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In considering the mechanism of transformation of epithelium to mesenchyme in the embryo, it is generally assumed that the ability to give rise to fibroblast-like cells is lost as epithelia mature. We reported previously that a definitive embryonic epithelium, that of the anterior lens, gives rise to freely migrating mesenchyme-like cells when suspended in type I collagen matrices. Here, we show that a highly differentiated epithelium that expresses cytokeratin changes to a vimentin cytoskeleton and loses thyroglobulin during epithelial-mesenchymal transformation induced by suspension in collagen gel. Using dispase and collagenase, we isolated adult thyroid follicles devoid of basal lamina and mesenchyme, and we suspended the follicles in 3D collagen gels. Cells bordering the follicle lumen retain epithelial polarity and thyroid phenotype, but basal cell surface organization is soon modified as a result of tissue multilayering and elongation of basal cells into the collagenous matrix. Cytodifferentiation, determined by thyroglobulin immunoreactivity, is lost as the basal epithelial cells move into the matrix after 3-4 days in collagen. By TEM, it can be seen that the elongating cells acquire pseudopodia, filopodia and mesenchyme-like nuclei and RER. Immunofluorescence examination of intermediate filaments showed that freshly isolated follicles and follicles cultured on planar substrata react only with anticytokeratin. However, all of the mesenchyme-like cells express vimentin and they gradually lose cytokeratin. These results suggest that vimentin may be necessary for cell functions associated with migration within a 3D matrix. The mesenchymal cells do not revert to epithelium when grown on planar substrata and the transformation of epithelium to mesenchyme-like cells does not occur within basement membrane gels. The results are relevant to our understanding of the initiation of epithelial-mesenchymal transformation in the embryo and the genetic mechanisms controlling cell shape, polarity and cytoskeletal phenotype.
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PMID:Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells. 246 Mar 6

The dramatic changes in morphology induced by nanomolar doses of tumor-promoting agents, especially in epithelial cells, have been noted previously (Driedger and Blumberg, 1980; Rifkin et al., 1979; Croop et al., 1980; Phaire-Washington et al., 1980; Ohuchi and Levine, 1980; Ojakian, 1981; Fey and Penman, 1984). This chapter shows the effect of the tumor promoter TPA on the underlying skeletal framework, which is involved in the maintenance of both cell and epithelial tissue morphology. It should be emphasized, however, that similar results are obtained for all the tumor promoters as well as for the complete, ultimate carcinogens examined so far. The organization of the cytoskeletal elements involved in these morphological changes is faithfully retained during the fractionation procedure employed here, as is evident from SEM and TEM analysis of Triton-extracted cells. A number of promoting agents have been compared, and the degree of disorganization viewed in these whole mounts appears to parallel the potency of the promoting agents as measured by other assays (Fey and Penman, 1984). Also, the inactive analogues of phorbol ester have no effect on cell structure (Rifkin et al., 1979; Ojakian, 1981; Fey and Penman, 1984). We suggest that the effect of TPA on the cytoskeleton occurs early as compared with many of the commonly studied biochemical responses and may indeed underlie many of the previously described cellular response to promoting agents, such as mitogenic stimulation. TPA-induced alterations in NM-IF scaffold occur in the absence of both protein and RNA synthesis (Fey and Penman, 1984). By contrast, plasminogen activator, stimulated by TPA (Wigler and Weinstein, 1976), is completely blocked by pretreatment with both cycloheximide and actinomycin D (Weinstein et al., 1977; Ojakian, 1981). Ornithine decarboxylase, another enzyme that is rapidly induced by tumor promoters, is inhibited by both cycloheximide and actinomycin D in the presence of TPA (O'Brien, 1976). Thus two of the early biochemical markers for tumor-promoter activity are separable from the induction of cytoskeletal alterations by TPA. One of the most striking features of the response to promoting agents is the adoption of the transformed phenotype, in which cells lose growth control and cease being organized into meaningful tissue structure. The alteration of desmosomal and junctional associations and the concomitant change in cytokeratin organization are clearly related to the breakdown of epithelial organization. The phenotype is completely reversible although it takes about 3 days for the mode line to reestablish normal morphology (data not shown).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The morphological oncogenic signature. Reorganization of epithelial cytoarchitecture and metabolic regulation by tumor promoters and by transformation. 307 72

A method is described for the sequential detergent and high ionic strength extraction of human amnion with the progressive enrichment of the intermediate filament (IF) cytoskeleton and its associated structures including hemidesmosomes (HD). TEM of the extracted epithelium in situ reveals IF bundles beneath the apical cell surface, around the nucleus and at the lateral edges of the cells where association with desmosomes occurs. IF bundles are also very prominent within basal cell processes where they loop through the cytoplasm adjacent to the HDs. A novel connecting filament network is observed running between the IFs and the hemidesmosomal dense plaque. The adjacent IF network contains both cytokeratin and vimentin, the latter revealed much more fully as a result of the extraction protocol. The hemidesmosomal plasma membrane contains integrin subunits alpha 6 and beta 4 and these are quantitatively retained as the basal cell surface during extraction, while nonjunctional plasma membrane is solubilised. Integrin beta 1 is found at the basolateral cell surface but, like actin, is extracted quantitatively and is not present in HDs. The extracted epithelial cells may be recovered by scraping and the IF network depolymerised to produce a particulate fraction containing short residual IFs, associated thin filaments and plaque material. This fraction contains immunoreactive cytokeratin and vimentin. Integrin alpha 6 beta 4 has been used as a biochemical criterion of the presence of HD material in the fraction. Both subunits are highly enriched. The fraction also contains the hemidesmosomal components HD1, BP230 and BP180. This method is likely to be useful in further characterisation of the HD.
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PMID:Studies of hemidesmosomes in human amnion: the use of a detergent extraction protocol for compositional and ultrastructural analysis and preparation of a hemidesmosome-enriched fraction from tissue. 757 27

To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique. The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml). triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml). To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed. The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase. We were able to maintain the cultured cells for 3 weeks or more. Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced. The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity. The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells. However, establishment of the cell line is needed for long-term study.
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PMID:Establishment of primary cell culture from stria vascularis explants. Morphological and functional characterization. 897 11

The ultrastructure of a cholangiocarcinoma cell line (HuCCA-l) originally established from an intrahepatic bile duct tumor of a patient seropositive for a liver fluke infection was studied by scanning (SEM) and transmission (TEM) electron miscroscopy. With the SEM, the surface of HuCCA-1 cells were found to be covered with microvilli. The size of these microvilli varied from cell to cell and they were irregularly distributed. The TEM clearly revealed the presence of cytokeratin filaments, an intracytoplasmic lumen, tight junctions at the apices and desmosomes at the lateral surfaces of neighboring cells, all of which are characteristics of adenocarcinoma cell origin. However, the tumor mass that developed in a nude mouse following subcutaneous injection of these cells was found to exhibit some morphological changes. Specifically, about 20-30% of the tumor cells, particularly those lining the base of the tumor tubules, exhibited electron dense tonofilaments typical of squamous cells. However, this alteration was reversible as the cell line (HuCCA-1Nu) derived from this nude mouse-passage did not exhibit any characteristics reminiscent of squamous cells. These observations are consistent with those occasionally found in human cases reported previously by other investigators. Altogether, the data showed that squamous transformation of adenocarcinoma cells can occur under appropriate conditions. It further showed that reversion to adenocarcinoma cells can occur when the microenvironment is changed.
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PMID:Ultrastructural characteristics of liver fluke associated human cholangiocarcinoma cell lines. 903 2

The overall architecture and structure of the perivascular space in the rat thymus were studied by light microscopy using silver-impregnated sections and sections stained immunohistochemically with anti-cytokeratin antibody, and by transmission and scanning electron microscopy (TEM and SEM). In silver-impregnated sections, the perivascular space was delimited by a thin sheath of delicate argyrophilic fibers from the thymic parenchyma in the cortico-medullary region and medulla. This space was continuous with the septal connective tissue, indicating that this was the connective tissue compartment rather than with the epithelial compartment of the parenchyma. In the medulla, the perivascular space widened at places, where the argyrophilic sheath was often discontinuous and the boundary between the perivascular space and parenchyma was indistinct. Lymphatics were located in the perivascular space of the corticomedullary region and sometimes in the wide perivascular space of the medulla. The presence of a thymic epithelial sheath surrounding the perivascular space was confirmed by light microscopy of anti-cytokeratin antibody immunostained sections and by TEM. SEM observations revealed three-dimensionally that the epithelial sheath lined by collagen fibrillar (i.e., argyrophilic) layer form a rather continuous tubular structure in the cortico-medullary region, while it often interrupted in the medulla. These findings indicated that the perivascular space (i.e., the connective tissue compartment) is extensively open to the parenchyma (i.e., the epithelial compartment) in some portions of the medulla, where medullary lymphocytes are probably freely exposed to blood borne substances similar to the peripheral lymphoid tissues.
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PMID:Three-dimensional ultrastructure of the perivascular space in the rat thymus. 916 92

The subapical region of umbrella cells in the urinary bladder contains a dense cytokeratin (CK) network. Yet, this network should enable a very intensive traffic of specific fusiform vesicles involved in alterations of the surface area of the apical membrane. Therefore, the cytokeratins should be organised in a way to be both mechanically strong and also passable for fusiform vesicles. The supramolecular organisation of the CKs in the subapical region of umbrella cells in mice was studied by conventional fluorescence, confocal laser microscopy, and TEM. It has been found that the first 150 to 300 nm under the apical membrane is filled with fusiform vesicles and only below that the CK network begins. The CK 7 and CK 20 compose a subapical network, which is created as an array of parallel trajectories pointing to the apical plasma membrane. The network is framed by a strong wall of CK, which is parallely aligned in neighbouring cells. The double labelling of the urothelial-specific membrane proteins, uroplakins, and CKs proved the presence of fusiform vesicles within these trajectories. The measurements proved that the trajectory diameter in the upper half of the network is smaller than in the lower half. The diameters of the trajectories in animals with distended bladders exceeded those in contracted bladders for 70%, which most likely facilitates the transport of fusiform vesicles to the apical membrane. Discovery of the subapical CK network elucidates the until now undescribed supramolecular organisation of CKs in the apical region of urothelial cells.
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PMID:Trajectorial organisation of cytokeratins within the subapical region of umbrella cells. 1237 41

Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.
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PMID:Quality evaluation analysis of bioengineered human skin. 1546 8