Keese Michael, Magdeburg Richard J, Herzog Torsten, Hasenberg Till, Offterdinger Martin, Pepperkok Rainer, Sturm Jörg W, Bastiaens Philippe I H
Surgical Clinic, University Hospital Mannheim, D-68167 Mannheim, Germany.
J Biol Chem. 2005 Jul 29;280(30):27826-31. doi: 10.1074/jbc.M504485200. Epub 2005 May 20.
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.
在肿瘤细胞中，由于自分泌信号传导或酪氨酸激酶活性增强，即使在没有外源性配体的情况下，受体酪氨酸激酶也可能出现高磷酸化水平。在此我们表明，通过检测分别与抗受体抗体和磷酸酪氨酸缀合的荧光团之间的荧光共振能量转移，可以对肿瘤细胞和组织中的内源性表皮生长因子受体（EGFR）的磷酸化状态进行定量成像。分析了五种不同的人类结肠直肠癌细胞系的EGFR活性和表达。所有细胞系在血清饥饿条件下均表现出基础EGFR磷酸化。用表皮生长因子（EGF）或过氧钒酸盐刺激后，磷酸化水平升高，这取决于各个细胞系中基础EGFR磷酸化的水平。这种基础活性与受体表达呈负相关。使用受体光漂白荧光共振能量转移成像方法，与相邻的健康组织相比，在切除的人类结肠直肠肿瘤样本中也发现EGFR的磷酸化状态明显更高。因此，EGFR磷酸化成像可能是研究受体酪氨酸激酶活性在恶性细胞生长中的作用的一种有价值的工具。
