Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: Correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level

Urokinase-type plasminogen activator (uPA) and one of its inhibitors, the PAI-1, are involved in the proteolytic cascade of matrix degradation during in vivo morphogenesis or metastasis. In the present study, we have characterized the in vitro morphological behavior of human normal and malignant mammary epithelial cells and determined the levels of uPA activity and PAI-1 during these events. Two-dimensional cultures in the presence of inductive fibroblast-conditioned medium (CM) allowed migration of HBL-100 cells and MDA-MB-231 cells. Normal human mammary epithelial cells (HMEC) and MCF-7 cells failed to migrate under these conditions. The epithelial cell migration correlated with an increase in the uPA activity whereas their immobility correlated with both increases in uPA activity and PAI-1 level. In three-dimensional cultures in collagen gel, fibroblasts or fibroblast CM induced branching tubular morphogenesis to HMEC, cord-like extensions to HBL-100 cells and a greater invasiveness ability to MDA-MB-231 cells. These events correlated with an increased uPA activity. In contrast, no morphological rearrangement was observed in MCF-7 cells and this correlated with both increases in uPA activity and PAI-1 level. Altogether, these results show that the in vitro mammary epithelial behavior is under the influence of mesenchymal inductive signals and is in agreement with modifications of uPA activity and PAI-1 levels. Our culture system gives a suitable model to study the mechanisms of mammary development and metastasis and to highlight the involvement of proteases and their inhibitors in cell-cell positioning and cell-matrix reorganization.     

Alcohol intoxication via the lung in rats]

1. The technique of chronic alcohol intoxication by inhalation of alcohol vapor was developed in rats. 2. The blood alcohol level value of rats staying in an alcohol-containing atmosphere increases (from 0 to 4 mg/l) in terms of the atmospheric alcohol level (from 0 to 20 mg/l). 3. The mean blood alcohol level of a group of animals maintained during 20 days in an atmosphere containing 15 mg/l of air, increases regularly during 7 days, and then decreases slowly. 4. Animals that are staying in an atmosphere with a regularly increasing alcohol level can breathe an air containing 20 mg/l of alcohol. This dose is early lethal when used in other animals from the beginning of treatment, what confirms the metabolic tolerance. 5. Withdrawal signs characterized by a central nervous system hyperexcitability are shown by animals which had a high blood alcohol level during 4 or 5 days, when they are back into the ambient atmosphere.     

Use of ethanol naturally enriched in carbon 13 for metabolic studies]

The ratio of carbon 13 to carbon 12 is higher in organic substances which originate from corn or cane, as compared to most higher plants. Cane alcohol or corn alcohol are enriched in carbon 13 as compared to common European foods. This discovery provided a new technique for alcohol metabolism studies with animals or men. The ratio of carbon 13 to carbon 12 is measured by mass spectrometry in the carbon dioxide expired after administration of cane or corn alcohol. This ratio increases significantly and reaches a maximum when the blood alcohol level has decreased considerably.     

Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies

β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.     

MAIT Cells Detect and Efficiently Lyse Bacterially-Infected Epithelial Cells

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells.     

LMP1-Induced Cell Death May Contribute to the Emergency of Its Oncogenic Property

Background and Objectives

Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) is linked to a variety of malignancies including Hodgkin''s disease, lymphomas, nasopharyngeal and gastric carcinoma. LMP1 exerts its transforming or oncogenic activity mainly through the recruitment of intracellular adapters via LMP1 C-terminal Transformation Effector Sites (TES) 1 and 2. However, LMP1 is also reported to elicit significant cytotoxic effects in some other cell types. This cytotoxic effect is quite intriguing for an oncogenic protein, and it is unclear whether both functional aspects of the protein are related or mutually exclusive.

Methodology and Principal Findings

Using different ectopic expression systems in both Madin-Darby canine kidney (MDCK) epithelial cells and human embryonic kidney HEK-293 cells, we observe that LMP1 ectopic expression massively induces cell death. Furthermore, we show that LMP1-induced cytotoxicity mainly implies LMP1 C-terminal transformation effector sites and TRADD recruitment. However, stable expression of LMP1 in the same cells, is found to be associated with an increase of cell survival and an acquisition of epithelial mesenchymal transition phenotype as evidenced by morphological modifications, increased cell mobility, increased expression of MMP9 and decreased expression of E-cadherin. Our results demonstrate for the first time that the cytotoxic and oncogenic effects of LMP1 are not mutually exclusive but may operate sequentially. We suggest that in a total cell population, cells resistant to LMP1-induced cytotoxicity are those that could take advantage of LMP1 oncogenic activity by integrating LMP1 signaling into the pre-existent signaling network. Our findings thus reconcile the apparent opposite apoptotic and oncogenic effects described for LMP1 and might reflect what actually happens on LMP1-induced cell transformation after EBV infection in patients.