1α (IL 1α) is a multifunctional cytokine 1α is 1α independent on the receptor sig 1α is responsible for 1α to the plasma membrane. 1α activates express κB, binds to 1α 1α 1α to the plasma membrane 1α to signal 1α is required for membrane 1α exter 1α anchoring 1α 1α 1α with tumor suppressor p53 following genotoxic stress is further described in human cell 1α coloca

Muitos genes têm sido indicados como responsivos ao estresse genotóxico, mas devido à necessidade de validação, a busca por marcadores gênicos continua. Vários genes são relacionados ao sistema ubiquitina-proteassomo (UPS), o qual é responsável pela remoção seletiva de proteínas, sendo que falhas no UPS têm sido relacionadas a doenças neurodegenerativas e ao câncer. Assim, o presente trabalho teve como objetivo a busca e confirmação de marcadores gênicos de resposta ao estresse genotóxico, por meio do estudo da expressão transcricional e protéica dos genes ERN1, EIF2AK3, GADD153 e TRAF2, visando à confirmação das respostas em linhagens de fibroblastos (GM07492A e AS405) e de glioblastoma (U87MG), sob tratamentos com peróxido de hidrogênio (H2O2) e Bleomicina (Blm). Foram utilizados o Ensaio Cometa, a análise de expressão gênica transcricional por qPCR em tempo real e de expressão gênica ao nível protéico (imunofluorescência). Os resultados mostraram que os tratamentos empregados foram capazes de induzir danos no DNA, sendo que a sensibilidade ao tratamento e a capacidade de recuperação das linhagens foi variável dependendo do agente testado. A análise de expressão gênica mostrou que GM07492A apresentou indução dos genes ERN1 e GADD153 após tratamento com H2O2 (resposta precoce, zero e 2 h) e Blm (durante todo pós-tratamento). A linhagem AS405 exibiu indução de ERN1 e GADD153 para H2O2, enquanto que para Blm foram induzidos os genes EIF2AK3 e GADD153. Para U87MG, a indução de EIF2AK3 pelo H2O2 ocorreu de modo tardio, enquanto GADD153 mostrou-se induzido após ambos os tratamentos. A proteína ERN1 apresentou expressão discreta e pontual, inclusive nos pontos onde não houve indução transcricional, indicando uma expressão basal. Essa proteína se expressou em GM07492A no tratamento com Blm em zero hora, diferentemente de AS405. Para U87MG tratada com H2O2 observou-se discreta expressão de ERN1, sendo mais evidente para Blm. Quanto à proteína GADD153, esta foi expressa em fibroblastos nos vários tempos analisados. No entanto, U87MG mostrou expressão nuclear apenas nas células tratadas, sendo mais evidente para H2O2 comparativamente à Blm. Assim, as alterações observadas nos perfis de expressão gênica são compatíveis com a indução de danos no DNA, indicando o envolvimento de genes do UPS nas respostas celulares ao estresse genotóxico. Em conjunto, os resultados estimulam uma avaliação mais detalhada desses genes como marcadores de resposta ao estresse e evidencia a sua importância no cenário da via UPS. / Many genes have been reported as responsive to genotoxic stress, but due to the need of validation, the search for genetic markers still continues. Several genes are related to the ubiquitin-proteasome system (UPS), which is responsible for the selective removal of proteins, and UPS failures have been associated to neurodegenerative diseases and cancer. Thus, this study aimed the search and confirmation of genetic markers that were responsive to genotoxic stress. For this, we evaluated the transcriptional or protein expression of the genes ERN1, EIF2AK3, GADD153 and TRAF2, seeking confirmation of responses in fibroblast cell lines (GM07492A and AS405) and glioblastoma (U87MG) under treatment with hydrogen peroxide (H2O2) and bleomycin (BLM). We used the Comet Assay, the transcriptional analysis of gene expression by quantitative real-time PCR and protein expression byimmunofluorescence. The results showed that the treatments employed were able to induce DNA damage, and that cell sensitivity to treatments and recovery capability of cell lines varied according to the tested agent. The gene expression analysis showed that GM07492A presented induction of ERN1 and GADD153 genes after treatment with H2O2 (early response, zero and 2 h) and Blm (throughout the post-treatment). The cell line AS405 showed induction of GADD153 and ERN1 after H2O2, whereas with Blm the genes induced were EIF2AK3 and GADD153. For U87MG, the induction of EIF2AK3 by H2O2 occurred at a later stage, while GADD153 was promptly induced after both treatments. The protein ERN1 showed discreet and punctual expression, even at time point without transcriptional induction, indicating a basal expression. This protein was expressed in GM07492A by treatment with Blm at zero hour, differently of AS405. For U87MG treated with H2O2, ERN1 showed a slight expression, being more evident for Blm. Regarding GADD153, protein expression was observed in fibroblasts at all time point. However, U87MG showed nuclear expression only in cells treated with H2O2, being more evident that in BLM-treated cells. Thus, the observed changes in gene expression profiles are consistent with the induction of DNA damage, which indicates the participation of UPS genes in cellular responses to genotoxic stress. Together, the results encourage further evaluation of these genes as markers of stress response, demonstrating its importance in the UPS acting scope.

Μηχανισμοί οι οποίοι εξασφαλίζουν τη σωστή διαδοχή των φάσεων του κυτταρικού κύκλου συμβάλλουν στη διασφάλιση της γονιδιωματικής σταθερότητας των κυττάρων. Η αδειοδότηση της αντιγραφής του DNA, η συγκρότηση επί των αφετηριών της αντιγραφής του DNA του προ-ανιγραφικού συμπλόκου, καθορίζει τη σωστή χρονικά και τοπικά έναρξη της αντιγραφής. Βασικό συστατικό αυτού του συμπλόκου είναι ο παράγοντας Cdt1. Η Geminin προσδένεται στο Cdt1, αναστέλοντας τη δράση του από την S μέχρι και την Μ φάση, παρεμποδίζοντας, έτσι, την αδειοδότηση της αντιγραφής. Παρά το οτι φυσική αλληλεπίδραση των δύο πρωτεϊνών έχει δειχθεί τόσο in vitro όσο και in vivo, προηγούμενες μελέτες δείχνουν οτι έκφραση των Cdt1 και Geminin εντοπίζεται σε διαφορετικές φάσεις του κυτταρικού κύκλου. Τα φυσιολογικά κύτταρα, ανάλογα με τα μηνύματα που δέχονται, είτε παραμένουν σε μιτωτικό κύκλο, είτε εξέρχονται από αυτόν προς φάση ηρεμίας (ή G0), διαφοροποίηση ή γήρανση. Αυστηρός συντονισμός των παραπάνω διαδικασιών είναι απαραίτητος προκειμένου να διασφαλιστεί η ομοιόσταση των πολύπλοκων δομών των ιστών των μεταζώων. Προηγούμενες μελέτες προτείνουν το σύστημα της αδειοδότησης της αντιγραφής του DNA ως έναν βασικό ρυθμιστή της εξόδου από τον κυτταρικό κύκλο και της επανεισόδου στη G1. Οι παράγοντες Cdt1 και Geminin ρυθμίζονται αρνητικά κατά την έξοδο των κυττάρων σε G0, ενώ έκφρασή τους χαρακτηρίζει διαιρούμενα κύτταρα. Σε αντίθεση με τις άλλες καταστάσεις εκτός κυτταρικού κύκλου, λίγα είναι γνωστά αναφορικά με τη ρύθμιση των Cdt1 και Geminin κατά την κυτταρική γήρανση. Στο πρώτο μέρος της διατριβής εστιαστήκαμε στη μελέτη του προτύπου έκφρασης των Cdt1 και Geminin κατά τη διάρκεια του κυτταρικού κύκλου πρωτογενών ανθρώπινων ινοβλαστών, και στη σύγκρισή του με εκείνο των καρκινικών κυττάρων. Διαπιστώσαμε οτι τόσο η ενδοκυτταρική εντόπιση όσο και η ικανότητα των Cdt1 και Geminin να εκφράζονται σε συγκεκριμένες φάσεις του κυτταρικού κύκλου, δεν διαφοροποιούνται στους πρωτογενείς φυσιολογικούς ινοβλάστες σε σχέση με κύτταρα που προέρχονται από καρκινικό ιστό. Επιπλέον, δείξαμε οτι ο παράγοντας Cdt1 εκφράζεται αποκλειστικά σε BrdU-αρνητικά κύτταρα, σε αντίθεση με την Geminin, η οποία δείχνει να συσσωρεύεται σταδιακά μετά την έναρξη της S φάσης, ενώ δεν εντοπίστηκε συνέκφραση των δύο πρωτεϊνών στο χρονικό παράθυρο της G1/S μετάβασης. Στο δεύτερο μέρος της εργασίας εστιαστήκαμε στη μελέτη της έκφρασης του παράγοντα αδειοδότησης Cdt1 και του αρνητικού ρυθμιστή αυτού, Geminin, κατά την είσοδο των κυττάρων σε κυτταρική γήρανση και εξετάσαμε την πιθανή λειτουργική εμπλοκή τους στην εξέλιξη του φαινομένου. Δείξαμε οτι, ενώ οι παράγοντες Cdt1 και Geminin διατηρούν τη σωστή ενδοκυτταρική εντόπιση και το σωστό πρότυπο έκφρασης κατά τη διάρκεια του κυτταρικού κύκλου, υφίστανται αρνητική ρύθμιση σε κύτταρα που εισέρχονται σε κυτταρική γήρανση, τόσο αναπαραγωγική όσο και πρόωρη, επαγόμενη από οξειδωτικό στρες. Το γεγονός οτι η μείωση της έκφρασης της Geminin προηγήθηκε της εμφάνισης του γηρασμένου φαινοτύπου, μας ώθησε στην περαιτέρω διερεύνιση του λειτουργικού ρόλου της Geminin στην επαγωγή της κυτταρικής γήρανσης. Για το σκοπό αυτό, απορρυθμίσαμε τα επίπεδα έκφρασης της Geminin σε πρωτογενή φυσιολογικά κύτταρα ανθρώπου και ποντικού, αξιοποιώντας την τεχνολογία του RNAi και ρετροϊικά συστήματα υπερέκφρασης γονιδίων αντίστοιχα. Δείξαμε οτι η μείωση της έκφρασης της Geminin σε ανθρώπινους ινοβλάστες (χρησιμοποιώντας siRNAs αλλά και pSUPER πλασμιδιακούς φορείς αποσιώπησης γονιδίων που κατασκευάστηκαν ειδικά για την Geminin) επάγει αύξηση της κυτταρικής γήρανσης της καλλιέργειας. Επιπλέον, κύτταρα που στερούνταν της έκφρασης της Geminin ήταν πιο επιρρεπή σε γήρανση επαγόμενη από οξειδωτικό στρες, σε σχέση με τα κύτταρα-μάρτυρες. Ετεροζυγώτες για το γονίδιο της Geminin εμβρυικοί ινοβλάστες ποντικού εμφάνιζαν μεγαλύτερα ποσοστά κυτταρικής γήρανσης σε σχέση με τους αντίστοιχους ινοβλάστες αγρίου τύπου. Αντίθετα, αύξηση των επιπέδων της Geminin σε αγρίου τύπου εμβρυικούς ινοβλάστες ποντικού προκάλεσε μείωση της εμφανιζόμενης γήρανσης. Τέλος, η μείωση των επιπέδων έκφρασης του παράγοντα αδειοδότησης Cdt1 σε ανθρώπινα κύτταρα ήταν, επίσης, σε θέση να επάγει ισχυρό φαινότυπο κυτταρικής γήρανσης. Συνοψίζοντας, τα αποτελέσματα μας αναδεικνύουν την κρισιμότητα του ισοζυγίου Cdt1:Geminin στα κύτταρα, και προτείνουμε οτι η διατάραξη της ισορροπίας αυτής είναι ικανή να επάγει κυτταρική γήρανση, μέσω διαδικασιών όπως η υπεραδειοδότηση ή η υποαδειοδότηση της αντιγραφής του DNA. / Genome integrity relies on the strict alternation of S and M phases of the cell cycle, so that one and only round of DNA replication takes place per cell cycle. This is achieved through replication licensing, which involves the formation of a multi-protein complex, the pre-replicative complex, onto origins of replication. Cdt1 is a crucial component of this complex and Geminin, a small protein shown to tightly bind Cdt1, inhibits its licensing function from S to M phase, when licensing is illegitimate. Although previous experimental evidence shows that Cdt1 and Geminin are expressed in different phases of the cell cycle, physical interaction between these two proteins has been demonstrated in vitro as well as in vivo. The fate of a normal cell is not perpetual division. Cells may exit the mitotic cell cycle to enter quiescence, to terminally differentiate or to senesce. These “out-of-cycle-states” must be strictly regulated in order to establish and maintain the hierarchical organization of complex tissues in metazoa. Replication licensing has been proposed to coordinate cell-cycle exit and re-entry in vitro and in metazoan tissues. Cdt1 and Geminin down-regulation during exit to quiescence supports the idea that their expression correlates with cell proliferation. In contrast to other out-of-cycle states, little is known about the regulation of Cdt1 and Geminin expression during cellular senescence. Senescence refers to the irreversible resting state of cells grown for succeeding passages in culture, as a response to DNA damage caused by telomeres erosion. Other stimuli, such as oxidative or oncogenic stress, may force mitotically competent cells to respond similarly, a phenomenon termed as Stress Induced Premature Senescence (SIPS). The first part of this work focused on the study of the expression patterns of Cdt1 and Geminin during the unperturbed cell cycle of primary human fibroblasts and compared to that of tumor-derived cell lines. The cell cycle specific expression and the intracellular localization of both proteins, as assessed at a single-cell level using indirect immunofluorescence and a new monoclonal antibody against Geminin, appear similar in primary fibroblasts compared to the cancer cells examined. Cdt1 is strictly expressed in BrdU-negative cells, whereas Geminin starts accumulating after S phase onset. The two proteins are, therefore, not co-expressed at the ”time-window” of G1/S transition of the cell cycle. We showed that Cdt1 levels, but not those of Geminin, are mainly regulated in a proteasome-dependent way during normal cell cycle of human primary and cancer cells. The second part of this work focused on the investigation of Cdt1 and Geminin during cellular senescence and their possible role in the establishment of the senescence phenotype. To this end, primary human fibroblasts were maintained in culture for succeeding passages in order to induce them to undergo replicative senescence. Alternatively, an H202-induced senescence protocol was applied to force cells to undergo premature senescence (Stress-Induced Premature Senescence/SIPS). We show that, although Cdt1 and Geminin retain their nuclear localization and are correctly expressed during specific phases of the cell cycle during both replicative and Η202-induced premature senescence, their expression levels are down-regulated. In SIPS-experiments, Geminin down-regulation is an early event during the establishment of the senescent-phenotype, as assessed by senescence-associated β-Galactosidase and BrdU incorporation assays. This prompted us to further examine Geminin’s functional significance in the establishment of cellular senescence. To achieve this, we interfered with Geminin expression levels in human and mouse cells. Using RNA interference techniques, we were able to show that Geminin depletion from human cells is able to induce a senescent-phenotype in a fraction of the treated culture. Similarly, Geminin-depleted human cells were more susceptible to Η202-induced premature senescence, compared to control cells. Heterozygotes for Geminin mouse embryonic fibroblasts were more prone to senescence compared to their control counterparts. In contrast, when Geminin was over-expressed in control mouse embryonic fibroblasts cultures, senescent phenotype was reduced. Finally, a strong senescent-phenotype was induced when the licensing regulator, Cdt1, was silenced within human cells. Taken together, we conclude that Cdt1:Geminin balance within cells is crucial, and when disturbed, is able to promote a senescent phenotype, possibly through a mechanism that involves over- or under-licensing of DNA replication.

Muitos genes têm sido indicados como responsivos ao estresse genotóxico, mas devido à necessidade de validação, a busca por marcadores gênicos continua. Vários genes são relacionados ao sistema ubiquitina-proteassomo (UPS), o qual é responsável pela remoção seletiva de proteínas, sendo que falhas no UPS têm sido relacionadas a doenças neurodegenerativas e ao câncer. Assim, o presente trabalho teve como objetivo a busca e confirmação de marcadores gênicos de resposta ao estresse genotóxico, por meio do estudo da expressão transcricional e protéica dos genes ERN1, EIF2AK3, GADD153 e TRAF2, visando à confirmação das respostas em linhagens de fibroblastos (GM07492A e AS405) e de glioblastoma (U87MG), sob tratamentos com peróxido de hidrogênio (H2O2) e Bleomicina (Blm). Foram utilizados o Ensaio Cometa, a análise de expressão gênica transcricional por qPCR em tempo real e de expressão gênica ao nível protéico (imunofluorescência). Os resultados mostraram que os tratamentos empregados foram capazes de induzir danos no DNA, sendo que a sensibilidade ao tratamento e a capacidade de recuperação das linhagens foi variável dependendo do agente testado. A análise de expressão gênica mostrou que GM07492A apresentou indução dos genes ERN1 e GADD153 após tratamento com H2O2 (resposta precoce, zero e 2 h) e Blm (durante todo pós-tratamento). A linhagem AS405 exibiu indução de ERN1 e GADD153 para H2O2, enquanto que para Blm foram induzidos os genes EIF2AK3 e GADD153. Para U87MG, a indução de EIF2AK3 pelo H2O2 ocorreu de modo tardio, enquanto GADD153 mostrou-se induzido após ambos os tratamentos. A proteína ERN1 apresentou expressão discreta e pontual, inclusive nos pontos onde não houve indução transcricional, indicando uma expressão basal. Essa proteína se expressou em GM07492A no tratamento com Blm em zero hora, diferentemente de AS405. Para U87MG tratada com H2O2 observou-se discreta expressão de ERN1, sendo mais evidente para Blm. Quanto à proteína GADD153, esta foi expressa em fibroblastos nos vários tempos analisados. No entanto, U87MG mostrou expressão nuclear apenas nas células tratadas, sendo mais evidente para H2O2 comparativamente à Blm. Assim, as alterações observadas nos perfis de expressão gênica são compatíveis com a indução de danos no DNA, indicando o envolvimento de genes do UPS nas respostas celulares ao estresse genotóxico. Em conjunto, os resultados estimulam uma avaliação mais detalhada desses genes como marcadores de resposta ao estresse e evidencia a sua importância no cenário da via UPS. / Many genes have been reported as responsive to genotoxic stress, but due to the need of validation, the search for genetic markers still continues. Several genes are related to the ubiquitin-proteasome system (UPS), which is responsible for the selective removal of proteins, and UPS failures have been associated to neurodegenerative diseases and cancer. Thus, this study aimed the search and confirmation of genetic markers that were responsive to genotoxic stress. For this, we evaluated the transcriptional or protein expression of the genes ERN1, EIF2AK3, GADD153 and TRAF2, seeking confirmation of responses in fibroblast cell lines (GM07492A and AS405) and glioblastoma (U87MG) under treatment with hydrogen peroxide (H2O2) and bleomycin (BLM). We used the Comet Assay, the transcriptional analysis of gene expression by quantitative real-time PCR and protein expression byimmunofluorescence. The results showed that the treatments employed were able to induce DNA damage, and that cell sensitivity to treatments and recovery capability of cell lines varied according to the tested agent. The gene expression analysis showed that GM07492A presented induction of ERN1 and GADD153 genes after treatment with H2O2 (early response, zero and 2 h) and Blm (throughout the post-treatment). The cell line AS405 showed induction of GADD153 and ERN1 after H2O2, whereas with Blm the genes induced were EIF2AK3 and GADD153. For U87MG, the induction of EIF2AK3 by H2O2 occurred at a later stage, while GADD153 was promptly induced after both treatments. The protein ERN1 showed discreet and punctual expression, even at time point without transcriptional induction, indicating a basal expression. This protein was expressed in GM07492A by treatment with Blm at zero hour, differently of AS405. For U87MG treated with H2O2, ERN1 showed a slight expression, being more evident for Blm. Regarding GADD153, protein expression was observed in fibroblasts at all time point. However, U87MG showed nuclear expression only in cells treated with H2O2, being more evident that in BLM-treated cells. Thus, the observed changes in gene expression profiles are consistent with the induction of DNA damage, which indicates the participation of UPS genes in cellular responses to genotoxic stress. Together, the results encourage further evaluation of these genes as markers of stress response, demonstrating its importance in the UPS acting scope.

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...

Nous avons identifié une nouvelle forme de phospho-S27-S33-Ku70 constitutivement surexprimée dans des cellules issues de la leucémie lymphocytaire chronique résistante à la chimiothérapie basée sur des agents alkylants de l’ADN et/ou analogues nucléotidiques. La protéine Ku70 est une protéine essentielle du maintien de la stabilité génomique par son rôle dans la réparation non-homologue (système NHEJ) des cassures double brin de l’ADN (CDB) et par sa fonction télomérique. Le laboratoire d’accueil a déjà démontré, in vitro et in vivo, dans les cellules LLC résistantes une altération de la réparation par le système NHEJ et un dysfonctionnement télomérique. Le travail de thèse a porté sur la caractérisation fonctionnelle de cette nouvelle forme phospho-S27-S33-Ku70. Pour ceci, nous avons utilisé des vecteurs d’expression permettant simultanément d’inhiber l’expression du Ku70 endogène (shRNA) et d’exprimer de façon épisomale différentes formes de Ku70 exogène. Ainsi, nous avons démontré : i) une stricte colocalisation de pS27-pS33-Ku70 avec les foyers γ-H2AX; ii) des cassures double brin (DSB) induisent la phosphorylation de S27-S33-Ku70 sous forme hétérodimère avec Ku80. Cette phosphorylation a lieu quelques minutes après le stress génotoxique et implique l'activité et l'interaction physique avec pS2056-DNA-PKcs, reliant ainsi pS27-pS33-Ku70 au système NHEJ ; iii) les cellules exprimant la forme sauvage exogène S27-S33-Ku70 ou la forme phosphomimétique E27-E33-Ku70 présentent une cinétique de réparation de l’ADN plus rapide que celle des cellules exprimant la forme mutée A27-A33-Ku70. Cependant, iv) la forme sauvage de Ku70 contribue à un niveau plus élevé d'aberrations structurales chromosomiques après la première division cellulaire suite à un stress génotoxique indiquant une infidélité lors de la réparation des dommages de l’ADN. En outre, les cellules exprimant A27-A33-Ku70 possèdent un index cellulaire plus élevé qui est corrélé avec une activation de la voie β-caténine. En adéquation avec sa surexpression dans la forme résistante de la LLC, l’ensemble de ces résultats suggère un rôle oncogénique de la forme phosphorylée de Ku70. Nous avons ensuite testé l’effet des nanodiamants hydrogénés (ND-H) dans des lignées exprimant différentes formes de Ku70. Grâce à leurs propriétés physico-chimiques les ND-H sont capables de potentialiser sous irradiation la production intracellulaire des espèces réactives de l’oxygène (ROS) et ainsi augmenter le taux des cassures (simple et double brin de l’ADN) et solliciter d’avantage le système de réparation de l’ADN. Nous observons que indépendamment de la forme exprimée de Ku70, ce double traitement induisait la sénescence cellulaire ; une découverte d’un intérêt à la fois fondamental (compréhension des voies apoptotiques vs senescence) et d’utilité pharmacologique potentielle. / We have identified a new form of phospho-S27-S33-Ku70 constitutively overexpressed in a subset of chronic lymphocytic leukemia (CLL) B cells resistant to apoptosis induced by DNA double strand breaks (DSB). Ku70 is one of the essential proteins involved in the maintenance of genomic stability through its role in DNA double strand break repair (non-homologous end-joining, NHEJ) and in telomeric protection.Laboratory previously established that resistant CLL cells disclose an upregulated NHEJ DNA repair and an impaired structure of telomeres. The goal of this thesis was to characterize the biological function(s) of this new form of Ku70. For this purpose we have constructed specific EBV-based vectors (siRNA / cDNA) enabling a simultaneous inhibition of endogenous Ku70 and an expression of different forms (mutated, wild, phosphomimetic at ser27-33) of Ku70 resistant to siRNA. Thus, we showed: i) a strict colocalisation of phospho-Ku70 with γ-H2AX foci; ii) that DSB induces the phosphorylation of Ku70 within minutes after genotoxic stress in heterodimer complex Ku70/Ku80. This phosphorylation necessitates both the physical interaction and the activity of pS2056-DNA-PKcs and/or ATM, linking phospho-Ku70 to NHEJ-mediated DNA DSB repair; iii) cells expressing mutated A27-A33-Ku70 exhibit a delayed G2/M cell cycle arrest, slower kinetic of DNA repair, lower level of genotoxic stress-induced chromosomal aberrations, and a higher cellular impedance correlated with translocation of transcriptional factor β-catenin from cytoplasmic membrane to the nucleus. Together, these data unveil an involvement of phospho-Ku70 in fast and inaccurate DNA repair; new paradigm for NHEJ regulation and to the control of resistance and maintenance of malignant cells.In parallel, we have initiated experimental approaches to explore other potential roles of phospho-Ku70. Especially, we were interested to determine whether it could play a role in an initiation of cell senescence induced by combined cells’ treatment by hydrogenated nanodiamonds (H-NDs) particles and ionizing irradiation. H-NDs exhibit positive surface charge in aqueous solutions allowing, when irradiated by photons, electrons’ emission and the release of reactive oxygen species (ROS) causing DNA damage. Effectively, we have established an intracellular increase of ROS that drive cell cycle arrest in G1/S in addition to the G2 arrest activated by irradiation alone. Finally, cells underwent the senescence process characterized byγ-galactosidaze activity, persistent large γ-H2AX foci and senescence-associated heterochromatinisation. Noteworthy, the senescence induced in this way occurred independently of Ku70 (ser27-ser33) status and irrespectively of cell resistance to genotoxic agents administrated alone; a finding of potential use in clinical trials.

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....

Cbf11 and Cbf12 proteins, the members of the CSL transcription factors family, are involved in a wide range of cellular processes in the fission yeast Schizosaccharomyces pombe - among other things they regulate cell adhesion and they have also been implicated in maintenance of genome integrity. At the level of the whole genome we previously identified target loci bound by CSL proteins in vivo. Many of them do not contain any consensus CSL-binding element. There are probably different DNA binding modes of the Cbf11/12 proteins and it has not been known what specific biological function is associated with the particular way of DNA binding. For the purpose of studying CSL DNA binding modes we have worked in this project on the implementation of the DNA binding mutation (DBM), which prevents direct DNA binding of CSL proteins to canonical motif in vitro, into the chromosomal locus of the cbf11 and cbf12 genes. Using the "ura4 selection system" we have successfully constructed the scar-less Cbf12-TAP and Cbf12DBM-TAP knock-ins, i.e. the strains without/with DBM in the open reading frame of Cbf12 where Cbf12 is C- terminally TAP-tagged and contains the intact 3'UTR. In our laboratory we have established the CRISPR/Cas9 system by which we have been able to prepare the Cbf11- TAP strain. We have failed to...

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...