O ciclo celular é controlado pela atividade das quinases dependentes de ciclinas (Ciclin-dependent kinases, CDKs). As CDKs são inativas como monômeros, e a sua ativação necessita da ligação às ciclinas, uma família diversa de proteínas cujos os níveis oscilam durante o ciclo celular, e fosforilação pela CAK (CDK-activating kinase) sobre um resíduo de treonina específico. As CDKs são capazes de fosforilar muitas proteínas que estão envolvidas nos eventos do ciclo celular, incluindo histonas e proteínas supressoras de tumores como pRb. Além da função de regulação positiva das ciclinas e CAK, muitas proteínas inibidoras de CDKs (CDK inhibitors, CKIs) têm sido descobertas, tais como p16, p21 e p28. Visto que, a desregulação das ciclinas e/ou alteração ou ausência de CKIs têm sido associadas com muitos cânceres, há um forte interesse em inibidores químicos de CDKs que possam ter uma função importante na descoberta de novas famílias de agentes anti-tumores. Vistoque, ATP é o autêntico co-fator da CDK2 este pode ser considerado como um \"pseudo-composto líder\" para a descoberta de inibidores de CDK2. Entretanto, há duas preocupações maiores a serem consideradas: composto contendo adenina são ligantes comuns para muitas enzimas nas células, desta forma, qualquer composto altamente carregado como ATP não será absorvido pelas células. Nós descrevemos aqui as estruturas determinadas por difração de raios-X da CDK2 em complexo com dois inibidores diferentes, descloro-flavopiridol (DFP) e Roscovitine. A estrutura do complexo binário CDK2-DFP foi resolvida por substituição molecular e refinada até um Rfactor=20,3% e a estrutura da CDK-2Roscovitine foi refinada até um Rfactor=18%. O descloro-flavopiridol é uma flavona com uma nova estrutura,comparável àquelas de flavonas polihidroxiladas. Estudos prévios mostraram que flavopiridol, um flavonóide, pode inibir cânceres de mama e de pulmão. O Roscovitine é um derivado de adenina e um potente inibidor de CDK2. A comparação das estruturas tridimensionais de CDK2-DFP e CDK2-Roscovitine com a de CDK2-ATP mostraram que o bolsão hidrofóbico de ligação de adenina tem a habilidade surpreendente de acomodar estruturas moleculares diferentes daquelas da ATP / Cell cycle progression is tightly controlled by the activity of ciclin-dependent kinases (CDKs). CDKs are inactive as monomers, and activation requires binding to cyclins, a diverse family of proteins whose levels oscillate during cell cycle, and phosphorilation by CDK-activating kinase (CAK) on a specific threonine residue. CDKs are able to phosphorylate many proteins that are in volvedin cell cycle events, including histones and tumor suppressor proteins like the retinoblastoma gene product pRb. In addition to the positive regulatory role of cyclins and CAK, many negative regulatory proteins (CDK Inhibitors, CIGs) have been discovered, such as p16, p21, and p28. Since deregulation of cyclins and/or alteration or absence ofCKIs have been associated with many cancers, there is strong interest in chemical inhibitors of CDKs that could play an important role in the discovery of new family of antitumor agents. Since ATP is the authentic cofactor of CDK2 it can be considered as a \"pseudo-lead compound\" for discovery of CDK2 inhibitors. However there are two major concerns: adenine containing compounds are common ligants for many enzymes in cells, thus, any adenine derivatives may inhibit many enzymes in the cells: second, any highly charged compounds such as ATP will prevent them from uptake by cells. We report here the x-ray structures of CDK2 in complex with two different inhibitors, deschloro-flavopiridol(DFP) and Roscovitine. The structure of the binary complex CDK2-DFP was solved by molecular replacement and refined to Rfactor = 20.3% and the structure ofCDK2-Roscovitine was refined to Rfactor = 18.0 %. The deschloro-flavopiridol(DFP) is a flavone with a novel structure, compared to that of polyhydroxylated flavones. Previous studies have shown that flavopiridol, a flavonoid, can inhibit growth of breast and lung carcinoma cell lines. The Roscovitine is an adenine derivative and a potent CDK2 inhibitor. The two inhibitors are competitive inhibitors for ATP binding to CDK2 and bind to the ATP binding pocket ofCDK2. The comparison of the three-dimensional structures of CDK2-DFP and CDK2-Roscovitine with the CDK2-ATP shows that the hydrophobic adenine-binding pocket has a surprising ability to accommodate molecular structures that are different from ATP.

Les kinases cycline-dépendantes (CDK) sont des protéines régulatrices essentielles du cycle cellulaire. Elles contrôlent la prolifération cellulaire et sont souvent déréglées dans les cancers. De nombreux inhibiteurs de CDKs ont été élaborés et sont actuellement le sujet d'essais cliniques. Bien que Cdk1 soit un régulateur essentiel de cycle cellulaire, Cdk2 n’est pas nécessaire pour la progression du cycle cellulaire, mais favorise la tumorigenèse. Par conséquent, Cdk2 est une cible thérapeutique prometteuse. L’utilisation des inhibiteurs de kinases pour modifier la prolifération cellulaire s’apparente à appliquer une sélection Darwinienne. Cette sélection peut être modélisée mathématiquement. Cette approche a montré que des avantages sélectifs, mêmes marginaux, peuvent être d'une importance majeure dans la compétition inter-cellulaire et la progression du cancer. Selon ce principe, nous avons fait l’hypothèse que le fait que la Cdk2 ait un rôle mineur dans la progression du cycle cellulaire lui confèrerait le statut de cible pertinente pour une thérapie du cancer. Selon cette hypothèse, son inhibition serait bien tolérée, permettant de réduire le niveau d’activité CDK et ainsi agir contre la prolifération déréglée des cellules. Nous avons supposé qu’au lieu d’éliminer entièrement les cellules les plus prolifératives, qui seraient les plus sensibles au traitement, il serait potentiellement intéressant de les exploiter pour concurrencer l’émergence des cellules résistantes, moins prolifératives. L'utilisation d'un traitement continu à faible dose avec les inhibiteurs Cdk2 pourrait permettre de maintenir cet équilibre. L'objectif de la thèse était d'étudier si Cdk2 confère un avantage prolifératif aux cellules cancéreuses, si les cellules peuvent développer une résistance aux inhibiteurs de CDKs, et si oui, déterminer quels étaient les mécanismes de résistance qui permettent de réduire le « fitness » des cellules prolifératives. Pour répondre à ces questions, nous avons généré des lignées cellulaires ayant des degrés variés de résistance à un inhibiteur spécifique de Cdk2 (inhibant également Cdk1 à des concentrations élevées). Nous avons caractérisé leur capacité à proliférer en comparaison avec des cellules parentales et des cellules isogéniques n’exprimant plus Cdk2 en raison d’un « knock-out » du gène. Bien que dans ces premières cellules le gène Cdk2 est retrouvé non muté et que l'expression de la protéine Cdk2 reste inaltérée, l'activité kinase de Cdk2 est diminuée. Les cellules résistantes à l’inhibiteur prolifèrent efficacement in vitro. Cependant, lors des expériences de compétition avec les cellules parentales, sensibles aux inhibiteurs, elles sont perdantes. Ceci montre que le développement d’une résistance à un inhibiteur de kinase entraîne un désavantage sélectif. Malgré une prolifération normale en l’absence de compétiteurs, ce désavantage est mis en évidence dans une population mixte, validant ainsi l’hypothèse de départ. Nous avons constaté que les Cdk2 KO et les cellules résistantes à l’inhibiteur (R50) ont un métabolisme altéré. Ces cellules sont sensibles à l'épuisement des nutriments et du glucose ainsi qu’à l'hypoxie, malgré un taux de consommation d'oxygène normal, ce qui indique une augmentation de la glycolyse aérobique. Les cellules R50 surexpriment la protéine Cdk6, ce qui peut contribuer à la résistance à l'inhibition Cdk2. De plus elles sont sensibles à l’inhibition des Cdk4/6, cibles référencées dans le traitement de certaines classes de cancer du sein. Enfin, les cellules Cdk2 KO présentent un point de contrôle de la phase S perturbé. Ces résultats suggèrent que des inhibiteurs pharmacologiques ciblant Cdk2 pourraient être synergique avec d’autres traitements, par exemple l’inhibition concomitante de la réplication de l'ADN, de la glycolyse, ou de Cdk6. Cela pourrait ainsi diminuer la prolifération des cellules cancéreuses et empêcher l’émergence d'une résistance thérapeutique. / Cyclin-dependent kinases (Cdk) are essential regulators of the cell cycle that support cell proliferation and are often deregulated in cancer. While Cdk1 is an essential regulator of the cell cycle, Cdk2 is not required for cell cycle progression but promotes tumorigenesis. Therefore, Cdk2 is a promising drug target. Many Cdk inhibitors have been developed and are currently undergoing clinical trials. Darwinian selection can be modelled mathematically, and such studies have shown that even marginal selective advantages can be of great importance in outcomes of cell-cell competition and cancer progression. We hypothesised that the non-essential role of Cdk2 for cell cycle progression may mean that it is a good target for cancer therapy as continual inhibition should be tolerated and should counteract deregulated cell proliferation in cancer. However, as with all chemotherapeutic agents, the development of clinical resistance is likely. We further hypothesized that applying a low-dose treatment with Cdk2 inhibitors should minimize chances of developing resistance, by maintaining competition between robustly proliferating cells that are sensitive to treatment, and resistant cells.The aim of the thesis was to investigate whether Cdk2 confers a proliferative advantage to cancer cells, whether cells can develop resistance to Cdk inhibitors, and if so, whether the mechanisms allowing resistance reduce cellular proliferative fitness.To answer these questions, we have created cell lines with varying degrees of resistance to a selective Cdk2 inhibitor (that at high doses, also inhibits Cdk1) and have characterised their proliferation capacity in comparison with parental cells and isogenic Cdk2 knockout cells. Although in these cells the Cdk2 gene is not mutated and the expression of Cdk2 protein remained unaltered, the kinase activity of Cdk2 is decreased. Similarly, Cdk2 gene knockout (Cdk2 KO) cells have reduced sensitivity to Cdk2 inhibition. Inhibitor-resistant cells proliferate efficiently but are outcompeted by parental, inhibitor-sensitive cells in competition experiments, confirming that inhibitor resistance entails a selective disadvantage. We found that the proliferation of both Cdk2 knockout and inhibitor-resistant (R50) cells is sensitive to nutrient and glucose depletion as well as hypoxia, despite a normal oxygen consumption rate, indicating increased aerobic glycolysis. R50 cells have highly upregulated Cdk6, which may contribute to resistance to Cdk2 inhibition. Moreover, they are sensitised to Cdk4/6 inhibition, which is currently authorised as a treatment for some classes of breast cancer. Finally, Cdk2 knockout cells have an impaired S-phase checkpoint. These results suggest that pharmacological inhibitors targeting Cdk2 might be synthetically lethal with other treatments, eg inhibition of DNA replication, of glycolysis, or of Cdk6. This might diminish cancer cell proliferation and prevent emergence of therapeutic resistance.

The cell cycle is an orchestrated mechanism ensuring cell division and differentiation to form multicellular organisms as well as to promote tissue homeostasis and regeneration. To secure correct cell division end ensure genome integrity for the next cell generation, the cell cycle must be strictly controlled. As part of this control cells have to adequately respond to the intra- and extracellular environment. Among the key molecules mediating the exchange of information from the surrounding environment are reactive oxygen species (ROS). ROS are small oxygen species, which control cellular signaling pathways through reductive-oxidative (redox) reactions with cellular proteins. For instance, mitogen signaling is sustained through ROS production by the NADPH oxidases in order to pass the information to proliferate or not to proliferate onto downstream cascades. Furthermore, cellular processes enabling proliferation and thus cell cycle progression require a level of high energy. Here, the cell cycle machinery meets mitochondria. Mitochondrial metabolism is the basis of aerobic respiration, the mechanism which supplies cells with energy and metabolites important for protein and DNA synthesis. By-products of mitochondrial metabolism as a result of incomplete reduction of oxygen are ROS molecules. Whether produced as metabolic by-product by mitochondria or as a growth factor stimulant by NADPH oxidases, many studies have demonstrated the broad influence of ROS on signaling pathways. When looking at ROS in context of cell division, ROS levels have been proposed to oscillate as cells progress through individual cell cycle phases. Perturbations of the cellular redox environment affect cell cycle progression and depending on the perturbation, may promote proliferation, cell cycle arrest or cell death. Thus, the interplay between redox mechanism and the cell cycle appears to be key to cell cycle decision making. Although the mechanisms of how ROS regulate proliferation-related proteins such as growth factor receptors or protein tyrosine phosphatases are known, the mechanisms of how ROS influence the cell cycle core machinery remain to be fully uncovered. To investigate the interplay between redox signaling and the cell cycle, I took advantage of approaches that allowed me to visualize and study changes in both systems at the same time. I visualized ROS dynamics in physiological and unperturbed conditions in non-transformed cells using redox specific dyes and indeed, observed that the levels of ROS oscillate during the cell cycle. ROS changes are characterized by basal levels in G1 phase and increased levels in S and G2 phases. My data provide evidence that ROS oscillations mainly originate from ROS produced by mitochondria. To investigate cause-consequence relations between ROS and the cell cycle I interfered with the cellular redox environment and studied the effect on cell cycle progression. Firstly, I discovered that the protein levels of NADPH oxidase 4 (NOX4), the enzyme producing ROS in response to mitogens, decrease shortly before cells enter S phase. Because NOX4 is constitutively active and its regulation is not known, this observation suggests that there is a mechanism of cell cycle-dependent NOX4 regulation that is important for entry into S phase. Secondly, I showed that reduction of ROS production by decreasing metabolites important for their production slowed proliferation due to prolonged S phase. This allowed me to establish that the main S phase regulator Cdk2 is a redox regulated cell cycle protein. Precisely, full phosphorylation of threonine 160 (T160) in the activatory segment of Cdk2, which is required for full Cdk2 activity was promoted by ROS derived from mitochondria. Furthermore, using a chemo-selective probe for cysteine oxidation I showed that Cdk2 is directly oxidized by ROS. Mutating the only surface exposed cysteine of Cdk2, C177, resulted in a change of Cdk2 binding to KAP, the phosphatase responsible for removing T160 phosphorylation. I found that only in reductive conditions KAP bound to Cdk2 resulting in Cdk2 dephosphorylation and thus reduced activity. In contrast oxidative conditions abolished the interaction between KAP and Cdk2. Thus, I propose a model of redox-dependent regulation of Cdk2 whereby the increase of mitochondrial ROS during S phase negatively regulates the Cdk2-KAP interaction to enable full activation of Cdk2 necessary for cells to rapidly progress through S phase. Altogether, in my thesis I investigated the link between mitochondrial ROS production and the cell cycle machinery and identified a mechanism of how increased levels of ROS drive the cell cycle. Furthermore, I outlined a potential cell cycle-dependent regulation of the NOX4 protein, which might provide a step towards understanding of its regulation. Thus, my thesis provides new views on the interplay between the redox system and the cell cycle machinery.:1. Introduction 1.1 Reactive oxygen species (ROS) 1.1.1 ROS and signal transduction 1.1.2 Antioxidant systems 1.1.3. Redox homeostasis, oxidative and reductive stress 1.2 ROS producing mechanisms 1.2.1 Mitochondria 1.2.2 NADPH oxidases 1.3 The cell cycle 1.4. ROS and the cell cycle 1.5 Aims of the thesis 2. Results 2.1 CellRox, a ROS sensitive dye, reveals redox changes during the cell cycle progression 2.2 Investigating the role of NADPH oxidases in cell cycle progression 2.2.1 General NOX inhibition causes defect in proliferation and suggests G1 phase delay 2.2.2 NOX4 and NOX1 specific inhibition causes a G1 delay or arrest 2.2.3 Specific down-regulation of NOX4 might have a negative impact on cell proliferation 2.2.4 NOX4 over-expression affects proliferation 2.2.5 NOX4 expression drops at G1 and S phase transition 2.3 Cell cycle dependent ROS oscillations correlate with mitochondria ROS production 2.4 Interference with mtROS decreases proliferation on the level of S phase 2.4.1 MitoTempo negatively affects proliferation and decreases population of EdU positive cells 2.4.2 Genetic interfering with mtROS production results in affected Cdk2 activation 2.5 Redox dependent Cdk2 activation via KAP binding 2.5.1 BTD labeling reveals Cdk2 as a direct target for oxidation 2.5.2 Preventing Cdk2 oxidation of cysteine 177 results in a drop of T160 phosphorylation 2.5.3 KAP binds to Cdk2 in a redox dependent manner 3. Discussion 3.1 ROS levels oscillate during the cell cycle in physiological cell culture conditions 3.2 Expression levels of NOX4 might determine the entry into S phase 3.3 Mitochondria are the main source of redox oscillations during the cell cycle 3.4 mtROS production contributes to Cdk2 activation and thus drives S phase progression 3.5 KAP phosphatase contributes to redox dependent regulation of Cdk2 3.6. Model of interconnection between the cellular redox environment and cell cycle regulation 4. Materials and methods 4.1 Cell culture 4.1.1 Cell lines 4.1.2 Cell treatments 4.1.3 Plasmids and cell line generation 4.1.4 RNA interference (RNAi) 4.1.5 EdU incorporation assay 4.2 Quantitative PCR (qPCR) 4.3 Protein studies 4.3.1 Cdk2-KAP/CAK interaction 4.3.2 Cdk2 sulfenylation by BTD labeling 4.4 SDS-PAGE and Western blot analyses 4.4.1 Total lysate preparation 4.4.2 SDS-PAGE 4.4.3 Western blotting 4.5 Flow cytometry analysis (FACS) 4.6 Hypoxia experiments 4.7 Microscopy 4.8 Automated image and data analysis 4.9 Statistical methods 5. Contributions 6. Bibliography 7. Acknowledgements 8. Appendix / Der Zellzyklus ist ein komplexer Mechanismus, der Zellteilung und Differenzierung in mehrzelligen Organismen, sowie die Homöostase und die Regeneration von Geweben gewährleistet. Um eine korrekte Zellteilung zu ermöglichen und die Integrität des Genoms für die nächste Zellgeneration sicherzustellen, muss der Zellzyklus streng kontrolliert werden. Im Rahmen dieser Kontrolle müssen die Zellen angemessen auf die intra- und extrazellulären Umgebungen reagieren. Zu den Schlüsselmolekülen, die den Austausch von Informationen aus der Umgebung vermitteln, gehören reaktive Sauerstoffspezies (ROS). ROS enthalten Sauerstoff und kontrollieren durch reduktiv-oxidative (Redox-) Reaktionen mit zellulären Proteinen viele verschiedene zelluläre Signalwege. So wird beispielsweise die Proliferation aufgrund von Wachstumssignalen durch die Produktion von ROS durch NADPH-Oxidasen ermöglicht, da sie die Information, ob eine Zellteilung stattfinden soll oder nicht, an nachgeschaltete Kaskaden weiterleiten. Darüber hinaus benötigen zelluläre Prozesse, die den Fortgang des Zellzyklus ermöglichen, ein hohes Energieniveau. Hier trifft die Zellzyklusmaschinerie auf die Mitochondrien. Der mitochondriale Stoffwechsel ist die Grundlage der aeroben Atmung, des Mechanismus, der die Zellen mit Energie und Metaboliten versorgt, die für die Protein- und DNA-Synthese notwendig sind. Als Nebenprodukte des mitochondrialen Stoffwechsels entstehen dabei häufig ROS, die aus der unvollständigen Reduktion von Sauerstoff resultieren. Unabhängig davon, ob sie als Nebenprodukte des Stoffwechsels in den Mitochondrien oder als wachstumsfördernde Substanzen in den NADPH-Oxidasen entstehen, viele Studien haben den weitreichenden Einfluss von ROS auf zahlreiche Signalwege gezeigt. Betrachtet man ROS im Zusammenhang mit der Zellteilung, so wird angenommen, dass die ROS-Konzentration mit dem Durchlaufen der einzelnen Zellzyklusphasen schwankt. Störungen der zellulären Redoxumgebung wirken sich auf den Verlauf des Zellzyklus aus und können je nach Störung Proliferation, Stillstand des Zellzykluses oder Zelltod fördern. Das Zusammenspiel zwischen Redox-Mechanismen und dem Zellzyklus scheint also der Schlüssel zur Entscheidungsfindung im Zellzyklus zu sein. Obwohl die Mechanismen, mit denen ROS essentielle Proteine wie Wachstumsrezeptoren oder Protein-Tyrosin-Phosphatasen regulieren, bekannt sind, sind die Mechanismen, mit denen ROS die Kernmaschinerie des Zellzyklus beeinflussen, noch nicht vollständig aufgeklärt. In der hier vorliegenden Arbeit nutzte ich daher Ansätze, die es mir ermöglichten, Veränderungen im Zellzklus, als auch Veränderungen in Redox-Signalen und deren Zusammenspiel gleichzeitig zu untersuchen. Ich habe die Dynamik reaktiver Sauerstoffspezies unter physiologischen und ungestörten Bedingungen in nichttransformierten Zellen mit redox-spezifischen Farbstoffen visualisiert und beobachtet, dass die ROS-Konzentrationen während des Zellzyklus oszillieren. Die Veränderungen der ROSKonzentrationen sind durch einen Grundwert in der G1-Phase und erhöhte Werte in der Sund G2-Phase gekennzeichnet. Meine Daten belegen, dass ROS-Oszillationen hauptsächlich von ROS herrühren, die von den Mitochondrien produziert werden. Um die Ursache-Folge-Beziehungen zwischen ROS und dem Zellzyklus zu untersuchen, habe ich in die zelluläre Redox-Balance eingegriffen und die Auswirkungen auf den Verlauf des Zellzyklus untersucht. Zunächst entdeckte ich, dass die Proteinkonzentration der NADPHOxidase 4 (NOX4), des Enzyms, das ROS als Reaktion auf Wachstumsfaktoren produziert, kurz vor dem Eintritt der Zellen in die S-Phase abnimmt. Da NOX4 konstitutiv aktiv ist und seine Regulierung nicht bekannt ist, deutet diese Beobachtung darauf hin, dass es einen zellzyklusabhängigen Mechanismus der NOX4-Regulierung gibt, der für den Eintritt in die S-Phase wichtig ist. Zweitens konnte ich zeigen, dass die Verringerung von Metaboliten zu einer Verringerung der ROS-Produktion führt, welches die Proliferation aufgrund einer verlängerten S-Phase verlangsamt. Dadurch konnte ich feststellen, dass Cdk2, der wichtigste S-Phasen-Regulator, ein redoxreguliertes Zellzyklusprotein ist. Genauer gesagt wurde die vollständige Phosphorylierung von Cdk2 am Threonin 160 (T160), welche für die volle Cdk2-Aktivität erforderlich ist, durch ROS aus Mitochondrien gefördert. Außerdem konnte ich mit einer chemo-selektiven Probe für Cysteinoxidation zeigen, dass Cdk2 direkt durch ROS oxidiert wird. Die Mutation des einzigen exponierten Cysteins von Cdk2, C177, führte zu einer Veränderung der Bindung von Cdk2 an KAP, die Phosphatase, die für die Aufhebung der T160-Phosphorylierung verantwortlich ist. Ich fand heraus, dass KAP nur unter reduktiven Bedingungen an Cdk2 bindet, was zu einer Dephosphorylierung von Cdk2 und damit zu einer verringerten Aktivität führt. Unter oxidativen Bedingungen hingegen wurde die Interaktion zwischen KAP und Cdk2 aufgehoben. Daher schlage ich ein Modell der redoxabhängigen Regulierung von Cdk2 vor, bei dem der Anstieg der mitochondrialen ROS während der S-Phase die Interaktion zwischen Cdk2 und KAP negativ reguliert, um eine vollständige Aktivierung von Cdk2 und damit eine erfolgreiche S-Phase zu ermöglichen. Insgesamt habe ich in meiner Dissertation die Verbindung zwischen mitochondrialer ROS-Produktion und der Zellzyklusmaschinerie untersucht und einen Mechanismus identifiziert, wie erhöhte ROS-Konzentrationen den Zellzyklus antreiben. Darüber hinaus habe ich die zellzyklusabhängige Regulierung des NOX4-Proteins aufgezeigt, was neue Einblicke zum Verständnis der Zellzykluskontrolle durch ROS gewährt. Somit bietet meine Arbeit neue, interessante Erkenntnisse für das Zusammenspiel zwischen dem Redoxsystem und der Zellzyklusmaschinerie.:1. Introduction 1.1 Reactive oxygen species (ROS) 1.1.1 ROS and signal transduction 1.1.2 Antioxidant systems 1.1.3. Redox homeostasis, oxidative and reductive stress 1.2 ROS producing mechanisms 1.2.1 Mitochondria 1.2.2 NADPH oxidases 1.3 The cell cycle 1.4. ROS and the cell cycle 1.5 Aims of the thesis 2. Results 2.1 CellRox, a ROS sensitive dye, reveals redox changes during the cell cycle progression 2.2 Investigating the role of NADPH oxidases in cell cycle progression 2.2.1 General NOX inhibition causes defect in proliferation and suggests G1 phase delay 2.2.2 NOX4 and NOX1 specific inhibition causes a G1 delay or arrest 2.2.3 Specific down-regulation of NOX4 might have a negative impact on cell proliferation 2.2.4 NOX4 over-expression affects proliferation 2.2.5 NOX4 expression drops at G1 and S phase transition 2.3 Cell cycle dependent ROS oscillations correlate with mitochondria ROS production 2.4 Interference with mtROS decreases proliferation on the level of S phase 2.4.1 MitoTempo negatively affects proliferation and decreases population of EdU positive cells 2.4.2 Genetic interfering with mtROS production results in affected Cdk2 activation 2.5 Redox dependent Cdk2 activation via KAP binding 2.5.1 BTD labeling reveals Cdk2 as a direct target for oxidation 2.5.2 Preventing Cdk2 oxidation of cysteine 177 results in a drop of T160 phosphorylation 2.5.3 KAP binds to Cdk2 in a redox dependent manner 3. Discussion 3.1 ROS levels oscillate during the cell cycle in physiological cell culture conditions 3.2 Expression levels of NOX4 might determine the entry into S phase 3.3 Mitochondria are the main source of redox oscillations during the cell cycle 3.4 mtROS production contributes to Cdk2 activation and thus drives S phase progression 3.5 KAP phosphatase contributes to redox dependent regulation of Cdk2 3.6. Model of interconnection between the cellular redox environment and cell cycle regulation 4. Materials and methods 4.1 Cell culture 4.1.1 Cell lines 4.1.2 Cell treatments 4.1.3 Plasmids and cell line generation 4.1.4 RNA interference (RNAi) 4.1.5 EdU incorporation assay 4.2 Quantitative PCR (qPCR) 4.3 Protein studies 4.3.1 Cdk2-KAP/CAK interaction 4.3.2 Cdk2 sulfenylation by BTD labeling 4.4 SDS-PAGE and Western blot analyses 4.4.1 Total lysate preparation 4.4.2 SDS-PAGE 4.4.3 Western blotting 4.5 Flow cytometry analysis (FACS) 4.6 Hypoxia experiments 4.7 Microscopy 4.8 Automated image and data analysis 4.9 Statistical methods 5. Contributions 6. Bibliography 7. Acknowledgements 8. Appendix

The radiosensitivity of tumour cells depends mainly on their capacity to maintain genomic integrity. This requires efficient repair of radiation-induced DNA double strand breaks, a process governed by the cell cycle. Based on their functions in cell cycle regulation and DNA damage repair, we hypothesised that targeting of CDK2 and CDK9 modifies cancer cell response to radiotherapy. Therefore, we evaluated the significance of CDK2 and CDK9 for the cellular radiation response in a panel of human head and neck squamous cell carcinoma (HNSCC) cell lines. In order to achieve our goal, we performed a series of experiments to measure several key parameters such as clonogenic radiation survival, cell cycling, DNA damage repair and apoptosis. We found that loss of CDK2 radiosensitises mouse embryonic fibroblasts (MEFs) as well as HNSCC two dimensional (2D) cell cultures. However, under more physiological three dimensional (3D) growth conditions in laminin-rich extracellular matrix, targeting of CDK2 failed to modulate the radiosensitivity of HNSCC cells. Moreover, CDK2 attenuated the repair of radiogenic double strand breaks (DSBs) in MEFs as well as SAS and FaDu HNSCC cells indicating a possible role of CDK2 in DNA damage repair. However, we found that CDK2 is dispensable for cell cycle and checkpoint regulation in response to irradiation in SAS and FaDu cells. Taken together, our results suggest that targeting of CDK2 may not provide a therapeutic benefit to overcome HNSCC cell resistance to radiotherapy. We also showed that depletion of CDK9 clearly enhances the radiosensitivity of HNSCC cultures. In addition, the ectopic expression of CDK9 has a radioprotective effect. These findings suggest a potential role of CDK9 in the radiation response of HNSCC cells. Moreover, our study indicates a possible role of CDK9 in the DNA damage repair response and cell cycling of HNSCC cells. Conclusively, on the basis of these data, targeting of CDK9 in addition to conventional radiotherapy might be a viable strategy to overcome cancer cell resistance.

The cell division cycles of differentiating cells are coordinated so as to generate sufficient numbers of mature cells. The cell cycle may also regulate the process of differentiation, in ways that are not well understood. We previously discovered that during erythropoiesis, the cell cycle is synchronized with a specific developmental switch, where erythroid progenitors known as colony-forming-unit-erythroid (CFU-e) transition from a self-renewal state to a state of erythroid terminal differentiation (ETD). This switch takes place during a single cell cycle S phase and is dependent on S-phase progression. My work shows that this S phase is unusual, in that it is shorter than S phase in preceding cycles, as a result of a global increase in replication fork speed. I found that the CDK inhibitor, p57KIP2, negatively regulates replication fork speed in self-renewing CFU-e, and its down-regulation at the switch to ETD results in S-phase shortening. p57KIP2-mediated inhibition of CDK2 is essential for CFU-e self-renewal. It exerts this effect by reducing replication stress and also reducing the probability of transition from CFU-e to ETD, promoting CFU-e self-renewal instead. CDK2 inhibiting drugs that mimic the action of p57KIP2 stimulate erythropoiesis both in vitro and in vivo, through expansion of the CFU-e pool. In addition to p57KIP2, E2f4 also regulates S-phase shortening and the efficiency of the CFU-e to ETD transition. Overall, my work shows that S-phase speed regulates a key erythroid cell fate decision, and suggests a possible translational application of CDK2 inhibiting drugs in the stimulation of erythropoiesis.

No description available.

The thesis specializes in the computational description of pharmaceutically important compounds. A substantial number of pharmaceutical drugs are small molecules that are bound to an active site of an enzyme by the "lock (binding site) and key (drug)" model through non-covalent interactions. The association of enzymes with drugs cause an increase or decrease in the activity of enzymes. The main topic is focused on the computational elucidation of the structural basis for the interactions of the purine-like compounds with the enzyme cyclin- dependent kinase 2 that belongs to the protein-kinase enzyme family. These enzymes play an important role in the cell cycle regulation; their increased activity significantly contributes to the loss of control over cell proliferation, which is one of the primary causes of cancer cell formation. The study describes the binding motifs of roscovitine, which shows an inhibitory effect on the function of cyclin-dependent kinases, and its analogues containing bioisosteric central heterocycles in the complex with cyclin-dependent kinase 2. The binding affinity between the cyclin-dependent kinase 2 enzyme and the inhibitors was quantified as calculated binding scores and evaluated in relation to the conformation of the optimized structures. The hybrid model combining the...

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We have identified a novel Cyclin, called Cyclin O, which is able to bind and activate Cdk2 in response to intrinsic apoptotic stimuli. We have focused on the study of Cyclin Oα and Cyclin Oβ, alternatively spliced products of the gene. Upon treatment with different stress stimuli, transfected Cyclin Oα accumulates in dense aggregations in the cytoplasm compatible with being Stress Granules (SGs). Furthermore, we have seen that Cyclin Oβ and a point mutant of the N-terminal part of the protein constitutively localize to the SGs. Although both alpha and beta isoforms are proapoptotic, only Cyclin Oα can bind and activate Cdk2. On the other hand, we have demonstrated that Cyclin O is upregulated by Endoplasmic Reticulum (ER) stress and is necessary for ER stress-induced apoptosis. Cyclin O activates specifically the PERK pathway and interacts with the PERK inhibitor protein p58IPK. Moreover, Cyclin O participates in the activation of other eIF2α kinases. We have also observed that a pool of Cyclin O is located in active mitochondria, suggesting a function of the protein linked to oxidative metabolism.Hemos identificado una nueva Ciclina, llamada Ciclina O, que es capaz de unirse y activar Cdk2 en respuesta a estímulos apoptóticos intrínsecos. Nos hemos centrado en el estudio de la Ciclina Oα y la Ciclina Oβ, productos de splicing alternativo del gen. En respuesta a diferentes tipos de estrés, la Ciclina Oα se acumula en agregaciones citoplásmicas densas que podrían corresponder a Gránulos de Estrés (SGs). Además, hemos visto que la Ciclina Oβ y un mutante puntual de la parte N-terminal de la proteína se localizan constitutivamente en los SGs. Aunque las dos isoformas alfa y beta son proapoptóticas, solo la Ciclina Oα es capaz de unirse y activar Cdk2. Por otro lado, hemos demostrado que los niveles de Ciclina O se incrementan en respuesta al estrés de Retículo Endoplásmico (RE) y que esta proteína es necesaria para la inducción de apoptosis dependiente de estrés de RE. La Ciclina O activa específicamente la vía de PERK e interacciona con la proteína inhibidora de PERK p58IPK. Además, la Ciclina O participa en la activación de otras quinasas de eIF2α. La Ciclina O se localiza en mitocondrias activas, lo que sugiere una función de la proteína ligada al metabolismo oxidativo.

L´apoptosi, o mort cel.lular programada, és un procés actiu que mobilitza els recursos cel.lulars amb l´objectiu de mantenir l´homeostasi de l´organisme a expenses del suïcidi de cèl.lules individuals. Diferents estudis han mostrat un increment de l´activiat d´algunes cdk, especialment Cdk1 i Cdk2, en correlació amb la progressió dels primers estadis apoptòtics. En el nostre laboratori l´estudi de l´apoptosi en timòcits, els quals no tenen una activitat cdk significativa degut a l´aturada del cicle cel.lular en G1, demostren que la inducció de l´activitat de Cdk2 després del tractament amb radiació gamma o amb glucocorticoides és necessària per l´inici de l´apoptosi. Mentre cap de les ciclines conegudes sembla ser la proteïna activadora de Cdk2 en apoptosi, en el nostre laboratori hem identificat un nou membre de la família de les ciclines, denominada Ciclina O, capaç d´activar aquesta kinasa in vivo en línies cel.lulars. L´expressió d´aquesta nova ciclina en el timus, i altres teixits, s´indueix ràpidament després del tractament amb radiació gamma i coincideix amb l´aparició de l´apoptosi. Aquests resultats posicionen la Ciclina O com a millor candidat a ser l´activador de Cdk2 necessari per induïr la mort cel.lular programada en el timus, i probablement també en altres òrgans. / The apoptosis, also called programmed cell death, is an active process able to use the cellular mechanisms to kill individual cells in order to keep the functional homeostasis of the whole organism. Different studies had shown a correlation between the first apoptotic events and the induction of some cdk proteins, particularly Cdk1 and Cdk2. The studies of thymocytes in our laboratory, wich lacks the most amount of cdk activity related to the cell cycle because of its arrest in G1, had shown that the induction of Cdk2 activity after the treatment with gamma radiation or glucocorticoids is a necessary step for the apoptosis induction. While any of the cyclins described at the moment seems to be the Cdk2 activator for apoptosis a new member of the cyclin family able to activate the kinase Cdk2 in vivo in cell lines has been identified in our laboratory. The expresion of this cyclin, known as Cyclin O, is quickly induced in the thymus after the treatment with gamma radiation and correlates with the induction of apoptosis. These results position Cyclin O as the best candidate to activate Cdk2 and inuce the programmed cell death in the thymus, and probably other tissues.