated fragments were directly sequenced on ABI 373A using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit , and all contained the same in-frame deletion (Δ666–809).DNA-free total RNA was prepared from breast tissue cryosections and from MCF7 or T-47D cells, and analyzed by RT-PCR using primary and nested primers spanning positions 1992–2932 of LRP5 mRNA as described 5) were distributed onto 35-mm dishes in DMEM/10% fetal bovine serum and harvested at 24 and 48 hours after siRNA transfection. The number of viable and nonviable cells were determined by using the NucleoCounter  or by using 96-well microplates (104 cells) and the cell proliferation reagent WST-1 . Cells were incubated for 96 hours without serum, with normal goat serum  or with the anti-LRP5 goat polyclonal antibody . Quantification of cytoplasmic histone-associated-DNA-fragments (mono- and oligonucleosomes) was done using the Cell Death Detection ELISA PLUS kit (Roche Diagnostics). Camptothecin at 0.1 µg/ml was used as positive control of apoptosis.MCF7 cells . The various siRNAs have been described previously 4 cells) with 0.15 µg TOPFLASH, 0.1 µg CMV-LacZ using 0.5 µl jetPEI (Poly-Plus-Transfection SAS). After 24 h, the cells were incubated further for 24 h without serum, with normal goat serum  or the anti-LRP5 goat polyclonal antibody . Western blotting and immunoprecipitation analyses were done as described MCF7 breast tumor cells were transfected with siRNA at least in triplicates at 2×105′-ATCGACTGTATCCCC GGGGC-3′; reverse, 5′-CACCACGCGCTGGCACACAA-3′; and probe, 5′-CGGACTGTG ACGCCATCTGCC TGC-3′. For 28S rRNA, the Ribosomal RNA Control Reagents (VIC probe) was used . The following PCR primers were used for DKK1: forward, 5′-TTCTCCCTCTTGAGTCCTTCTG-3′; and reverse, 5′-AGGAGTTCACTGCATTTGGAT-3′. PCR reactions were performed on MyiQ Single-Color Real-Time PCR Detection System  using the TaqMan PCR core Reagent Kit (Applied Biosystems) or the iQ SYBR Green Supermix . Each cDNA sample was analyzed in triplicate. Standard curves for the expressed genes were established by amplifying a purified PCR fragment covering the sites for probes and primers.cDNA from siRNA transfected MCF7 cells was prepared as described above. The following mRNA-specific PCR primers and labeled probe (5′FAM-sequence-3′TAMRA) were used for quantitative real-time RT-PCR analysis. For LRP5tot: forward, 6 cells for 24 hours. The animals were monitored every day and sacrificed after 5 weeks. The animal experiments was approved by the Uppsala University board of animal experimentation and was performed according to the United Kingdom Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia Two to three week old female Fox Chase severe combined immunodeficient mice (SCID) were used . The mice were anesthetized with isoflurane  during the manipulations. Both flanks of each animal were injected subcutaneously  with MCF7 cells together (1∶1) with BD Matrigel Matrix , after transfection of 10t test was used for all statistical analyses. Values are presented as arithmetrical mean±SEM. A p value of <0.05 was considered significant.Unpaired


N-cadherin is responsible for this adhesion under normal physiological conditions. The role of cadherins in adverse cardiac pathology is less clear. We studied the hearts of the stroke-prone spontaneously hypertensive (SHRSP) rat as a genetic model of cardiac hypertrophy and compared them to Wistar–Kyoto control animals. Western blotting of protein homogenates from 12-week old SHRSP animals indicated that similar levels of β, γ-, and α-catenin and T, N and R-cadherin were expressed in the control and SHRSP animals. However, dramatically higher levels of E-cadherin were detected in SHRSP animals compared to controls at 6, 12 and 18 weeks of age. This was confirmed by quantitative Taqman PCR and immunohistochemistry. E-cadherin was located at the intercalated disc of the myocytes in co-localisation with connexin 43. Adenoviral overexpression of E-cadherin in rat H9c2 cells and primary rabbit myocytes resulted in a significant reduction in myocyte cell diameter and breadth. E-cadherin overexpression resulted in re-localisation of β-catenin to the cell surface particularly to cell–cell junctions. Subsequent immunohistochemistry of the hearts of WKY and SHRSP animals also revealed increased levels of β-catenin in the intercalated disc in the SHRSP compared to WKY. Therefore, remodelling of the intercalated disc in the hearts of SHRSP animals may contribute to the altered function observed in these animals.The structural integrity of cardiac cells is maintained by the Ca E-cadherin is expressed in most epithelia throughout the body, while N-cadherin expression is more restricted to specific cell types such as muscle and neurones The SHRSP is a genetically bred model of hypertension with a high incidence of cerebrovascular events and cardiac remodelling 22.1Inbred colonies of SHRSP and Wistar–Kyoto (WKY) rats were maintained at the University of Glasgow as described previously 2.2−∆∆ct) method and is shown as fold change 6, 12 and 18-week old SHRSP animals and WKY controls were euthanized and hearts removed. Tissue was divided and processed for protein homogenates, RNA extraction and immunohistochemistry. For western blotting, proteins were electrophoresed and transferred onto Hybond-P membrane , and blocked with 10% (wt/vol) skimmed milk powder in TBS-T  Tween-20). The membrane was incubated for 1 h at 37 °C with the following antibodies, N-cadherin, E-cadherin, R-cadherin, T-cadherin, β-catenin, γ-catenin, α-catenin and GAPDH as the loading control . Proteins were visualised using an ECL detection system . For quantification of mRNA levels, quantitative Real-Time PCR was used. Heart RNA was extracted from 12-week old rats using RNeasy kits , treated with DNase Free , and quantified using Nanodrop . Normalisation was confirmed by performing real-time PCR  of Actβ  with comparable threshold cycles. Taqman probe for E-cadherin (Rn00580109_m1-labelled FAM) were multiplexed with Actβ (4352340E-labelled VIC). Expression of E-cadherin relative to Actβ was derived using the comparative  cells were counted, and adenoviral infection was performed during plating of the myocytes at 3 × 104 rod-shaped cells into Petri dishes with either Ad:E-cadherin or Ad:LacZ with an indicated multiplicity of infection (MOI). Myocytes were cultured in supplemented M199 medium (Sigma) for 48 h. The lysate of cells transfected with Ad–E-cadherin were subjected to western immunoblotting as before. Infection of H9c2 and isolated rabbit myocytes with increasing doses of Ad:E-cadherin allowed for the effects of dose response on hypertrophy to be assessed by the calculation of cell volume. Cell length was measured 48 h post-transfection. Samples of the cell suspension were placed on a glass slide and measured with a graticule in the eye-piece of the microscope and cell volume calculated (n = 50 per group).New Zealand White rabbits (2–2.5 kg) were euthanized by administration of an intravenous injection of 500 IU heparin together with an overdose of sodium pentobarbitone (100 mg/kg). Hearts were removed and perfused retrogradely  with a nominally Ca2.5LacZ at an MOI of 30. 48 h later the cells were washed in PBS and fixed with 10% formaldehyde. Fixed cells were incubated with primary anti-E-cadherin, N-cadherin and β-catenin antibodies or matched mouse IgG non-immune control  followed by detection with FITC conjugated universal secondary antibody. The cells were mounted with vectashield with DAPI and visualised by fluorescent microscopy.H9c2 cells were infected with either the Ad:E-cadherin or Ad:2.6LacZ at an MOI of 30. Following 48 h, the cells were harvested in RIPA buffer. Equal protein concentrations of the lysed cells were incubated with either β-catenin-agarose conjugated antibody or IgG-agarose conjugated control at 4 °C overnight. A separate sample of Ad-transduced cells was incubated overnight without antibody and was used to quantify the total level of E-cadherin. The cell lysate was then washed with RIPA buffer and loading buffer added. The cell sample was then probed for E-cadherin as described before.H9c2 cells were grown in a monolayer and infected with either Ad:E-cadherin or Ad:2.7t-test. Statistical analysis was performed in Prism version 4.0 . For all tests, P < 0.05 is considered statistically significant. The results represent mean values and SEM of the data.Comparisons were made using the student's 3p = 0.03). This suggests a transcriptional control mechanism that leads to E-cadherin overexpression in the SHRSP rather than post-transcriptional control. Interestingly, following amplification of 5′ and 3′ regions of the rat E-cadherin gene we observed a single nucleotide polymorphism in the 5′ non-coding region (exon 1) between the SHRSP and WKY . Of the two known SNPs, the SNP positioned in the 5′ non-coding region of exon 1 was different between WKY and SHRSP animals. This may have a further impact on the effect of dysregulated E-cadherin expression in the SHRSP.Although levels of the other cadherins and catenins appeared to be unaltered, distribution of β-catenin was different. In E-cadherin overexpressing cells, β-catenin was detected at cell–cell junctions and in association with E-cadherin, which may restrict WNT/β-catenin signalling. In heart development, β-catenin plays a biphasic role in cardiomyocyte differentiation; initially in committing mesenchymal cells to the cardiac lineage, while at later stages a down-regulation is required for differentiation The accumulation of β-catenin in intercalated discs is thought to play a role in determining the previously described plasmalemma rigidification in δ-SG null cardiomyocytes We have shown for the first time that increased E-cadherin and β-catenin are dysregulated in the hearts of the SHRSP and therefore may contribute in the pathology of cardiac disease. This association of aberrant pathology with E-cadherin/β-catenin has however been observed in animal models of cancer such as colon cancer 2+ concentration, E-cadherin has been shown to assemble and increase the function of connexin 43 To evaluate the effect of E-cadherin on cardiac function, we transfected adult rabbit ventricular myocytes with E-cadherin adenovirus and measured their fractional shortening in response to field stimulation. The cells overexpressing E-cadherin maintained normal function (data not shown). Dependent on the intracellular CaDysregulation of the intercalated disc in the SHRSP model may therefore result in alterations in structure and cell-signalling which may contribute to the aberrant structure and function observed in these animals. Together, these findings may provide an insight into the pathology observed in heart disease.


The adenomatous polyposis coli (APC) gene and its expressed product are highly studied because of its role as a tumour-suppressor protein. Inherited mutations in APC lead to the condition known as familial adenomatous polyposis (FAP), which predisposes the affected individuals to colorectal cancer. Furthermore, mutations in APC are found in the majority of sporadic cases of colon cancer. There have been many published studies concerning the cellular localisation of APC, this being fundamental to our understanding of its function, but there has also been much concern over the specificity of certain commercially available antibodies to APC. Here we report that the widely used antibody APC(N15) demonstrates a strong interaction with the Ku80 subunit of the Ku heterodimer under defined experimental conditions. Based on the data presented here, we suggest that APC(N15) is not suitable for many applications used for the study of APC. Adenomatous polyposis coli (APC) is a large multifunctional tumour-suppressor protein. It is central to many processes including developmental regulation and it is mutated in most cases of sporadic colon cancer. Germline mutations in APC lead to the condition known as familial adenomatous polyposis (FAP), which predisposes affected individuals to colorectal cancer , which has been widely used for a number of applications (SW480 cells (ECACC) were grown in DMEM/10% FBS, HCT116 cells (ECACC) were grown in McCoy's 5a medium/10% FBS.5 cells) in 40 mm tissue culture dishes. Cells were washed in PBS, fixed with 4% paraformaldehyde for 20 min at RT, washed in PBS before being permeabilised in 0.2% Triton X-100 for 15 min at RT. After a further PBS wash, cells were blocked in PBS–5%FBS for 1 h at RT. Cells were incubated with primary antibodies diluted in PBS–5%FBS for 30–60 min at 37°C, rinsed in PBS (×3) for 5 min before incubation for 30–60 min at 37°C in secondary antibodies diluted in PBS–5%FBS. Following secondary antibody incubation, cells were washed (×3) in PBS and stored in PBS for imaging using a Zeiss Axioplan 2 and LSM510 confocal microscope. Antibodies and dilutions are as follows: APC(N15) 1 : 75 (Santa-Cruz Biotechnology), Ku(p80) 1 : 50 (Oncogene).Cells were seeded  Triton, 1 mM AEBSF, 2 μg ml−1 aprotinin, 10 μM bestatin, 10 μM E64, 1 UM pepstatin, 100 μM leupeptin, 1 mM sodium ortho vanadate) and cell extracts resolved by SDS-PAGE. Gels were electrophoetically transferred onto PVDF membrane. Blots were probed with antibodies diluted as follows: APC(N15) 1 : 1000 , Ku80 1 : 200 .Whole-cell lysate was prepared . MS protein analysis was carried out by Micromass using electrospray MS and MS/MS on a Micromass Q-TOF2 mass spectrometer. All data were processed automatically by means of Protein Lynx software, protein identification was achieved by analysis with ProteinLynx Global Server version 1.0.In an effort to isolate APC and its binding partners, the antibody APC(N15) was incubated with whole-cell lysate from SW480 colon carcinoma cells. SW480 cells contain a truncated version of APC of 150 kDa as a result of a stop codon caused by frameshift mutation. Protein A–agarose beads were used to pull down the antibody–protein complex allowing protein analysis by SDS-PAGE. Following silver staining, two bands of 70 and 80 kDa showed a reproducible enrichment; however, there did not appear to be an equivalent band of the expected size for APC  antibody. To address this, an IP from SW480 whole-cell lysate was performed using an antibody to Ku80. The resulting immunoprecipitate was split into two and resolved by SDS-PAGE, each sample alongside an aliquot of purified DNA–PK complex (which includes Ku70 and Ku80), as positive control. One set was visualised by silver staining, while the other was transferred onto PVDF membrane and immunoblotted first with the Ku80 antibody and then APC(N15). The silver-stained gel revealed two enriched bands of 70 and 80 kDa (data not shown), identical in appearance to those pulled down by APC(N15). The immunoblot demonstrated that both the Ku80 and APC(N15) antibodies detected the Ku80 subunit , demonstrates a strong interaction with the Ku80 subunit component of the Ku heterodimer. This antibody is not suitable for immunoprecipitation of its intended antigen, although it is extremely efficient at affinity purifying the Ku heterodimer. As others have commented  for immunohistochemistry  and Ku80  is unreliable because of crossreactivity and is therefore not suitable for immunodetection of APC.The adenomatous polyposis coli (APC) gene and its expressed product are highly studied because of its role as a tumour-suppressor protein. Here we report that the widely used APC antibody (N15) demonstrates a strong interaction with the Ku80 subunit of the Ku heterodimer.


The purpose of this study is to determine the point (number of procedures during training) beyond which ductal endoscopy is successfully performed.Breast ductal endoscopy is a relatively new diagnostic method with ever growing importance in the work-up of patients with bloody nipple discharge. The ability to perform ductal endoscopy is very important and useful for breast fellows. Learning curve in breast ductal endoscopy remains a ex vivo model was adopted. Fellows were trained on 20 surgical specimens derived from modified radical mastectomy for breast cancer. The target of the education program was to acquire proficiency in performing ductoscopy. The achievement of four consecutively successful ductal endoscopies was determined as the point beyond which proficiency had been achieved. The number of procedures needed for the achievement of proficiency as defined above ranged between 9 and 17 procedures. The median value was 13 procedures; i.e. 50% of trainees had achieved proficiency at the 13th procedure or earlier.Ten breast fellows received training in our Breast Unit. For the training process, an These pilot findings point to approximately 13 procedures as a point beyond which ductal endoscopy is successfully performed; studies on a larger number of fellows are nevertheless needed. Further research, focusing on the learning curves of different training models of ductal endoscopy, seems desirable. Breast ductal endoscopy is a relatively new diagnostic method which is rapidly gaining ground with the advent of new generation endoscopies. Patients with bloody nipple discharge stand to benefit most from this method, due to its enhanced visualization of previously undetected areas of the breast ductal system ,2.terra incognita, since no systematic studies have addressed this topic. Various training models have been documented in the literature based on either ex vivo or in vivo specimens, but to date questions still remain [Indeed, the ability to perform ductal endoscopy is important and useful for breast fellows. Unfortunately, the learning curve in breast ductal endoscopy remains a l remain ; is it aIn this study, 10 breast fellows received training in our Breast Unit. All breast fellows had a minimum of three years experience in general surgery and in minimally invasive breast procedures.ex vivo model was adopted. Fellows were trained on 20 surgical specimens derived from modified radical mastectomy for breast cancer. Within 20 minutes of excision, free of any form of fixation and under adequate illumination, the specimens were mounted on a flat surface to simulate the anatomical position on a supine body and submitted fresh to ductoscopy   Figure .The target of the education program was to acquire proficiency in performing ductoscopy. The achievement of four consecutively successful ductal endoscopies was determined as the point beyond which proficiency had been achieved.Ductal endoscopy was considered successful when it encompassed all the following:i) the ability to perform mapping of the ductal system, recording the location and number of ducts cannulated, taking care to not cannulate the same duct more than once. Additionally, the ability to record the distance of scope advancement and the number of bifurcations passed for each duct.ii) the ability to locate and identify accessible intraductal lesions.For each fellow the point of proficiency (i.e. the achievement of four consecutively successful ductoscopies) was noted.Informed consent was obtained by all participants in the study. Approval was obtained by the local Institutional Review Board.The number of procedures needed for the achievement of proficiency as defined above ranged between 9 and 17 procedures. The median value was 13 procedures; i.e. 50% of trainees had achieved proficiency at the 13th procedure or earlier. The success rate of the ten fellows at each attempt is depicted in Figure ex vivo training of the fellows seems indispensable to prevent both complications and discomfort.Ductal endoscopy is a novel method of ever growing importance in the work-up of patients with bloody nipple discharge. Despite its technical feasibility ,5, ductaex vivo specimens is considerably more complex than on live patients due to less distensible ducts rendering intraductal lesions more difficult to identify. In addition, the adoption of different definitions or settings may diversify the number of procedures needed for qualification.Our results indicate that approximately 13 procedures are warranted to guarantee a successful ductal endoscopy. For the optimal interpretation of the result one should bear in mind that ductal endoscopy on per se. The definition integrates a variety of skills ; in the present study, 'success' pointed to the accomplishment of all aspects of the procedure. As a result, no separate rates  have been provided. Indeed, future studies on each component may be particularly interesting, as they may point to the most demanding steps of the procedure.An important limitation of the study pertains to the definition of success/failure Notwithstanding, this manuscript highlights the necessity of further studies focusing on the learning curves of different training models of ductal endoscopy. It remains to envisage comparative protocols assessing the efficacy and rapidness of training programs as well as the fellows' perception and attitudes towards the former.Breast ductal endoscopy is a relatively new diagnostic method which is rapidly gaining ground with the advent of new generation endoscopies. The ability to perform ductal endoscopy is important and useful for breast fellows. These pilot findings point to approximately 13 procedures as a point beyond which ductal endoscopy is successfully performed; studies on a larger number of fellows are nevertheless needed. Further research, focusing on the learning curves of different training models of ductal endoscopy, seems desirable.The authors declare that they have no competing interests.FZ conceived the idea, designed the study and wrote the manuscript. TNS wrote the manuscript and performed the descriptive statistics. GG, EP, DC, GB, JF and GF participated in the design of the study, made substantial contributions to the acquisition of data and also helped in drafting the manuscript. AP revised the manuscript critically for important intellectual content and made substantial contribution in the interpretation of data. KJB revised the manuscript for important intellectual content and participated in the design of the study. GCZ conceived the idea, designed the study, revised the manuscript for important intellectual content and has given final approval of the version to be published


The molecular pathway that controls cardiogenesis is temporally and spatially regulated by master transcriptional regulators such as NKX2-5, Isl1, MEF2C, GATA4, and β-catenin. The interplay between these factors and their downstream targets are not completely understood. Here, we studied regulation of β-catenin and GATA4 by NKX2-5 in human fetal cardiac myocytes.β-catenin and GATA4 genes. Using promoter-luciferase reporter assay combined with mutational analysis of the NKEs we demonstrated that the identified NKX2-5 binding sites were essential for the suppression of β-catenin, and upregulation of GATA4 by NKX2-5.Using antisense inhibition we disrupted the expression of NKX2-5 and studied changes in expression of cardiac-associated genes. Down-regulation of NKX2-5 resulted in increased β-catenin while GATA4 was decreased. We demonstrated that this regulation was conferred by binding of NKX2-5 to specific elements (NKEs) in the promoter region of the This study suggests that NKX2-5 modulates the β-catenin and GATA4 transcriptional activities in developing human cardiac myocytes. To test if β-catenin and GATA4 are regulated by NKX2-5, endogenous NKX2-5 expression was knocked down by expressing antisense NKX2-5 RNA (XKN) in human fetal ventricular myocytes. This study shows that β-catenin and GATA4 transcription factors are regulated by NKE sequences in the promoter region of these genes. In addition, we confirm direct physical interactions between NKX2-5 and NKEs in the promoters of β-catenin and GATA4 as demonstrated by electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase promoter assays. This study supports the essential role of NKX2-5 in maintaining the cardiac gene expression program and suggests direct regulation of β-catenin and GATA4 by NKX2-5 in human cardiomyocytes.During early development myocardial cells within primary and secondary heart fields form under influence of bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and proteins from Wnt family, that initiate expression of a cascade of cardiac-associated transcription factors including NKX2-5, Isl1, MEF2C, GATA4, and β-catenin and their downstream targets (reviewed in Identification of NKX2-5 binding sites in the promoters of β-catenin and GATA4 genesβ-catenin and GATA4 genes were searched for NKX2-5-binding consensus sequence (NKE), TNAAGTG β-catenin gene (CTNNB1) GATA4 gene was performed and a candidate NKEs in position -1540 was identified. The identified NKEs are located in the regions partially conserved between human and mouse when the promoter sequences are aligned using rVISTA  using nuclear extract from retrovirally-transduced C2C12 cells overexpressing NKX2-5 th exon of β-catenin (CTNNB1-E) was used as negative control in ChIP analysis. The DNA harboring the candidate NKE sequences in the promoters of GATA4 and β-catenin genes were immunopreceipitated  are decreased to approximately 80% of that in the wild type mice. An approximately 20% increase in β-catenin transcript level was detected. Similarly, Gata4 RNA level was also increased  revealed an increase in β-catenin RNA which was in agreement with our in vitro data. A recent study has shown that activation of Wnt/β-catenin downregulates Hdac1, a suppressor of Nkx2-5 and GATA4 in cardiomyocytes Nkx2-5 transcription factor regulates multiple aspects of cardiac cell structure, function, and development Nkx2-5 promoter Gata4 is genetically upstream of Nkx2-5. Accordingly, the expression of GATA4 is upregulated prior to Nkx2-5 in P19CL6 cardiomyocyte model in vitro cultured ventricular myocytes with antisense NKX2-5 in this study, however, led to a reduction in GATA4 level suggesting that NKX2-5 positively regulates the expression of GATA4, at least in cultured cardiac myocytes, and therefore, not only does GATA4 regulate NKX2-5, but the converse regulation exists as well. GATA4 RNA level in Nkx2-5+/− embryos, however, was unexpectedly upregulated. We reason that this upregulation represents a complex regulation of GATA4 transcription factor during development. Since GATA4 begins to express in cardiac progenitor cells prior to Nkx2-5 Moreover, our study indicated the direct regulation of GATA4 by NKX2-5 in human fetal cardiac myocytes. The role of GATA4 in early cardiogenesis is clearly established in vitro cell culture model of fetal cardiac myocytes, such as the one used in this study, in combination with antisense RNA or siRNA technology Overall, our study proposes a model where Nkx2-5 negatively regulates β-catenin and positively regulates GATA4 in cardiomyocytes, as depicted in 2.Human fetal hearts at gestational ages 20–22 (n = 5) weeks were collected in IMDM+20% FBS. Ventricles were separated and the fat and connective tissues were removed before mincing. The tissue was then digested by incubating in enzyme solution  for 10 min at 37°C and passed through a filter. These steps were repeated 2–3 times and the cells were pelleted by centrifugation. The cell mixture was resuspended in the media and the number of live cells was counted. Fibroblasts were separated from other cardiac cells using anti-fibroblast antibody-conjugated magnetic beads purchased from Miltenyi Biotech. Inc., according to manufacturer's instruction. Cells were cultured in IMDM+20% FBS for 3–5 days at 37°C and 5% COMouse neonatal cardiomyocytes were prepared using similar protocol. Isolated cells obtained after tissue digestion were pre-plated for 1 hr to allow attachment of fibroblasts to the plate. Cells not adhered were then seeded on plates coated with 0.1% gelatin and cultured in DMEM+15% FBS for 24–48 hrs before they were used in experiments.Fetal cardiac cells were derived from terminated pregnancies under an approved protocol by the Hospital for Sick Children Research Ethics Board. Written consent was obtained from mothers who participated in the study. The use of mice in this study was also approved by an institutional ethics board.Immunofluorescence and western blot analyses were performed according to the standard procedures. Myosin heavy chain antibody (MF-20) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Other antibodies were purchased: PCNA (DAKO), GAPDH , β-catenin (Upstate Biotech.) and Nkx2-5, GATA4, and MEF2C (Santa Cruz Biotechnology). For western blot analysis one to three plates of cardiac myocytes, for each experimental condition, were lysed with RIPA buffer and the samples were separately loaded on SDS-PAGE gel and blotted.agactggtcgactgccaccatgttcc and agagtcagggatcctagttgaggtg, and digested with SalI/BamHI. NKX2-5 cDNA was cloned into SalI/BglII site of pAdTrack-CMV (Stratagene Inc.) in forward and reverse orientations. Manufacturer's instructions were followed to produce the virus. The virus titer and multiplicity of infection (MOI) were estimated using HEK-293 cells and by calculating the number of GFP+ cells. The recombinant adenoviruses were used at different MOIs.Human NKX2-5 cDNA was amplified from fetal heart RNA by RT-PCR using one-step RT-PCR kit (Qiagen) and using primers: aacctgcgtcgaccaccatgttcc and cggagaaaggatcccaggcctgcg and cloning the cDNA into NotI/SalI sites of pAdTrack-CMV.Adenoviral vector to express mouse Nkx2-5 was constructed by amplifying mouse Nkx2-5 cDNA using primers: http://www.cbrc.jp/research/db/TFSEARCH.html) was used to find putative NKX2-5 binding elements in 2kb upstream of the transcriptional initiation sites of human GATA4 (accession no. AC090790) and β-catenin genes (accession no. AY463360.1).TFSEARCH (http://genome.lbl.gov/vista/index.shtml) was used to examine the similarity between human and mouse sequence in the 5′-flanking region of each gene.rVISTA , Cx43 , and Gapdh . Expression levels determined in each assay were normalized against Gapdh. Data were analyzed by relative quantitation method using standard curve.Mouse neonatal cardiomyocytes were prepared as described above. Cells were transfected with adenovirus expressing mouse Nkx2-5 at 1 MOI and cultured in growth media for 24 hrs. Cells were then treated with mouse Wnt-3A (R&D Systems), at a concentration of 100 ng/mL for 16 hrs in media containing 1% serum. RNA was isolated using Trizol (Invitrogen) and cDNA was synthesized. Gene expression was assayed using either pre-designed Taqman assays or oligonucleotides with SYBR Green. The following sets of primers were used: Nkx2-5 , Gapdh  Axin2 (tataagaattaacctgcagacagcgctctg) and GATA4 (NKE-G: agaagaaaccctaagtgtgtcgcccccagc) promoters were synthesized and annealed. The annealed oligonucleotides were biotinylated using 3′ end DNA labeling kit (Pierce) and incubated with 5 µg of nuclear extracts for 20 min to allow binding. The mixtures were analyzed by gel electrophoresis on 5% PAGE in 0.5× TBE buffer. The membrane was hybridized with HRP-conjugated straptavidin (Pierce) for detection of DNA-protein complex according to the manufacturer's instruction.EMSA was performed using Lightshift Chemiluminescent EMSA kit (Pierce). Nuclear extracts were prepared from C2C12 myoblasts expressing either human Nkx2-5 or GFP agtaagaatgcggctcttgc), (gagacagaaccctccccatt), CTNNB1-P: (tcgacaaacgtcaattttgc), (tcgattaagcagcctccaat), GATA4-P: (cccttcagccttaagttcc), (gcgacacacttagggtttcttc), and CTNNB1-E (cacccggaggtgatttacaca), (gcctggctctgatctccatgt). Amplification condition was 94°C- 4 min, 94°C- 30 sec, 50°C-30 sec, 72°C- 30 sec, for 35 cycles.ChIP was performed according to a published protocol gagtccgaagagctcgcagattgg), GR2 (ggcgcaaactcgaggaaaggaaac), and BF2 (ggcagttgagctcttaccacttata), BR2 (ggctgtgaactctcgagtagaacg) and cloned into the SacI/XhoI sites of pGL3-promoter plasmid (Promega). Plasmids were sequenced to confirm the correct DNA sequence. COS7 cells were grown in DMEM+10%FBS and co-transfected with pCMV-lacZ plasmid and either pGL3-GATA4 or pGL3-βcatenin constructs using FuGENE 6 (Roche Applied Science).Promoter regions containing the candidate NKX2-5-binding sites in β-catenin (CTNNB1) and GATA4 promoters were amplified using primers GF2 (PstI sites) and primers on both sides of the promoter sequence and surrounding the cloning site of pGL3 vector: pGL3-1 (ctagcaaaataggctgtcccag) or pGL3-2 (ccaagcttacttagatcgcaga). The amplified DNA fragments were then digested with either SacI/PstI or PstI/XhoI and subcloned into the SacI/XhoI sites of the pGL3-promoter vector. Oligonucleotides used to amplify mutated constructs: mNKE1: aattggaggctgctgcagatagctttctcta, mNKE2: ttgatacctagtgactgcaggaaccagataa, mNKE3: ctataagaattaactgcagacagcgctctgg, and their complementary sequences. To make mNKEΔ, the mNKE3 construct was cut with PstI and SacI and blunted with Klenow DNA polymerase. The DNA fragment encompassing the promoter sequence downstream of mNKE3 site was eluted from gel and cloned into the SmaI site of pGL3-promoter.Mutation or deletion of Nkx2-5-binding elements was carried out by amplifying the promoter from the wild type reporter construct using oligonucleotides harboring modified DNA sequences  to detect levels of Nkx2-5 , Gata4 , β-catenin , and Gapdh  RNAs. Data were analyzed by relative quantitation method using standard curve.Animal procedures were performed under approved institutional protocol. Heterozygote P<0.05 was used to indicate statistical significance.Results are expressed as mean±SD unless otherwise indicated. Statistical significance was determined by one-way ANOVA.


Here, we have investigated the expression of key components of β-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca2+ receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both β-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These β-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.In most colorectal tumours, APC mutation stabilises  Our studies  and thosβ-catenin activity, when normal controls have been lost through β-catenin mutation as occurs sometimes in colorectal cancer  pathway  (2+) pathway is more difficult to define, particularly in non-embryonic tissues. It may employ a distinct subset of FZDs, notably FZD3, FZD4 and FZD6   , would b2+-response pathways are expressed at an early stage in tumour formation. These inductions are coordinated in adenomas and correlate with β-catenin stabilisation and nuclear accumulation of phospho-c-Jun. Stabilisation of β-catenin in cultured cells using lithium chloride induced NKD1 (Wnt/PCP pathway) expression but had no effect on FZD3 (Wnt/Ca2+ pathway) levels, while Wnt3a conditioned medium caused induction of both targets, indicating that β-catenin is responsible for some but not all of the changes seen in adenomas.In this paper, we show that NKD1, FZD3 and FZD6 transcripts are all induced significantly in colorectal adenomas compared to matched normal tissue, indicating that both the Wnt/PCP and the Wnt/Caβ-catenin stabilisation is sufficient to induce Wnt/PCP components but that a Wnt signal is required to induce expression of the Wnt/Ca2+ receptors, indicating that the expression profile of Wnt/Ca2+ receptors seen in adenomas requires β-catenin-independent signalling.These data show that Adenoma and carcinoma samples were obtained with individual patient consent, following the local ethics committee guidelines for resected specimens. Separate ethical approval was given for analysis of paraffin blocks (LREC No. 2003/277).All adenomas were from the rectum and sigmoid colon. The 14 matched samples were from patients with a median age 63 years (range 56–65 years), and included five adenomas less than 10 mm in diameter, five tubular adenomas and nine tubulovillous lesions. Unmatched polyps included six adenomas under 10 mm in size, two were simple tubular lesions and the median age was 61 years. The unmatched normal tissue was from patients with a median age of 63 years (range 60–65 years).Eighteen primary colorectal cancers were collected with matched normal mucosal samples. The median age of the cohort was 74 years (range 43–91 years). In 10 cases, the primary cancer was at the splenic flexure or proximal colon, and 3 were rectal cancers. Six cancers had lymph node metastases at the time of diagnosis, and two had liver metastases.μg of DNase-treated total RNA using Ready-To-Go You-Prime First-Strand Beads  and random hexamers .Total RNA was extracted using TRI reagent  according to the manufacturer's instructions. First-strand cDNA was synthesised from 2 μl, containing 1 × BIOTAQ NH4-based reaction buffer , 1 mM Mg2Cl, 2 mM dNTPs (Amersham Pharmacia Biotech), 250 ng of each primer, 0.5 U of BIOTAQ DNA polymerase (BIOLINE) and 1 μl of cDNA. Amplification involved an initial step of 2 min at 94°C, followed by 35 cycles of 94°C for 10 s, 58°C for 20 s and 72°C for 30 s, with a final extension of 5 min at 72°C.Oligonucleotide primers to amplify NKD1 and FZDs 3, 4, 6 and 10 were designed using Primer3 software  and syntμl. Each reaction contained 12.5 μl of 2 × TaqMan Universal PCR Master Mix (PE Applied Biosystems), 90 nM keratin 8 (KRT8) and target gene primers, 150 nM target gene TaqMan probe, 175 nM KRT8 TaqMan probe and 1 μl of cDNA. Cycling conditions were an initial step at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for15 s and 60°C for 1 min. Results from the target genes were normalised to the epithelial cell-specific gene KRT8 as described previously  and normal samples .Oligonucleotide primers and TaqMan probes  were desμm) were dewaxed, rehydrated and endogenous peroxidase activity blocked by incubation in 10% H2O2 in methanol for 10 min. Microwave antigen retrieval was undertaken for 1 h. Sections were incubated overnight with primary antibodies recognising β-catenin  or phospho-c-Jun  at a dilution of 1 : 300. After washing with PBS, sections were incubated with biotinylated goat anti-mouse/rabbit IgG  according to the manufacturer's instructions for 30 min. Serial PBS washing and incubation with streptavidin-peroxidase conjugate (Dako) was undertaken prior to incubation with diaminobenzidine tetrahydrochloride (Sigma). Sections were counterstained with haematoxylin (BDH), dehydrated and analysed on a light microscope.The Streptavidin–biotin indirect immunoperoxidase method was performed as described previously .The normal small intestine epithelial cell line FHs74Int  was cultured using Hybri-Care medium (ATCC) supplemented with 1.5 g lWnt3a conditioned medium was produced from mouse L-cells (ATCC) stably expressing the recombinant protein and used to treat HeLa cells. Parallel experiments were performed with medium conditioned by the parental L-cells to exclude the effects of other secreted activities.Where appropriate, statistical analysis was performed using Fishers exact test.P=0.001).We devised conventional RT–PCR assays for NKD1 and each of the human FZD sequences and used these to analyse RNA extracted from a series of adenomas and control normal mucosa samples. P=0.003 and 0.007 respectively). Frizzleds 4, 6 and 10 showed less obvious changes, with a tendency to become expressed in the adenomas, the increased incidence of FZD6 just reaching significance (P<0.04). The most striking change found, however, was that for the Wnt/Ca2+ receptor FZD3, which was expressed in 13 of 19 adenomas, but only in 2 of 13 normal tissues (P=0.005).Significant changes were also apparent in the analyses performed on a series of 19 adenomas for the FZDs, and these are summarised in Since the qualitative review of expression indicated induction of NKD1, FZD3 and FZD6 in colorectal adenomas, we developed quantitative real-time RT–PCR assays for each of these to determine the extent of the induction. Transcript levels were compared with levels of KRT8, an epithelial cell-specific marker, to control for the variation in stromal and inflammatory cell components between dysplastic and normal epithelium . PerformFrizzleds 3 and 6 were also induced in most adenomas, with a median of 6.5- and 4.6-fold, respectively. Frizzled 3 was induced in 11 of 14 adenomas by a factor of 1.5–31. Frizzled 6 was measured in a subset of 12 of the 14 adenomas and was found to be induced in 10 of these.2+ pathways compared to normal epithelium.These data show upregulation of key components of both the Wnt/PCP and the Wnt/Caβ-catenin and identified this in all but two cases (M and N). These were the same individuals that lacked induction of NKD1, FZD3 and FZD6 mRNAs, indicating that unregulated β-catenin signalling is required to induce these transcripts.Although most colorectal tumours harbour APC mutations, some develop through alternative routes , to stabilise β-catenin  was striking and indicates that incoming signals from Wnt growth factors would be directed towards the Wnt/PCP response in these tumours. Our demonstration of c-Jun activation in adenomas supports this. The importance of c-Jun in colorectal adenoma formation, through its interaction with β-catenin and TCF4, has been demonstrated in animal models  was substantially lower than the 135-fold we saw in adenomas. The reason for the difference observed between the degree of change seen in adenomas and in cancers is not entirely clear. The many differences between adenomas and cancers may lead to a reduction in dependence on these reorganised Wnt pathways as the disease progresses. Although genetic changes (e.g. APC mutations) cannot be reversed during tumour progression, other effects may show plasticity when selection pressure is lost due to mutations in other pathways. To explore this, we shall first have to define the outcomes of 2+ signalling to have a role in early colorectal tumorigenesis is presently unclear. Signalling in this pathway has been associated with the progression in some tumours (The potential for Wnt/Ca2+ signalling outcomes in this cohort until we can identify unambiguous markers of a response. Studies in more tractable systems have shown that Wnt5a and Wnt11 action leads to a loss of cell adhesion, stimulates PKC and CamKII, and antagonises the β-catenin pathway (2+-dependent PKC isoforms are present in colorectal adenomas, but are less abundant in proliferating areas (2+ signalling through PKC and proliferation of adenoma cells. Studies with purified Wnt5a indicated that calcium release is not a universal outcome of signalling, but demonstrated receptor-dependent antagonism of β-catenin's transcriptional activity in the presence of the ROR2 co-receptor (β-catenin-dependent transcription is a hallmark of colorectal tumours, β-catenin antagonism must be incomplete.One disadvantage of studying these events in adenomas is the scarcity of material available, which limits the number of parallel analyses that can be performed on a single sample. For this reason, we have chosen not to explore Wnt/Careceptor  emphasisβ-catenin using lithium treatment led to NKD1 expression but failed to influence Wnt/Ca2+ receptors, while Wnt growth factor treatment induced FZD3 transcripts to the degree seen in adenomas. In early tumorigenesis, Wnt signalling is enhanced relative to normal epithelium as a result of antagonist suppression, and this is likely to be the origin of the elevated FZDs3 and 6 expression we identified in adenomas.In cultured cells, stabilisation of β-catenin stabilisation providing a negative feedback to Wnt stimulation. In tumour cells lacking APC function, this homoeostatic loop fails to complete, leading to sustained expression and establishment of alternative Wnt-response pathways. The epigenetic suppression of secreted Wnt antagonists seen in adenomas (In normal cells, NDK1 and FZD3/6 are expressed transiently to repress further β-catenin-independent Wnt-response pathway components we have shown distinguish early tumour cells from normal tissue. These early tumours are potentially unstable and susceptible to regression. If β-catenin-independent Wnt signalling is shown to contribute to progression, blockade of these events may provide a novel approach to preventing cancer development.The high levels of