Background Lung cancer often exhibits molecular changes such as the overexpression
Background Lung cancer often exhibits molecular changes such as the overexpression of the ErbB1 gene. lapse. Results We investigated the activation and function of EGFR in the A549 and HK2 lung cancer cell lines which contain 3 and 6 copies of ErbB1 respectively. The expression of EGFR was lower in the HK2 cell line. EGFR was activated after stimulation with EGF in both cell lines but this activation did not promote differences in Elvucitabine cellular proliferation when compared to control cells. Inhibiting EGFR with AG1478 did not modify cellular proliferation confirming previous data. However we observed morphological alterations changes in microfilament organization and increased cell migration upon EGF stimulation. However these effects did not seem to be consequence of an epithelial-mesenchymal transition. Conclusion EGFR expression did not appear to be associated to the ErbB1 gene copy number and neither of these aspects appeared to affect cell proliferation. However EGFR activation by EGF resulted in cell migration stimulation in both cell lines. ANOVA ANOVA (multiple comparisons by Tukey) and hybridization. The nuclei were visualized by interference contrast (DIC). (B) The … Figure 2 Cellular localization expression and mRNA levels of EGFR. (A) Immunofluorescence was performed with an antibody against EGFR (green). The nuclei were stained with propidium iodide (red). EGFR was identified at the cell membrane of both cell types and … The lower levels of EGFR labeling in the cytoplasm suggest that the HK2 cell line presents a lower concentration of EGFR. Therefore we investigated whether there were differences in the levels of protein expression. Western blotting experiments demonstrated that the HK2 cells manifested reduced receptor expression levels compared to the A549 cells (Figure?2B and C). Quantitative RT-PCR revealed that levels of ErbB1 messenger RNA were higher in the A549 cells than the HK2 cells (Figure?2D). Determination of the cellular localization and activation status of EGFR after EGF stimulation A549 cells exhibited significant changes in EGFR distribution after EGF stimulation. The localization of EGFR to the cell borders was Elvucitabine altered and the receptor was located in numerous small agglomerates dispersed in cytoplasm with the appearance of vesicles and in clusters near the nucleus (Figure?3A). HK2 cells presented some possible cytoplasmic vesicles but compared to A549 cells the considerably fewer of these structures were detected (Figure?3A). After EGF stimulation EGFR was located at the cell borders only in HK2 cells (data not shown). Figure 3 Detection of the EGFR cellular distribution after EGF stimulation. (A) Cells were cultured in medium containing 10% FCS and treated with EGF (100 ng/ml) for one hour. EGFR (green) was detected in small and numerous vesicle-like agglomerates dispersed … The Golgi apparatus was detected by immunofluorescence using an antibody against golgin. The histogram in Figure?3B presents the intensity of the green (EGFR) and red (golgin) signals in the cytoplasm at the selected locations and it indicates where signals are co-localized. The EGFR labeling co-localizes with the golgin immunolocalization in the vesicle-like structures in A549 cells while HK2 cells did not present this co-localization (Figure?3B). The phosphorylated form of EGFR (p-EGFR) was analyzed by immunofluorescence in control and EGF-stimulated cells. The A549 control cells did not present p-EGFR labeling but in the EGF-stimulated cells Elvucitabine p-EGFR could be identified at vesicle-like structures in the cytoplasm (Figure?4A). The pattern of p-EGFR labeling was similar to that of EGFR after EGF stimulation (Figure?3A) suggesting that some of the vesicle-like structures Rabbit Polyclonal to TAIP-12. in EGF-stimulated cells likely contain p-EGFR. The HK2 cells presented a different pattern: the phosphorylated form of EGFR was at the borders of control cells and inside vesicle-like structures in EGF-stimulated cells (Figure?4A). Figure 4 Location and expression of p-EGFR in A549 and HK2 cell lines. (A) Cells were cultured in medium containing 10% FCS (control) or 10% FCS Elvucitabine and EGF (100 ng) for one hour. The labeling for p-EGFR is shown in green and the.