Glioma Cell Death: Cell–Cell Interactions and Signalling Networks

The prognosis for patients with malignant gliomas is poor, but improvements may emerge from a better understanding of the pathophysiology of glioma signalling. Recent therapeutic developments have implicated lipid signalling in glioma cell death. Stress signalling in glioma cell death involves mitochondria and endoplasmic reticulum. Lipid mediators also signal via extrinsic pathways in glioma cell proliferation, migration and interaction with endothelial and microglial cells. Glioma cell death and tumour regression have been reported using polyunsaturated fatty acids in animal models, human ex vivo explants, glioma cell preparations and in clinical case reports involving intratumoral infusion. Cell death signalling was associated with generation of reactive oxygen intermediates and mitochondrial and other signalling pathways. In this review, evidence for mitochondrial responses to stress signals, including polyunsaturated fatty acids, peroxidising agents and calcium is presented. Additionally, evidence for interaction of glioma cells with primary brain endothelial cells is described, modulating human glioma peroxidative signalling. Glioma responses to potential therapeutic agents should be analysed in systems reflecting tumour connectivity and CNS structural and functional integrity. Future insights may also be derived from studies of signalling in glioma-derived tumour stem cells.     

Biochemistry of Direct Cell−Cell Interactions. Signaling Factors Regulating Orchestration of Cell Populations

Biochemical mechanisms for the orchestration of cell populations are discussed in view of direct cell?cell inter-actions and composition of the intercellular medium. In our works of the last 20 years, we used circahoralian (ultradian) rhythm of protein synthesis as a marker of cell interactions. Experiments in cell cultures are described; some influences on the organism native medium were performed. Information is presented on the signaling membrane factors that trigger a cascade of processes in the cytoplasm and lead to the orchestration of cell activity in vitro and in vivo. Among these factors are blood serum neurotransmitters, gangliosides, and some hormones. Studying protein synthesis kinetics allowed us to understand the importance of maintaining the constant levels of signaling factors in mammalian blood. The literature on protein phosphorylation as a key process of cell organization is reviewed. The persistence of the organizing signal for several days is described as a type of cell “memory”. It seems promising to extend the area for application of direct cell?cell interactions (respiration of cells, proliferation, etc.) to study possibilities of epigenetic regulation. It is important to continue the studies on the mechanisms of biochemical action of the known drugs as signaling factors.     

Epithelial Cell Adhesion Molecule (Ep-CAM) Modulates Cell–Cell Interactions Mediated by Classic Cadherins

The contribution of noncadherin-type, Ca²⁺-independent cell–cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca²⁺-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM–positive transfectants behave like cells with a decreased strength of cell–cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM–cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of α- and β-catenins decreased in cells overexpressing Ep-CAM. While the total β-catenin content remains unchanged, a reduction in total cellular α-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell–cell adhesions diminish, Ep-CAM–mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell–cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell–cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.Tissue and organ morphogenesis can be viewed as the result of interactions of various cell populations. One important type of intercellular interaction involved in the processes of tissue morphogenesis, morphogenetic movements of cells, and segregation of cell types, are adhesions mediated by cell adhesion molecules (Steinberg and Pool, 1982; Edelman, 1986; Cunningham, 1995; Takeichi, 1995; Gumbiner, 1996). Except for their direct mechanical role as interconnectors of cells and connectors of cells to substrates, cell adhesion molecules are also believed to be responsible for a variety of dynamic processes including cell locomotion, proliferation, and differentiation. There is also evidence that the adhesion systems within a cell may act as regulators of other cell adhesions, thereby offering a means of signaling that is relevant for rearrangements in cell or tissue organization (Edelman, 1993; Rosales et al., 1995; Gumbiner, 1996).In many tissues, a critical role in the maintenance of multicellular structures is assigned to cadherins, a family of Ca²⁺-dependent, homophilic cell–cell adhesion molecules (Takeichi, 1991, 1995; Gumbiner, 1996). In epithelia this critical role belongs to E-cadherin, which is crucial for the establishment and maintenance of epithelial cell polarity (McNeil et al., 1990; Näthke et al., 1993), morphogenesis of epithelial tissues (Wheelock and Jensen, 1992; Larue et al., 1996), and regulation of cell proliferation and programmed cell death (Hermiston and Gordon, 1995; Hermiston et al., 1996; Takahashi and Suzuki, 1996; Wilding et al., 1996; Zhu and Watt, 1996). Expression of different types of classic cadherin molecules (Nose et al., 1988; Friedlander et al., 1989; Daniel et al., 1995), and even quantitative differences in the levels of the same type of cadherin (Steinberg and Takeichi, 1994), may be responsible for segregation of cell types in epithelial tissues. The phenotype of epithelial cells may be modulated by expression of combinations of different types of cadherins (Marrs et al., 1995; Islam et al., 1996). However, cadherins represent only one of the intercellular adhesion systems that are present in epithelia, along with adhesion molecules of the immunoglobulin superfamily, such as carcinoembryonic antigen (Benchimol et al., 1989), and others. The actual contribution of Ca²⁺-independent nonjunctional adhesion molecules to the formation and maintenance of the epithelial tissue architecture and epithelial cell morphology is not clear.We have recently demonstrated that a 40-kD epithelial glycoprotein, which we have designated epithelial cell adhesion molecule (Ep-CAM)¹ (Litvinov et al., 1994a ), may perform as a homophilic, Ca²⁺-independent intercellular adhesion molecule, capable of mediating cell aggregation, preventing cell scattering, and directing cell segregation. This type I transmembrane glycoprotein consists of two EGF-like domains followed by a cysteine-poor region, a transmembrane domain, and a short (26-amino acid) cytoplasmic tail, and is not structurally related to the four major types of CAMs, such as cadherins, integrins, selectins, and the immunoglobulin superfamily (for review see Litvinov, 1995). Ep-CAM demonstrates adhesion properties when introduced into cell systems that are deficient in intercellular adhesive interactions (Litvinov et al., 1994a ). However, the participation of the Ep-CAM molecule in supporting cell–cell interactions of epithelial cells was not evident (Litvinov et al., 1994b ).Most epithelial cell types coexpress E-cadherin (and sometimes other classic cadherins) and Ep-CAM (for review see Litvinov, 1995) during some stage of embryogenesis. In adult squamous epithelia, which are Ep-CAM negative, de novo expression of this molecule is associated with metaplastic or neoplastic changes. Thus, in ectocervical epithelia, expression of Ep-CAM occurs in early preneoplastic lesions (Litvinov et al., 1996); most squamous carcinomas of the head and neck region are Ep-CAM positive (Quak et al., 1990), and basal cell carcinomas are Ep-CAM positive in contrast to the normal epidermis (Tsubura et al., 1992).In many tumors that express Ep-CAM heterogeneously, an Ep-CAM–positive cell population may be found within an Ep-CAM–negative cell population, with both cell types expressing approximately equal levels of cadherins, as illustrated in Fig. ​Fig.11 A by a case of basal cell carcinoma. In glandular tissues such as gastric epithelium, which are low/ negative for Ep-CAM, expression of Ep-CAM is related to the development of early stages of intestinal metaplasia (our unpublished observation). Even in tissues with relatively high Ep-CAM expression, such as colon, the development of polyps is accompanied by an increase in Ep-CAM expression (Salem et al., 1993). In intestinal metaplasia one may observe Ep-CAM–positive cells bordering morphologically identical normal cells that are Ep-CAM–negative (as illustrated in Fig. ​Fig.11 B) Ep-CAM–positive cells bordering Ep-CAM–negative epithelial cells may also be found in some normal tissues such as hair follicles (Tsubura et al., 1992). Open in a separate windowFigure 1Examples of Ep-CAM expression by some cells within the E-cadherin–positive cell population. (A) Heterogeneous expression of Ep-CAM in a basal cell carcinoma, as detected by immunofluorescent staining with mAb 323/A3 to Ep-CAM (green fluorescence); the red fluorescence indicates the expression of E-cadherin (mAb HECD-1). (B) The de novo expression of Ep-CAM in gastric mucosa in relation to the development of intestinal metaplasia; immunohistochemical staining with mAb 323/A3. Note the bordering Ep-CAM–positive and –negative cells. Bars, 30 μM.From the examples presented, an increased or de novo expression of Ep-CAM is often observed in epithelial tissues in vivo. Expression of an additional molecule that may participate in cell adhesion in the context of other adhesion systems may have certain effects on the cell–cell interactions. Therefore, we have investigated whether the increased/de novo expression of Ep-CAM in epithelial cells (a) has any impact on interactions of positive cells with the parental Ep-CAM–negative cells, and (b) modulates in any way intercellular adhesive interactions of cells interconnected by E-cadherin, which is the major morphoregulatory molecule in epithelia.Here we demonstrate that expression of Ep-CAM by some cells in a mixed cell population expressing classical cadherins induces segregation of the Ep-CAM–positive cells from the parental cell population due to a negative effect on cadherin junctions caused by expression of Ep-CAM. The cadherin-modulating properties observed for Ep-CAM suggest a role for this molecule in the development of a proliferative and metaplastic cell phenotype, and probably in the development and progression of malignancies.     

Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?

Cell surface analysis often requires manipulation of cells prior to examination. The most commonly employed procedures are centrifugation at different speeds, changes of media during washing or final resuspension, desiccation (either air drying for contact angle measurements or freeze-drying for sensitive spectroscopic analysis, such as X-ray photoelectron spectroscopy), and contact with hydrocarbon (hydrophobicity assays). The effects of these procedures on electrophoretic mobility, adhesion to solid substrata, affinity to a number of Sepharose columns, structural integrity, and cell viability were systematically investigated for a range of model organisms, including carbon- and nitrogen-limited Psychrobacter sp. strain SW8 (glycocalyx-bearing cells), Escherichia coli (gram-negative cells without a glycocalyx), and Staphylococcus epidermidis (gram-positive cells without a glycocalyx). All of the cell manipulation procedures severely modified the physicochemical properties of cells, but with each procedure some organisms were more susceptible than others. Considerable disruption of cell surfaces occurred when organisms were placed in contact with a hydrocarbon (hexadecane). The majority of cells became nonculturable after air drying and freeze-drying. Centrifugation at a high speed (15,000 × g) modified many cell surface parameters significantly, although cell viability was considerably affected only in E. coli. The type of washing or resuspension medium had a strong influence on the values of cell surface parameters, particularly when high-salt solutions were compared with low-salt buffers. The values for parameters obtained with different methods that allegedly measure similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of the in situ microbial cell surface as closely as possible. There is an urgent need for new, reliable, nondestructive, minimally manipulative cell surface analysis techniques that can be used in situ.     

Cell Stem Cell揭示受精卵DNA去甲基化重要机制

<正>2014年9月11日,Cell Stem Cell在线发表了中国科学院上海生科院生物化学与细胞生物学研究所徐国良研究组、李劲松研究组和北京大学生命科学学院生物动态光学成像中心汤富酬研究组的最新研究成果,该研究发现小鼠早期胚胎中母源和父源基因组在单细胞的受精卵阶段均会发生大规模的DNA主动和被动去甲基化,并且DNA双加氧酶Tet3介导了主动去甲基化的发生,而糖苷酶     

Molecular Bases of Cell–Cell Junctions Stability and Dynamics

Epithelial cell–cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell–cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell–cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell–cell junctions.Cell–cell junction formation and remodeling occur repeatedly throughout development. Epithelial cells are linked by apical adherens junctions (AJs) that rely on the cadherin-catenin-actin module. Cadherins, of which epithelial E-cadherin (E-cad) is the most studied, are Ca²⁺-dependent transmembrane adhesion proteins forming homophilic and heterophilic bonds in trans between adjacent cells. Cadherins and the actin cytoskeleton are mutually interdependent (Jaffe et al. 1990; Matsuzaki et al. 1990; Hirano et al. 1992; Oyama et al. 1994; Angres et al. 1996; Orsulic and Peifer 1996; Adams et al. 1998; Zhang et al. 2005; Pilot et al. 2006). This has long been attributed to direct physical interaction of E-cad with β-catenin (β-cat) and of α-catenin (α-cat) with actin filaments (for reviews, see Gumbiner 2005; Leckband and Prakasam 2006; Pokutta and Weis 2007). Recently, biochemical and protein dynamics analyses have shown that such a link may not exist and that instead, a constant shuttling of α-cat between cadherin/β-cat complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al. 2005; Yamada et al. 2005). Regardless of the exact nature of this link, several studies show that AJs are indeed physically attached to actin and that cadherins transmit cortical forces exerted by junctional acto-myosin networks (Costa et al. 1998; Sako et al. 1998; Pettitt et al. 2003; Dawes-Hoang et al. 2005; Cavey et al. 2008; Martin et al. 2008; Rauzi et al. 2008). In addition, physical association depends in part on α-cat (Cavey et al. 2008) and additional intermediates have been proposed to represent alternative missing links (Abe and Takeichi 2008) (reviewed in Gates and Peifer 2005; Weis and Nelson 2006). Although further work is needed to address the molecular nature of cadherin/actin dynamic interactions, association with actin is crucial all throughout the lifespan of AJs. In this article, we will review our current understanding of the molecular mechanisms at work during three different developmental stages of AJs biology: assembly, stabilization, and remodeling, with special emphasis on the mechanical forces controlling AJs integrity and development.     

Cell Adhesion: Sorting out Cell Mixing with Echinoid?

Cadherins control intercellular adhesion in epithelial cells. This property relies on the ability to recruit actin filaments at adherens junctions via beta-catenin and alpha-catenin. A recent study shows that Echinoid, a member of the immunoglobulin domain containing protein family, is a modulator of intercellular adhesion in Drosophila that controls cell sorting.