The homologous recombination (HR) pathway contributes to genome integrity by mediating double strand break (DSB) repair using a homologous DNA sequence as a template. In mammals Rad51 and Brca2 are molecules central to this process. Little is known about HR repair in Dictyostelium. However, research previously conducted on DSB repair using this organism has shown that DSB repair pathways are highly conserved when compared to humans. This encouraged study of HR in this organism. In this study, through a bioinformatics search I have identified putative orthologues of most human HR proteins and most interestingly of BRCA2, which cannot be found in other lower eukaryotes used as models for DSB repair, such as the budding yeast S.cerevisiae. Brcp, the Dictyostelium BRCA2 ortholog, shows similar domain structure when compared to BRCA2-related proteins identified in other organisms. To verify the implication of HR proteins in DSB repair, I developed a method to monitor recruitment of DNA repair proteins on chromatin upon DSB induction. Findings of this study suggest that both Brcp and Rad51 get recruited to chromatin upon DSB induction and are therefore implicated in DSB repair in Dictyostelium. To further study Brcp function and based on findings suggesting that disruption of brcp might be lethal, I developed a novel system for specific and conditional depletion of endogenous Dictyostelium proteins. Utilizing this system, I conducted phenotypic studies in a strain depleted of Brcp to examine its role in DNA repair. Overall this study shows that the HR pathway in Dictyostelium shows great similarity to vertebrates, making Dictyostelium an appealing model for the study of DSB repair and specifically HR.

Platinum-based chemotherapeutics are used for the treatment of a wide range of cancers and a number of attempts have been made toward developing compounds with better cellular stability and similar or enhanced cytotoxicity as compared to their predecessors. The first part of the work reported here focuses on the cellular effects of the metabolically stable dinuclear platinum compound, BBR3610-DACH. Comet assay showed this compound to form interstrand crosslinks, a highly toxic DNA lesion in HCT116 cells, at equimolar concentrations to its parental compound, BBR3610. Cell cycle studies showed that BBR3610-DACH causes G1/S and G2/M cell cycle arrest with S phase depletion, which was p21 dependent and partially p53 dependent in contrast to BBR3610 which showed initial S phase accumulation followed by a classical G2/M arrest. BBR3610-DACH-induced G1/S and G2/M cell cycle arrest interestingly was found to be independent of the DNA damage response mediated via the activation of ATM and ATR kinases. Also, the cell cycle arrest culminated in apoptosis, although apparently through a non-canonical pathway. The second project explores the cellular effects of trans-4-NBD which is a fluorescent derivative of transplatin. Like cisplatin, trans-4-NBD induced interstrand crosslinks in HCT116 cells as detected by the comet assay. Treatment with trans-4-NBD showed a G2/M arrest in HCT116 cells and a transient S phase accumulation in A2780 cells, with a marked increase in p53 and p21 protein levels. A robust apoptotic response is also seen via caspase activation and PARP cleavage in both the cell lines. Finally, the focus is shifted toward the nucleolar targeting platinum complex, TriplatinNC. Confocal studies in TriplatinNC-treated HCT116 and A2780 cells showed disruption of rRNA transcription as an early event followed by a robust G1 cell cycle arrest. Apoptotic induction was observed with the onset of cellular morphological changes and apparent caspase activation which was independent of the p53 status of the cells. Overall, these studies explore novel platinum based compounds that show promising anti-cancer activities by affecting various facets of cellular signaling.

Hypoxia (low physiological O₂ levels) is a characteristic of solid tumours. It not only alters the chemical microenvironment of a tumour but initiates a number of mechanisms which enable cells to cope and thrive under these conditions, resulting in therapy-resistant and aggressive tumours. The replication stress induced by severe hypoxia activates a DNA damage response which involves the kinases ATR and Chk1. Moreover, periods of hypoxia are often followed by reoxygenation, which induces DNA damage. Chk1 inhibitors have been used to potentiate chemotherapy with cytotoxic agents and have recently been proposed as single agents in tumours with high levels of replication stress. However, inhibition of Chk1 also affects normal DNA replication, cell cycle progression and DNA repair. The herein presented study chose known inhibitors of Chk1 and, with methods of synthetic organic chemistry, modified them into agents to selectively target hypoxic cells. Three different Chk1 inhibitors were selected and bioreductive analogues synthesised which were evaluated in chemical, biochemical and cellular assays. We found a convenient route to access a precursor of the bioreductive 2-nitroimidazole group and established a three-step protocol for the testing of bioreductive drugs. This protocol allows us to determine whether a bioreductive drug undergoes reduction and prodrug activation. In addition, bioreductive Chk1 inhibitors were shown to induce DNA damage and cellular toxicity in a hypoxia-selective fashion. While reduction of the prodrugs occurred in all three cases, fragmentation was always the rate-limiting step. We propose that the use of bioreductive Chk1 inhibitors is a promising strategy to target the most therapy-resistant tumour fraction while sparing normal tissue.

Introduction Les lésions induites par les rayons UV peuvent causer des blocages dans la réplication de l'ADN. Ces dommages sont éliminés par le processus moléculaire très conservé de réparation par excision de nucléotides (NER). Nous avons précédemment démontré que la protéine ATR, une kinase majeure impliquée dans le stress réplicatif, est requise pour une NER efficace, et ce exclusivement durant la phase S. Des résultats subséquents ont suggéré que ce prérequis n’était pas lié à la réponse induite par ATR, mais plutôt d’une conséquence globale causée par la présence de stress réplicatif. En ce sens, nous mettons l’emphase qu’après irradiation UV, le complexe RPA joue un rôle crucial dans l'activation des mécanismes de NER ainsi que dans le redémarrage des fourches de réplication bloquées. Hypothèses: En général, les mutations qui confèrent une augmentation du stress réplicatif engendrent une séquestration excessive du facteur RPA aux fourches de réplication bloquées ce qui réduit son accessibilité pour le NER. Méthodes et résultats: Le modèle de la levure a été choisi pour vérifier cette hypothèse. Nous avons développé un essai de NER spécifique à chacune des phases du cycle cellulaire pour démontrer que les cellules déficientes en Mec1, l’homologue d’ATR, sont défectives dans la réparation par excision de nucléotides spécifiquement en phase S. De plus, plusieurs autres mutants de levure, caractérisés par un niveau de dommages spontanés élevé, ont aussi exhibé un défaut similaire. Ces mutants ont démontré une fréquence et une intensité de formation de foyers de RPA plus élevée. Finalement, une diminution partielle de RPA dans les levures a induit un défaut significatif dans le NER spécifiquement durant la phase S. Conclusion: Nos résultats supportent la notion que la séquestration de RPA aux fourches de réplication endommagées durant la phase S prévient son utilisation pour la réparation par excision de nucléotides ce qui inhibe fortement l'efficacité de réparation. Cette étude chez la levure facilite l’élucidation du phénomène analogue chez l’humain et, ultimement, comprend des implications majeures dans la compréhension du mécanisme de développement des cancers UV-dépendants. / Introduction Replication-blocking UV-induced DNA adducts are removed by the highly-conserved nucleotide excision repair (NER) pathway. We previously demonstrated that ATR kinase, a preeminent responder to replicative stress, is required for efficient NER exclusively during S phase. Subsequent data suggested that this requirement is not a manifestation of ATR signaling per se, but rather more broadly reflects some consequence of replication stress. In this respect we emphasize that after UV treatment, heterotrimeric RPA plays essential and independent roles in both NER and restart of blocked replication forks. Hypothesis: In general, mutations which confer increased replicative stress engender excessive sequestration of RPA at blocked replications forks which in turn reduces the availability of this factor for NER. Methods and Results: The powerful yeast model was chosen to address this hypothesis. We developed a cell-cycle-specific DNA repair assay to demonstrate that cells deleted for Mec1, the yeast ATR homolog, are defective in S phase-specific NER. Moreover several other yeast mutants, i.e., ones characterized by elevated levels of spontaneous DNA damage, exhibited a similar defect. These mutants also displayed increased frequency and intensity of RPA focus formation. Finally, degron-mediated depletion of RPA in wild-type cells resulted in significant inhibition of NER uniquely during S. Conclusion: Our data support the notion that RPA sequestration at blocked replication forks in yeast undergoing high levels of replication stress abrogates NER during S phase. This work in yeast facilitates elucidation of the analogous phenomenon in humans and, ultimately, harbours implications for undestanding the mechanism of UV-associated skin cancer development.

The myelodysplastic syndrome (MDS) is a group of hematopoietic clonal disorders resulting in the inefficient production of myeloid lineage blood cells, with the prevalence of patients older than 65 years. One of the possible treatment options for MDS is 5- azacytidine and 5-aza-2'-deoxycytidine therapy. These compounds have been shown to cause the induction of cell-cycle arrest, cell differentiation and/or apoptosis. The in vitro experiments with 5-aza-2'-deoxycytidine indicated that this compound causes the premature cellular senescence, a state of the irreversible cell-cycle arrest. We have asked, whether 5-azacytidine, as a molecule with similar structure, is capable of causing the same effect. This treatment strategy could be beneficial in case that the negative pro- inflammatory effect of senescent cells on their surroundings can be nullified. In this thesis we have shown that 5-azacytidine induces DNA damage response, which is described as a fundamental event for the onset of the cell senescence. We tested 5- azacytidine treated HeLa cells for several markers of the cell senescence - the increase of the β-galactosidase activity, the PML and PML nuclear bodies and the formation of persistent DNA damage signaling lesions - albeit all these markers were positive, it was the very low increase in...

The frequent cause of failure of prostate carcinoma radiotherapy and chemotherapy is the emergence of resistance and a progress into the essentially incurable metastatic form of disease. Although the mechanisms of the radioresistance and chemoresistance are still not well understood, recent studies indicate that transcription factor Snail, a key mediator of the epithelial-mesenchymal transition and subsequent metastasis formation, plays a critical role in the development of the chemoresistance and radioresistance in the tumor cells. As the activation of the optimal DNA damage response pathway is the determining factor for the cell survival after chemotherapy and radiotherapy, we hypothesized the role of Snail in the transcription regulation of these processes. In this study, we first analyzed the relationship between Snail and ATM kinase, as the ATM was recently reported to regulate stability of Snail by its phosphorylation. Although, we observed a modest effect of ATM inhibition on Snail levels after cancer cells exposure to ionizing radiation, we did not fully reproduced the recently published findings. Furthermore, we evaluated the role of Snail in transcription regulation of cyclin-dependent kinase inhibitor p21waf1/cip1 . Our data point towards the suppressive role of Snail in p21waf1/cip1...

Organisms such as mammals need tissue renewal as an important process for maintenance of their viability. Because proliferation is essential also for tumourigenesis, cells need tumour-suppressor mechanisms to protect organism against cancer. Cellular senescence, the permanent state of cell-cycle arrest, features one of these intrinsic barriers against tumourigenesis after DNA damage and understanding of this process may lead to finding of novel therapeutic targets and to optimization of chemotherapy for patients with cancer. In the first part of the PhD thesis, we investigated activation of JAK/STAT signalling pathway in drug-induced senescence. We used genotoxic drugs like aphidicolin, camptothecine, 5-bromo- 2'-doexyuridin, etoposide or thymidine to induce premature senescence in normal and cancer cells. All this chemicals were able to persistently activate JAK/STAT signalling in monitored cells. Activation of STATs was accompanied with up-regulation of expression of interferon-stimulated genes (ISGs), such as MX1, IRF1, IRF7 and PML. Since IRF1 and IRF7 can be directly involved in stimulation of the IFN genes, we show activated expression as well as secretion of IFNbeta and IFNgamma, but not IFNalpha in drug-induced senescent cells. Furthermore, an inhibition of JAK1 as a major kinase of STAT...

A proteína Cdc42 (Cell Division Cycle 42) é um membro da família das Rho GTPases, sinalizadores intracelulares conhecidos pelo seu papel na regulação do citoesqueleto. Essa proteína e capaz de ciclar entre um estado ativo (ligado à GTP) e um estado inativo (ligado à GDP) e essa ativação é modulada por diversas proteínas, conhecidas como GEFs (guanine nucleotide-exchange factors), GAPs (GTPase-activating proteins) e GDIs (guanine nucleotide-dissociation inhibitors). Trabalhos recentes têm demonstrado um papel de Cdc42 na apoptose e na senescência, respostas relacionadas e comumente desencadeadas por estresse genotóxico. Neste contexto este trabalho procurou identificar interações de Cdc42 com outras proteínas, que podem ou não estar envolvidas nos mecanismos de resposta ao dano do DNA. Para isso foram utilizadas as linhagens celulares HeLa e MRC-5 submetidas a tratamento com radiação ultravioleta tipo C, a fim de provocar danos no DNA. Foram realizados dois diferentes tratamentos em cada uma das linhagens com diferentes tempos de incubação pós radiação UV, visando a busca de proteínas envolvidas em uma resposta rápida ou tardia ao dano causado. Os lisados celulares desses tratamentos foram submetidos ao pull-down com proteínas recombinantes GST, GST-Cdc42WT (Selvagem) e GST-Cdc42V12 (Mutação constitutivamente ativa). As proteínas purificadas foram digeridas e submetidas à análise por espectrometria de massa e os dados obtidos foram utilizados para a construção de redes de interação proteica. Dentre as proteínas identificadas as que despertaram maior atenção foram: Proibitina-2 (PHB2) encontrada nas amostras incubadas por 48 horas pós irradiação e Cullina-4A (CUL4A) e P53, encontradas em amostra incubada por 5 minutos pós radiação. Essas proteínas possuem papéis em apoptose e reparo de DNA e foram observadas em posições muito próximas de Cdc42 nas redes de interação, fazendo delas interessantes alvos para futuras validações de interação proteica por análises experimentais distintas / The Cdc42 protein (Cell Division Cycle 42) is a member of the Rho family of GTPases, intracellular signalling molecules well known for their role in the cytoskeleton regulation. This protein cycles between an active state (GTP-bound) and an inactive state (GDP-bound) and this regulation is modulated by proteins known as GEFs, GAPs and GDIs. Recent studies demonstrated roles for Cdc42 in apoptosis and senescence, cellular responses commonly triggered by genotoxic stress. This work sought to identify Cdc42 interactions with other proteins that possibly involved in response to DNA damage mechanisms. To reach this aims we used HeLa and MRC-5 cell lines submitted to treatments with ultraviolet C radiation to induce DNA damage. Two experimental conditions were used in each cell line with different times and doses post UV irradiation in order to search for proteins involved in either rapid or delayed response to the installed DNA damage. Cell lysates obtained from these treatments were subjected to pull-down experiments using recombinant proteins GST, GST-Cdc42-WT (Wild type) and GST-Cdc42-V12 (constitutively active mutant). Purified proteins were digested by trypsin, analyzed by mass spectrometry and th obtained data were used for the construction of protein-protein interaction (PPI) networks. Among the identified proteins those that seem more relevant to the aims of this project were: Prohibitin-2 (PHB2), found in samples incubated 48 hours post irradiation; Cullin-4A (CUL4A) and P53, found in samples incubated 5 minutes after radiation. These proteins have roles in apoptosis and DNA repair and were observed in close proximity to Cdc42 in PPI networks, making them interesting targets for future validation by different experimental approaches

Bisher wurde davon ausgegangen, dass der IKK-komplex durch Regulation des Transkriptionsfaktors NF-kappaB die stressinduzierte Expression von Zielgenen steuert. Im Rahmen der hier vorgelegten Dissertation konnte jedoch gezeigt werden, dass der IKK-Komplex unabhängig von seiner Rolle in der NF-kappaB-Aktivierung die Stabilität einer Vielzahl von mRNAs kontrolliert. Mittels der Kombination von Ko-Immunopräzipitationsstudien und SILAC-MS konnte die induzierte Interaktion der regulatorischen Untereinheit des IKK-Komplexes IKKgamma mit dem Gerüstprotein EDC4 (Enhancer of Decapping 4) nachgewiesen werden. EDC4 ist eine essentielle Komponente sogenannter zytoplasmatischer „Processing Bodies“ (P-Bodies). Diese fungieren als Depots für die Speicherung von mRNAs, aber auch als Orte der mRNA-Degradation und der miRNA-vermittelten Repression spezifischer Zielgene. Die Interaktion von IKKgamma mit EDC4 konnte durch verschiedene Stimuli induziert werden. Dazu zählen DNA-Schäden durch Doppelstrangbrüche, aber auch die Aktivierung von Oberflächenrezeptoren durch TNFalpha und IL-1beta. EDC4 dient darüber hinaus als Substrat der Kinase IKKbeta. Mittels Massenspektrometrie und Kinaseassays konnten vier IKK-abhängige Phosphorylierungsstellen identifiziert werden. Die IKK-vermittelte Phosphorylierung von EDC4 ist essentiell für die Regulation von mRNAs und die damit verbundene Bildung der zytoplasmatischen P-Bodies. Diese Befunde konnten sowohl in stabilen induzierbaren Zelllinien, mittels transienter Transfektion und durch den Gebrauch von Kinaseinhibitoren in primären als auch in Krebszelllinien bestätigt werden. mRNA-Stabilitätsassays und eine RNA-Seq Analyse bestätigten die stressinduzierten Änderungen in den Halbwertszeiten spezifischer Transkripte und offenbarten einen gemeinsamen Regulationsmechanismus des IKK-Komplexes mit EDC4. / The IKK complex is deemed to regulate gene expression through the activation of the transcription factor NF-kappaB. Here I describe an NF-kappaB-independent function of the IKK complex in regulating mRNA stability across different cell types and stimuli. A SILAC-MS screen for interaction partners of the regulatory subunit IKKgamma revealed an inducible interaction with Enhancer of mRNA Decapping 4 (EDC4). EDC4 is an essential component of cytoplasmic processing bodies (P-bodies). P-bodies function as sites of mRNA storage, degradation and miRNA-mediated silencing. Interaction between IKKgamma and EDC4 can be induced by various stimuli, including DNA damage, TNFalpha and IL-1beta. EDC4 was identified as a novel IKK substrate and four IKKbeta phosphorylation sites were determined by mass spectrometry and in kinase assays. Stable inducible cell lines, transient transfection and kinase inhibitors were used in different human cancer and in primary cell lines and demonstrated that phosphorylation of EDC4 by IKK is essential for formation of P-Bodies in response to numerous stimuli. mRNA stability assays confirmed stress-induced changes in the half-life of target mRNAs and revealed common regulation of mRNA stability by IKK and EDC4. The transcriptome-wide reach of this joint regulation was assessed via RNA-Seq analysis.

Les G-quadruplexes (G4) sont des structures non canoniques des acides nucléiques qui peuvent être formés dans des régions d’ADN ou d’ARN riches en guanines. Les ligands G4 (LG4), sont des molécules capables d’interagir et de stabiliser les structures G4, qui présentent de nombreuses propriétés anti-cancéreuses. Nous avons travaillé avec le LG4 20A, appartenant à la famille des triarylpyridines, qui stabilise efficacement les structures G4 in vitro. Les objectifs de ce travail ont été de déterminer les mécanismes moléculaires et cellulaires responsables des effets anti-prolifératifs du 20A dans des cellules cancéreuses. Dans cette étude, nous avons montré que le 20A induit un arrêt de la croissance cellulaire de cellules en culture et dans un modèle de xénogreffe tumorale, grâce à l’induction de la sénescence et de la mort cellulaire par apoptose. Ces réponses sont associées à l’activation de la voie des réponses aux dommages à l’ADN (DDR) via la kinase ATM, qui favorise l’autophagie (un processus catabolique) et la sénescence, tout en protégeant les cellules de l’apoptose. De plus, nous avons observé que le 20A induit un échec de la cytokinèse, conduisant à l’accumulation de cellules binucléées qui présentent une résistance à la mort cellulaire. De façon inattendue, nous avons trouvé que le 20A s’accumule dans les lysosomes, induisant une augmentation de la taille de ces derniers. La combinaison du 20A et de l’agent lysomotropique chloroquine, potentialise de façon importante la perméabilisation de la membrane lysosomale (LMP) et la mort cellulaire. En particulier, cette combinaison sensibilise de façon notable ces cellules binucléées à la mort cellulaire. L’ensemble de ces résultats révèle une relation entre les processus de mort cellulaire et de sénescence induits par le LG4 20A, et les voies de DDR et lysosomales. Ces régulations devraient être prises en considération lors de l’utilisation d’agents antiprolifératifs susceptibles d’interférer avec les fonctions lysosomales. / G-quadruplexes (G4) are unusual nucleic acid structures that can be formed by guanine-rich DNA and RNA. Through their ability to stabilize G4 structures, G4 ligands (G4L) have been described to display potent anticancer properties. Here, we studied the G4L 20A belonging to the triarylpyridine family of compounds that have the ability to efficiently bind to and stabilize G4 structures in vitro. The objectives of this work were to determine the molecular and cellular mechanisms responsible for the anti-proliferative effects of 20A in cancer cells. In this study, we showed that 20A causes cancer cell growth arrest in cell culture and a mice tumour xenograft model, through induction of senescence and apoptotic cell death. These cellular responses are associated with the induction of the DNA damage response pathway (DDR), in particular ATM activation, which promotes the induction of both autophagy (a lysosomal catabolic pathway) and senescence, while protecting cells against apoptosis. Furthermore, we found that 20A induces failure of cytokinesis which results in the accumulation of binucleated cells that display marked resistance to 20A-induced cell death. Unexpectedly, we found that 20A accumulates in the lysosomal compartment and causes lysosome enlargement. The combination of a lysosomotropic agent, chloroquine, and 20A promotes a significant induction of lysosomal membrane permeabilization (LMP) and a robust cell death. In particular, this combination significantly sensitizes binucleated cells to cell death. Altogether, our results uncover the relationship of the DDR and lysosomal pathways to cell death and senescence induced by the G4L 20A. Such regulation should also be taken into account when using antiproliferative drugs susceptible to interfere with the lysosomal functions.