La sénescence cellulaire est un mécanisme de suppression cellulaire caractérisé par un arrêt irréversible du cycle cellulaire. Les cellules sénescentes ont également un secrétome spécifique qui influe sur les cellules voisines, pouvant induire leur entrée prématurée en sénescence, l'apoptose, la prolifération cellulaire et le développement tumoral. Dans ce contexte, l'objectif de ma thèse était d'identifier une nouvelle protéine sécrétée par les fibroblastes sénescents et de caractériser ses fonctions dans les cellules sénescentes et dans les cellules tumorales. Nous avons mis en évidence la protéine WNT16B. Elle est surexprimée dans plusieurs modèles de sénescence cellulaire obtenus à partir de fibroblastes pulmonaires. Dans un modèle de souris transgénique, WNT16B est également associée avec l'accumulation de cellules sénescentes dans les lésions précancéreuses pulmonaires. WNT16B est impliqué dans le mécanisme de sénescence cellulaire. En effet, l'inhibition de WNT16B prévient l'entrée en sénescence, en empêchant l'activation de p53 et l'expression de p21/WAF1, et donc l'arrêt du cycle cellulaire.Nous avons finalement associé l'expression de WNT16B avec la présence de cellules sénescentes dans des tumeurs prélevées chez des patients atteints de carcinomes pulmonaires non à petites cellules. Cette expression est corrélée avec une meilleure survie pour les patients ayant reçu un traitement chimiothérapeutique. Ce dernier résultat fait donc le lien entre traitement, sénescence cellulaire et survie, et illustre le rôle potentiel des marqueurs des cellules sénescentes pour le suivi des patients. Globalement, ces travaux ont donc non seulement permis d'identifier WNT16B comme nouveau marqueur des cellules sénescentes, mais ils ont également ouvert des perspectives d'utilisation de ce marqueur pour l'identification, le traitement et le suivi des patients atteints d'un cancer. / Cellular senescence is a tumor suppression mechanism consisting of an irreversible cell cycle arrest. Senescent cells also express a specific secretome that plays a role in the microenvironment. It may induce premature senescence, apoptosis, cellular proliferation or tumor development in the neighboring cells. In this context, my thesis aimed to identify and characterize a new senescence-associated secreted protein, before highlighting its role in tumorigenesis. We identified the WNT16B protein. It was overexpressed in various cellular senescence models obtained from lung fetal fibroblasts. Moreover, WNT16B expression was associated with the accumulation of senescent cells in precancerous lesions in a transgenic mice model. WNT16B is also involved in the senescence machinery. Indeed, WNT16B inhibition prevented the senescence onset through inactivation of the p53/p21 pathway, thus stopping the cell cycle progression. Finally, we observed a correlation between the WNT16B expression and the occurrence of senescent tumoral cells in NSCLC samples. This expression was also correlated with a better patient outcome after chemotherapeutical treatment. Thus, this last result linked the treatment efficiency, the occurrence of senescence and the patient survival. Overall, my thesis established elevated WNT16B levels as a novel biomarker for senescence that might be useful for the identification, the treatment follow-up and the prognosis of patient outcome.

Premature cellular senescence is the process of permanent cell cycle arrest in response to various inducers, such as DNA damage, oxidative stress, chemotherapy agents, and irradiation. Senescent cells produce and secrete numbers of cytokines, chemokines, growth factors, which compose specific senescence-associated secretory phenotype (SASP). Senescence is considered to be an important barrier against tumor progression. On the other hand, senescent cells can also exert protumorigenic effects in their microenvironment. Based on this concept, the major aim of this thesis was to determine tumor cells senescence in terms of different inducers, namely chemotherapeutic agent docetaxel (DTX) and cytokines IFNγ and TNFα, and to demonstrate the role of immunotherapy in senescent cells elimination. Our results show that DTX-induced senescent cells can exert a tumor-promoting effect when co-injected with proliferating cells in mice. Importantly, we demonstrate that IL-12-based immunotherapy suppresses senescence-accelerated tumor growth. These results suggest that IL-12-based immunotherapy can be effectively used in anti-tumor therapy mainly in a case when the microenvironment is altered by the presence of tumor senescent cells. On the other hand, the data we obtained in vitro show that bystander or...

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Background: Ageing is associated with age-related disease and it has been divided into 12 hallmarks and cellular senescence is one of them. Cellular senescence increases with age and has different pathological inducing aspects in the tissue. Hyperbaric oxygen therapy is used in the clinic to treat different pathological conditions and has emerged as a possible intervention for the reduction of senescent cells. Reducing senescent cells could be a way to reduce the effects of ageing and therefore possibly in the future treat age-related diseases. Aim: This systematic literature review aims to investigate articles researching the effect of HBO exposure on senescent cells and the properties of senescent cells. Methods: PubMed database was used in this systematic literature review. Exclusion and inclusion criteria were specified using the PECO format. A search plan was created using both MeSH words and free text words. An article search was performed twice on 11 th September 2023 and 20 th November 2023. The exclusion and inclusion criteria were used to filter the search results and for the full-text review. Result: 6 articles in total were included in this study. 2 of those included articles were conducted on humans while the rest were conducted on animals or animal cells. Two studies showed that HBOT decreases the number of senescent cells, one study showed that the senescent cell markers and SASP were decreased post-HBOT, two studies showed that the properties of senescent cells were decreased post-HBOT while one study showed the contrary with an increased senescent cell properties. Conclusion: The result of this systematic literature review suggests that there is an association between HBOT and a decrease in senescent cells or its properties. More research is needed though to better understand the relationship between HBOT and the effect it imposes on senescent cells.

Aging is a complicated and multifactorial phenomenon. Model systems involving the induction of replicative senescence in cultured cells have been indispensable in elucidating some of the mechanisms underlying this complex process. An understanding of how and why cellular senescence occurs is thus critical to the field of aging research. While there is much correlative evidence to suggest a connection between poly(ADP-ribose) (PAR) and mammalian longevity, no studies have been done to explore a possible role for PARP-1 — the enzyme responsible for synthesis of 90% of cellular PAR — in mechanisms of senescence. Furthermore, many techniques currently used for analysis of protein poly(ADP-ribosyl)ation are fraught with imprecision. We therefore sought to address these issues both by developing methods for the unambiguous analysis of poly(ADP-ribosyl)ation by mass spectrometry, and by exploring the role of PARP-1 in nicotinamide-mediated cellular lifespan extension. Due to the challenges introduced by PAR's biochemical characteristics, successful mass spectrometric analysis of poly(ADP-ribosylation) will require the use of techniques to reduce the mass, charge, and heterogeneity of the polymer, as well as methods to enrich for poly(ADP- ribosyl)ated protein. To this end, we evaluated the effectiveness of several approaches, including ammonium sulfate fractionation, boronate affinity chromatography, snake venom phosphodiesterase digestion, manipulation of PARP-1 reaction conditions, and immobilized metal affinity chromatography (IMAC) for the preparation of poly(ADP-ribosyl)ated protein samples prior to MS analysis using both MALDI-TOF and Q-TRAP LC-MS. Based on this work, we developed a three-tiered scheme that may provide the first ever identification of poly(ADP- ribosyl)ated peptides from full-length wild-type PARP-1 by mass spectrometry. Past work in our laboratory has demonstrated that nicotinamide (NAM), a component of vitamin B3, significantly extends the replicative lifespan of human fibroblasts. In order to help elucidate the role of PARP-1 in cellular senescence, we then analyzed the poly(ADP-ribosyl)ation response of aging cells undergoing NAM-mediated lifespan extension. While NAM is a known PARP-1 inhibitor, we found that oxidative stress-induced poly(ADP- ribosyl)ation is increased, not decreased, in NAM-treated cells. We propose that supplemented NAM is taken up by the NAD salvage pathway, ultimately leading to increased cellular NAD and extending replicative lifespan by both preventing PARP-mediated NAD depletion and upregulating SIRT1. We further propose that the demonstrated protective effects of NAM treatment in a number of disease models are due not to PARP-1 inhibition as is commonly assumed, but to upregulation of NAD salvage. / Ph. D.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / After the progressive telomere shortening in successive cell divisions, normal somatic cells undergo a growth arrest called cellular senescence that occurs due to incomplete DNA replication. Senescence can also be activated by various types of stressful stimuli, including aberrant oncogenic signaling, oxidative stress and DNA damage. Senescent cells have limited proliferative capacity and seems to play an important role in tumorigenesis. They are also involved in the inflammation associated with aging and cancer progression. The process of senescence vary significantly between cells, but the different paths for the aging, however, converge to p53 and pRB. The network simulation is based on the model proposed by Porath using a Boolean model to represent the state of activation of genes involved, including the p16-pRb and p53-p21 pathways. The simulation includes 23 nodes representing the genes of the regulatory network where one of them represents the activation of the senescent state as a result of network processing. Experiments with human fibroblasts indicate that inactivation of both genes, p53 and pRB is necessary to block senescence. The simulations confirms that these pathways are able to trigger senescence independently. The simulation shows that pRb is essential to maintain the senescent state even when p16 and p53 are switched off, but the simultaneous inactivation of both p53 and pRB blocks senescence. In addition, the simulation shows that inactivation of the p16-pRb pathway is not essential to preserve the senescent state, however when p53-p21 pathway is inactivated, the senescent state is preserved. / Após o progressivo encurtamento dos telômeros em sucessivas divisões celulares, as células somáticas normais se submetidas a uma parada do crescimento chamado senescência celular que ocorre devido à replicação incompleta do DNA. A senescência também pode ser ativada por diversos tipos de estímulos estressantes, incluindo sinalização oncogênica aberrante, estresse oxidativo e danos ao DNA. Células senescentes têm capacidade proliferativa limitada e parecem desempenhar um papel importante na tumorigênese. Elas também estão envolvidas na inflamação associada com o envelhecimento e progressão do câncer. As vias de senescência variam significativamente entre as células, mas os caminhos diversos para a senescência, no entanto, convergem para p53 e pRb. A simulação é baseada no modelo proposto por Porath usando um modelo booleano para representar o estado de ativação dos genes envolvidos, incluindo as vias p16-pRb e a p53-p21. A simulação inclui 23 nós representando os genes da rede regulatória onde um deles representa o estado celular senescente que pode assumir estados Verdadeiro ou Falso como resultado do processamento de rede Experiências com fibroblastos humanos indicam que a inativação de ambos os genes, p53 e pRb, é necessária para bloquear a senescência. As simulações confirmam que essas vias são capazes de acionar a senescência independentemente. A simulação mostra que pRb é essencial para a manutenção do estado senescente mesmo se p16 e p53 forem desligados, no entanto a inativação simultânea de ambos p53 e pRb bloqueia senescência. Além disso, a simulação mostra que a inativação da via p16-pRb não é essencial para preservar o estado senescente, no entanto, quando a via p53-p21 é inativada, o estado senescente é preservado.

In recent years, a great effort has been deployed towards a better understanding of the molecular changes in cells and in the bone marrow (BM) environment that contribute to the development and progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Among others, the aberrant hematopoietic stem cells in MDS often display increase in DNA double strand breaks, genomic instability with common loss or rearrangement of chromosomes and an ineffective response to DNA damage, a phenomenon that has been linked to the onset of cellular senescence. Additionally, the BM microenvironment can become more pro-inflammatory. In our effort to better understand the contribution of the BM microenvironment on MDS progression, we analyzed the expression profiles of cytokines in the BM microenvironment in all stages of MDS/AML and found several proinflammatory cytokines that increase with disease progression. Also, by repeated sampling of patients over the course of 5-azacytidine therapy, we were able to assess the changes in the proinflammatory cytokine milieu with the progression of the disease. Additionally, we aimed to identify the candidate markers for the improvement of MDS prognosis. We focused on naturally occurring germline polymorphism of NAD(P)H dehydrogenase (quinone 1) gene (NQO1*2)...

Zelluläre Seneszenz wird als terminaler Zellzyklusarrest definiert, der mit dem Altern und funktionellen Verlust von Geweben verknüpft ist. Eine Seneszenzreaktion wird ebenso durch Onkogene und zytotoxischen Stress verursacht. Die Ausführung des Seneszenzprogramms wird durch eine zeitlich hochdynamische Aktivität von Transkriptionsfaktoren (TF) bedingt. Interessanterweise kann die Zelllinienzugehörigkeit einer Zelle durch die Expression von linien-aberranten TF überschrieben werden. Die vorliegende Arbeit untersucht Chemotherapie-induzierte Seneszenz (TIS) in Bcl-2 überexprimierenden, deshalb vor Apoptose geschützten, murinen Eµ-Myc B-Zell Lymphomen in An- oder Abwesenheit der Seneszenz-essentiellen Histonmethyltransferase Suv39h1. Analysen auf Transkriptom- und auf Proteinebene ergeben dabei, dass in einer Seneszenz-spezifischen Weise die TF AP-1, PU.1 und C/EBPβ induziert werden, welche normalerweise für die Funktion und Entwicklung myeloischer Zelllinien bedeutend sind. Dementsprechend korreliert der Seneszenzzustand mit Transkripten, Oberflächenmarkern und einer enzymatischen Funktion der myeloischen Linie. Indem die identifizierten TFs heruntergeschaltet oder überexprimiert werden, wird ihre direkte Beteiligung an der Linienuntreue der TIS Lymphome demonstriert. TIS-Kapazität wird als für den Erfolg von Krebstherapie günstige Eigenschaft betrachtet, da sie zu einem Wachstumsblock führt. Nichtsdestotrotz können sich verweilende TIS Zellen krebsbiologisch auch nachteilig auswirken. Anhand von murinen und humanen, klinisch annotieren Transkriptomdatensätzen kann hier in beiden Spezies ein myeloisch verschobenes, Linienuntreue anzeigendes Genexpressionsprofil mit einer besseren Überlebensprognose korreliert werden. Die vorliegenden Befunde legen nahe, dass die Modulation von TF Aktivitäten in Seneszenz einen potentiellen therapeutischen Angriffspunkt darstellt, um den für den Therapieerfolg nützlichen Zweig des TIS Phänotyps zu befördern. / Cellular senescence is regarded as an irreversible cell cycle arrest associated with tissue aging and its functional decline. A senescence response is also evoked by oncogenic and cytotoxic stress. The execution of the senescence program relies on a highly dynamic sequence of transcription factor (TF) activities. Interestingly, cell lineage commitment can be overridden by the expression of lineage-aberrant TFs. This thesis examines chemotherapy-induced senescence (TIS) in Bcl-2 overexpressing, thus apoptosis-protected, murine Eμ-Myc B-cell lymphomas with or without the senescence-essential histone methyltransferase Suv39h1. Transcriptome as well as protein level analyses reveal senescence-specific induction of the TFs AP-1, PU.1 and C/EBPβ which are typically crucial for myeloid lineage commitment and function. Correspondingly, the senescent state associates with myeloid lineage transcripts, surface markers and enzymatic function, reminiscent of, but not equal to a transdifferentiation phenotype. By knocking down and overexpressing the identified TFs, we demonstrate their direct involvement in the lineage infidelity of TIS lymphomas. TIS-capacity is viewed as beneficial to cancer therapy outcome due to its block on proliferation. However, lingering TIS cells can also be detrimental due to the acquisition of latent stemness properties or tumor-protective remodeling of their microenvironment. By interrogating murine and human, clinical course-annotated transcriptome data sets, an association between a myeloid-skewed, lineage infidelity indicating gene expression profile and better tumor prognosis is established in both species. The presented findings suggest that modulation of the senescent TF activities could be therapeutically exploited to foster the cancer patient-beneficial branch of the TIS phenotype.

La sénescence est un mécanisme de défense antiprolifératif dont la cellule est munie afin de prévenir l’accumulation de mutations pouvant mener à sa transformation et l’éventuel développement d’une tumeur. Ce programme consiste en un arrêt permanent du cycle cellulaire. Il peut être activé par de nombreux stimuli tels que le raccourcissement des télomères, le stress oxydatif, ou l’expression d’un oncogène constitutivement actif. Sa régulation requiert l’activation de protéines appelées des suppresseurs de tumeur dont les plus importants sont p53 et RB. De manière plus spécifique, les sénescences induites par l’expression des oncogène RASV12 ou STAT5A(1*6) sont respectivement caractérisées par l’augmentation de l’expression des protéines PML et CHES1/FOXN3. Le but de cette thèse est, dans un premier temps, de mettre en évidence le mécanisme de régulation de la sénescence par PML. PML est un suppresseur de tumeur dont l’expression dans des cellules diploïdes normales est suffisante pour induire la sénescence. Cette protéine forme des corps nucléaires sphériques au sein desquels est recruté, parmi d’autres molécules, la protéine du rétinoblastome RB. RB est un régulateur négatif du cycle cellulaire capable de lier et inhiber les facteurs de transcription E2F dont les gènes cibles sont nécessaires à la prolifération. Nos travaux démontrent que le mécanisme d’induction de la sénescence par PML implique le recrutement du complexe RB/E2F aux corps de PML afin de renforcer l’inhibition de l’activité des E2F par RB. Également, les E2F sont recrutés aux corps de PML en compagnie de leurs promoteurs ce qui favorise la formation d’hétérochromatine au niveau de ces gènes, aidant à leur répression et donc à l’établissement de la sénescence. D’autre part, cette thèse a pour but de caractériser le rôle de CHES1/FOXN3 dans la régulation du cycle cellulaire. CHES1 est un facteur de transcription de la famille des Forkheads. Son expression dans des cellules cancéreuses provoque un ralentissement de leur prolifération. Afin de comprendre son mécanisme de fonctionnement, une analyse sur micropuce d’ADN de l’expression des gènes de cellules cancéreuses exprimant CHES1 a été réalisée. Cette analyse a montré que, dans la cellule, CHES1 joue un rôle de répresseur transcriptionnel. Plus précisément, CHES1 réprime, entre autres, l’expression de gènes nécessaires à la synthèse des protéines tels que PIM2 et DYRK3. De manière intéressante, la réexpression de PIM2 dans des cellules cancéreuses exprimant CHES1 permet de rétablir partiellement la prolifération cellulaire. Également, l’analyse sur micropuce a révélé que CHES1 régule l’expression de nombreux gènes impliqués dans la formation des cilia dont l’une des fonctions semble être de moduler la synthèse protéique. Pris ensemble, ces résultats suggèrent que le mécanisme antitumoral de CHES1 consiste en une inhibition de la synthèse de protéines. / Senescence is an antiproliferative defense mechanism that protects the cell against the accumulation of mutations and, eventually, her transformation and the development of a tumor. This program consists of a permanent cell cycle arrest. It can be activated by many stimuli, such as telomere shortening, oxidative stress, or the expression of a constitutively active oncogene. Senescence regulation requires activation of proteins called tumor suppressors which the most important are p53 and RB. More specifically, senescences induced by the expression of oncogenic RASV12 or STAT5A (1 * 6) are respectively characterized by increased expression of the proteins PML and CHES1/FOXN3. The purpose of this thesis is, firstly, to identify the mechanism of regulation of senescence by PML. PML is a tumor suppressor whose expression in normal diploid cells is sufficient to induce senescence. This protein forms spherical nuclear bodies in which is recruited, among other molecules, the retinoblastoma protein RB. RB is a negative regulator of the cell cycle due to its capacity to bind and inhibit the E2F transcription factors whose target genes are necessary for proliferation. Our work demonstrates that the mechanism of induction of senescence by PML involves the recruitment of the complex RB/E2F to the PML body to enhance the inhibition of E2F activity by RB. Also, the E2Fs are recruited to PML bodies together with their promoters which promotes the formation of heterochromatin in these genes, helping their repression and thus the establishment of senescence. On the other hand, this thesis aims to characterize the role of CHES1/FOXN3 in regulating the cell cycle. CHES1 is a transcription factor of the Forkheads family which expression in cancer cells causes a growth reduction. To understand the antitumoral mechanism of CHES1, a microarray analysis of cancer cells expressing CHES1 was performed. This analysis shows that CHES1 is a transcriptional repressor. Specifically, CHES1 represses, among others, the expression of genes required for the synthesis of proteins such as PIM 2 and DYRK3. Interestingly, re-expression of PIM 2 in cancer cells expressing CHES1 partially restores cell proliferation. Also, microarray analysis revealed that CHES1 regulates the expression of many genes involved in the formation of cilia which one of the functions seems to be to modulate protein synthesis. Taken together, these results suggest that the antitumor mechanism of CHES1 involves inhibition of protein synthesis.

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...