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

As sirtuínas 1-7 (SIRT) constituem uma família altamente conservada de desacetilases de histonas que, de modo geral, participam da regulação da longevidade em diversos organismos, modulando a resposta celular frente ao stress oxidativo e promovendo mecanismo de reparo de DNA, parada do ciclo celular, estabilidade telomérica, senescência e apoptose celulares. O envolvimento das SIRTs no processo tumorigênico tem sido bastante investigado, contudo ainda não existe descrição do estudo desses genes nos adenomas hipofisários. O objetivo desse estudo foi avaliar a expressão gênica das SIRT1-7 nos somatotropinomas e adenomas hipofisários clinicamente não funcionantes (ACNF) e sua relação com o tamanho e a invasividade do tumor. A expressão das sirtuínas foi ainda correlacionada à expressão dos marcadores de senescência CDKN1A (p21) e CDKN2A (p16) e do proto-oncogene PTTG (pituitary tumor transforming gene). Foram selecionados 68 pacientes, 37 somatotropinomas e 31 portadores de ACNF. Desses casos, 33 apresentavam tumores invasivos e 35 eram não invasivos. A quantificação do RNAm das SIRT1-7, CDKN1A, CDKN2A e PTTG foi realizada nas amostras tumorais pela técnica de PCR em tempo real utilizando o método de quantificação relativa 2-??Ct. A hiperexpressão da SIRT1 foi observada em 86,5% dos somatotropinomas versus 41,9% dos ACNF (P < 0.01), não sendo observada perda de expressão desse gene. A SIRT3 foi mais hipoexpressa nos ACNF em relação aos somatotropinomas (77,4% e 40,5%, respectivamente; P < 0.01). A SIRT4 foi hipo e hiperexpressa, respectivamente, em 45,2% e 12,9% dos ACNF e 16,2% e 24,3% dos somatotropinomas (P=0.03). A hipoexpressão da SIRT7 também foi maior nos ACNF (67,7%) versus somatotropinomas (32,4%; P=0.01) e, para ambos os subtipos, o percentual de casos apresentando hiperexpressão desse RNAm foi baixo. O padrão de expressão das SIRT2 e 5 não diferiu entre os subtipos tumorais e não se mostrou alterado em relação ao pool de hipófises normais. Não foi observada diferença estatisticamente significante na expressão dos genes das sirtuínas entre os grupos de tumores invasivos e não invasivos. Contudo, a expressão das SIRT1 e 3 foi relacionada ao tamanho tumoral; nos casos com hiperexpressão da SIRT1 a média do maior diâmetro tumoral foi 2.4 ± 1.1 enquanto nos pacientes com expressão normal foi de 3.3 ± 1.3 (P < 0.01). Já os casos com perda de expressão da SIRT3 apresentaram tumores maiores (3.1 ± 1.2) em relação aos casos com expressão normal (2.2 ± 1.1; P < 0.01). A expressão de todas as SIRTs apresentou correlação positiva moderada (SIRT1-5,7) ou forte (SIRT6) com a expressão do CDKN1A. Uma correlação positiva foi observada também em relação a expressão do CDKN2A. Contudo, essa foi fraca e presente apenas para as SIRTs 3-5. Em relação ao PTTG, foi observado apenas uma fraca correlação com a expressão da SIRT1 e SIRT3. Em conclusão, esses resultados sugerem que a hiperexpressão de SIRT1 e a hipoexpressão das SIRTs 3, 4 e 7 podem estar relacionadas ao processo tumorigênico nos somatotropinomas e ACNFs, respectivamente e, em especial as SIRT1 e 3, ao controle da proliferação celular nesses adenomas / Sirtuins 1-7 (SIRT) are a highly conserved family of histone deacetylases. In general, these proteins are involved in the regulation of longevity in several organisms, modulating the cellular response to oxidative stress. SIRTs can also regulate DNA repair, telomeric stability, cell senescence and apoptosis. Due to their functions, there is a growing interest in the role of sirtuins in tumorigenesis. However, these genes were not investigated in pituitary tumors so far. In this study, SIRT1-7 gene expression was evaluated in somatotropinomas and nonfunctioning pituitary adenomas (NFPA) and related to tumor size and invasiveness. SIRT1-7 expression was also correlated with cellular senescence markers CDKN1A (p21) e CDKN2A (p16) and with the proto-oncogene PTTG (pituitary tumor transforming gene). Sixty-eight patients were selected, 37 with somatotropinomas and 31 with NFPA. Tumor invasion was observed in 33 patients. SIRT1-7, CDKN1A, CDKN2A and PTTG mRNA levels was evaluated from pituitary tumor samples by the real-time PCR using 2-??Ct relative quantification. Pronounced differences in SIRT1, 3, 4 and 7 expressions were identified between somatotropinomas and NFPA. Overexpression of SIRT1 was observed in 86.5% of somatotropinomas versus 41.9% of NFPA (P < 0.01) whereas underexpression was not detected. SIRT3 was more underexpressed in NFPA than somatotropinomas (77.4% and 40.5%, respectively, P < 0.01). SIRT4 was under and overexpressed, respectively, in 45.2% and 12.9% of NFPA and 16.2% and 24.3% of somatotropinomas (P=0.03). SIRT7 underexpression was also higher in NFPAs (67.7%) versus somatotropinomas (32.4%; P=0.01) with few cases showing overexpression. SIRT2 and 5 expressions did not differ between tumors subtypes and was not altered in relation to the normal pituitary gland pool. No statistically significant difference was observed in the expression of these genes between invasive and non-invasive tumor groups. However, SIRT1 and 3 expressions were related to tumor size. Mean of the largest tumor diameter was 2.4 ± 1.1 and 3.3 ± 1.3 (P < 0.01) in adenomas with SIRT1 over- and normal expression, respectively. On the other hand, cases with SIRT3 underepression exhibited larger tumors (3.1 ± 1.2) compared to cases with SIRT3 normal expression (2.2 ± 1.1, P < 0.01). Moderated (SIRT1-5.7) or strong (SIRT6) positive correlation was observed between sirtuins and CDKN1A expression. A weak correlation was observed with respect to CDKN2A expression and SIRTs 3-5. Regarding PTTG mRNA, only a weak correlation with SIRT1 and SIRT3 expression was observed. In conclusion, these results suggest that overexpression of SIRT1 and underexpression of SIRTs 3, 4 and 7 could be related to the tumorigenic process in somatotropinomas and NFPAs, respectively. SIRT1 and 3 could also play a role in control of pituitary adenomas cell proliferation

La sénescence cellulaire est une réponse à un stress des cellules de mammifère caractérisée par un arrêt durable du cycle cellulaire. Celle-ci peut être déclenchée par un dysfonctionnement des télomères, des stress génotoxiques et l’activation d’oncogènes. La sénescence constitue une puissante ligne de défense contre le développement de cancers et intervient aussi dans le vieillissement. Les cellules en sénescence réorganisent leur génome par l’assemblage en hétérochromatine sous forme de SAHFs (senescence-associated heterochromatin foci). Nous avons mis en évidence que la désacétylation globale de H4-K16Ac par la désacétylase SIRT2 est impliquée dans l’assemblage de l’hétérochromatine en sénescence. De plus, nous avons identifié une accumulation de variants d’histones H2A et H2B spécifiquement dans des cellules en sénescence présentant des dommages persistants à l’ADN. Ces variants d’histone pourraient avoir des fonctions spécifiques dans ces cellules et pourraient représenter un biomarqueur du vieillissement in vivo.Mes travaux apportent des éléments pour la compréhension des rôles de l’information épigénétique dans la sénescence cellulaire. / Cellular senescence is a stress response of mammalian cells characterized by a stable cell proliferation arrest. It can be triggered by telomere dysfunction, genotoxic stress and oncogene activation. Cellular senescence acts as a natural barrier against cancer development and is involved in ageing. Senescent cells reorganize their genome by the assembly of chromatin into senescence-associated heterochromatin foci (SAHF). We showed that SIRT2-mediated global deacetylation of H4-K16Ac is involved in heterochromatin assembly in senescence. Moreover, we identified the accumulation with time of specific H2A and H2B variants in senescence triggered by persistent DNA damage signaling. These histone variants could have specific functions in senescent cells and could be a useful ageing biomarker in vivo.This work provides novel insights into chromatin modification and epigenetic regulation in cellular senescence.

El síndrome de Down constituye la cromosomopatía más frecuente que ocurre en uno de cada 700 a 1000 nacimientos y está causado por la trisomía completa o por una parte del cromosoma 21 humano (HSA21). Aún se desconoce cómo la presencia del cromosoma 21 extra da lugar al fenotipo característico de este síndrome. En este sentido la participación de las especies reactivas de oxígeno (ROS) ha sido propuesta como uno de los mecanismos que intervienen en la patogénesis del mismo. Dicho mecanismo se fundamenta en la sobreexpresión de al menos 16 genes del HSA21 relacionados con el metabolismo de las especies reactivas de oxígeno (ROS) y con la generación de energía mitocondrial. Uno de estos genes es el que codifica una importante enzima del sistema antioxidante celular, el gen SOD1, propuesto como potencial culpable del estrés oxidativo inusual en los individuos con SD. En condiciones normales, los ROS, producidos in vivo principalmente por la respiración aeróbica, se eliminan de la célula por la acción de las enzimas antioxidantes, superóxido dismutasa (SOD), catalasa (CAT) y glutatión peroxidasa (GPx). La Cu/Zn superóxido dismutasa (SOD1) convierte el radical superóxido a peróxido de hidrógeno, el cual es eliminado por la glutatión peroxidasa y/o catalasa a agua y oxígeno. La sobreexpresión de la SOD1 puede producir un desequilibrio en la relación de las enzimas antioxidantes (SOD1, GPx y CAT) generando estrés oxidativo y podría resultar en el daño oxidativo a biomoléculas tales como ácidos grasos poliinsaturados en los lípidos de las membranas, proteínas esenciales y el DNA, ya que existe una variabilidad en los niveles de enzimas antioxidantes dentro de la población con SD, lo que puede estar relacionado con una desregulación compleja que afecte no sólo a los genes del HSA21 sino también en otros cromosomas. Así, el daño celular puede ser inducido por los ROS y asociarse a algunas de las alteraciones celulares en el SD, causando diversas patologías y conducir a un envejecimiento prematuro. Se obtuvieron 18 muestras de fibroblastos primarios fetales humanos, 9 de ellos con síndrome de Down (FT21) y 9 controles (FC), en los cuales se evaluó la disminución de la capacidad endógena antioxidante debido a la sobreexpresión de la SOD1, causando un exceso en la producción intracelular de ROS y el origen prematuro de estrés oxidativo asociado al daño oxidativo a lípidos y proteínas, así como a una disfunción mitocondrial. Se analizaron varios marcadores de senescencia celular con la finalidad de contribuir al conocimiento de un nuevo aspecto de la patología de este síndrome, el envejecimiento prematuro. Estos mecanismos fisiopatológicos podrían estar relacionados con la aparición y establecimiento de la senescencia celular prematura en los fibroblastos con trisomía del cromosoma 21 (FT21). / Down syndrome is the most common chromosomal disorder that occurs in 1 in 700 to 1,000 births and is caused by trisomy full or part of human chromosome 21 (HSA21). It is still unknown how the presence of the extra chromosome 21 results in the phenotype of this syndrome. In this sense the involvement of reactive oxygen species (ROS) has been proposed as one of the mechanisms involved in the pathogenesis of same. This mechanism is based on the overexpression of at least 16 genes related HSA21 metabolism of reactive oxygen species (ROS) and mitochondrial energy generation. One of these genes encoding is an important cellular antioxidant enzyme system, the SOD1 gene, proposed as a potential culprit unusual oxidative stress in individuals with DS. Under normal conditions, the ROS produced in vivo mainly by aerobic respiration of the cells are removed by the action of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). The Cu/Zn superoxide dismutase (SOD1) converts the superoxide radical to hydrogen peroxide, which is eliminated by glutathione peroxidase and/or catalase into water and oxygen. SOD1 overexpression can produce an imbalance in the ratio of antioxidant enzymes (SOD1, GPx and CAT) generating oxidative stress and may result in oxidative damage to biomolecules such as polyunsaturated fatty acids in the membrane lipids, proteins and essential DNA, since there is a variability in the levels of antioxidant enzymes in the DS population, which can be related to a complex deregulation affects not only Hsa21 genes but also on other chromosomes. Thus, cell damage can be induced by ROS and associated with some of the cellular changes in the DS, causing various diseases and lead to premature aging. Eighteen samples were obtained from primary human fetal fibroblasts, 9 with Down syndrome (TF21) and 9 normal (NF), which was evaluated in decreasing endogenous antioxidant capacity due to overexpression of the SOD1, causing excess in intracellular production of ROS and oxidative stress origin associated premature oxidative damage to lipids and proteins, as well as mitochondrial dysfunction. We analyzed several markers of cellular senescence in order to contribute to the knowledge of a new aspect of the pathology of this syndrome, premature aging. These pathophysiological mechanisms may be related to the emergence and development of premature cellular senescence in fibroblasts with trisomy 21 (FT21).

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.

Evidências demonstram uma associação entre o aumento dos níveis de estresse crônico e o desenvolvimento de diferentes patologias, promovendo um desgaste do organismo que acelera a taxa de declínio das funções fisiológicas levando a senescência celular. A possibilidade de identificar potenciais biomarcadores em estágios iniciais de exposição a condições adversas e acompanhá-los ao longo da progressão clínica, podem torná-los ferramentas extremamente úteis no esclarecimento de diversas desordens. As sequências finais dos cromossomos, denominadas telômeros, representam um novo biomarcador de senescência celular. São responsivos a mudanças ambientais e parecem ter um papel essencial no ajuste das respostas fisiológicas e socioambientais. O encurtamento dos telômeros ao longo do ciclo vital está associado a diferentes variáveis de estilo de vida, estresse oxidativo ou psicológico e doenças crônicas, sugerindo que o comprimento dos telômeros pode ser reconhecido como um bom indicador do estado geral de saúde e da idade biológica dos indivíduos. Dessa forma, o objetivo desse estudo foi padronizar e estabelecer uma técnica para a determinação do comprimento relativo de telômeros por qPCR (através da razão T/S), a partir de três tecidos diferentes, e validar a técnica em três desordens diferentes em relação a suas respectivas populações controle. A análise dos resultados demonstrou que foi possível padronizar a detecção do comprimento relativo de telômeros em leucócitos e em células mononucleares de sangue periférico (PBMC), baseado na técnica de qPCR. Utilizamos amostras de três coortes diferentes para validar nossa padronização de qPCR. Na primeira coorte não observamos diferenças no comprimento de telômeros em leucócitos de crianças ex-prematuras e seus controles (teste Mann-Whitney, P = 0,5219). Todavia, crianças portadoras de asma severa apresentaram telômeros de leucócitos significativamente mais curtos que as crianças portadoras de asma leve ou do grupo controle (teste Kruskal-Wallis, P = 0,0426). Ainda, detectamos um encurtamento significativo de telômeros entre o grupo asma severa, quando comparadas ao grupo controle e ao grupo asma leve (pós-teste de Dunn, P < 0,05). Além disso, observamos uma diminuição do comprimento de telômeros em PBMC de adultos portadores de obesidade severa e o seu grupo controle (teste Mann-Whitney, P = 0,0006). Esse efeito foi significativo e se manteve presente após ajuste para idade (teste Mann-Whitney, P = 0,026). Também observamos uma correlação inversa significativa entre a o comprimento de telômeros e a idade de indivíduos portadores de obesidade (r = -0,4108, P = 0,0116) e o IMC de ambos os grupos (r = -0,4174, P = 0,0005). De acordo com a análise dos resultados desse estudo, em consonância com dados obtidos na literatura, é possível inferir que telômeros podem se tornar uma importante ferramenta para auxiliar em estudos sobre as bases moleculares do desenvolvimento de diversas patologias, com a finalidade de elucidar os mecanismos que possam acelerar o processo de envelhecimento. / Evidences demonstrate an association between increased levels of chronic stress and development of various pathologies, promoting damage to the organism which accelerates the decline of physiological functions leading to cellular senescence. The ability to identify biomarkers in early stages of exposure to adverse conditions and accompany them along clinical progression can make them extremely useful tools to elucidate several disorders. Telomeres, the ends of linear chromosomes, represent a new biomarker of cellular senescence. They are responsive to environmental changes and appear to play an essential role in the setting of physiological and social responses. The telomere shortening over the life cycle is associated with different lifestyle variables, oxidative or psychological stress and chronic diseases, suggesting that telomere length can be recognized as a good indicator of general health and biological age of individuals. Thus, the aim of this study was to establish a technique to detect relative telomere length by qPCR (T/S ratio) from three different tissues and validate the technique in three different disorders in relation to their respective controls. The results demonstrated that it was possible to standardize the detection of the relative telomere length in leukocytes and in PBMC using a qPCR-based method. We used samples from three different cohorts to validate our qPCR. We didn't observed a difference in telomere length in leukocyte from ex-preterm children and control (Mann-Whitney test, P = 0.5219). The severe asthma group had significantly shorter telomeres when compared to the control and mild asthma group (Kruskal-Wallis test, P = 0,0426) and . There was a significant difference when severe asthma were compared to diferença significativa entre o grupo asma severa, quando comparada ao asma leve (teste pos-hoc de Dunn, P < 0,05). We also observed a significant difference in telomere length in PBMC in adults with obesity when compared to controls (Mann-Whitney test, P = 0.0006). This effect was still present, and remained so after adjustment for age (Mann-Whitney test, P = 0.026). We also observed a significant inverse correlation between the telomere length and adults with obesity (r = -0.4108, P = 0.0116), and BMI in both groups (r = -0.4174, P = 0.0005). According to the analysis of this study and in line with data from the literature, we can infer that telomeres might become important tools to support studies on the molecular basis of the development of various diseases, in order to elucidate mechanisms that might accelerate the aging process.

Evidências demonstram uma associação entre o aumento dos níveis de estresse crônico e o desenvolvimento de diferentes patologias, promovendo um desgaste do organismo que acelera a taxa de declínio das funções fisiológicas levando a senescência celular. A possibilidade de identificar potenciais biomarcadores em estágios iniciais de exposição a condições adversas e acompanhá-los ao longo da progressão clínica, podem torná-los ferramentas extremamente úteis no esclarecimento de diversas desordens. As sequências finais dos cromossomos, denominadas telômeros, representam um novo biomarcador de senescência celular. São responsivos a mudanças ambientais e parecem ter um papel essencial no ajuste das respostas fisiológicas e socioambientais. O encurtamento dos telômeros ao longo do ciclo vital está associado a diferentes variáveis de estilo de vida, estresse oxidativo ou psicológico e doenças crônicas, sugerindo que o comprimento dos telômeros pode ser reconhecido como um bom indicador do estado geral de saúde e da idade biológica dos indivíduos. Dessa forma, o objetivo desse estudo foi padronizar e estabelecer uma técnica para a determinação do comprimento relativo de telômeros por qPCR (através da razão T/S), a partir de três tecidos diferentes, e validar a técnica em três desordens diferentes em relação a suas respectivas populações controle. A análise dos resultados demonstrou que foi possível padronizar a detecção do comprimento relativo de telômeros em leucócitos e em células mononucleares de sangue periférico (PBMC), baseado na técnica de qPCR. Utilizamos amostras de três coortes diferentes para validar nossa padronização de qPCR. Na primeira coorte não observamos diferenças no comprimento de telômeros em leucócitos de crianças ex-prematuras e seus controles (teste Mann-Whitney, P = 0,5219). Todavia, crianças portadoras de asma severa apresentaram telômeros de leucócitos significativamente mais curtos que as crianças portadoras de asma leve ou do grupo controle (teste Kruskal-Wallis, P = 0,0426). Ainda, detectamos um encurtamento significativo de telômeros entre o grupo asma severa, quando comparadas ao grupo controle e ao grupo asma leve (pós-teste de Dunn, P < 0,05). Além disso, observamos uma diminuição do comprimento de telômeros em PBMC de adultos portadores de obesidade severa e o seu grupo controle (teste Mann-Whitney, P = 0,0006). Esse efeito foi significativo e se manteve presente após ajuste para idade (teste Mann-Whitney, P = 0,026). Também observamos uma correlação inversa significativa entre a o comprimento de telômeros e a idade de indivíduos portadores de obesidade (r = -0,4108, P = 0,0116) e o IMC de ambos os grupos (r = -0,4174, P = 0,0005). De acordo com a análise dos resultados desse estudo, em consonância com dados obtidos na literatura, é possível inferir que telômeros podem se tornar uma importante ferramenta para auxiliar em estudos sobre as bases moleculares do desenvolvimento de diversas patologias, com a finalidade de elucidar os mecanismos que possam acelerar o processo de envelhecimento. / Evidences demonstrate an association between increased levels of chronic stress and development of various pathologies, promoting damage to the organism which accelerates the decline of physiological functions leading to cellular senescence. The ability to identify biomarkers in early stages of exposure to adverse conditions and accompany them along clinical progression can make them extremely useful tools to elucidate several disorders. Telomeres, the ends of linear chromosomes, represent a new biomarker of cellular senescence. They are responsive to environmental changes and appear to play an essential role in the setting of physiological and social responses. The telomere shortening over the life cycle is associated with different lifestyle variables, oxidative or psychological stress and chronic diseases, suggesting that telomere length can be recognized as a good indicator of general health and biological age of individuals. Thus, the aim of this study was to establish a technique to detect relative telomere length by qPCR (T/S ratio) from three different tissues and validate the technique in three different disorders in relation to their respective controls. The results demonstrated that it was possible to standardize the detection of the relative telomere length in leukocytes and in PBMC using a qPCR-based method. We used samples from three different cohorts to validate our qPCR. We didn't observed a difference in telomere length in leukocyte from ex-preterm children and control (Mann-Whitney test, P = 0.5219). The severe asthma group had significantly shorter telomeres when compared to the control and mild asthma group (Kruskal-Wallis test, P = 0,0426) and . There was a significant difference when severe asthma were compared to diferença significativa entre o grupo asma severa, quando comparada ao asma leve (teste pos-hoc de Dunn, P < 0,05). We also observed a significant difference in telomere length in PBMC in adults with obesity when compared to controls (Mann-Whitney test, P = 0.0006). This effect was still present, and remained so after adjustment for age (Mann-Whitney test, P = 0.026). We also observed a significant inverse correlation between the telomere length and adults with obesity (r = -0.4108, P = 0.0116), and BMI in both groups (r = -0.4174, P = 0.0005). According to the analysis of this study and in line with data from the literature, we can infer that telomeres might become important tools to support studies on the molecular basis of the development of various diseases, in order to elucidate mechanisms that might accelerate the aging process.

As sirtuínas 1-7 (SIRT) constituem uma família altamente conservada de desacetilases de histonas que, de modo geral, participam da regulação da longevidade em diversos organismos, modulando a resposta celular frente ao stress oxidativo e promovendo mecanismo de reparo de DNA, parada do ciclo celular, estabilidade telomérica, senescência e apoptose celulares. O envolvimento das SIRTs no processo tumorigênico tem sido bastante investigado, contudo ainda não existe descrição do estudo desses genes nos adenomas hipofisários. O objetivo desse estudo foi avaliar a expressão gênica das SIRT1-7 nos somatotropinomas e adenomas hipofisários clinicamente não funcionantes (ACNF) e sua relação com o tamanho e a invasividade do tumor. A expressão das sirtuínas foi ainda correlacionada à expressão dos marcadores de senescência CDKN1A (p21) e CDKN2A (p16) e do proto-oncogene PTTG (pituitary tumor transforming gene). Foram selecionados 68 pacientes, 37 somatotropinomas e 31 portadores de ACNF. Desses casos, 33 apresentavam tumores invasivos e 35 eram não invasivos. A quantificação do RNAm das SIRT1-7, CDKN1A, CDKN2A e PTTG foi realizada nas amostras tumorais pela técnica de PCR em tempo real utilizando o método de quantificação relativa 2-??Ct. A hiperexpressão da SIRT1 foi observada em 86,5% dos somatotropinomas versus 41,9% dos ACNF (P < 0.01), não sendo observada perda de expressão desse gene. A SIRT3 foi mais hipoexpressa nos ACNF em relação aos somatotropinomas (77,4% e 40,5%, respectivamente; P < 0.01). A SIRT4 foi hipo e hiperexpressa, respectivamente, em 45,2% e 12,9% dos ACNF e 16,2% e 24,3% dos somatotropinomas (P=0.03). A hipoexpressão da SIRT7 também foi maior nos ACNF (67,7%) versus somatotropinomas (32,4%; P=0.01) e, para ambos os subtipos, o percentual de casos apresentando hiperexpressão desse RNAm foi baixo. O padrão de expressão das SIRT2 e 5 não diferiu entre os subtipos tumorais e não se mostrou alterado em relação ao pool de hipófises normais. Não foi observada diferença estatisticamente significante na expressão dos genes das sirtuínas entre os grupos de tumores invasivos e não invasivos. Contudo, a expressão das SIRT1 e 3 foi relacionada ao tamanho tumoral; nos casos com hiperexpressão da SIRT1 a média do maior diâmetro tumoral foi 2.4 ± 1.1 enquanto nos pacientes com expressão normal foi de 3.3 ± 1.3 (P < 0.01). Já os casos com perda de expressão da SIRT3 apresentaram tumores maiores (3.1 ± 1.2) em relação aos casos com expressão normal (2.2 ± 1.1; P < 0.01). A expressão de todas as SIRTs apresentou correlação positiva moderada (SIRT1-5,7) ou forte (SIRT6) com a expressão do CDKN1A. Uma correlação positiva foi observada também em relação a expressão do CDKN2A. Contudo, essa foi fraca e presente apenas para as SIRTs 3-5. Em relação ao PTTG, foi observado apenas uma fraca correlação com a expressão da SIRT1 e SIRT3. Em conclusão, esses resultados sugerem que a hiperexpressão de SIRT1 e a hipoexpressão das SIRTs 3, 4 e 7 podem estar relacionadas ao processo tumorigênico nos somatotropinomas e ACNFs, respectivamente e, em especial as SIRT1 e 3, ao controle da proliferação celular nesses adenomas / Sirtuins 1-7 (SIRT) are a highly conserved family of histone deacetylases. In general, these proteins are involved in the regulation of longevity in several organisms, modulating the cellular response to oxidative stress. SIRTs can also regulate DNA repair, telomeric stability, cell senescence and apoptosis. Due to their functions, there is a growing interest in the role of sirtuins in tumorigenesis. However, these genes were not investigated in pituitary tumors so far. In this study, SIRT1-7 gene expression was evaluated in somatotropinomas and nonfunctioning pituitary adenomas (NFPA) and related to tumor size and invasiveness. SIRT1-7 expression was also correlated with cellular senescence markers CDKN1A (p21) e CDKN2A (p16) and with the proto-oncogene PTTG (pituitary tumor transforming gene). Sixty-eight patients were selected, 37 with somatotropinomas and 31 with NFPA. Tumor invasion was observed in 33 patients. SIRT1-7, CDKN1A, CDKN2A and PTTG mRNA levels was evaluated from pituitary tumor samples by the real-time PCR using 2-??Ct relative quantification. Pronounced differences in SIRT1, 3, 4 and 7 expressions were identified between somatotropinomas and NFPA. Overexpression of SIRT1 was observed in 86.5% of somatotropinomas versus 41.9% of NFPA (P < 0.01) whereas underexpression was not detected. SIRT3 was more underexpressed in NFPA than somatotropinomas (77.4% and 40.5%, respectively, P < 0.01). SIRT4 was under and overexpressed, respectively, in 45.2% and 12.9% of NFPA and 16.2% and 24.3% of somatotropinomas (P=0.03). SIRT7 underexpression was also higher in NFPAs (67.7%) versus somatotropinomas (32.4%; P=0.01) with few cases showing overexpression. SIRT2 and 5 expressions did not differ between tumors subtypes and was not altered in relation to the normal pituitary gland pool. No statistically significant difference was observed in the expression of these genes between invasive and non-invasive tumor groups. However, SIRT1 and 3 expressions were related to tumor size. Mean of the largest tumor diameter was 2.4 ± 1.1 and 3.3 ± 1.3 (P < 0.01) in adenomas with SIRT1 over- and normal expression, respectively. On the other hand, cases with SIRT3 underepression exhibited larger tumors (3.1 ± 1.2) compared to cases with SIRT3 normal expression (2.2 ± 1.1, P < 0.01). Moderated (SIRT1-5.7) or strong (SIRT6) positive correlation was observed between sirtuins and CDKN1A expression. A weak correlation was observed with respect to CDKN2A expression and SIRTs 3-5. Regarding PTTG mRNA, only a weak correlation with SIRT1 and SIRT3 expression was observed. In conclusion, these results suggest that overexpression of SIRT1 and underexpression of SIRTs 3, 4 and 7 could be related to the tumorigenic process in somatotropinomas and NFPAs, respectively. SIRT1 and 3 could also play a role in control of pituitary adenomas cell proliferation

Evidências demonstram uma associação entre o aumento dos níveis de estresse crônico e o desenvolvimento de diferentes patologias, promovendo um desgaste do organismo que acelera a taxa de declínio das funções fisiológicas levando a senescência celular. A possibilidade de identificar potenciais biomarcadores em estágios iniciais de exposição a condições adversas e acompanhá-los ao longo da progressão clínica, podem torná-los ferramentas extremamente úteis no esclarecimento de diversas desordens. As sequências finais dos cromossomos, denominadas telômeros, representam um novo biomarcador de senescência celular. São responsivos a mudanças ambientais e parecem ter um papel essencial no ajuste das respostas fisiológicas e socioambientais. O encurtamento dos telômeros ao longo do ciclo vital está associado a diferentes variáveis de estilo de vida, estresse oxidativo ou psicológico e doenças crônicas, sugerindo que o comprimento dos telômeros pode ser reconhecido como um bom indicador do estado geral de saúde e da idade biológica dos indivíduos. Dessa forma, o objetivo desse estudo foi padronizar e estabelecer uma técnica para a determinação do comprimento relativo de telômeros por qPCR (através da razão T/S), a partir de três tecidos diferentes, e validar a técnica em três desordens diferentes em relação a suas respectivas populações controle. A análise dos resultados demonstrou que foi possível padronizar a detecção do comprimento relativo de telômeros em leucócitos e em células mononucleares de sangue periférico (PBMC), baseado na técnica de qPCR. Utilizamos amostras de três coortes diferentes para validar nossa padronização de qPCR. Na primeira coorte não observamos diferenças no comprimento de telômeros em leucócitos de crianças ex-prematuras e seus controles (teste Mann-Whitney, P = 0,5219). Todavia, crianças portadoras de asma severa apresentaram telômeros de leucócitos significativamente mais curtos que as crianças portadoras de asma leve ou do grupo controle (teste Kruskal-Wallis, P = 0,0426). Ainda, detectamos um encurtamento significativo de telômeros entre o grupo asma severa, quando comparadas ao grupo controle e ao grupo asma leve (pós-teste de Dunn, P < 0,05). Além disso, observamos uma diminuição do comprimento de telômeros em PBMC de adultos portadores de obesidade severa e o seu grupo controle (teste Mann-Whitney, P = 0,0006). Esse efeito foi significativo e se manteve presente após ajuste para idade (teste Mann-Whitney, P = 0,026). Também observamos uma correlação inversa significativa entre a o comprimento de telômeros e a idade de indivíduos portadores de obesidade (r = -0,4108, P = 0,0116) e o IMC de ambos os grupos (r = -0,4174, P = 0,0005). De acordo com a análise dos resultados desse estudo, em consonância com dados obtidos na literatura, é possível inferir que telômeros podem se tornar uma importante ferramenta para auxiliar em estudos sobre as bases moleculares do desenvolvimento de diversas patologias, com a finalidade de elucidar os mecanismos que possam acelerar o processo de envelhecimento. / Evidences demonstrate an association between increased levels of chronic stress and development of various pathologies, promoting damage to the organism which accelerates the decline of physiological functions leading to cellular senescence. The ability to identify biomarkers in early stages of exposure to adverse conditions and accompany them along clinical progression can make them extremely useful tools to elucidate several disorders. Telomeres, the ends of linear chromosomes, represent a new biomarker of cellular senescence. They are responsive to environmental changes and appear to play an essential role in the setting of physiological and social responses. The telomere shortening over the life cycle is associated with different lifestyle variables, oxidative or psychological stress and chronic diseases, suggesting that telomere length can be recognized as a good indicator of general health and biological age of individuals. Thus, the aim of this study was to establish a technique to detect relative telomere length by qPCR (T/S ratio) from three different tissues and validate the technique in three different disorders in relation to their respective controls. The results demonstrated that it was possible to standardize the detection of the relative telomere length in leukocytes and in PBMC using a qPCR-based method. We used samples from three different cohorts to validate our qPCR. We didn't observed a difference in telomere length in leukocyte from ex-preterm children and control (Mann-Whitney test, P = 0.5219). The severe asthma group had significantly shorter telomeres when compared to the control and mild asthma group (Kruskal-Wallis test, P = 0,0426) and . There was a significant difference when severe asthma were compared to diferença significativa entre o grupo asma severa, quando comparada ao asma leve (teste pos-hoc de Dunn, P < 0,05). We also observed a significant difference in telomere length in PBMC in adults with obesity when compared to controls (Mann-Whitney test, P = 0.0006). This effect was still present, and remained so after adjustment for age (Mann-Whitney test, P = 0.026). We also observed a significant inverse correlation between the telomere length and adults with obesity (r = -0.4108, P = 0.0116), and BMI in both groups (r = -0.4174, P = 0.0005). According to the analysis of this study and in line with data from the literature, we can infer that telomeres might become important tools to support studies on the molecular basis of the development of various diseases, in order to elucidate mechanisms that might accelerate the aging process.

Therapy resistance of malignant cells represents the main reason responsible for the failure of cancer therapy. The growth of malignant cells at primary tumour sites but most importantly the dissemination of tumour cells and their growth at secondary sites, are the main reasons why patients eventually succumb to the disease. Even novel immune-based therapies find their limitation in most tumour types. The therapy resistance is mediated by the tumour cells but also by other cellular components of the tumour microenvironment. Understanding the tumour cells mechanisms and the tumour microenvironment features responsible for therapy resistance enables the development of novel therapeutic strategies. Here, we show that ionizing irradiation, 5-azacytidine, and IFNγ treatments induced expression of suprabasin (SBSN) and therapy-resistant low-adherent phenotype in cancer cells. Knockdown of SBSN resulted in suppression of the phenotype. Next, we identified aberrantly elevated SBSN in the bone marrow of a subgroup of myelodysplastic syndromes (MDS) patients. SBSN was expressed by myeloid-derived suppressor cells (MDSCs) and showed significant anti-correlation with T cell abundance and CCL2 levels, hence promises a prognostic value in clinical use. We compiled the most of the relevant knowledge of SBSN...