| 1. |
- Björkelund, Hanna, et al.
(författare)
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Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with (125)I-EGF
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:9, s. e24739-
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Tidskriftsartikel (refereegranskat)abstract
- The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer (R) Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of (125)I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0-3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8-2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF - EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with (125)I-EGF.
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| 2. |
- Björkelund, Hanna, 1983-, et al.
(författare)
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Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach
- 2013
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Ingår i: Molecular and Clinical Oncology. - : Spandidos Publications. - 2049-9469 .- 2049-9450. ; 1:2, s. 343-352
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression and aberrant activity of the epidermal growth factor (EGF) have been observed in various cancer types, rendering it an important target in oncology research. The interaction between EGF and its receptor (EGFR), as well as subsequent internalization, is complex and may be affected by various factors including tyrosine kinase inhibitors (TKIs). By combining real‑time binding curves produced in LigandTracer® with internalization assays conducted at different temperatures and with different TKIs, the processes of ligand binding, internalization and excretion was visualized. SKOV3 cells had a slower excretion rate compared to A431 and U343 cells, and the tested TKIs (gefitinib, lapatinib, AG1478 and erlotinib) reduced the degree of internalization. The kinetic analysis of the binding curves further demonstrated TKI‑dependent balances of EGFR monomer and dimer populations, where lapatinib promoted the monomeric form, while the other TKIs induced dimers. The dimer levels were found to be associated with the apparent affinity of the EGF‑EGFR interaction, with EGF binding stronger to EGFR dimers compared to monomers. This study analyzed how real‑time molecular interaction analysis may be utilized in combination with perturbations in order to understand the kinetics of a ligand‑receptor interaction, as well as some of its associated intracellular processes. Our multiple‑temperature and ‑inhibitor assay setup renders it possible to follow the EGFR monomer, dimer and internalized populations in a detailed manner, allowing for a new perspective of the EGFR biology.
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| 3. |
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| 4. |
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| 5. |
- Gedda, Lars, et al.
(författare)
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Evaluation of Real-Time Immunohistochemistry and Interaction Map as an Alternative Objective Assessment of HER2 Expression in Human Breast Cancer Tissue
- 2013
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Ingår i: Applied immunohistochemistry & molecular morphology (Print). - 1541-2016 .- 1533-4058. ; 21:6, s. 497-505
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Tidskriftsartikel (refereegranskat)abstract
- Immunohistochemical study (IHC) is a critical tool in the clinical diagnosis of breast cancer. One common assessment is the expression level of the HER2 receptor in breast cancer tissue samples with the aim of stratifying patients for applicability of the therapeutic antibody Herceptin. In this study, we aimed to investigate whether a novel assay, real-time IHC combined with Interaction Map analysis, offers the possibility of objective assessment of HER2 expression. Interaction Map presents real-time interaction data as a collection of peaks on a surface, and it was performed on 20 patient tissue samples previously scored for HER2 expression. The result shows that the relative weight of the peaks in the maps contains novel information that could discriminate between high and low HER2 expression in an operator-independent manner (P<0.001). We conclude that the real-time IHC assay has a promising potential to complement conventional IHC and may improve the precision in the future clinical diagnostics of breast cancer.
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| 6. |
- Gedda, Magnus, 1978-
(författare)
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Contributions to 3D Image Analysis using Discrete Methods and Fuzzy Techniques : With Focus on Images from Cryo-Electron Tomography
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With the emergence of new imaging techniques, researchers are always eager to push the boundaries by examining objects either smaller or further away than what was previously possible. The development of image analysis techniques has greatly helped to introduce objectivity and coherence in measurements and decision making. It has become an essential tool for facilitating both large-scale quantitative studies and qualitative research. In this Thesis, methods were developed for analysis of low-resolution (in respect to the size of the imaged objects) three-dimensional (3D) images with low signal-to-noise ratios (SNR) applied to images from cryo-electron tomography (cryo-ET) and fluorescence microscopy (FM). The main focus is on methods of low complexity, that take into account both grey-level and shape information, to facilitate large-scale studies. Methods were developed to localise and represent complex macromolecules in images from cryo-ET. The methods were applied to Immunoglobulin G (IgG) antibodies and MET proteins. The low resolution and low SNR required that grey-level information was utilised to create fuzzy representations of the macromolecules. To extract structural properties, a method was developed to use grey-level-based distance measures to facilitate decomposition of the fuzzy representations into sub-domains. The structural properties of the MET protein were analysed by developing a analytical curve representation of its stalk. To facilitate large-scale analysis of structural properties of nerve cells, a method for tracing neurites in FM images using local path-finding was developed. Both theoretical and implementational details of computationally heavy approaches were examined to keep the time complexity low in the developed methods. Grey-weighted distance definitions and various aspects of their implementations were examined in detail to form guidelines on which definition to use in which setting and which implementation is the fastest. Heuristics were developed to speed up computations when calculating grey-weighted distances between two points. The methods were evaluated on both real and synthetic data and the results show that the methods provide a step towards facilitating large-scale studies of images from both cryo-ET and FM.
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| 7. |
- Gedda, Magnus, 1978-
(författare)
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Heuristics for grey-weighted distance computations
- 2010
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Ingår i: Symposium on Image Analysis, Uppsala, March 11-12. Proceedings SSBA 2010..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- With new imaging techniques and computing power come increasing demands on the computation cost of image analysis algorithms. Grey-weighted distance transforms are traditionally computed using graph-based region-growing algorithms. These algorithms expand nodes in all directions by evolving an isotropic wave front. When calculating point-to-point distances, the isotropic propagation can be unnecessarily costly since it expands many nodes in directions away from the goal node. By introducing a heuristic to guide the search, the number of excessive nodes can be decreased. Here we introduce heuristics for computing Distance on Curved Spaces and Weighted Distance on Curved Spaces. We also examine the impact of these heuristics together with a heuristic previously proposed for fuzzy distance computations. The results show that the number of nodes expanded in point-to-point grey-weighted distances can be decreased by up to ~79%.
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| 8. |
- Gedda, Magnus, 1978-, et al.
(författare)
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Three-Dimensional Tracing of Neurites in Fluorescence Microscopy Images Using Local Path-Finding
- 2010
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Ingår i: 2010 IEEE International Conference On Acoustics, Speech And Signal Processing, 2010. ICASSP 2010. - 9781424442966 ; , s. 646-649
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Konferensbidrag (refereegranskat)abstract
- Neurite tracing in 3D neuron images is important when it comes to analysing the growth and functionality of nerve cells. The methods used today are either of high complexity, limiting throughput, or semi-automatic, i.e., requiring user interaction. This makes them unsuitable for analysis where high throughput is needed. In this work we propose a method designed for low complexity and void of user interaction by using local path-finding. The method is illustrated on both phantom and real data, and compared with a widely used commercial software package with promising results.
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| 9. |
- Leja, Justyna, et al.
(författare)
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Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells
- 2011
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Ingår i: Gene Therapy. - : Springer Science and Business Media LLC. - 0969-7128 .- 1476-5462. ; 18:11, s. 1052-1062
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Tidskriftsartikel (refereegranskat)abstract
- We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackie-adenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5 while it transduces normal hepatocytes at about 50% of Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systematic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.
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