上皮増殖因子(EGF)によるヒト口腔扁平上皮癌細胞の運動能促進作用とその細胞内情報伝達経路に関する研究
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Enhancement effects of EGF on the motility of human oral squamous cell carcinoma (SCC) cells and the intracellular signal transduction pathway of EGF were investigated. Five human oral SCC cells (SAS, Ca9-22, HSC-2, HSC-3, HSC-4,) were treated with EGF (1-lOOng/ml) and motilities of these cells were measured with phagokinetic track assay. The EGF significantly enhanced the motilities of SAS, Ca9-22, and HSC-3 cells in a dose dependent fashion; in this experiment (assay) the motility of SAS and HSC-2 cells was significantly higher than that of the other cells without EGF stimulation. The culture supernatants of each cell was hervested and the collected media were analyzed with immuno blotting using EGF monoclonal antibodies (MoAb). There was a large amount of EGF (7.8-15.6ng/ml) in the SAS cell medium, and the EGF-producing ability corresponded to the motility in the absence of EGF-stimulation. To show the EGF autocrine loop in SAS cells, anti-EGF MoAb, exhibiting neutralizing activity and anti-EGF receptor (EGFR) MoAb were added to the phagokinetic track assay. This MoAb treatment significantly reduced the motility. Erbstatin analog, which specifically inhibits the phosphorilation of EGFR, was added to the phagokinetic assay to confirm the EGF autocrine stimulation through the EGF receptor. Erbstatin analog significantly reduced the motility at concentrations higher than 2μM. These results indicate that EGF autocrine stimulation is due to switching through the EGFR. To show the intracellular signal transduction pathway of EGF, s-1 clone cells, the most sensitive clone for EGF stimulation, were established from Ca9-22 cells. When s-1 clone cells were treated with phorbol 12-myristate 13-acetate (PMA), the motility was enhanced like with EGF stimulation. The intracellular localization of PKC was examined with a confocal laserscan microscope. Chronological changes of localization in EGF-stimulated s-1 cells were observed following indirect immunostaining with FITC conjugated antibodies. The expression of PKC was enhanced, and translocation of PKC to the membrane area was observed at 30 minutes after EGF stimulation. To show the signal transduction of EGF-induced motility, inhibition assays of motilities were carried out using blocking agents. To inhibit tyrosine phosphorelation, the Erbstatin analog Psi-tectorigenin was used to inhibit phosphatidylinositol (PI) turnover. Calphostin C was used to inhibit protein kinase C (PKC), and Wortmannin was used to inhibit phosphatidylinositol 3-kinase (PI3K). These blocking agents were completely inhibited the motilities enhanced by EGF. The results strongly suggest that, when EGF binds to EGFR, tyrosin phosphorelation first occurs at the intracellular domain of the EGFR and that PKC is subsequently activated through the activation of PLCy arising from erbB homodimer and/or activation of PI3K, arising from erbBS heterodimer.
- 1997-06-30
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