Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines:: Local stimulation using magnetic microspheres as assessed by quantitative digital microscopy

被引:15
作者
Friedländer, E
Arndt-Jovin, DJ
Nagy, P
Jovin, TM
Szöllsi, J
Vereb, G
机构
[1] Univ Debrecen, Med & Hlth Sci Ctr, Res Ctr Mol Med, Dept Biophys & Cell Biol, H-4012 Debrecen, Hungary
[2] Max Planck Inst Biophys Chem, Dept Mol Biol, D-37077 Gottingen, Germany
关键词
paramagnetic microspheres; ErbB tyrosine kinases; epidermal growth factor receptor; ErbB2; receptor phosphorylation; localized signal spreading; confocal laser scanning microscopy;
D O I
10.1002/cyto.a.20173
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Background: ErbB2 (HER-2), a member of the epidermal growth factor (EGF) receptor family, is a class I transmembrane receptor tyrosine kinase. Although erbB2 has no known physiologic ligand, it can form complexes with other members of the family and undergo transactivation of its very potent kinase activity, thereby initiating downstream signaling and cell proliferation. ErbB2 is a frequent pathologic marker in ductal invasive breast carcinomas and is targeted by using a specific humanized monoclonal antibody, trastuzumab (Herceptin). The antibody is effective in only 20% to 50% of erbB2-positive tumors, and this resistance, as yet poorly understood, constitutes a major therapeutic challenge. Methods: Magnetic microspheres coated with ligands or antibodies are widely used for separation of proteins and cells and allow localized, high intensity, and precisely timed Stimulation of cells. We used EGF- and trastuzumab-covered paramagnetic microspheres, quantitative confocal laser scanning microscopy, and digital image processing to investigate the (trans)activation of and local signal propagation from erbB1 and erbB2 on trastuzumab sensitive and resistant carcinoma cell lines expressing these receptors at high levels. Results: On A431 cells expressing high levels of endogenous erbB1 and transfected erbB2-mYFP (A4-erbB2mYFP F4 cell line), EGF-coupled-microspheres activated erbB1 and transactivated erbB2-mYFP. In two other cell lines with comparable erbB2 expression but lower levels of erbB1, EGF microspheres transactivated erbB2 less efficiently. Trastuzumab in solution activated erbB2 on A4-erbB2-mYFP and the trastuzumab sensitive SKBR-3 cells, but only negligibly on the resistant JIMT-1 cells that showed a 10 times higher & for the antibody. Nevertheless, pronounced erbB2 activation and tyrosine phosphorylation could be detected after stimulation with trastuzumab-coupled microspheres in all cell lines, although transactivation of erbB1 was negligible. Receptor phosphorylation was restricted to the immediate proximity of the microspheres, i.e., receptor clusters external to these locations remained inactive. Conclusion: ErbB1 ligand and erbB2 specific antibody attached to magnetic microspheres are efficient tools in assessing erbB activation, localized signal propagation, and erbB heterodimer formation. Trastuzumab coupled to microspheres is more efficient at accessing erbB2 and activating it than trastuzumab in solution. (c) 2005 International Society for Analytical Cytology.
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收藏
页码:161 / 171
页数:11
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