Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells
| Autor: | A W, de Feijter, D F, Matesic, R J, Ruch, X, Guan, C C, Chang, J E, Trosko |
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| Rok vydání: | 1996 |
| Předmět: |
Microinjections
Staining and Labeling Blotting Western Gap Junctions Gene Expression Epithelial Cells Cell Communication Oncogenes Isoquinolines Transfection Epithelium Rats Inbred F344 Rats Mice Cell Transformation Neoplastic Liver Transduction Genetic Connexin 43 Mutation Animals Microscopy Phase-Contrast Phosphorylation Cells Cultured |
| Zdroj: | Molecular carcinogenesis. 16(4) |
| ISSN: | 0899-1987 |
| Popis: | Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection-dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disrupted gap-junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed by src, neu, ras, and myc/ras all displayed reduced degrees of GJIC, reduced levels of membrane-associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src- and neu-transformed cells, but not in the ras- and myc/ras-transformed cells. Nuclei isolated from WB-neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB-neo control cells, supporting the idea that the nuclear-localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC-deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane-localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane-localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein. Cells expressing raf, previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf-expressing cells into a mouse showed a marked reduction in GJIC and plasma membrane-associated Cx43 immunostaining. These data suggest that altered localization of the gap-junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastically transformed rat liver epithelial cells. |
| Databáze: | OpenAIRE |
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