Alzheimer dementia caused by a novel mutation located in the APP C-terminal intracytosolic fragment.

Since the first report showing that Alzheimer disease (AD) might be caused by mutations in the amyloid precursor protein gene (APP), 20 different missense mutations have been reported. The majority of early-onset AD mutations alter processing of APP increasing relative levels of Abeta42 peptide, either by increasing Abeta42 or decreasing Abeta40 peptide levels or both. In a diagnostic setting using direct sequence analysis, we identified in one patient with familial early-onset AD a novel mutation in APP (c.2172G>C), predicting a K724N substitution in the intracytosolic fragment. The mutation is located downstream of the epsilon-cleavage site of APP and is the furthermost C-terminal mutation reported to date. In vitro expression of APP K724N cDNA showed an increase in Abeta42 and a decrease in Abeta40 levels resulting in a near three-fold increase of the Abeta42/Abeta40 ratio. Further, in vivo amyloid positron emission tomography (PET) imaging revealed significantly increased cortical amyloid deposits, supporting that in human this novel APP mutation is likely causing disease.

Influence of tangeretin on tamoxifen's therapeutic benefit in mammary cancer.

BACKGROUND: Tamoxifen and the citrus flavonoid tangeretin exhibit similar inhibitory effects on the growth and invasive properties of human mammary cancer cells in vitro; furthermore, the two agents have displayed additive effects in vitro. In this study, we examined whether tangeretin would enhance tamoxifen's therapeutic benefit in vivo. METHODS: Female nude mice (n = 80) were inoculated subcutaneously with human MCF-7/6 mammary adenocarcinoma cells. Groups of 20 mice were treated orally by adding the following substances to their drinking water: tamoxifen (3 x 10(-5) M), tangeretin (1 x 10(-4) M), tamoxifen plus tangeretin (3 x 10(-5) M plus 1 x 10(-4) M), or solvent. RESULTS AND CONCLUSIONS: Oral treatment of mice with tamoxifen resulted in a statistically significant inhibition of tumor growth compared with solvent treatment (two-sided P = .001). Treatment with tangeretin did not inhibit tumor growth, and addition of this compound to drinking water with tamoxifen completely neutralized tamoxifen's inhibitory effect. The median survival time of tumor-bearing mice treated with tamoxifen plus tangeretin was reduced in comparison with that of mice treated with tamoxifen alone (14 versus 56 weeks; two-sided P = .002). Tangeretin (1 x 10(-6) M or higher) inhibited the cytolytic effect of murine natural killer cells on MCF-7/6 cells in vitro, which may explain why tamoxifen-induced inhibition of tumor growth in mice is abolished when tangeretin is present in drinking water. IMPLICATIONS: We describe an in vivo model to study potential interference of dietary compounds, such as flavonoids, with tamoxifen, which could lead to reduced efficacy of adjuvant therapy. In our study, the tumor growth-inhibiting effect of oral tamoxifen was reversed upon addition of tangeretin to the diet. Our data argue against excessive consumption of tangeretin-added products and supplements by patients with mammary cancer during tamoxifen treatment.

Transition from the noninvasive to the invasive phenotype and loss of alpha-catenin in human colon cancer cells.

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.

The alphaE-catenin gene (CTNNA1) acts as an invasion-suppressor gene in human colon cancer cells.

The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the alphaE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second alphaE-catenin allele. The alphaE-catenin gene is therefore, an invasion-suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes.

Functional downregulation of the E-cadherin/catenin complex leads to loss of contact inhibition of motility and of mitochondrial activity, but not of growth in confluent epithelial cell cultures.

When epithelial cells reach confluency in vitro, a number of energy-requiring activities such as growth and motility are contact-inhibited. We investigated the possible role of the E-cadherin/catenin complex, which acts as an invasion suppressor, in contact inhibition. Three strategies for modulation of the complex were used. Firstly, the cell-cell adhesion and signal transduction functions of E-cadherin were neutralized immunologically in human MCF-7/6 mammary carcinoma cells possessing a complete complex. Secondly, the effect of E-cadherin transfection in E-cadherin negative cell lines was investigated. Thirdly, alpha-catenin deficient variants of the human HCT-8/S11 colon carcinoma cell line were compared with their parent cells. In confluent cultures functional downregulation of the E-cadherin/catenin complex did not alter cell growth nor saturation density. This was shown by cell number counts, protein staining assays, cell cycle analysis, proliferation markers (Ki67 and Proliferating Cell Nuclear Antigen) and apoptosis assays. However, confluent cells with a functionally deficient complex showed positional instability and enhanced succinate dehydrogenase-mediated mitochondrial 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl) tetrazolium bromide (MTT) conversion, as compared to cells with an active complex. Our data indicate that contact inhibition of motility and of mitochondrial enzyme activity, but not of growth is regulated by the E-cadherin/catenin complex in epithelial cells.

Inhibition of tamoxifen's therapeutic benefit by tangeretin in mammary cancer.

Tangeretin, a molecule present in citrus fruits and in certain 'natural' menopausal medications, is an effective tumour growth and invasion inhibitor in vitro of human MCF 7/6 breast cancer cells. However, when added to the drinking water of MCF 7/6 tumour-bearing mice it neutralises the beneficial tumour-suppressing effect of tamoxifen. Tangeretin reduces the number of natural killer cells. This may explain why the beneficial suppressive effect of tangeretin on MCF 7/6 cell proliferation in vitro is completely counteracted in vivo.

Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay.

MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.

Internalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16.

Cytokines and other paracrine or autocrine factors functionally modulate the invasion-suppressor and signal-transducing E-cadherin/catenin complex. We have used conditioned medium from human squamous carcinoma COLO 16 cells (CM COLO 16) as a source of such factors to modulate the E-cadherin/catenin complex in human breast carcinoma MCF-7 cells. CM COLO 16 induces scattering of MCF-7/AZ, but not of MCF-7/6 cells on tissue culture plastic substratum, and reduces aggregation of MCF-7/AZ cells in suspension. Insulin-like growth factor I counteracts this reduction of aggregation. Confocal laser scanning microscopy of immunocytochemical stainings shows loss of the honeycomb pattern of E-cadherin, alpha-catenin and beta-catenin, and internalization of those elements. Cell surface biotinylation shows a decrease in membrane-bound E-cadherin. Immunoprecipitation and cell fractionation show that the composition of the complex is maintained. Interleukin-1, interleukin-6, granulocyte-monocyte colony stimulating factor, stem cell factor, scatter factor/hepatocyte growth factor and transforming growth factor-beta, added separately to MCF-7/AZ cells, could not mimic the effects of CM COLO 16. Neither could we find evidence that the 80 kDa extracellular fragment of E-cadherin is implicated in scattering of MCF-7/AZ cells. This fragment is present in CM COLO 16, but it is also produced by the MCF-7/AZ cells themselves, even at higher levels. Our data point toward cytoplasmic internalization induced by paracrine factors as one of the downregulating mechanisms for the E-cadherin/catenin complex.

Anti-invasive activity of alkaloids and polyphenolics in vitro.

Invasiveness, the ability of certain tumour cells to migrate beyond their natural tissue boundaries, often leads to metastasis, and usually determines the fatal outcome of cancer. The need for anti-invasive agents has led us to search for possibly active compounds among alkaloids and polyphenolics. One hundred compounds were screened in an assay based on the confrontation of invasive human MCF-7/6 mammary carcinoma cells with fragments of normal embryonic chick heart in vitro. Anti-invasive activity was frequently found among chalcones having a prenyl group. Six compounds were found to inhibit invasion when added to the culture medium at concentrations as low as 1 microM. For at least three of them the anti-invasive effect could be associated with a cytotoxic effect on the MCF-7/6 cells, but not on the heart tissue. This selective cytotoxicity was substantiated by different methods, such as histology and growth assays (volume measurements, cell counts, MTT and sulforhodamine B assays). The anti-invasive effects of the compounds could neither be ascribed to induction of apoptosis nor to the promotion of cell-cell adhesion. Our data indicate that among the alkaloids and polyphenolics a number of molecules can inhibit growth and invasion of human mammary cancer cells via selective cytotoxicity.