WNT-5A, a key player in embryonic development and post-natal homeostasis, has been associated with a myriad of pathological conditions including malignant, fibroproliferative and inflammatory disorders. Previously, we have identified WNT-5A as a transcriptional target of TGF-β in airway smooth muscle cells and demonstrated its function as a mediator of airway remodeling. Here, we investigated the molecular mechanisms underlying TGF-β-induced WNT-5A expression. We show that TGF-β-activated kinase 1 (TAK1) is a critical mediator of WNT-5A expression as its pharmacological inhibition or siRNA-mediated silencing reduced TGF-β induction of WNT-5A. Furthermore, we show that TAK1 engages p38 and c-Jun N-terminal kinase (JNK) signaling which redundantly participates in WNT-5A induction as only simultaneous, but not individual, inhibition of p38 and JNK suppressed TGF-β-induced WNT-5A expression. Remarkably, we demonstrate a central role of β-catenin in TGF-β-induced WNT-5A expression. Regulated by TAK1, β-catenin is required for WNT-5A induction as its silencing repressed WNT-5A expression whereas a constitutively active mutant augmented basal WNT-5A abundance. Furthermore, we identify Sp1 as the transcription factor for WNT-5A and demonstrate its interaction with β-catenin. We discover that Sp1 is recruited to the WNT-5A promoter in a TGF-β-induced and TAK1-regulated manner. Collectively, our findings describe a TAK1-dependent, β-catenin- and Sp1-mediated signaling cascade activated downstream of TGF-β which regulates WNT-5A induction. In the WNT/Ca2+ signaling, binding of WNT ligands to FZD or non-FZD receptors activates calcium-dependent signaling molecules, including protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and nuclear factor of activated T-cell (NFAT), whereas the WNT/PCP pathway involves activation of the RhoA signaling or c-Jun N-terminal Kinases (JNKs) via small Rho-GTPases WNT-5A is a member of the Wingless/integrase 1 (WNT) family of secreted glycoproteins. There are 19 WNT ligands known in humans that act through 10 Frizzled (FZD) receptors, low-density lipoprotein receptor-related protein (LRP) 5/6 co-receptors and many non-FZD receptors, including ROR1, ROR2, RYK WNT-5A is a crucial signaling molecule which primarily acts through non-canonical WNT signaling and plays key roles in embryonic development and post-natal homeostatic processes Furthermore, WNT-5A has been very closely associated with fibrosis. Increased amount of WNT-5A is reported in lung fibroblasts of pulmonary fibrosis patients where it regulates proliferation and promotes cell viability 2+ concentration We have recently reported increased WNT-5A expression in asthmatic airway smooth muscle cells While our knowledge about the involvement of WNT-5A in various physiological and pathological processes is evolving rapidly along with the identification of novel inducers, the understanding of mechanisms regulating WNT-5A expression and homeostasis remains poor. In this study, we have investigated the molecular mechanisms involved in TGF-β-induced WNT-5A expression using airway smooth muscle cells as model system.TGF-β is a pleiotropic cytokine with functions as diverse as embryonic development and maintenance of adult tissue homeostasis to regulating stem cell renewal, cell fate determination and cellular proliferation TAK1, first identified as a mitogen-activated kinase kinase kinase (MAP3K) activated by TGF-β, is a critical regulator in inflammatory, immune and stress response signaling WNT-5A and demonstrate a link between TAK1, β-catenin and Sp1.Here, we investigated the molecular mechanisms involved in TGF-β-induced WNT-5A expression using airway smooth muscle cells as 1] airway smooth muscle cells are key structural and functional component of airways and major contributor of airway remodeling in asthma and 2] TGF-β upregulates WNT-5A expression in these cells. We examined the participation of various TGF-β-activated pathways and demonstrate that TAK1 via p38 and JNK mediates WNT-5A expression. Further, we determined an unanticipated role for β-catenin in WNT-5A expression and describe its regulation by TAK1. Finally, we identify Sp1 as the transcription factor for 1 and rat anti-WNT-5A antibody were from R&D systems . siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology . Rabbit anti-phospho-Thr183/Tyr185-SAPK/JNK antibody and rabbit anti-phospho-Thr180/Tyr182-p38 MAPK (D3F9) antibody were obtained from Cell Signaling Technology . Mouse anti-total β-catenin antibody was from BD Biosciences  and mouse anti-active β-catenin antibody (clone 8E7) was obtained from Millipore . Cycloheximide, IGEPAL CA-630, HRP-conjugated goat anti-mouse antibody and HRP-conjugated goat anti-rabbit antibody were obtained from Sigma . Human β-catenin and non-targeting siRNA were procured from Qiagen . X-tremeGENE siRNA and X-tremeGENE DNA HP transfection reagents were purchased from Roche Applied Science . LL-Z1640-2 was obtained from Bioaustralis . Y-27632 dihydrochloride, LY294002 hydrochloride, SB203580, SP600125 and Mithramycin A were from Tocris  and SIS3 and Bisindolylmaleimide I (BIM) were purchased from Calbiochem . All other chemicals were of analytical grade.Recombinant human TGF-βThree human airway smooth muscle cell lines, immortalized by human telomerase reverse transcriptase (hTERT) Airway smooth muscle cells were grown to ∼90% confluence in 6-well cluster plates and transfected with 200 pmol of specific siRNA in serum and antibiotic free DMEM with X-tremeGENE siRNA transfection reagent. Control transfections were performed using a non-targeting control siRNA. After 6 hours of transfection, medium was replaced with DMEM supplemented with antibiotics and ITS for a period of 42 hours before TGF-β stimulation.Airway smooth muscle cells grown to ∼90% confluence in 6-well cluster plates were transfected with 1 µg of mutant S33Y-β-catenin plasmid  q: amplification cycle number) method. The amount of target gene was normalized to the endogenous reference gene 18S ribosomal RNA (ΔCq). Relative differences were determined using the equation 2(−ΔΔCq). Primers used to analyze gene expression are: WNT-5A Fwd 5′- GGGTGGGAACCAAGAAAAAT -3′ and Rev 5′- TGGAACCTACCCATCCCATA -3′; TAK1 Fwd 5′- CTTGGATGGCACCTGAAG -3′ and Rev 5′- CAGGCTCTCAATGGGCTTAG -3′; Collagen IαI Fwd 5′- AGCCAGCAGATCGAGAACAT -3′ and Rev 5′- TCTTGTCCTTGGGGTTCTTG -3′; Fibronectin Fwd 5′- TCGAGGAGGAAATTCCAATG -3′ and Rev 5′- ACACACGTGCACCTCATCAT -3′; β-catenin Fwd 5′- CCCACTAATGTCCAGCGTTT -3′and Rev 5′- AATCCACTGGTGAACCAAGC -3′; CUTL1 Fwd 5′- GCTGTTGCTGGAGAAGAACC -3′and Rev 5′- GGTCTTTCCCTTTCCTCCTG -3′; TCF4 Fwd 5′-CGTAGACCCCAAAACAGGAA -3′and Rev 5′- TCCTGTCGTGATTGGGTACA -3′; ETS1 Fwd 5′- CCAATCCAGCTATGGCAGTT -3′and Rev 5′- TTCCTCTTTCCCCATCTCCT -3′; Sp1 Fwd 5′- GGAGAGCAAAACCAGCAGAC -3′ and Rev 5′- AAGGTGATTGTTTGGGCTTG -3′ and 18S rRNA Fwd 5′- CGCCGCTAGAGGTGAAATTC -3′and Rev 5′- TTGGCAAATGCTTTCGCTC -3′.Total RNA was extracted using the Nucleospin RNAII kit  as per the manufacturer's instructions. Equal amounts of total RNA were then reverse transcribed using the Reverse Transcription System . 1 µl of 1∶2 diluted cDNA was subjected to real-time PCR, which was performed with the Illumina Eco Personal QPCR System  using FastStart Universal SYBR Green Master (Rox) from Roche Applied Science . Real time PCR was performed with denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds and extension at 72°C for 30 seconds for 40 cycles followed by 10 minutes at 72°C. Real time PCR data was analyzed using the comparative cycle threshold  in consultation with earlier reports 7 airway smooth muscle cells were fixed in formaldehyde to final concentration of 1% for 10 minutes and then stopped by adding glycine. Cross-linked chromatin was digested using Micrococcal nuclease at 37°C for 20 minutes followed by a brief sonication to generate 200–500 bp DNA fragments. Sheared chromatin was incubated with anti-Sp1 (PEP2) X TransCruz reagent  or normal rabbit antibody (IgG) as negative control and precipitated using Protein G-agarose beads. Immunoprecipitated chromatin complexes were washed sequentially in Low- and High- salt wash buffers and protein-DNA cross-links were reversed in presence of Proteinase K at 65°C for 4 hours. DNA fragments were purified using the spin columns supplied in the kit as per recommendations. 2 µl of DNA from each sample was used as a template for PCR amplification. PCR was performed with denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds and extension at 72°C for 30 seconds for 40 cycles followed by 10 minutes at 72°C using primers designed to amplify the region encompassing putative Sp1 binding sites on WNT-5A promoter A Fwd 5′- ACAGGATCGCGTGGAAATCT -3′and Rev 5′- GAAGCTGCCCACCTCCTC -3′.ChIP analysis was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Agarose Beads) from Cell Signaling Technology  as per manufacturer's instructions. Briefly, 1×10Control and WNT-3A conditioned medium from L-cells were prepared as described previously The whole cell extracts were either prepared as described previously 3VO4, 1 mM PMSF and 1 mM β-glycerophosphate) on ice, scraped and collected in a microfuge. The collected lysate was further incubated at 4°C with constant rotation for 4 hours. Lysates were then cleared by centrifugation at 18000 g for 10 min at 4°C and supernatant was collected. Protein concentrations were measured using the BCA assay (Pierce) and 500 µg of protein lysate was incubated with 2 µg anti-Sp1 antibody overnight at 4°C. Immunocomplexes were then incubated with 30 µl of Protein A-agarose slurry for 4 hours with constant rotation at 4°C. Protein A-agarose-bound immunocomplexes were precipitated by centrifugation at 4000 g for 5 min at 4°C and washed three times with lysis buffer. Finally, 2X Laemmli buffer was added to the precipitates and heated for 5 min at 95°C. The heated lysates were cleared by centrifugation at 4000 g for 5 min, supernatant collected and stored at −20°C until further use.For co-immunoprecipitation assay, airway smooth muscle cells were washed twice with ice-cold PBS and lysed in 1% IGEPAL buffer  using enhanced chemiluminescence reagents and were quantified by densitometry using Genetools software.t-test, by 1-way ANOVA or by 2-way ANOVA, followed by Student-Newman Keuls or Bonferroni multiple comparisons test, where appropriate. Differences were considered to be statistically significant when p<0.05.Values reported for all data are represented as mean ± SEM. The statistical significance of differences between means was determined on log transformed data by Student's TGF-β activates multiple pathways, both SMAD-dependent and -independent, downstream of its receptor. We targeted key pathways to identify the signaling cascades involved in WNT-5A expression by TGF-β in airway smooth muscle cells. We observed that pharmacological inhibition of SMAD3 , Rho-associated protein kinase (ROCK) , PI3K , glycogen synthase kinase (GSK) -3  and PKC , failed to reduce WNT-5A induction by TGF-β . SurprisNotably, inhibition of TAK1 by LL-Z1640-2 attenuated WNT-5A mRNA expression in a dose-dependent manner with significant reduction at 0.5 µM and 1 µM by ∼75% and ∼91%, respectively . ConsistTo further validate the role of TAK1 in WNT-5A induction, we employed TAK1-specific siRNA. Transfection of airway smooth muscle cells with TAK1 siRNA significantly repressed TAK1 transcripts to ∼30% of the baseline expression in both the unstimulated and TGF-β-stimulated airway smooth muscle cells in comparison to non-targeting siRNA transfected cells . In agreWe have previously reported a role for WNT-5A in TGF-β-induced ECM production Collectively, our data suggest that TAK1 specifically mediates TGF-β-induced WNT-5A production.Next, we investigated the signaling mechanisms downstream of TAK1 activation which could be involved in WNT-5A induction by TGF-β in airway smooth muscle cells. TAK1 activates JNK and p38 pathways in multiple systems Our data therefore suggest that TAK1 mediates TGF-β-induced p38 and JNK kinases activation which can redundantly mediate the downstream effects of TAK1 on WNT-5A induction.de novo protein synthesis is involved in TGF-β-induced WNT-5A expression in airway smooth muscle cells. Interestingly, while the presence of cycloheximide increased basal WNT-5A mRNA abundance (fold-induction 2.2±0.26); it significantly attenuated TGF-β-induced augmentation in WNT-5A transcript levels by ∼44% . This S33Y-β-catenin mutant has a serine to tyrosine substitution at amino acid position 33 rendering it unphosphorylatable by GSK-3 and therefore resistant to proteasomal degradation. Transfection of airway smooth muscle cells with S33Y-β-catenin lead to enhanced expression of total β-catenin in the cell . InteresAs β-catenin stabilization is a hallmark of canonical WNT signaling activation, we hypothesized that canonical WNT signaling can also increase WNT-5A expression. To test this hypothesis, we stimulated airway smooth muscle cells with WNT-3A conditioned medium. Remarkably, WNT-3A conditioned medium led to a 2-fold induction in WNT-5A transcript levels in airway smooth muscle cells when compared to control conditioned medium .Our data therefore suggest a central role for β-catenin in WNT-5A induction.Having confirmed the role of β-catenin, we got interested in the link between TAK1 and β-catenin in WNT-5A expression. As both TAK1 and β-catenin are required for WNT-5A induction, we first investigated for possible cross-regulation. To address this, we studied β-catenin stability in the presence of LL-Z1640-2. Interestingly, we observed that the TGF-β-induced increase in total β-catenin abundance was significantly suppressed in the presence of LL-Z1640-2 by ∼68% .Next, we investigated whether p38 and JNK are involved in TAK1-mediated β-catenin regulation. Of note, while JNK inhibition had no effect, inhibition of p38 significantly attenuated TGF-β-induced total β-catenin protein abundance by ∼70% in comparison to TGF-β in airway smooth muscle cells . AccordiWe were intrigued by the contrasting results that while β-catenin is required for TGF-β-induced WNT-5A expression, the reduction in total β-catenin by p38 inhibition, though substantial, totally failed to affect WNT-5A transcript levels  and 4B. Collectively, our data suggest that TAK1 signaling mediates regulation of β-catenin via p38 and JNK.in silico analysis of both the human WNT-5A promoter A and B as described in the Materials and Methods section which predicted binding sites for various transcription factors on both the promoters A (We next sought to determine the transcription factor(s) employed by TGF-β to induce WNT-5A expression in airway smooth muscle cells. WNT-5A has two alternative promoters-A and B. To identify the potential transcription factors, we did moters A  and B  totally abrogated TGF-β-induced expression of WNT-5A mRNA . In line with the role of TAK1 in WNT-5A induction, the TGF-β-induced Sp1 recruitment to WNT-5A promoter A was abrogated in the presence of TAK1 inhibitor LL-Z1640-2 (We next performed chromatin immunoprecipitation (ChIP) assay and validated the direct binding of Sp1 to WNT-5A promoters. Consistent with the role of Sp1 in WNT-5A induction as deduced from Mithramycin A and Sp1 siRNA, we confirmed binding of Sp1 on to TGF-β . Of note-Z1640-2 WNT-5A promoter via TAK1 in response to TGF-β in airway smooth muscle cells.Our data, therefore, suggest that Sp1 is required for WNT-5A expression and is recruited to As we observed that both Sp1 and β-catenin are required for WNT-5A induction via TAK1, we sought to investigate the functional link between these findings. β-Catenin can function as transcriptional co-activator and partner with various transcription factors to regulate gene expression. We therefore determined whether β-catenin physically interacts with Sp1. Indeed, a co-immunoprecipitation assay using whole cell extracts from airway smooth muscle cells demonstrated that Sp1 associates with β-catenin . InteresIn summary, our data demonstrate that TGF-β promotes β-catenin/Sp1 interaction.In the present study, we have delineated the signaling mechanisms driving TGF-β-induced WNT-5A expression in airway smooth muscle cells. To the best of our knowledge, this is the first report describing a signaling cascade consisting of TAK1, β-catenin and Sp1 that regulates WNT-5A expression. We demonstrate that TAK1 activity is required for WNT-5A expression in response to TGF-β stimulation and provide evidence for the involvement of β-catenin in this process which, in turn, is regulated by TAK1 signaling. We further identify Sp1 as transcription factor for WNT-5A and demonstrate its interaction with β-catenin in airway smooth muscle cells. We provide evidence that Sp1 is recruited to the WNT-5A promoter in response to TGF-β, a phenomenon regulated by TAK1 activity. Collectively, our study identifies a novel pathway involved in WNT-5A regulation, thus, providing an understanding of mechanisms governing WNT-5A homeostasis.WNT-5A plays a key role in wide range of developmental and postnatal processes and derailed WNT-5A homeostasis has been widely implicated in myriad of pathological situations MAPKs including p38 and JNK are downstream effectors of TAK1 in many cell types TGF-β/SMAD constitutes the principle signaling axis in TGF-β responses β-Catenin, the canonical WNT signaling effector, constitutes an important component in TGF-β signaling in airway smooth muscle cells TGF-β engages a two pronged mechanism to increase the cytosolic abundance of β-catenin in airway smooth muscle cells- first, it inactivates GSK-3, the key upstream mediator of β-catenin degradation and second, it induces transcriptional upregulation of β-catenin Altered expression patterns of WNT-5A and β-catenin have been implicated in various disorders, for instance, fibrosis. Enhanced expression and increased nuclear localization of β-catenin have been shown in idiopathic pulmonary fibrosis (IPF) WNT-5A gene generates two very identical transcripts by utilization of alternative transcription start sites of which the corresponding upstream sequences are termed as promoter A and B WNT-5A promoter A has been suggested to be more active in human and murine fibroblasts in silico analysis of WNT-5A promoters which revealed multiple putative transcription factor binding sites on both the promoters. Our WNT-5A promoter screen predicted previously described transcription factor binding sites underlining its accuracy. Silencing of CUTL1 or ETS1 failed to affect WNT-5A induction in airway smooth muscle cells suggesting a cell-specific transcriptional program regulating WNT-5A expression. Our observations regarding involvement of β-catenin in WNT-5A induction lead us to target TCF4, the most common binding partner of β-catenin. However, TCF4 knock-down didn't effect WNT-5A induction in our system suggesting that β-catenin does not utilize TCF4 for mediating WNT-5A induction.Transcriptional upregulation of WNT-5A has been reported in several studies. The WNT-5A promoter have been predicted earlier WNT-5A promoter screen. We used Mithramycin A and specific siRNA to deduce the role of Sp1 in WNT-5A induction. Mithramycin A is a highly selective inhibitor of Sp1 which competes for DNA binding with Sp1 and attenuates its recruitment on promoters WNT-5A promoter in TGF-β-dependent manner. Furthermore, we identified TAK1 as upstream regulator of TGF-β-induced recruitment of Sp1 as LL-Z1640-2 treatment reduced Sp1 binding to WNT-5A promoter in airway smooth muscle cells. This is in contrast with earlier reports where TAK1 has been shown to negatively regulate Sp1 activity in keratinocytes and lung adenocarcinoma cells Sp1, a member of Specificity protein/Kruppel-like family of transcription factors, is ubiquitously expressed and involved in regulating expression of a wide array of genes starting from early embryonic phase and extending throughout the life span filamin A promoter il-10 promoter in human monocytes platelet-activating factor acetylhydrolase (PAF AH) promoter in murine and human immune cells urokinase-type plasminogen activator (uPA) gene promoter Sp1 activity is influenced by multiple post-translational modifications governing its DNA binding activity and protein stability Both β-catenin and Sp1 can associate with various transcription factors and co-activators in a cell- and stimulus-dependent manner to mediate their cellular responses. However, the interaction between β-catenin and Sp1 has been shown to be counteractive and indirect. For instance, constitutive activation of WNT/β-catenin signaling in mouse brain represses Sp1 target gene expression via upregulation of Sp5, a Sp1 repressor protein In airway smooth muscle cells, TAK1 mediates cell phenotype and cigarette smoke-induced inflammation. A study from our group has shown that TAK1-mediates PDGF induced activation of ERK1/2, leading to airway smooth muscle cell proliferation and reduction in contractile proteins In conclusion, our present study describes a novel signaling cascade comprising of TAK1, β-catenin and Sp1 in TGF-β-induced WNT-5A expression in airway smooth muscle cells. We deduce the molecular pathway regulating WNT-5A expression which can have implications in various physiological and pathological situations involving WNT-5A. Moreover, our study also provides a mechanistic insight intertwining TAK1, β-catenin and Sp1 which, perhaps, has a much wider applicability extending to other cell- and tissue types and processes involving these factors. Our data suggest that TAK1 regulates TGF-β-induced WNT-5A expression by two simultaneous but linked mechanisms – 1] it augments expression of β-catenin which, in turn, partners with Sp1, perhaps, finalizing a transcriptional complex and 2] it promotes binding of Sp1 transcriptional complex to WNT-5A promoter thereby allowing WNT-5A transcription. Interestingly, therapeutic tools for targeting TAK1 Figure S1Signaling cascades in TGF-β-induced WNT-5A expression. (A-E) Airway smooth muscle cells were either left unstimulated  or stimulated with TGF-β (2 ng/ml) in the presence or absence of SIS3 (3 µM), Y27632 (1 µM), LY294002 (3 µM), SB216763 (10 µM) or BIM (3 µM) for 24 hours. Expression of WNT-5A mRNA was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to vehicle basal. Data represent mean ± SEM of 3-8 independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to vehicle basal, # p<0.05, ## p<0.01, ### p<0.001 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.(TIF)Click here for additional data file.


This study was designed to assess the clinical value of magnifying endoscopy combined with EUS for estimating the invasion depth of colorectal tumors. We studied 168 colorectal adenomas and carcinomas that were sequentially examined by conventional endoscopy followed by magnifying endoscopy and EUS in the same session to evaluate invasion depth. Endoscopic images obtained by each technique were reassessed by 3 endoscopists to determine whether endoscopic resection  or colectomy  was indicated. The accuracy of differential diagnosis was compared among the examination techniques. The rate of correct differential diagnosis according to endoscopic examination technique was similar. The proportion of lesions that were difficult to diagnose was significantly higher for EUS (15.5%) than for conventional endoscopy and magnifying endoscopy. Among lesions that could be diagnosed, the rate of correct differential diagnosis was the highest for EUS (89.4%), but did not significantly differ among three endoscopic examination techniques. When it is difficult to evaluate the invasion depth of colorectal tumors on conventional endoscopy alone, the combined use of different examination techniques such as EUS may enhance diagnostic accuracy in some lesions. Endoscopic examinations have an important role in the differential diagnosis of benign and malignant colorectal tumors (adenomas and carcinomas), as well as in accurate estimation of the depth of invasion and selection of the treatment. In particular, “early” colorectal cancer with invasion confined to the mucosa or the submucosa is a borderline lesion for the selection of either endoscopic resection or colectomy. It is thus essential to accurately evaluate the depth of tumor invasion on the basis of endoscopic findings. The invasion depth of colorectal cancer is basically estimated on conventional endoscopy combined with chromoendoscopy, as needed. If the depth of tumor invasion is difficult to estimate on conventional endoscopy alone, however, additional examinations such as magnifying endoscopy to assess pit patterns and endoscopic ultrasonography (EUS) are performed. However, few studies have examined the extent to which these detailed examination techniques improve the accuracy of estimating the depth of invasion. We studied colorectal tumors that were sequentially examined by conventional endoscopy followed by EUS and magnifying endoscopy in the same session to evaluate invasion depth. The accuracy of evaluating the depth of tumor invasion was then compared among these endoscopic examination techniques to determine whether the additional use of in-depth procedures improved the accuracy of estimating the depth of tumor invasion, thereby facilitating the selection of treatment. From January 2002 through April 2007, we sequentially examined 168 colorectal tumors (166 patients) by conventional endoscopy, followed by magnifying endoscopy to evaluate the pit pattern, and EUS to estimate the depth of tumor invasion at the request of the patients' attending physicians. After endoscopic resection or surgical operation, the invasion depth of all lesions was determined histopathologically. Lesions that were difficult to assess on conventional endoscopy because of factors such as inadequate bowel preparation and high-grade intestinal peristalsis were excluded. μm (slight invasion), and 39 had an invasion depth of 1000 μm or greater (massive invasion). The most common lesion location was the rectum (74 lesions), followed by the sigmoid colon, transverse colon, and ascending colon. The macroscopic type of the tumors was classified according to the Paris endoscopic classification [The invasion depth of the colorectal tumors was classified according to the Japanese classification of colorectal carcinoma, issued by the Japanese Society for Cancer of the Colon and Rectum . There wfication  and systFor bowel preparation before colonoscopy, oral intestinal lavage (polyethylene glycol) was mainly performed. As premedication, scopolamine butylbromide (10 mg) or glucagon (1 mg) was given intramuscularly to suppress intestinal peristalsis. We used colonoscopy with magnifying function . All colonoscopic examinations, including magnifying endoscopy and EUS, were performed by a single endoscopist who had at least 20 years of experience in colonoscopy. The number of years of experience in detailed evaluations was 17 for EUS and 7 for magnifying endoscopy. During conventional endoscopy, most lesions were also examined by chromoendoscopy, performed by spraying the mucosa with 0.2% indigo carmine dye. Before colonoscopy, patients were given a detailed explanation of the examination objectives, methods, and possible complications. Written-informed consent for colonoscopic examination was obtained from all patients. After the completion of conventional endoscopy, the intestine near the tumor was filled with deaerated water that had been warmed to about body temperature, and EUS was performed to evaluate the depth of invasion. An ultrasound probe with a frequency of 20 MHz  or a 3-dimensional ultrasound probe with a frequency of 20 MHz  was used. On EUS, the normal wall of the colon is basically visualized as a 5-layer structure. From the luminal side, the hyperechoic first layer and hypoechoic second layer correspond to the mucosa, the hyperechoic third layer to the submucosa, the hypoechoic fourth layer to the muscularis propria, and the hyperechoic fifth layer to the subserosa or serosa (adventitia) . The invI pit patterns were further classified as mildly irregular or severely irregular on the basis of structural and arrangement irregularities of pits, pit density, and stromal staining between pits. On the basis of the results of previous studies examining the relation between pit patterns and tumor invasion depth [L, IV, or VI mildly irregular pit patterns were considered to be indicated for endoscopic resection . Tumors with type VI severely irregular or type VN pit patterns were considered to be indicated for colectomy . After the completion of conventional endoscopy and EUS, magnifying colonoscopic examination was performed to evaluate pit patterns. The tumor was washed with water to remove any mucus, sprayed with 0.2% indigo carmine dye, and examined by magnifying endoscopy at a magnification of 80 to 100 times to evaluate pit patterns. If pit patterns could not be accurately evaluated on indigo carmine staining alone, 0.05% crystal violet stain was concurrently applied. Pit patterns of the colorectal tumors were evaluated according to Kudo's classification . Type VIon depth –10, tumoThree endoscopists who had no information on the histopathological findings of tumors reassessed the invasion depth of the colorectal tumors. All 3 endoscopists had at least 10 years of experience in colonoscopy and at least 5 years of experience in EUS and magnifying endoscopy. The number of years of experience did not differ appreciably according to examination technique among the 3 endoscopists. Conventional endoscopic, magnifying endoscopic, and EUS images were reviewed for lesions presented in random order. The lesions were divided into 2 groups on the basis of the estimated depth of invasion: lesions for which endoscopic resection was indicated  and those for which colectomy was indicated . If the evaluation made by each endoscopist was consistent with the histopathological diagnosis for the resected specimen, the endoscopic diagnosis was classified as a correct diagnosis. If the evaluation did not agree with the histopathological diagnosis, the endoscopic diagnosis was classified as a misdiagnosis. If the invasion depth was difficult to evaluate on the basis of the presented endoscopic images, the endoscopic diagnosis was classified as a misdiagnosis. If at least 2 endoscopists made the same diagnosis, that diagnosis was considered the final diagnosis for the lesion. The accuracy of differential diagnosis and the frequency of difficult-to-diagnose lesions were retrospectively compared among conventional endoscopy, magnifying endoscopy, and EUS. For lesions that were considered by all 3 endoscopists to have an assessable invasion depth, the diagnostic accuracy was compared among the examination techniques. Our institutional review board approved the study protocol.P values of less than 0.05 were considered to indicate statistical significance. StatView software  was used for statistical analysis.Numerical data are expressed as means ± standard deviation. The chi-square test and Fisher's exact test were used to compare frequencies among groups. The rate of correctly diagnosing lesions for which endoscopic resection was indicated  was 83.2% (104/125 lesions) on conventional endoscopy, 83.2% (104/125) on magnifying endoscopy, and 81.6% (102/125) on EUS. The rate of correctly diagnosing lesions for which colectomy was indicated  was 76.7% (33/43 lesions) on conventional endoscopy, 79.1% (34/43) on magnifying endoscopy, and 79.1% (34/43) on EUS. The overall accuracy of differential diagnosis was similar for conventional endoscopy (81.5%), magnifying endoscopy (82.1%), and EUS (81.0%) .  The percentage of lesions that were evaluated by at least 1 of the 3 endoscopists to be difficult to diagnose on endoscopic images was 3.0% for conventional endoscopy, 4.8% for magnifying endoscopy, and 15.5% for EUS . The freThe number of lesions for which the invasion depth was considered assessable by all 3 endoscopists was 163 for conventional endoscopy, 160 for magnifying endoscopy, and 142 for EUS. The rate of correct diagnosis among lesions with assessable endoscopic images was highest for EUS (89.4%), followed by magnifying endoscopy (85.6%) and conventional endoscopy (82.8%). The diagnostic accuracy of EUS was the highest, but did not significantly differ among three endoscopic examination techniques . N pit pattern) in part of the tumor  should then be selected.  Among colorectal cancers, mucosal cancer can be resected endoscopically because there is no risk of metastasis. In particular, the recent development of techniques such as endoscopic submucosal dissection and endoscopic piecemeal mucosal resection has enabled the endoscopic resection of even large lesions –13. Subm lesions . In contThe invasion depth of colorectal cancer is generally evaluated on endoscopic examination. The basic procedure is conventional endoscopy. The depth of invasion is estimated on the basis of tumor diameter and macroscopic findings, as well as other characteristics of the tumor surface, such as a cracked or distended appearance, friability, spread, and fold convergence , 19. As S, IIIL, and IV with adenomatous polyps; and type V with cancer. Type V pit patterns can be further classified into type VI and type VN. Intype VN, the pit structure has been lost and is amorphous, suggesting submucosal cancer with massive invasion [I is subclassified into type VI with mild irregularity and type VI with severe irregularity on the basis of findings such as narrowed pit lumens, irregular margins,unclear outlines, and decreased or absence of stromal staining between pits [I pit patterns were subdivided into type VI with mild irregularity and type VI with severe irregularity. The presented tumors were then reevaluated to decide whether endoscopic resection  or colectomy  was indicated. The evaluation of pit patterns on magnifying endoscopy is useful for differentiating neoplastic from nonneoplastic colorectal polyps , 24, as invasion . Type VIeen pits . The foreen pits –10. In oMany studies have reported that EUS is useful for estimating the invasion depth of colorectal cancer –30. In pPit patterns on magnifying endoscopy and EUS findings have been confirmed to be useful for evaluating the invasion depth of colorectal tumors. However, few studies have compared the diagnostic accuracy of conventional endoscopy, magnifying endoscopy, and EUS in large numbers of lesions. Some studies have reported that the diagnostic accuracy of EUS is superior to that of magnifying endoscopy , 32, wheThe present study compared the diagnostic accuracies of conventional endoscopy, magnifying endoscopy, and EUS by reviewing endoscopic images to estimate the invasion depth of mainly early colorectal tumors and thereby select the treatment method. Because the macroscopic appearance and disease stage of tumors can differ on endoscopic examinations performed at different times, we only studied lesions that were sequentially examined by conventional endoscopy, magnifying endoscopy, and EUS in the same session. Moreover, to ensure that invasion depth was objectively evaluated, 3 endoscopists who were blinded to the histopathological diagnosis of the tumors reviewed the endoscopic images.P = 0.0978). Among gastrointestinal endoscopic examinations, the depth of invasion is estimated on the basis of changes of the tumor surface on conventional endoscopy and magnifying endoscopy. In contrast, with EUS the entire lesion can be visualized in vertical slices, allowing the invasion depth to be objectively evaluated on the basis of changes in wall structure. When only lesions with interpretable endoscopic images as assessed by all 3 endoscopists were evaluated, the diagnostic accuracy of EUS was the highest, but did not differ from that of magnifying endoscopy and tended to be higher than that of conventional endoscopy (However, the diagnosis of colorectal tumors on EUS has several limitations. Histologically, the presence of inflammation or fibrosis around the tumor invasion front may lead to overestimation of the depth of invasion . In addiThis study compared the accuracy of estimating the invasion depth of mainly colorectal LST among 3 different endoscopic techniques. The invasion depth was correctly diagnosed on conventional endoscopy combined with chromoendoscopy for more than 80% of lesions. The relatively high diagnostic accuracy of conventional endoscopy may be attributed to the following factors: a high proportion of lesions were adenomas and mucosal cancers, for which it is relatively easy to estimate the invasion depth; the endoscopists who performed the examinations and estimated the invasion depth were well experienced. Because many conventional endoscopic findings used to evaluate invasion depth are subjective, diagnostic accuracy may largely depend on the knowledge and experience of the endoscopist. A previous study has reported that the accuracy of estimating the invasion depth of colorectal cancer on conventional endoscopy is negatively affected if the examination is performed by an inexperienced endoscopist .I pit patterns [Among in-depth evaluations of colorectal tumors, the assessment of pit patterns on magnifying endoscopy, especially the classification of type Vpatterns , is ofte


The NF-κB growth pathway is constitutively activated in many cancers but its activation mechanism is unclear in most cases. We show that PHLPP2 interacts with IKKβ kinase, decreases its phosphorylation and the subsequent NF-κB activation in cancer cells. PHLPP2 is progressively lost in glioma and colorectal cancer and acts as a bona fide tumor suppressor, depending on IKKβ expression in cells. Physiologically, IKKβ activation by growth factors requires the formation of the Bcl10-MALT1 ubiquitin-ligase complex leading to NEMO/IKKγ non-degradative ubiquitination and IKKβ phosphorylation. PHLPP2 opposes the formation of this complex through interaction with Bcl10 and competitive displacement of MALT1 from Bcl10. Conversely, PHLPP2 loss enhances Bcl10-MALT1 complex formation, NEMO ubiquitination and subsequent IKKβ phosphorylation, resulting in increased NF-κB-dependent transcription of multiple target genes. Our results reveal PHLPP2 as a new biomarker of cancer progression, and implicate it as major negative regulator of NF-κB signaling. Studies of signaling pathways controlling cancer cell proliferation, migration and therapy resistance enabled better diagnosis and treatment by the discovery of progression markers and therapeutic targets. The transcription nuclear factor κB (NF-κB) pathway is one of the major pathways activated in cancer that promotes transformation, survival, proliferation, metastasis and chemoresistance of most types of cancer cells . In unstThe homologous pleckstrin-homology (PH)-domain leucine-rich-repeat protein phosphatases PHLPP1-2) were recently described as survival/proliferation suppressors in various cancers  were rec. InitialWe examined PHLPP2 in glioma and colorectal cancer (CRC), and documented its progressive loss. By undertaking a convergent systems approach to detect PHLPP2-associated proteins and PHLPP2-regulated proliferation/survival pathways, we identified PHLPP2 as a constitutive negative regulator of NF-κB signaling, through interaction and inhibition of IKKβ.An initial analysis of PHLPP2 expression in tumors of increasing grade was performed on 21 glioma samples and showed decreased PHLPP2 expression in glioblastoma compared to lower grade gliomas Fig.. SurvivaTo establish PHLPP2 as bona fide tumor suppressor, we examined its effect on tumor growth and invasion. PHLPP2 expression was inversely correlated with cell proliferation in a panel of five glioblastoma lines . Stable To identify the signaling pathway controlled by PHLPP2, we explored putative interacting proteins by LC-MS/MS following immunoprecipitation of endogenous PHLPP2 from D54 cells . In PHLPWe confirmed these findings with overexpressed Myc-PHLPP2 or untagged PHLPP1 splice isoforms and FLAG-tagged IKKα and IKKβ wild-type or kinase-dead (KD) forms in 293T cells . WhereasWe examined the influence of PHLPP2-IKKβ in proliferation. IKKβ knockdown led to dose-dependent decreased proliferation Fig., effect The NF-κB canonical pathway is upregulated by various stimuli that lead to IKKβ activation by phosphorylation on Ser177 and Ser181 , 10. MorAlternatively, PHLPP2 knockdown by two independent shRNAs induced higher and more sustained IKKβ phosphorylation than control shRNA in two different cell lines Fig.. ReconstThe progressive PHLPP2 loss in tumors Fig. promptedIKKβ is phosphorylated by either TAK1 or MEKK3 kinases. NEMO, which directly associates with IKKβ, connects IKKβ to its kinase either by binding to nondegradative K63-linked polyubiquitin chains or by being itself polyubiquitinated with K63-linked chains. The complex inducing NEMO K63-linked-polyubiquitination is formed of Bcl10 and mucosa-associated lymphoid-tissue-1 (MALT1) protein, and is absolutely required for IKKβ activation following PMA stimulation -14. BecaThe phosphorylation by IKKβ of the p65 NF-κB subunit on Ser536 is required for efficient transcriptional activity of NF-κB and is commonly used as marker for increased NF-κB activity in tumor samples . The levBcl10 or MALT1 rearrangements in MALT lymphomas, or mutations, polymorphisms and deletion of IκBα-encoding NFΚBIA gene in lymphoid malignancies but also in glioblastoma and CRC [With the advent of personalized cancer therapy, the discovery of markers for disease staging, progression and response to therapy is front line. We characterized PHLPP2 as a marker of tumor progression in two cancer types, glioma and CRC, and linked its tumor suppressor functions to the inhibition of the NF-κB pathway. In somatic cancers, the activation of NF-κB is common, but only some cases can be explained by known genetic alterations, such as  and CRC , 17. In The control of IKKβ by PHLPP2 does not appear to involve direct dephosphorylation of IKKβ by PHLPP2, but rather relies on inhibition of NEMO ubiquitination by direct displacement of MALT1 from Bcl10 Fig.. As NEMOThe full significance of the constitutive interaction between PHLPP2 and IKKβ is not entirely clear. This interaction could function as a PHLPP2 reservoir, as forced IKKβ knockdown results in MALT1 displacement from Bcl10 most likely by PHLPP2 shift into the complex. However, it is possible that PHLPP2 may directly mask the phosphorylation sites on IKKβ or, alternatively, function as an anchor that brings other components into the larger IKK activation complex. PHLPP2 has been shown to interact with three deubiquitinases that have not been involved so far in NF-kB activation . FurtherIn conclusion, the identification of PHLPP2 as a bona fide growth and invasion suppressor through inhibitory molecular interactions within the IKKβ and MALT1-Bcl10 complexes clarifies the contribution of PHLPP2 to tumor progression and provides a mechanism for the ubiquitous activation of the NF-κB pathway in tumors.Glioma frozen samples comprise grade II oligodendroglioma, grade III anaplastic astrocytoma, oligoastrocytoma or oligodendroglioma, and grade IV glioblastoma. All patients were recorded in M.D. Anderson Cancer Center between 1991 and 2011. CRC frozen specimens with pathologist annotations as normal, deep tumor (carcinoma), and tumor-edge (adenoma), and paraffin-embedded specimens were obtained from the CRC Bank at M.D. Anderson Cancer Center. Surgeries were performed between 1992 and 2007, and no patient received prior therapy. These samples and the paraffin-embedded glioma tissue microarray (TMA) were previously described , 19, 20.TMA immunohistochemistry with PHLPP2 antibody  and deconvolution immunoflorescence with PHLPP2  and IKKβ  antibodies, were performed as described . The ort293T, HELA and glioblastoma cell lines LN229, LN18, D54, U251 and A172 were authenticated by the Cell Line Fingerprinting Core of Brain Tumor Center at M.D. Anderson Cancer Center using short tandem repeat profiling with GenomeLab Human Primer Set , as described [Cell lysis, immunoprecipitation immunoblotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were described , 23. AntThe NF-κB-driven luciferase-reporter assay was described .6 control or PHLPP2-depleted LN229 cells were grown to 60-70% confluence, serum-starved for 16 h, and treated or not with 200ng/ml phorbol 12-myristate 13-acetate (PMA)  for 5 h. Total RNA was isolated by using RNeasy Mini Kit , according to the manufacturer's instructions. 1¼g total RNA was reverse transcribed, using an RT2 First Strand Kit (SA Biosciences). The resulting cDNA was subjected to SYBR-Green based quantitative PCR (SA Biosciences) following manufacturer's protocol. RT2 Profiler PCR 96-well arrays were run on a Chrom4 system for real-time PCR detection (Biorad Laboratories). Data analysis was performed using the manufacturer's integrated web-based software package using cycle threshold (Ct)-based fold-change calculations.Based on commercially-available quantitative real-time PCR microarrays from SA Biosciences, Frederick, MD, we designed a growth pathway transcription target microarray  customizKaplan-Meier survival analysis using Log-rank (Mantel-Cox) test was plotted with GraphPad Prism program . ImageJ program was used for densitometric analysis . Data are representative of at least two or three independent experiments with similar results. Silencing data were derived from independent sets of lentiviral/retroviral infections with two different shRNAs. Numerical data were examined for normality of distribution and expressed as mean±SEM, unless mentioned otherwise. Two-tailed t-test with or without Welch's correction for variances significantly different and one-way analysis of variance were used to analyze the differences between groups. Statistical significance was considered for P<0.05. Confidence intervals for all tests were 95%.


The adult cerebellum is composed of several distinct cell types with well defined developmental origins. However, the molecular mechanisms that govern the generation of these cell types are only partially resolved. Wnt/β-catenin signalling has a wide variety of roles in generation of the central nervous system, though the specific activity of this pathway during cerebellum development is not well understood. Here, we present data that delineate the spatio-temporal specific pattern of Wnt/β-catenin signaling during mouse cerebellum development between E12.5 and P21. Using the BAT-gal Wnt/β-catenin reporter mouse, we found that Wnt/β-catenin activity is present transiently at the embryonic rhombic lip but not at later stages during the expansion of cell populations that arise from there. At late embryonic and early postnatal stages, Wnt/β-catenin activity shifts to the cerebellar ventricular zone and to cells arising from this germinal centre. Subsequently, the expression pattern becomes progressively restricted to Bergmann glial cells, which show expression of the reporter at P21. These results indicate a variety of potential functions for Wnt/β-catenin activity during cerebellum development. The cerebellum forms as a result of a highly regulated programme of cell specification, proliferation, differentiation and migration (reviewed in The rhombic lip gives rise to the entire complement of glutamatergic neurons that populate the IGL. The first glutamatergic neurons born are the projection neurons of the deep cerebellar nuclei (DCN). In the mouse these arise between embryonic day (E) 10.5 and E12.5 Distinct from the rhombic lip, the ventricular zone gives rise to all cerebellar cells of the γ-aminobutyric acid (GABA)ergic, and glial lineages. The first of these, the Purkinje cells, are born from E10.5 then migrate radially towards the pial surface of the cerebellum and settle as a distinct monolayer of cells around the time of birth (E19.5-E20) −/−Wnt1 mutant mice completely lack or exhibit a severely underdeveloped cerebellum APC that constitutively activate Wnt/β-catenin signalling have a greatly elevated risk of developing medullobastoma in vivo evidence supports this These tightly coordinated developmental processes rely on the spatio-temporal specific activity of several key signalling pathways. The sonic hedgehog (Shh) signalling pathway, for example, is the main mitogenic factor driving GPC proliferation within the EGL In order to investigate possible roles played by Wnt/β-catenin signalling during development of the cerebellum, we used the BAT-gal reporter transgenic mouse strain lacZ gene under the control of β-catenin/T cell factor (TCF) responsive elements LacZ transgene) from E12.5 to E14.5 in cerebellum sections from BAT-gal+ embryos. β-galactosidase expression was observed in a progressively restricted pattern  mirror those of the LacZ expression.Since β-galactosidase protein is a stable protein and can persist in tissue after dization . The expdization , althougin situ hybridisation for Axin2. Axin2 encodes a negative feedback inhibitor of the Wnt/β-catenin signalling pathway. It is a direct target of TCF/LEF-mediated transcription and is therefore widely used as a readout of Wnt/β-catenin signalling Axin2  indicating that the β-gal+ cell types identified at that late stage may no longer be responding to a Wnt/β-catenin signal and could be maintaining detectable levels of the β-galactosidase protein from when they last transduced a Wnt/β-catenin signal.We next examined patterns of BAT-gal reporter expression at later postnatal stages. By P5, all of the layers that make up the mature cerebellum  can be identified - along with the transient EGL. We found β-gal+ cells in each of these layers, except for the EGL . A similacZ mRNA , 4B, furAs Wnt/β-catenin signalling is commonly associated with the control of cell proliferation, we wanted to determine whether the BAT-gal expression pattern correlated with proliferation between P5 and P10 (the most highly proliferative period of cerebellar development). We therefore performed double immunofluorescence staining for β-galactosidase and PCNA. A number of PCNA+ cells were seen in each layer at P5 , becominWe next sought to identify the specific cell type(s) within the postnatal cerebellum in which Wnt/β-catenin signalling is active. The locations of the β-gal+ cell population identified at each postnatal stage examined suggested a number of possible cell types, including interneuron and glial progenitors within the WM, granule cells, astrocytes, oligodendrocytes and interneurons within the IGL, Purkinje cells and Bergmann glia within the PCL and interneurons within the ML. To determine the identity of the β-gal+ cells we performed double immunofluorescence experiments between β-galactosidase and a number of marker proteins known to label specific cerebellar cell types.The transcription factor Pax2 is expressed in committed cerebellar interneurons after their exit from the ventricular zone and prior to their terminal differentiation The β-gal+ cells in the IGL, PCL and ML could also be migratory post-mitotic granule cells, exiting the EGL towards their final destination in the IGL. However, double immunofluorescence between β-galactosidase and NeuN, a marker for post-mitotic granule cells Many β-gal+ cells were clearly localised to the PCL, suggesting the possibility that Purkinje cells may be responding to Wnt/β-catenin signalling. However, no colocalisation was observed between β-galactosidase and calbindin, a marker that clearly identifies Purkinje cells from P10, at either P10  or P21 . InteresTo determine whether Wnt/β-catenin signalling indeed marks a population of glial cells, we performed double immunofluorescence with s100β, a marker for Bergman glia and other astrocytes from P10 In this study we have investigated the distribution of Wnt/β-catenin signalling during development of the cerebellum from E12.5 to P21 primarily using the BAT-gal Wnt reporter mouse strain Our experiments revealed expression of the BAT-gal reporter at the rhombic lip at E12.5 and early EGL at E14.5. The rhombic lip gives birth to projection neurons of the deep cerebellar nuclei from E10.5 to E12.5 Wnt1 at the rhombic lip and at the isthmus Wnt1 leads to a severe developmental phenotype of the cerebellum, most likely due to a failure to maintain the isthmus Wnt1, and its proven role as a key signalling molecule in this area, it is possible that Wnt1 activity is responsible for the active Wnt/β-catenin signalling at the embryonic isthmus and rhombic lip identified in our experiments. However, it remains to be established whether additional Wnt genes are expressed in this area.Consistent with this, a number of studies have identified expression of While active Wnt/β-catenin signalling was observed in the early migrating GPCs at E14.5 , this waAdditionally, the absence of NeuN expression in any of the β-gal+ cells observed from P5-P21 demonstrates that Wnt/β-catenin signalling is also not active in the migration of terminally differentiated GCs from the EGL to the IGL. , 4E, 5D.At E18.5 and P1, many β-gal+ cells were seen in the ventricular zone , althougFurther to the identification of β-gal+ cells at the ventricular zone, we also observed a population of β-gal+ cells within all other layers of the cerebellum (excluding the EGL) from E18.5 through to P21. Lack of colocalisation between β-galactosidase and Pax2 , 4E, 5C,LacZ mRNA identified at all stages except for P21 supports a potential role for Wnt/β-catenin signalling during development, and would suggest that residual β-galactosidase protein has been identified at P21.The remaining alternative is that the β-gal+ cell population identified within the developing cerebellum are glia. Oligodendrocytes are thought to arise from extra-cerebellar tissue Interestingly, the lack of β-galactosidase expression at the ventricular zone at E14.5 indicates that Wnt/β-catenin signalling is not involved in the birth of the Bergmann glia but may be potentially involved in its further development and maturation. This is consistent with the postnatal dynamic transformation of Bergmann glia alongside dendritogenesis and synaptogenesis of Purkinje cells Axin2 expression in the lower rhombic lip  and by the Home Office on 6th November 2008. Animal husbandry was in accordance with the UK Animals (Scientific Procedures) Act 1986 regulations. To minimise animal suffering, pregnant dams were culled by cervical dislocation under terminal anaesthesia according to the Code of Practice for Humane Killing of Animals under Schedule 1 to the Animals (Scientific Procedures) Act 1986 issued by the Home Office. The day the vaginal plug was detected was designated E0.5 and the day of birth as P0. BAT-gal mice were maintained on a C57BL/6J genetic background and were genotyped as described previously in situ hybridisation was cryoprotected in 30% sucrose/PBS overnight before embedding in 30% sucrose/OCT (1∶1) and snap freezing. Frozen sections were cut on a cryostat at 14 µm. At least two animals were analysed at each age.Embryos were collected at E12.5 and E18.5 and pups between P1 and P21. Whole E12.5 embryos, heads from E14.5 embryos and brains dissected from E18.5 embryos were immersion fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) overnight at 4°C. P1, 5, 10 and 21 pups were anaesthetised with Avertin and transcardially perfused with PFA, followed by tissue dissection and overnight immersion fixation in fresh PFA. Embryonic and postnatal tissue for immunohistochemistry and immunofluorescence were all processed following standard conditions Immunohistochemistry and immunofluorescence were performed according to standard protocols. Antigen retrieval was achieved by microwaving sections at full power in 10 mM citrate buffer (pH6.0) for five minutes followed by 15 minutes at medium power. Primary antibodies were rabbit anti-β-galactosidase , mouse anti-β-galactosidase , mouse anti-PCNA , mouse anti-Pax2 , mouse anti-NeuN , mouse anti-Calbindin , mouse anti-s100β . Secondary non-fluorescent antibodies were biotinylated anti-rabbit IgG .For single immunohistochemistry experiments the dark brown signal was revealed after incubation with the ABC kit (Vector), followed by a diaminobenzidine (DAB) and hydrogen peroxide reaction using the DAB detection kit (Vector). For double immunofluorescence experiments, all primary antibodies were detected using goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies conjugated to Alexa fluor 568 or 488 dyes  respectively. Appropriate controls were used in all cases by incubating sections with all but the primary antibodies. No staining was observed under these conditions.In situ hybridisation on frozen section was performed as described previously LacZ and Axin2 antisense riboprobes A Leica brightfield microscope connected to a Leica DFC 480 digital camera was used to capture images of DAB labelled sections. A Leica brightfield microscope connected to a Leica DFC360Fx camera was used to capture images of fluorescently labelled sections.


Previously, we found that β-galactoside α2,6-sialyltransferase , an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration.We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA.We found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by BACE 1 inhibited integrin β1 sialylation and migration by Golgi-anchored form of ST6Gal I.Our results suggest that soluble ST6Gal I, possibly in cooperation with the Golgi-bound form, may participate in cancer progression and metastasis prior to being secreted from cancer cells. ST6Gal I  GlcNAc: α2,6 sialyltransferase) is an important glycosyltransferase that adds a sialic acid residue to the terminal position on N-linked oligosaccharides ,4 GlcNAc. Recent Metastasis represents an obligatory step in cancer progression. A variety of molecules contribute to cancer progression and metastasis , and manβ-site amyloid precursor protein-cleaving enzyme (BACE) is a membrane-bound aspartic protease that cleaves the amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease ,28. ImpoIn the present study, we examined IR-induced cleavage and solubilization of ST6Gal I, which is released into the cell culture media of colon cancer cell lines, and sought to identify the protease involved in cleaving ST6Gal I after exposure to IR. We found that BACE1 could be the secretase responsible for IR-induced cleavage of ST6Gal I, and showed that BACE1 mediated cleavage of ST6Gal I decreased ST6Gal I -mediated cancer cell migration. Additionally, the soluble form of ST6Gal I possesses sialyltransferase enzymatic activity, but unlike the Golgi-associated form, did not sialylate integrin β1 to a significant degree, suggesting that soluble ST6Gal I, possibly in cooperation with the Golgi-bound form, may participate in cancer progression and metastasis before being secreted from cancer cells, independent of intergrin β1-sialylation.SW480 and SW48 human colorectal carcinoma cells were grown in RPMI media supplemented with heat-inactivated 10% fetal bovine serum and antibiotics. CT26 mouse colorectal carcinoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum and antibiotics.For transient transfection experiments, expression constructs of C-terminally Flag-tagged wild-type  ST6Gal I and N-terminally deleted (HA-tagged) ST6Gal I  were created as described previously . An N-te137Cs γ-ray source  at a dose rate of 3.81 Gy/min.Cells were exposed to γ-irradiation using a Sambucus Nigra lectin; Vector Laboratories, Burlingame, CA, USA), and protein-lectin complexes were precipitated with avidin-coated protein A-agarose (Sigma).Protein levels were detected using the following commercial antibodies: anti-integrin β1 ; anti-phospho-p130CAS, anti-phospho-Src, anti-HA, and anti-Myc ; anti-Flag (Sigma); and anti-ST6Gal I and anti-BACE-1 . For detection of sialylated proteins, cell lysates were incubated with biotinylated SNA (Maackia amurensis (FITC-MAA) lectin  for detection of α2,6 and α2,3 sialylation, respectively. After staining, fluorescence intensity was analyzed by fluorescence-activated cell sorting (FACS).Cells were detached with trypsin/EDTA at the indicated times and stained with fluorescein isothiocyanate (FITC)-conjugated SNA (FITC-SNA) or Total RNA was isolated with TRI reagents (Molecular Research Center), and reverse transcription was done using Omniscript transcriptase (Qiagen) under the following thermocycling conditions: 30 cycles of 95°C for 5 min (denaturing), 60°C for 30 s , and 72°C for 30 seconds (extension). The primer sequences used were as follows: human BACE1, 5'-GGT GGA GAT CAA TGG ACA GG-3' (sense) and 5'-CGT GGA TGA CTG TGA GAT GG-3' (antisense); mouse Bace1, 5'-GCA GAC CCA CAT TCC GAA CA-3' (sense) and 5'-GCC ACT GTC CAC GAT GCT CTT-3' (anti-sense); and GAPDH, 5'-CAT GGA GAA GGC TGG GGC TCA TTT-3' (sense) and 5'-CGC CAG TAG AGG CAG GGA TGA TGT-3' (antisense).Cell migration assays were performed using a Boyden chamber as previously described . Cells wCell culture media were concentrated using Centricon centrifugal filter devices  and then analyzed for soluble ST6Gal I levels using an α2,6 sialyltransferase sandwich enzyme-linked immunosorbent assay (ELISA) kit , according to the manufacturer's protocol.t-test for comparisons between two means. The null hypothesis was rejected in cases where p-values were < 0.05.Data are expressed as means ± standard deviations (SDs). Statistical significance was determined using Student's -/-) by transiently transfecting these cells with expression vectors for Golgi-anchored  of ST6Gal I and exposing them to IR. As shown in Figure In our previous study, we found that IR exposure induces ST6Gal I cleavage and produces a soluble form of ST6Gal I in SW480 colon cancer cells . To suppIt was previously demonstrated that replacement of ST6Gal I residues Leu37 and Lys40 with alanine significantly decreases BACE1-mediated cleavage of ST6Gal I . TherefoY410) and sialylation of integrin β1 to a greater degree than did the WT form. Next, we further examined the effects of BACE1 on ST6Gal I cleavage by co-transfecting SW480 cells with BACE1 and WT or L37A/K40A forms of ST6Gal I. These experiments showed that the Golgi-anchored WT proform was decreased to a greater extent by co-transfection of BACE1 compared with the non-cleavable L37A/K40A form  form of p130CAS  grant funded by the Korean government , and by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs (grant code: A100627). This work was also supported by the Ewha Global Top5 Grant 2011 of Ewha Womans University.


Multidrug resistance (MDR) is an unfavorable factor causing the failure of treatments against cancer cells. The most important and thoroughly studied mechanism involved in MDR is the active efflux of chemotherapeutic agents through membrane drug transporters. There is growing evidence that Norcantharidin (NCTD), a water-soluble synthetic small molecule derivative of naturally occurring cantharidin from the medicinal insect blister beetle , is capable of chemoprevention and tumor inhibition. We summarize investigations into the modulation of self-renewal pathways and MDR in CSCs by NCTD. This review may aid in further investigation of using NCTD to develop more effective strategies for cancer treatment to reduce resistance and recurrence.Cancer stem cells (CSCs) existing in human cancers have been demonstrated to be a major cause of cancer treatment resistance, invasion, metastasis, and relapse. Self-renewal pathways, Wnt/ After developing resistance to a single drug or a class of drugs, cancer cells show cross-resistance to other functionally and structurally unrelated drugs, causing the failure of treatments against cancer cells . This phMylabris phalerata Pallas) [in vivo but not granulocytopenia induced by most chemotherapeutics [Norcantharidin , a water-soluble synthetic small molecule, is a demethylated analog of cantharidin  . The mol Pallas) . The mos Pallas) . It is c Pallas) –22. CTD apeutics . This unapeutics . NCTD caapeutics , 23, andapeutics . The cytapeutics –27. Moreapeutics  and overapeutics . in vitro [NCTD decreased hepatic leukemia factor (HLF) protein levels, a gene implicated in hematopoietic stem cell (HSC) regulation, and induced apoptosis in the acute myeloid leukemia (AML) cell line MV4-11 by modulating the expression of several molecules that govern survival pathways, including HLF, SLUG, NFIL3, and c-myc, thereby inducing p53 and the mitochondrial caspase cascade that explores the ability of NCTD to target stem cells . NCTD enin vitro . Their rin vitro .in vivo and in vitro [Besides inhibiting cancer cells, NCTD also affects normal cells. NCTD inhibits peripheral blood mononuclear cell (PBMC) proliferation with a 50% inhibitory concentration (IC (50)) 42.1 ± 2.3 microM without direct cytotoxicity or the arrest of cell-cycle progression in the cells . NCTD moin vitro , 49. Epiin vitro . On highIn this paper we review the current understanding of NCTD, which has cancer treatment potential, with a focus on overcoming MDR and CSC self-renewal characteristics .β-catenin pathway modulates cell proliferation, migration, apoptosis, differentiation, and stem cell self-renewal [β-Catenin participates in two distinct functions in the cell. Membrane-localized β-catenin is a protein adhesive that with E-cadherin maintains cell-cell adhesion [β-catenin cooperates with the transcription factors T cell factor/lymphoid enhancer factor (TCF/LEF) as a transcription activator, which eventually leads to activation of Wnt target genes such as c-Jun, c-Myc, fibronectin, and cyclin D1 [The Wnt/-renewal –53. β-Caadhesion . Cytoplayclin D1 –60.β-catenin pathway. Cimmino et al. [β-catenin signaling pathway contributes to refractory and relapsed leukemia. Chuang et al. [β-catenin pathway. They note that NCTD (50 microM) inhibits the proliferation of Jurkat cells with dominant beta-catenin signaling by 64% in a concentration-dependent manner. In CT26 colorectal adenocarcinoma cells, NCTD decreases the adhesive ability of CT26 cells and shows a downregulation of several cadherin-catenin adhesion molecules in vitro. It could reduce both the pulmonary metastatic capacity of CT26 cells and prolong the survival time of the tumor-bearing mice [Increasing evidence supports the ability of NCTD to inhibit the Wnt/o et al.  reportedg et al.  also coning mice   signaling pathway plays a major role as regulator of cell differentiation, tissue polarity and cell proliferation , 62. Thepoptosis –67. Hh ppoptosis  or cancepoptosis . The plasma VEGF levels of tumor-bearing mice, migration, and capillary-like tube formation of HUVECs are suppressed by NCTD with potential antimetastasis and antiangiogenesis . Chen etβ-catenin, notch signaling, and the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. These signaling molecules are activated by G-protein-coupled receptors, such as Frizzled or Smo [β-catenin [β, regulates the molecules involved in Hh signaling [β3-catenin signaling [There are crosstalks between hedgehog signaling, Wnt/d or Smo , 71. The-catenin . The stu-catenin . The molignaling , but theignaling . Taken tβ-catenin pathway. The PI3-Kinase/Akt pathway acts as a survival signal and plays a key role in the regulation of apoptotic events. The PI3-Kinase/Akt pathway is important in regulating the mammary stem/progenitor cells by promoting β-catenin downstream through phosphorylation of GSK-3β. Activated Akt was shown to be able to phosphorylate Ser9 on GSK-3β, which may decrease the activity of GSK-3β, thereby stabilizing β-catenin [κB) survival pathway [κB can promote tumorigenesis and is linked to cell invasion and metastasis. The suppression of NF-κB activation is effective in the prevention and treatment of cancer [The PI3-Kinase/Akt pathway links to the Wnt/-catenin , 76. Akt pathway . NF-κB cf cancer . More reports have demonstrated the direct or synergistic role of PI3-kinase/Akt activation in mediating the biological effects of hedgehog signaling –82. GeneκB signaling. It has been suggested that overexpression of Shh is activated by NF-κB in pancreatic cancer and pancreatic cancer cell proliferation is accelerated by NF-κB in part through Shh overexpression [κB target gene and mapped a minimal NF-κB consensus site to position +139 of the Shh promoter [Likewise, the Shh pathway is linked to transcription factor NF-pression . Kaspercpromoter . κB pathway in diverse cellular situations [κB subunits [κB promoter activity [Notch signaling is known to control cell proliferation and apoptosis to modulate the development of many organs . A numbetuations –97. Specsubunits , 98, andactivity .κB increases the migration of cancer cell lines such as human lung cancer A549 cells [κB expression in human breast cancer MDA-MB-231 cells [μg/mL) without affecting cell viability [κB DNA-binding activities, leading to matrix metalloproteinases (MMP)-9 downregulation and u-plasminogen activator (PA) expression to reduce the invasion of hepatocellular carcinoma (Huh7) cells [Activation of PI3K/Akt and NF-49 cells  and huma49 cells . NCTD do31 cells . Moreoveiability . Activatiability . Similariability . NCTD ca7) cells . It suggMultidrug resistance (MDR) is an unfavorable factor causing the failure of treatments against cancer cells . It occuNCTD is found to inhibit the P-gp  and the The experimental demonstration of CSCs in several human tumors in recent years promises a new cellular target for anticancer drug discovery , 112–114β-catenin pathway initiates a signaling cascade critical in both normal development and the initiation and progression of cancer [β-catenin/TCF inhibitors, most of them belonging to low molecular-weight inhibitors, downregulate the expression of β-catenin/TCF-responsive genes and disrupt the interaction of element-binding protein (CBP) with β-catenin [β-catenin/TCF complexes directly [β-catenin activation [β-catenin signaling [β-catenin pathways.The Wnt/f cancer –121. Var-catenin  or disrudirectly . Similartivation  and inhiignaling . These dEpithelial cell adhesion molecule (EPCAM) is highly expressed in numerous solid tumors, and it has recently been shown to be expressed on tumor-initiating cells from breast, prostate, colon, and pancreatic cancer , 124. ThRadiation sensitization is one of the important directions to develop anticancer agents for radiotherapy or chemoradiation therapy. Shh signaling has been discovered as a mechanism rendering cancer cells resistant to chemoradiation. In clinical practice, the expression of Ptch or Gli-1 has been significantly associated with a poor prognosis for oral cavity cancer patients . Cancer Safety issues are worth noting in efforts to develop chemotherapy-enhancing or radiation sensitization agents aimed at eliminating CSCs. Research efforts should be oriented toward avoiding, or at least minimizing, the inhibition of crucial mechanisms for normal stem cell maintenance. NCTD has pharmacological potential in the treatment of CSCs. The beneficial effects of NCTD include the modulation of CSC self-renewal pathways, overcoming multidrug resistance and as radiation sensitizer. Although the mechanisms are not clearly addressed in the reviewed publications, the results indicate that further evaluation of NCTD is warranted. In particular, the mechanisms of action by which NCTD modulates CSC characteristics should be clarified. Preclinical studies to pave the way for clinical trials may eventually enable scientists to discover more effective strategies for cancer treatment to reduce resistance and recurrence and, eventually, to improve survival of cancer patients.


This paper focuses on recent experience in setting up an endoscopy unit in a large regional hospital. The mix of endoscopy in three smaller hospitals, draining into the large hospital endoscopy unit, has enabled the authors to comment on practical and achievable steps towards creating best practice endoscopy in the regional setting. The challenges of using what is available from an infrastructural equipment and personnel setting are discussed. In a fast moving field such as endoscopy, new techniques have an important role to play, and some are indeed cost effective and have been shown to improve patient care. Some of the new techniques and technologies are easily applicable to smaller endoscopy units and can be easily integrated into the practice of working endoscopists. Cost effectiveness and patient care should always be the final arbiter of what is essential, as opposed to what is nice to have. Close cooperation between referral and peripheral centers should also guide these decisions. In Australia, it is probably easier to define what a teaching hospital is than a community hospital. A community hospital can be defined by what it is not, rather than what it is. It is not a tertiary referral center where research, training, and university affiliations are of primary importance. There are however many larger community hospitals in Australia where registrars rotate. Medical and nursing students are to be found even in small hospitals. A tertiary hospital refers more to the scope of hospital referral, so, for instance, a large hospital in Sydney to which several smaller hospitals send their difficult patients could rightly be called a tertiary hospital. There are however many large community hospitals which drain smaller hospitals, and they in turn would send patients that they could not manage to large university hospitals. The authors' hospital in Nambour, Queensland represents an interesting mix of practice. The endoscopy department conducts outreach endoscopy in 3 peripheral hospitals serving small towns of less than 30 000 people, upto 70 km from the base hospital. The base hospital has 400 beds and is a community hospital. We have very recently extended our endoscopy unit, transforming it from a single theatre complex-based room, into a standalone endoscopy unit, with dedicated endoscopy nursing staff and two large endoscopy suites. Within 5 years a new teaching hospital will be built, and the hospital district will become a tertiary center. These changes have enabled us to ponder on community-based endoscopy service in Australia, and on what is needed to upgrade to a teaching hospital endoscopic service.Endoscopy is a fast moving field, where new techniques are constantly being instituted, resulting in better patient care and the need for less surgical intervention. Here we will review the major hurdles and the benefits in providing selected top end endoscopic service to a community hospital. The major hurdles to provision of excellent endoscopic services are infrastructure, personnel, and equipment.Infrastructure is defined as the area in which endoscopy occurs, the patient flow to and from such area, as well as the recovery space. The majority of community hospitals in Australia use theatre complex space to perform endoscopy. In addition, surgeons with an interest in endoscopy perform most of their endoscopic procedures in this setting, often placing endoscopy patients on lists where other procedures are performed. This practice does not necessarily result in poor quality endoscopy, but dedicated endoscopists who perform a high volume of polypectomies in the hospital setting have the lowest rate of missed postcolonoscopy colorectal cancer . 2 insufflation results in easier recovery from colonoscopy [In a nondedicated endoscopy room, storage of endoscopic equipment, training, and proficiency of endoscopy nurses, and patient turnover are not optimal. By definition, endoscopy is not a sterile procedure, and the culture of a sterile theatre environment and nursing protocols associated with it tend to clash with efficient endoscopic turnover. Sharing recovery space with postsurgical patients is often also a problem. Recovery protocols for postsurgical patients are not optimal for endoscopy patients where conscious sedation results in quick patient recovery. In addition, the use of COonoscopy . Recent Hence, the creation of dedicated endoscopy units with their own personnel is probably the first step which needs to be taken to improve endoscopic standards and implement new techniques , 5. PatiThere are no clear data regarding this, but a minimum of three dedicated endoscopists would be needed to justify the funding commitment of a dedicated unit. The techniques employed in the unit, would have to depend on the competence and training of the endoscopists. The ability to perform new techniques depends on having the right equipment, but more importantly having endoscopists with the confidence and training to perform these techniques safely. In general, new techniques are pioneered in teaching hospitals and often diffuse into the community setting when trainees become consultants in community hospitals. In small hospitals with stable staffing complements, this diffusion of knowledge does not occur quickly. In general, consultant endoscopists in smaller hospitals in Australia are senior and job mobility is minimal. Training of these individuals is difficult, since they are often clinically committed and cannot leave the hospital for long periods of time to master new techniques. Important teaching occurs at endoscopy forums, and new techniques such as endoscopic submucosal resection of large polyps  can be aThe equipment needed to provide acceptable endoscopy is closely related with the expertise and preference of the endoscopists. An optimal suite of equipment would include high definition and recent generation endoscopes, preferably with narrow band or equivalent image enhancement capabilities. However, somewhat counterintuitively, a recent meta-analysis failed to document significantly improved adenoma detection when standard and high definition colonoscopes were compared, emphasizing that endoscopic competence is probably more important than equipment .With endoscope numbers, redundancy needs to be built in for unexpected events, such as sterilizer failure or scope breakage. Endoscopy lists are fluid, and urgent cases may be unexpectedly added, so a ready-to-go colonoscope and gastroscope should always be on standby. As shown in This section will focus on what type of services a community endoscopy unit should provide, which services are “nice to have”, and which are rather left to teaching hospitals. There is no literature to guide these decisions, but enthusiasm and expertise of the endoscopic personnel and budgets are the determining factors. We will provide a guide for which technologies may fit into the community hospital setup and what the benefits are. Emergency procedures are unplanned and often after hours, consisting mostly of upper gastrointestinal bleeds . CommuniMost competent endoscopists can apply clips and bands, use coagulation devices, and inject adrenaline to control bleeding. The extensive use of anticlotting agents has added a new dimension to upper and lower GI bleeding and the acute control of this. Mortality remains worryingly high particularly for variceal bleeds (15%) . This wiNew additions to control bleeding include over-the-scope clip devices (OTSC) , 17 and No elective endoscopic task is more important than the early detection and removal of polyps in the colon. Institution of screening services such as FOB testing has been shown to increase endoscopy workload in community hospitals by 118% . SurveilEndoscopy units, whether large or small, must achieve maximal detection of polyps, with particular focus on proximal lesions and particular care must be taken to find flat lesions and sessile serrated adenomas  which coRetroflexion and third eye retroscopes have attempted to address polyps hidden in folds, particularly in the ascending colon. Cecal retroflexion is achievable in 94.4% of patients, with no reported complications in this series . An addiSimilar results can be achieved with third eye retroscopes, although cost is an issue here. A recent report highlighted the success of this technology, showing an increased adenoma detection rate of 11%, and impressively a third more large (>10 mm) adenomas were discovered . SomewhaOther techniques of increasing adenoma detection have focused on withdrawal time, but a recent report specifically addressing this issue has failed to show marked differences , and unsChanging the appearance of the mucosa is now accepted as a method of increasing adenoma detection. Two methods exist: dye staining and equipment settings such as narrow band imaging. Dye stains increase precision of diagnosis in Barrett's esophagus  and ulceNarrow band imaging (NBI) uses filters to emphasize blue-coloured light, thus accentuating vascular structures and can accurately discriminate between hyperplastic and adenomatous polyps . DysplasThe concept of an optical biopsy, as opposed to a tissue biopsy is appealing . In concThe problem with NBI is that the field depth is much reduced when compared to white light. Inevitably white light is used to see an abnormality, and then interrogation with NBI assists in confirming the pathology. It certainly is a “nice to have” for endoscopists who use it regularly, and training endoscopists to use NBI is not daunting , but theThe next level of mucosal evaluation is confocal laser endomicroscopy, which uses laser to visualize cellular structures in the mucosa, producing real-time histology of suspicious lesions . The mosEndoscopist skill is therefore of greater importance than updating equipment in community hospitals.Accurate diagnosis of small-bowel pathology is now achievable. Radiological techniques, particularly MR enterography, allow excellent visualization of the bowel, without the danger of radiation. The best evaluation of intraluminal pathology is capsule enteroscopy. Sensitivity and specificity are very high for any structural lesion of the small bowel , 60. SetThe problem with small-bowel evaluation comes when pathology is detected and intervention is necessary. A patient with iron deficiency anemia may have small-bowel vascular ectasia which is amenable to Argon plasma coagulation, or a polyp which can be removed endoscopically. There are three ways of accessing the small bowel: double balloon enteroscopy, single-balloon enteroscopy, and spiral enteroscopy. Double balloon is the established technology and achieves a higher completion rate and diagnostic yield than single balloon enteroscopy . Spiral What role should these small-bowel techniques play in community endoscopy units? Capsule endoscopy would probably be the first choice, since it is easy, affordable, safe, and diagnostically accurate. If small-bowel pathology is diagnosed, it would be essential to perform therapeutic procedures, and the choice between the three systems may be challenging. Although double balloon is the more established technology, it is only available in the Fujinon system. It would therefore be expensive to acquire this if other endoscopy platforms are being used already. Olympus has a single-balloon system which probably would access most small-bowel pathology. If the capsule showed very distal disease, referral for double balloon could be undertaken, or single balloon attempted with recognition that failure and subsequent referral may occur. Spiral enteroscopy has the advantage of using different endoscopic platforms, but its role has not been sufficiently defined to make recommendations yet. More data using all three systems needs to be assessed before the role of each technology can be defined. Ironically the greatest advance in ERCP in the last 10 years is the development of MRCP, which has completely dispensed with the need for diagnostic ERCP. The absolute number of ERCP has thus come down dramatically, but interventional ERCP, and particularly removal of common bile duct stones in patients with cholangitis, is a life-saving procedure, which should be available in most large community-based hospitals. Techniques have not really changed in 10 years. The interplay between EUS and ERCP in challenging patients is an interesting development, but probably something for the large academic units with enthusiastic experts.Endoscopic ultrasound is an established technique, which is probably most useful in assessment and intervention of pancreaticobiliary disease. The difficulty in training consultants in this has been discussed. A recent publication from a large community hospital in Hong Kong documents an uptake of 2 to 3 patients per week, but importantly describes use of this technique for staging of esophageal and gastric cancer . This wo2 insufflation. Although described more than 30 years ago [2 insufflation. One technical advance which has improved colonoscopy, both for patients and proceduralists, is COears ago , recent ears ago . PatientNurse sedationists , 69 and Unsedated colonoscopy is practiced in some community settings, but low patient acceptance rate (56.2%), and substantially lower completion rates would suggest that this is not an option in most community units .In summary, the main hurdles in implementing an excellent endoscopic service in Australia are in our opinion budgetary constraints and personnel and infrastructural inadequacy. There is no doubt that the first and most crucial step to improving endoscopy is the creation of dedicated endoscopy units, preferably removed from the surgical theatre environment. There are also major bureaucratic obstacles to creating such a unit, and even if money is available, an enthusiastic team has to drive the process. Although recruiting and retaining dedicated luminal endoscopists are an important component to the success of this endeavor, the other foundation issues such as equipment, nursing staff, booking personnel, patient flow, and recovery protocols are more difficult to put into place. The techniques which could be easily introduced in community hospitals in western countries are latest generation endoscopes, newer clipping devices, retroflexion, chromoendoscopy, and small-bowel capsule endoscopy. Some methods of accessing the small bowel would be useful, although not essential. There are some relatively cheap modifications, which fit onto most endoscopes which would enable most units to perform small-bowel interventions. The endoscopic skills needed for this do not materially differ from those used in gastroscopy and colonoscopy, so training for these is not a major hurdle. Cost-benefit analysis and helpful discussion with hospital administrators are important in deciding what is absolutely essential to providing a top-class endoscopy service and what should be referred elsewhere. Emergency endoscopy procedures are obviously judged differently from more elective procedures. The final hurdle to good regional endoscopy lies in the endoscopist. There is no doubt that regional endoscopy services can be as good as large academic units , and the