NAP1L1: A Novel Human Colorectal Cancer Biomarker Derived From Animal Models of Apc Inactivation.

INTRODUCTION: Colorectal cancer (CRC) is the second leading cause of cancer death worldwide and most deaths result from metastases. We have analyzed animal models in which Apc, a gene that is frequently mutated during the early stages of colorectal carcinogenesis, was inactivated and human samples to try to identify novel potential biomarkers for CRC. MATERIALS AND METHODS: We initially compared the proteomic and transcriptomic profiles of the small intestinal epithelium of transgenic mice in which Apc and/or Myc had been inactivated. We then studied the mRNA and immunohistochemical expression of one protein that we identified to show altered expression following Apc inactivation, nucleosome assembly protein 1-like 1 (NAP1L1) in human CRC samples and performed a prognostic correlation between biomarker expression and survival in CRC patients. RESULTS: Nap1l1 mRNA expression was increased in mouse small intestine following Apc deletion in a Myc dependant manner and was also increased in human CRC samples. Immunohistochemical NAP1L1 expression was decreased in human CRC samples relative to matched adjacent normal colonic tissue. In a separate cohort of 75 CRC patients, we found a strong correlation between NAP1L1 nuclear expression and overall survival in those patients who had stage III and IV cancers. CONCLUSION: NAP1L1 expression is increased in the mouse small intestine following Apc inactivation and its expression is also altered in human CRC. Immunohistochemical NAP1L1 nuclear expression correlated with overall survival in a cohort of CRC patients. Further studies are now required to clarify the role of this protein in CRC.

APC2 is critical for ovarian WNT signalling control, fertility and tumour suppression.

BACKGROUND: Canonical WNT signalling plays a critical role in the regulation of ovarian development; mis-regulation of this key pathway in the adult ovary is associated with subfertility and tumourigenesis. The roles of Adenomatous polyposis coli 2 (APC2), a little-studied WNT signalling pathway regulator, in ovarian homeostasis, fertility and tumourigenesis have not previously been explored. Here, we demonstrate essential roles of APC2 in regulating ovarian WNT signalling and ovarian homeostasis. METHODS: A detailed analysis of ovarian histology, gene expression, ovulation and hormone levels was carried out in 10 week old and in aged constitutive APC2-knockout (Apc2-/-) mice (mixed background). Statistical significance for qRT-PCR data was determined from 95% confidence intervals. Significance testing was performed using 2-tailed Student's t-test, when 2 experimental cohorts were compared. When more were compared, ANOVA test was used, followed by a post-hoc test (LSD or Games-Howell). P-values of < 0.05 were considered statistically significant. RESULTS: APC2-deficiency resulted in activation of ovarian WNT signalling and sub-fertility driven by intra-ovarian defects. Follicular growth was perturbed, resulting in a reduced rate of ovulation and corpora lutea formation, which could not be rescued by administration of gonadotrophins. Defects in steroidogenesis and follicular vascularity contributed to the subfertility phenotype. Tumour incidence was assessed in aged APC2-deficient mice, which also carried a hypomorphic Apc allele. APC2-deficiency in these mice resulted in predisposition to granulosa cell tumour (GCT) formation, accompanied by acute tumour-associated WNT-signalling activation and a histologic pattern and molecular signature seen in human adult GCTs. CONCLUSIONS: Our work adds APC2 to the growing list of WNT-signalling members that regulate ovarian homeostasis, fertility and suppress GCT formation. Importantly, given that the APC2-deficient mouse develops tumours that recapitulate the molecular signature and histological features of human adult GCTs, this mouse has excellent potential as a pre-clinical model to study ovarian subfertility and transitioning to GCT, tumour biology and for therapeutic testing.

Spatiotemporal regulation of liver development by the Wnt/ß-catenin pathway.

While the Wnt/ß-catenin pathway plays a critical role in the maintenance of the zonation of ammonia metabolizing enzymes in the adult liver, the mechanisms responsible for inducing zonation in the embryo are not well understood. Herein we address the spatiotemporal role of the Wnt/ß-catenin pathway in the development of zonation in embryonic mouse liver by conditional deletion of Apc and ß-catenin at different stages of mouse liver development. In normal development, the ammonia metabolising enzymes carbamoylphosphate synthetase I (CPSI) and Glutamine synthetase (GS) begin to be expressed in separate hepatoblasts from E13.5 and E15.5 respectively and gradually increase in number thereafter. Restriction of GS expression occurs at E18 and becomes increasingly limited to the terminal perivenous hepatocytes postnatally. Expression of nuclear ß-catenin coincides with the restriction of GS expression to the terminal perivenous hepatocytes. Conditional loss of Apc resulted in the expression of nuclear ß-catenin throughout the developing liver and increased number of cells expressing GS. Conversely, conditional loss of ß-catenin resulted in loss of GS expression. These data suggest that the Wnt pathway is critical to the development of zonation as well as maintaining the zonation in the adult liver.

Organoids as a Model for Colorectal Cancer.

Modelling human diseases in in vitro systems is undisputedly an invaluable research tool, yet there are many limitations. Some of those limitations have been overcome through the introduction of organoid culture systems, which have revolutionised colorectal cancer research and enabled an array of new experimental techniques. This 3D system models the physiology, shape, dynamics and cell make-up of the intestinal epithelium producing a relevant and highly adaptable model system. The increased functional relevance of this model compared to the use of 2D cancer cell lines makes it an invaluable tool for both basic and translational research. As the limitations of this system are being overcome to make high-throughput assays possible, it is clear that organoids are becoming a mainstay of colorectal cancer research. This review aims to explore the advantages and limitations of this system and discusses the future directions enabled by this model.

Secreted HMGB1 from Wnt activated intestinal cells is required to maintain a crypt progenitor phenotype.

BACKGROUND AND AIMS: Colorectal cancer (CRC) arises via multiple genetic changes. Mutation of the tumour suppressor gene APC, a key regulator of Wnt signalling, is recognised as a frequent early driving mutation in CRC. We have previously shown that conditional loss of Apc within the murine small intestine (Apcfloxmice) results in acute Wnt signalling activation, altered crypt-villus architecture and many hallmarks of neoplasia. Our transctipomic profiling (Affymetrix Microarrays) and proteomic profiling (iTRAQ-QSTAR) of Apc-deficient intestine inferred the involvement of High Mobility Group Box 1 (Hmgb1) in CRC pathogenesis. Here we assess the contribution of HMGB1 to the crypt progenitor phenotype seen following Apc loss. RESULTS: Elevated HMGB1 was confirmed in intestinal epithelia and serum following conditional loss of Apc. Treatment of Apcflox mice with anti-HMGB1 neutralising antibody significantly reduced many of the crypt progenitor phenotypes associated with Apc loss; proliferation and apoptosis levels were reduced, cell differentiation was restored and the expansion of stem cell marker expression was eradicated. METHODS: Hmgb1 levels in intestinal epithelia and serum in Apcflox and ApcMin mice were assessed using qRT-PCR, Western blot and ELISA assays. The functional importance of elevated extracellular Hmgb1 was assessed using an anti-HMGB1 neutralising antibody in Apcflox mice. CONCLUSIONS: HMGB1 is expressed and secreted from intestinal epithelial cells in response to Wnt signalling activation. This secreted HMGB1 is required to maintain nearly all aspects of the crypt progenitor phenotype observed following Apc loss and add to the body of accumulating evidence indicating that targeting HMGB1 may be a viable novel therapeutic approach.

E-cadherin can limit the transforming properties of activating ß-catenin mutations.

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the pancreas) have activating mutations in ß-catenin (CTNNB1). We have compared the dynamics and the potency of ß-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of ß-catenin took much longer to achieve Wnt deregulation and acquire a crypt-progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of ß-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of ß-catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E-cadherin and a higher number of E-cadherin:ß-catenin complexes at the membrane. Reduction in E-cadherin synergised with an activating mutation of ß-catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of ß-catenin that is required to drive transformation, and E-cadherin can act as a buffer to sequester mutated ß-catenin.

Hunk/Mak-v is a negative regulator of intestinal cell proliferation.

BACKGROUND: Conditional deletion of the tumour suppressor gene Apc within the murine intestine results in acute Wnt signalling activation. The associated over-expression of a myriad of Wnt signalling target genes yields phenotypic alterations that encompass many of the hallmarks of neoplasia. Previous transcriptomic analysis aimed at identifying genes that potentially play an important role in this process, inferred the Hormonally upregulated Neu-associated kinase (HUNK/Mak-v/Bstk1) gene as a possible candidate. Hunk is a SNF1 (sucrose non fermenting 1)-related serine/threonine kinase with a proposed association with many different tumour types, including colorectal cancer. METHODS: Here we describe the generation of a novel Hunk kinase deficient mouse which has been used to investigate the involvement of Hunk-kinase activity in intestinal homeostasis and tumourigenesis. RESULTS: We show that in the morphologically normal intestine, Hunk-kinase negatively regulates epithelial cell proliferation. However, the increase in cell proliferation observed in the Hunk kinase deficient intestine is counteracted by increased cell migration, thereby maintaining intestinal homeostasis. Using qRT-PCR, we further demonstrate that Hunk is significantly over-expressed in Apc deficient / Wnt-signalling activated intestinal tissue. Using the classical intestinal tumourigenesis Apc (Min) mouse model we show that loss of Hunk-kinase activity significantly reduced tumour initiation rates in the small intestine. However, an accompanying increase in the size of the tumours counteracts the impact this has on overall tumour burden or subsequently survival. CONCLUSIONS: In the intestinal setting we demonstrate that Hunk has a role in normal intestinal proliferation and homeostasis and, although it does not alter overall survival rates, activity of this kinase does impact on tumour initiation rates during the early stages in tumourigenesis in the small intestine.

Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC).

Colorectal cancer (CRC) is the fourth most common cause of cancer-related death worldwide. Accurate non-invasive screening for CRC would greatly enhance a population's health. Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas, leading to Wnt signalling pathway activation. Acute conditional transgenic deletion of Apc in murine intestinal epithelium (AhCre(+)Apc(fl)(/)(fl)) causes phenotypic changes similar to those found during colorectal tumourigenesis. This study comprised a proteomic analysis of murine small intestinal epithelial cells following acute Apc deletion to identify proteins that show altered expression during human colorectal carcinogenesis, thus identifying proteins that may prove clinically useful as blood/serum biomarkers of colorectal neoplasia. Eighty-one proteins showed significantly increased expression following iTRAQ analysis, and validation of nine of these by Ingenuity Pathaway Analysis showed they could be detected in blood or serum. Expression was assessed in AhCre(+)Apc(fl)(/)(fl) small intestinal epithelium by immunohistochemistry, western blot and quantitative real-time PCR; increased nucelolin concentrations were also detected in the serum of AhCre(+)Apc(fl)(/)(fl) and Apc(Min)(/)(+) mice by ELISA. Six proteins; heat shock 60kDa protein 1, Nucleolin, Prohibitin, Cytokeratin 18, Ribosomal protein L6 and DEAD (Asp-Glu-Ala-Asp) box polypeptide 5,were selected for further investigation. Increased expression of 4 of these was confirmed in human CRC by qPCR. In conclusion, several novel candidate biomarkers have been identified from analysis of transgenic mice in which the Apc gene was deleted in the intestinal epithelium that also showed increased expression in human CRC. Some of these warrant further investigation as potential serum-based biomarkers of human CRC.

Conditional disruption of Axin1 leads to development of liver tumors in mice.

BACKGROUND & AIMS: Mutations in components of the Wnt signaling pathway, including ß-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. METHODS: Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of ß-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. RESULTS: Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G(2)/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of ß-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of ß-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. CONCLUSIONS: Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of ß-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC.

Entopic overexpression of Ascl2 does not accelerate tumourigenesis in ApcMin mice.

OBJECTIVE: Expression of the Wnt target gene ASCL2 is elevated in 78% of intestinal neoplasia datasets (Oncomine), suggesting a role for deregulated ASCL2 in the aetiology of intestinal tumourigenesis. Furthermore, ectopic expression of Ascl2 has previously been shown to lead to hyperplasia in the mouse. However, elevated levels of ASCL2 does not have an impact on the overall survival or recurrence-free survival rates in colorectal cancer patients. Here the authors use a novel mouse model to analyse the role of Ascl2 in intestinal tumourigenesis and address the controversy surrounding the relevance of this gene to the aetiology of colorectal cancer. DESIGN: The authors have generated a mouse possessing a transgene carrying the Ascl2 gene together with its endogenous promoter and regulatory regions, thereby elevating Ascl2 expression in an authentic manner. The authors have further intercrossed these Ascl2 overexpressers to the classic Apc(Min) model, to study the consequence of elevated Ascl2 expression in intestinal tumourigenesis. RESULTS: Here the authors genetically demonstrate that elevated expression of Ascl2 in a Wnt signalling dependent manner specifically in the stem cell compartment of the intestine neither increases tumour formation nor diminishes survival in a well established intestinal tumour model, the Apc(min) mouse. CONCLUSION: The authors conclude that ectopic expression of Ascl2 is more important in the aetiology of neoplasia than overexpression of Ascl2.

LITAF mediation of increased TNF-α secretion from inflamed colonic lamina propria macrophages.

Dysregulation of TNF-α in lamina propria macrophages (LPM) is a feature of inflammatory bowel diseases (IBD). LPS-Induced-TNF-Alpha-Factor (LITAF) is a transcription factor that mediates TNF-α expression. To determine whether LITAF participates in the mediation of TNF-α expression in acutely inflamed colonic tissues, we first established the TNBS-induced colonic inflammation model in C57BL/6 mice. LPM were harvested from non-inflamed and inflamed colonic tissue and inflammatory parameters TNF-α and LITAF mRNA and protein levels were measured ex-vivo. LPM from TNBS-treated mice secreted significantly more TNF-α at basal state and in response to LPS than LPM from untreated mice (p<0.05). LITAF mRNA and protein levels were elevated in LPM from TNBS compared with untreated animals and LPS further increased LITAF protein levels in LPM from inflamed tissue (P<0.05). To further confirm the role of LITAF in acutely inflamed colonic tissues, TNBS-induced colonic inflammation was produced in LITAF macrophage specific knockout mice (LITAF mac -/- mice) and compared to wild type (WT) C57BL/6. Twenty four hours following TNBS administration, colonic tissue from LITAF mac -/- mice had less MPO activity and reduced colonic TNF-α mRNA then WT C57BL/6 mice (p<0.05). LPM harvested from LITAF mac -/- secreted significantly less TNF-α in response to LPS than wild type (WT) C57BL/6 (p<0.05). This study provides evidence that LITAF contributes to the regulation of TNF-α in LPM harvested following acute inflammation or LPS treatment paving the way for future work focusing on LITAF inhibitors in the treatment of TNF-α-mediated inflammatory conditions.

N-acetyl-l-cysteine decreases intra-abdominal adhesion formation through the upregulation of peritoneal fibrinolytic activity and antioxidant defenses.

BACKGROUND: Intraperitoneal adhesions occur in more than 94% of patients after abdominal surgery. Mechanisms that decrease oxidative stress and upregulate peritoneal fibrinolysis reduce adhesions. N-acetyl-l-cysteine (NAC) is a clinically relevant antioxidant whose effect on peritoneal fibrinolysis and ability to decrease adhesions has not been established. The aims of this study were to determine if NAC reduces adhesions and to characterize its potential mechanism(s) of action. METHODS: Male Wistar rats (n = 92) received 0.9% saline (OP Control), intraperitoneal NAC (150 mg/kg, OP + NAC), or oral NAC (1200 mg/kg) twice daily on preoperative day 1, day of operation, and postoperative day 1. Adhesions were induced on the day of operation using our previously described ischemic button model. Animals were killed on postoperative day 7 for adhesion scoring. Peritoneal tissue and fluid from the intraperitoneal NAC group were measured at 24 hours for fibrinolytic activity, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), total glutathione, and 8-isoprostane (8-IP). The effect of NAC on tPA and PAI-1 production was tested in vitro in human mesothelial cells. The effect of NAC on intestinal wound healing was measured using colonic anastomotic burst pressures. RESULTS: Intraperitoneal NAC reduced adhesions by 53% (P < .001) compared to OP Controls without affecting anastomotic wound healing. NAC increased the tPA/PAI-1 protein ratio and peritoneal fibrinolytic activity by 69% and 127%, respectively, compared to OP Controls (P < .05). NAC did not restore total glutathione levels in peritoneal adhesion tissue but decreased 8-IP by 46% and 65% (P < .05) in peritoneal tissue and fluid, respectively, compared to OP Controls. Human mesothelial cells incubated with NAC exhibited a concentration-dependent increase in the tPA/PAI-1 ratio, which supported in vivo observations (P < .05). Oral NAC did not decrease adhesions. CONCLUSION: NAC administered intraperitoneally decreased adhesion formation while upregulating peritoneal fibrinolytic activity and antioxidant defenses without affecting normal anastomotic wound healing. These data suggest a potential new therapeutic use for NAC in adhesion prevention.

Rectal epithelial cell mitosis and expression of macrophage migration inhibitory factor are increased 3 years after Roux-en-Y gastric bypass (RYGB) for morbid obesity: implications for long-term neoplastic risk following RYGB.

BACKGROUND: Rectal epithelial cell mitosis and crypt size, as well as expression of proinflammatory genes including macrophage migration inhibitory factor (MIF), are increased 6 months after Roux-en-Y gastric bypass (RYGB) in morbidly obese patients. Tests were carried out to determine whether these putative colorectal cancer risk biomarkers remained elevated long term after RYGB, and the mechanistic basis, as well as the functional consequences, of Mif upregulation in intestinal epithelial cells was investigated. METHODS: Rectal mucosa and blood were obtained a median of 3 years after RYGB from the original cohort of patients with RYGB (n = 19) for crypt microdissection, real-time PCR, immunohistochemistry for MIF and immunoassay of proinflammatory markers. Immunohistochemistry for Mif and bromodeoxyuridine labelling were performed on AhCre⁺ mouse and Apc(Min/⁺) mouse (with and without functional Mif alleles) intestine, respectively. RESULTS: Rectal epithelial cell mitosis and crypt size remained elevated 3 years after RYGB compared with preoperative values (1.7- and 1.5-fold, respectively; p < 0.05). There was a 40-fold (95% CI 13 to 125) increase in mucosal MIF transcript levels at 3 years associated with increased epithelial cell MIF protein levels. Conditional Apc loss in AhCre⁺ mice led to increased epithelial cell Mif content. Mif deficiency in Apc(Min/⁺) mice was associated with a combined defect in intestinal epithelial cell proliferation and migration, which was reflected by the longitudinal clinical data. CONCLUSIONS: Mucosal abnormalities persist 3 years after RYGB and include elevation of the protumorigenic cytokine MIF, which is upregulated following Apc loss and which contributes to intestinal epithelial cell homeostasis. These observations should prompt clinical studies of colorectal neoplastic risk after RYGB.

Cyclin D2-cyclin-dependent kinase 4/6 is required for efficient proliferation and tumorigenesis following Apc loss.

Inactivation of the Apc gene is recognized as the key early event in the development of sporadic colorectal cancer (CRC), where its loss leads to constitutive activation of ß-catenin/T-cell factor 4 signaling and hence transcription of Wnt target genes such as c-Myc. Our and other previous studies have shown that although cyclin D1 is required for adenoma formation, it is not immediately upregulated following Apc loss within the intestine, suggesting that proliferation following acute Apc loss may be dependent on another D-type cyclin. In this study, we investigated the expression and functional relevance of cyclin D2 following Apc loss in the intestinal epithelium. Cyclin D2 is upregulated immediately following Apc loss, which corresponded with a significant increase in cyclin-dependent kinase 4 (CDK4) and hyperphosphorylated Rb levels. Deficiency of cyclin D2 resulted in a reduction in enterocyte proliferation and crypt size within Apc-deficient intestinal epithelium. Moreover, cyclin D2 dramatically reduced tumor growth and development in Apc(Min/+) mice. Importantly, cyclin D2 knockout did not affect proliferation of normal enterocytes, and furthermore, CDK4/6 inhibition also suppressed the proliferation of adenomatous cells and not normal cells from Apc(Min/+) mice. Taken together, these results indicate that cyclin D-CDK4/6 complexes are required for the efficient proliferation of cells with deregulated Wnt signaling, and inhibiting this complex may be an effective chemopreventative strategy in CRC.

The efficacy of a hyaluronate-carboxymethylcellulose bioresorbable membrane that reduces postoperative adhesions is increased by the intra-operative co-administration of a neurokinin 1 receptor antagonist in a rat model.

BACKGROUND: Bioresorbable membranes composed of hyaluronic acid and carboxymethylcellulose (HA/CMC) are the most effective method to prevent intra-abdominal adhesions; however, their efficacy may be limited to the site of application. Previous studies in our laboratory have shown that the intraperitoneal administration of a neurokinin-1 receptor antagonist (NK-1RA) reduces adhesions; however, the co-administration of HA/CMC plus an NK-1RA has not been studied. METHODS: Adhesions were induced in rats by creating ischemic buttons on the peritoneum. Rats received NK-1RA, HA/CMC, HA/CMC+NK-1RA or saline intraperitoneally at surgery. The HA/CMC was applied either bilaterally over all ischemic buttons or unilaterally over half the ischemic buttons. Animals were sacrificed and adhesions quantified at 7 days. Peritoneal fluid was collected at 24 hours to measure peritoneal tissue plasminogen activator (tPA) activity using a bioassay. RESULTS: The bilateral placement of HA/CMC alone reduced adhesions by 62% (P < .05) while the NK-1RA when administered alone reduced adhesions by 45% (P < .05), both groups compared with saline controls. The bilateral placement of HA/CMC+ NK-1RA decreased adhesions by 86% (P < .05) compared with saline controls and by 70% (P < .05) compared with either HA/CMC or NK-1RA alone. Unilateral application of HA/CMC resulted in a 41% decrease (P < .05) in adhesions where placed compared with the distal unprotected buttons in the same animal. However, the unilateral placement of HA/CMC+NK-1RA reduced adhesions by nearly 75% (P < .05) at the site of HA/CMC application compared with HA/CMC + saline, and by 45% (P < .05) at the distal unprotected buttons compared with saline controls. HA/CMC and the NK-1RA alone as well as HA/CMC+NK-1RA increased peritoneal tPA activity by 124%, 432%, and 192%, respectively (P < .05) compared with saline controls. CONCLUSION: The co-administration of HA/CMC plus NK-1RA not only increases the efficacy of the membrane at the site of application, but significantly reduces adhesions formation at distal unprotected sites. This combination may represent an emerging concept in more effective adhesion prevention throughout the peritoneum.

Liver zonation occurs through a beta-catenin-dependent, c-Myc-independent mechanism.

BACKGROUND AND AIMS: The Wnt pathway has previously been shown to play a role in hepatic zonation. Herein, we have explored the role of 3 key components (Apc, beta-catenin, and c-Myc) of the Wnt pathway in the zonation of ammonia metabolizing enzymes. METHODS: Conditional deletion of Apc, beta-catenin, and c-Myc was induced in the livers of mice and the expression of periportal and perivenous hepatocyte markers was determined by polymerase chain reaction, Western blotting, and immunohistochemical techniques. RESULTS: Under normal circumstances, the urea cycle enzyme carbamoylphosphate synthetase I (CPS I) is present in the periportal, intermediate, and the first few layers of the perivenous zone. In contrast, glutamine synthetase (GS)--and nuclear beta-catenin--is expressed in a complementary fashion in the last 1-2 cell layers of the perivenous zone. Conditional loss of Apc resulted in the expression of nuclear beta-catenin and GS in most hepatocytes irrespective of zone. Induction of GS in hepatocytes outside the normal perivenous zone was accompanied by a reduction in the expression of CPS I. Deletion of beta-catenin induces a loss of GS and a complementary increase in expression of CPS I irrespective of whether Apc is present. Remarkably, deletion of c-Myc did not perturb the pattern of zonation. CONCLUSIONS: It has been shown that the Wnt pathway is key to imposing the pattern of zonation within the liver. Herein we have addressed the relevance of 3 major Wnt pathway components and show critically that the zonation is c-Myc independent but beta-catenin dependent.

Practical limitations of bioresorbable membranes in the prevention of intra-abdominal adhesions.

INTRODUCTION: Intra-abdominal adhesions are a significant source of postoperative morbidity. Bioresorbable barriers composed of hyaluronic acid and carboxymethylcellulose (HA/CMC) reduce adhesion formation by physically separating injured or healing peritoneal surfaces. To assess whether the efficacy of a physical barrier can extend beyond the site of application, we evaluated the effectiveness of an HA/CMC barrier in preventing adhesions distal to the site of placement. METHODS: Adhesions were induced in rats by creating peritoneal ischemic buttons on either side of a midline incision. An HA/CMC barrier (Seprafilm Genzyme) was intraoperatively placed either under the midline incision, unilaterally over half the ischemic buttons, or bilaterally over all ischemic buttons. Control buttons received no HA/CMC. On day 7 adhesions were scored. In similar experiments, peritoneal fluid was collected at 24 h to assess the effects of HA/CMC on tissue plasminogen activator activity. RESULTS: Placement of HA/CMC under the midline incision did not reduce adhesion formation to distal ischemic buttons (72 +/- 7%) compared to controls (80 +/- 8%). Unilateral placement of HA/CMC significantly (p < 0.05) reduced adhesion formation to those ischemic buttons over which the barrier was applied (35 +/- 7%) compared to both contralateral (83 +/- 9%) and control (80 +/- 8%) ischemic buttons. The bilateral application of HA/CMC also significantly (p < 0.05) reduced adhesion formation to all ischemic buttons compared to controls (22 +/- 7% vs. 66 +/- 7%, respectively). HA/CMC did not affect peritoneal tPA activity. CONCLUSIONS: Effective adhesion reduction by the physical barrier HA/CMC appears to be limited to the site of application in this rat model. Despite the presence of a bioresorbable membrane at predicted sites of adhesion formation in the peritoneal cavity, adhesions readily form to distal unprotected sites.

Inhibitory effects of a neurokinin-1 receptor antagonist on postoperative peritoneal adhesion formation.

Intra-abdominal adhesions are a costly, long-term sequela of abdominal surgeries. They occur in up to 94% of patients following abdominal operation and cause significant postoperative morbidity including difficult reoperative surgeries, small bowel obstructions, and infertility. The pathophysiology of adhesion formation remains poorly defined, and a uniformly effective method of adhesion prevention does not exist. Research focused on understanding the mechanisms underlying adhesion formation is essential for the development of safe and effective therapeutic approaches to adhesion prevention. The proinflammatory peptide substance P (SP), known to participate in inflammatory and wound-healing events, may contribute to the early processes of adhesion formation. SP is the most widely studied ligand of the neurokinin-1 receptor (NK-1R), and we have determined in a rat model that intraoperative administration of an NK-1R antagonist, CJ-12-255 (Pfizer), that blocks ligand binding to the NK-1R, significantly reduces adhesion formation. It also has been determined that animals administered the NK-1R antagonist intraperitoneally have increased peritoneal fibrinolytic and matrix metalloproteinase activities, and reduced levels of oxidative stress postoperatively, all of which may contribute to the observed reduction in adhesion formation. Studies suggest that intra-abdominal adhesion formation begins within hours of surgery and that the regulation of fibrin deposition, and degradation is of key importance. A pharmacologic agent, such as an NK-1R antagonist, administered at the time of surgery that could augment postoperative peritoneal fibrinolytic activity without compromising wound healing, would be a beneficial tool in the prevention of postoperative adhesions.

B-catenin deficiency, but not Myc deletion, suppresses the immediate phenotypes of APC loss in the liver.

Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear beta-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors beta-catenin and c-Myc. beta-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being beta-catenin-dependent but c-Myc-independent in the liver.