Titel: | Regulation of DNA Repair by EGF Receptor Signaling After X-Irradiation | Sonstige Titel: | Regulation der DNA-Reparatur durch das EGF Rezeptor-Signaling nach ionisierender Strahlung | Sprache: | Englisch | Autor*in: | Myllynen, Laura | Schlagwörter: | DNA repair; EGFR signaling; proteome analytic; regulation; phosphorylation | Erscheinungsdatum: | 2014 | Tag der mündlichen Prüfung: | 2013-12-06 | Zusammenfassung: | In many tumors, the epidermal growth factor receptor (EGFR) is overexpressed or deregulated. This is associated with therapy resistance, especially against radiation therapy, and poor prognosis. Consequently, EGFR inhibitors are used to achieve a radiosensitization. This sensitization is assumed to result mostly from a suppression in the repair of radiation-induced DNA double-strand breaks (DSB), as non- and incorrectly repaired DSBs lead to lethal chromosome aberrations that eventually cause cell inactivation. However, the molecular mechanisms responcible for this suppressed DSB repair are not yet fully understood. In particular, it is not known whether the regulation of DSB repair by EGFR occurs in all tumors, or whether it is only seen in a few tumor cells with the expression of distinct regulatory factors. As a consequence, so far no biomarkers are available that could predict the response of an individual tumor to EGFR inhibition. The aim of this study was to investigate whether the regulation of DSB repair by EGFR is a general mechanism or whether it depends on specific parameters. In a second part, it is shown that mass spectrometry can be used to unveil the molecular mechanisms of this regulation and might help to identify biomarkers for responding and non-responding tumor cell lines. The regulation of DSB repair by EGFR was examined in the bronchial carcinoma cell line (NSCLC) A549, which is p53 wt and K-Ras mutated, with both proteins being reported to be important for DSB repair. It is shown that the activity of both overall DSB repair and non-homologous end-joining (NHEJ) are enhanced by EGFR stimulation, regardless of the ligand used. In turn the activity of both processes is reduced when EGFR is blocked. However, a regulation of DSB repair was also observed for a second NSCLC cell line (H1299) which -in contrast to A549- is mutated in p53 but wt for K-Ras. Additionally, the EGFR was also found to regulate homologous recombination (HR), which is the second major repair pathway for the repair of DSBs. Therefore, these data indicate that regulation of DSB repair by EGFR is a general mechanism which does not depend on specific mutations and is not restricted to a specific pathway. In the second part of this work, a mass spectrometric (MS) approach coupled with stable isotope labeling by amino acids in cell culture (SILAC) was established to study the molecular mechanism of this regulation. These experiments were performed with the two head and neck squamous cell carcinoma (HNSCC) cell lines UTSCC5 and SAS as these cell lines display a clear difference in EGFR expression and signaling as well as in the EGFR-dependent regulation of DSB repair. Analysing chromatin-bound proteins upon EGFR inhibition by MS, no significant alterations in chromatin binding were observed. In contrast, clear effects were seen when analyzing the phosphorylated nuclear proteins. Overall, 16 DNA repair proteins were detected in UTSCC5 cells and 20 in SAS cells. In both cell lines, four of these showed a reduced phosphorylation after EGFR inhibition, and two of these proteins (PARP1 and GAPDH) were regulated in both UTSCC5 and SAS cells. These results indicate that the EGFR effectively regulates the repair of DSBs and that this is a conserved mechanism. A mass spectrometric approach was successfully established to analyse DNA repair proteins. In contrast to chromatin-bound proteins, the analysis of nuclear phosphoproteins revealed EGFR-dependent regulation mechanism. Therefore, this method represents a promising tool to further elucidate mechanisms involved in the EGFR-dependent regulation of DSB repair and to identify markers for tumors which will respond positively to EGFR targeted therapy or other new targeted therapeutics. |
URL: | https://ediss.sub.uni-hamburg.de/handle/ediss/5261 | URN: | urn:nbn:de:gbv:18-65752 | Dokumenttyp: | Dissertation | Betreuer*in: | Dikomey, Ekkehard (Prof. Dr.) |
Enthalten in den Sammlungen: | Elektronische Dissertationen und Habilitationen |
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Datei | Beschreibung | Prüfsumme | Größe | Format | |
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Dissertation.pdf | 17779e61db6ec11b448f8964b8d088cb | 13.44 MB | Adobe PDF | Öffnen/Anzeigen |
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