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Nek1 - developmental involvement in DNA repair and role as a target in radiotherapy

Freund, Isabel (2022):
Nek1 - developmental involvement in DNA repair and role as a target in radiotherapy. (Publisher's Version)
Darmstadt, Technische Universität,
DOI: 10.26083/tuprints-00021560,
[Ph.D. Thesis]

Abstract

Organisms are inevitably exposed to ionizing radiation (IR) which is emitted by various natural sources such as decaying radionuclides. Since its discovery in the 19th century, IR has become a highly relevant tool, especially in the field of medicine where it is used for diagnostic procedures and the treatment of tumors. However, its property to alter the structure of the exposed matter by breaking chemical bonds threatens the integrity of an important molecule that presents the fundamental prerequisite of life, namely DNA. Exposing DNA to IR results in different types of lesions of which the DNA double-strand break (DSB) represents the most detrimental. Since DSBs can also result from several endogenous processes, cells evolved certain mechanisms to minimize the harmful impact of this lesion on their genomic integrity, collectively termed DNA damage response (DDR). The DDR consists of highly coordinated signaling pathways that allow for damage detection, cell cycle arrest, and damage repair. While originally studied for their involvement in ciliogenesis, centrosome organization, and mitosis, the members of the never-in mitosis-gene A (NIMA) related kinase (Nek) family increasingly move into the focus of DDR research as nearly all have functions in related processes. However, one member, Nek1, stands out in this context due to its multifunctional role in the DDR, including the regulation of cell cycle checkpoints, apoptosis, and DNA repair. In the following work, the results of two projects are presented, each highlighting a different aspect of Nek1 as an important kinase of the DNA damage response. The first project is based on research conducted by the Löbrich lab, which identified Nek1 as a regulator of a factor required for the successful execution of the DSB repair pathway "Homologous Recombination" (HR), namely Rad54. Subsequent in vivo studies surprisingly revealed that Nek1 is important for HR in adult mice but not in embryos, which exhibit a normal repair behavior despite a Knock-Out mutation in the Nek1 gene. The objective of this project was therefore to further characterize the apparent differential regulation of HR during development. To this end, embryonic and adult fibroblast lines were isolated from different mice strains and analyzed for DSB repair and differences in gene expression. The collected data suggest that the kinases required for the activation of Rad54 indeed change in response to an organism’s developmental stage: Nek1 exclusively controls Rad54’s activation in adult cells whereas Nek3 and Nek5 can redundantly activate Rad54 in embryonic cells. Thus, this study not only consolidates previous findings of the Löbrich lab but is also the first to report a developmental change in the regulation of HR and to associate Nek3 and Nek5 with DNA repair. Considering its multifunctionality in the DDR, pharmacological inactivation of Nek1 has the potential to significantly improve the treatment of cancer by e.g. radiotherapy. The Rödel lab confirmed this assumption by demonstrating that depleting Nek1 significantly sensitizes two different cancer cell lines to single-dose irradiation as shown by their reduced ability to form colonies. Since further experiments revealed that Nek1-depleted cancer cells are still capable of inducing a functional G2/M checkpoint, the second project of this work investigated the extent to which fractionated irradiation can enhance the radiosensitizing effect of Nek1 depletion. Cancer cells were therefore subjected to three fractionation regimes, in which a total radiation dose of 6 Gy was applied in three small fractions either every 2 h, 6 h, or 24 h, and evaluated for cell cycle behavior as well as colony-forming ability. Indeed, the 6 h interval tremendously increases the radiosensitivity of Nek1-depleted cells beyond the level observed for single-dose irradiations while Nek1-proficient cells are less affected. This finding has been additionally strengthened in in vivo xenograft studies. Taken together, this work strengthens Nek1 as a promising target in cancer therapy. It further demonstrates that the efficacy of fractionated radiotherapy can be significantly increased if the employed regime is adapted to the cycle rate of cancer cells and takes the cell cycle-dependent function of Nek1 as an HR factor into account.

Item Type: Ph.D. Thesis
Erschienen: 2022
Creators: Freund, Isabel
Status: Publisher's Version
Title: Nek1 - developmental involvement in DNA repair and role as a target in radiotherapy
Language: English
Abstract:

Organisms are inevitably exposed to ionizing radiation (IR) which is emitted by various natural sources such as decaying radionuclides. Since its discovery in the 19th century, IR has become a highly relevant tool, especially in the field of medicine where it is used for diagnostic procedures and the treatment of tumors. However, its property to alter the structure of the exposed matter by breaking chemical bonds threatens the integrity of an important molecule that presents the fundamental prerequisite of life, namely DNA. Exposing DNA to IR results in different types of lesions of which the DNA double-strand break (DSB) represents the most detrimental. Since DSBs can also result from several endogenous processes, cells evolved certain mechanisms to minimize the harmful impact of this lesion on their genomic integrity, collectively termed DNA damage response (DDR). The DDR consists of highly coordinated signaling pathways that allow for damage detection, cell cycle arrest, and damage repair. While originally studied for their involvement in ciliogenesis, centrosome organization, and mitosis, the members of the never-in mitosis-gene A (NIMA) related kinase (Nek) family increasingly move into the focus of DDR research as nearly all have functions in related processes. However, one member, Nek1, stands out in this context due to its multifunctional role in the DDR, including the regulation of cell cycle checkpoints, apoptosis, and DNA repair. In the following work, the results of two projects are presented, each highlighting a different aspect of Nek1 as an important kinase of the DNA damage response. The first project is based on research conducted by the Löbrich lab, which identified Nek1 as a regulator of a factor required for the successful execution of the DSB repair pathway "Homologous Recombination" (HR), namely Rad54. Subsequent in vivo studies surprisingly revealed that Nek1 is important for HR in adult mice but not in embryos, which exhibit a normal repair behavior despite a Knock-Out mutation in the Nek1 gene. The objective of this project was therefore to further characterize the apparent differential regulation of HR during development. To this end, embryonic and adult fibroblast lines were isolated from different mice strains and analyzed for DSB repair and differences in gene expression. The collected data suggest that the kinases required for the activation of Rad54 indeed change in response to an organism’s developmental stage: Nek1 exclusively controls Rad54’s activation in adult cells whereas Nek3 and Nek5 can redundantly activate Rad54 in embryonic cells. Thus, this study not only consolidates previous findings of the Löbrich lab but is also the first to report a developmental change in the regulation of HR and to associate Nek3 and Nek5 with DNA repair. Considering its multifunctionality in the DDR, pharmacological inactivation of Nek1 has the potential to significantly improve the treatment of cancer by e.g. radiotherapy. The Rödel lab confirmed this assumption by demonstrating that depleting Nek1 significantly sensitizes two different cancer cell lines to single-dose irradiation as shown by their reduced ability to form colonies. Since further experiments revealed that Nek1-depleted cancer cells are still capable of inducing a functional G2/M checkpoint, the second project of this work investigated the extent to which fractionated irradiation can enhance the radiosensitizing effect of Nek1 depletion. Cancer cells were therefore subjected to three fractionation regimes, in which a total radiation dose of 6 Gy was applied in three small fractions either every 2 h, 6 h, or 24 h, and evaluated for cell cycle behavior as well as colony-forming ability. Indeed, the 6 h interval tremendously increases the radiosensitivity of Nek1-depleted cells beyond the level observed for single-dose irradiations while Nek1-proficient cells are less affected. This finding has been additionally strengthened in in vivo xenograft studies. Taken together, this work strengthens Nek1 as a promising target in cancer therapy. It further demonstrates that the efficacy of fractionated radiotherapy can be significantly increased if the employed regime is adapted to the cycle rate of cancer cells and takes the cell cycle-dependent function of Nek1 as an HR factor into account.

Place of Publication: Darmstadt
Collation: 86 Seiten
Divisions: 10 Department of Biology
10 Department of Biology > Radiation Biology and DNA Repair
Date Deposited: 13 Jul 2022 12:21
DOI: 10.26083/tuprints-00021560
URL / URN: https://tuprints.ulb.tu-darmstadt.de/21560
URN: urn:nbn:de:tuda-tuprints-215607
PPN:
Referees: Löbrich, Prof. Dr. Markus ; Rödel, Prof. Dr. Franz
Refereed / Verteidigung / mdl. Prüfung: 10 June 2022
Alternative Abstract:
Alternative abstract Language

Organismen sind unweigerlich ionisierender Strahlung (engl. Ionizing Radiation, IR) ausgesetzt, die von verschiedenen natürlichen Quellen wie beispielsweise zerfallenden Radionukliden emittiert wird. Seit ihrer Entdeckung im 19. Jahrhundert ist die IR vor allem in der Medizin für diagnostische und therapeutische Verfahren zu einem äußerst wichtigen Instrument geworden. Ihre Eigenschaft, chemische Strukturen und Bindungen in der bestrahlten Materie aufzubrechen, bedroht jedoch die Integrität eines besonderen Moleküls, das die Grundvoraussetzung für die Lebensfähigkeit eines jeden Organismus darstellt, die DNA. Eine Strahlenexposition der DNA führt zu verschiedenen Arten von Schäden, von denen der Doppelstrangbruch (DSB) die schwerwiegendste Läsion darstellt. Da DSBs auch als Folge verschiedener endogener Prozesse entstehen können, haben Zellen verschiedene Mechanismen entwickelt, um die schädlichen Auswirkungen dieser Läsion auf ihre genomische Integrität zu minimieren. Diese DNA-Schadensantwort (engl. DNA damage response, DDR) besteht aus koordinierten Signalwegen, die Schadenserkennung, Unterbrechung des Zellzyklus und Reparatur der DNA ermöglichen. Die Mitglieder der never in mitosis-gene A (NIMA) related kinase (Nek)-Familie wurden ursprünglich für ihre Beteiligung in der Ciliogenese, der Zentrosomenorganisation und der Mitose untersucht, rücken jedoch zunehmend in den Fokus der DDR-Forschung, da fast alle eine Schlüsselrolle in entsprechenden Prozessen spielen. Ein Mitglied, Nek1, sticht in diesem Zusammenhang aufgrund seiner multifunktionalen Aufgaben in der DDR (Regulierung von Zellzyklus-Checkpoints, Apoptose und DNA-Reparatur) hervor. In der folgenden Arbeit werden die Ergebnisse zweier Projekte vorgestellt, die jeweils einen anderen Aspekt von Nek1 als wichtige Kinase der DNA-Schadensantwort beleuchten. Das erste Projekt basiert auf Forschungsarbeiten der AG Löbrich, in denen Nek1 als Regulator eines Faktors identifiziert wurde, der für die erfolgreiche Durchführung des DSB-Reparatur-weges "Homologe Rekombination" (HR) essentiell ist, Rad54. Nachfolgende in vivo Studien ergaben das überraschende Ergebnis, dass Nek1 zwar für die HR in adulten Mäusen von Bedeutung ist, nicht aber für Embryonen, die trotz Knock-Out-Mutation im Nek1-Gen ein normales Reparaturverhalten aufweisen. Das Hauptziel dieses Teilprojekts der vorgelegten Arbeit war es daher, die offensichtlich unterschiedliche Regulierung der HR während der murinen Entwicklung weiter zu charakterisieren. Zu diesem Zweck wurden embryonale und adulte Fibroblastenlinien aus verschiedenen Mausstämmen isoliert und im Hinblick auf DSB-Reparatur sowie Genexpression analysiert. Die gesammelten Daten deuten darauf hin, dass sich die Kinasen, die für die Aktivierung von Rad54 erforderlich sind, tatsächlich in Abhängigkeit vom Entwicklungsstadium eines Organismus ändern: Nek1 kontrolliert die Aktivierung von Rad54 ausschließlich in adulten Zellen, während Nek3 und Nek5 Rad54 in embryonalen Zellen redundant aktivieren können. Diese Studie konsolidiert also nicht nur frühere Ergebnisse der AG Löbrich, sondern ist vielmehr die erste, die über eine entwicklungsbedingte Veränderung in der Regulierung der HR berichtet und Nek3 sowie Nek5 mit der DNA-Reparatur in Verbindung bringt. In Anbetracht seiner Multifunktionalität in der DDR besitzt die pharmakologische Inaktivierung von Nek1 das Potenzial, die Behandlung von Krebs durch z.B. eine Strahlentherapie deutlich zu verbessern. Die AG Rödel bestätigte diese Annahme, indem sie nachwies, dass die Depletion von Nek1 zwei verschiedene Krebszelllinien signifikant für Einzeldosenbestrahlung sensibilisiert, was sich in einer verminderten Fähigkeit zur Koloniebildung äußerte. Da weitere Experimente zeigten, dass Nek1-depletierte Krebszellen einen funktionsfähigen G2/M-Kontrollpunkt induzieren können, untersuchte das zweite Projekt dieser Arbeit, inwiefern eine fraktionierte Bestrahlung die Strahlen-sensitivierende Wirkung eines Nek1-Verlusts verstärken kann. Krebszellen wurden daher drei fraktionierten Bestrahlungsschemata unterzogen, bei denen eine Gesamtdosis von 6 Gy in drei kleinen Fraktionen entweder alle 2 h, 6 h oder 24 h verabreicht wurde. Die Analyse des Zellzyklusverhalten und des klonogenen Überlebens ergab, dass das 6 h Intervall tatsächlich die Radiosensitivität von Nek1-depletierten Zellen über das bei Einzeldosen beobachtete Maß hinaus erhöhte, während Nek1-profiziente Zellen weniger betroffen waren. Dieses Ergebnis konnte in in vivo Xenotransplantationsstudien zusätzlich bestätigt werden. Insgesamt konsolidiert dieses Teilprojekt Nek1 als vielversprechendes Ziel in der Krebstherapie. Sie zeigt darüber hinaus, dass die Wirksamkeit einer fraktionierten Strahlentherapie deutlich erhöht werden kann, wenn das verwendete Bestrahlungsintervall an die Zyklusrate der Krebszellen angepasst ist und die zellzyklusabhängige Funktion von Nek1 als HR-Faktor berücksichtigt wird.

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