Rozprawy doktorskie na temat „Genotoxic stress response”

The enteric nervous system (ENS) constitutes a gut-intrinsic network of interconnected ganglia which control multiple aspects of gastrointestinal activity, including motility, secretion and blood flow. Most enteric neurons and glia are derived from a relatively small population of neural crest (NC) cells which originate primarily from the vagal NC and migrate ventromedially to invade the foregut mesenchyme. Within the gut microenvironment, ENS progenitors receive signals that allow them to survive, proliferate, differentiate and migrate extensively, giving rise to a uniformly distributed population of enteric neurons and glia. So far, most developmental studies have explored the behaviour of ENS progenitors within the gut wall, but little is known about the properties of NC cells prior to foregut invasion. To explore the spatiotemporal dynamics of ENS development, we conditionally inactivated Geminin (Gem), a mouse gene with fundamental roles in genome integrity and cell fate decisions. Gem deletion from pre-enteric NC cells results in extensive DNA damage followed by P53-mediated apoptotic cell death and intestinal aganglionosis. In contrast, ablation of Gem from enteric NC cells has minimal effects on ENS development. To determine whether ENS lineages display a stage-specific sensitivity to generic genotoxic stress, we monitored the in vivo response of NC cells to γ-irradiation at different developmental stages. While both pre-enteric and enteric NC cells where characterised by robust DNA damage response, pre-enteric NC cells displayed a far greater apoptotic response relative to their enteric counterparts. These experiments reveal a previously unknown spatiotemporal sensitivity of NC cell lineages to genotoxic stress and provide an experimental paradigm for understanding the mechanisms by which DNA damage from genetic or environmental insults leads to congenital ENS deficits. Our studies allow us to draw general conclusions as to how DNA damage and fundamental developmental processes are integrated in vertebrate embryos.

Introduction: miR-16 and miR-26a have been identified as key effectors of the p53 pathway in response to genotoxic stress. This work is focused on preliminary elucidation of regulatory mechanisms by which p53 controls expression of miR-16 and miR-26a and characterisation of their gene targets involved in the p53 network. Methods: Microarray expression analysis of miR-16 and miR-26a was followed by Q-PCR to confirm these miRNAs dependence on p53. We analysed the transcriptional regulation of these miRNAs by p53 via luciferase assay and ChIP assay. We investigated these miRNAs contribution to p53-dependent response to genotoxic stress. To validate miR-16 and miR-26a targets (Cyclin E, CHK1, and WEE1) we employed Q-PCR, western blotting and luciferase assay. We also analysed the transcriptional regulation of Cyclin E by SET9 via luciferase assay. Results: High miR-16 and miR-26a expression are associated with increased cancer survival. p53-dependent and -independent regulatory mechanisms exist for miR-16 and miR-26a, and p53 controls expression of miRNAs on several levels. p53 recruits Drosha complex to miR-16 and miR-26a to facilitate the processing of these miRNAs. miR-26a cooperates with p53 to induce apoptosis and miR-16 enhances p53-mediated cell cycle arrest. miR-16 and miR-26a regulates CHK1 and WEE1, in the presence or absence of p53. miR-16 also reduces Cyclin E levels, in the presence and absence of p53. SET9 controls expression of Cyclin E on a transcriptional level. Conclusions: Our results showed that in response to DNA damage, miR-16 and miR-26a expression levels are controlled by p53-dependent and p53-independent mechanisms, potentially involving other stress-response transcription factors such as E2F1. Our data also confirms that miR-16 and miR-26a directly target Cyclin E, CHK1, and WEE1 for down-regulation. Additonally, SET9 directly controls Cyclin E expression. Reduced CHK1 and WEE1 levels leads to decreased G2/M arrest, and reduced Cyclin E levels results in increased G1/S arrest. As a consequence, apoptosis occurs.

La réplication fidèle de l’ADN au cours du cycle cellulaire est essentielle au maintien de l’intégrité du génome à travers les générations. Toutefois, de nombreux éléments peuvent perturber et compromettre la réplication et donc cette intégrité. La mitomycine C (MMC) est une molécule génotoxique utilisée en chimiothérapie. Elle forme des liaisons covalentes entre les deux brins d’ADN, ce qui est un obstacle à la bonne réplication de l’ADN. La rencontre de la fourche de réplication avec une liaison covalente entre les deux brins d’ADN va aboutir à une cassure double brin. Escherichia coli (E.coli) est un modèle d’étude très étendu car facile d’utilisation, permettant d’aborder des notions complexes. E coli possède divers mécanismes pour réparer ces lésions dont le régulon SOS. Le régulon SOS est un ensemble de gènes sous contrôle d’un promoteur réprimé par la protéine LexA. En réponse à des dommages à l’ADN, LexA est dégradé et les gènes du régulon sont activés.En utilisant une technique de biologie moléculaire qui permet de quantifier l’interaction entre deux chromatides sœurs restées cohésives derrière la fourche de réplication (étape appelée cohésion des chromatides sœurs), nous avons montré qu’en réponse à des cassures double brin générées par la MMC, la cohésion entre les chromatides sœurs nouvellement répliquées est maintenue. Ce phénomène est dépendant de RecN, une protéine induite de façon précoce dans le régulon SOS. RecN est une protéine de type SMC (structural maintenance of chromosomes), un groupe de protéines impliqué dans la dynamique et la structure du chromosome. En parallèle, des techniques de microscopie confocale et de marquage du chromosome par des protéines fluorescentes ont permis de montrer que la protéine RecN est impliquée dans une condensation globale du nucléoide suite à un traitement par la MMC. Cette condensation du nucléoide s’accompagne d’un rapprochement des chromatides sœurs ségrégées. Ces deux phénomènes, médiés par RecN pourraient permettre une stabilisation globale des nucléoides et favoriser l’appariement des chromatides sœurs pour permettre la recombinaison homologue.De façon intéressante, l’inhibition de Topoisomérases de type II (Topoisomerase IV et Gyrase) permettent de restaurer le phénotype d’un mutant recN en viabilité et en cohésion des chromatides sœurs. Les Topoisomérases sont des protéines qui prennent en charge les liens topologiques générés par la réplication et la transcription). Les liens topologiques non éliminés par les Topoisomerases permettraient de garder les chromatides sœurs cohésives et favoriser la réparation, même en l’absence de RecN.De plus, une expérience de RNA seq (séquençage de tout le transcriptome de la bactérie) a révélé que dans un mutant recN, le régulon SOS est moins induit que dans les cellules sauvages. Ceci va de pair avec une déstructuration des foci de réparation RecA. Il est possible que le rapprochement des chromatides sœurs médié par RecN permettrait de stabiliser le filament RecA et donc l’induction du SOS.L’ensemble de ces résultats suggère que RecN, une protéine de type SMC, permet de maintenir la cohésion entre les chromatides sœurs nouvellement répliquées, favorisant la réparation de cassures double brins par recombinaison homologue
Maintaining genome integrity through replication is an essential process for the cell cycle. However, many factors can compromise this replication and thus the genome integrity. Mitomycin C is a genotoxic agent that creates a covalent link between the two DNA strands. When the replication fork encounters the DNA crosslink, it breaks and creates a DNA double strand break (DSB). Escherichia coli (E.coli) is a widely used model for studying complex DNA mechanisms. When facing a DNA DSB, E. coli activates the SOS response pathway. The SOS response comprises over 50 genes that are under the control of a LexA-repressed promoter. Upon a DSB induction, RecA, a central protein of the SOS response will trigger the degradation of LexA and all the SOS genes will be expressed.We have developed a novel molecular biology tool that reveals contacts between sister chromatids that are cohesive. It has been shown in the lab (Lesterlin et al. 2012) that during a regular cell cycle, the two newly replicated sister chromatids stay in close contact for 10 to 20 min before segregating to separate cell halves thanks to the action of Topoisomerase IV. This step is called sister chromatid cohesion. We have used this molecular biology tool to study sister chromatid cohesion upon a genotoxic stress induced by mitomycin C (MMC). We have shown that sister chromatid cohesion is maintained and prolonged when the cell is facing a DSB. Moreover, this sister chromatid cohesion is dependent on RecN, an SOS induced structural maintenance of chromosome-like (SMC-like) protein. In the absence of RecN, the proximity between both sister chromatids is lost and this has a deleterious effect on cell viability. By tagging the chromosome with fluorescent proteins, we have revealed that RecN can also mediated a progressive regression of two previously segregated sister chromatids and this is coordinated with a whole nucleoid compaction. Further studies showed that this genome compaction is orderly and is not the result of a random compaction in response to DNA damage.Interestingly, inhibiting TopoIV in a recN mutant fully restores viability and sister chromatid cohesion suggesting that RecN’s action is mainly structural. Preserving cohesion through precatenanes is sufficient to favor repair and cell viability even in the absence of RecN.An RNA-seq experiment in a WT strain and a recN mutant revealed that the whole SOS response is downregulated in a recN mutant. This suggests that RecN may have an effect on the induction of the SOS response and thus RecA filament formation. This is in good agreement with the change in RecA-mcherry foci formation we observed. In the WT strain, the RecA-mcherry foci are defined as described in previous work. However, in the recN, the RecA-mcherry foci seemed to form bundle like structures. These RecA bundles were previsously described by Lesterlin et al. in the particular case of a DSB occurring on a chromatid that has already been segregated from its homolog. This could mean that in the absence of recN, the sister chromatids segregate and RecA forms bundle like structures in order to perform a search for the intact homologous sister chromatid.Altogether, these results reveal that RecN is an essential protein for sister chromatid cohesion upon a genotoxic stress. RecN favors sister chromatid cohesion by preventing their segregation. Through a whole nucleoid rearrangement, RecN mediates sister chromatid regression, favoring DNA repair and cell viability

Interferon regulatory factor 3 (IRF3) plays an important role in activating the innate immune response in a variety of conditions, including viral infection. As well as regulating the immune response to viruses, IRF3 is involved in regulating cellular functions including apoptosis. Apoptosis and the inflammatory response to viral infection are very different; therefore, it is obvious that IRF3 plays dramatically different roles in the cell depending on the conditions. We previously identified a non-activating phosphorylation of IRF3 in response to adenovirus (Ad) in which Serine-173 is phosphorylated. In addition to Ad infection, IRF3- S173 is phosphorylated in response to genotoxic stresses including ultraviolet (UV) irradiation and etoposide. In this study, I show that this phosphorylation event is involved in a variety of processes including protein stability, cell survival and IRF3 regulation. Thus, phosphorylation of IRF3-S173 is a novel and important event in a complex regulatory pathway of an integral protein.

Esophageal cancer is the third most common digestive tract malignancy. Along with surgery, genotoxic drugs (e.g. cisplatin) and radiotherapy are the mainstays of treatment for this disease. Environmental factors and environmental stress-induced responses contribute to esophageal tumorigenesis and chemoresistance. Studying key molecules in stress-induced signal pathway can help unravel the underlying mechanisms and discover rational therapeutic targets. Cyclin D1 is DNA damage response protein. Genotoxic stress induces rapid cyclin D1 degradation and the molecules mediating this response are cell-type dependent. The first part of this study investigated the changes of cyclin D1 expression in response to genotoxic stress in immortalized esophageal epithelial cells, which are experimental models commonly used to study the early events of cancer development. The results showed that cyclin D1 underwent rapid proteasomal degradation before p53-induced p21 accumulation, which substantiates that cyclin D1 plays a role in eliciting cell cycle arrest very early in the DNA damage response. FBXO31 and FBX4, two F-box proteins previously reported to mediate cyclin D1 degradation, were found to be accumulated and unchanged, respectively, after ionizing irradiation in immortalized esophageal epithelial cells and esophageal squamous cell carcinoma (ESCC) cell lines. Yet, knockdown of FBXO31 did not rescue rapid cyclin D1 degradation upon UV or ionizing irradiation. This led to the hypothesis that accumulation of FBXO31 may have novel functions beyond mediating cyclin D1 degradation in cells responding to genotoxic stress. The second part of this study explored the function of FBXO31 in genotoxic stress response. The accumulation of FBXO31 in cancer cells after exposure to various genotoxic stresses was found to coincide with p38 deactivation, giving the clue that FBXO31 may negatively regulate this important pathway. Further studies revealed that knockdown of FBXO31 resulted in sustained activation of stress-activated MAPKs (SAPKs) p38 and JNK, as well as increase in UV-induced cell apoptosis, whereas overexpression of FBXO31 had opposite effects. The inhibitory role of FBXO31 on SAPK activation and apoptosis was confirmed by shRNA rescue experiments. Consistent with the observed anti-apoptotic effect, soft agar, colony formation and in vivo xenograft experiments showed that FBXO31 had oncogenic function in ESCC. Moreover, in vitro and in vivo results showed that knockdown of FBXO31 could sensitize ESCC cells to cisplatin treatment. The mechanism underlying the inhibition of SAPKs by FBXO31 was investigated in the third part of this study. Co-immunoprecipitation results showed that FBXO31 could interact with MKK6 (a p38 activator), but not p38, JNK1, or other MAP2Ks. FBXO31 was found to be co-localized with MKK6 in the cytoplasm. Mapping of interaction domains of FBXO31 revealed that aa 115-240 and aa 351-475 were responsible for binding to MKK6. Further study found that binding of FBXO31 to MKK6 could facilitate the K48-linked polyubiquitination and degradation of MKK6. Taken together, the results of this study showed that FBXO31 accumulation upon genotoxic stress can promote the degradation of MKK6 via K48-linked ubiquitination, thereby inhibiting SAPK activation and protecting cancer cells from genotoxic stress-induced apoptosis. FBXO31 may be a potentially useful therapeutic target to overcome chemoresistance in cancer therapy.
published_or_final_version
Anatomy
Doctoral
Doctor of Philosophy

Cancer cells can exist in a reversible state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely due to dormant cells escape frontline treatment. Dormant AML cells have been identified in the bone marrow (BM) endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. As the first step in this project, we developed an in vitro model of AML cell dormancy by exploiting these features. Following a preliminary investigation of four AML cell lines, the CD34+CD38- line TF1-a was selected for in-depth investigation. TF1-a showed significant inhibition of proliferation, with features of dormancy and stemness, in response to 72 hours of TGFB1+mTOR inhibitor treatment (mTOR pathway inhibition mimics major effects of nutrient scarcity) without affecting cell viability or inducing differentiation of these cells. Secondly, whole human genome gene expression profiling using Affymetrix microarray strips (HuGene2.1ST) was conducted to molecularly characterise the dormancy-induced TF1-a cells. As a result, we identified 240 genes which were significantly up-regulated by at least twofold, including genes involved in adhesion, stemness, chemoresistance, tumor suppression and genes involved in canonical cell cycle regulation. The most upregulated gene was osteopontin (17.1fold). Immunocytochemistry of BM biopsies from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular BM. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABCC3 and CDKN2B) as well as little-known ones (e.g. ITGB3, BTG2 and PTPRU), are potential therapeutic targets in AML. AML cells which are identified in the bone marrow (BM) endosteal region are likely to survive chemotherapy for several reasons including the low concentration of the drug delivered to this poorly perfused area of the BM. We hypothesised that these cells might induce dormancy features that help them escaping chemotherapy. Moreover, little is known regarding the molecular changes in those AML cells surviving genotoxic stress. The third aim of this study was to investigate the AML cells surviving genotoxic stress. Therefore, we developed and characterised an in vitro model of prolonged sub-lethal genotoxicity in AML cells by utilising the TF1-a cells treated with etoposide for 6 days. TF1-a cells survived this conditioning with significant inhibition of proliferation and limited damage and apoptosis. The molecular signature of these cells was characterized using GEP of the whole human genome and was compared to that of the dormancy-induced TF1-a cells in an aim to identify genes commonly up-regulated in both scenarios that might act as possible drivers of dormancy in AML cells residing in a sub-lethal genotoxic environment. In this context, 31 genes were significantly commonly upregulated in both scenarios including ITGB3, SLFN5, C15orf26 and GRAP2 which are candidates for in-depth investigation in AML. To sum up, in this study we developed and molecularly characterised in vitro models of dormancy and sub-lethal genotoxicity in AML cells with stemness characteristics through novel approaches that took bone marrow microenvironmental features into consideration. These features likely contribute to the resistance of the residual sub-population of AML cells that causing disease relapse. The current models helped to overcome the obstacles facing the in-depth studying of these rare AML cells due to the difficulty in obtaining them from clinical samples. Finally, the molecular findings of this study paved the way to potentially important future directions of research that could help to achieve the ultimate goal of eradicating AML cells and preventing relapse.

The DNA damage response (DDR) forms a signaling cascade rapidly activated upon exposure to genotoxic stress. The DDR safeguards genomic integrity by halting cell cycle progression to allow repair of damaged DNA or by inducing cell death. Myc proteins are key regulators of cell proliferation that transcriptionally control the cell cycle machinery. Amplification of N-myc in neuroblastomas (MNA-NB) is associated with abrogation of the regulatory mechanisms that normally prevent aberrant cell proliferation and the interplay between N-myc and the DDR was here analysed. Initially, an association between N-myc and the cdk inhibitor p21 was investigated in MNA-NB as a possible mechanism by which p21 is functionally suppressed in these cells. Although an N-myc/p21 interaction was not observed, MNA-NB cells appear to express short N-myc isoforms with the potential to associate with p21. Expression of N-myc rendered Rat-1 cells resistant to the cell cycle block imposed by serum starvation but these cells were not able to bypass a G1 arrest imposed by ectopic p21 suggesting N-myc does not abolish p21 activity through regulation at the protein level. Analysis of the N-myc response to DNA damage in MNA-NB cells revealed that N-myc is downregulated in a proteasome-dependent manner in response to UVC or a UV-mimetic carcinogen, 4NQO. This effect was not reproduced with other agents such as IR which like UVC were found to repress cyclin D1 expression likely indicating that alternative DDR signaling pathways differently regulate N-myc. N-myc was found to interact with the DDB2 subunit of the damaged-DNA binding (DDB) complex, a substrate receptor for the DDB1-Cul4ADDB2 E3 ligase. The DDB complex has been implicated in UV-induced protein ubiquitylation suggesting it may play a role in the N-myc response to UVC radiation. These findings highlight the complexities of the DDR and uncover potentially important mechanisms of cell cycle control through regulation of N-myc.

Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed March 13, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.

Une exposition prénatale aux radiations ionisantes est associée au développement de pathologies neurodéveloppementales liées à l’induction de dommages à l’ADN dans les cellules souches et progéniteurs neuraux (CSPN). Ainsi, la stabilité génétique des CSPN est cruciale pour le développement et l’homéostasie du cerveau. Cependant, des altérations génomiques au niveau des CSPN au cours du développement pourraient promouvoir la diversité neuronale. XLF est un composant de la voie de réparation d’ADN par NHEJ (pour Non-Homologous End-Joining). Nous avons montré une augmentation de l’instabilité des CSPN dans le cerveau embryonnaire des souris Xlf-/- qui pourrait perturber la neurogenèse au cours du développement, et ainsi être responsable d’altérations comportementales identifiées chez ces souris à l’âge adulte. A l’aide d’approches cytogénétiques, nous avons comparés la stabilité chromosomique des CSPN et des fibroblastes embryonnaires murins (MEF) exposés à un stress génotoxique aigue (irradiation γ) ou chronique (incorporation de thymidine tritiée dans l’ADN). Nos résultats démontrent que les CSPN maintiennent leur intégrité génétique de façon plus efficace que les MEF. En effet, les CSPN semblent avoir de meilleures capacités de réparation des dommages à l’ADN que les MEF, ce qui leur permet de développer une réponse adaptative à un stress génotoxique chronique. Cette réponse adaptative implique XLF et agit conjointement avec les points de contrôle du cycle cellulaire et l'apoptose pour préserver la stabilité du génome et éliminer les cellules endommagées. L’ensemble de nos résultats apporte la démonstration d’une réponse robuste aux dommages de l'ADN dans les CSPN et souligne l'importance de XLF lors du développement du cerveau
Prenatal exposure to ionizing radiation has been associated with many neurodevelopmental disorders due to the DNA damage induced in neural stem and progenitors cells (NSPC). Thus, genetic stability of NSPC is crucial for brain development and homeostasis. Nevertheless, genomic alterations occurring during development in NSPC may have a potential impact on the physiological neuronal diversity. XLF is a component of the NHEJ (Non-Homologous End-Joining) repair pathway. Here, we show that NSPC from Xlf-/- embryos exhibit increased chromosome instability, leading to premature neurogenesis and consequently neurobehavioral disorders. Using cytogenetic approaches, we compared the chromosome stability of mouse embryonic NSPC and fibroblasts (MEF) exposed to acute (γ-irradiation) or chronic (incorporation of tritiated thymidine into DNA) genotoxic stress. Our results demonstrate the higher capacity of NSPC as compared to MEF to maintain their genomic integrity. We evidenced that NSPC have more efficient DNA repair activity than MEF, allowing them to develop an adaptive response to chronic genotoxic stress. This adaptive response involves XLF and acts together with apoptosis and cell cycle checkpoints to preserve the stability of the genome and to eliminate damaged cells. Altogether, our results provide new insights into the robust DNA damage response in NSPC and highlight the importance of Xlf during brain development

Polymers of ADP-ribose (PAR) are rapidly synthesized by poly(ADPribose) polymerases (PARPs) and rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) following genotoxic stress. Since PAR metabolism plays an important role in cell fate determination following genotoxic stress, enzymes involved in PAR metabolism potentially represent promising therapeutic targets for modulating diseases of inappropriate cell proliferation or death. PARP-1 has been well validated and several PARP-1 inhibitors are currently being evaluated in clinical trials for cancer and ischemia treatment. In contrast, the biological function of PARG is still poorly understood. Due to low abundance of protein levels in mammalian cells and its unique substrate, PARG potentially represents another attractive target for pathological conditions mentioned above. PARG-Δ2,3 cells derived from homozygous PARG-Δ2,3 mice with targeted disruption of exons 2 and 3 of the PARG gene are used in this dissertation. The nuclear isoform PARG60 in PARG-Δ2,3 cells lacks the putative regulatory domain A compared to the nuclear isoform PARG110 in wild type cells. We report in this dissertation that PARG-Δ2,3 cells accumulate less PAR in spite of more rapid depletion of NAD following treatment with N-methyl- N’- Nitro-N-Nitrosoguanidine (MNNG). The estimation of PARP and PARG activity in intact cells shows increased activity of both enzymes in PARG-Δ2,3 cells following MNNG treatment, indicating the important role of domain A in the regulation of PARG and PARP activity under these conditions. Following MNNG treatment, PARG-Δ2,3 cells show reduced formation of XRCC1 foci, decreased H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. The altered PAR metabolism and defective cellular responses related to DNA repair in PARG-Δ2,3 cells may contribute to increased sensitivity of these cells to MNNG. Studies presented in this dissertation clearly demonstrate the important role of PARG110 in PAR metabolism and cellular responses to genotoxic stress, and thus provide supportive data for the validation of PARG as a promising potential therapeutic target.

The ability of organisms to sense and respond to challenges to their genome integrity is key to survival. In particular, the ability to detect and respond to double-stranded DNA breaks (DSBs) is of fundamental importance as not only are DSBs potentially lethal as they can trigger apoptosis, but there is also the potential for the loss of genetic information. The response to DSBs is well conserved across Eukaryotes and comprises two stages: detection of the break and subsequent remedial action. The remedial action involves cell cycle arrest, DNA repair, and, if repair cannot be effected, possible apoptosis. Whilst many of the key components, especially in the initial detection of the break, are conserved there are also differences between plants and animals in some of the main components and their roles. In this thesis I have proposed an overall framework for the cellular response to DSBs in plants and have proposed two candidate genes, TCP20 and SOG1, as novel plant specific activators in this response. Their suitability has been addressed by considering their activation and their downstream targets. I have shown that TCP20 is necessary for growth arrest observed in shoot apical meristems after exposure to genotoxic stress. I have also shown that activation of one of the key targets of TCP20, CYCB1;1 requires TCP20 and that a key TCP20 binding motif in the promoter of CYCB1;1 is necessary for the up-regulation of CYCB1;1 in response to genotoxic stress. This motif is over-represented in the promoters of many of the genes involved in DNA damage repair, suggesting that TCP20 plays a role in the co-ordination of the cellular response to DSBs.

Em estudos de proteção ao genoma contendo lesões de C. crescentus, foi feita uma varredura em uma biblioteca clones mutados pelo transposon Tn5 e sensíveis à luz UV-B e MMS. Dos 249 clones selecionados conseguimos identificar mutações em 102 genes, distribuídos em dez categorias funcionais, incluindo metabolismo de DNA. Além de genes já descritos como atuantes na defesa de genoma a lesões, os dados ainda adicionam funções potenciais para outros genes. Investigação mais detalhada indica que vários dos genes identificados podem afetar o estresse e ciclo celular, podendo estar relacionados a respostas do tipo pontos de checagem. Além disso, verificamos que mutantes para genes envolvidos em reparo excisão de nucleotídeos são mais sensíveis à H2O2, indicando que nessa bactéria, este sistema de reparo atua também em lesões oxidativas no DNA. Os dados obtidos são pioneiros no estabelecimento de funções para vários genes de C. crescentus e de outras alfa-proteobactérias.
This work aimed to identify genes related to DNA damage protection in C. crescentus, by means of screening a Tn5 -mutated library for clones sensitive to UV-B light and MMS. Out of 249 selected clones, we were able to identify mutations in 102 genes, classified in ten different functional categories, including DNA metabolism. In addition to genes already described as playing a role in genome defense to lesions, the results provide new potential functions to several other genes. More detailed investigation indicates also that the mutations in some of these genes may also affect cell stress and cell cycle, being potentially involved in check-point responses. Moreover, mutants for nucleotide excision repair genes were also found to be sensitive to H2O2, indicating that this DNA repair system also acts in DNA oxidative damage. These data are the first establishing a role in genome protection for several genes in C. crescentus, as well as other alpha-proteobacteria.

Insects have evolved various physiological responses to cope with stressors such as pathogens, toxins and environmental factors. It is known that the responses resulting from infection or DNA damage share some of the same pathways. Exposure of Drosophila melanogaster and the dung beetle Euoniticellus intermedius to stress led to changes in the expression of proteins involved in metabolism, development, protein degradation, mRNA processing and stress responses. Stress responses in D. melanogaster are well characterised. However, the role played by Drosophila p53 (Dmp53) and a member of the retinoblastoma binding protein 6 (RBBP6) family, Snama, are unknown. Snama has been proposed to play a role in Dmp53 regulation. Following DNA damage we investigated the role of Dmp53 and Snama. Flies recovering from camptothecin treatment display a glycolytic flux, involving a metabolic shift, different to that observed in cancer cells. Camptothecin treatment leads to an increase in the mortality of both sexes. Furthermore, females show a specific decrease in fecundity which is due to an increase in Dmp53 dependent apoptosis in the ovaries and is accompanied by a depletion of Snama and an increase in Dmp53 transcripts. Expression data indicated that Dmp53 activity may be largely regulated at the protein level. Bypassing glycolysis through methyl pyruvate supplementation led to differential expression of Dmp53 and Snama and improved reproduction and embryonic development. These results highlight differences between the metabolic strategies used by cancerous and non-cancerous cells which may be exploited in future chemotherapies. While immune responses amongst insect orders are evolutionarily conserved, many remain uncharacterised. To investigate the immune system of an organism that lives in a microbe rich environment, E. intermedius was infected with the fungal pathogen Beauveria bassiana. This resulted in decreased lifespan and fecundity. Homologs of proteins involved in the immune response of insects were identified in E. intermedius, including a member of the Toll family of proteins, an insect defensin (present in the hemolymph) as well as a homolog of the serine protease Persephone. These results show that immune signalling pathways are conserved in this dung beetle.

Promyelocytic leukemia protein (PML) is a tumour suppressor which is frequently downregulated in human tumours. PML plays a role in many cellular processes including DNA damage response, senescence and apoptosis and is mainly localized in special structures called PML nuclear bodies (PML NBs). The nucleolus is a key nuclear compartment, where transcription of ribosomal DNA and biogenesis of ribosomes take place. The nucleolus is also called a stress sensor because of its role, for instance, in stabilization of tumour suppressor p53. Localization of PML to the nucleolar periphery appears to be prominent after disturbance of nucleolar functions - for example inhibition of rRNA transcription or processing. Thus the relationship between the nucleolus and PML nuclear bodies may be important for cellular response to stress. However, the role of PML nucleolar associations in nucleolar function including mechanism of formation of these structures remain unclear. Here we characterised PML nucleolar structures and mechanism of their formation. We showed that formation of PML nucleolar structures is not caused by replication stress, is not dependent on any specific phase of cell cycle and is not caused by DNA damage response but is induced by topological stress due to inhibition of toposiomerase function....

The dissertation deals with a cell response to genotoxic stress, specifically to anti-cancer treatments with a genotoxic mechanism of action. In principle, cells can respond to these perturbing stimuli in several ways: in case of severe DNA damage, they usually undergo apoptosis or enter senescence. In case of minor DNA damage, or upon defective checkpoint mechanisms, they may continue the cell cycle, either with successfully repaired DNA or with mutations of various kind. Thanks to selection pressure, the mutations that provide cells with a certain growth advantage under conditions of continuing genotoxic stress, gradually accumulate and render the tumor treatment-resistant. In my thesis, I focus on several aspects of this whole process. First, I participated in a characterization of a radioresistant and anoikis-resistant population of prostate cancer cells. This population was generated by irradiating cells 35 times by 2 Gy, a regime used in clinics. After this treatment, a population of low-adherent cells emerged that demonstrated increased expression of EMT- and stem cell markers. The low-adherent state of these cells was maintained by Snail signaling and their anoikis resistance by ERK1/2 signaling. Interestingly, after a protracted period of time, these cells were able to re-adhere and...

RNA granules are conserved membraneless mRNP complexes that play an important role in determining mRNA fate by affecting translation repression and mRNA decay. Processing bodies (P-bodies) harbor enzymes responsible for mRNA decay and proteins involved in modulating translation. Although many proteins have been identified to play a role in P-body assembly, a bonafide disassembly factor remains unknown. We observed that Sbp1 with the help of its RGG-motif promotes P-body disassembly in S. cerevisiae. This study provides an example of the role of low complexity sequence in RNA granule disassembly. We have further explored the role of human RGG-motif containing proteins in regulation of translation. Here we studied LSM14A (human homolog of Scd6) in maintaining RNA granule dynamics and regulation of mRNA. We observe that LSM14A binds to human eIF4G and plays an important role in regulating the translation of certain mRNAs in response to genotoxic stress. Overall using a combination of yeast and human cell culture system, this study provides critical insight into the role of conserved low complexity sequences.

The promyelocytic leukemia protein (PML) is a tumour suppressor. It has been reported that PML interaction with the p53 protein is involved in the activation of cell cycle checkpoints and, when persistent, may lead to the premature onset of cellular senescence. Cellular senescence is a state of permanent cell growth arrest that is associated with characteristic morphological and metabolic changes and persistent DNA damage signalling. Importantly, PML nuclear bodies coassociate with persistent DNA damage foci in senescent cells; however, the role of this interaction is still obscure. My goal was to characterize the role of PML in DNA damage response (DDR) and the induction of premature cellular senescence after genotoxic stress, namely X-radiation, using both siRNA-mediated PML knock down (PML KD) and complete PML knock out (PML KO) in human cells. The dynamics of DNA damage foci, levels of various proteins involved in DDR, and proliferation rate were measured in both PML KD and KO cells. No significant changes in the formation of DNA damage foci, activated DDR (p53 and Chk2), activated p21CIP1/WAF1 cyclin-dependent kinase inhibitor, senescent morphology, and SA-β-galactosidase activity in PML KO cells were observed. However, PML KO cells displayed higher levels of retinoblastoma protein (Rb) and...

Thesis (Ph. D.)--Columbia University, 2005.
Department: Cellular, Molecular and Biophysical Studies. Includes bibliographical references (leaves 103-107). Proquest Digital Dissertations Online ; Subscription required. Also available via the World Wide Web;