Increased Pontin expression is a potential predictor for outcome in sporadic colorectal carcinoma.

Colorectal cancer is one of the leading causes of cancer-related death worldwide. Molecular biomarkers could help to predict patient outcome and to identify patients who benefit from adjuvant therapy. Pontin and Reptin are ATPases which are involved in transcriptional regulation, DNA damage repair and regulation of cell proliferation. Many interaction partners of Pontin and Reptin such as ß-catenin and c-myc are important factors in carcinogenesis. We hypothesized that Pontin and Reptin expression may be a negative predictor for survival in colorectal carcinoma. Specimens from 115 patients with primary colon adenocarcinomas UICC stage III and primary rectal adenocarcinomas UICC stage II and III curatively resected at the Department of Surgery, Charité Berlin, were evaluated. Clinical follow-up data were complete and mean follow-up time of patients was 51.8 months. We evaluated the expression of Pontin, Reptin and Ki-67 by immunohistochemistry. Patients with Pontin-positive carcinomas showed no differences in recurrence-free survival (p=0.109) and overall survival (p=0.197). There were no differences in Reptin-positive carcinomas and Ki-67-positive carcinomas in recurrence-free survival (p=0.443 and p=0.160) and overall survival (p=0.477 and p=0.687). Patients with Pontin-positive colorectal carcinomas receiving adjuvant therapy had a significantly worse recurrence-free survival (p=0.008) and overall survival (p=0.011) than Pontin-negative patients with adjuvant therapy. In UICC stage III, Pontin-positive colorectal carcinomas had a significantly worse recurrence-free survival (p=0.028). Pontin-positivity seems to be a negative predictor for response to adjuvant therapy in colorectal cancer patients and may help to identify patients with adverse outcome in advanced tumor stages.

NALP expression in Paneth cells provides a novel track in IBD signaling.

PURPOSE: Paneth cells are part of the innate mucosal immunity of the gut with possible regulatory function. This study intends to identify the gene expression pattern of the orthotopic and metaplastic Paneth cells, searching for differences between metaplastic occurrence between Crohn's disease and ulcerative colitis. METHODS: Paneth cells were collected in RNAse-free conditions via micro dissection. RNA isolation and super amplification was followed by microarray analysis of whole genome expression activity of the orthotopic and metaplastic Paneth cells. Immunohistology of beta-catenin and Frizzled-5 receptor was performed. RESULTS: Histological analysis showed no morphological or secretory change (Frizzled-5 receptor and beta-catenin) in orthotopic and metaplastic Paneth cells. Microarray analysis indicated an increased, but not mutant activation of Wnt/beta-catenin signaling and firstly showed expression of NALP 1, 7, 8 and 11 in metaplastic Paneth cells. CONCLUSIONS: Paneth cells might play a NALP-mediated role in the pathogenesis of IBD.

Association between activation of atypical NF-kappaB1 p105 signaling pathway and nuclear beta-catenin accumulation in colorectal carcinoma.

Recent studies have demonstrated that increased expression of coding region determinant-binding protein (CRD-BP) in response to beta-catenin signaling leads to the stabilization of beta-TrCP1, a substrate-specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IkappaBalpha and activation of canonical nuclear factor-kappaB (NF-kappaB) pathway. Here, we show that the noncanonical NF-kappaB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with beta-catenin-mediated increased expression of CRD-BP and beta-TrCP1. In the carcinoma tissues exhibiting high levels of nuclear beta-catenin the phospho-p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF-kappaB pathway. Knockdown of CRD-BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF-kappaB inhibitory proteins, p105 and IkappaBalpha. Furthermore decreased NF-kappaB binding activity was observed in CRD-BP siRNA-transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF-kappaB1 p105 signaling in colorectal carcinoma might be attributed to beta-catenin-mediated induction of CRD-BP and beta-TrCP1.

Inflamed pouch mucosa possesses altered tight junctions indicating recurrence of inflammatory bowel disease.

BACKGROUND AND AIMS: The etiology of pouchitis after coloproctomucosectomy with ileal pouch-anal anastomosis in patients with ulcerative colitis is still unknown. Beside changes in luminal antigens, the immunological predisposition is assumed to be responsible. In previous electrophysiological studies, we showed that mucosal barrier and transport function in pouchitis is markedly reduced. Thus, the aim of the present study was to analyze barrier function on the molecular level. MATERIAL AND METHODS: Pouch biopsies of 36 ulcerative colitis patients were analyzed. Time points were (1) intraoperative immediately prior to ileal pouch-anal anastomosis (n = 13), (2) >1 year after ileostomy closure (pouch, n = 12), and (3) during pouchitis (n = 11). Control terminal ileum biopsies were obtained from eight patients undergoing hemicolectomy due to carcinoma. Expression of tight junction proteins was analyzed by Western blotting and confocal laser-scanning microscopy. To elucidate effects on epithelial barrier properties, impedance spectroscopy was performed in miniaturized Ussing chambers. RESULTS: In pouchitis, epithelial resistance was markedly reduced compared to non-inflamed pouch and control ileum. Expression of tight junction proteins claudin-1, 3, 4, 5, and 7 and occludin revealed differential expression regulation with the tightening tight junction protein claudin-1 being decreased and an increase of the pore-forming claudin-2, whereas other claudins remained constant. Morphometry indicated the mucosal surface to be unchanged. CONCLUSION: Pouchitis is characterized by a selective change of tight junction proteins in favor of opening the epithelial tight junction and, thus, the paracellular pathway, which contributes to the inflammatory process. This resembles changes in inflammatory bowel disease (IBD) and indicates IBD recurrence in pouchitis.

Expression of catalytic proteasome subunits in the gut of patients with Crohn's disease.

BACKGROUND AND PURPOSE: Activation of the transcription factor NF-kappaB by proteasomes and subsequent nuclear translocation of cytoplasmatic complexes play a crucial role in the intestinal inflammation. Proteasomes have a pivotal function in NF-kappaB activation by mediating degradation of inhibitory IkappaB proteins and processing of NF-kappaB precursor proteins. This study aims to analyze the expression of the human proteasome subunits in colonic tissue of patients with Crohn's disease. MATERIALS AND METHODS: Thirteen patients with Crohn's disease and 12 control patients were studied. The expression of immunoproteasomes and constitutive proteasomes was examined by Western blot analysis, immunoflourescence and quantitative real-time PCR. For real-time PCR, AK2C was used as housekeeping gene. RESULTS: The results indicate the influence of the intestinal inflammation on the expression of the proteasomes in Crohn's disease. Proteasomes from inflamed intestine of patients with Crohn's disease showed significantly increased expression of immunosubunits on both protein and mRNA levels. Especially, the replacement of the constitutive proteasome subunit beta1 by inducible immunosubunit beta1i was observed in patients with active Crohn's disease. In contrast, relatively low abundance of immunoproteasomes was found in control tissue. CONCLUSIONS: Our data demonstrate that in contrast to normal colonic tissue, the expression of immunoproteasomes was evidently increased in the inflamed colonic mucosa of patients with Crohn's disease. Thus, the chronic intestinal inflammation process in Crohn's disease leads to significant alterations of proteasome subsets.

Permanently increased mucosal permeability in patients with backwash ileitis after ileoanal pouch for ulcerative colitis.

OBJECTIVE: Backwash ileitis (BI) has not been identified as a risk factor for pouchitis. The aim of this study was to investigate the barrier function of the ileoanal pouch depending on the presence of BI. The incidence of pouchitis in a population of ulcerative colitis patients with BI is also reported. MATERIAL AND METHODS: Biopsies were taken from 80 patients with ulcerative colitis: a) terminal ileum prior to pouch creation (pre-IAP); b) 16 months after ileostomy closure (intact pouch); and c) during pouchitis. Patients were stratified into the BI group and the non-BI (ØBI) group. Barrier function was determined in Ussing-chambers as epithelial resistance by impedance analysis and as mannitol permeability from (3)H-mannitol fluxes. Na(+)-glucose co-transport was measured as a change in short-circuit current (I(SC)) after addition of glucose. Relative risk of developing pouchitis was calculated by corrected chi(2) test. RESULTS: In 13/21 (BI/ØBI) pre-IAP patients, 23/37 (BI/ØBI) with an intact pouch, and 35/7 (BI/ØBI) with pouchitis, epithelial resistance in BI/ØBI was 13.5+/-1.6/14.3+/-0.9 Omega.cm(2) for pre-IAP, 12.7+/-1.3/16.8+/-1.2 Omega x cm(2) (p<0.05 BI versus ØBI) for the intact pouch, and 10.1+/-1.1/9.9+/-1.8 Omega x cm(2) for pouchitis (p<0.05 BI versus ØBI with an intact pouch). No differences were found for electrogenic chloride secretion and active Na(+)-glucose co-transport between BI/ØBI in the three groups. In patients with BI, pouchitis was more common (35 versus 7 patients, odds ratio 33.0 (95% CI 8.3-143.9; p<0.0001)). CONCLUSIONS: Ulcerative colitis patients with BI show impaired barrier function in the further course of the ileoanal pouch. Thus, BI has a long-term impact on epithelial barrier function.

Epithelial to mesenchymal transition: expression of the regulators snail, slug, and twist in pancreatic cancer.

PURPOSE: Epithelial to mesenchymal transitions are vital for tumor growth and metastasis. Several inducers of epithelial to mesenchymal transition are transcription factors that repress E-cadherin expression, such as Snail, Slug, and Twist. In this study, we aimed to examine the expression of these transcription factors in pancreatic cancer. EXPERIMENTAL DESIGN: The expression of Snail, Slug, and Twist was detected by immunohistochemistry in tissue samples from patients with pancreatic ductal adenocarcinoma. Five human pancreatic cancer cell lines (AsPC-1, Capan-1, HPAF-2, MiaPaCa-2, and Panc-1) were analyzed by reverse transcription-PCR, real-time PCR, and Western blotting. An orthotopic nude mouse model of pancreatic cancer was applied for in vivo experiments. RESULTS: Seventy-eight percent of human pancreatic cancer tissues showed an expression of Snail, and 50% of the patients displayed positive expression of Slug. Twist showed no or only weak expression. Snail expression was higher in undifferentiated cancer cell lines (MiaPaCa-2 and Panc-1) than in more differentiated cell lines (Capan-1, HPAF-2, AsPC-1). Expression of Slug was detected in all cell lines with different intensities. Twist was not expressed. After exposure to hypoxia, the Twist gene was activated in all five pancreatic cancer cell lines. CONCLUSIONS: The transcription factors Snail and Slug are expressed in pancreatic cancer but not in normal tissue, suggesting a role in the progression of human pancreatic tumors. Twist, activated by hypoxia, may play an important role in the invasive behavior of pancreatic tumors.

Increased bacterial permeation in long-lasting ileoanal pouches.

BACKGROUND AND AIMS: Bacterial overgrowth appears to play an important role in the pathogenesis of ileoanal pouches. Therefore, the capability of bacterial permeation and its determinants is of great interest. The aim of this study was to examine bacterial permeation in the ileoanal pouch and to correlate the results with the degree of inflammation, the epithelial resistance, the mucosal transport function, and the age of the ileoanal pouches. MATERIALS AND METHODS: Biopsies were taken from 54 patients before colectomy (n = 13; preileal pouch-anal anastomosis [IPAA]), and closure of ileostomy (n = 7; deviation), <1 year after closure of ileostomy (n = 8; intact pouch I), >1 year after closure of ileostomy (n = 16; intact pouch II), in the case of pouchitis (n = 11), and in 11 controls. Tissues were mounted in a miniaturized Ussing chamber. Escherichia coli was added to the mucosal side of the Ussing chamber, and the permeation was proven by serosal presence of E. coli. Epithelial and subepithelial resistance was determined by transmural impedance analysis. Active Na-glucose cotransport and active Cl secretion were measured. Specimens were analyzed by fluorescent in situ hybridization with oligonucleotide probes targeting the bacterial 16s ribosomal RNA. The bacteria in and on the tissue were enumerated. RESULTS: Bacterial permeation occurred in 2 of 13 pre-IPAA, 2 of 7 deviations, 0 of 8 intact pouch I, 9 of 16 intact pouch II, 5 of 11 pouchitis specimens, and 0 of 11 ileum controls. The frequency of bacterial permeation in the intact pouch II group is higher than in the intact pouch I group (P < 0.001). Epithelial resistance, mannitol fluxes, electrogenic chloride secretion, sodium-glucose cotransport of the bacterially permeated specimens versus nonpermeated of the intact pouch II group, and the pouchitis group and subepithelial resistance remained unchanged. Intramural bacteria could be detected by fluorescence in situ hybridization mainly in long-lasting pouches, but there was no correlation with bacterial permeation. CONCLUSIONS: The long-lasting ileoanal pouch is associated with increased bacterial permeability. This is not correlated with a disturbed function of the pouch mucosa but could be a precursor of pouchitis.