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Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 1, e16536- p.Article in journal (Refereed) Published
Abstract [en]

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of (125)I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the (125)I-EGF - EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K(D)≈200 pM on SKBR3 cells to K(D)≈8 nM on A431 cells. The (125)I-EGF - EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the (125)I-EGF - EGFR affinity, in particular when the cells are starved. The (125)I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.

Place, publisher, year, edition, pages
2011. Vol. 6, no 1, e16536- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-147027DOI: 10.1371/journal.pone.0016536ISI: 000286834300081PubMedID: 21304974OAI: oai:DiVA.org:uu-147027DiVA: diva2:399739
Available from: 2011-02-23 Created: 2011-02-23 Last updated: 2013-02-11Bibliographically approved
In thesis
1. Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
Open this publication in new window or tab >>Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now.

This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates.

In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 854
Keyword
Heterogeneity, Kinetics, EGFR, HER2, LigandTracer, Interaction Map, Internalization, Specificity
National Category
Medical Biotechnology Cell and Molecular Biology
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-183872 (URN)978-91-554-8570-2 (ISBN)
Public defence
2013-02-15, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-01-24 Created: 2012-11-05 Last updated: 2013-02-11Bibliographically approved

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