E-cadherin germline missense mutations have been shown to be responsible for significant loss of protein activity. A new cytoplasmic E-cadherin germline missense mutation (V832 M) was recently identified in a hereditary diffuse gastric cancer (HDGC) Japanese family. This E-cadherin mutant was cloned in a Chinese hamster ovary cell model system and functionally characterized, in terms of aggregation and invasion. Cells expressing the germline V832M mutant fail to aggregate and invade into collagen, supporting the pathogenic role of this germline missense mutation in gastric cancer. We also tested the ability of this mutation to activate the TCF-LEF trascriptional activity, in comparison with three other E-cadherin missense mutations (T340A, A634V and A617T), associated to loss of E-cadherin function. All the E-cadherin mutants reduced TCF-LEF activation to a similar extent as the wild-type protein, suggesting that the oncogenic effect of the E-cadherin mutants is unlikely to be transmitted through a b-catenin-dependent activation of the WNT pathway. Oncogene (2003Oncogene ( ) 22, 5716-5719. doi:10.1038 Keywords: E-cadherin; hereditary diffuse gastric cancer; mutations; cell-cell adhesion; invasion; TCF-LEF E-cadherin is a transmembrane glycoprotein localized at the adherens junctions of epithelial cells, where it mediates homophilic, Ca 2 þ -dependent cell-adhesion (Shore and Nelson, 1991;Shapiro et al., 1995;Pertz et al., 1999). The adhesion process is accomplished by homophilic interactions between extracellular E-cadherin domains, leading to the formation of zipper-like structures (Pertz et al., 1999). The E-cadherin intracellular domain interacts with catenins, assembling the celladhesion complex. b-Catenin and g-catenin compete for the same binding site at the E-cadherin cytoplasmic tail, directly linking the adhesion complex to the cytoskeleton through a-catenin (Christofori and Semb, 1999). These interactions represent a prerequisite not only for cell-cell adhesion, but also for inhibition of cell motility and invasion (Chen et al., 1997). The dual role of b-catenin, as a component of the cell-cell adhesion complex as well as transcriptional coactivator in the WNT growth factor signal pathway has been extensively characterized (Cavallaro et al., 2002). In the absence of WNT, phosphorylation by glycogen synthase kinase-3b (GSK-3b) targets b-catenin for degradation in the adenomatous polyposis coli (APC)-GSK-3b complex. It was reported that APC and/or GSK-3b inactivation as well as mutations of AXIN and bcatenin itself (Bienz and Clevers, 2000) leads to an accumulation of high level of b-catenin into the cytoplasm; b-catenin could translocate into the nucleus and bind to a member of the TCF-LEF-1 family of transcription factors, activating gene expression (Polakis, 2000).Aberrant E-cadherin protein expression has been reported in several types of human cancer (Becker et al., 1994;Gayther et al., 1998;Guilford et al., 1998;Machado et al., 2001;Van Aken et al., 2001;Thiery, 2002). Despite what has been observe...

Neurotensin(8–13) analogues were biologically stabilised by replacement of the peptide bond between amino acids 8 and 9 by the reduced ψ(CH2‐NH) isostere. Diethylenetriaminepentaacetic acid (DTPA) analogues for In‐111 labelling and 2‐bromo‐phenyl‐acetyl analogues for radioiodination, showed receptor affinities in the nanomolar range in combination with a biological half life in human plasma up to 275 minutes. Biodistribution studies in male Wistar rats of metabolically stabilised and non‐stabilised 111In‐DTPA‐NT(8–13) analogues showed a major clearance from the blood through the kidneys. 125I‐Labelled neurotensin (8–13) analogues showed accumulation up to 2.2% of the injected dose per g tissue in the liver which might be an important disadvantage when diagnosis of tumours in the gut is aimed. Neurotensin(8–13) analogues labelled with In‐111 or I‐123 may act as new potential peptidergic radiopharmaceuticals for SPET diagnosis of neurotensin receptor (NTR) positive tumours. Copyright © 1999 John Wiley & Sons, Ltd.

Invasion of malignant MO4 cells into embryonic chick heart fragments in an organ culture assay was arrested for at least 7 days when the temperature was lowered to 28 degrees C. Prolonged culturing of MO4 cells at 28 degrees C on tissue culture substrates showed no recuperation of fucose incorporation into cell surface glycopeptides. However, invasion was restored after 10 days of organ culture in confrontation with chick heart tissue at 28 degrees C. A histoautoradiographic study showed that the regained capability to invade was accompanied by an increase in fucose labeling of the MO4 cells in the invading areas. At 28 degrees C the incorporation of [3H]fucose into total cell protein was drastically reduced, whereas [3H]leucine incorporation as a measure for protein synthesis was less affected. Cell surface glycopeptides, metabolically labeled with either fucose or glucosamine at 28 degrees C, showed a time-dependent decrease in the incorporation of fucose but not of glucosamine and no changes in overall size distribution. Low temperature did not reduce fucosyltransferase activity but the relative accumulation of fucose-1-P suggested inhibited conversion towards GDP-fucose. Moreover, mouse L cells which were incapable of invading chick heart tissue appeared also deficient in fucose incorporation, owing to low levels of fucosyltransferase activity. According to the results, fucosylation of surface carbohydrates may be required for invasive capacity and restored in MO4 cells invading at 28 degrees C by metabolic cooperation with the host tissue.

The involvement of phosphoinositide 3-kinases class IA (PI3K-alpha and -beta) in cancer cell proliferation, survival, motility, and invasiveness is now well established. However, the possible contribution of the class IB PI3Kgamma in cancer cell transformation remains to be explored. In this study, we have stably transfected the PI3Kgamma-deficient human colon cancer cell line HCT8/S11 with expression vectors encoding either wild-type PI3Kgamma, its plasma membrane targeted form CAAX-PI3Kgamma, or the PI3Kgamma lipid and protein kinase-dead mutant (CAAX-K832R). We provide evidence that the constitutively active CAAX-PI3Kgamma variant induced collagen type I invasion in HCT8/S11 cells through disruption of cell-cell adhesion, with no apparent impact on cell proliferation and motility. The proinvasive activity of CAAX-PI3K-gamma was abolished by pharmacological inhibitors targeting PI3-K activities (wortmannin), Rho-GTPases, and the Rho-Rho kinase axis (C3T exoenzyme and Y27632, respectively). Conversely, the wild-type PI3Kgamma and its double mutant CAAX-K832R were ineffective on cancer cell invasion measured under control or stimulated conditions operated with the proinvasive agents leptin and intestinal trefoil factor. Taken together, our data indicate that PI3Kgamma exerts transforming functions via several mechanisms in human colon epithelial cancer cells, including alterations of homotypic cell-cell adhesion and induction of collagen type I invasion through canonical proinvasive pathways.

Despite its intensive use in adjuvant breast cancer therapy for more than 30 years, the exact mechanisms of action of tamoxifen have not yet been fully characterized. Tamoxifen was recently shown to restore the Ecadherin function of human breast cancer MCF7/6 cells and to suppress their invasive phenotype. Because tamoxifen interacts with targets implicated in Ca 2+ homeostasis, we explored the possibility that the restoration of E-cadherin function in MCF7/6 cells induced by this drug could be affected by Ca 2+ modulators. Two different Ca 2+ channel antagonists (verapamil and nifedipine) potentiated the effect of tamoxifen on E-cadherin function, as evaluated with a fast cell aggregation assay. These molecules decreased the tamoxifen concentration needed to restore the E-cadherin function from 10 -6 M to 10 -7 M. When incubated with a Ca 2+ channel agonist, Bay K8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoro-methylphenyl)-pyridine-5-carboxylate), the effect of tamoxifen on E-cadherin function was completely abolished. These results demonstrate that the restoration of the E-cadherin function induced by tamoxifen depends, at least in part, on a Ca 2+ pathway, and support the evidence of an effect of tamoxifen on Ca 2+ -dependent mechanisms. Our data also suggest that Ca 2+ channel modulators could make it possible to decrease the dose of tamoxifen administered to patients without reducing the therapeutic effects.