SMART MODE:

NORMAL GENOMIC

• Ling Y, Zhong Y, Perez-Soler R
• Disruption of cell adhesion and caspase-mediated proteolysis of beta- and gamma-catenins and APC protein in paclitaxel-induced apoptosis.
• Mol Pharmacol. 2001; 59: 593-603
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• Cell adhesion is important in the regulation of cell proliferation, migration, survival, and apoptosis. The major components of cell adhesion are the cadherin family of proteins, alpha-, beta- and gamma-catenins, and cytoskeletons. In addition, beta-catenin, when associated with adenomatous polyposis coli (APC) protein, an oncosuppressor, is implicated in the regulation of beta-catenin/APC-related signaling pathways. To examine the correlation between impairment of cell adhesion events and apoptosis, we used human non-small-cell lung cancer H460 and H520 cell lines as models to determine whether paclitaxel-induced apoptosis is associated with disruption of the components of cell adhesion and their functions. Paclitaxel treatment resulted in cells rounding up and losing contact with their neighboring cells, suggesting that the drug does indeed affect cell adhesion and related events. Western blot analysis revealed that paclitaxel caused a time- and concentration-dependent cleavage of beta-catenin, gamma-catenin, and APC protein, but not alpha-catenin or E-cadherin. These cleavages of beta-catenin and gamma-catenin were apoptosis-dependent, not mitosis-dependent. Paclitaxel treatment led to the proteolysis and activation of caspase-3 and -7, but not caspase-1. Furthermore, paclitaxel-induced apoptosis and cleavage of beta-catenin and gamma-catenin were inhibited by the pan-caspase inhibitor Z-VAD-FMK and partially inhibited by the caspase-3 inhibitor Z-DEVD-FMK but were not affected by the caspase-1 inhibitor AC-YVAD-CMK. Although the pan-caspase inhibitor blocked the cleavage of beta-catenin as well as DNA fragmentation, it did not affect paclitaxel-induced M-phase arrest and only partially prevented cell-growth inhibition. Biochemical studies revealed that cleaved beta-catenin was detected only in the Triton X-100 insoluble fraction, suggesting that it might localize in nuclear and/or membrane structures. Interestingly, the paclitaxel-induced beta-catenin fragment lost its ability to bind to E-cadherin, alpha-catenin, or APC protein and to serve as a substrate for tyrosine kinase. All our data demonstrate that the caspase-mediated cleavage of beta-catenin, gamma-catenin, and APC protein might contribute to paclitaxel-induced apoptosis.

• Kobielak A, Kobielak K, Trzeciak WH
• A novel isoform of human lymphoid enhancer-binding factor-1 (LEF-1) gene transcript encodes a protein devoid of HMG domain and nuclear localization signal.
• Acta Biochim Pol. 2001; 48: 221-6
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• Lymphoid enhancer-binding factor-1 (LEF-1), a member of the high mobility group (HMG) family of proteins, regulates expression of T-cell receptor-alpha gene and is one of the key regulatory molecules in the epithelial-mesenchymal interactions during embryonic development. Among others, LEF-1 regulates expression of cytokeratin genes involved in formation of hair follicles and the gene encoding the cell-adhesion molecule E-cadherin. Transcription factor LEF-1, which acts as a dimer, binds beta-catenin and is involved in signal transduction by the wnt pathway. We have cloned and sequenced a novel isoform of human LEF-1 gene transcript. This isoform encodes a truncated protein devoid of HMG domain and nuclear localization signal but retaining beta-catenin binding domain. This isoform might either act in a dominant-negative manner by interfering with native LEF-1, or might bind beta-catenin in the cytosol, which would result in attenuation of the signals transmitted by the LEF-beta-catenin pathway.

• Ritchie TC et al.
• Developmental expression of catenins and associated proteins during submucosal gland morphogenesis in the airway.
• Exp Lung Res. 2001; 27: 121-41
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• 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.

• Reyes-Muigica M et al.
• Truncated DCC reduces N-cadherin/catenin expression and calcium-dependent cell adhesion in neuroblastoma cells.
• Lab Invest. 2001; 81: 201-10
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• The deleted in colorectal cancer (DCC) protein is important in the pathway guidance of cells and cell processes during neural development, and DCC has also been implicated in the aberrant cellular migrations of neuroblastoma dissemination. We attempted to further define DCC protein function by the overexpression of full-length and truncated DCC constructs in a human neuroblastoma cell line. Overexpression of the truncated DCC protein resulted in a less epithelioid morphology. This was accompanied by decreases in expression of N-cadherin and alpha- and beta-catenin by immunoblot and Northern blot analysis. Levels of desmoglein were relatively less affected, whereas endogenous DCC protein levels were increased in the truncated transfectants. N-cadherin immunofluorescence was consistent with the immunoblot studies and localized the protein to the cytoplasm and sites of cell-cell contact. Cell aggregation studies demonstrated diminished calcium-dependent aggregation in the truncated transfectants. In conclusion, overexpression of a truncated DCC protein in neuroblastoma cells resulted in the loss of an epithelioid morphology, diminished expression of N-cadherin and alpha- and beta-catenin, and diminished calcium-dependent cell adhesion. These studies provide the first evidence of an apparent functional link between DCC and N-cadherin/catenin-dependent cell adhesion.

• Natarajan L, Witwer NE, Eisenmann DM
• The Divergent Caenorhabditis elegans beta-Catenin Proteins BAR-1, WRM-1 and HMP-2 Make Distinct Protein Interactions but Retain Functional Redundancy in Vivo.
• Genetics. 2001; 159: 159-72
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• beta-Catenins function both in cell adhesion as part of the cadherin/catenin complex and in Wnt signal transduction as transcription factors. Vertebrates express two related proteins, beta-catenin and plakoglobin, while Drosophila has a single family member, Armadillo. Caenorhabditis elegans expresses three beta-catenin-related proteins, BAR-1, HMP-2, and WRM-1, which are quite diverged in sequence from each other and other beta-catenins. While BAR-1 and WRM-1 are known to act in Wnt-mediated processes, and HMP-2 acts in a complex with cadherin/alpha-catenin homologs, it is unclear whether all three proteins retain the other functions of beta-catenin. Here we show that BAR-1, like vertebrate beta-catenin, has redundant transcription activation domains in its amino- and carboxyl-terminal regions but that HMP-2 and WRM-1 also possess the ability to activate transcription. We show via yeast two-hybrid analysis that these three proteins display distinct patterns of protein interactions. Surprisingly, we find that both WRM-1 and HMP-2 can substitute for BAR-1 in C. elegans when expressed from the bar-1 promoter. Therefore, although their mutant phenotypes and protein interaction patterns strongly suggest that the functions of beta-catenin in other species have been segregated among three diverged proteins in C. elegans, these proteins still retain sufficient similarity to display functional redundancy in vivo.

• Huber AH, Stewart DB, Laurents DV, Nelson WJ, Weis WI
• The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover.
• J Biol Chem. 2001; 276: 12301-9
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• Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.

• Wakita K, Tetsu O, McCormick F
• A mammalian two-hybrid system for adenomatous polyposis coli-mutated colon cancer therapeutics.
• Cancer Res. 2001; 61: 854-8
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• Colon cancer cells frequently lose expression of the tumor suppressor adenomatous polyposis coli (APC). As result, beta-catenin accumulates and activates transcription of Tcf-responsive genes. Here we describe a novel mammalian two-hybrid system that selectively kills APC-mutated cells. This system consists of GAL4/beta-catenin, VP16/Tcf4, and a gene that is transcribed when GAL4 and VP16 associate. In APC-mutated human colon cancer cells, such as SW480, GAL4/beta-catenin accumulates, and in the presence of VP16/Tcf4, induces high levels of expression of the reporter gene. Expression of wild-type APC reduced GAL4/beta-catenin and intact beta-catenin levels and inhibited reporter gene expression. In colon cancer cells such as SW48 that have wild-type APC, GAL4/beta-catenin was degraded, and expression levels of the output gene were low. Replacement of the reporter gene with a suicide gene resulted in selective killing of SW480 cells. This system may be applicable for broader use of gene therapy by targeting diseases that involve protein degradation.

• Huber AH, Weis WI
• The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin.
• Cell. 2001; 105: 391-402
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• As a component of adherens junctions and the Wnt signaling pathway, beta-catenin binds cadherins, Tcf family transcription factors, and the tumor suppressor APC. We have determined the crystal structures of both unphosphorylated and phosphorylated E-cadherin cytoplasmic domain complexed with the arm repeat region of beta-catenin. The interaction spans all 12 arm repeats, and features quasi-independent binding regions that include helices which interact with both ends of the arm repeat domain and an extended stretch of 14 residues which closely resembles a portion of XTcf-3. Phosphorylation of E-cadherin results in interactions with a hydrophobic patch of beta-catenin that mimics the binding of an amphipathic XTcf-3 helix. APC contains sequences homologous to the phosphorylated region of cadherin, and is likely to bind similarly.

• Meigs TE, Fields TA, McKee DD, Casey PJ
• Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release.
• Proc Natl Acad Sci U S A. 2001; 98: 519-24
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• The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.

• Chen RH, McCormick F
• Selective targeting to the hyperactive beta-catenin/T-cell factor pathway in colon cancer cells.
• Cancer Res. 2001; 61: 4445-9
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• Many colon cancers suffer mutations in either the adenomatous polyposis coli or beta-catenin genes that lead to stabilization of beta-catenin and activation of downstream T-cell factor (Tcf) target genes. We have developed a novel approach targeting colon cancer cells based on their aberrant beta-catenin/Tcf signaling pathway. A recombinant adenovirus, in which an apoptosis gene fadd is under the control of the promoter containing Tcf-responsive elements, selectively and efficiently kills colon cancer cells in which the beta-catenin/Tcf pathway is hyperactivated. Our data therefore provide a conceptual proof that aberrantly activated Wnt/beta-catenin/Tcf pathways can be used to selectively target colon cancers.

• Loureiro JJ, Akong K, Cayirlioglu P, Baltus AE, DiAntonio A, Peifer M
• Activated armadillo/beta-catenin does not play a general role in cell migration and process extension in Drosophila.
• Dev Biol. 2001; 235: 33-44
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• Human beta-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that beta-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of beta-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivo in Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/beta-catenin, and that Armadillo/beta-catenin does not play a general role in inhibiting cell migration or process extension. Copyright 2001 Academic Press.

• Pellman D
• Cancer. A CINtillating new job for the APC tumor suppressor.
• Science. 2001; 291: 2555-6

• Lin YM et al.
• Identification of AF17 as a downstream gene of the beta-catenin/T-cell factor pathway and its involvement in colorectal carcinogenesis.
• Cancer Res. 2001; 61: 6345-9
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• To elucidate the molecular mechanism of colorectal carcinogenesis, we have been attempting to isolate genes involved in the beta-catenin/T-cell factor pathway. In the experiments reported here, analysis by cDNA microarray indicated that AF17, a fusion partner of the MLL gene in acute leukemias with t(11;17)(q23;q21), was transactivated according to accumulation of beta-catenin. Expression of AF17 was significantly enhanced in 8 of the 12 colorectal cancer tissues examined. Introduction of a plasmid designed to express AF17 stimulated growth of NIH3T3 cells, and fluorescence-activated cell sorter analysis indicated that the AF17 regulation of cell-cycle progression was occurring mainly at the G(2)-M transition. Our results suggest that the AF17 gene product is likely to be involved in the beta-catenin-T-cell factor/lymphoid enhancer factor signaling pathway and to function as a growth-promoting, oncogenic protein. These findings should aid development of new strategies for diagnosis, treatment, and prevention of colon cancers and acute leukemias by clarifying the pathogenesis of these conditions.

• Roh H, Green DW, Boswell CB, Pippin JA, Drebin JA
• Suppression of beta-catenin inhibits the neoplastic growth of APC-mutant colon cancer cells.
• Cancer Res. 2001; 61: 6563-8
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• Mutations involving the adenomatous polyposis coli (APC) tumor suppressorgene/beta-catenin signaling pathway have been identified in the majority of colon carcinomas. However, the role of aberrant beta-catenin signaling in the neoplastic growth of APC-mutant colon cancer cells has not been directly studied. To address this question, antisense oligonucleotides have been used to specifically down-regulate beta-catenin expression in APC-mutant human colon carcinoma cells. Antisense-mediated suppression of beta-catenin inhibits the in vitro proliferation, anchorage-independent growth, and cellular invasiveness of APC-mutant human colon carcinoma cells. The systemic administration of beta-catenin antisense oligonucleotides down-regulates beta-catenin expression in vivo in human colon cancer xenografts in nude mice. Such treatment inhibits the tumorigenic growth of colon cancer xenografts and can completely eradicate tumors in some treated animals. These studies formally demonstrate the critical role of beta-catenin signaling in the neoplastic growth of APC-mutant colon cancer cells and suggest that strategies targeting beta-catenin may be of use in the therapy of colon cancer.

• Bauer A et al.
• Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity.
• EMBO J. 2000; 19: 6121-30
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• In Wnt-stimulated cells, beta-catenin becomes stabilized in the cytoplasm, enters the nucleus and interacts with HMG box transcription factors of the lymphoid-enhancing factor-1 (LEF-1)/T-cell factor (TCF) family, thereby stimulating the transcription of specific target genes. We recently identified Pontin52 as a nuclear protein interacting with beta-catenin and the TATA-box binding protein (TBP), suggesting its involvement in regulating beta-catenin-mediated transactivation. Here, we report the identification of Reptin52 as an interacting partner of Pontin52. Highly homologous to Pontin52, Reptin52 likewise binds beta-catenin and TBP. Using reporter gene assays, we show that the two proteins antagonistically influence the transactivation potential of the beta-catenin-TCF complex. Furthermore, we demonstrate the evolutionary conservation of this mechanism in DROSOPHILA: dpontin and dreptin are essential genes that act antagonistically in the control of Wingless signalling in vivo. These results indicate that the opposite action of Pontin52 and Reptin52 on beta-catenin-mediated transactivation constitutes an additional mechanism for the control of the canonical Wingless/Wnt pathway.

• Ninomiya I et al.
• Alteration of beta-catenin expression in esophageal squamous-cell carcinoma.
• Int J Cancer. 2000; 85: 757-61
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• beta-catenin regulates cadherin-mediated cell-cell adhesion and also functions as a signaling molecule. In this study, we examined the expression pattern of E-cadherin, alpha-catenin and beta-catenin in 22 cases of esophageal squamous-cell carcinoma by Western-blot analysis. Expression of E-cadherin, alpha-catenin and beta-catenin was lower in carcinomas than in normal esophageal mucosa in 4 cases (18.2%) for E-cadherin, 6 cases (27.3%) for alpha-catenin and 9 cases (40.9%) for beta-catenin. Expression of beta-catenin was not always correlated with that of E-cadherin. Over-expression of beta-catenin was observed in 3 cases (13.6%). Of 3 cases that presented with over-expression of beta-catenin, 2 showed cytoplasmic staining by immunohistochemistry. Nuclear localization of beta-catenin was observed in one case that had higher beta-catenin level in tumor tissue (1.4-fold higher than normal mucosa). The genomic DNA sequences of the beta-catenin and the APC gene were analyzed. No mutation of the beta-catenin gene was observed in any cases. Silent mutation of the APC gene was found in all the cases that showed over-expression or nuclear localization of the beta-catenin protein. These results indicate that alterations of the cadherin-catenin complex may play an important role in a sub-set of esophageal carcinogenesis. Furthermore, it is suggested that beta-catenin over-expression is not caused by genetic alteration of either the beta-catenin or the APC gene.

• Clevers H
• Armadillo takes the APC shuttle.
• Nat Cell Biol. 2000; 2: 1778-1778

• Rowan AJ et al.
• APC mutations in sporadic colorectal tumors: A mutational "hotspot" and interdependence of the "two hits".
• Proc Natl Acad Sci U S A. 2000; 97: 3352-7
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• Although APC mutations occur at a high frequency in colorectal cancers, few studies have performed a comprehensive analysis by screening the whole gene for mutations and assessing allelic loss. APC seems to act as a tumor-suppressor gene in a "nonclassical" fashion: data from familial adenomatous polyposis (FAP) show that the site of the germ-line mutation determines the type of "second hit" in FAP tumors, and simple protein inactivation is selected weakly, if at all. In this study, we screened the entire coding region of APC for mutations and assessed allelic loss in a set of 41 colorectal cancer cell lines. Of 41 cancers, 32 (83%) showed evidence of APC mutation and/or allelic loss. We identified several APC mutations and found a "hotspot" for somatic mutation in sporadic colorectal tumors at codon 1,554. Our results suggest that APC mutations occur in the great majority of colorectal cancers, the exceptions almost all being RER+ tumors, which may substitute for altered APC function by mutations in beta-catenin and/or at other loci. When combined with previously published data, our results show that there is interdependence of the "two hits" at APC in sporadic colorectal tumors as well as in FAP. APC mutations in the "mutation cluster region," especially those close to codon 1,300, are associated with allelic loss, whereas tumors with mutations outside this region tend to harbor truncating mutations. The causes of this phenomenon are probably selection for retained N-terminal and lost C-terminal APC functions, effects on beta-catenin levels, and APC protein stability.

• Matsuda M
• Roles of beta-catenin in somitogenesis in rat embryos.
• In Vitro Cell Dev Biol Anim. 2000; 36: 593-9
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• We studied the roles of beta-catenin in somitogenesis using immunostaining and antisense experiments in rat embryos. High levels of beta-catenin appeared transiently in the developing rat somites. Initially, beta-catenin accumulation was observed in the core cells of presomitic cell aggregates and then in the lumen of epithelial vesicles. Subsequently, it was confined to the dermomyotomes and their lumen and then the myotomes. High levels of cyclin D1 were observed in the core cells, in the lumen of epithelial vesicles, in myotomes, and in mesenchymal sclerotomes. When embryos were cultured in medium supplemented with beta-catenin antisense oligodeoxynucleotide (ODN), the accumulation of beta-catenin, but not of cyclin D1, in the nascent somites and dermomyotomes was suppressed, while the number of somites was the same as that observed in control embryos. The number of myosin-positive somites and the amount of myosin per somite in embryos treated with the antisense ODN were lower than those in controls. These results suggested that beta-catenin promotes development of myotomal cells during somitogenesis. The function of beta-catenin in the development of myotomes may not be correlated to cyclin D1.

• Stewart DB, Barth AI, Nelson WJ
• Differential regulation of endogenous cadherin expression in Madin-Darby canine kidney cells by cell-cell adhesion and activation of beta -catenin signaling.
• J Biol Chem. 2000; 275: 20707-16
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• Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin-associated cytoplasmic proteins alpha- and beta-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997) J. Biol. Chem. 272, 29652-29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of beta-catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active beta-catenin mutants (DeltaGSK, DeltaN90, and DeltaN131). In these cells, while levels of E-cadherin, alpha- and beta-catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of DeltaGSK-beta-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post-translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype.

• Iwamoto M, Ahnen DJ, Franklin WA, Maltzman TH
• Expression of beta-catenin and full-length APC protein in normal and neoplastic colonic tissues.
• Carcinogenesis. 2000; 21: 1935-40
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• Mutations of the APC gene are thought to be early events in the process of colorectal carcinogenesis. Although the complete function(s) of the APC gene product is not known, it has been shown that the APC protein interacts with beta-catenin in a multi-protein complex to regulate the level of expression of beta-catenin. Loss of normal APC protein function can lead to an accumulation of beta-catenin in the cytosol and the nucleus. Immunohistochemical methods were used to determine the relationship between APC and beta-catenin protein expression in human colonic tissues (150 normal, 9 hyperplastic, 58 adenomas and 83 carcinomas) and 12 paired samples of normal and cancer tissue in mouse colon. In all samples of normal human and mouse colonic mucosa and in human hyperplastic polyps both APC and beta-catenin immunoreactivity were present in colonocytes. APC expression was cytoplasmic, with maximal immunoreactivity in the goblet cells. beta-Catenin expression was predominantly localized to the plasma membrane, with no nuclear immunoreactivity. APC immunoreactivity was absent in all of the mouse adenocarcinomas and 83% of the human colon cancers. All of the human and mouse carcinomas had nuclear and cytoplasmic beta-catenin expression. In contrast, only 29% of the 58 colonic adenomas were completely negative for APC immunoreactivity. Regardless of the presence or absence of APC, all of the adenomas had cytoplasmic and nuclear beta-catenin immunoreactivity. Many colonic adenomas retain expression of full-length APC protein whereas it is usually lost in colorectal cancers. Regardless of the status of APC protein expression, beta-catenin protein was found in the cytoplasm and nucleus of all neoplastic colonic mucosa. The dissociation between loss of expression of APC and accumulation of beta-catenin in the nucleus suggests that inactivation of both alleles of the APC gene may not be required for beta-catenin nuclear accumulation in colonic adenomas.

• Giannini AL, Vivanco MM, Kypta RM
• Analysis of beta-catenin aggregation and localization using GFP fusion proteins: nuclear import of alpha-catenin by the beta-catenin/Tcf complex.
• Exp Cell Res. 2000; 255: 207-20
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• beta-Catenin plays essential roles in cell adhesion, by associating with cadherins, and as a signaling molecule, by interacting with the Tcf/LEF-1 family of transcription factors. In order to study the protein-protein interactions of beta-catenin in living cells, we fused it to green fluorescent protein (GFP). GFP-beta-catenin was incorporated into cell junctions but also accumulated in the nucleus, where it formed rod-like structures. The carboxyl-terminal armadillo repeats of GFP-beta-catenin were sufficient for nuclear localization, but formation of rods required the armadillo repeats and sequences in both the amino- and the carboxyl-terminal domains. Rod formation was prevented by coexpression of N-cadherin, APC, and Tcf-4, which bind to the armadillo repeats of beta-catenin, but not by coexpression of alpha-catenin, although alpha-catenin expression did prevent accumulation of beta-catenin in the nucleus. Interestingly, when alpha-catenin, beta-catenin, and Tcf-4 were coexpressed they colocalized in the nucleus, and this correlated with a decrease in beta-catenin/Tcf-dependent transcriptional activity. These results indicate that binding of beta-catenin to Tcf-4 overrides the function of alpha-catenin to sequester beta-catenin in the cytoplasm and suggest that alpha-catenin can regulate beta-catenin signaling in the nucleus.

• Schlosshauer PW et al.
• APC truncation and increased beta-catenin levels in a human breast cancer cell line.
• Carcinogenesis. 2000; 21: 1453-6
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• Mutations in the Adenomatous Polyposis Coli (APC) tumor suppressor gene or the beta-catenin gene are present in most colon cancers and less frequently in other tumor types. In this study, we screened 24 human breast cancer cell lines and three immortalized human breast epithelial cell lines for alterations in beta- and gamma-catenin and APC by western blotting, protein truncation assay and DNA sequence analysis. In one cell line (DU 4475), an APC mutation was identified (E1577stop) that resulted in expression of truncated APC. This mutation was associated with elevated cytosolic beta-catenin levels, probably due to loss of APC function, as in colon cancers. No mutations were found in exon 3 of the beta- or gamma-catenin genes. We conclude that APC mutations and beta-catenin upregulation may occur with low frequency in human breast cancer cells.

• Tucker EL, Pignatelli M
• Catenins and their associated proteins in colorectal cancer.
• Histol Histopathol. 2000; 15: 251-60
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• Colorectal cancer is the second most common cause of cancer mortality in the western world. Colorectal cancer has been well studied, and the genetic steps involved in the adenoma to carcinoma sequence have been well elucidated. The first genetic alteration, found in 85% of adenomas, are mutations in the adenomatous polyposis coli (APC) gene. However, the consequences of this and the exact function of APC in the colon is not fully understood. It has been suggested that APC could function through its regulation of beta-catenin, an ubiquitous cytoskeletal protein with multiple binding specificities resulting in diverse functions including cell growth, adhesion, and migration. Any change in these associations may play a role in colorectal cancer development and progression.

• Norris AL et al.
• Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation.
• Eur J Cancer. 2000; 36: 525-32
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• Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.

• Wijnhoven BP, Dinjens WN, Pignatelli M
• E-cadherin-catenin cell-cell adhesion complex and human cancer.
• Br J Surg. 2000; 87: 992-1005
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• BACKGROUND: The E-cadherin-catenin complex plays a crucial role in epithelial cell-cell adhesion and in the maintenance of tissue architecture. Perturbation in the expression or function of this complex results in loss of intercellular adhesion, with possible consequent cell transformation and tumour progression. Recently, much progress has been made in understanding the interaction between the different components of this protein complex and how this cell-cell adhesion complex is modulated in cancer cells. METHODS: This is an update of the role of the E-cadherin-catenin complex in human cancers. It emphasizes new features and the possible role of the complex in clinical practice, discussed in the light of 165 references obtained from the Medline database from 1995 to 1999. RESULTS: More evidence is now appearing to suggest that disturbance in protein-protein interaction in the E-cadherin-catenin adhesion complex is one of the main events in the early and late steps of cancer development. An inverse correlation is found between expression of the E-cadherin-catenin complex and the invasive behaviour of tumour cells. Therefore, E-cadherin-catenin may become a significant prognostic marker for tumour behaviour. Besides its role in establishing tight cell-cell adhesion, beta- catenin plays a major role in cell signalling and promotion of neoplastic growth. This suggests its dual role as a tumour suppressor and as an oncogene in human cancers. CONCLUSION: Recent developments show that the E-cadherin-catenin complex is more than a 'sticky molecular complex'. Further studies may yield greater insight into the early molecular interactions critical to the initiation and progression of tumours. This should aid the development of novel strategies for both prevention and treatment of cancer.

• Brabletz T, Jung A, Dag S, Reu S, Kirchner T
• [beta-Catenin induces invasive growth by activating matrix metalloproteinases in colorectal carcinoma]
• Verh Dtsch Ges Pathol. 2000; 84: 175-81
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• beta-catenin was shown to be a major oncoprotein in colon cancer development. Its oncogenic function as a transcriptional activator is upregulated by mutations in the APC tumor suppressor gene, leading to a constitutive activation of the proliferation-associated genes c-myc and cyclin D. The aim of this study was to demonstrate a role of APC-mutations and dysregulated beta-catenin also for the progression of colorectal cancer, by identifying new target genes of beta-catenin associated with tumor invasion and metastasis. Potential invasion genes regulated by beta-catenin and its DNA binding partner TCF4 were identified by a computer search for the consensus DNA binding sequence in relevant promoter regions. Specific DNA binding was confirmed by gel shift assays. Functional importance of beta-catenin for the activation of identified genes was determined by luciferase reporter assays. The significance was demonstrated by coexpression of nuclear beta-catenin and the identified target genes by immunohistochemistry. Among other invasion genes, we identified the matrix metallo proteinases MMP-7 and MMP-1 activated by beta-catenin in the tumor cells. MMP-7 is an important factor for invasion and metastasis and overexpressed in 75% of colon carcinomas. The significance for human colon cancer development was demonstrated by a correlated overexpression of beta-catenin and the MMPs, beginning in large, severely dysplastic adenomas. Our results explain the high percentage of MMP-7 overexpression in colorectal tumors and the resulting activation of invasive growth. Moreover by identifying dysregulated beta-catenin as a transcriptional activator of MMPs and other invasion factors, we demonstrated an important role of mutated APC not only for early steps but also for the progression of colorectal carcinogenesis.

• Groden J
• Touch and go: mediating cell-to-cell interactions and Wnt signaling in gastrointestinal tumor formation.
• Gastroenterology. 2000; 119: 1161-4

• Weidner WJ, Waddell DS, Sillman AJ
• Low levels of cadmium chloride alter the immunoprecipitation of corneal cadherin-complex proteins.
• Arch Toxicol. 2000; 74: 578-81
• Display abstract

• The effect of cadmium chloride on the immunoprecipitation of cadherin and the associated adherens junctional proteins, alpha- and beta-catenin, was examined in isolated bullfrog (Rana catesbeiana) corneas utilizing Western blot and enhanced chemoluminescent techniques. Application of either 1.0 microM or 75.0 microM CdCl2 to the corneal endothelium for 2 h markedly decreased the immunoprecipitation of cadherins as compared to paired control corneas. Immunoprecipitation of alpha-catenin was increased in response to both doses of CdCl2, while the immunoprecipitation of beta-catenin was little changed by either cadmium dose. There is accumulating evidence that cadmium may increase epithelial paracellular permeability by interfering with cadherin complex activity at intercellular junctions. The present study suggests that inorganic cadmium in low micromolar concentrations may decrease the integrity of the corneal endothelium, at least in part through a similar mechanism involving disruption of junctional cadherin complex function.

• Kuhnen C et al.
• APC and beta-catenin in alveolar soft part sarcoma (ASPS)--immunohistochemical and molecular genetic analysis.
• Pathol Res Pract. 2000; 196: 299-304
• Display abstract

• Apart from its role in cell-adhesion, beta-catenin is regarded as an oncoprotein, the cytoplasmic level of which is regulated by APC as a tumor suppressor protein. Changes of chromosome 5q, the region that includes the APC-gene, are known to be important in the pathogenesis of fibromatosis; however, little is known about the significance of APC and beta-catenin in other mesenchymal tumors. Therefore, we used immunohistochemistry and DNA-analysis to investigate four cases of alveolar soft-part sarcoma (ASPS) as a mesenchymal tumor with a distinct histologic appearance. In three cases of ASPS the APC-gene product was found to have strong nuclear expression and only faint cytoplasmic staining. Beta-catenin showed a partly membranous, partly strong intracytoplasmic expression. No gene mutations for APC and beta-catenin were detected in any of the four cases. These investigations suggest that, apart from their function in carcinogenesis and fibromatoses, APC and beta-catenin play a role in the pathogenesis of soft tissue tumors such as ASPS. The significance of a striking nuclear accumulation of non-mutated, virtually functionally active APC-tumor suppressor protein has not yet been investigated. A nuclear function of APC in ASPS in down-regulating nuclear transcription processes linked to overexpression of beta-catenin, as is known in colorectal carcinogenesis, may be hypothesized.

• Hiscox S, Jiang WG
• Association of the HGF/SF receptor, c-met, with the cell-surface adhesion molecule, E-cadherin, and catenins in human tumor cells.
• Biochem Biophys Res Commun. 1999; 261: 406-11
• Display abstract

• Tumour cell metastatic potential is significantly enhanced following treatment with HGF/SF, the ligand for the c-met receptor tyrosine kinase. Following c-met activation in tumour cells, phosphorylation of beta-catenin occurs, together with loss of intercellular adhesion and a gain in the motile and invasive nature of the cell. In this study we show that c-met is co-localised with beta-catenin and E-cadherin at regions of cell-cell contact in human colon cancer (HRT18 and HT115) and two breast cancer (MCF7 and MDA MB 231) cell lines. Immunoprecipitation studies demonstrated an association between c-met and members of the cadherin adhesion complex in these epithelial tumour cells, along with the membrane tyrosine protein phophatase, PTPmu. We conclude that the HGF/SF receptor, c-met, together with members of the cadherin/catenin cell-cell adhesion system and PTPmu, may form part of a protein complex in E-cadherin positive tumour cells that acts to regulate intercellular adhesion following HGF/SF stimulation.

• El-Hariry I, Jordinson M, Lemoine N, Pignatelli M
• Characterization of the E-cadherin-catenin complexes in pancreatic carcinoma cell lines.
• J Pathol. 1999; 188: 155-62
• Display abstract

• E-cadherin and its associated cytoplasmic proteins alpha-, beta-, and gamma-catenins play important roles in cell adhesion and signal transduction, as well as in maintenance of the structural and functional organization of polarized epithelial cells. In this study, the expression, distribution, and complex assembly of catenins with E-cadherin was analysed at the steady state in a panel of human pancreatic adenocarcinoma cell lines (BxPc3, HPAF, T3M4, and PaTuII cell lines). The expression and subcellular distribution were determined by western blotting and immunocytochemistry. Co-immunoprecipitation and cross-linking studies were performed to examine the complex assembly in both Triton X-100 (TX-100)-soluble and -insoluble fractions. In BxPc3 and T3M4 cells, E-cadherin exists in two complexes, one with alpha- and gamma-catenin, and the other with beta-catenin alone. In HPAF cells there are two complexes, one consisting of E-cadherin with alpha- and beta-catenin, and another of E-cadherin with gamma-catenin. In PaTuII cells, there is only a single complex of E-cadherin with alpha-catenin and gamma-catenin. Modification of E-cadherin-catenin complexes in HPAF and PaTuII cells was associated with loss of membranous E-cadherin immunolocalization. The common denominator is impaired beta-catenin association with either E-cadherin (PaTuII) or alpha-catenin (BxPc3 and T3M4). This may suggest the presence of distinct mechanisms that modulate the assembly of each complex, which could disturb the tumour suppressor function of E-cadherin and the catenins.

• Soler C, Grangeasse C, Baggetto LG, Damour O
• Dermal fibroblast proliferation is improved by beta-catenin overexpression and inhibited by E-cadherin expression.
• FEBS Lett. 1999; 442: 178-82
• Display abstract

• Several recent studies have shown that proteins of the cadherin-catenin complex are not only involved in cell-cell adhesion but also in the proliferation and differentiation processes. For the first time, we investigated the effect of the quantity of cytoplasmic beta-catenin on dermal fibroblast proliferation by overexpressing human beta-catenin in human dermal fibroblasts. Our results show that dermal fibroblasts overexpressing normal beta-catenin or a stabilized beta-catenin mutant have a higher growth rate than control fibroblasts. Moreover, when confluence is reached, the number of fibroblasts is increased when the cells overexpress beta-catenin suggesting a role for beta-catenin in the regulation of contact growth arrest. Finally, by comparing proliferation in normal dermal fibroblasts and dermal fibroblasts expressing E-cadherin we observed a negative regulatory effect of E-cadherin expression on fibroblast proliferation. These data demonstrate the involvement of beta-catenin and cadherin in the dermal fibroblast proliferation process and in contact growth arrest.

• da Costa LT et al.
• CDX2 is mutated in a colorectal cancer with normal APC/beta-catenin signaling.
• Oncogene. 1999; 18: 5010-4
• Display abstract

• The majority of human colorectal cancers have elevated beta-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of beta-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and beta-catenin and no demonstrable beta-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These findings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive effects.

• Omer CA, Miller PJ, Diehl RE, Kral AM
• Identification of Tcf4 residues involved in high-affinity beta-catenin binding.
• Biochem Biophys Res Commun. 1999; 256: 584-90
• Display abstract

• The N-termini of members of the T-cell factor (Tcf) and lymphocyte-enhancement factor (Lef) protein families bind to beta-catenin, forming bipartite transcription factors which regulate expression of genes involved in organismal development and the growth of normal and malignant colon epithelium. Elevated levels of Tcf4:beta-catenin are found in colon tumor cells with mutations in the adenomatous polyposis coli (APC) gene. The elevated levels of Tcf4:beta-catenin result in increased transcription of genes, including c-myc, important for the growth of these tumor cells. Here we analyze the interaction between beta-catenin and Tcf4 and show that the N-terminal 53 amino acids of Tcf4 bind with high affinity to beta-catenin. We show that this high-affinity interaction involves multiple contact points including Tcf4 Asp-16, which is essential for beta-catenin binding. In addition to Tcf/Lef family members, beta-catenin binds to APC and cadherins. We found that the binding of beta-catenin to Tcf4, APC, or E-cadherin was mutually exclusive. These results are discussed with regard to how beta-catenin interacts with its binding partners and to the potential for identifying specific, small molecule inhibitors of these interactions.

• Bierkamp C, Schwarz H, Huber O, Kemler R
• Desmosomal localization of beta-catenin in the skin of plakoglobin null-mutant mice.
• Development. 1999; 126: 371-81
• Display abstract

• Plakoglobin, a protein belonging to the Armadillo-repeat gene family, is the only component that adherens junctions and desmosomes have in common. Plakoglobin null-mutant mouse embryos die because of severe heart defects and may exhibit an additional skin phenotype, depending on the genetic background. Lack of plakoglobin affects the number and structure of desmosomes, resulting in visible defects when cells are subjected to increasing mechanical stress, e.g. when embryonic blood starts circulating or during skin differentiation. By analysing plakoglobin-negative embryonic skin differentiation in more detail, we show here that, in the absence of plakoglobin, its closest homologue, beta-catenin, becomes localized to desmosomes and associated with desmoglein. This substitution may account for the relatively late appearance of the developmental defects seen in plakoglobin null-mutant embryos. beta-catenin cannot, however, fully compensate a lack of plakoglobin. In the absence of plakoglobin, there was reduced cell-cell adhesion, resulting in large intercellular spaces between keratinocytes, subcorneal acantholysis and necrosis in the granular layer of the skin. Electron microscopic analysis documented a reduced number of desmosomes, and those present lacked the inner dense plaque and had fewer keratin filaments anchored. Our analysis underlines the central role of plakoglobin for desmosomal assembly and function during embryogenesis.

• Blaker H, Hofmann WJ, Rieker RJ, Penzel R, Graf M, Otto HF
• Beta-catenin accumulation and mutation of the CTNNB1 gene in hepatoblastoma.
• Genes Chromosomes Cancer. 1999; 25: 399-402
• Display abstract

• Hepatoblastoma is a rare malignant tumor of the liver that occurs in children at an average age of 2 to 3 years. Epidemiologic studies have shown an increased frequency of this tumor type in families affected by adenomatous polyposis coli. In addition to the epidemiologic data, molecular genetic studies suggest that inactivation of the APC tumor suppressor may be involved in hepatoblastoma tumorigenesis. A major function of APC is the downregulation of beta-catenin, a transcription-activating protein with oncogenic potential. In an ongoing immunohistochemical study of beta-catenin expression in sporadic cases of tumor types that are associated with adenomatous polyposis coli, we observed increased beta-catenin levels in the cytoplasm and in the nuclei of three investigated hepatoblastomas. Sequencing of exon 3 of the beta-catenin gene (CTNNB1) revealed an activating mutation in one of the tumor samples. Our data indicate for the first time that beta-catenin accumulation may play a role in the development of hepatoblastoma and that activating mutations of the beta-catenin gene may substitute biallelic APC inactivation in this tumor type. Genes Chromosomes Cancer 25:399-402, 1999.

• Kucerova D, Sloncova E, Tuhackova Z, Uhlirova M, Kos M, Sovova V
• Changes of E-cadherin and beta-catenin levels during induced differentiation of colorectal carcinoma cells.
• Int J Mol Med. 1999; 4: 541-4
• Display abstract

• Following the finding of a great increase in cell-cell adhesion in several colorectal carcinoma cell lines after induced differentiation, the expression of E-cadherin-catenin complexes was analyzed. The sensitivity of cell lines to the differentiation induced by sodium butyrate differed. Nevertheless, all cells growing for 5 days in the medium containing 2 mM sodium butyrate changed their morphology and adherent properties. The expression of E-cadherin and catenins participating in its function were analyzed. A significant increase in E-cadherin level after butyrate treatment was found in HT29 and LS174T cell lines only. However, a high decrease in beta-catenin level was detected in all cell lines treated with butyrate. Further analysis showed regulation of beta-catenin at the level of mRNA.

• Morin PJ
• beta-catenin signaling and cancer.
• Bioessays. 1999; 21: 1021-30
• Display abstract

• Since its discovery as a protein associated with the cytoplasmic region of E-cadherin, beta-catenin has been shown to perform two apparently unrelated functions: it has a crucial role in cell-cell adhesion in addition to a signaling role as a component of the Wnt/wg pathway. Wnt/wg signaling results in beta-catenin accumulation and transcriptional activation of specific target genes during development. It is now apparent that deregulation of beta-catenin signaling is an important event in the genesis of a number of malignancies, such as colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, and prostate cancer. beta-catenin mutations appear to be a crucial step in the progression of a subset of these cancers, suggesting an important role in the control of cellular proliferation or cell death. The APC/beta-catenin pathway is highly regulated and includes players such as GSK3-beta, CBP, Groucho, Axin, Conductin, and TCF. c-MYC and cyclin D1 were recently identified as a key transcriptional targets of this pathway and additional targets are likely to emerge. Published 1999 John Wiley & Sons, Inc.

• Behrens J
• Cadherins and catenins: role in signal transduction and tumor progression.
• Cancer Metastasis Rev. 1999; 18: 15-30
• Display abstract

• Cadherins are transmembrane cell-cell adhesion molecules which are connected to the cytoskeleton by association with the cytoplasmic proteins, alpha-, beta-, and, gamma-catenin (plakoglobin). Beta-catenin has an additional role in the wnt signal transduction pathway in which it transmitts signals to the cell nucleus in complexes with transcription factors of the LEF-1/TCF family. The cell adhesion function of the epithelial E-cadherin is frequently disturbed in carcinomas either by downregulation or by mutation of the E-cadherin/catenin genes. The signaling function of beta-catenin is activated in tumors by mutations of beta-catenin or of the tumor suppressor gene product APC. In this review I will give an introduction to the structure and function of the cadherin/catenin complex and summarize findings which support a decisive role of these components in the development of cancer.

• Debruyne P, Vermeulen S, Mareel M
• The role of the E-cadherin/catenin complex in gastrointestinal cancer.
• Acta Gastroenterol Belg. 1999; 62: 393-402
• Display abstract

• Cancer is a genetic disease. The unstable genome of cancer cells causes tumour progression through multiple alterations in suppressor and promoter genes, leading to loss of homeostatic and gain of oncogenic functions. Invasion is the critical step in the acquisition of malignancy. It implicates a continuous molecular conversation of the cancer cells with other cells and with the extracellular matrix in which adhesion molecules are crucial. One of these, E-cadherin, is discussed in the present review. E-cadherin is a transmembrane glycoprotein that forms a complex with cytoplasmic proteins, termed catenins because they link E-cadherin to the actin cytoskeleton. E-cadherin/catenin-mediated intercellular adhesion and communication is mainly homophylic homotypic. There is compelling evidence from experiments in vitro as well as in vivo to accept that the E-cadherin/catenin complex acts as an invasion suppressor. The mechanism of this action is not only through cell-cell adhesion but also through transduction of signals to the cell's motility system. In the replication error positive human colon cancer cell line HCT-8, the alpha E-catenin gene CTNNA1 is an invasion suppressor gene. Here, the transition from the non-invasive to the invasive state was prevented by introduction into the unstable non-invasive cells of either an extra CTNNA1 or a wild type hMSH6 mismatch repair gene. beta-catenin also participates at a complex which comprises the adenomatous polyposis cancer protein APC. In colorectal cancer, mutation of either APC or beta-catenin is oncogenic. Downregulation of the E-cadherin/catenin complex may occur in several ways amongst which are gene mutations, methylation of 5'CpG dinucleotides within the promotor region of E-cadherin, tyrosine phosphorylation of beta-catenin, cell surface expression of proteoglycans sterically hindering E-cadherin and proteolytic release of fragments from the extracellular part of E-cadherin. Upregulation of the E-cadherin/catenin complex has been realized with a series of agents, some of which can be used therapeutically. In most human gastrointestinal cancers the E-cadherin/catenin or related complexes are disturbed and this underscores their pivotal role in the progression of these tumours. Mutations of the E-cadherin gene, including germline mutations, occur in diffuse gastric carcinoma, CpG methylation around the promotor region of E-cadherin in hepatocellular carcinomas and mutations of the APC tumour suppressor gene or in the beta-catenin oncogene in most colorectal cancers. The literature agrees about the disturbance of immunohistochemical patterns of E-cadherin and catenin expression in gastrointestinal cancers. Conflicting opinions do, however, exist about the prognostic value of such immunohistochemical aberrations. We doubt that immunohistochemistry of E-cadherin or catenins add prognostic value to the already used histological grading systems. In our opinion the major benefit from understanding of the E-cadherin/catenin-mediated pathways of invasion will be the development of new anti-invasive treatment strategies.

• Jaiswal AS, Narayan S
• Protein synthesis inhibitor-mediated stability of adenomatous polyposis coli mRNA levels in HCT-116 colon cancer cells.
• Int J Oncol. 1999; 14: 1045-8
• Display abstract

• We examined the effect of a protein synthesis inhibitor, cycloheximide (CHX) on the adenomatous polyposis coli (APC) mRNA levels in HCT-116 colon cancer cell line. The HCT-116 cells were treated with different concentrations of CHX for 15 h. APC, p53 and beta-actin mRNA levels were determined by Northern blotting. Results showed that APC and beta-actin mRNA levels were significantly increased in a dose-dependent manner after CHX treatment. The p53 mRNA levels were moderately increased. The increase in APC mRNA levels after CHX treatment was due to increase in its stability instead of transcription. These results provide a model for CHX-induced APC mRNA stabilization and its implication in cell cycle arrest and cell survival studies.

• Wijnhoven BP, Pignatelli M
• E-cadherin-catenin: more than a "sticky" molecular complex.
• Lancet. 1999; 354: 356-7

• Romagnolo B et al.
• Intestinal dysplasia and adenoma in transgenic mice after overexpression of an activated beta-catenin.
• Cancer Res. 1999; 59: 3875-9
• Display abstract

• Mutations in the adenomatous polyposis coli gene or activating mutations in the beta-catenin gene itself are thought to be responsible for the excessive beta-catenin signaling involved in intestinal carcinogenesis. We generated transgenic mice that expressed large amounts of a NH2-terminally truncated mutant beta-catenin (deltaN131beta-catenin) in the intestine. These mice had multifocal dysplastic lesions in the small intestine, reminiscent of the early lesions observed in the mouse models of familial adenomatous polyposis. The number of apoptotic cells in the villi of these transgenic mice was 3-4-fold higher than in nontransgenic mice. Expression of the truncated beta-catenin mutant in the kidney led to the development of severe polycystic kidney disease. Our findings support the concept that deregulation of the beta-catenin signaling pathway is the major oncogenic consequence of adenomatous polyposis coli mutations in intestinal neoplasia.

• Kimura Y et al.
• Cytoplasmic beta-catenin in esophageal cancers.
• Int J Cancer. 1999; 84: 174-8
• Display abstract

• beta-Catenin has 2 distinct roles in E-cadherin-mediated cell adhesion and carcinogenesis through APC gene mutation. One occurs at cell-adhesion sites, where cadherins become linked to the actin-based cytoskeleton. The others occur in the cytoplasm and nuclei and are thought to regulate cell transformation. We studied these different beta-catenins and evaluated their significance in carcinogenesis. Fresh surgical specimens were obtained from 22 patients with squamous-cell carcinoma of the esophagus. beta-Catenin in the free soluble fraction and the insoluble fraction was immunoblotted separately. At the same time, its localization was observed by immuno-histochemical techniques. In the normal esophageal epithelium, 91% of beta-catenin was detected in the insoluble fraction and beta-catenin staining occurred at the cell membrane, in co-existence with E-cadherin. In cancerous tissues, the amount of soluble beta-catenin was significantly (about 4-fold) higher than in normal tissues. Also, in cancerous tissues with higher amounts of soluble beta-catenin, immuno-histochemical techniques revealed the presence of beta-catenin in the cytoplasm and nuclei, as well as in the cell membrane. However, in samples with lower amounts of beta-catenin, expression was found only at the cell boundaries. The amount of soluble beta-catenin was not associated with the clinico-pathological grading of the tumors. Our results show that the accumulation of free soluble beta-catenin in the cytoplasm and nuclei frequently occurs during carcinogenesis of the squamous epithelium of the esophagus.

• Hecht A, Litterst CM, Huber O, Kemler R
• Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA-binding protein in vitro.
• J Biol Chem. 1999; 274: 18017-25
• Display abstract

• beta-Catenin, a member of the family of Armadillo repeat proteins, plays a dual role in cadherin-mediated cell adhesion and in signaling by Wnt growth factors. Upon Wnt stimulation beta-catenin undergoes nuclear translocation and serves as transcriptional coactivator of T cell factor DNA-binding proteins. Previously the transactivation potential of different portions of beta-catenin has been demonstrated, but the precise location of transactivating elements has not been established. Also, the mechanism of transactivation by beta-catenin and the molecular basis for functional differences between beta-catenin and the closely related proteins Armadillo and Plakoglobin are poorly understood. Here we have used a yeast system for the detailed characterization of the transactivation properties of beta-catenin. We show that its transactivation domains possess a modular structure, consist of multiple subelements that cover broad regions at its N and C termini, and extend considerably into the Armadillo repeat region. Compared with beta-catenin the N termini of Plakoglobin and Armadillo have different transactivation capacities that may explain their distinct signaling properties. Furthermore, transactivating elements of beta-catenin interact specifically and directly with the TATA-binding protein in vitro providing further evidence that a major function of beta-catenin during Wnt signaling is to recruit the basal transcription machinery to promoter regions of Wnt target genes.

• Krufka A, Johnson RG, Wylie CC, Heasman J
• Evidence that dorsal-ventral differences in gap junctional communication in the early Xenopus embryo are generated by beta-catenin independent of cell adhesion effects.
• Dev Biol. 1998; 200: 92-102
• Display abstract

• Gap junctional communication (GJC) is regulated in the early Xenopus embryo and quantitative differences in junctional communication correlate with the specification of the dorsal-ventral axis. To address the mechanism that is responsible for regulating this differential communication, we investigated the function of beta-catenin during the formation of the dorsal-ventral axis in Xenopus embryos by blocking its synthesis with antisense oligodeoxynucleotides. This method has previously been shown to reduce the level of beta-catenin in the early embryo, prior to zygotic transcription, and to inhibit the formation of the dorsal axis (Heasman et al., 1994, Cell 79, 791-803). We show here that antisense inhibition of beta-catenin synthesis also reduces GJC among cells in the dorsal hemisphere of 32-cell embryos to levels similar to those observed among ventral cells. Full-length beta-catenin mRNA can restore elevated levels of dorsal GJC when injected into beta-catenin-deficient oocytes, demonstrating the specificity of the beta-catenin depletion with the antisense oligonucleotides. Thus, endogenous beta-catenin is required for the observed differential GJC. This regulation of GJC is the earliest known action of the dorsal regulator, beta-catenin, in Xenopus development. Two lines of evidence, presented here, indicate that beta-catenin acts within the cytoplasm to regulate GJC, rather than through an effect on cell adhesion. First, when EP-cadherin is overexpressed and increased adhesion is observed, embryos display both a ventralized phenotype and reduced dye transfer. Second, a truncated form of beta-catenin (i.e., the ARM region), that lacks the cadherin-binding domain, restores dorsal GJC to beta-catenin-depleted embryos. Thus, beta-catenin appears to regulate GJC independent of its role in cell-cell adhesion, by acting within the cytoplasm through a signaling mechanism.

• Shibamoto S, Higano K, Takada R, Ito F, Takeichi M, Takada S
• Cytoskeletal reorganization by soluble Wnt-3a protein signalling.
• Genes Cells. 1998; 3: 659-70
• Display abstract

• BACKGROUND: Wnt-3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt-3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles. RESULTS: We prepared mouse L cells transfected with Wnt-3a cDNA, and found that their beta-catenin protein level was up-regulated. When conditioned medium (CM) was collected from cultures of the transfectants and added to nontransfected L cells, the beta-catenin level of the latter was also increased. Approximately 50% of the Wnt-3a proteins synthesized by the transfectants were secreted into the CM in a soluble form. These secreted Wnt-3a proteins formed an activity gradient in the environment surrounding the transfectants. Then, we studied whether Wnt-3a had any effect on cellular behaviour in vitro. When the CM containing Wnt-3a (W3a-CM) was added to cultures of C57MG mammary epithelial cells, their morphology was altered to exhibit closer intercellular contacts. Immunostaining for various adhesion and cytoskeletal proteins showed that the actin-microfilamental system was re-organized by the W3a-CM treatment. It induced a directional alignment of actin stress fibres and other actin-associated proteins. Moreover, villin, localized only at the perinuclear regions in untreated C57MG cells, was re-distributed to the leading edges of the cells, co-localizing with F-actin, in the presence of Wnt-3a. CONCLUSION: Our findings suggest that Wnt-3a protein, in the soluble form, can act to re-organize cytoskeletal structures.

• Sharma VA, Logan J, King DS, White R, Alber T
• Sequence-based design of a peptide probe for the APC tumor suppressor protein.
• Curr Biol. 1998; 8: 823-30
• Display abstract

• BACKGROUND: Proteins form specific associations, but predictive rules for protein pairing are generally unknown. Here, we describe amino-acid sequence patterns capable of mediating specific pairing of a widespread protein motif: the parallel, dimeric, alpha-helical coiled coil. The pairing rules were tested by designing a 54-residue peptide (anti-APCp1) that is predicted to dimerize preferentially with a coiled-coil sequence from the adenomatous polyposis coli (APC) tumor suppressor protein. RESULTS: As judged by circular dichroism, ultracentrifugation and native gel electrophoresis, anti-APCp1 formed a specific, helical, dimeric complex with the target APC coiled coil. On western blots of APC fragments expressed in Escherichia coli, the designed peptide detected a pattern of bands identical to the pattern detected by an antibody directed against the APC coiled coil. Peptide-mediated precipitation experiments showed that anti-APCp1 bound and sequestered wild-type and mutant APC proteins in extracts of human colon cancer cell lines. In addition, binding of the designed peptide preserved native APC-beta-catenin complexes. CONCLUSIONS: These biochemical experiments demonstrate that the anti-APC peptide preferentially forms a heterodimeric coiled coil with mutant and full-length APC proteins. The specificity of the designed peptide is sufficient to support several applications that commonly use antibodies. The observed specificity of anti-APCp1 validates the pairing rules used as the basis for the probe design, and it suggests that residues in the core positions of coiled coils help impart pairing selectivity.

• Ozawa M, Kemler R
• Altered cell adhesion activity by pervanadate due to the dissociation of alpha-catenin from the E-cadherin.catenin complex.
• J Biol Chem. 1998; 273: 6166-70
• Display abstract

• Leukemia cells (K562) that grow as non-adhesive single cells and have no endogenous cadherin were transfected with an E-cadherin expression vector, and cell clones stably expressing E-cadherin on their surface were established. The expression of E-cadherin induced the up-regulation of catenins, and E-cadherin became associated with catenins. The transfected cells grew as floating aggregates. Cell aggregation was Ca2+-dependent and was inhibited by E-cadherin antibodies. The aggregates dissociated into single cells on the addition of pervanadate. Pervanadate caused a dramatic augmentation of the phosphorylation of E-cadherin, beta-catenin, and gamma-catenin (plakoglobin), but alpha-catenin was not detectably phosphorylated. After pervanadate treatment, beta-catenin and gamma-catenin migrated more slowly on gel electrophoresis, suggesting changes in their conformations due to eventual changes in their phosphorylation levels. In the treated cells, a significant amount of alpha-catenin was dissociated from the E-cadherin.catenin complex. Aggregates of cells expressing an E-cadherin chimeric molecule covalently linked with alpha-catenin were not dissociated on pervanadate treatment, supporting the idea that the dissociation of alpha-catenin from the complex underlies the observed E-cadherin dysfunction.

• Parker HR, Li Z, Sheinin H, Lauzon G, Pasdar M
• Plakoglobin induces desmosome formation and epidermoid phenotype in N-cadherin-expressing squamous carcinoma cells deficient in plakoglobin and E-cadherin.
• Cell Motil Cytoskeleton. 1998; 40: 87-100
• Display abstract

• Pg is a homologue of beta-catenin and Armadillo, the product of the Drosophila segment polarity gene and has been shown to have both adhesive and signaling functions. It interacts with both classic and desmosomal cadherins. Pg interaction with the desmosomal cadherins is essential for desmosome assembly. Its precise role in the classic cadherin complexes is unclear, although Pg-E-cadherin interaction appears to be necessary for the formation of desmosomes. In addition to cadherins in adhesion complexes, Pg interacts with a number of proteins involved in regulation of cell differentiation and proliferation such as Lef-1/Tcf-1 transcription factors and the tumor suppressor protein APC. In this study, we have introduced Pg cDNA into SCC9 cells, a Pg- and E-cadherin-deficient squamous cell carcinoma line, which also lacks desmosomes. These cells have both alpha-catenin and beta-catenin, display unusual expression of N-cadherin, and have the typical fibroblastic phenotype of transformed cells. Pg-expressing SCC9 cells (SCC9P) formed desmosomes. Desmosome formation coincided with the appearance of an epidermoid phenotype, with increased adhesiveness and a contact-dependent decrease in growth. Biochemical characterization of SCC9P cells showed an increase in the expression and stability of N-cadherin and a decrease in level and stability of beta-catenin, without any apparent effects on alpha-catenin. These results show that, in the absence of E-cadherin, Pg can efficiently use N-cadherin to induce desmosome formation and epidermoid phenotype. They also suggest a role for Pg as one of the regulators of the intracellular beta-catenin levels and underscore the pivotal role of this protein in regulating cell adhesion and differentiation.

• Vermeulen SJ, Chen TR, Speleman F, Nollet F, Van Roy FM, Mareel MM
• Did the four human cancer cell lines DLD-1, HCT-15, HCT-8, and HRT-18 originate from one and the same patient?
• Cancer Genet Cytogenet. 1998; 107: 76-9
• Display abstract

• Four human colon cancer cell lines, HCT-8, HRT-18, DLD-1, and HCT-15, with an epithelioid morphotype reproducibly formed alpha-catenin-deficient round cells. Using DNA fingerprinting, we found that these four cell lines have an identical genetic background. Our finding strongly suggests a genetic background for the reproducible loss of alpha-catenin and the ensuing acquisition of invasiveness in all four cell lines.

• Tesco G, Kim TW, Diehlmann A, Beyreuther K, Tanzi RE
• Abrogation of the presenilin 1/beta-catenin interaction and preservation of the heterodimeric presenilin 1 complex following caspase activation.
• J Biol Chem. 1998; 273: 33909-14
• Display abstract

• beta-Catenin has previously been shown to interact with presenilin 1 (PS1) in transfected cells. Here we report that beta-catenin co-immunoprecipitates with the endogenous C-terminal fragment of presenilin 1 (PS1-CTF) but not with the endogenous CTF of presenilin 2 (PS2-CTF) in H4 human neuroglioma cells. During staurosporine (STS)-induced cell death, beta-catenin and PS1-CTF undergo a caspase-mediated cleavage. After 12 h of STS treatment, the beta-catenin.PS1-CTF interaction is abrogated. While PS1-CTF immunoprecipitated with all caspase-cleaved species of beta-catenin, beta-catenin holoprotein did not co-immunoprecipitate with the "alternative" caspase-derived PS1-CTF (PS1-aCTF). Thus, the abrogation of the beta-catenin.PS1-CTF complex was due to caspase cleavage of PS1-CTF. beta-Catenin co-immunoprecipitated with PS1-NTF, but only when PS1-NTF was associated with PS1-CTF. Even though PS1-NTF.CTF complex stability was not altered by caspase cleavage, its ability to bind beta-catenin was abolished. Thus, while the PS1-NTF.CTF complex is preserved after caspase cleavage, it may no longer be fully functional.

• Nelson RW, Gumbiner BM
• Beta-catenin directly induces expression of the Siamois gene, and can initiate signaling indirectly via a membrane-tethered form.
• Ann N Y Acad Sci. 1998; 857: 86-98
• Display abstract

• Beta-catenin is shown to directly induce the expression of siamois (a homeobox-containing gene involved in axial patterning) in a cell-autonomous, protein synthesis independent manner. Siamois can thus be considered a direct target of beta-catenin signaling in Xenopus. Expression of a portion of the armadillo repeat region of beta-catenin via a membrane-tethered fusion protein is shown to give similar levels of siamois induction and axis duplication as a free, untethered form. Reduction of endogenous free beta-catenin levels by overexpression of C-cadherin leads to complete inhibition of signaling by the membrane-tethered repeat region. Since the membrane-tethered repeat region is unlikely to be bound up by C-cadherin, these results show that the membrane-tethered beta-catenin relies on endogenous beta-catenin for signaling. We propose that the membrane-tethered construct acts by titrating a cytoplasmic inhibitor of beta-catenin signaling.

• Chitaev NA, Troyanovsky SM
• Adhesive but not lateral E-cadherin complexes require calcium and catenins for their formation.
• J Cell Biol. 1998; 142: 837-46
• Display abstract

• We examined intercadherin interactions in epithelial A-431 cells producing endogenous E-cadherin and recombinant forms of E-cadherin tagged either by myc or by flag epitopes. Three distinct E-cadherin complexes were found. The first is a conventional E-cadherin-catenin complex consisting of one E-cadherin molecule linked either to beta-catenin/alpha-catenin or to plakoglobin/alpha-catenin dimers. The second is a lateral E-cadherin complex incorporating two E-cadherin- catenin conventional complexes combined in parallel fashion via dimerization of the NH2-terminal extracellular domain of E-cadherin. The third complex is likely to contain two E-cadherin-catenin conventional complexes derived from two opposing cells and arranged in an antiparallel fashion. Formation of the antiparallel but not lateral complex strictly depends on extracellular calcium and E-cadherin binding to catenins. Double amino acid substitution Trp156Ala/Val157Gly within the extracellular NH2-terminal E-cadherin domain completely abolished both lateral and antiparallel inter-E-cadherin association. These data support an idea that the antiparallel complex has the adhesion function. Furthermore, they allow us to suggest that antiparallel complexes derive from lateral dimers and this complex process requires catenins and calcium ions.

• White P, Aberle H, Vincent JP
• Signaling and adhesion activities of mammalian beta-catenin and plakoglobin in Drosophila.
• J Cell Biol. 1998; 140: 183-95
• Display abstract

• The armadillo protein of Drosophila and its vertebrate homologues, beta-catenin and plakoglobin, are implicated in cell adhesion and wnt signaling. Here, we examine the conservation of these two functions by assaying the activities of mammalian beta-catenin and plakoglobin in Drosophila. We show that, in the female germ line, both mammalian beta-catenin and plakoglobin complement an armadillo mutation. We also show that shotgun mutant germ cells (which lack Drosophila E-cadherin) have a phenotype identical to that of armadillo mutant germ cells. It therefore appears that armadillo's role in the germ line is solely in a complex with Drosophila E-cadherin (possibly an adhesion complex), and both beta-catenin and plakoglobin can function in Drosophila cadherin complexes. In embryonic signaling assays, we find that plakoglobin has no detectable activity whereas beta-catenin's activity is weak. Surprisingly, when overexpressed, either in embryos or in wing imaginal disks, both beta-catenin and plakoglobin have dominant negative activity on signaling, an effect also obtained with COOH-terminally truncated armadillo. We suggest that the signaling complex, which has been shown by others to comprise armadillo and a member of the lymphocyte enhancer binding factor-1/T cell factor-family, may contain an additional factor that normally binds to the COOH-terminal region of armadillo.

• Ozawa M
• Identification of the region of alpha-catenin that plays an essential role in cadherin-mediated cell adhesion.
• J Biol Chem. 1998; 273: 29524-9
• Display abstract

• alpha-Catenin is an intrinsic component of the cadherin adhesion complex and is a 102-kDa protein with multiple interaction sites, including homodimerization sites, and binding sites for beta- and gamma-catenin (plakoglobin), alpha-actinin, and actin. Besides the binding to beta- or gamma-catenin, it is unknown, however, which interaction is critical for the function of cadherins. By expressing a series of E-cadherin-alpha-catenin chimeric molecules on leukemia cells (K562), we have identified the region of alpha-catenin that confers aggregation inducing activity to nonfunctional tail-less E-cadherin. The region has been mapped to the carboxyl-terminal 295 amino acids of alpha-catenin. Consistent with this result, expression in alpha-catenin-deficient cells (DLD-1/Delta alpha) of a mutant alpha-catenin molecule consisting of the amino-terminal beta-/gamma-catenin-binding site and the carboxyl-terminal cell adhesion region identified in the above experiments induced E-cadherin-mediated cell aggregation and compaction. Cells expressing E-cadherin chimeric molecules with the homologous carboxyl-terminal region of vinculin, which contains the actin-binding site of vinculin, did not, however, aggregate as strongly as ones expressing E-cadherin-alpha-catenin chimeric molecules.

• Roe S, Koslov ER, Rimm DL
• A mutation in alpha-catenin disrupts adhesion in clone A cells without perturbing its actin and beta-catenin binding activity.
• Cell Adhes Commun. 1998; 5: 283-96
• Display abstract

• Cadherin mediated cell-cell adhesion requires cytoplasmic connections to the cytoskeleton mediated by alpha-catenin. Original descriptions of the catenins, as well as our own in vitro studies, have suggested that this connection was mediated by the interaction of alpha-catenin to actin. Loss of adhesion in the human colon carcinoma cell line "Clone A" is the result of an internal deletion mutation of 158 residues near the N-terminus of the protein resulting in an 80 kD mutated protein. Transfection of these cells with the full length protein restores the normal adhesive phenotype. We have characterized this mutant protein in efforts to understand the normal function of alpha-catenin and, in particular, the region deleted in the Clone A mutant. Co-precipitation experiments using whole cell lysates indicate that the mutant form of alpha-catenin binds beta-catenin and plakoglobin, and can form a structural complex with E-cadherin via these interactions. Actin co-sedimentation assays show that the recombinant mutant binds and bundles F-actin and binds both actin and beta-catenin simultaneously, as seen with wild type alpha-catenin. These results suggest that the stabilization of the E-cadherin-catenin complex may be mediated by factors beyond its direct interaction with actin. We conclude that a region near the N-terminus of alpha-catenin mediates additional interactions between the adhesive complex and the cytoskeleton that are critical for functional adhesion.

• Efstathiou JA et al.
• Intestinal trefoil factor controls the expression of the adenomatous polyposis coli-catenin and the E-cadherin-catenin complexes in human colon carcinoma cells.
• Proc Natl Acad Sci U S A. 1998; 95: 3122-7
• Display abstract

• Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance mucosal healing and epithelial restitution in vivo. In this study, we evaluated the effect of recombinant TFF3 (rTFF3) stimulation on the expression and cellular localization of the epithelial (E)-cadherin-catenin complex, a prime mediator of Ca2+ dependent cell-cell adhesion, and the adenomatous polyposis coli (APC)-catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 (10(-9) M and 10(-8) M) for 20-24 hr led to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression was down-regulated. The expression of APC, alpha-catenin and beta-catenin also was decreased in HT29 cells, with a translocation of APC into the nucleus. No change in either cell adhesion or in the expression of E-cadherin, the catenins, and APC was detected in SW480 cells. In addition, TFF3 induced DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibited the effects of TFF3. Our results indicate that by perturbing the complexes between E-cadherin, beta-catenin, and associated proteins, TFF3 may modulate epithelial cell adhesion, migration, and survival.

• Miskevich F, Zhu Y, Ranscht B, Sanes JR
• Expression of multiple cadherins and catenins in the chick optic tectum.
• Mol Cell Neurosci. 1998; 12: 240-55
• Display abstract

• Cadherins form a large family of homophilic cell adhesion molecules that are involved in numerous aspects of neural development. The best-studied neural cadherin, N-cadherin, is concentrated at synapses made by retinal axons in the chick optic tectum and is required for the arborization of retinal axons in their target (retinorecipient) laminae. By analogy, other cadherins might mediate arborization or synaptogenesis in other tectal laminae. Here we consider which cadherins are expressed in tectum, which cells express them, and how their expression is regulated. First, using N-cadherin as a model, we show that synaptic input regulates both cadherin gene expression and the subcellular distribution of cadherin protein. Second, we demonstrate that N-, R-, and T-cadherin are each expressed in distinct laminar patterns during retinotectal synaptogenesis and that N- and R- are enriched in nonoverlapping synaptic subsets. Third, we show that over 20 cadherin superfamily genes are expressed in the tectum during the time that synapses are forming and that many of them are expressed in restricted groups of cells. Finally, we report that both beta-catenin and gamma-catenin (plakoglobin), cytoplasmic proteins required for cadherin signaling, are enriched at synapses and associated with N-cadherin. However, beta- and gamma-catenins are differentially distributed and regulated, and form mutually exclusive complexes. This result suggests that cadherin-based specificity involves multiple cadherin-dependent signaling pathways as well as multiple cadherins.

• Daniel JM, Reynolds AB
• Tyrosine phosphorylation and cadherin/catenin function.
• Bioessays. 1997; 19: 883-91
• Display abstract

• Cadherin-mediated cell-cell adhesion is perturbed in protein tyrosine kinase (PTK)-transformed cells. While cadherins themselves appear to be poor PTK substrates, their cytoplasmic binding partners, the Arm catenins, are excellent PTK substrates and therefore good candidates for mediating PTK-induced changes in cadherin behavior. These proteins, p120ctn, beta-catenin and plakoglobin, bind to the cytoplasmic region of classical cadherins and function to modulate adhesion and/or bridge cadherins to the actin cytoskeleton. In addition, as demonstrated recently for beta-catenin, these proteins also have crucial signaling roles that may or may not be related to their effects on cell-cell adhesion. Tyrosine phosphorylation of cadherin complexes is well documented and widely believed to modulate cell adhesiveness. The data to date, however, is largely correlative and the mechanism of action remains unresolved. In this review, we discuss the current literature and suggest models whereby tyrosine phosphorylation of Arm catenins contribute to regulation or perturbation of cadherin function.

• Denk C, Hulsken J, Schwarz E
• Reduced gene expression of E-cadherin and associated catenins in human cervical carcinoma cell lines.
• Cancer Lett. 1997; 120: 185-93
• Display abstract

• Cell-cell adhesion mediated by E-cadherin is often lost or disturbed in human carcinomas. For regular adhesive function, E-cadherin has to form complexes with peripheral cytoplasmic catenins which are multifunctional proteins that are also involved in signal transduction and growth regulation. We have analyzed the expression levels of the genes encoding alpha-catenin, beta-catenin and plakoglobin in correlation to the E-cadherin expression levels in cell lines derived from human cervical carcinomas. Reduced mRNA and protein levels were detected for plakoglobin, whereas alpha- and beta-catenin showed only reduced protein (but not mRNA) levels. The alterations in catenin gene expression were often associated with absent or reduced E-cadherin. The findings indicate that a reduction of catenin gene expression may contribute to the development of cervical carcinomas.

• Lilien J, Balsamo J, Hoffman S, Eisenberg C
• beta-Catenin is a target for extracellular signals controlling cadherin function: the neurocan-GalNAcPTase connection.
• Curr Top Dev Biol. 1997; 35: 161-89

• Bonneton C, Larue L, Thiery JP
• [Current data on the role of APC protein in the origin of colorectal cancer]
• Bull Cancer. 1997; 84: 1053-60
• Display abstract

• The adenomatous polyposis coli (APC) gene has been found to be mutated during the development of sporadic colorectal cancers as well as in familial adenomatous polyposis (FAP). These conditions result from initially somatic and germ line mutations respectively. In both cases, the expressed protein is truncated at its carboxyterminal region. Investigations into the role of wild-type APC have led to a better understanding of the importance of mutations in the genesis and progression of adenomas. APC was shown to regulate cell growth and cell death, to bind beta-catenin, and to colocalize with microtubules. APC truncation was therefore hypothesized to alter cell multiplication and cells are no longer able to undergo apoptosis. Owing to its beta-catenin binding, APC can modify the pool of beta-catenin which is in part utilized in the assembly of adherens junctions and in nuclear signalling. Truncated APC is unable to regulate this pool thereby altering adhesion and cell signalling. Finally, APC involvement in microtubule-dependent locomotion may explain some changes in cell movement which are observed in adenomas. The establishment of murine mutants and of normal and malignant intestinal cell cultures have allowed to assess biochemical and physiological properties of APC and its putative role in the genesis of colorectal carcinogenesis. Moreover, these experimental models have suggested a variety of possible therapeutic approaches.

• White RL
• Colon cancer. Molecular biology of the APC protein.
• Pathol Biol (Paris). 1997; 45: 240-4
• Display abstract

• The discovery of a tumor suppressor gene opens a new pathway to discovery of the fundamental mechanisms that underlie tumor initiation and progression. An inherited tumor suppressor gene is of special interest in that it defines a step in the tumorigenesis pathway that can be rate limiting in development of that tumor type. In the case of colon cancer, we were fortunate in identifying an inherited tumor suppressor gene, the APC gene, that plays a major etiologic role in both the inherited disease, familial adenomatous polyposis (FAP), and in sporadic colon polyps. Characterization of the molecular biology of that gene, and the underlying mechanisms that result in the development of colon tumors, could provide new approaches to both colon cancer diagnostics, therapeutics and chemopreventives. We have embarked, therefore, on a series of exploratory studies designed to provides clues to possible functional roles for the APC protein. We have found through immunocytochemistry that APC protein is distributed throughout the cell, in both the cytoplasm and nucleus. Furthermore, within the nucleus much of the APC protein seems associated with the nucleoli. The cytoplasmic label is distributed in a punctate pattern, with concentrations at the leading edge of migrating cells at the ends of microtubules. Furthermore, following an extraction of the cells that leaves behind primarily cytoskeletal and nuclear scaffold structures, we see strong APC staining of these structures. The yeast two-hybrid system has offered a number of potentially interacting partners for APC, including a new binding site for alpha-tubulin. These results, and others recent discoveries concerning APC, suggest a rather global role for APC protein, modulating cellular activity and signal transduction pathway from the cell periphery to the nucleus.

• Bresalier RS
• The gatekeeper has many keys: dissecting the function of the APC gene.
• Gastroenterology. 1997; 113: 2009-10

• Finnemann S, Mitrik I, Hess M, Otto G, Wedlich D
• Uncoupling of XB/U-cadherin-catenin complex formation from its function in cell-cell adhesion.
• J Biol Chem. 1997; 272: 11856-62
• Display abstract

• Xenopus XB/U-cadherin forms functional complexes with mouse alpha- and beta-catenins and p120(cas) when expressed in murine L-TK- fibroblasts. These cells were stably transfected with cDNAs encoding different cytoplasmic XB/U-cadherin mutants, each partially deleted in the different parts of the 38 most carboxyl-terminal amino acids. The binding of p120(cas) was not affected by carboxyl-terminal deletions, confirming its binding to a region more amino-terminal and distinct from the catenins. alpha- and beta-catenins associate with truncated XB/U-cadherins if either 19 amino acid half of the cadherin 38 amino acid tail is present, indicating that the site of catenin interaction is upstream of the deletions. However, for adhesive function of XB/U-cadherin constructs, the most carboxyl-terminal 19 amino acids are essential; if these amino acids are deleted, cadherin-catenin complexes unable to mediate cell-cell adhesion are formed. Nonadhesive complexes are solubilized by mild detergent, whereas functional complexes are stable. Provided that detergent stability of cadherin-catenin complexes is taken as a measure of their cytoskeletal association, our results give first evidence that cytoskeletal stabilization occurs independent of cadherin-catenin complex formation and requires the 19-amino acid cadherin carboxyl terminus.

• Neufeld KL, White RL
• Nuclear and cytoplasmic localizations of the adenomatous polyposis coli protein.
• Proc Natl Acad Sci U S A. 1997; 94: 3034-9
• Display abstract

• Mutation of the adenomatous polyposis coli (APC) gene is an early step in the initiation of colon cancer. Because the distribution pattern of a protein within the cell can provide important clues as to function, we have used a combination of immunofluorescence microscopy and biochemical fractionation to determine the location of APC protein in epithelial cells. Immunofluorescence microscopy placed full-length APC protein in both the nucleus and the cytoplasm. The nuclear APC protein was concentrated in discrete subnuclear regions, including nucleoli, whereas the cytoplasmic APC protein concentrated at the leading edge of migrating cells. Colocalization of APC protein with rRNA confirmed a nucleolar localization. These immunocytochemical findings have been supported by cell fractionation, which demonstrated that full-length APC protein was located in both the membrane/cytoskeletal and the nuclear fractions.

• Bullions LC, Notterman DA, Chung LS, Levine AJ
• Expression of wild-type alpha-catenin protein in cells with a mutant alpha-catenin gene restores both growth regulation and tumor suppressor activities.
• Mol Cell Biol. 1997; 17: 4501-8
• Display abstract

• Recent studies indicate that disruption of the E-cadherin-mediated cell-cell adhesion system is frequently associated with human cancers of epithelial origin. Reduced levels of both E-cadherin and the associated protein, alpha-catenin, have been reported in human tumors. This report describes the characterization of a human ovarian carcinoma-derived cell line (Ov2008) which expresses a novel mutant form of the alpha-catenin protein lacking the extreme N terminus of the wild-type protein. The altered form of alpha-catenin expressed in Ov2008 cells fails to bind efficiently to beta-catenin and is localized in the cytoplasm. Deletion mapping has localized the beta-catenin binding site on alpha-catenin between amino acids 46 and 149, which encompasses the same region of the protein that is deleted in the Ov2008 variant. Restoration of inducible expression of the wild-type alpha-catenin protein in these cells caused them to assume the morphology typical of an epithelial sheet and retarded their growth in vitro. Additionally, the induction of alpha-catenin expression in Ov2008 cells injected into nude mice attenuated the ability of these cells to form tumors. These observations support the classification of alpha-catenin as a growth-regulatory and candidate tumor suppressor gene.

• Ilyas M, Tomlinson IP
• The interactions of APC, E-cadherin and beta-catenin in tumour development and progression.
• J Pathol. 1997; 182: 128-37
• Display abstract

• Much progress has been made in identifying genes mutated during the development of colorectal carcinoma. Mutation of the APC gene in particular appears to be fundamental for colorectal tumour initiation. In contrast, loss of expression of E-cadherin appears to be a late event, which may be important in the development of invasion. Recent clarification of the function of APC, however, has shown that it exists in equilibrium with beta-catenin and E-cadherin. This review discusses the function of these molecules, their interactions, and how APC mutations may alter the equilibrium with beta-catenin and E-cadherin. It is argued that these changes cause aberrant architectural development of tissue, which results in loss of growth control. It is this escape from growth control, rather than acquisition of cell-autonomous growth, which results in the initial development of adenomas. The role of the E-cadherin-catenin unit in colorectal tumour invasion is discussed and the evidence is reviewed for the involvement of APC and E-cadherin in tumours arising from non-intestinal epithelia.

• Torres M et al.
• An alpha-E-catenin gene trap mutation defines its function in preimplantation development.
• Proc Natl Acad Sci U S A. 1997; 94: 901-6
• Display abstract

• Catenins are proteins associated with the cytoplasmic domain of cadherins, a family of transmembrane cell adhesion molecules. The cadherin-catenin adhesion system is involved in morphogenesis during development and in the maintenance of the integrity of different tissue types. Using a gene trap strategy, we have isolated a mouse mutation for the gene encoding the alpha-E-catenin. This form of the alpha-catenin appears frequently coexpressed with E-cadherin in epithelial cell types. The mutation obtained eliminates the carboxyl-terminal third of the protein but nevertheless provokes a complete loss-of-function phenotype. Homozygous mutants show disruption of the trophoblast epithelium (the first differentiated embryonic tissue), and development is consequently blocked at the blastocyst stage. This phenotype parallels the defects observed in E-cadherin mutant embryos. Our results show the requirement of the alpha-E-catenin carboxy terminus for its function and represent evidence of the role of the alpha-E-catenin in vivo, identifying this molecule as the natural partner of the E-cadherin in trophoblast epithelium.

• Hazan RB, Kang L, Roe S, Borgen PI, Rimm DL
• Vinculin is associated with the E-cadherin adhesion complex.
• J Biol Chem. 1997; 272: 32448-53
• Display abstract

• Cadherins mediate calcium-dependent cell-cell adhesion, and this activity is regulated by cytoplasmic interactions between cadherins, catenins, and the actin-based cytoskeleton. alpha-Catenin plays a critical role in the transmembrane anchorage of cadherins, and deletion of alpha-catenin has been shown to inactivate cadherin-mediated adhesion, resulting in a nonadhesive phenotype. Here we show that serum starvation increases E-cadherin expression and induces E-cadherin-dependent adhesion in the MDA-MB-468 breast cancer cell line. This adhesion occurred despite a lack of alpha-catenin expression, which was caused by mutations in the alpha-catenin gene. Coprecipitation analysis suggests that this adhesion may be mediated by cytoplasmic connections from cadherins to the cytoskeleton involving vinculin. A high level of vinculin associated with E-cadherin immunoprecipitates was observed in MDA-MB-468 cells. In contrast, vinculin was not detected in E-cadherin complexes in the A431 and MCF-7 epithelial carcinoma cell lines, which express alpha-catenin. However, in reciprocal immunoprecipitations using anti-vinculin antibodies, E-cadherin associated strongly with vinculin in MDA-MB-468 cells and, to a lesser extent, in A431 and MCF-7 cells. These results suggest that both alpha-catenin and vinculin may be present in the adhesion complex. To test the hypothesis that vinculin associates with E-cadherin complexes via beta-catenin, excess recombinant beta-catenin or alpha-catenin fusion protein was added to MDA-MB-468 cell lysates. Both specifically inhibited the coprecipitation of E-cadherin with vinculin, suggesting competition for the same binding site. These results suggest that vinculin plays a role in the establishment or regulation of the cadherin-based cell adhesion complex by direct interaction with beta-catenin.

• Polakis P
• The adenomatous polyposis coli (APC) tumor suppressor.
• Biochim Biophys Acta. 1997; 1332: 12747-12747
• Display abstract

• Defects in the APC gene are inarguably linked to the progression of colon cancers that arise both sporadically and through the transmission of germline mutations. Genetic evidence from humans and mouse models suggest that APC is a classic tumor suppressor in that both alleles likely require inactivation for tumor growth to ensue. Nearly all of the mutations, germline and somatic, result in premature termination of the single polypeptide chain, normally consisting of 2843 amino acids. Several definable motifs have now been mapped to the linear amino acid sequence of the APC polypeptide. These include an oligomerization domain, armadillo repeats, binding sites for beta-catenin, the human discs large protein, microtubules, and other proteins of unknown function. Inactivation of APC in cancer is likely due to loss of function(s) normally associated with the deleted protein structure.

• Oda H, Uemura T, Takeichi M
• Phenotypic analysis of null mutants for DE-cadherin and Armadillo in Drosophila ovaries reveals distinct aspects of their functions in cell adhesion and cytoskeletal organization.
• Genes Cells. 1997; 2: 29-40
• Display abstract

• BACKGROUND: DE-cadherin is an epithelial cadherin in Drosophila, and forms adherens junctions by associating with Armadillo (beta-catenin). To investigate its role in oogenesis, we generated germ-line clones homozygous for a null mutation in shotgun (shg) encoding this molecule, and examined their phenotypes, comparing with those of armadillo (arm) mutants. RESULTS: In the wild-type ovaries, DE-cadherin was expressed by both the germ-line and somatic derivatives, colocalizing with Armadillo. In the shg mutant ovaries in which the mutation was restricted to the germ line, germ cells were rounded, and generated gaps between themselves, suggesting that their surface adhesiveness was reduced or lost. However, the positioning of germ cells in the egg chamber was normal. Two groups of somatic follicle cells--the border cells and centripetal follicle cells--frequently migrated along incorrect pathways, indicating that DE-cadherin is required for their appropriate migration. Notably, the shg phenotypes were distinct from those of arm null mutants. Intercellular adhesion appeared to be less severely affected by arm than by the shg mutation, and the actin-based cytoskeleton and cell arrangement were disorganized only in the arm mutants. CONCLUSIONS: These findings suggest that DE-cadherin is critical for cell-cell adhesion, and functional to a certain extent without Armadillo, whereas Armadillo is required for cytoskeletal organization and for the control of cell positioning. We therefore propose that the molecular complex of DE-cadherin and Armadillo which is present in normal cells is endowed with multiple functions derived from each molecule.

• Koslov ER, Maupin P, Pradhan D, Morrow JS, Rimm DL
• Alpha-catenin can form asymmetric homodimeric complexes and/or heterodimeric complexes with beta-catenin.
• J Biol Chem. 1997; 272: 27301-6
• Display abstract

• The cadherin-based transmembrane cell-cell adhesive complex is thought to be composed of a cadherin molecule, a beta-catenin, and an alpha-catenin, which connects the complex to the cytoskeleton. The precise stoichiometry of this complex remains uncertain. We have used a series of recombinant molecules and biophysical techniques to assess the multimeric state of human alpha- and beta-catenin in vitro and then visualized them by electron microscopy after rotary shadowing. Calculated solution molecular masses are 213 kDa for alpha-catenin, 73 kDa for beta-catenin, and 186 kDa for both. This suggests that alpha-catenin exists as a homodimer in solution, beta-catenin is a monomer, and when both are present, they form alpha/beta-catenin heterodimers. Co-precipitation and surface plasmon resonance assays localize the site of alpha-catenin dimerization to the NH2-terminal 228 amino acids. This region encompasses a high-affinity (Kd = 100 nM) binding site for beta-catenin that lies between residues 54 and 157. We anticipate that the oligomeric state of alpha-catenin and the relative stoichiometry of the components in the membrane adhesion complex will be dynamic and regulated by beta-catenin, cell adhesion, and probably other factors as well.

• Yamamoto M, Bharti A, Li Y, Kufe D
• Interaction of the DF3/MUC1 breast carcinoma-associated antigen and beta-catenin in cell adhesion.
• J Biol Chem. 1997; 272: 12492-4
• Display abstract

• The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in human breast carcinomas. The functional role of DF3 is unknown. The present studies demonstrate that DF3 associates with beta-catenin. Similar findings have been obtained for gamma-catenin but not alpha-catenin. DF3, like E-cadherin and the adenomatous polyposis coli gene product, contains an SXXXXXSSL site that is responsible for direct binding to beta-catenin. The results further demonstrate that interaction of DF3 and beta-catenin is dependent on cell adhesion. These findings and the role of beta-catenin in cell signaling support a role for DF3 in the adhesion of epithelial cells.

• Miller JR, McClay DR
• Changes in the pattern of adherens junction-associated beta-catenin accompany morphogenesis in the sea urchin embryo.
• Dev Biol. 1997; 192: 310-22
• Display abstract

• beta-Catenin was originally identified biochemically as a protein that binds E-cadherin in cultured cells and that interaction was later shown to be essential for cadherin function. Independently, armadillo, the beta-catenin homolog in Drosophila melanogaster, was identified as a segment polarity gene necessary for the transduction of wingless (Wnt) signals during embryonic and larval development. Recently, several investigations have also shown that beta-catenin plays a critical role in axial patterning of early Xenopus, zebrafish, and mouse embryos. In these systems, the localization of beta-catenin to the plasma membrane, cytosol, and nucleus is predictive of its role in cell adhesion and signaling. In order to examine the potential role of beta-catenin in regulating cell adhesion during embryogenesis, we cloned beta-catenin in the sea urchin Lytechinus variegatus and characterized its subcellular distribution in cells undergoing morphogenetic movements. Indicative of a role in the establishment and maintenance of cell adhesion, beta-catenin is associated with lateral cell-cell contacts and accumulates at adherens junctions from cleavage stages onward. At gastrulation, changes in junctional beta-catenin localization accompany several morphogenetic events. The epithelial-mesenchymal conversion that characterizes the ingression of both primary and secondary mesenchyme cells coincides with a rapid and dramatic loss of adherens junction-associated beta-catenin. In addition, epithelial cells in the archenteron display a significant decrease in adherens junction-associated beta-catenin levels as they undergo convergent-extension movements. These data are consistent with a role for beta-catenin in regulating cell adhesion and adherens junction function during gastrulation in the sea urchin embryo.

• Nakamura Y
• Cleaning up on beta-catenin.
• Nat Med. 1997; 3: 499-500

• Shapiro L
• The multi-talented beta-catenin makes its first appearance.
• Structure. 1997; 5: 1265-8
• Display abstract

• beta-catenin plays a central part in cell adhesion as a structural component of the cadherin complex. In a seemingly disparate role, it is also important in embryo patterning, and now has emerged as a leading actor in carcinogenesis. beta-catenin achieves its diverse functions by interacting with many partners. The recent structure of the core domain from beta-catenin suggests how this talented molecule can achieve its many functions.

• Clevers H, van de Wetering M
• TCF/LEF factor earn their wings.
• Trends Genet. 1997; 13: 485-9
• Display abstract

• Factors of the TCF/LEF HMG domain family (TCFs) exist in vertebrates, Drosophila melanogaster and Caenorhabditis elegans. It has very recently become evident that TCFs interact with the vertebrate WNT effector beta-catenin to mediate axis formation in Xenopus. Likewise, Armadillo (the Drosophila ortholog of beta-catenin) is genetically upstream of a Drosophila TCF in the Wingless pathway. Upon Wingless/Wnt signaling, Armadillo/beta-catenin associate with nuclear TCFs and contribute a trans-activation domain to the resulting bipartite transcription factor. The cytoplasmic tumor-suppressor protein APC binds to beta-catenin causing its destruction. In APC-deficient colon carcinoma cells, beta-catenin accumulates and is constitutively complexed with TCF factors. In APC-positive colon carcinomas and melanomas, dominant mutations in beta-catenin render it indestructable, providing an alternative mechanism to activate transcription of TCF target genes inappropriately. So, transcriptional activation of TCF target genes by beta-catenin appears to be a central event in development and cellular transformation.

• Rubenstein A, Merriam J, Klymkowsky MW
• Localizing the adhesive and signaling functions of plakoglobin.
• Dev Genet. 1997; 20: 91-102
• Display abstract

• Plakoglobin (PKG) is a major component of cell-cell adhesive junctions. It is also closely related to the Drosophila segment polarity gene product armadillo and can induce a WNT-like neural axis duplication (NAD) phenotype in Xenopus [Kamovsky and Klymkowsky, 1995.] To define the regions of PKG involved in cell adhesion and inductive signaling, we examined the behavior of mutated forms of PKG in Xenopus. Deletion of amino acids 22 through 39 (in the Xenopus PKG sequence increased the apparent stability of the polypeptide within the embryo and increased its ability to induce a WNT-like, NAD phenotype when expressed in the vegetal hemisphere. The N-terminal "head" and first 6 "ARM" repeats of PKG, or the C-terminal "tail" and the last 3 "ARM" repeats, could be removed without destroying the remaining polypeptide's ability to induce a NAD phenotype. The nuclear localization of mutant PKGs, however, was not strictly correlated with the ability to induce a NAD phenotype, i.e., some inactive polypeptides still accumulate in nuclei. Removal of PKG's head and first ARM repeat, which includes its alpha-catenin binding site, resulted in a polypeptide that, when expressed in the embryo, generated alpha dramatic cell adhesion defect. Removal of the next three ARM repeats abolished this adhesion defect, suggesting that the polypeptide no longer competes effectively with endogenous catenins for binding to cadherins. Expression of a form of PKG truncated after the 5th ARM repeat produced a milder cell adhesion defect, whereas expression of a polypeptide truncated after the 8th ARM repeat had little apparent effect on cellular adhesion. Based on these observations, we conclude that functions related to stability and cellular adhesion reside in the N-terminal region of the polypeptide, whereas the ability to induce a NAD phenotype lies within repeats 6-10 of the central region. The function(s) of the C-terminal domain of PKG remain uncertain at this time.

• Vermeulen SJ et al.
• Mutation of alpha-catenin results in invasiveness of human HCT-8 colon cancer cells.
• Ann N Y Acad Sci. 1997; 833: 186-9

• Fagotto F, Funayama N, Gluck U, Gumbiner BM
• Binding to cadherins antagonizes the signaling activity of beta-catenin during axis formation in Xenopus.
• J Cell Biol. 1996; 132: 1105-14
• Display abstract

• beta-Catenin, a cytoplasmic protein known for its association with cadherin cell adhesion molecules, is also part of a signaling cascade involved in embryonic patterning processes such as the determination of the dorsoventral axis in Xenopus and determination of segment polarity in Drosophila. Previous studies suggest that increased cytoplasmic levels of beta-catenin correlate with signaling, raising questions about the need for in- teraction with cadherins in this process. We have tested the role of the beta-catenin-cadherin interaction in axis formation. Using beta-catenin deletion mutants, we demonstrate that significant binding to cadherins can be eliminated without affecting the signaling activity. Also, depletion of the soluble, cytosolic pool of beta-catenin by binding to overexpressed C-cadherin completely inhibited beta-catenin-inducing activity. We conclude that binding to cadherins is not required for beta-catenin signaling, and therefore the signaling function of beta-catenin is independent of its role in cell adhesion. Moreover, because beta-catenin signaling is antagonized by binding to cadherins, we suggest that cadherins can act as regulators of the intracellular beta-catenin signaling pathway.

• Huber O, Korn R, McLaughlin J, Ohsugi M, Herrmann BG, Kemler R
• Nuclear localization of beta-catenin by interaction with transcription factor LEF-1.
• Mech Dev. 1996; 59: 3-10
• Display abstract

• Vertebrate beta-catenin and Drosophila Armadillo share structural similarities suggesting that beta-catenin, like Armadillo, has a developmental signaling function. Both proteins are present as components of cell adherens junctions, but accumulate in the cytoplasm upon Wingless/Wnt signaling. beta-Catenin has axis-inducing properties like Wnt when injected into Xenopus blastomeres, providing evidence for participation of beta-catenin in the Wnt-pathway, but until now no downstream targets for beta-catenin have been identified. Here we demonstrate that beta-catenin binds to the HMG-type transcription factor lymphoid enhancer factor-1 (LEF-1), resulting in a nuclear translocation of beta-catenin both in cultured mouse cells and after ectopic expression of LEF-1 in two-cell mouse embryos. LEF-1/beta-catenin complexes bind to the promoter region of the E-cadherin gene in vitro, suggesting that this interaction could regulate E-cadherin transcription. As shown for beta-catenin, ectopic expression of LEF-1 in Xenopus embryos caused duplication of the body axis, indicating a regulatory role for a LEF-1-like molecule in dorsal mesoderm formation.

• Takayama T et al.
• Beta-catenin expression in human cancers.
• Am J Pathol. 1996; 148: 39-46
• Display abstract

• Cell-cell adhesion in tissue is mainly regulated by homotypic interaction of cadherin molecules, which are anchored to the cytoskeleton via cytoplasmic proteins, including alpha- and beta-catenin. Although we previously demonstrated that alpha-catenin is crucial for cadherin function in vivo, little is known about the role of beta-catenin. We examined the expression of beta-catenin in human carcinoma samples along with normal tissue (esophagus, stomach, and colon) by immunostaining using our antibody for beta-catenin. Normal epithelium strongly expressed beta-catenin. However, beta-catenin expression was frequently reduced in primary tumors of the esophagus (10 of 15, 67%), stomach (9 of 19, 47%), and colon (11 of 22, 50%). From an immunoprecipitation study, we found that beta-catenin forms a complex with E-cadherin not only in the normal epithelium but also in cancerous tissues. In coexpression patterns of E-cadherin and beta-catenin, 43 (77%) of the 56 tumors showed a similar expression of both molecules, whereas the other 13 tumors (23%) showed positive staining for E-cadherin and reduced expression of beta-catenin. These findings suggest that beta-catenin forms a complex with E-cadherin in vivo and down-regulation of beta-catenin expression is associated with malignant transformation.

• Aberle H, Schwartz H, Hoschuetzky H, Kemler R
• Single amino acid substitutions in proteins of the armadillo gene family abolish their binding to alpha-catenin.
• J Biol Chem. 1996; 271: 1520-6
• Display abstract

• Analysis of the calcium-dependent cell adhesion molecule E-cadherin has led to the identification of catenins, which are necessary for cadherin function. Growing evidence that cadherins and catenins are subjected to genetic alterations in carcinogenesis makes it especially important to understand protein-protein interactions within the cadherin-catenin complex. Here we report the identification and analysis of the alpha-catenin binding site in plakoglobin (gamma-catenin). Using N- and C-terminal truncations of plakoglobin, we identified a domain of 29 amino acids necessary and sufficient for binding alpha-catenin. The alpha-catenin binding site is fully encoded within exon 3 of plakoglobin but only partially represented in Armadillo repeat 1. This suggests that exons rather than individual Arm repeats encode functional domains of plakoglobin. Site-directed mutagenesis identified residues in the alpha-catenin binding site indispensable for binding in vitro. Analogous mutations in beta-catenin and Armadillo had identical effects. Our results indicate that single amino acid mutations in the alpha-catenin binding site of homologs of Armadillo could prevent a stable association with alpha-catenin, thus affecting cadherin-mediated adhesion.

• Orsulic S, Peifer M
• An in vivo structure-function study of armadillo, the beta-catenin homologue, reveals both separate and overlapping regions of the protein required for cell adhesion and for wingless signaling.
• J Cell Biol. 1996; 134: 1283-300
• Display abstract

• Armadillo, the Drosophila homologue of vertebrate beta-catenin, plays a pivotal role both in Wingless signaling and in assembly of adherens junctions. We performed the first in vivo structure-function study of an adherens junction protein, by generating and examining a series of Armadillo mutants in the context of the entire animal. We tested each mutant by assaying its biological function, its ability to bind proteins that normally associate with Armadillo in adherens junctions, its cellular localization, and its pattern of phosphorylation. We mapped the binding sites for DE-cadherin and alpha-catenin. Although these bind to Armadillo independently of each other, binding of each is required for the function of adherens junctions. We identified two separate regions of Armadillo critical for Wingless signaling. We demonstrated that endogenous Armadillo accumulates in the nucleus and provide evidence that it may act there in transducing Wingless signal. We found that the Arm repeats, which make up the central two-thirds of Armadillo, differ among themselves in their functional importance in different processes. Finally, we demonstrated that Armadillo's roles in adherens junctions and Wingless signaling are independent. We discuss the potential biochemical role of Armadillo in each process.

• Senda T et al.
• The tumor suppressor protein APC colocalizes with beta-catenin in the colon epithelial cells.
• Biochem Biophys Res Commun. 1996; 223: 329-34
• Display abstract

• The APC gene is mutated in familial adenomatous polyposis and sporadic colorectal tumors. The product of this gene is a 300 kDa cytoplasmic protein associated with catenin. In the present study, we examined the subcellular localization of the APC protein and beta-catenin in the mouse colon by double-labeling immunocytochemistry. While the APC protein was localized in the lateral and apical cytoplasm and in microvilli of the epithelial cells, beta-catenin was present exclusively in the lateral cytoplasm. Double-labeling-immunoelectron microscopy demonstrated precise colocalization of the APC protein and beta-catenin along the lateral plasma membrane. These results suggest that the APC protein functions in cooperation with beta-catenin in the lateral cytoplasm but has other functions independent of beta-catenin in the apical cytoplasm and in microvilli.

• Huber O, Bierkamp C, Kemler R
• Cadherins and catenins in development.
• Curr Opin Cell Biol. 1996; 8: 685-91
• Display abstract

• Cadherins and catenins represent key molecules during development. Recent findings demonstrate the involvement of cadherins and catenins in signaling pathways. In a working hypothesis, signaling via beta-catenin regulates the epithelial-mesenchymal transition in vertebrate development.

• Behrens J et al.
• Functional interaction of beta-catenin with the transcription factor LEF-1.
• Nature. 1996; 382: 638-42
• Display abstract

• The cytoplasmic proteins beta-catenin of vertebrates and armadillo of Drosophila have two functions: they link the cadherin cell-adhesion molecules to the cytoskeleton, and they participate in the wnt/wingless signal pathway. Here we show, in a yeast two-hybrid screen, that the architectural transcription factor LEF-1 (for lymphoid enhancer-binding factor) interacts with beta-catenin. In mammalian cells, coexpressed LEF-1 and beta-catenin form a complex that is localized to the nucleus and can be detected by immunoprecipitation. Moreover, LEF-1 and beta-catenin form a ternary complex with DNA that splays an altered DNA bend. Microinjection of LEF-1 into XenoPus embryos induces axis duplication, which is augmented by interaction with beta-catenin. Thus beta-catenin regulates gene expression by direct interaction with transcription factors such as LEF-1, providing a molecular mechanism for the transmission of signals, from cell-adhesion components or wnt protein to the nucleus.

• Morin PJ, Vogelstein B, Kinzler KW
• Apoptosis and APC in colorectal tumorigenesis.
• Proc Natl Acad Sci U S A. 1996; 93: 7950-4
• Display abstract

• Tumors result from disruptions in the homeostatic mechanisms that regulate cell birth and cell death. In colon cancer, one of the earliest manifestation of this imbalance is the formation of polyps, caused by somatic and inherited mutations of the adenomatous polyposis coli (APC) tumor suppressor gene in both humans and mice. While the importance of APC in tumorigenesis is well documented, how it functions to prevent tumors remains a mystery. Using a novel inducible expression system, we show that expression of APC in human colorectal cancer cells containing endogenous inactive APC alleles results in a substantial diminution of cell growth. Further evaluation demonstrated that this was due to the induction of cell death through apoptosis. These results suggest that apoptosis plays a role not only in advanced tumors but also at the very earliest stages of neoplasia.

• Peifer M
• Regulating cell proliferation: as easy as APC.
• Science. 1996; 272: 974-5

• Akiyama T
• [The APC gene]
• Nippon Rinsho. 1996; 54: 955-9
• Display abstract

• The tumor suppressor gene APC is mutated in most cases of familial adenomatous polyposis (FAP) and sporadic colorectal tumors. The product of the APC gene is a 300 kDa protein present in the cytoplasm as a homodimer. Interestingly, the APC protein is known to interact with the adherence junction protein catenin, suggesting that APC may be involved in cell adhesion. More recently we have demonstrated that overexpression of APC blocks cell cycle progression from the G0/G1 to the S phase.

• Pai LM, Kirkpatrick C, Blanton J, Oda H, Takeichi M, Peifer M
• Drosophila alpha-catenin and E-cadherin bind to distinct regions of Drosophila Armadillo.
• J Biol Chem. 1996; 271: 32411-20
• Display abstract

• Adherens junctions are multiprotein complexes mediating cell-cell adhesion and communication. They are organized around a transmembrane cadherin, which binds a set of cytoplasmic proteins required for adhesion and to link the complex to the actin cytoskeleton. Three components of Drosophila adherens junctions, analogous to those in vertebrates, have been identified: Armadillo (homolog of beta-catenin), Drosophila E-cadherin (DE-cadherin), and alpha-catenin. We carried out the first analysis of the interactions between these proteins using in vitro binding assays, the yeast two-hybrid system, and in vivo assays. We identified a 76-amino acid region of Armadillo that is necessary and sufficient for binding alpha-catenin and found that the N-terminal 258 amino acids of alpha-catenin interact with Armadillo. A large region of Armadillo, spanning six central Armadillo repeats, is required for DE-cadherin binding, whereas only 41 amino acids of the DE-cadherin cytoplasmic tail are sufficient for Armadillo binding. Our data complement and extend results obtained in studies of vertebrate adherens junctions, providing a foundation for understanding how junctional proteins assemble and a basis for interpreting existing mutations and creating new ones.

• Wong MH, Hermiston ML, Syder AJ, Gordon JI
• Forced expression of the tumor suppressor adenomatosis polyposis coli protein induces disordered cell migration in the intestinal epithelium.
• Proc Natl Acad Sci U S A. 1996; 93: 9588-93
• Display abstract

• Mutations of the human adenomatosis polyposis coli (APC) gene are associated with the development of familial as well as sporadic intestinal neoplasia. To examine the in vivo function of APC, 129/Sv embryonic stem (ES) cells were transfected with DNA encoding the wild-type human protein under the control of a promoter that is active in all four of the small intestine's principal epithelial lineages during their migration-associated differentiation. ES-APC cells were then introduced into C57BL/6-ROSA26 blastocysts. Analyses of adult B6-ROSA26<-->129/Sv-APC chimeric mice revealed that forced expression of APC results in markedly disordered cell migration. When compared with the effects of forced expression of E-cadherin, the data suggest that APC-catenin and E-cadherin-catenin complexes have opposing effects on intestinal epithelial cell movement/adhesiveness; augmentation of E-cadherin-beta-catenin complexes produces a highly ordered, "adhesive" migration, whereas augmentation of APC-beta-catenin complexes produces a disordered, nonadhesive migratory phenotype. We propose that APC mutations may promote tumorigenesis by increasing the relative activity of cadherin-catenin complexes, resulting in enhanced adhesiveness and functional anchorage of initiated cells within the intestinal crypt. Our studies also indicate that chimeric mice generated from B6-ROSA26 blastocysts and genetically manipulated ES cells should be useful for auditing gene function in the gastrointestinal tract and in other tissues.

• Haegel H, Larue L, Ohsugi M, Fedorov L, Herrenknecht K, Kemler R
• Lack of beta-catenin affects mouse development at gastrulation.
• Development. 1995; 121: 3529-37
• Display abstract

• Molecular analysis of the cadherin-catenin complex elucidated the central role of beta-catenin in this adhesion complex, as it binds to the cytoplasmic domain of E-cadherin and to alpha-catenin. beta-Catenin may also function in signalling pathways, given its homology to the gene product of the Drosophila segment polarity gene armadillo, which is known to be involved in the wingless signalling cascade. To study the function of beta-catenin during mouse development, gene knock-out experiments were performed in embryonic stem cells and transgenic mice were generated. beta-Catenin null-mutant embryos formed blastocysts, implanted and developed into egg-cylinder-stage embryos. At day 7 post coitum, the development of the embryonic ectoderm was affected in mutant embryos. Cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity. No mesoderm formation was observed in mutant embryos. The development of extraembryonic structures appeared less dramatically or not at all affected. Our results demonstrate that, although beta-catenin is expressed rather ubiquitously, it is specifically required in the ectodermal cell layer.

• Trent JM, Wiltshire R, Su LK, Nicolaides NC, Vogelstein B, Kinzler KW
• The gene for the APC-binding protein beta-catenin (CTNNB1) maps to chromosome 3p22, a region frequently altered in human malignancies.
• Cytogenet Cell Genet. 1995; 71: 343-4
• Display abstract

• beta-Catenin is one of the E-cadherin associated proteins involved in the process of cellular adhesion. It has recently been shown to interact with the APC protein whose gene is known to be mutated in the germline of familial adenomatous polyposis patients. This interaction implies that beta-catenin is a potential regulator of the APC gene. The localization of the human beta-catenin gene (CTNNB1) to chromosome 3p22, by fluorescent in situ hybridization (FISH), has linked the gene to a region that is frequently altered in several human malignancies. The location of the gene and the protein interactions suggest the importance of beta-catenin in the etiology of various human cancers.

• Jou TS, Stewart DB, Stappert J, Nelson WJ, Marrs JA
• Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex.
• Proc Natl Acad Sci U S A. 1995; 92: 5067-71
• Display abstract

• The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that beta-catenin is the linker protein between E-cadherin and alpha-catenin and that E-cadherin does not bind directly to alpha-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of alpha-catenin are independent binding sites for beta-catenin. In addition to beta-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike beta-catenin, p120 does not bind alpha-catenin in vitro, although a complex of p120 and endogenous alpha-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and alpha-catenin revealed two catenin pools in cell lysates: an approximately 1000- to approximately 2000-kDa complex bound to E-cadherin and an approximately 220-kDa pool that did not contain E-cadherin. Only beta-catenin in the approximately 220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.

• Su LK et al.
• APC binds to the novel protein EB1.
• Cancer Res. 1995; 55: 2972-7
• Display abstract

• Mutations of the APC gene play a critical role in both sporadic and familial forms of colorectal cancer. The vast majority of these mutations result in the loss of the carboxyl terminus of the protein. To further elucidate the function of APC, we searched for cellular proteins that associate with its carboxyl terminus. One million human cDNA clones were screened with the use of the interaction trap two-hybrid system, and 67 clones were found to have a phenotype suggestive of an APC-interacting protein. Nucleotide sequence analysis revealed that 48 of these clones were derived from a single novel named EBI. The association of APC and EB1 proteins was confirmed with in vitro binding assays. mAbs against EB1 were then produced and used to demonstrate the association of APC and EB1 in vivo. The EB1 gene was predicted to encode a 268-amino acid protein without significant homology to proteins with known function. However, searches of nucleotide databases did identify evidence for at least two related human genes and a yeast homologue. This conservation suggests an essential function for EB1 that might provide clues to the mechanism through which APC suppresses colonic neoplasia.

• Daniel JM, Reynolds AB
• The tyrosine kinase substrate p120cas binds directly to E-cadherin but not to the adenomatous polyposis coli protein or alpha-catenin.
• Mol Cell Biol. 1995; 15: 4819-24
• Display abstract

• The tyrosine kinase substrate p120cas (CAS), which is structurally similar to the cell adhesion proteins beta-catenin and plakoglobin, was recently shown to associate with the E-cadherin-catenin cell adhesion complex. beta-catenin, plakoglobin, and CAS all have an Arm domain that consists of 10 to 13 repeats of a 42-amino-acid motif originally described in the Drosophila Armadillo protein. To determine if the association of CAS with the cadherin cell adhesion machinery is similar to that of beta-catenin and plakoglobin, we examined the CAS-cadherin-catenin interactions in a number of cell lines and in the yeast two-hybrid system. In the prostate carcinoma cell line PC3, CAS associated normally with cadherin complexes despite the specific absence of alpha-catenin in these cells. However, in the colon carcinoma cell line SW480, which has negligible E-cadherin expression, CAS did not associate with beta-catenin, plakoglobin, or alpha-catenin, suggesting that E-cadherin is the protein which bridges CAS to the rest of the complex. In addition, CAS did not associate with the adenomatous polyposis coli (APC) tumor suppressor protein in any of the cell lines analyzed. Interestingly, expression of the various CAS isoforms was quite heterogeneous in these tumor cell lines, and in the colon carcinoma cell line HCT116, which expresses normal levels of E-cadherin and the catenins, the CAS1 isoforms were completely absent. By using the yeast two-hybrid system, we confirmed the direct interaction between CAS and E-cadherin and determined that CAS Arm repeats 1 to 10 are necessary and sufficient for this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

• Shibamoto S et al.
• Association of p120, a tyrosine kinase substrate, with E-cadherin/catenin complexes.
• J Cell Biol. 1995; 128: 949-57
• Display abstract

• p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E-cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E-cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E-cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin-catenin adhesion system.

• Brady-Kalnay SM, Rimm DL, Tonks NK
• Receptor protein tyrosine phosphatase PTPmu associates with cadherins and catenins in vivo.
• J Cell Biol. 1995; 130: 977-86
• Display abstract

• The extracellular segment of the receptor-type type protein tyrosine phosphatase PTPmu, possesses an MAM domain, an immunoglobulin domain, and four fibronectin type-III repeats. It binds homophilically, i.e., PTPmu on the surface of one cell binds to PTPmu on an apposing cell, and the binding site lies within the immunoglobulin domain. The intracellular segment of PTPmu has two PTP domains and a juxtamembrane segment that is homologous to the conserved intracellular domain of the cadherins. In cadherins, this segment interacts with proteins termed catenins to mediate association with the actin cytoskeleton. In this article, we demonstrate that PTPmu associates with a complex containing cadherins, alpha- and beta-catenin in mink lung (MvLu) cells, and in rat heart, lung, and brain tissues. Greater than 80% of the cadherin in the cell is cleared from Triton X-100 lysates of MvLu cells after immunoprecipitation with antibodies to PTPmu; however, the complex is dissociated when lysates are prepared in more stringent, SDS-containing RIPA buffer. In vitro binding studies demonstrated that the intracellular segment of PTPmu binds directly to the intracellular domain of E-cadherin, but not to alpha- or beta-catenin. Consistent with their ability to interact in vivo, PTPmu, cadherins, and catenins all localized to points of cell-cell contact in MvLu cells, as assessed by immunocytochemical staining. After pervanadate treatment of MvLu cells, which inhibits cellular tyrosine phosphatase activity including PTPmu, the cadherins associated with PTPmu are now found in a tyrosine-phosphorylated form, indicating that the cadherins may be an endogenous substrate for PTPmu. These data suggest that PTPmu may be one of the enzymes that regulates the dynamic tyrosine phosphorylation, and thus function, of the cadherin/catenin complex in vivo.

• Boman BM et al.
• Radioimmunoassay of the APC gene product using antibodies against its middle and carboxyl regions.
• Biochem Biophys Res Commun. 1995; 206: 909-15
• Display abstract

• A radioimmunoassay (RIA) has been developed for the adenomatous polyposis coli protein (APC). High-avidity rabbit polyclonal antibodies were produced against synthetic peptides corresponding to amino acids 1865-1881 (APC-1) and to amino acids 1336-1350 (APC-2) in APC's 2844 amino acid sequence. Both antibodies were utilized in RIA to evaluate full-length APC that is present in the insoluble particulate fraction of cell lysates. High salt extraction, often employed for coiled-coil type protein preparations, was found to be useful for extraction of APC from lysates of normal colonic epithelium. Proteolytic digestion of high salt extracts increased antibody reactivity toward both epitopes, suggesting that APC-1 and APC-2 antigenic sites are partially concealed due to APC's involvement in multiprotein complexes. Thus, RIA using our antibodies will provide a valuable tool for APC protein purification and in studies for elucidating APC's biologic function.

• Groden J et al.
• Response of colon cancer cell lines to the introduction of APC, a colon-specific tumor suppressor gene.
• Cancer Res. 1995; 55: 1531-9
• Display abstract

• The APC gene, mutations in which are responsible for the inherited colon cancer syndrome adenomatous polyposis coli (APC), is described as a tumor suppressor gene. A full-length, wild-type APC gene was introduced by transfection into three human colon carcinoma cell lines, each characterized for mutations at loci involved in colon tumor formation. The response of each cell line to the introduction of APC differed with the genotype of the cell line. Some of the cell clones derived from these transfections displayed altered morphologies; some showed suppression of tumorigenicity based on growth in soft agar and tumor formation in nude mice. One cell line, SW480, could not be stably transfected with the APC gene. These results provide the first direct evidence that the APC gene can alter the transformation properties of colon carcinoma cells.

• Kawanishi J, Kato J, Sasaki K, Fujii S, Watanabe N, Niitsu Y
• Loss of E-cadherin-dependent cell-cell adhesion due to mutation of the beta-catenin gene in a human cancer cell line, HSC-39.
• Mol Cell Biol. 1995; 15: 1175-81
• Display abstract

• Detachment of cell-cell adhesion is indispensable for the first step of invasion and metastasis of cancer. This mechanism is frequently associated with the impairment of either E-cadherin expression or function. However, mechanisms of such abnormalities have not been fully elucidated. In this study, we demonstrated that the function of E-cadherin was completely abolished in the human gastric cancer cell line HSC-39, despite the high expression of E-cadherin, because of mutations in one of the E-cadherin-associated cytoplasmic proteins, beta-catenin. Although immunofluorescence staining of HSC-39 cells by using an anti-E-cadherin antibody (HECD-1) revealed the strong and uniform expression of E-cadherin on the cell surface, cell compaction and cell aggregation were not observed in this cell. Western blotting (immunoblotting) using HECD-1 exhibited a 120-kDa band which is equivalent to normal E-cadherin. Northern (RNA) blotting demonstrated a 4.7-kb band, the same as mature E-cadherin mRNA. Immunoprecipitation of metabolically labeled proteins with HECD-1 revealed three bands corresponding to E-cadherin, alpha-catenin, and gamma-catenin and a 79-kDa band which was apparently smaller than that of normal beta-catenin, indicating truncated beta-catenin. The 79-kDa band was immunologically identified as beta-catenin by using immunoblotting with anti-beta-catenin antibodies. Examination of beta-catenin mRNA by the reverse transcriptase-PCR method revealed a transcript which was shorter than that of normal beta-catenin. The sequencing of PCR product for beta-catenin confirmed deletion in 321 bases from nucleotides +82 to +402.(ABSTRACT TRUNCATED AT 250 WORDS)

• Bhattacharya G, Boman BM
• Phosphorylation of the adenomatous polyposis coli protein and its possible regulatory effects in cells.
• Biochem Biophys Res Commun. 1995; 208: 103-10
• Display abstract

• The adenomatous polyposis coli (APC) gene is etiologically associated with familial adenomatous polyposis and gastrointestinal malignancies, but its cellular function and role in tumorigenesis are unclear. Recent reports indicate that wild-type, but not mutant, APC gene product (APC) is associated with and promotes the assembly of cytoskeletal microtubules in vitro, suggesting that this mechanism has importance in tumor development. Because other microtubule-associated proteins (MAPs) undergo phosphorylation in their normal functioning, we postulated that APC is a phosphoprotein. HCT116 cells, containing full-length APC protein, were [32P]-prelabeled, and a 300-kDa band corresponding to phosphorylated APC was immunoprecipitated using each of three different anti-APC antibodies. High voltage electrophoresis of [32P]-labeled APC showed the presence of phospho-serine and phospho-threonine residues. Further immunoprecipitation analyses showed phosphorylation of i) full-length APC in human lymphoblastoid cells and ii) carboxyl-truncated APC in SW480 and DiFi colon carcinoma cells. Thus, APC is probably a phosphoprotein in normal and malignant tissues. We hypothesize a mechanism whereby phosphorylation of APC may play a regulatory role in its interaction with microtubules. This may involve phosphorylation of (Ser/Thr)-Pro amino acid motifs in APC's basic domain. We propose that deletion of this domain disrupts APC binding to microtubules, explaining how APC mutations are linked to cancer development.

• Miyashiro I et al.
• Subcellular localization of the APC protein: immunoelectron microscopic study of the association of the APC protein with catenin.
• Oncogene. 1995; 11: 89-96
• Display abstract

• Mutations in the APC gene are linked to the development of sporadic colorectal tumors as well as to familial adenomatous polyposis. Recently, the APC protein was reported to associated with catenins, proteins that bind to the cell adhesion molecule E-cadherin. In the present study, we examined the distribution and localization of the APC protein and alpha -catenin in the normal mouse intestine by light and immunoelectron microscopy using specific antibodies. The APC protein was found to be localized in microvilli and in the apical and lateral cytoplasm of the epithelial cells, whereas alpha-catenin was detected only in the lateral cytoplasm. Double-labeling immunoelectron microscopy showed colocalization of the APC protein with alpha-catenin in the lateral cytoplasm, especially along the lateral plasma membrane, although a certain portion of the APC protein in this region was distributed independently of alpha-catenin. These results suggest that a portion of the APC protein localized in the lateral cytoplasm of intestinal epithelial cells functions in cooperation with catenins, whereas the APC protein in microvilli and in the apical cytoplasm has other functions independent of catenins.

• Shinoura N et al.
• Expression of N-cadherin and alpha-catenin in astrocytomas and glioblastomas.
• Br J Cancer. 1995; 72: 627-33
• Display abstract

• We examined levels of mRNA and protein for N-cadherin, the predominant cadherin in neural tissues, and mRNA levels for the cadherin-associated protein, alpha-catenin, in a series of gliomas and in glioblastoma cell lines. mRNA levels for N-cadherin and alpha-catenin were significantly higher in glioblastomas than in low-grade astrocytomas or normal brain, while the levels of intact N-cadherin protein were similar in glioblastomas, low-grade astrocytomas and brain. In addition, there was no consistent relationship between invasiveness and expression of N-cadherin and alpha-catenin in highly invasive vs minimally invasive tumours within the same histopathological grade. To assess further the relationship between cadherin expression and neural tumour invasion, we measured N-cadherin expression, calcium-dependent cell adhesion and motility of several glioblastoma cell lines. While all N-cadherin-expressing lines were adhesive, no correlation was seen between the level of N-cadherin expression and cell motility. Together, these findings imply that, in contrast to the role played by E-cadherin in carcinomas, N-cadherin does not restrict the invasion of glioblastomas.

• Ozawa M, Terada H, Pedraza C
• The fourth armadillo repeat of plakoglobin (gamma-catenin) is required for its high affinity binding to the cytoplasmic domains of E-cadherin and desmosomal cadherin Dsg2, and the tumor suppressor APC protein.
• J Biochem (Tokyo). 1995; 118: 1077-82
• Display abstract

• Plakoglobin is a member of a protein family with a repeated amino acid motif, the armadillo repeat, and is a cytoplasmic protein found in both adherens junctions and desmosomes. Plakoglobin has been shown to form distinct complexes with cadherins or desmosomal cadherins. Also, plakoglobin has been shown to complex with APC, the tumor suppressor gene product. Recently we isolated a cDNA clone encoding plakoglobin lacking the fourth armadillo repeat of the original 13-repeat protein [Ozawa et al. (1995) J. Biochem. 118, 836-840]. In this study, we established an in vitro assay system to study the molecular interaction of plakoglobin with cadherins, the APC gene product, and alpha-catenin. Establishment of the system and cloning of an alternate form of plakoglobin cDNA allowed us to examine the biological activity of plakoglobin lacking the fourth armadillo repeat. Experiments with the bacterially expressed 12-repeat plakoglobin revealed that the protein binds to E-cadherin, desmoglein (Dsg2), and APC with lower affinity than the 13-repeat form does. Consistent with the observation that the affinity of alpha-catenin for these two alternate forms was similar, we found amino acid residues 104 to 145 of plakoglobin, the residues present in both isoforms, are sufficient for its binding to alpha-catenin.

• Vermeulen SJ et al.
• Transition from the noninvasive to the invasive phenotype and loss of alpha-catenin in human colon cancer cells.
• Cancer Res. 1995; 55: 4722-8
• Display abstract

• Loss of epithelioid organization in carcinoma cell lines has been related to invasiveness and poor differentiation of tumors. We investigated the invasion in vitro of various human colon cancer cell lines. Most cell lines were noninvasive into chick heart fragments, and this correlated with an epithelioid morphotype. Only cell lines COLO320DM, SW620, and variants of HCT-8 and DLD-1 were invasive and nonepithelioid. We examined in these cell lines whether invasiveness was related to changes in the structure and function of the E-cadherin/catenin complex. E-cadherin functions as an invasion suppressor and as a cell-cell adhesion molecule when linked to the cytoskeleton via alpha-catenin plus beta- or gamma-catenin. All noninvasive cell lines showed E-cadherin linked to these catenins. The E-cadherin-dependent cell-cell adhesion function in these cell lines was demonstrated by two assays in vitro. It was interesting that all invasive cell lines showed a dysfunctional E-cadherin/catenin complex. COLO320DM, SW480, and SW620 cells were defective in E-cadherin expression, whereas the invasive variants of HCT-8 and DLD-1 lacked the alpha-catenin protein. From clonal epithelioid HCT-8 cultures with functional E-cadherin/catenin complexes, we subcloned, repeatedly, round cell variants that were again invasive and expressed no alpha-catenin protein. Our data suggest that reproducible transformations toward a more invasive phenotype in HCT-8 cells are associated with down-regulation of alpha-catenin. The mechanisms of this transformation and the level of alpha-catenin down-regulation are currently investigated.

• Rubinfeld B, Souza B, Albert I, Munemitsu S, Polakis P
• The APC protein and E-cadherin form similar but independent complexes with alpha-catenin, beta-catenin, and plakoglobin.
• J Biol Chem. 1995; 270: 5549-55
• Display abstract

• The tumor suppressor APC protein associates with the cadherin-binding proteins alpha- and beta-catenin. To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins. beta-Catenin directly binds to APC at high and low affinity sites. alpha-Catenin cannot directly bind APC but associates with it by binding to beta-catenin. Plakoglobin, also known as gamma-catenin, directly binds to both APC and alpha-catenin and also to the APC-beta-catenin complex, but not directly to beta-catenin. beta-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 amino acid repeat sequences. The APC binding site on beta-catenin may be discontinuous since neither the carboxyl- nor amino-terminal halves of beta-catenin will independently associate with APC, although the amino-terminal half independently binds alpha-catenin. The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins. Thus, APC forms distinct heteromeric complexes containing combinations of alpha-catenin, beta-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.

• Fagotto F, Gumbiner BM
• Beta-catenin localization during Xenopus embryogenesis: accumulation at tissue and somite boundaries.
• Development. 1994; 120: 3667-79
• Display abstract

• beta-catenin is a cytoplasmic protein associated with cadherin adhesion molecules and has been implicated in axis formation in Xenopus (McCrea, P. D., Brieher, W. M. and Gumbiner, B. M. (1993) J. Cell Biol. 127, 477-484). We have studied its distribution in Xenopus embryos by immunofluorescence on frozen sections. Consistent with its function in cell-cell adhesion, beta-catenin is present in every cell. However, high levels are expressed in certain regions and different tissues of the embryo. No simple correlation appears to exist between the levels of beta-catenin with the expected strength of adhesion. High levels of beta-catenin were found in regions undergoing active morphogenetic movements, such as the marginal zone of blastulae and gastrulae. This suggests that high expression of beta-catenin could be involved in dynamic adhesion events. Surprisingly, beta-catenin also accumulates on plasma membranes that probably do not establish direct or strong contacts with other cells. In particular, high amounts of beta-catenin are found transiently at boundaries between tissue anlagen and at the intersomitic boundaries. This unexpected pattern of beta-catenin expression raises the possibility that this molecule participates in developmental processes, perhaps independently of its classical role in cell-cell adhesion.

• Browne SJ, Williams AC, Hague A, Butt AJ, Paraskeva C
• Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific low-molecular-weight form is associated with apoptosis in vitro.
• Int J Cancer. 1994; 59: 56-64
• Display abstract

• APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1-3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36-96% depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC was also lost, though a 95-kDa protein distinct from that in apoptotic cells was observed. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival.

• Peifer M, Sweeton D, Casey M, Wieschaus E
• wingless signal and Zeste-white 3 kinase trigger opposing changes in the intracellular distribution of Armadillo.
• Development. 1994; 120: 369-80
• Display abstract

• wingless/wnt-1 signaling directs cell fate during development. Genetic analysis in Drosophila identified genes that may encode components of the wingless signal transduction system. Drosophila Armadillo, homolog of vertebrate beta-catenin, is required for wingless signaling. Unlike armadillo RNA, Armadillo protein accumulates non-uniformly in different cells of each embryonic segment. We found that cells alter their intracellular distribution of Armadillo in response to Wingless signal, accumulating increased levels of cytoplasmic Armadillo relative to those of membrane-associated protein. Levels of cytoplasmic Armadillo are also regulated by Zeste-White 3 kinase. Analysis of double mutants demonstrates that Armadillo's role in wingless signaling is direct, and that Armadillo functions downstream of both wingless and zeste-white 3. We present a model for the role of Armadillo stripes in transduction of wingless signal.

• Butz S, Kemler R
• Distinct cadherin-catenin complexes in Ca(2+)-dependent cell-cell adhesion.
• FEBS Lett. 1994; 355: 195-200
• Display abstract

• Catenins are peripheral cytoplasmic proteins originally identified in association with the mouse epithelial cell adhesion molecule E-cadherin. Molecular cloning and primary structure analysis demonstrated that alpha-catenin is homologous to vinculin and the beta-catenin is homologous to human plakoglobin and the Drosophila gene product armadillo. With the use of peptide-specific anti plakoglobin antibodies were confirm here that plakoglobin is a component of the cadherin-catenin complex and that it is most likely identical to gamma-catenin. We show that plakoglobin binds directly to E-cadherin. We consolidate the biochemical evidence for the existence of two distinct and separable E-cadherin-catenin complexes in the same cell. One complex is composed of E-cadherin, alpha- and beta-catenin, the other of E-cadherin, alpha-catenin and plakoglobin. A similar distinct association with catenins is also found for other cadherins. Comparison of different cell lines revealed that the relative amounts of the two complexes vary depending on cell types.

• Hulsken J, Birchmeier W, Behrens J
• E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton.
• J Cell Biol. 1994; 127: 2061-9
• Display abstract

• beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.

• Oyama T et al.
• A truncated beta-catenin disrupts the interaction between E-cadherin and alpha-catenin: a cause of loss of intercellular adhesiveness in human cancer cell lines.
• Cancer Res. 1994; 54: 6282-7
• Display abstract

• Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with catenins, a group of cytoplasmic proteins including alpha- and beta-catenins. While alpha-catenin has been demonstrated to be crucial for cadherin function, the role of beta-catenin is not yet fully understood. In this study, we analyzed the cadherin-catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of beta-catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the beta-catenin gene. On the other hand, these cells expressed E-cadherin, alpha-catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated beta-catenin but not with alpha-catenin, and antibodies against beta-catenin did not copurify alpha-catenin. However, the recombinant fusion protein containing wild-type beta-catenin precipitated alpha-catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with alpha-catenin, and that this defect results from the mutation in beta-catenin. Thus, it is most likely that the association between E-cadherin and alpha-catenin is mediated by beta-catenin, and that this process is blocked by NH2-terminal deletion in beta-catenin. These findings indicate that genetic abnormality of beta-catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers.

• Hinck L, Nelson WJ, Papkoff J
• Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-catenin binding to the cell adhesion protein cadherin.
• J Cell Biol. 1994; 124: 729-41
• Display abstract

• Wnt-1 homologs have been identified in invertebrates and vertebrates and play important roles in cellular differentiation and organization. In Drosophila, the products of the segment polarity genes wingless (the Wnt-1 homolog) and armadillo participate in a signal transduction pathway important for cellular boundary formation in embryonic development, but functional interactions between the proteins are unknown. We have examined Wnt-1 function in mammalian cells in which armadillo (beta-catenin and plakoglobin) is known to bind to and regulate cadherin cell adhesion proteins. We show that Wnt-1 expression results in the accumulation of beta-catenin and plakoglobin. In addition, binding of beta-catenin to the cell adhesion protein, cadherin, is stabilized, resulting in a concomitant increase in the strength of calcium-dependent cell-cell adhesion. Thus, a consequence of the functional interaction between Wnt-1 and armadillo family members is the strengthening of cell-cell adhesion, which may lead to the specification of cellular boundaries.

• Hulsken J, Behrens J, Birchmeier W
• Tumor-suppressor gene products in cell contacts: the cadherin-APC-armadillo connection.
• Curr Opin Cell Biol. 1994; 6: 711-6
• Display abstract

• Various structural components of intercellular junctions have recently been found to represent (or be related to) products of tumor-suppressor genes. The tumor-suppressor gene product adenomatous polyposis coli (APC) binds to beta 2-catenin (homologous to the product of Drosophila armadillo), which is cytoplasmically associated with the cell adhesion molecule E-cadherin.

• Burchill SA
• The tumour suppressor APC gene product is associated with cell adhesion.
• Bioessays. 1994; 16: 225-7

• Rosenthal E
• Identification of homologues to beta-catenin/plakoglobin/armadillo in two invertebrates, Urechis caupo and Tripneustes gratilla.
• Biochim Biophys Acta. 1993; 1173: 337-41
• Display abstract

• beta-Catenin and plakoglobin are intracellular proteins that participate in cell-cell adhesion, probably through interaction with the cadherin family of transmembrane adhesion proteins. They are also homologous to the segment polarity gene, armadillo, from Drosophila. I have cloned and sequenced armadillo/beta-catenin/plakoglobin homologues from two other invertebrates, Urechis caupo and Tripneustes gratilla, and shown that the mRNA is present in oocytes, eggs and early embryos. In Urechis, the mRNA is particularly abundant in oocytes, but is not translated until after fertilization. These results provide further indications that cell adhesion proteins play a key role during embryogenesis.

• Peifer M, Orsulic S, Sweeton D, Wieschaus E
• A role for the Drosophila segment polarity gene armadillo in cell adhesion and cytoskeletal integrity during oogenesis.
• Development. 1993; 118: 1191-207
• Display abstract

• The epithelial sheet is a structural unit common to many tissues. Its organization appears to depend on the function of the multi-protein complexes that form adherens junctions. Elegant cell biological experiments have provided support for hypotheses explaining the function of adherens junctions and of their components. These systems, however, lack the ability to test function within an entire organism during development. The realization that the product of the Drosophila segment polarity gene armadillo is related to the vertebrate adhesive junction components plakoglobin and beta-catenin led to the suggestion that armadillo might provide a genetic handle to study adhesive junction structure and function. An examination of the potential function of Armadillo in cell-cell adhesive junctions was initiated using the Drosophila ovary as the model system. We examined the distribution of Armadillo in the Drosophila ovary and demonstrated that this localization often parallels the location of cell-cell adhesive junctions. The consequences of removing armadillo function from the germ-line cells of the ovary were also examined. Germ-line armadillo mutations appear to disrupt processes requiring cell adhesion and integrity of the actin cytoskeleton, consistent with a role for Armadillo in cell-cell adhesive junctions. We have also used armadillo mutations to examine the effects on ovarian development of altering the stereotyped cell arrangements of the ovary. The implications of these results for the role of adhesive junctions during development are discussed.

• McCrea PD, Brieher WM, Gumbiner BM
• Induction of a secondary body axis in Xenopus by antibodies to beta-catenin.
• J Cell Biol. 1993; 123: 477-84
• Display abstract

• We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.

• Gumbiner BM, McCrea PD
• Catenins as mediators of the cytoplasmic functions of cadherins.
• J Cell Sci Suppl. 1993; 17: 155-8
• Display abstract

• The catenins are polypeptides that bind to the conserved cytoplasmic tail of cadherins and are required for cadherin function. alpha-Catenin is related to vinculin and seems to be required for the interaction of cadherins with the actin cytoskeleton. beta-Catenin is homologous to armadillo, a segment polarity gene in Drosophila that participates in developmental signaling. Recent findings indicate that beta-catenin also participates in developmental signaling and embryonic patterning in Xenopus laevis. At least a portion of the electrophoretic band migrating at the position of gamma-catenin consists of plakoglobin, a desmosomal and zonula adherens protein that has high sequence similarity to beta-catenin and armadillo. The catenins may be involved in the regulation of cadherin function during tissue morphogenesis and tumorigenesis.

• Moon RT, DeMarais A, Olson DJ
• Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement.
• J Cell Sci Suppl. 1993; 17: 183-8
• Display abstract

• Wnt genes encode secreted glycoproteins, and, because of their homology with the Drosophila segment polarity gene wingless, are likely to play important roles as modulators of local intercellular signalling during embryonic development. Although little is known of the mechanisms by which Wnts signal in an autocrine or paracrine manner, it is increasingly clear that cells can respond rapidly to Wnt signals in the absence of transcription, and that these responses may include changes in cell adhesion and cell movement. We review recent evidence from studies on Xenopus laevis and other systems, which demonstrate that (1) a subset of Wnts modulate gap junctional permeability, which may be a reflection of changes in cadherin-mediated cell adhesion, (2) embryos express beta-catenin and plakoglobin, which are homologs of the armadillo gene products, known to be involved in the wingless signalling pathway, and known to be found at cell junctions, and (3) overexpression of specific Wnts in Xenopus embryos leads to clear changes in cell behavior and movement.

• Breen E, Clarke A, Steele G Jr, Mercurio AM
• Poorly differentiated colon carcinoma cell lines deficient in alpha-catenin expression express high levels of surface E-cadherin but lack Ca(2+)-dependent cell-cell adhesion.
• Cell Adhes Commun. 1993; 1: 239-50
• Display abstract

• Studies on several different types of carcinomas, with the notable exception of colon carcinoma, have shown that poorly differentiated tumors are frequently deficient in E-cadherin dependent cell-cell adhesion. In this study, we examined Ca(2+)-dependent cell-cell adhesion in colon carcinoma cell lines. Five poorly differentiated (Clone A, MIP 101, RKO, CCL 222, CCL 228) and four moderately-well differentiated (CX-1, CCL 235, DLD-2, CCL 187) colon carcinoma cell lines were assayed for their ability to form cell-cell aggregates and for their levels of E-cadherin expression. All of the poorly differentiated cell lines exhibited low levels of Ca(2+)-dependent cell-cell aggregation, in contrast to the moderately-well differentiated cell lines. Contrary to most previous studies, however, we observed that three of the five poorly differentiated cell lines examined expressed E-cadherin by FACS analysis and immunoprecipitation using an E-cadherin mAb. In fact, two of these cell lines expressed a 3- to 4-fold higher level of E-cadherin than that found in the moderately-well differentiated cell lines. mRNA levels for E-cadherin, as evaluated by both RT-PCR and Northern hybridization, corresponded to the levels of protein expression in each of the cell lines. Immunoprecipitation with an E-cadherin mAb, which is known to co-precipitate the catenins, demonstrated that the three poorly differentiated cell lines expressing E-cadherin did not co-precipitate alpha-catenin, although all of the moderately-well differentiated cell lines expressed both alpha- and beta-catenin. RT-PCR confirmed the absence of the alpha-catenin mRNA from two of these cell lines. Stable expression of an alpha-catenin cDNA in one of the poorly differentiated cell lines lacking alpha-catenin expression resulted in a 5-fold increase in its level of Ca(2+)-dependent cell-cell aggregation, providing evidence that alpha-catenin is directly responsible for the loss of cell-cell adhesion in some cell lines. The alpha-catenin transfectants also exhibited a marked reduction in migration on collagen I. These data indicate that loss of alpha-catenin expression, as well as E-cadherin expression, can lead to a phenotype associated with poorly differentiated colon carcinomas.

• Oda T, Kanai Y, Shimoyama Y, Nagafuchi A, Tsukita S, Hirohashi S
• Cloning of the human alpha-catenin cDNA and its aberrant mRNA in a human cancer cell line.
• Biochem Biophys Res Commun. 1993; 193: 897-904
• Display abstract

• Cadherin and catenin compose cell adhesion complex and are indispensable for tight cell-cell adhesion. Dysfunction of this adhesion complex causes dissociation of cancer cells from primary tumor nodules, thus possibly contributing to cancer invasion and metastasis. In this report, we present the human alpha-catenin sequence. Human alpha-catenin showed extensive homology with that of mouse, i.e., 91.8% and 99.3% at the nucleic acid and amino acid levels, respectively, indicating that this molecule has been evolutionarily conserved in mammals. Characterization of the mRNA sequence of alpha-catenin in PC9 was also carried out, and two distinct abnormal sequences, i.e., one of 957 bp deletion resulting in a 319-amino-acid deletion and another of 761 bp deletion resulting in a frameshift, were identified. These deletions were probably produced by an error of RNA splicing, presenting one possible mechanism for the loss of intact alpha-catenin expression.

• Peifer M
• Cancer, catenins, and cuticle pattern: a complex connection.
• Science. 1993; 262: 1667-8

• Troyanovsky SM, Eshkind LG, Troyanovsky RB, Leube RE, Franke WW
• Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage.
• Cell. 1993; 72: 561-74
• Display abstract

• To examine the potential of cytoplasmic portions ("tails") of desmosomal cadherins for assembly of desmosome plaque structures and anchorage of intermediate filaments (IFs), we transfected cultured human A-431 carcinoma cells, abundant in desmosomes and cytokeratin IFs, with constructs encoding chimeric proteins in which the transmembranous region of connexin 32 had been fused with tails of desmocollin (Dsc) or desmoglein (Dsg). The results show that the tail of the long splice form a of Dsc, but not its shorter splice form b, contains sufficient information to recruit desmoplakin and plakoglobin to connexon membrane paracrystals (gap junctions) and to form a novel kind of plaque at which cytokeratin IFs attach. By contrast, chimeras containing a Dsg tail, which accumulated in the plasma membrane, showed a dominant-negative effect: they not only were unable to form gap junction structures and plaques but also led to the disappearance of all endogenous desmosomes and the detachment of IFs from the plasma membrane.

• Smith KJ et al.
• The APC gene product in normal and tumor cells.
• Proc Natl Acad Sci U S A. 1993; 90: 2846-50
• Display abstract

• The APC gene has been found to be mutated during the development of sporadic colorectal tumors as well as in the germ line of familial adenomatous polyposis patients. To facilitate the characterization of both normal and mutant APC protein, a series of monoclonal and polyclonal antibodies specific for the APC protein was produced. When lymphoblastoid cell lines derived from seven familial adenomatous polyposis patients with known mutations were analyzed by Western blot, an approximately 300-kDa protein corresponding to the predicted size of full-length APC was detected in all 7 cell lines. In addition, truncated APC proteins corresponding to the product of the known mutated alleles could be detected in 4 of the 7 lines. Similar analysis of 23 colon carcinoma and 9 adenoma cell lines revealed truncated proteins in 24 (75%) of the cell lines. Moreover, 26 (81%) of the colon tumor lines were totally devoid of the normal, full-length protein. In contrast, Western blot analysis of 40 cell lines derived from sporadic tumors of other organs detected only full-length APC. Immunohistochemical analysis of APC in normal colonic mucosa revealed cytoplasmic staining with more intense staining in the basolateral margins of the epithelial cell. This staining was markedly increased in the upper portions of the crypts, suggesting an increased level of expression with maturation. These studies provide some initial clues to the function of the cytoplasmic protein APC and demonstrate the feasibility of identifying APC mutations by direct analysis of the APC protein.

• Su LK, Johnson KA, Smith KJ, Hill DE, Vogelstein B, Kinzler KW
• Association between wild type and mutant APC gene products.
• Cancer Res. 1993; 53: 2728-31
• Display abstract

• Germline mutations of the APC gene are responsible for familial adenomatous polyposis, an autosomal dominant inherited predisposition to colorectal tumors. Mutation of the APC gene is also an early, if not initiating, event for sporadic colorectal tumorigenesis. In both cases, almost all of the currently identified mutations of APC result in the truncation of the protein. In this study, we demonstrate that truncated APC proteins can associate with the wild type APC in vivo. Using in vitro expression and immunoprecipitation, we show that the first 171 residues of APC are sufficient for APC oligomerization and that the first 45 amino acids of APC is necessary for this interaction. These results indicate that most mutant APC proteins should be able to bind to wild type APC protein and perhaps inactivate it in a dominant negative manner.

• Peifer M, McCrea PD, Green KJ, Wieschaus E, Gumbiner BM
• The vertebrate adhesive junction proteins beta-catenin and plakoglobin and the Drosophila segment polarity gene armadillo form a multigene family with similar properties.
• J Cell Biol. 1992; 118: 681-91
• Display abstract

• Three proteins identified by quite different criteria in three different systems, the Drosophila segment polarity gene armadillo, the human desmosomal protein plakoglobin, and the Xenopus E-cadherin-associated protein beta-catenin, share amino acid sequence similarity. These findings raise questions about the relationship among the three molecules and their roles in different cell-cell adhesive junctions. We have found that antibodies against the Drosophila segment polarity gene armadillo cross react with a conserved vertebrate protein. This protein is membrane associated, probably via its interaction with a cadherin-like molecule. This cross-reacting protein is the cadherin-associated protein beta-catenin. Using anti-armadillo and antiplakoglobin antibodies, it was shown that beta-catenin and plakoglobin are distinct molecules, which can coexist in the same cell type. Plakoglobin interacts with the desmosomal glycoprotein desmoglein I, and weakly with E-cadherin. Although beta-catenin interacts tightly with E-cadherin, it does not seem to be associated with either desmoglein I or with isolated desmosomes. Anti-armadillo antibodies have been further used to determine the intracellular localization of beta-catenin, and to examine its tissue distribution. The implications of these results for the structure and function of different cell-cell adhesive junctions are discussed.

• Butz S, Stappert J, Weissig H, Kemler R
• Plakoglobin and beta-catenin: distinct but closely related.
• Science. 1992; 257: 1142-4

• Ozawa M, Kemler R
• Molecular organization of the uvomorulin-catenin complex.
• J Cell Biol. 1992; 116: 989-96
• Display abstract

• The Ca(2+)-dependent cell adhesion molecule uvomorulin is a member of the cadherin gene family. Its cytoplasmic region complexes with structurally defined proteins termed alpha-, beta-, and gamma-catenins. Here we show that A-CAM (N-cadherin), another member of this gene family, also associates with catenins suggesting that this complex formation may be a general property of the cadherins. For uvomorulin it has been found that this association with catenins is of crucial importance for the adhesive function, but little is known about the molecular organization of the uvomorulin-catenin complex. Using a combination of biochemical analyses we show that a single complex is composed of one molecule of uvomorulin, one or two molecules of beta-catenin, and one molecule of alpha-catenin. Furthermore, beta-catenin seems to interact more directly with uvomorulin. In pulse-chase experiments beta-catenin is already associated with the 135-kD uvomorulin precursor molecule but the assembly of the newly synthesized alpha-catenin into the complex is only detected around the time of endoproteolytic processing.

• DeMarais AA, Moon RT
• The armadillo homologs beta-catenin and plakoglobin are differentially expressed during early development of Xenopus laevis.
• Dev Biol. 1992; 153: 337-46
• Display abstract

• Plakoglobin and beta-catenin are cytoplasmic proteins associated with the intracellular plaques of cell adhesive junctions. While plakoglobin is present in both adherens junctions and desmosomes, beta-catenin is associated with the cadherins that accumulate only in adherens junctions. Both beta-catenin and plakoglobin are homologs of armadillo, a Drosophila segment polarity gene that is considered to be in the wingless signaling pathway. We have characterized the expression and distribution of both plakoglobin and beta-catenin in Xenopus embryos. As shown by RNA blot analysis, beta-catenin and plakoglobin transcripts are present in fertilized eggs and in embryos through to tadpole stage. Whole-mount in situ hybridization indicates that both genes are expressed in the dorsal ectoderm and mesoderm of tailbud- and tadpole-stage embryos and that beta-catenin is expressed in the midbrain. Both plakoglobin and beta-catenin polypeptides are present during early Xenopus development; however, differences exist in the timing of maximal expression. Plakoglobin is present in the fertilized egg, increases in abundance by neurula stage, then declines at the tailbud and tadpole stages. beta-Catenin, recognized by an anti-arm antibody, is also present in the fertilized egg and in blastula-stage embryos. However, beta-catenin continues to be detected at the neurula, tailbud, and tadpole stages when levels of plakoglobin decline. The presence of multiple homologs of armadillo in Xenopus embryos and the differences in their patterns of expression suggest distinct roles for these proteins in processes affected by cell adhesion.

• Herrenknecht K, Ozawa M, Eckerskorn C, Lottspeich F, Lenter M, Kemler R
• The uvomorulin-anchorage protein alpha catenin is a vinculin homologue.
• Proc Natl Acad Sci U S A. 1991; 88: 9156-60
• Display abstract

• The cytoplasmic region of the Ca(2+)-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed alpha, beta, and gamma catenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against alpha catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that alpha catenin is complexed with other cadherins. In immunofluorescence tests, alpha catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk- cells, alpha catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of alpha catenin at membrane areas of cell contacts. We also have cloned and sequenced murine alpha catenin. The deduced amino acid sequence reveals a significant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.

• McCrea PD, Turck CW, Gumbiner B
• A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin.
• Science. 1991; 254: 1359-61
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• Three cytoplasmic proteins, called catenins, bind to the cytoplasmic tail of the epithelial cell-cell adhesion molecule E-cadherin. The complementary DNA sequence was determined for the 92-kilodalton beta catenin of Xenopus laevis. The sequence is homologous to mammalian plakoglobin, a protein of desmosomal and zonula adherens cell junctions, and to the plakoglobin homolog in Drosophila melanogaster, the product of the segment polarity gene armadillo. A monoclonal antibody to bovine plakoglobin recognizes the analogous beta catenin in the Madin-Darby canine kidney (MDCK) cell line. Armadillo plakoglobin may link E-cadherin to the underlying actin cytoskeleton at cell-cell junctions; the E-cadherin-catenin protein complex may also participate in the transmission of developmental information.

• Koch PJ, Walsh MJ, Schmelz M, Goldschmidt MD, Zimbelmann R, Franke WW
• Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.
• Eur J Cell Biol. 1990; 53: 1-12
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• Monoclonal antibodies to the constitutive desmosomal glycoprotein desmoglein were characterized whose epitopes are located intracellularly, i.e., in the cytoplasmic portion of this molecule, and contribute to the structure of the desmosomal plaque. Using one of these antibodies (DG3.10), a peptide was isolated from a proteolytic digest of desmoglein purified from isolated bovine muzzle demosomes, and its amino acid sequence was determined. In comparisons of this sequence with the amino acid sequence of desmoglein as deduced from the sequence of cDNA clones from the same tissue, encompassing most of approximately 7.6 kb mRNA and the complete coding region of 959 residues (calculated molecular weight approximately 102,400), the DG3.10 epitope was identified in a region starting 163 amino acids before the carboxy terminus in the first of four consecutive repeats of a homologous element of 29 +/- 1 amino acids. This topological information, together with the identification of a single hydrophobic region of sufficient length to provide a transmembrane segment and of several extended regions showing high sequence homology to various cadherins, has allowed the construction of a model of the molecular organization of desmoglein. We conclude that desmoglein is a member of the cadherin family of cell adhesion glycoproteins which is characterized by an unusually long cytoplasmic domain which exceeds those of the cadherins by more than 275 amino acids, contains special repetitive elements and spans the desmosomal plaque at least once.

• Wheelock MJ
• Catenin association with E-cadherin changes with the state of polarity of HT-29 cells.
• Exp Cell Res. 1990; 191: 186-93
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• Cadherins are transmembrane glycoproteins that mediate the calcium-dependent adhesion of cells to one another. It has been reported that at least two and probably more proteins associate with cadherins in various systems. These proteins have been called catenins. HT-29 cells can be manipulated to express either a polar or a nonpolar phenotype, depending on the growth conditions. We have taken advantage of this feature of HT-29 cells to explore the role catenins may play in cadherin-mediated adhesion. In this paper we report that several catenins co-immune-precipitate with E-cadherin in cultured human cells (HT-29 and JAR PR497) and that the nature of the complex of proteins varies with the physiological state of the HT-29 cells. In addition, we show data suggesting that the proteins that associate with calcium-dependent adhesion molecules may represent a group of proteins, some of which are present in all cells and some of which are cell-type specific.

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