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Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell line
Author(s) -
Alokail Majed S.
Publication year - 2004
Publication title -
cell biochemistry and function
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.933
H-Index - 61
eISSN - 1099-0844
pISSN - 0263-6484
DOI - 10.1002/cbf.1186
Subject(s) - transfection , epidermal growth factor receptor , cancer research , epidermal growth factor , cell culture , breast carcinoma , gene , ductal carcinoma , receptor , carcinoma , biology , chemistry , breast cancer , medicine , cancer , genetics
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 μg of pcDNA3‐EGFR containing the full‐length human EGFR promoter or 1 μg of the vectors alone (pcDNA3). The transfected cells were transferred into a 25‐cm 2 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°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.