Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice.

Integrins are heterodimeric transmembrane glycoproteins which are engaged in a variety of cellular functions, such as adhesion, migration and differentiation1. The integrin alpha 6 beta 4 is expressed on squamous epithelia, on subsets of endothelial cells, immature thymocytes and on Schwann cells and fibroblasts in the peripheral nervous system. In stratified epithelia, alpha 6 beta 4 is concentrated in specialised adhesion structures, called hemidesmosomes, which are implicated in the stable attachment of the basal cells to the underlying basement membrane by connecting the intermediate filaments with the extracellular matrix. The nature of the interactions between the various hemidesmosomal proteins, that lead to the formation of hemidesmosome is poorly understood. To study the contribution of the integrin alpha 6 beta 4 in hemidesmosome formation and their anchoring properties, we inactivated the beta 4 gene in mice by targeted gene disruption. Homozygous beta 4 null mice died shortly after birth and displayed extensive detachment of the epidermis and other squamous epithelia. The dramatically reduced adhesive properties of the skin was accompanied by the absence of hemidesmosomes at the basal surface of keratinocytes. No evidence was found for impaired T-cell development, nor for defects in myelination in the peripheral nervous system.

Cre-loxP-mediated inactivation of the alpha6A integrin splice variant in vivo: evidence for a specific functional role of alpha6A in lymphocyte migration but not in heart development.

Two splice variants of the alpha6 integrin subunit, alpha6A and alpha6B, with different cytoplasmic domains, have previously been described. While alpha6B is expressed throughout the development of the mouse, the expression of alpha6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, alpha6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of alpha6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of alpha6A in embryonic stem cells, and the deletion resulted in the expression of alpha6B in all tissues that normally express alpha6A. We show that alpha6A-/- mice develop normally and are fertile. The substitution of alpha6A by alpha6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in alpha6A-/- mice. However, the substitution of alpha6A by alpha6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin-ligand interactions, was not affected in the alpha6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of alpha6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.

The hepatocyte growth factor/Met pathway in development, tumorigenesis, and B-cell differentiation.

This article summarizes the structure, signal transduction and physiologic functions of the HGF/Met pathway, as well as its role in tumor growth, invasion, and metastasis. Moreover, it highlights recent studies indicating a role for the HGF/Met pathway in antigen-specific B-cell development and B-cell neoplasia.

Effects of adriamycin on lipid polymorphism in cardiolipin-containing model and mitochondrial membranes.

The effects of the anti-tumor drug adriamycin on lipid polymorphism in cardiolipin-containing model membranes and in isolated inner mitochondrial membranes has been examined by 31P-NMR. Adriamycin binding does not affect the macroscopic structure or local order in the phosphate region of cardiolipin liposomes. In cardiolipin liposomes and in cardiolipin-phosphatidylcholine (1:1) liposomes, the drug inhibits the ability of Ca2+ to induce the hexagonal HII phase. Adriamycin interaction with both dioleoylphosphatidylethanolamine-cardiolipin (2:1) and dioleoylphosphatidylethanolamine-phosphatidylserine (1:1) liposomes results in structural phase separation into a liquid-crystalline hexagonal HII phase for the phosphatidylethanolamine and a liquid-crystalline lamellar phase for the negatively charged phospholipid. Combined high-resolution 31P-NMR, electron microscopy and light scattering studies reveal the prominent fusion capacity of adriamycin towards cardiolipin-phosphatidylcholine small unilamellar vesicles. Addition of Ca2+ to total rat liver inner mitochondrial membrane lipids, dispersed in excess buffer, results in hexagonal HII formation for part of the phospholipids. By contrast, the original bilayer structure is completely conserved when the above experiment is performed in the presence of adriamycin. 31P-NMR spectra of isolated inner mitochondrial membranes are indicative of a bilayer organization for the majority of the phospholipids. Approximately 15% of the signal intensity originates from phospholipids which experience isotropic motion. Adriamycin addition almost completely eliminates the latter spectral component. In the absence of adriamycin, Ca2+ addition greatly increases the percentage of the phospholipids giving rise to an isotropic signal possibly indicating the formation of non-lamellar lipid structures. Adriamycin which specifically binds to cardiolipin (K. Nicolay et al. (1984) Biochim. Biophys. Acta 778, 359-371) completely blocks the Ca2+-induced structural reorganization of the lipids in this membrane.

The interaction of adriamycin with cardiolipin in model and rat liver mitochondrial membranes.

The interaction of adriamycin with cardiolipin in model membranes and in various membrane preparations derived from rat liver mitochondria was studied and the results are analyzed in the light of a possible specific interaction between adriamycin and cardiolipin. It was found that adriamycin binds to cardiolipin-containing model membranes with a fixed stoichiometry of two drug molecules per cardiolipin. Furthermore, the extent of drug complexation by mitochondria and mitoplasts (inner membrane plus matrix) is in reasonable agreement with their cardiolipin content. In contrast, adriamycin-binding curves of inner membrane ghosts and submitochondrial particles reveal considerable association to an additional site, presumably RNA. The evidence for the potential importance of RNA as a target comes from experiments on outer membranes and microsomes which both appear to bind substantial amounts of adriamycin. Removal of the major part of the RNA associated with these fractions by EDTA treatment is accompanied by a dramatic reduction of binding capacity. We propose that endogenous RNA present in mitochondria and mitoplasts is not accessible for adriamycin at low concentrations of the drug due to the presence of an intact lipid barrier. This potential site comes to expression in ghosts and submitochondrial particles, due to the absence of an intact lipid bilayer and due to the inside-out orientation of the limiting membrane, respectively. Electron microscopical studies show that adriamycin induces dramatic changes in mitochondrial morphology, similar to the uncoupler-induced effects described by Knoll and Brdiczka (Biochim. Biophys. Acta 733, 102-110 (1983). Adriamycin has an uncoupling effect on mitochondrial respiration and oxidative phosphorylation. The concentration dependence of this effect correlates with the adriamycin-binding curve for mitochondria which implies that only bound adriamycin actively inhibits respiration.

CD44 regulates arteriogenesis in mice and is differentially expressed in patients with poor and good collateralization.

BACKGROUND: Arteriogenesis refers to the development of collateral conductance arteries and is orchestrated by circulating monocytes, which invade growing collateral arteries and act as suppliers of cytokines and growth factors. CD44 glycoproteins are involved in leukocyte extravasation but also in the regulation of growth factor activation, stability, and signaling. Here, we explored the role of CD44 during arteriogenesis. METHODS AND RESULTS: CD44 expression increases strongly during collateral artery growth in a murine hind-limb model of arteriogenesis. This CD44 expression is of great functional importance, because arteriogenesis is severely impaired in CD44-/- mice (wild-type, 54.5+/-14.9% versus CD44-/-, 24.1+/-9.2%, P<0.001). The defective arteriogenesis is accompanied by reduced leukocyte trafficking to sites of collateral artery growth (wild-type, 29+/-12% versus CD44-/-, 18+/-7% CD11b-positive cells/square, P<0.01) and reduced expression of fibroblast growth factor-2 and platelet-derived growth factor-B protein. Finally, in patients with single-vessel coronary artery disease, the maximal expression of CD44 on activated monocytes is reduced in case of impaired collateral artery formation (poor collateralization, 1764+/-572 versus good collateralization, 2817+/-1029 AU, P<0.05). CONCLUSIONS: For the first time, the pivotal role of CD44 during arteriogenesis is shown. The expression of CD44 increases during arteriogenesis, and the deficiency of CD44 severely impedes arteriogenesis. Maximal CD44 expression on isolated monocytes is decreased in patients with a poor collateralization compared with patients with a good collateralization.

A novel beta 1 integrin isoform produced by alternative splicing: unique expression in cardiac and skeletal muscle.

The mRNA's of several integrin subunits are alternatively spliced in the region encoding cytoplasmic domains, that may potentially provide alternative integrin-cytoskeleton interactions and transmembrane signaling pathways. We identified a novel cytoplasmic tail variant of the human beta 1 subunit by reverse transcriptase polymerase chain reaction. This fourth beta 1 variant, named beta 1D, is specific for skeletal and cardiac muscle. The determined genomic organization of the 3'-region of the human beta 1 gene reveals that beta 1D is produced by alternative splicing of mRNA. In addition, we show that the expression of beta 1D is developmentally regulated during murine myoblast differentiation, suggesting a role for beta 1D in myogenesis.

Stimulation by melanocortins of neurite outgrowth from spinal and sensory neurons in vitro.

Using ELISAs for B-50/GAP43 and neurofilament (NF), we tested ACTH(1-24), alpha-MSH, ACTH(4-10), and an ACTH(4-9) analogue (ORG2766) for their ability to induce sprouting and neuritogenesis from spinal and sensory neurons. Dissociated fetal rat spinal cord neurons or neonatal rat dorsal root ganglion (DRG) cells were cultured with peptide and assayed after 24, 48, or 96 h. In spinal neurons, alpha-MSH and ACTH(1-24) induced the expression of B-50 dose dependently. After 24 h alpha-MSH had a stimulatory effect (from 10 nM onwards), with a maximum at 100 microM (36% increase). After 96 h the maximal effect of 100 microM alpha-MSH on B-50/GAP43 was lower (19%). ACTH(1-24) (100 microM) stimulated B-50/GAP43 by 19%. Neurofilament levels (96 h) were elevated maximally by 64% at 100 microM alpha-MSH. In DRG neurons a bell-shaped dose-response curve was found for alpha-MSH, the maximal effect being observed after 48 h at 100 nM: 54% for B-50/GAP43 and 22% for NF. In both culture systems neither ACTH(4-10) nor ORG2766 was effective. We conclude that alpha-MSH stimulates the expression of B-50/GAP43 (sprouting) and the formation of NF (neurite elongation) and may therefore be considered a neurotrophic factor.

Trophic influences of alpha-MSH and ACTH4-10 on neuronal outgrowth in vitro.

Slices of foetal spinal cords in culture were used to establish possible trophic effects of alpha-melanocyte stimulating hormone (alpha-MSH) and a fragment of the adrenocorticotropic hormone (ACTH4-10) on the outgrowth of neurites from spinal neurons. The spinal cord slices were treated with peptides over a wide concentration range. Using monoclonal antibodies against (subunits of) neurofilament followed by immunofluorescence, we could show that the extension consisted mainly of axons. After 5 and 7 days, outgrowth was quantified with 2 different techniques, namely by visual scoring under phase contrast and by means of an ELISA for neurofilament protein. Both methods yielded the same dose-response profile. Both alpha-MSH and ACTH4-10 stimulated the formation of neurites in a dose-dependent manner, with a maximal stimulatory effect at 0.001-0.01 nM (ACTH4-10) or 0.1-1.0 nM (alpha-MSH). The maximal effect of the peptides was 30-40% compared to controls. We conclude that alpha-MSH and ACTH4-10 stimulate axonal outgrowth from foetal spinal cord slices in vitro in a dose-dependent way.

Targeted gene disruption: applications in neurobiology.

In classical gene inactivation approaches by homologous recombination in embryonic stem cells, the resulting knockout mice are genotypically homogeneous. The inactivation of a gene in the complete organism may sometimes lead to early embryonic lethality. The observation that bacterial recombinases can drive site-specific recombination in mammalian cells has allowed for spatiotemporally controlled genetic modifications. Thus, conditional gene inactivation can be achieved in a specific subset of cells, leaving the rest of the organism genotypically unchanged. Another application of bacterial recombinases is the generation of exon-specific knockout mice, allowing for the analysis of the role of tissue-specific splice variants. A combination of the above-mentioned bacterial recombinase technique with inducible promoter systems permits the investigator to choose precisely the onset of recombination. An extension of the above-mentioned techniques is the combination of the bacterial recombinase technique with adenovirus-based technology, which would open vast possibilities of tissue-specific genetic modifications in a controlled time frame.

Cultivation of rat fetal spinal cord slices in a semi-solid medium: a new approach to studying axonal outgrowth and regeneration.

A soft agar culture system was used for the cultivation of spinal cord slices with the purpose of improving the evaluation of the dynamics of axonal outgrowth and development. Slices of the spinal cord of 15-day-old fetal Wistar rats were cultured in a 0.5% agar culture medium. The sprouting and outgrowth of axons from the slices was observed at 6-24-h intervals. The morphology and growth rates of axons could be easily investigated by light microscopy. Quantification of growth parameters of individual neurites is made easy because no cells migrate out of the slices, so that the outgrowth is not masked by migrating neurons, fibroblasts, glial cells etc. The axons had well-developed growth cones, comparable to those observed in liquid medium; the daily growth rate was on average 318 microns during the 6 days of observation, with a maximum of 1050 microns per day. Back-labelling with a fluorescent dye (DiI) indicated that the longest neurites originated from motoneurons. Our experiments show that axons can develop and grow in a soft agar medium without the need for adding any growth promoting factor or substrate molecule.

The expression of B-50/GAP-43 during development of rat spinal neurons in culture is regulated by interneuronal contact.

The neuron-specific phosphoprotein B-50 (GAP-43) is associated with neuritogenesis during development and regeneration. We monitored B-50 in cultured spinal neurons (fetal rat) with an enzyme-linked immunoadsorbent assay. B-50 levels increased from 24 to 72 h, then decreased. Other cultures, fixed at 24 h intervals and incubated with anti-B-50 immunoglobulins and fluorescent conjugates, showed that B-50 was present in somata after 24 h, but mainly in neurites after 48 h; after 72-96 h neurons migrated into clusters and B-50 was detected only in free neurites at the perimeter of the culture. We conclude that B-50 expression is down-regulated by neurite-cell or cell-cell contact.

MET signalling in primary colon epithelial cells leads to increased transformation irrespective of aberrant Wnt signalling.

It has been shown that in hereditary and most sporadic colon tumours, components of the Wnt pathway are mutated. The Wnt target MET has been implicated in the development of colon cancer. Here, we show that overexpression of wild-type or a constitutively activated form of MET in colon epithelial cells leads to increased transformation irrespective of Wnt signalling. Fetal human colon epithelial cells without aberrant Wnt signalling were transfected with wild-type or mutated MET constructs. Expression of these constructs leads to increased phosphorylation of MET and its downstream targets PKB and MAPK. Upon stimulation with HGF, the expression of E-cadherin is downregulated in wild-type MET-transfected cells, whereas cells expressing mutated MET show low E-cadherin levels independent of stimulation with ligand. This implies a higher migratory propensity of these cells. Furthermore, fetal human colon epithelial cells expressing the mutated form of MET have colony-forming capacity in soft agar, while cells expressing wild-type MET show an intermediate phenotype. Subcutaneous injection of mutated MET-transfected cells in nude mice leads to the formation of tumours within 12 days in all mice injected. At this time point, mock-transfected cells do not form tumours, while wild-type MET-transfected cells form subcutaneous tumours in one out of five mice. We thus show that MET signalling can lead to increased transformation of colon epithelial cells independent of Wnt signalling and in this way could play an essential role in the onset and progression of colorectal cancer.

Serum from patients with amyotrophic lateral sclerosis induces the expression of B-50/GAP-43 and neurofilament in cultured rat fetal spinal neurons.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons in the spinal cord, brain stem, and cortex. Cultures of fetal rat spinal cord cells were used to test sera from ALS patients (ALS sera) on their ability to influence the expression of the neuron-specific phosphoprotein B-50/GAP-43. Neurons were treated with ALS sera, sera of age-matched controls (CON sera), or sera of patients with autonomic neuropathy (AUTO sera) and fixed after 24 or 96 h. The levels of B-50 and neurofilament (NF) protein were assayed with an enzyme-linked immunoadsorbent assay (ELISA). No toxic effects of the ALS sera were observed. It appeared that after 24 h, both B-50 and NF levels were elevated in the ALS sera-treated cells by 12 and 11%, respectively. After 96 h, the B-50 level was 19% higher than in CON sera-treated neurons, and the NF level was 29% higher. AUTO sera did not differ from CON sera. The stimulating effect of ALS sera was absent if the sera were heated at 56 degrees C for 30 min. We conclude that ALS serum induces the expression of B-50 and the subsequent axonal outgrowth and maturation in vitro. This induction might be a reflection in vitro of the processes underlying the collateral sprouting responses observed in ALS patients.

Mutagenesis of the amino-terminal glycine to alanine in Gs alpha subunit alters beta gamma-dependent properties and decreases adenylylcyclase activation.

Proteolytic removal and genetic deletion of the amino-terminal domain of G protein alpha subunit have shown that this region is necessary for interaction with beta gamma subunits. In the alpha subunits which undergo myristoylation, myristoylation of the amino-terminal glycine modulates the affinity of alpha subunit for the beta gamma complex. To determine the role of the same glycine in nonmyristoylated alpha subunits, we substituted it for alanine in Gs alpha and characterized the properties of the mutated chain G2A Gs alpha. The mutant could still bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) as revealed by its resistance to trypsin proteolysis and was able to interact with the membrane. However, G2A Gs alpha was a poor substrate for cholera toxin-catalyzed ADP-ribosylation either in the soluble form or when membrane-associated. Addition of beta gamma subunits increased the sedimentation rate of G2A Gs alpha in sucrose gradients. Binding experiments performed on cyc- membranes reconstituted by G2A Gs alpha showed that the GTP-induced shift of isoproterenol affinity for the beta-adrenergic receptors was reduced. On the same membranes, isoproterenol, GTP gamma S and NaF were 2-fold less effective for activating adenylylcyclase when compared to cyc- membranes reconstituted by Gs alpha. This differential stimulation of adenylylcyclase was not due to an affinity change for the effector but to a decrease in the maximal activation. Thus the G2A substitution affected beta gamma-dependent properties on reconstituted membranes such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation and we propose that the decreased activation of adenylylcyclase might result from the same defect. Although not essential for association with beta gamma subunits, the amino-terminal glycine of nonmyristoylated Gs alpha might play a modulatory role in this interaction.

alpha3beta1 and alpha6beta4 integrin receptors for laminin-5 are not essential for epidermal morphogenesis and homeostasis during skin development.

Continuous regeneration and homeostasis of the stratified epidermis requires coordinated regulation of cell proliferation, cell differentiation, and cell survival. Integrin-mediated cell adhesion to the extracellular matrix has important roles in regulating each of these processes. Integrins alpha3beta1 and alpha6beta4 are both receptors on epidermal keratinocytes for the basement membrane protein laminin-5, the major ligand for epidermal adhesion in mature skin. Ablation in mice of either alpha3beta1 or alpha6beta4, through null mutation of the gene encoding the alpha3, alpha6, or beta4 integrin subunit, results in epidermal blistering of varying severity. Our previous studies showed that, despite blistering, differentiation and stratification of the epidermis appeared essentially normal in mice that lacked either alpha3beta1 or alpha6beta4. However, these studies did not definitively address the specific developmental importance of each integrin, since they may have overlapping and/or compensatory functions. Given the individual importance of alpha3beta1 or alpha6beta4 in maintaining the dermo-epidermal junction in mature skin, we sought to determine the importance of these integrins for embryonic skin development and epidermal morphogenesis. In the current study, we analyzed skin development in mutant embryos that completely lack both integrins alpha3beta1 and alpha6beta4. Although alpha3beta1/alpha6beta4-deficient embryos displayed epidermal blistering by stage E15.5 of development, they also retained regions of extensive epidermal adhesion to the basement membrane through stage E16.5, indicating alternative adhesion mechanisms. Apoptosis was induced in detached epidermis of alpha3beta1/alpha6beta4-deficient embryos, exemplifying vividly the importance of epithelial attachment to the basement membrane for cell survival. However, apoptotic cells were completely absent from attached epidermis of alpha3beta1/alpha6beta4-deficient embryos, showing that epithelial adhesion that occurred independently of alpha3beta1 and alpha6beta4 also protected cells from apoptosis. Remarkably, in the absence of the known laminin-5 binding integrins (alpha3beta1, alpha6beta4, and alpha6beta1), keratinocytes retained the capacity to proliferate in the epidermis, and epidermal stratification and skin morphogenesis appeared normal prior to blister formation. These findings show that while alpha3beta1 and alpha6beta4 are both required for integrity of the dermo-epidermal junction, neither one is essential for epidermal morphogenesis during skin development.

Tumour necrosis factor-alpha, interferon-gamma and interferon-beta exert antiviral activity in nervous tissue cells.

The individual and synergistic antiviral effects of cytokines released by infiltrating immune cells or by cells of the nervous system may play an important role in inhibiting virus spread during infections of the central nervous system (CNS). We examined the antiviral activity against the neurotropic pseudorabies virus (PRV) of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and combinations of these cytokines, as compared to that of IFN-beta, in rat nervous tissue cells. PRV replicated efficiently in all neural cell types tested, including neurons, astrocytes and oligodendrocytes. The inhibitory effects were determined by quantifying the inhibition of virus plaque formation, yields of infectious virus at various times after infection and synthesis of viral proteins. At a low m.o.i., IFN-gamma and IFN-beta inhibited viral plaque formation in all cell types; TNF-alpha was effective only in astrocytes but showed synergy with IFN-gamma. At a higher m.o.i., IFN-beta inhibited yields of infectious virus more effectively than IFN-gamma, whereas TNF-alpha had no effect on virus yields and was only marginally synergistic with the antiviral activity of IFN-gamma. The yield-reduction assays correlated well with cytokine-induced inhibition of viral protein synthesis. Our results show that both IFN-gamma and IFN-beta can induce a state of antiviral resistance in neural cells whereas TNF-alpha is effective only in astrocytes at low m.o.i.; they suggest an antiviral role of cytokines in the immune response to virus infections of the CNS.

Heterogeneity of mesothelioma cell lines as defined by altered genomic structure and expression of the NF2 gene.

Germ-line mutations in the neurofibromatosis 2 (NF2) gene cause a susceptibility to the development of schwannoma and meningioma, 2 mostly benign tumors of neural crest origin. Bi-allelic inactivation of this gene has been observed in sporadic schwannomas and meningiomas. The NF2 gene may also be somatically inactivated in human malignant mesotheliomas (HMMs). Surprisingly, patients with an NF2 germ-line mutation have not been reported to be at an increased risk for this highly invasive tumor of mesodermal origin. To investigate in HMMs the silencing mechanism of the NF2 gene, we have analyzed its structure and expression in a series of 18 cell lines derived from HMMs. NF2 gene alterations were identified at a genomic level in 7 cell lines and were associated with a marked decrease in the concentration of the NF2 transcript. This decrease was also observed in 4 additional cell lines with no identified NF2 mutation. The 11 cell lines presented evidence suggesting deletion of one NF2 allele. None of these enabled the detection of normal or truncated forms of the NF2 protein by immunoprecipitational immunoblot analyses. In the 7 remaining cell lines, NF2 mRNA and NF2 protein were easily detectable. Among the latter, 4 lines were heterozygous for several chromosome 22 microsatellite loci, suggesting the presence of 2 NF2 alleles. Taken together, our data indicate that silencing of the NF2 gene is restricted to a subset of mesothelioma cell lines. The availability of established cell lines with different characterized NF2 status provides a powerful tool to explore the mechanism by which the NF2 protein exerts its tumor suppressive activity.

Sulindac targets nuclear beta-catenin accumulation and Wnt signalling in adenomas of patients with familial adenomatous polyposis and in human colorectal cancer cell lines.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive potential against colorectal carcinomas (CRCs). Inhibition of cyclooxygenase (COX)-2 underlies part of this effect, although COX-2-independent mechanisms may also exist. Nonsteroidal anti-inflammatory drugs appear to inhibit the initial stages of the adenoma-carcinoma sequence, suggesting a link to the APC/beta-catenin/TCF pathway (Wnt-signalling pathway). Therefore, the effect of the NSAID sulindac on nuclear (nonphosphorylated) beta-catenin and beta-catenin/TCF-mediated transcription was investigated. Nuclear beta-catenin expression was assessed in pretreatment colorectal adenomas and in adenomas after treatment with sulindac from five patients with familial adenomatous polyposis (FAP). Also, the effect of sulindac sulphide on beta-catenin/TCF-mediated transcription was studied. Adenomas of FAP patients collected after treatment with sulindac for up to 6 months showed less nuclear beta-catenin expression compared to pretreatment adenomas of the same patients. Sulindac sulphide abrogated beta-catenin/TCF-mediated transcription in the CRC cell lines DLD1 and SW480, and decreased the levels of nonphosphorylated beta-catenin. As a result, the protein levels of the positively regulated TCF targets Met and cyclin D1 were downregulated after sulindac treatment. This study provides in vivo and in vitro evidence that nuclear beta-catenin localisation and beta-catenin/TCF-regulated transcription of target genes can be inhibited by sulindac. The inhibition of Wnt-signalling provides an explanation for the COX-2-independent mechanism of chemoprevention by NSAIDs.