Increased variability in Apc(Min)/+ intestinal tissue can be measured with microultrasound.

Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic.

Combined changes in Wnt signaling response and contact inhibition induce altered proliferation in radiation-treated intestinal crypts.

Curative intervention is possible if colorectal cancer is identified early, underscoring the need to detect the earliest stages of malignant transformation. A candidate biomarker is the expanded proliferative zone observed in crypts before adenoma formation, also found in irradiated crypts. However, the underlying driving mechanism for this is not known. Wnt signaling is a key regulator of proliferation, and elevated Wnt signaling is implicated in cancer. Nonetheless, how cells differentiate Wnt signals of varying strengths is not understood. We use computational modeling to compare alternative hypotheses about how Wnt signaling and contact inhibition affect proliferation. Direct comparison of simulations with published experimental data revealed that the model that best reproduces proliferation patterns in normal crypts stipulates that proliferative fate and cell cycle duration are set by the Wnt stimulus experienced at birth. The model also showed that the broadened proliferation zone induced by tumorigenic radiation can be attributed to cells responding to lower Wnt concentrations and dividing at smaller volumes. Application of the model to data from irradiated crypts after an extended recovery period permitted deductions about the extent of the initial insult. Application of computational modeling to experimental data revealed how mechanisms that control cell dynamics are altered at the earliest stages of carcinogenesis.

Opposing actions of the synapse-associated protein of 97-kDa molecular weight (SAP97) and Disrupted in Schizophrenia 1 (DISC1) on Wnt/ß-catenin signaling.

It has been suggested that synapse-associated protein of 97-kDa molecular weight (SAP97) is a susceptibility factor for childhood and adult neuropsychiatric disorders. SAP97 is a scaffolding protein that shares direct and indirect binding partners with the Disrupted in Schizophrenia 1 (DISC1) gene product, a gene with strong association with neuropsychiatric disorders. Here we investigated the possibility that these two proteins converge upon a common molecular pathway. Since DISC1 modifies Wnt/ß-catenin signaling via changes in glycogen synthase kinase 3 beta (GSK3ß) phosphorylation, we asked if SAP97 impacts Wnt/ß-catenin signaling and GSK3ß phosphorylation. We find that SAP97 acts as inhibitor of Wnt signaling activity and can suppress the stimulatory effects of DISC1 on ß-catenin transcriptional activity. Reductions in SAP97 abundance also decrease GSK3ß phosphorylation. In addition, we find that over expression of DISC1 leads to an increase in the abundance of SAP97, by inhibiting its proteasomal degradation. Our findings suggest that SAP97 and DISC1 contribute to maintaining Wnt/ß-catenin signaling activity within a homeostatic range by regulating GSK3ß phosphorylation.

Defining the role of APC in the mitotic spindle checkpoint in vivo: APC-deficient cells are resistant to Taxol.

Mutations in the adenomatous polyposis coli (APC) tumour suppressor are the key initiating event of colorectal cancer. Although the control of WNT signalling is well established as a central tumour-suppressive function, the significance of APC in regulating chromosome instability is less well established. In this study, we test whether APC-deficient cells have a functional spindle assembly checkpoint (SAC) in vivo by examining the response of these cells to Taxol and Vinorelbine. We also show for the first time that APC deficiency compromises the arrest response to Taxol in vivo. This effect is independent of the role that APC has in WNT signalling. At higher levels of Taxol, APC-deficient cells arrest as efficiently as wild-type cells. Importantly, this dose of Taxol strongly suppresses intestinal tumourigenesis in models of benign (APC(Min/+) mouse) and invasive (AhCreER(+)APC(fl/+)PTEN(fl/fl)) cancer. In contrast to intestinal enterocytes with a general SAC defect because of Bub1 (budding uninhibited by benzimidazole 1) deletion, APC-deficient enterocytes arrest equivalently to wild type when treated with Vinorelbine. This suggests that the failed arrest in response to Taxol is because of a specific defect in microtubule stabilization following Taxol treatment rather than a general role of the APC protein in the mitotic spindle checkpoint. In summary, this study clarifies the role of APC as a mitotic spindle checkpoint protein in vivo and shows that APC-deficient cells have a compromised response to Taxol.

Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy.

Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 mum within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of

Prognostic and therapeutic implications of Apc mutations in colorectal cancer.

The adenomatous polyposis coli gene (Apc) is mutated in most colorectal cancers. The multifunctional character of the Apc protein in the regulation of beta-catenin-mediated gene transcription and cytoskeletal proteins has been well described. An important question is how this protein affects the behaviour of cells within a tumour and how its mutational status influences the prognosis for these tumours. Here we provide an overview of the functions of Apc and examine how this information can be used in the prognosis and development of directed therapy in colorectal cancer.

Relationship between the role of the adenomatous polyposis coli protein in colon cancer and its contribution to cytoskeletal regulation.

A unique feature of colon cancer is that truncation mutations in the APC (adenomatous polyposis coli) gene are common to most tumours. The high penetrance of APC mutations, especially in gut epithelium, supports the idea that APC may be involved in a number of the processes that govern the normal maintenance of this tissue: differentiation, migration, proliferation and apoptosis. Indeed, APC is involved in the regulation of beta-catenin and it also is an important regulator of the cytoskeleton. Thus mutations in APC lead to the accumulation of beta-catenin, which causes changes in differentiation, and they also produce changes in cytoskeletal organization, which results in altered cell migration and disrupted mitotic spindles. The function of APC in cytoskeletal organization is related to its effect on microtubules and F-actin. Depleting APC from cultured cells leads to changes in cytoskeletal organization. In addition, N-terminal fragments of APC, like those commonly found in tumours, compromise cell migration in Dictyostelium and in early developing chicken embryos. Consistent with the idea that such dominant effects are normally balanced by interactions within the full-length molecule, protein interactions of N-terminal fragments expressed in tumour cells can be altered by binding to C-terminal regions of APC commonly lost in tumours. This review summarizes effects of APC on the cytoskeleton and discusses how these functions of APC may contribute to its role in cancer.

The adenomatous polyposis coli protein: in the limelight out at the edge.

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial and sporadic colonic tumours. APC is best known for its role in regulating beta-catenin, an important mediator of cell adhesion and a transcriptional activator. However, recent studies indicate that APC has additional roles in cytoskeletal regulation. It binds to microtubules directly and indirectly. Furthermore, indirect connections between APC and the actin cytoskeleton have also been described. Here, we integrate recent information describing the association between APC and the cytoskeleton to illustrate how this multifaceted protein might link different cytoskeletal elements to each other and to cellular signaling pathways.

A role for the Adenomatous Polyposis Coli protein in chromosome segregation.

Mutations in the Adenomatous Polyposis Coli (APC) gene are responsible for familial colon cancer and also occur in the early stages of sporadic colon cancer. APC functions in the Wnt signalling pathway to regulate the degradation of beta-catenin (reviewed in refs 1-3). APC also binds to and stabilizes microtubules in vivo and in vitro, localizes to clusters at the ends of microtubules near the plasma membrane of interphase cells, and is an important regulator of cytoskeletal function. Here we show that cells carrying a truncated APC gene (Min) are defective in chromosome segregation. Moreover, during mitosis, APC localizes to the ends of microtubules embedded in kinetochores and forms a complex with the checkpoint proteins Bub1 and Bub3. In vitro, APC is a high-affinity substrate for Bub kinases. Our data are consistent with a role for APC in kinetochore-microtubule attachment and suggest that truncations in APC that eliminate microtubule binding may contribute to chromosomal instability in cancer cells.

Developmental expression of catenins and associated proteins during submucosal gland morphogenesis in the airway.

Although lymphoid enhancer binding factor-1 (Lef-1) plays an obligatory role in airway submucosal gland (SMG) development, its expression alone is not an adequate signal for initiating gland morphogenesis. Because Lef-1 forms a bipartite transcription factor with beta-catenin to mediate wnt pathway signaling, we investigated the expression of beta-catenin and associated proteins during SMG development with both in situ hybridization and immunocytochemistry. Unexpectedly, high levels of E-cadherin mRNA were expressed by cells in developing gland buds from the earliest stages through subsequent differentiation into mature glands. In contrast, a decreased level of E-cadherin immunoreactivity in stage I gland bud cells suggested that post-translational modulation of E-cadherin protein levels may play a critical role in early stages of gland morphogenesis. Adenomatous polyposis coli (APC) mRNA was expressed relatively weakly in the developing ferret trachea, but higher levels of protein staining were observed throughout the cytoplasm of gland buds and surface epithelial cells. B-Catenin mRNA was abundantly expressed throughout the tracheal epithelium and at the highest levels in primordial gland buds. B-Catenin protein localized to the basolateral membranes of all airway epithelial cell types. However, no detectable increases in nuclear or cytoplasmic staining were associated with gland buds, as would be expected if beta-catenin served as a transcriptional cofactor for Lef-1 in gland morphogenesis. Additional studies demonstrated the gamma-catenin distribution to be remarkably similar to that of beta-catenin, whereas alpha-catenin staining was more diffuse in the cytoplasm of airway epithelial and gland bud cells. These descriptive results do not rule out a role for wnt signaling in SMG development , but provide no evidence that beta-catenin, or gamma-catenin, is a cofactor in Lef-1 regulation of SMG development.

Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation.

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.

A GSK3-binding peptide from FRAT1 selectively inhibits the GSK3-catalysed phosphorylation of axin and beta-catenin.

The Axin-dependent phosphorylation of beta-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects beta-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and beta-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of beta-catenin phosphorylation.

The adenomatous polyposis coli protein.

Mutations in the adenomatous polyposis coli (APC) gene are associated with most colorectal cancers. The APC protein has been implicated in many aspects of tumour development. This article will discuss recent data suggesting that APC may have multiple functions in the cell. First, APC is a component of the Wnt signalling pathway; second, APC may have a role in cell migration; finally, APC may regulate proliferation and apoptosis.

Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways.

Cadherins play important roles in cell-cell adhesion during tissue differentiation. Cadherins are linked to the actin cytoskeleton by catenins (beta-catenin/armadillo, plakoglobin, and alpha-catenin). Recent results show that beta-catenin also binds to another cytoskeletal complex containing the adenomatous polyposis coli protein and microtubules, and interacts with several signaling pathways that include tyrosine kinases and phosphatases and Wnt/Wingless. Interplay between these cytoskeletal complexes and signaling pathways may regulate morphogenesis.

The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration.

Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.

Beta-catenin: a common target for the regulation of cell adhesion by Wnt-1 and Src signaling pathways.

Beta-catenin is a cytosolic protein originally identified through its association with the cadherin class of cell-adhesion proteins. However, recent studies have demonstrated that there are cadherin-independent pools of beta-catenin and that beta-catenin binds at least one other protein, the product of the tumor-suppressor gene APC. Furthermore, beta-catenin is the target of two signal transduction pathways mediated by the proto-oncogenes src and wnt-1. This raises the possibility that beta-catenin plays a pivotal role in balancing cellular responses to both adhesive and proliferative signals.

Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.

Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed alpha-, beta-, and gamma-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/alpha-catenin, and either beta-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, beta-catenin, and plakoglobin cosedimented as complexes. alpha-Catenin was not associated with these complexes after synthesis, but a subpopulation of alpha-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin/beta-catenin, and cadherin/plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of alpha-catenin. Subsequently, > 90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells.

The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta-catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100-insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.

Epithelial cell adhesion and development of cell surface polarity: possible mechanisms for modulation of cadherin function, organization and distribution.

Epithelial cell adhesion is principally regulated by calcium-dependent cell adhesion proteins, termed cadherins. Recent studies indicate that cadherin function is modulated by a class of proteins, termed catenins, that bind to the cytoplasmic domain of cadherin. Here we review the evidence that catenins regulate cadherin function in cell-cell adhesion, and discuss their role in initiating cell surface polarity in epithelial cells.