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J. Biol. Chem., Vol. 280, Issue 30, 27826-27831, July 29, 2005

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Imaging Epidermal Growth Factor Receptor Phosphorylation in Human Colorectal Cancer Cells and Human Tissues^*

Michael Keese¶, Richard J. Magdeburg, Torsten Herzog, Till Hasenberg, Martin Offterdinger, Rainer Pepperkok, Jörg W. Sturm, and Philippe I. H. Bastiaens||

From the Surgical Clinic, University Hospital Mannheim, D-68167 Mannheim, Germany and the European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany

In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.

Received for publication, April 25, 2005

^* This work was supported in part by the Forschungsfond der Fakultät für klinische Medizin, Universität Heidelberg and by the Bluemel Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported in part by the European Union Grant "Insight Inside" 11421.

^|| To whom correspondence should be addressed.: Cell Biology and Biophysics Program, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel.: 49-6221-387407; Fax: 49-6221-387242; E-mail: bastiaen{at}embl.de.

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