title: Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell line.
abstract: 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.
authors.affiliation: Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia. msalokail@yahoo.com
authors.forename: Majed S
authors.initials: MS
authors.lastname: Alokail
mesh_terms: D001943:Breast Neoplasms; D044584:Carcinoma, Ductal; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D014162:Transfection / Q000379:methods*
chemical_list: D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3