phalloidine and Cell-Transformation--Viral

In the current study we examined if the multifunctional cytokine TGF-beta1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD).. RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-beta1 and Cx43 in HCD-cells. The effect of TGF-beta1 and high glucose (25 mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-beta1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry.. At 24 hrs, high glucose (25 mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-beta1 (2 ng/ml) at low glucose (5 mM). Both high glucose and TGF-beta1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-beta. Furthermore, high glucose-evoked changes were inhibited by a TGF-beta1-specific monoclonal antibody. Mannitol (25 mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-beta1 evoked changes in Cx43 expression were biphasic. An early (4-8 hr) transient decrease in expression was followed by an increase in protein expression (12-24 hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24 hrs coincided with a TGF-beta1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells.. High glucose evoked a TGF-beta1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.

Topics: Actins; Cell Communication; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Connexin 43; Cytosol; Dose-Response Relationship, Drug; Fluorescent Dyes; Glucose; Humans; Immunohistochemistry; Kidney Tubules, Collecting; Phalloidine; Proteins; Rhodamines; RNA, Messenger; Simian virus 40; Transforming Growth Factor beta1; Up-Regulation

The cytoskeletons of two established chick embryo cell (CEC) lines were examined by fluorescence and electron microscopy and compared with those of control cells and cells transformed by Rous sarcoma virus (RSV). In normal CEC, many stress fibers were observed. On the other hand, stress fibers were disorganized in nontransformed spontaneously established CEC, non-tumorigenic CEC partially transformed with a chemical carcinogen, and tumorigenic RSV-transformed CEC. In the normal CEC, actin filaments formed several bundles along the processes of the cell. Stereo-images of the peripheral region revealed bundles of filaments which were located along the attached side to the substrate. A fine well preserved network of filaments was also observed. On the other hand, in spontaneously established, partially transformed and RSV-transformed CEC, a fine network of filaments, but no actin cables, was found. These results support previous evidence that the cytoskeletal changes themselves are not directly related to the transformation or tumorigenicity of cells.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Cytoskeleton; Detergents; Microscopy, Electron; Microscopy, Fluorescence; Phalloidine

Actin in cultured fibroblasts is organized into a complex set of fibers. Patterns of organization visualized with antibody to actin are similar but not identical to those visualized with fluorescein isothiocyanate-phalloidin (Fl-phalloidin), a chemical that binds to F-actin polymer with a dissociation constant of 2.7 X 10(-7) M [Wulf, E., Deboben, A., Bautz, F. A., Faulstich, H. & Wieland, T. (1979) Proc. Natl. Acad. Sci. USA 76, 4498-4502]. Fl-phalloidin reveals that transformed cells have fewer, finer, and shorter F-actin-containing structures than do normal cells. Two-color fluorescence microscopy of single cells reveals that F-actin staining by Fl-phalloidin picks out the cytoskeletal cables more sharply than does antibody to actin, due to a reduced intracellular background fluorescence. This improved resolution permits sorting of cellular Fl-phalloidin patterns into four classes ranging in organization from 90% of the cytoplasm occupied by large cables to the absence of detectable cables. Reproducible differences in pattern distributions between normal and transformed cell lines have been quantitated. Fl-phalloidin together with rhodamine-based indirect antibody to simian virus 40 tumor antigen reveals a direct relationship between the degree of pattern change and simian virus 40 nuclear antigen expression in intermediate transformed 3T3 cell lines [Risser, R. & Pollack, R. (1974) Virology 59, 477-489].

Topics: Actins; Animals; Antigens, Neoplasm; Antigens, Viral; Cell Transformation, Viral; Cytoskeleton; Fluoresceins; Humans; Mice; Phalloidine; Rats; Simian virus 40