Epidermal growth factor receptor (EGFR) is activated by autocrine growth factors in many types of tumours, including breast tumours. This receptor has been linked to a poor prognosis in breast cancer and may promote proliferation, migration, invasion, and cell survival as well as inhibition of apoptosis. Human breast ductal carcinoma MCF7 cells were transfected using FuGENE 6 with 1 microg of pcDNA3-EGFR containing the full-length human EGFR promoter or 1 microg of the vectors alone (pcDNA3). The transfected cells were transferred into a 25-cm2 flask containing growth medium and G418. Confluent cultures were lysed, total protein levels measured and electrophoresed. The electrophoresed samples were transferred to nitrocellulose and incubated overnight at 4 degrees C with either anti-EGFR or anti-phospho-ERK and immunoreactive bands were visualized using HRP-linked secondary antibody. We created a model system of EGFR overexpression in MCF7 clones with stably transfected pcDNA3/EGFR plasmid. These cells have been shown to promote substantial phosphorylation of both ERK1 and ERK2. The high level of EGFR and ERK1/2 phosphorylation was not seen in the pcDNA3 vector control cells or in non-transfected cells. In this article we describe successful transient transfection experiments on MCF7 cells using the FuGENE 6 Transfection Reagent. The overexpression of EGFR could be a mediated stress response and a survival signal that involves ERK1 and ERK2 phosphorylation.
Copyright 2004 John Wiley & Sons, Ltd.