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Das Ziel der vorliegenden Arbeit war es, Mechanismen zu identifizieren, die an der Induktion von Zellwachstum und/oder Zelltod von Kardiomyozyten beteiligt sind. Zunächst wurde ein Modell entwickelt, das es erlaubt, genregulatorische Elemente in vivo zu identifizieren. Es wurden die verschiedenen Variablen, die die Expression in vivo ins Myokard injizierter Reportergenplasmide regulieren, analysiert. Es stellte sich heraus, daß die Injektion von Reportergenkonstrukten ins Myokard des Hundes ein ausgezeichnetes Modell darstellt zur Analyse der Genregulation im Myokard großer Säugetierspezies. Mit Hilfe dieses Modells gelang durch Injektion von ANF (atrialer natriuretischer Faktor)-Promotorkonstrukten mit anschließendem aortic banding die Identifizierung einer AP1-Bindungsstelle im Promotor des ANF-Gens als cis-regulatorisches Element, das für die Aktivierung des ANF-Gens bei der Druckhypertrophie verantwortlich ist. Zur Identifizierung von Faktoren, die für den Zellzyklusarrest von Kardiomyozyten verantwortlich sind, wurde der ubiquitär exprimierte Transkriptionsfaktor E2F-1 in isolierte Kardiomyozyten mittels adenoviralem Gentransfer eingebracht. Die Überexpression von E2F-1 in Kardiomyozyten führte zur Induktion von programmiertem Zelltod (Apoptose). Die Apoptose wurde in Anwesenheit von Insulin-like Growth Factor-I (IGF-I) supprimiert und es konnte nun gesteigerte DNA-Synthese beobachtet werden. Es zeigte sich weiterhin, daß die Zellzyklusinhibitoren p21CIP1 und p27KIP1 eine besondere Rolle bei der Aufrechterhaltung des Zellzyklusarrestes von Kardiomyozyten spielen, denn in der Anwesenheit von IGF-I verschwanden in Kardiomyozyten, die E2F-1 exprimierten, diese Faktoren aus den Komplexen, die sie mit Zyklinen und zyklin-abhängigen Kinasen (cdks) bilden. Um zu verstehen, über welche Faktoren Apoptose in Kardiomyozyten induziert und über welche intrazellulären Signalwege sie vermittelt wird, wurden isolierte Kardiomyozyten mit freien Sauerstoffradikalen (ROS) exponiert, von denen bekannt ist, daß sie in bestimmten Zellen in einem bestimmten Dosisbereich Apoptose erzeugen können. Obwohl beides, H2O2 und O2-, zur Induktion von Apoptose in Kardiomyozyten führt, werden jeweils unterschiedliche intrazelluläre Signalwege aktiviert. So führt H2O2 zur Freisetzung von mitochondrialem Cytochrom C, was mit einer Translokation von Bax an die Mitochondrien und seiner Interaktion mit dem anti-apoptotischen Faktor Bcl-2 einhergeht. Dies führt zur Aktivierung der Caspase 3. O2- hingegen führt zur Aktivierung der Caspase 6 und Spaltung von Lamin A. / The aim of the study was to elucidate mechanisms controlling death and growth of cardiomyocytes. First, a model was developed suitable to identify regulatory gene sequences in vivo. Multiple variables controlling expression of reportergene constructs injected into the heart were investigated. The results showed that injection of reportergene constructs into the heart of dogs is an appropriate model to analyse regulation of gene expression in vivo in large mammals. Using this approach reportergene constructs harboring the promoter of the ANF (atrial natriuretic factor)-gene were injected in dog hearts which were subjected to pressure overload by aortic banding. Serial mutations of the promoter region revealed an AP-1 like sequence to be of importance for the induction of this gene in pressure overload hypertrophy. In order to identify factors responsible for the cell cycle arrest of cardiomyocytes the transcription factor E2F-1 was overexpressed in isolated cardiomyocytes using adenoviral gene transfer. In cardiomyoccytes the overexpression of E2F1- was followed by apoptosis. Apoptosis was suppressed in the presence of insulin-like growth factor-I (IGF-I) and the re-induction of DNA synthesis could be observed. Cyclin dependent inhibitors (cdi) p21CIP1 and p27KIP1 appear to play an important role in the maintenance of the cell cycle arrest in cardiomyocytes, since these factors dissappeared from cyclin complexes in the presence of IGF-I. In order to understand how apoptosis is induced in cardiomyocytes and which intracellular signalling cascades may be involved, isolated cardiomyocytes were exposed to reactive oxygen species (superoxide anion (O2-) or hydrogen peroxide (H2O2)). Both O2- and H2O2 induced apoptosis in cardiomyocytes dose-dependently. However, different intracellular signalling cascades were activated. Cytochrome C was released by H2O2, but not by O2-. Release of cytochrome c was followed by translocation of Bax from the cytosol to mitochondria where it was interacting with anti-apoptotic Bcl-2 leading to the subsequent activation of caspase-3. O2- lead to an activation of caspase-6 which was followed by the cleavage of lamin A.

Selbstreproduktion ist eines der Kennzeichen aller lebenden Organismen. Der Zellzyklus dient der Selbstreproduktion in einzelligen Organismen. In mehrzelligen Organismen ist der Zellzyklus darüber hinaus für andere lebenswichtige Prozesse, einschließlich Immunreaktionen, unerlässlich. In dieser Arbeit wird eine Methode entwickelt mit der die Dauer der Zellzyklus Phasen bestimmt werden kann. Kenntnis über die Zellzyklusphasendauer ermöglicht vorherzusagen, wie schnell eine Population von proliferierenden Zellen wachsen wird, oder wie viele neue Zellen pro Stunde in einem Gewebe geboren werden. Im Kapitel 1 dieser Arbeit wird ein Zellzyklusmodell aufgestellt und mit experimentellen Bromdesoxyuridin Daten verglichen. Die Analyse zeigt, dass das Modell gut die experimentelle Kinetik beschreibt, hebt jedoch auch hervor dass einige der Parameter nicht identifiziert werden können. Dieses Problem wird in Kapitel 2 bearbeitet, wo zwei Ansätze erforscht werden, um den Informationsgehalt der Experimente zu erhöhen. In einem ersten Ansatz wird die Theorie der Versuchsplanung angewendet, um optimale Versuchspläne zu bestimmen. In einem zweiten Ansatz wird das übliche Bromdesoxyuridin Protokoll durch ein zweites Nukleosid erweitert. Beide Methoden verbessern in silico erheblich die Genauigkeit und Präzision der Abschätzungen. Im dritten Kapitel wird die Methodik in der Analyse der Keimzentrumsreaktion angewendet. Ein erheblicher Zufluss von Zellen in die dunkle Zone von Keimzentren wird vorhergesagt, und die Ansicht einer extrem schellen Zellteilung im Keimzentrum erscheint in dem Modell als ein Artefakt der Zellmigration. / Self-reproduction is one of the distinguishing marks of living organisms. The cell cycle is the underlying process by which self-reproduction is accomplished in single-celled organisms. In multi-cellular organisms, the cell cycle is in addition indispensable for other vital processes, including immune reactions. In this thesis a method is developed that allows to estimate the time it takes for a dividing cells to complete the CC phases. Knowledge of the CC phase durations allows to predict, for example, how fast a population of proliferating cells will grow in size, or how many new cells are born per hour in a given tissue. In Chapter 1 of this thesis, a cell cycle model with delays and variability in the completion times of each phase is developed. Analytical solutions are derived to describe a common experimental technique used for cell cycle analysis, namely pulse labeling with bromodeoxyuridine (BrdU). Comparison with data shows that the model reproduces closely measured cell cycle kinetics, however also reveals that some of the parameter values cannot be identified. This problem is addressed in Chapter 2. In a first approach, the framework of D-optimal experimental designs is employed, in order to choose optimal sampling schemes. In a second approach, the prevailing protocol with a single nucleoside is modified by adding a second nucleoside analog pulse. Both methods are tested and the results suggest that experimental design can significantly improve parameter estimation. In Chapter 3, the model is applied to the germinal center reaction. A substantial influx of cells into the dark zone of germinal centers is predicted. Moreover the wide-held view of rapid proliferation in germinal centers, appears, under this model, as an artifact of cell migration.

Ziel der vorliegenden Arbeit war, die pathogenetische Bedeutung von dynamischen Zellzyklusveränderungen nach transienter fokaler Ischämie zu analysieren. Als in-vivo-Modell des humanen Schlaganfalls wurde eine 30-minütige Fadenokklusion der A. cerebri media (MCAo) in der Maus gewählt. In diesem Modell findet ein zeitlich verzögerter selektiver Zelluntergang von striatalen Projektionsneuronen bei relativer Aussparung von Gliazellen und Interneuronen statt. Somit entspricht dieses Modell dem humanpathologischen Konzept der "elektiven Parenchymnekrose". Als in-vitro-Modell wurde eine 90-minütige Sauerstoff-Glukose-Deprivation (OGD) der primären neuronalen Zellkultur eingesetzt. Die Experimente wurden parallel in vivo und in vitro durchgeführt. Wir konnten zeigen, daß alle adulten striatalen Neurone den zellzyklushemmenden CDK4-Inhibitor p16INK4a in vivo exprimierten. Die spezifische Herunterregulierung dieses Inhibitors nach 30-minütiger "milder" zerebraler Ischämie war ein frühzeitiger und zuverlässiger Indikator des verzögerten neuronalen Zellunterganges. Der Verlust von p27Kip1, eines weiteren CDK-Inhibitors, ging dem Zelluntergang in der primären neuronalen Zellkultur nach OGD voraus. Der Verlust von CDK-Inhibitoren wurde von der Hochregulierung des Cyclin D1 begleitet. Cyclin D1 wurde in den Zellkern transloziert. Dieser Translokation folgte eine Aktivierung der Cyclin-abhängigen Kinase 2 (CDK2). Wir postulieren, daß die Herunterregulierung der CDK-Inhibitoren das initiierende Ereignis für die Zellzyklusaktivierung darstellt. Die Mehrzahl der Neurone wurde noch vor dem Eintritt in die S-Phase apoptotisch, wenngleich eine kleine Fraktion noch vor ihrem Untergang DNA synthetisierte. Die Behandlung mit dem synthetischen CDK-Inhibitor Olomoucine schützte die primäre neuronale Zellkultur signifikant vor OGD. In der Zusammenschau weisen diese Ergebnisse darauf hin, daß die Zellzyklusaktivierung nach fokaler transienter Ischämie kausal mit der Schadensprogression verknüpft ist. Die pharmakologische Inhibition dieser Vorgänge könnte -unter Berücksichtigung der Nebenwirkungen der jeweiligen Pharmaka- einen therapeutischen Ansatz im Rahmen der akuten Schlaganfallbehandlung liefern. / Following mild ischemic insults many neurons undergo delayed neuronal death. Aberrant activation of the cell cycle machinery is thought to contribute to apoptosis in a variety of conditions including ischemia. We demonstrate that loss of endogenous cyclin dependent kinase (Cdk) inhibitor p16INK4a is an early and reliable indicator of delayed neuronal death in striatal neurons after mild cerebral ischemia in vivo. Loss of p27Kip1, another Cdk inhibitor, precedes cell death in neocortical neurons subjected to oxygen glucose deprivation in vitro. The loss of Cdk inhibitors is followed by upregulation of cyclin D1, activation of Cdk2, and subsequent cytoskeletal disintegration. The majority of neurons undergoes cell death prior to entering S-phase; albeit a small number (~1%) do progress to the S-phase prior to their death. Treatment with Cdk inhibitors significantly reduces cell death in vitro. These results show that alteration of cell cycle regulatory mechanisms is a prelude to delayed neuronal death in focal cerebral ischemia and that pharmacological interventions aimed at neuroprotection may be usefully directed at cell cycle regulatory mechanisms.

Der pRB/E2F-Signalweg ist ein zentraler Regulator der Proliferationskontrolle in Säugerzellen, der in fast allen auftretenden Tumoren dereguliert ist. Durch unterschiedliche Mutationen in Komponenten dieses Signalwegs kommt es letzten Endes zu einer erhöhten Aktivität der E2F-Transkriptionsfaktoren und somit zu einer verstärkten Transkription von E2F-Zielgenen in diesen Tumoren. Um die molekularen Mechanismen der Rolle von E2F3 in der Zellzykluskontrolle und der Tumorigenese besser zu verstehen, wurden in dieser Arbeit per GST-Pulldown mit anschließender Massenspektrometrie neue potenzielle Interaktions-partner von E2F3 identifiziert. Ein identifizierter Interaktionspartner war die SNF2-ähnliche Helikase HELLS. HELLS interagiert in vitro und in vivo spezifisch mit der Marked Box-Domäne von E2F3, aber nicht mit anderen untersuchten E2F-Transkriptionsfaktoren, wie durch GST-Interaktionsstudien und Ko-Immunpräzipi-tationsexperimente demonstriert werden konnte. Durch Chromatin-Immunpräzipitation konnte zusätzlich gezeigt werden, dass E2F3 für die Rekrutierung von HELLS an E2F-regulierte Promotoren wie z. B. CDC6 oder p107 verantwortlich ist. Die shRNA-vermittelte Depletion von HELLS führte zu einer stark verminderten Induktion von allen untersuchten E2F-Zielgenen nach Serumstimulation und einem verspäteten Eintritt in die S-Phase der HELLS-depletierten Zellen, was zeigt, dass HELLS essenziell für die Induktion von E2F-Zielgenen ist. Bei der immunhistochemischen Untersuchung der E2F3- und HELLS-Expression in humanen Prostatakarzinomen zeigte sich, dass sowohl E2F3 als auch HELLS in späten aggressiven Stadien dieser Tumore sehr stark exprimiert sind, jedoch nur sehr schwach in den weniger aggressiven Tumoren. Diese Versuche zeigen, dass es sich bei HELLS um einen neuen Bestandteil des pRB/E2F-Signalwegs handelt, der eventuell in der Entstehung gewisser Tumorarten eine Rolle spielt und somit ein neues potenzielles Ziel für neuartige Krebstherapien darstellt. / The pRB/E2F pathway is a key regulator of proliferation in mammalian cells and is commonly mutated in human tumors. These mutations in the components of the pRB/E2F pathway lead to deregulated activity of the E2F transcription factors resulting in increased expression of E2F target genes. To further understand the molecular mechanisms of E2F3 in cell cycle control and its role in tumorigenesis new interaction partners for E2F3 were identified in the course of this thesis with the help of a GST-Pulldown approach coupled to mass spectrometric analysis. One of the identified interaction partners was the SNF2-like helicase HELLS. With the help of GST-interaction studies and Co-Immunoprecipitation assays it could be demonstrated that HELLS interacts specifically with E2F3 via its Marked Box domain but does not bind to the other investigated E2F transcription factors. HELLS could be detected at E2F target genes like p107 and CDC6 in vivo with the help of Chromatin-Immunoprecipitation assays. Furthermore, the forced recruitment of E2F3 to E2F target genes led to an enhanced binding of HELLS to these promotors suggesting that HELLS is recruited to E2F target genes via protein-protein interaction with E2F3. The shRNA-mediated depletion of HELLS led to a strongly reduced induction of E2F target genes and a delay in S-phase entry, showing that HELLS is essential for the induction of E2F target genes. During the immunohistochemical analysis of human prostate cancer specimens it became evident that both E2F3 and HELLS are strongly expressed in the more aggressive late stages but only weakly expressed in the early stages of this tumor type. These findings demonstrate that HELLS is a new component of the E2F/pRB pathway which might play a role in the development of certain tumors and might represent a new target for novel cancer therapies.

No description available.

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The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4′-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4′,7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 μmol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (ΔΨm) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Eukaryotic cells have to divide constantly in order to promote the growth of certain organs, to replace dying or damaged cells, or to set up an entire organism. These essential processes are called mitosis in the case of somatic cell division. Mitotic cell division is the process during which chromosomes, centrosomes, and microtubules (MTs) are involved to set up a bipolar structure called the “mitotic spindle”. This bipolar spindle is formed by MTs, which are presumably mainly organized from the centrosomes. However, more data are being published that suggest MTs nucleation can occur from other MTs or even a chromosome surface. These biopolymers are built from α/β-tubulin heterodimers and can dynamically grow and shrink to exert forces necessary for chromosome segregation. Previous studies of spindles during mitosis have allowed the identification of different MT classes based on their plus-ends interaction with different cellular target sites. One of the MT classes is the kinetochore microtubules (KMTs), which physically connect chromosomes and centrosomes (i.e. spindle pole) via a specialized protein structure termed the “kinetochore”. This kinetochore-to-spindle pole connection has been studied in many organisms. In budding yeast, this connection is established by only a single KMT. In contrast, multiple KMTs bind to each mammalian kinetochore and form an MT bundle also called “k-fiber”. The ultrastructural architecture of the mammalian k-fiber connection is not well documented. Currently, different models concerning the nature of the kinetochore-to-spindle pole connection via k-fibers are discussed in the literature, i.e. a direct, semi-direct or indirect connection. The widely accepted ‘direct’ model proposes that all k-fibers of the mammalian spindle are formed through tight bundles of up to 20 KMTs, with all MT minus ends associated with the centrosome. However, it is necessary to understand the k-fibers structure in order to interpret its role during chromosome segregation. Here the architecture of the k-fiber was studied in human HeLa, U2OS and RPE-1 cell lines, as these different types of cells have been widely used in studies of mitosis. This thesis aimed to systematically investigate the characteristics of mammalian k-fibers and their attachment to the kinetochore within mammalian metaphase spindles. For that, the ultrastructure of mitotic spindles and k-fibers were analyzed using serial-section electron tomography primarily in HeLa cells. Furthermore, the spindle ultrastructure was compared by electron tomography to metaphase spindles in both U2OS and RPE-1 cells. Electron tomographic analysis of the mitotic spindle in HeLa cells revealed that the kinetochore-to-spindle pole connection is formed by k-fibers consisting of ~9 KMTs. Moreover, the data revealed that not all KMTs in k-fibers are directly associated with one of the spindle poles. Instead, KMT ends were located along the length of k-fibers indicating strongly for a semi-direct connection between the kinetochores and the spindle poles. Unexpectedly, by correlating the k-fiber ultrastructure with its position in the mitotic spindle, it can be demonstrated that the k-fiber structure varied depending on the position on the metaphase plate. It can also be shown that k-fibers located in the center of the metaphase plate had a tendency to form straighter and more bundled k-fibers. In contrast, k-fibers associated with the periphery of the metaphase plate had a more loose and disorganized structure resembling a fusiform shape. Furthermore, additional analysis of U2OS and RPE-1 cells indicated ultrastructural differences between the different cell lines. Mainly, differences between HeLa and RPE-1 cells were observed. K-fibers observed in RPE-1 cells showed a lower curvature and overall a more bundled ultrastructure compared to HeLa or U2OS cells. However, due to the small sample size for U2OS and RPE-1 cells, the results have to be confirmed in future experiments to conclude that there are indeed functional and structural differences in the k-fiber organization in different mammalian cell lines. Taken together, this work presents the first detailed quantitative ultrastructural analysis of KMTs in whole spindles in three different human cell lines. The data revealed that the currently favored direct model of k-fiber ultrastructure is oversimplified and needs to be corrected in terms of the k-fibers interaction with the spindle pole and the surrounding MT network within the mitotic spindle. The data here will serve as a structural basis for further analyses of mutant situations and contribute to our understanding of the overall organization and function of MTs in mitotic spindles.:Summary 6 Zusammenfassung 8 List of figures 10 List of tables 13 List of abbreviations and symbols 14 1 Introduction 19 1.1 The morphology of the mitotic spindle 21 1.1.1 Centrosomes 22 1.1.2 Microtubules 23 1.2 Kinetochores, KMTs and k-fibers 28 1.2.1 A brief history of k-fiber formation in mammalian cells 30 1.2.2 Models of the k-fiber ultrastructure in mammalian cells 32 2 Aims of this thesis 35 3 Materials and methods 36 3.1 Materials 37 3.1.1 Mammalian cell lines 37 3.1.2 Chemicals 38 3.1.3 Instrumentation and materials 40 3.1.4 Solutions and buffers 44 3.1.5 Software 46 3.2 Methods 47 3.2.1 Handling of cell cultures 47 3.2.2 Custom-designed incubation chambers 49 3.2.3 Specimen preparation for electron microscopy 51 3.2.4 Quality assessment of samples, acquisition of the tomographic data, and the 3D reconstruction 59 3.2.5 Ultrastructural analysis of MTs in mitotic spindles 62 3.2.6 Ultrastructural analysis of the k-fiber organization 70 4 Results 76 4.1 Initial characterization of mammalian mitotic spindles 77 4.2 Ultrastructure of KMTs 84 4.3 Curvature and tortuosity of KMTs 91 4.4 Ultrastructure of k-fibers 98 4.5 Effect of metaphase position on the k-fiber ultrastructure 102 5 Discussion 110 5.1. Comparison of data sets from different cell lines 111 5.2. Establishing a data analysis pipeline for the analysis of KMTs 113 5.3 Ultrastructural characterization of KMTs and k-fibers in HeLa cells 114 5.3.1 K-fibers have an unexpectedly low number of KMTs 115 5.3.2 Semi-direct kinetochores-to-spindle pole connection 117 5.3.3 Shape of k-fibers 121 5.4 Positional effect on the k-fiber shape 124 5.5 Comparison of k-fiber ultrastructure in different mammalian cells 127 5.6 Outlook 130 References 133 Appendix 1 149 Appendix 2 150 Appendix 3 151 Appendix 4 152 Acknowledgments 153

Cell cycle (CC) and TP53 regulatory networks are frequently deregulated in cancer. While numerous genome-wide studies of TP53 and CC-regulated genes have been performed, significant variation between studies has made it difficult to assess regulation of any given gene of interest. To overcome the limitation of individual studies, we developed a meta-analysis approach to identify high confidence target genes that reflect their frequency of identification in independent datasets. Gene regulatory networks were generated by comparing differential expression of TP53 and CC-regulated genes with chromatin immunoprecipitation studies for TP53, RB1, E2F, DREAM, B-MYB, FOXM1 and MuvB. RNA-seq data from p21-null cells revealed that gene downregulation by TP53 generally requires p21 (CDKN1A). Genes downregulated by TP53 were also identified as CC genes bound by the DREAM complex. The transcription factors RB, E2F1 and E2F7 bind to a subset of DREAM target genes that function in G1/S of the CC while B-MYB, FOXM1 and MuvB control G2/M gene expression. Our approach yields high confidence ranked target gene maps for TP53, DREAM, MMB-FOXM1 and RB-E2F and enables prediction and distinction of CC regulation. A web-based atlas at www.targetgenereg.org enables assessing the regulation of any human gene of interest.