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PubMed2004

The actin cytoskeleton-associated protein zyxin acts as a tumor suppressor in Ewing tumor cells.

Changes in cell architecture, essentially linked to profound cytoskeleton rearrangements, are common features accompanying cell transformation. Supporting the involvement of the microfilament network in tumor cell behavior, several actin-binding proteins, including zyxin, a potential regulator of actin polymerization, may play a role in oncogenesis. In this work, we investigate the status of zyxin in Ewing tumors, a family of pediatric malignancies of bone and soft tissues, which are mainly associated with a t(11;22) chromosomal translocation encoding the EWS-FLI1 oncoprotein. We observe that EWS-FLI1-transformed murine fibroblasts, as well as human Ewing tumor-derived SK-N-MC cells, exhibit a complete disruption of their actin cytoskeleton, retaining very few stress fibers, focal adhesions and cell-to-cell contacts. We show that within these cells, zyxin is expressed at very low levels and remains diffusely distributed throughout the cytoplasm, instead of concentrating in actin-rich dynamic structures. We demonstrate that zyxin gene transfer into EWS-FLI1-transformed fibroblasts elicits reconstitution of zyxin-rich focal adhesions and intercellular junctions, dramatic reorganization of the actin cytoskeleton, decreased cell motility, inhibition of anchorage-independent growth and impairment of tumor formation in athymic mice. We observe similar phenotypic changes after zyxin gene transfer in SK-N-MC cells, suggesting that zyxin has tumor suppressor activity in Ewing tumor cells.

Actin Cytoskeleton↗
PubMed1990

Differential effects of renal carcinogens and tumor promoters on growth promotion and inhibition of gap junctional communication in two rat renal epithelial cell lines.

To evaluate the effects of renal carcinogens and tumor promoters on gap junctional intercellular communication (IC) and colony formation, two rat kidney epithelial cell lines were examined in vitro. The NRK-52E line was obtained from the American Type Culture Collection and the NK-4 line was established by us from male F344/NCr rats. The NRK-52E line was co-cultured on a confluent monolayer of lethally irradiated NRK-52E cells to determine colony-forming ability (cloning efficiency) and clonal expansion (average surface area of the colonies, SA) and the NK-4 line was cultured on collagen gel. Streptozotocin (STZ), a renal carcinogen, and trisodium nitrilotriacetate monohydrate (NTANa3), a renal carcinogen and tumor promoter, showed mild growth-enhancing effects on both cell lines, as determined by significant increases in SA, but not in cloning efficiency (CE). Sodium barbital (BBNa), a renal tumor promoter, had similar effects, while phenobarbital sodium (PBNa), a non-renal tumor promoter, lacked such activity. The effects of these compounds on gap junctional function were investigated by the Lucifer Yellow dye transfer microinjection assay using monolayers of NRK-52E and NK-4 cells. We found that STZ, NTANa3 and BBNa inhibited IC in NRK-52E cells, in accordance with their reported tumorigenic and/or tumor-promoting activity in vivo, while PBNa did not. Thus, we could present these findings in NRK-52E cells as an in vitro model to examine the potential tissue-specific effects of renal carcinogens and tumor promoters in vitro. We found, however, that other growth control mechanisms beside IC may play a role, because although the NK-4 line did not possess normal IC, the tumor promoters studied had identical growth-promoting effects on colony formation and expansion in both this cell line and the NRK-52E line.

Acetates↗
PubMed1983

Oligodendroglioma-like cells (clear cells) in ependymoma.

A brain tumor of a 22-year-old man was composed mostly of round cells with perinuclear halos (clear cells), forming clusters intersected by small blood vessels. In some areas, the tumor cells showed perivascular arrangement and epithelial pattern. Phosphotungstic-acid hematoxylin stain and immunoperoxidase stain for glial fibrillary acidic protein (GFAP) technique failed to stain the clear cells. Electron microscopy of the clear cells revealed them to be classical ependymoma cells with well developed intercellular junctions, microvilli and cilia. As no reporters in the past showed the evidence to clarify the nature of the clear cells, this case is considered a good example to support the viewpoint that the clear cells (oligodendroglioma-like cells) commonly observed in ependymomas are in reality ependymoma cells. It is stressed that the diagnosis of "mixed glioma" or "oligoependymoma" should be made with sufficient caution despite the recent advances of GFAP technique.

Adult↗
PubMed1982

Factors controlling the time of onset of the migration of neural crest cells in the fowl embryo.

Transmission electron microscopy of fowl embryos during the 7-10 h preceding migration of trunk-level neural crest (NC) cells revealed extracellular material near the NC-cells. In contrast to the cells of the neural tube, the basal surfaces of NC-cells possessed projections, and were neither contiguous nor covered by a complete basal lamina. The apical zones of NC-cells showed intercellular junctions at the stage of neural-fold fusion, but such junctions were absent in some NC-cells 5 h before migration. The basal laminae of the neural tube and the ectoderm were fused lateral to the NC before migration. In vitro, NC-cell migration commenced immediately when neural anlagen were explanted onto fibronectin-rich matrices, but only when the neural anlagen were from a level where migration had commenced in vivo. Migration was delayed 4-8 h when premigratory-level explants were used. Short-term cell-adhesion assays showed that NC-cells of both premigratory and migratory levels could adhere to fibronectin-rich matrices and to collagen gels, but only migratory NC-cells could be detached from the neural anlage. The results suggest that the precise schedule of the onset of NC-cell migration correlates with a decrease in the intercellular adhesion of NC-cells.

Animals↗
PubMed1984

Relationship between intercellular communication and radiosensitivity of human tumor xenografts.

Micro-electrode techniques permit the detection of electrical coupling between adjacent cells if they are connected by intercellular junctions (gap junctions). This technique was applied to four human tumors xenografted onto nude mice. In three of the tumors, which showed a 'contact resistance' after irradiation in vivo, electrical coupling could be established. No coupling was found in the other tumor, which did not exhibit contact resistance. These results are similar to those obtained recently with cultured spheroids of mammalian cell lines in which only the electrically coupled cell types developed contact resistance to ionizing radiation.

Adenocarcinoma↗
PubMed1984

Morphometry of the ultrastructure of the blood-joint barrier in the rabbit knee.

The ultrastructure of the barrier to water and solute exchange between blood and a synovial cavity was studied morphometrically in five rabbit knees. The synovial surface consisted of 80% synoviocytes and 20% interstitium in direct contact with synovial fluid. The surface intercellular gaps were wide (0.1-5.5 microns). A rich network of capillaries lay just beneath the surface, the commonest (modal) depth of the capillary perimeter being 5 microns at its nearest point. Of these capillaries, 91% were fenestrated in adipose synovium, 44% in areolar synovium and 21% in posterior synovium; the remainder were continuous capillaries. The number of fenestrae per fenestrated capillary was low (approximately 0.5 micron-1, circumference: approximately 6 microns-2). The fenestrae (width 52 nm; membrane thickness 3-4 nm) occupied approximately 1% of the fenestrated endothelial surface-area. Nuclear segments and intercellular junctions were roughly twice as frequent in continuous capillaries as in fenestrated capillaries of the same circumference, indicating that continuous endothelial cells are of smaller surface area than fenestrated cells. The basement membranes of continuous and fenestrated synovial capillaries were relatively thick (98 and 110 nm respectively), perhaps supporting the synovial endothelium during joint motion. In both fenestrated and continuous capillaries the nuclei were preferentially oriented away from the synovial surface (92 and 67% respectively), which presumably facilitates exchange with the surface.

Animals↗
PubMed1975

The aortic intima. II. Repair of the aortic lining after mechanical denudation.

This article reports the ultrastructure of the aortic lining during the repair of mechanically denuded aortic intima in the rat. Three main features were observed: a) Although platelets form a pavement on the exposed components of the aortic intima, platelet thrombi do not form on the denuded surface. b) During the first weeks after injury, a temporary false endothelial lining is formed by modified intimal smooth muscle cells. While the modified smooth muscle cells do not constitute a continuous cell layer, they are like true endothelial cells in that platelets do not adhere to the cell membrane of either cell type. c) A continuous layer of true endothelial cells is formed within 2 months after the original injury. Even after reestablishment of a continuous endothelium, however, abnormalities persist in the form of incompletely formed intercellular junctions. This abnormal endothelium is associated with areas of intimal smooth muscle cell proliferation. These observations are compatible with two alternative interpretations of the role of endothelial injury in the intimal proliferation seen following injury to the vessel wall: a) persistent defects in the endothelium may result in proliferation of underlying arterial smooth muscle cells or b) the proliferation, in converse, may in some manner delay the healing process of the overlying endothelium.

Animals↗
PubMed1985

Gastrulation in the sea urchin embryo is accompanied by the rearrangement of invaginating epithelial cells.

The second phase of gastrulation in the sea urchin embryo, secondary invagination, involves a dramatic elongation of the tube-like gut rudiment. The cells in the wall of the rudiment, which are organized as a monolayered epithelium, change their arrangement during this process. The number of cells in the wall of the gut rudiment at any given level along its long axis decreases markedly as determined by light microscopy of serial cross sections and by scanning electron microscopy, an observation that can be accounted for only if some of the cells exchange nearest neighbors during secondary invagination. Transmission electron microscopy reveals that cell rearrangement takes place despite the continued presence of typical intercellular junctional complexes. In addition to undergoing rearrangement, the cells in the wall of the gut rudiment change their shape during secondary invagination, becoming more flattened. These data raise the possibility that mechanisms other than the contraction of the filopodia of the presumptive secondary mesenchyme cells contribute to the second phase of invagination in the sea urchin embryo. In addition, the observation that cells in the wall of the gut rudiment undergo rearrangement during secondary invagination provides additional evidence that epithelial sheets can exhibit fluid-like properties during morphogenesis.

Animals↗
PubMed1999

Nephrin localizes to the slit pore of the glomerular epithelial cell.

BACKGROUND: Recognition that mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement, and nephrotic range proteinuria emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. This article reports the development of reagents necessary to study the biology of nephrin in mouse, and describes the initial characterization of the nephrin protein. METHODS: A cDNA including the full-length mouse nephrin open reading frame was cloned and sequenced. Immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin was developed. RESULTS: Nephrin identified in mouse glomerular extract was found to be a glycoprotein with an apparent molecular mass of 185 kDa. As detected by indirect immunofluorescence microscopy and immunogold electron microscopy, nephrin was located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalized to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible. CONCLUSION: As a putative cell adhesion molecule of the immunoglobulin superfamily, nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm.

Age Factors↗
PubMed1989

Structural and molecular biology of the eye lens membranes.

Lens transparency is associated with a unique design in tissue development and architecture. The fiber plasma membrane has domains which link with the cytoskeleton, thus maintaining cell shape. Other membrane regions form processes which interlock adjacent lens fibers, and intercellular junctions contain transmembrane pores which allow passage of metabolites between cells. Much interest has recently focused on the study of lens membrane structure and function, mainly because membrane dysfunction may be associated with cataract formation. This article reviews what is known about the structure of membrane domains, about the identification of domain-specific proteins, and describes current attempts to relate these results to function. Much of the presently available data is controversial, and an attempt will be made to reconcile them in revised models and testable hypotheses.

Animals↗
PubMed2002

Rapid translocation of polarized MDCK cell monolayers by Leptospira interrogans, an invasive but nonintracellular pathogen.

Pathogenic spirochetes of the genus Leptospira are a major cause of human zoonotic infectious disease worldwide. After gaining entry through the skin, the organism causes disease by hematogenously disseminating to multiple organs. The mechanism by which it penetrates the mammalian cell barriers to disseminate is not well understood. In this study, we used a low-passage-number isolate of Leptospira interrogans to elucidate the invasive potential of this spirochete. Quantification of bacteria by dark-field microscopy revealed that pathogenic spirochetes were able to translocate through polarized MDCK cell monolayers at a rate significantly greater than that of nonpathogenic Leptospira or a recognized invasive bacterial pathogen, Salmonella: In contrast to Salmonella, L. interrogans did not alter transepithelial electrical resistance during cell translocation. Both transmission and scanning electron microscopy revealed tight association of the extracellular spirochetes with the host cell plasma membrane, without membrane perturbations suggestive of cytoskeletal rearrangement. Spirochetes were not observed within intercellular junctions or membrane-bound compartments inside cells. They were found within the cytoplasm of only 8% of the counted cells. These results indicate that Leptospira is an invasive but not a facultative intracellular organism. We propose that the rapid translocation of mammalian cells by pathogenic Leptospira is a mechanism designed to evade killing by host cells that permits the organism to quickly reach the bloodstream and disseminate to multiple organs.

Animals↗
PubMed1980

Synovial sarcoma of the abdominal wall. Light microscopic, histochemical and electron microscopic investigations.

A synovial sarcoma of the abdominal wall in a 56-year old woman showed the typical features of this tumor type. Histologically a characteristic biphasic cellular pattern with epithelium-like cell complexes and sarcomatous spindle cell areas was found. The histochemical examination revealed that tumor cells synthesize glycoproteins and weakly acid glycosaminoglycans (mainly hyaluronic acid). Electron microscopically the tumor cells in epithelium-like cell islets were sometimes arranged in gland-like formations with microvilli at the luminal side, specialized intercellular junctions and a peripheral basement membrane-like condensation of the ground substance. There was no fundamental cytological difference between cells of epithelium-like and spindle cell areas. Generally the tumor cells imitated cells of the synovial membrane and we found no evidence for origin from cells of the nerve sheath. Because of the submicroscopic relationship and histochemical similarities of synovial sarcomas and mesotheliomas we suggest that they should be united in a group of sarcomas with possible biphasic cellular pattern, while preserving their clinicopathologic definition.

Abdominal Muscles↗
PubMed1976

Aggregation-induced alterations in fibroblast morphology. An ultrastructural study.

The different stages during aggregation of diploid human skin fibroblasts have been examined by transmission and scanning electron microscopy. As a result of aggregation, fibroblasts form a complex tissue configuration. Numerous intercellular junctions can be observed, while the cells remain polygonal and do not develop an organised intracellular cytoskeleton. Cell division occurs only rarely. After aggregation, signs of progressive auto-digestion develop. Adhesion to a substrate results in outgrowth of the cells and monolayer formation, even when extensive cell damage had occurred. The morphology of fibroblasts in aggregates and in the monolayers, from which they were derived is compared and the contribution of the aggregate system to the study of fibroblast behavior is discussed.

Cell Adhesion↗
PubMed2003

Low T cell receptor expression and thermal fluctuations contribute to formation of dynamic multifocal synapses in thymocytes.

Mature T cell activation and selection of immature T cells (thymocytes) are both initiated by binding of T cell receptor (TCR) molecules on the surface of T cells to MHC peptide (MHCp) molecules on the surface of antigen-presenting cells. Recent experiments have shown that the spatial pattern of receptors and ligands in the intercellular junction (synapse) is different during thymocyte selection compared with mature T cell activation. Using a statistical mechanical model, we show that lower TCR expression in thymocytes contributes to effecting these differences. An analogy with the phase behavior of simple fluids helps clarify how, for low TCR expression, thermal fluctuations lead to the dynamic synapse patterns observed for thymocytes. We suggest that a different synapse pattern resulting from lower TCR expression, which could mediate differential signaling, may be the reason why TCR expression level is low in thymocytes.

Animals↗
PubMed1985

Primary neuroendocrine (Merkel cell?) carcinoma of the skin. I. A clinicopathologic and ultrastructural study of 43 cases.

The clinical and pathologic features of 43 cases of primary neuroendocrine carcinoma of the skin are reported. These tumors arise in the dermis and subcutaneous tissues of elderly individuals. The head and neck are the most common primary sites followed by the lower and upper extremities and trunk. Characteristic histologic features include round cells with scanty amphophilic cytoplasm and vesicular nuclei with multiple small nucleoli. The cells are arranged in sheets, solid nests, or anastomosing trabeculae. Collections of perinuclear intermediate filaments, cytoplasmic dense-core membrane-bound secretory granules, complex intercellular junctions, and cytoplasmic spinous processes are the principal fine structural features. These ultrastructural findings are similar to those of the normal cutaneous Merkel cell. The natural history of this neoplasm is characterized by local recurrence in 30% of cases, regional lymph node metastases in 65% of cases, and distant metastases in 40%. One-third of the patients were dead because of their tumors. Treatment of extensive local or distant metastatic disease with chemotherapy or radiotherapy resulted in only short-term palliative response.

Adult↗
PubMed1987

[Role of pathological dedifferentiation in the level of exfoliation of elements of the multilayer epithelium of the uterine cervix].

In cervical dysplasias, dissociation of intercellular junctions occurs to a degree that depends directly on the severity of the dysplastic process which involves the entire epithelium from the basement membrane to the surface layer. The present cytologic studies have demonstrated that as the severity of dysplasia increases, smears taken from exfoliative cells of the stratified squamous epithelium show proportional decreases in the number of cells from the upper epithelial layers and increases in that of cells from the lower levels. The findings obtained indicate that morphologic characterization of exfoliative cellular material may help in the early cytologic diagnosis of cervical disorders.

Adult↗
PubMed2005

Tight junctions, from tight intercellular seals to sophisticated protein complexes involved in drug delivery, pathogens interaction and cell proliferation.

Tight junctions (TJ) are intercellular seals that regulate the passage of ions and molecules through the paracellular pathway. This property allows epithelial and endothelial sheets to function as barriers between different body compartments. Although TJ were identified by electron microscopy since the 1950's, the analysis in recent years of their molecular composition, has depicted TJ as signaling complexes involved in a wide spectrum of physiological and pathological processes, such as cell growth and differentiation, cancer and metastasis, and pathogen interaction. This new knowledge has in turn given rise to novel strategies for enhancing the paracellular flux of poorly absorbed therapeutics.

Animals↗
PubMed1992

Epithelioid sarcoma: an electron-microscopic, immunohistochemical and DNA flow cytometric analysis.

Eight epithelioid sarcomas (ES) were studied by electron microscopy, immunohistochemistry, and DNA flow cytometry. Ultrastructurally, the tumour cells showed desmosome-like intercellular junctions and numerous microvilli, in addition to whorled arrangements of intermediate filaments. Tumour cells were positive for epithelial membrane antigen, cytokeratin, and vimentin, and negative for carcinoembryonic antigen and desmin. All seven cases examined by flow cytometry showed diploid or hyperploid (near diploid) DNA content. This seems to correspond to the relatively long clinical course and low-grade malignant nature of ES. Although the histogenesis of ES is still uncertain, the results of this study suggest that it is a tumour of primitive mesenchymal cells with the capacity to show epithelial differentiation.

Adult↗