4 Abstract Senescence is a specific cell state distinquished by cessation of cell division and proliferation and changes in gene expression. Normal cells enter senescence after distinct number of cell divisions or in case of an unrepairable damage. Senescence in cancer cells can be induced by subliminal stress as sublethal treatment with certain drugs. Senescent cancer cells persist in the tissue and may secrete a number of factors and nutrients affecting surrounding cells. Senescence can thus change the response of cancer cells to various apoptogens during cancer therapy. In this study, we focused on the elucidation of presumed differences between normal proliferating and senescent cancer cells in their response to selected apoptogens. Implementing bromodeoxyuridine (BrdU)-mediated replication stress in cancer cells derived from pancreatic (PANC-1) or mesothelioma (H28) tumors, we efficiently forced these cells to acquire senescent phenotype. We document that these senescent cells gain higher resistance to combined TRAIL and homoharringtonine (HHT) treatment and enhance sensitivity to other apoptogens such as FasL, camptothecin and mVES. These cells also showed increased expression of anti-apoptotic protein c-FLIP in senescent cells and changes in the expression of some Bcl-2 family proteins....

World population is ageing and in Europe the situation is especially alarming. In the next two decades the ratio of people over 65 years of age is expected to double. This threatens long-term sustainability of welfare systems. The debate about future developments has been going on since the turn of the century. Based on this debate the European Union defined improving the inclusion of people over 50 as the main solution to this problem. The means to achieving this goal have been identified at the level of the labour market, disease prevention and lifelong learning. The specific instruments are: 1) increasing the participation of older workers in the labour market by increasing retirement age, introducing penalties for early retirement, promoting retraining of older and increasing retraining efficiency, 2) restructuring of pension systems and provision of social services and 3) strengthening participation of older people in society, applying the principles of active aging at all levels of social life and increasing the quality and variety of lifelong learning opportunities. This paper analyses the situation of persons over 50 years of age in the Czech Republic in terms of their position in the labour market, participation in social life and quality of life in the context of the strategic concept of...

Eine seneszente Zelle erfährt proteotoxischen Stress durch einen Prozess, der als “Seneszenz-assoziierter sekretorischer Phänotyp (SASP)” bezeichnet wird. SASP Faktoren involvieren vermehrte Produktion von Cytokinen und Chemokinen, die die Proteinhomöostase stören. Das führt zu Stress im Endoplasmatischen Retikulum (ER), Unfolded Protein Response (UPR) sowie erhöhter Autophagie in seneszenten Zellen. In dieser Arbeit zeigen wir, dass Therapie-induzierte Seneszenz (TIS), ausgelöst durch Adriamycin (ADR) oder Cyclophosphamid (CTX), zu einer erhöhten Expression von Alzheimer-assoziierten Genen führt. Das ist allerdings nur der Fall, wenn TIS mit SASP und Proteotoxizität korreliert. Außerdem zeigen wir, dass transgene Mäuse mit Alzheimer ebenso Eigenschaften von Seneszenz aufweisen. Mutationen in Genen wie Amyloid-Precursor-Protein (APP) und Presenilin 1 (PSEN1) erzeugen nicht nur Alzheimer, sondern verstärken auch Seneszenz. In APPPS1+/- transgenen Mäusen treten seneszente Zellen zusammen mit Amyloid β (Aβ) Plaques auf. Gleichfalls führt die Überexpression von APP in der Neuroblastom Zelllinie SH-SY5Y zu einer Erhöhung von Seneszenz-Markern, einschließlich ER Stress und Autophagie-assoziierten Lysosomen. Diese Beobachtungen implizieren einen Zusammenhang zwischen Seneszenz und Alzheimer. Wir präsentieren Proteotoxizität als gemeinsamen Nenner dieser beiden Pathologien. Allerdings kommen wir zu dem Ergebnis, dass APP und PSEN1 keine Regulatoren von TIS sind. APP trägt zur TIS-assoziierten Proteotoxizität bei, da ein knock-down von APP zu einer Reduzierung des ER Stress führt. Außerdem, können seneszente Zellen mittels Blockierung der APP Spaltung und somit Bildung von Aβ Plaques durch den Gamma-Sekretase Inhibitor Semagacestat, selektiv getötet werden. Das deutet auf ein senolytisches Potential hin. Allerdings hat Semagacestat keinen Einfluss auf die Expression von ER Stress Marker. Dies suggeriert, dass APP in Eμ-myc Lymphomen Aβ-unabhängig zu ER Stress beiträgt, während diese in Alzheimer eine zentrale Rolle in der Phathophysiologie spielen. / A senescent cell experiences proteotoxic stress in consequence to senescence-associated secretory phenotype (SASP). SASP factors involve increased production of cytokines and chemokines, which overload and disrupt protein homeostasis. This induces endoplasmic reticulum (ER) stress, unfolded protein response (UPR) as well as elevated autophagic-lysosomal burden in senescent cells, further promoting the formation of misfolded proteins. In this thesis we found, in Eμ-myc transgenic mice lymphomas, therapy-induced senescence (TIS) by Adriamycin or Cyclophosphamide treatment leads to an up regulation of genes involved in Alzheimer’s disease (AD). However, this is true only when TIS correlates with SASP, leading to proteotoxicity. On the other hand, we demonstrate that transgenic AD inflicted mice also present with features of senescence. Mutations of genes such as amyloid precursor protein (APP) and presenilin1 (PSEN1), not only engender AD, but also augment senescence. In APPPS1+/- transgenic mice, the senescent cells co-occur with amyloid β (Aβ) plaques. Likewise, in neuroblastoma cell line SH-SY5Y, APP overexpression results in upregulation of senescence markers, including markers for ER stress and autophagic-lysosomal burden. These observations imply that there is a crosstalk between senescence and AD. We demonstrate proteotoxicity as a common denominator between the two pathologies. However, we found that APP and PSEN1 are not regulators of TIS. APP contributes to proteotoxic stress associated with TIS as knocking down of APP leads to a reduction of ER stress. Inhibiting Aβ formation by targeting the processing of APP using Semagacestat- a gamma-secretase inhibitor, selectively targets senescent cells resulting in cell death. This indicates its senolytic potential. However, Semagacestat has no impact on the expression of ER stress markers. This suggested that in Eμ-myc lymphomas, APP contributes to ER stress in an Aβ-independent fashion, unlike in AD, where Aβ is considered central to its pathophysiology.

Diversas pesquisas têm reportado que o aumento da produtividade da cultura do milho se deve muito mais à tolerância a estresses abióticos do que a aumentos per se. O déficit hídrico é um dos mais importantes fatores envolvidos na redução de produtividade nessa cultura, mas devido à dificuldade de seleção direta para tolerância à seca, são empregados caracteres secundários como o caráter stay green ou senescência retardada de folhas e colmos. Todavia, estudos sobre a herança desse caráter são escassos na literatura. Em vista disso, este trabalho teve como objetivo estudar a herança deste caráter em milho tropical, utilizando o delineamento III (Comstock e Robinson, 1952). A partir do cruzamento entre as linhagens endogâmicas L08-05F e L38-05D, foram obtidas 100 progênies F2:3, as quais foram retrocruzadas com cada um dos parentais, resultando em 200 progênies de retrocruzamento. Estas progênies foram avaliadas em dez ambientes, com duas repetições por ambiente, em Piracicaba SP, utilizando o delineamento ?-látice. A avaliação do stay green foi feita em dez plantas competitivas por parcela, 120 dias após a semeadura, utilizando uma escala de notas variando de 1 a 5, em que 1 correspondeu às plantas com todas as folhas acima e pelo menos duas folhas verdes abaixo da espiga e 5 à planta com todas as folhas secas. O caráter florescimento feminino foi avaliado e utilizado como covariável para ajuste das variações de maturação das progênies, e as médias das parcelas foram utilizadas para as análises. A estimativa da variância aditiva foi muito superior à de dominância e a variância da interação aditiva por ambiente apresentou maior magnitude que a variância aditiva. O tipo de ação gênica foi de dominância parcial, mas devido ao desequilíbrio de ligação da população, este pode estar superestimado. O coeficiente de herdabilidade em nível de plantas apresentou baixa magnitude (~25%) enquanto que em nível de médias a magnitude foi elevada (~70%). Estes resultados indicam que os efeitos aditivos são mais importantes que os dominantes no controle do caráter stay green e que os efeitos aditivos não são consistentes nos diversos ambientes de avaliação. Assim, o nível de heterose deve apresentar baixa magnitude, e a seleção fenotípica não pode ser realizada em nível de plantas, mas baseada em médias de experimentos com repetições, conduzidas em diversos ambientes para minimizar os efeitos destes e suas interações com os genótipos. A linhagem L08-05F apresentou maior nível de stay green que a linhagem L38-05D e, portanto, pode ser utilizada como fonte de alelos favoráveis para serem transferidos para outras linhagens visando aumentar o nível deste caráter em linhagens que apresentarem baixos níveis de senescência retardada. / Several researches have reported that the increase in maize productivity could be attributed to the tolerance to abiotic stresses rather than to an increase on a plant productivity per se. Moisture stress is one of the most important stresses that reduce the maize productivity, but because of the difficulties for the direct selection for tolerance to moisture stress secondary traits as delayed senescence, also known as stay green trait, has been employed for this purpose. However, there is limited information reported on the inheritance of this trait. Thus, the objective of this research was to study the inheritance of the stay green trait in maize using the design III (Comstock and Robinson, 1952). One hundred F2:3 progenies were developed from a population derived from the cross between the inbred lines L08-05F and L38-05D; and these progenies were backcrossed to the inbred parents giving rise to 200 backcrossed progenies. These progenies were evaluated in ten environments, with two replications per environment, in Piracicaba City, São Paulo State, using the experimental design ?-lattice. The stay green trait was recorded in ten competitive plants per plot 120 days after sowing, using a scale with scores from 1 to 5, where score 1 was assigned to the plants with all leaves above the ear and at least two leaves below the ear green, and score 5 was assigned to plants with all leaves senescent. The trait days to silking was also recorded and used as covariate to adjust the maturation variation of the backcrossed progenies, and the mean of the plots was used for analysis. The estimate of the additive variance was quite larger than the dominance variance, the additive x environment variance was quite larger than the additive variance, and the average level of dominance was partial dominance, but since the population was in linkage disequilibrium the level of dominance may be overestimated. The heritability coefficient on a plant level was of low magnitude (~25%) while that one on a mean level presented high magnitude (~70%). These results indicated that the additive effects were more important than the dominance effects for the control of the stay green trait, and that the additive effects are not consistent across the environments. Therefore, the level of heterosis should present low magnitude, and the phenotypic selection should not be conducted on a plant level; instead selection should be based on the means of replicated experiments assessed in several environments to minimize their effects and the genotype by environment interactions. The inbred line L08-05F presented higher level of stay green than the inbred L38-05D and thus the former inbred could be used as a source of favorable alleles to be transferred to others inbreds to increase the level of this trait in inbreds that have low levels of delayed senescence.

Introdução: o processo fisiológico de envelhecimento traduz-se em diversas alterações estruturais do sistema musculoesquelético. Estas, por sua vez, acarretam em modificações funcionais que se repercutem na dependência do senescente, determinando a diminuição da sua qualidade de vida. Das estratégias existentes para atenuar os efeitos da senescência o treinamento de potência tem sido apontado como preferido. Existem, contudo, indícios de que um treinamento baseado na potencialização da ação do ciclo alongamento-encurtamento seja uma melhor escolha. Hipóteses do estudo: pelos resultados obtidos em intervenções similares, hipotetiza-se que as participantes do protocolo de intervenção proposto apresentarão um aumento da densidade mineral óssea, do volume muscular, da capacidade funcional e melhora de alguns parâmetros biomecânicos da marcha, bem como da sua qualidade de vida. Objetivos: o objetivo geral do presente trabalho é, portanto, propor um modelo de treinamento fundamentado na potencialização da ação do ciclo alongamento-encurtamento e averiguar os seus efeitos em parâmetros selecionados da morfologia osteomuscular, capacidade funcional e qualidade de vida de idosas. Materiais e Métodos: 21 idosas sedentárias (66,9±4,2 anos) executaram o protocolo proposto durante 20 semanas, tendo os efeitos na densidade mineral óssea de fêmur, coluna, tíbia e rádio; efeitos na composição corporal, na força, no equilíbrio, na marcha, na flexibilidade e na qualidade de vida comparados aos efeitos obtidos pelo grupo controle (N=17, 65,0±3,4 anos), que não alterou o seu nível de atividade física. O protocolo de intervenção, composto por onze exercícios de força realizados com o intuito de potencializar a ação do ciclo alongamento-encurtamento, que apresentava duas modalidades de salto (salto vertical com contramovimento e drop jump), exigia a realização da fase concêntrica na maior velocidade possível. Resultados: quando comparado com o grupo controle, o grupo experimental apresentou alterações significantes (p<0,05) na densidade mineral óssea de coluna (g=1,06) e sua microarquitetura (g=0,80), na microarquitetura da tíbia (g=0,82), na força máxima (g=2,39) e potência (g=1,38) de extensores de joelho, na velocidade máxima de marcha (g=0,96), na flexibilidade de membros inferiores (g=1,05) e superiores (g=0,86) e no domínio Atividades passadas, presentes e futuras da qualidade de vida (g=1,08). Conclusão: os resultados apontam para a eficácia da proposta de intervenção, apresentando-se como uma nova estratégia para atenuar e até mesmo reverter algumas perdas estruturais e funcionais impostas pelo processo de envelhecimento / Introduction: the physiological aging process induces several structural changes in the musculoskeletal system. These, in turn, result in functional changes that are reflected in the senescent dependency, determining the reduction in their quality of life. Power training has been identified as ideal to mitigate the effects of aging. However, there are indications that an intervention based on the potentiation of the stretch-shortening cycle action is a better choice. Study hypotheses: the participants of the proposed intervention will increase their bone mineral density, muscle volume, functional capacity and will show some improvement in their gait, as well as in their quality of life. Purposes: the main objective of this study was to propose a training model based in the potentiation of the stretch-shortening cycle action and assess its effects on selected parameters of musculoskeletal morphology, functional capacity and quality of life of elderly women. Methods: 21 sendentary elderly women (66.9 ± 4.2 years) performed the proposed intervention protocol for 20 weeks and the effects on bone mineral density of the femur, spine, tibia and radio; effects on body composition, strength, balance, gait, flexibility and quality of life were compared to the effects obtained by the control group (N = 17, 65.0 ± 3.4 years) that did not change their level of physical activity. The program was composed by eleven strength exercises performed in order to enhance stretch-shortening cycle action, had two jump exercises (vertical jump with countermovement and drop jump) and had the concentric phase of the movements performed as fast as possible. Results: when compared to the control group the experimental group showed significant changes (p <0.05) in bone mineral density of the spine (g = 1.06) and its microarchitecture (g = 0.80), the microarchitecture of the tibia (g = 0.82), the knee extensors maximum force (g = 2.39) and power (g = 1.38), the maximum walking speed (g = 0.96), the lower (g = 1.05) and upper (g = 0.86) limbs flexibility and in the domain past, present and future activities of the quality of life (g = 1.08). Conclusion: the results point to the effectiveness of the proposed intervention, suggesting it as a new strategy to slow down and even reverse some structural and functional losses imposed by the aging process

FGFs compreendem um grande família de 24 proteínas, participando de processos chaves nos mais variados tecidos, tendo funções parácrina, autócrina e intrácrina, regulando mitogênese, diferenciação celular, morfogênese e cicatrização. Mas, a relação estrutura-função dos FGFs é pobremente entendida. O membro protótipo desta família é o FGF-2, que apresenta quatro isoformas moleculares incluindo a forma de 18 kDa que é secretada e se liga aos receptores específicos (FGFRs) e dispara uma complexa sinalização. As outras isoformas, de alto peso molecular (21, 22 e 22,5 kDa) são expressas por códons alternativos (CUG) e permanecem no interior da célula interagindo com parceiros moleculares desconhecidos. Para antecipar mecanismos e parceiros do FGF-2 HMW foi realizada modelagem molecular desta isoforma que mostrou: uma estrutura do N-terminal da proteína com motivo &#946;&#8594;&#945;&#8594&#946; e manutenção do barril &#946;. A busca por parceiros intracelulares, foi realizada através da técnica do duplo hibrido de levedura, usando um biblioteca de cDNA de cérebro de rato. Foram encontrados 4 possíveis parceiros: BRD2, UBE2I, BRPF1, PC4. Todas essas interações foram confirmadas através do crescimento da levedura em meio sem histidina, produção de &#946;-galactosidase e ensaios de \"pull-down\" com GST. Analises por FACS confirmam que FGF2 não causa apoptose em células adrenais tumorais Y1 de camundongo, mas promovem um acumulo de células na fase S com bloqueio do ciclo celular e da proliferação, configurando uma forma de senescência. Resultados com as células humanas HEK-ER:Ras permitem fazer a seguinte generalização: FGF2 induz senescência em células malignas transformadas pelos oncogenes raso A superexpressão da proteína de fusão FGF-2(18kDa):protA, mas não a da FGF-2(22,5 kDa):protA, protege a célula Y1 da senescência induzida por FGF-2. Por outro lado, a superexpressão destas mesmas isoformas de FGF-2 fusionadas à proteína A em células imortalizadas Balb3T3 não causou transformação celular e nem alterou a resposta mitogênica destas células ao FGF-2 recombinante adicionado ao meio de cultura. Células Y1 quando tratadas com FGF-2 recombinante produz ROS intracelular e libera anions superóxido no meio extracelular. Além disso, o anti-oxidante NAC protege estas células da indução de senescência induzida por FGF-2, sugerindo que ROS pode ser intermediário no disparo de senescência por FGF-2. / FGFs comprise a large fami1y of 24 proteins that play key roles in a number of tissues as local paracrine, autocrine and intracrine regulators of mitogenesis, cellular differentiation, organ morphogenesis and tissue repair. Structure-function relationship among FGFs is still poorly understood. FGF-2, the fami1y prototype member, exists as four molecular species. The 18 kDa form is released to the extracellular milieu and binds to specific receptors (FGFR), initiating a complex array of signals. Other isoforms of higher molecular weights (21, 22 and 22,5 kDa) are translated from alternative codons (CUG) and remain inside of the cell interacting with unknown partners. Aiming to anticipate mechanisms and partners, we modeled the FGF2-HMW molecule, showing that the protein displays &#946;&#8594;&#945;&#8594&#946; motif in the N-terminal region and maintains the &#946;-barrel structure common to ali FGFs. By the yeast two-hybrid method, using a cDNA rat brain library, we found four possible partners for FGF2-HMW: BRD2, UBE2I, BRP1 and PC4. Ali partners were confirmed by yeast growth without histidine, production of &#946;-galactosidase and \"pull-down\" assays with GST. FACS analyses confirmed that FGF2 does not cause apoptosis in mouse Y1 adrenal tumor cells. But, FGF2 inhibited S phase progression blocking cell cycle and proliferation, characterizing a form of senescence. In addition, results obtained with the human HEK-ER:Ras cells support the following general statement: FGF2 triggers senescence in malignant cells transformed by ras oncogenes. Ectopic expression of the fusion protein FGF-2(18 kDa):protA, but not of FGF-2(22,s kDa):protA, protected Y1 cells senescence induced by FGF-2. On the other hand, ectopic expression of FGF-2 isoforms fusioned to protA in Balb3T3 immortalized cells did not cause transformation and neither modified the mitogenic response of this cell to recombinant FGF2. Recombinant FGF-2 stimules Y1 cells to produce intracellular ROS and to release superoxide anions into intracellular medium. Moreover, the ROS scavenger NAC protect Y1 cells from senescence induced by FGF-2, suggesting that ROS may be mediate senescence triggering induced by FGF-2.

Entre todos os cânceres de pele, o melanoma está entre os menos comuns, mas é responsável pela maior parte das mortes. No caso da doença metastática, não há um tratamento satisfatório capaz de prolongar a vida do paciente. Isso leva à necessidade de novas estratégias e de novos tratamentos que possam reverter a quimiorresistência do tumor. Entre as proteínas que têm seu perfil de expressão modificado no melanoma está a proibitina, cuja expressão aumenta durante a progressão tumoral. Proibitina é uma chaperona mitocondrial pertencente a uma família de proteínas que possuem um resíduo hidrofóbico SPFH, que confere a ela uma capacidade de ancoragem e de organização de espaços em membranas. Além disso, no compartimento nuclear, é um inibidor da família de fatores de transcrição E2F, juntamente com a proteína retinoblastoma (Rb). Em melanomas, proibitina localiza-se no citoplasma, associada à mitocôndria, e no núcleo. No citoplasma, proibitina faz parte da resposta a diversas drogas, como cisplatina, dacarbazina, temozolamida, vimblastina e tunicamicina pode estar relacionado com o aumento de espécies reativas de oxigênio (ROS), já que essas drogas podem de alguma forma induzir ROS intracelular. O aumento de expressão de proibitina, nesse contexto, poderia fazer parte de uma resposta protetora da mitocôndria, o que em última análise protegeria a célula contra a morte celular, já que a inibição de proibitina sensibiliza a célula ao tratamento com cisplatina ou tunicamicina. Além disso, o estresse provocado pela privação de soro fetal bovino em linhagens de melanoma leva ao aumento de expressão de proibitina e é acompanhado pela indução de ROS. No núcleo, proibitina esta colocalizada com MCM5 e MCM7, mas não MCM2. A inibição de proibitina leva ao aumento de expressão de metaloproteinases de matriz extracelular, não só em melanomas, mas também em linhagens de câncer de mama e de câncer de pulmão. Ainda, proibitina parece estar relacionada com o fenômeno da transição epitélio mesênquima, já que a inibição de proibitina leva ao aumento de expressão de marcadores mesenquimais como N-caderina e vimentina e a perda de expressão de marcadores epiteliais, como a E-caderina. Outras funções controladas por E2F1 que proibitina pode estar modulando são a capacidade de E2F1 induzir reparo de DNA devido a lesões causadas por radiação UVB e a indução de senescência. A inibição de proibitina em linhagem de câncer de pulmão protegeu a célula contra o dano genotóxico causado pela radiação UVB, pelo aumento da proteína de reparo de DNA Gadd45a, que é induzida por E2F1. Ainda, a inibição de proibitina diminuiu a quantidade de células senescência induzida por adriamicina em linhagens de melanoma. Ainda, a expressão de proibitina responde a fatores do microambiente tumoral como TGF?, IL4 e LPS juntamente com INF? e, além disso, têm sua expressão diminuída durante a maturação de macrófagos. Esses resultados mostram que proibitina pode atuar protegendo o tumor ou bloqueando vias importantes para seu desenvolvimento, dependendo da sua compartimentalização subcelular / Among all skin cancers, melanoma is the least common, but is responsible for most deaths. In metastatic disease, no satisfactory treatment can prolong the patient\'s life. This leads to the need for new strategies and new treatments that may reverse tumor chemoresistance. Among proteins that have their expression profile altered in melanoma is prohibitin whose expression increases during tumor progression. Prohibitin is a mitochondrial chaperone belonging to a family of proteins which possess a hydrophobic residue SPFH, which gives it a capacity for anchorage and organization in membrane regions. Furthermore, in the nuclear compartment, prohibitin is an inhibitor of the E2F transcription factor family, together with the retinoblastoma protein (Rb). In melanomas, prohibitin is located in the cytoplasm, associated to the mitochondria, and inside the nucleus. In the cytoplasm, prohibitin is part of the response to various drugs such as cisplatin, dacarbazine, temozolomide, vinblastine and tunicamycin and may be associated with increased reactive oxygen species (ROS), since these drugs can somehow induce intracellular ROS. Prohibitin overxpression in this context could be part of a protective response of the mitochondria, which ultimately protect cells against death, as prohibitin inhibition sensitizes cells to cisplatin or tunicamycin treatment. Moreover, the stress caused by deprivation of fetal bovine serum in melanoma cell lines leads to prohibitin overexpression and is accompanied by ROS induction. In the nucleus, prohibitin is colocalized to MCM5 and MCM7, but not to MCM2. Inhibition of prohibitin leads to increased expression of matrix metalloproteinases, not only on melanomas but also on breast cancer and lung cancer cell lines. Further, prohibitin appears to be related to the phenomenon of epithelial mesenchymal transition, since prohibitin inhibition leads to increased expression of mesenchymal markers such as N-cadherin and vimentin and loss of expression of epithelial markers, such as E-cadherin. Other functions controlled by E2F1 that are modulated by prohibitin include be the ability of E2F1 to induce DNA repair against UVB radiation and the induction of cellular senescence. Inhibition of prohibitin in lung cancer cell line protected against genotoxic damage caused by UVB radiation, due to DNA repair protein Gadd45a overexpression, which is induced by E2F1. Also, prohibitin inhibition decreased the amount of cell senescence induced by adriamycin in melanoma cell line. Further, prohibitin expression is triggered by tumor microenvironmental factors such as TGF?, IL4, and LPS together with INF? and, in addition, prohibitin expression decreases during macrophages maturation. These results show that prohibitin may act protecting the tumor or blocking pathways important for its development, depending on its subcellular compartment distribution

Zelluläre Seneszenz beschreibt einen terminalen Zellzyklus-Arrest. Nach zellulärem Stress u. a. durch aktivierte Onkogene oder DNA-schädigende Chemotherapie wird Seneszenz induziert und kann so zur Tumorsuppression bzw. zum Behandlungserfolg beitragen. Vor kurzem wurde gezeigt, dass der Transkriptionsfaktor NF-kappaB – welcher bisher vor allem durch seine onkogenen Funktionen mit Krebs in Verbindung gebracht wurde - bei der Seneszenz-assoziierten Zytokinausschüttung mitwirkt und den seneszenzten Phänotyp möglicherweise sogar verstärkt, wodurch NF-kappaB potentiell eine tumorsuppressive Rolle zukäme. Ziel dieser Arbeit ist die Untersuchung des NF-kappaB-Signalweges in Seneszenz und Therapie. In der vorliegenden Arbeit zeige ich die deutliche Aktivierung von NF-kappaB nach Therapie-induzierter Seneszenz (therapy-induced senescence, TIS) und erhöhte Expression NF-kappaB-regulierter Zytokine. TIS ist vor allem in vivo mit starker Aktivität des NF-kappaB-Signalweges assoziiert und von selbiger abhängig. Primäre Eµ-myc-transgene Mauslymphome wurden nach ihrer endogenen NF-kappaB-Aktivität klassifiziert bzw. mit inhibierenden und aktivierenden NF-kappaB-Konstrukten modifiziert, welche auch in diffusen großzelligen B-Zell Lymphomen (diffuse large B-cell lymphoma, DLBCL) als natürlich vorkommende Mutationen gefunden wurden. Über einen neuartigen „Cross-Species“-Vergleich wurden Bcl2-hochexprimierende Keimzentrums-B-Zell-DLBCL (germinal center B-cell type, GCB) als klinisch relevante Gruppe identifiziert, welche nach NF-kappaB-Hyperaktivierung signifikant besser auf Therapie ansprach. Diese Ergebnisse zeigen eine kontextspezifische, d. h. von „onkogenen Netzwerken“ abhängige Rolle des NF-kappaB Signalweges unter Chemotherapie. Diese Information könnte für künftige klinische Studien bedeutsam sein, da sie Bedingungen aufzeigt, unter denen NF-kappaB als Vermittler einer erwünschten Therapie-induzierten Seneszenzantwort eher nicht inhibiert werden sollte. / Cellular senescence is a terminal cell-cycle arrest program that is executed in response to cellular stresses, such as activated oncogenes or DNA-damaging anti-cancer chemotherapy, where it serves as a tumor-suppressive mechanism or contributes to treatment outcome, respectively. Recently, transcription factor NF-kappaB which has long been linked to cancer development primarily through its oncogenic functions, has been postulated to participate in a senescence-associated and possibly senescence-reinforcing cytokine response, thereby suggesting a tumor-restraining role for NF-kappaB. The aim of my PhD project was to understand the role of the NF-kappaB pathway in senescence and cancer treatment outcome. In this thesis, I show markedly elevated NF-kappaB activity upon therapy-induced senescence (TIS), associated with strong upregulation of NF-kappaB-controlled cytokines. TIS is associated with and depends on hyper-activated NF-kappaB signaling. By characterization and genetic engineering of primary mouse lymphomas according to distinct NF-kappaB-related oncogenic networks reminiscent of diffuse large B-cell lymphoma (DLBCL) subtypes, Bcl2-overexpressing germinal center B-cell-like (GCB) DLBCL were identified as a clinically relevant subgroup with significantly superior outcome when NF-kappaB is hyperactive. These results demonstrate the context-dependent role of NF-kappaB signaling in cancer therapy and unveil oncogenic scenarios in which NF-kappaB hyperactivity unexpectedly accounts for superior long-term outcome to therapy. This finding has significant ramifications for future clinical trials that aim at inhibiting NF-kappaB activity based on the assumption of its detrimental impact on treatment outcome.

Apoptose und Seneszenz sind stress-responsive, genetisch verankerte „Failsafe“- Mechanismen, welche die Zelle vor maligner Transformation schützen. Onkogenes Ras induziert zelluläre Seneszenz über den p16/Retinoblastoma (Rb)-Signalweg und führt dabei zu einem permanenten Zellzyklusarrest - das tumorsuppressive Potential von Seneszenz in vivo bleibt jedoch bis heute fraglich. In seneszenten Zellen ist die Expression von S-Phase relevanten Gene durch die lokale Ausbildung von Heterochromatin, bzw. der Methylierung von Histon H3 an Lysin 9 (H3K9me) blockiert. Dies lässt vermuten, dass Seneszenz ein epigenetische kontrollierter Prozess ist und von Proteinen wie der Rb-assozierte Histonmethyltransferase Suv39h1 reguliert wird. In der vorliegenden Arbeit konnte gezeigt werden, dass Eµ-N-Ras transgene Mäuse mit heterozygoten Läsionen im Suv39h1 oder p53 Lokus aggressive T-Zell Lymphome entwickeln, die gegen Suv39h1, bzw. p53-Expression selektieren. Im Gegensatz dazu entwickeln N-Ras-transgene Wildtyp-Tiere („Kontrollen“) vorrangig nicht-lymphoide Tumoren und sterben signifikant später. In primären Lymphozyten induziert onkogenes Ras einen Suv39h1-abhängigen, H3K9me-assoziierten Proliferationsarrest und kann dadurch Lymphomgenese verhindern. Suv39h1-defiziente Lymphomzellen wachsen exponentiell und sind, entgegen p53 defizienten Zellen, sensitiv gegenüber Adriamycin-induzierten Zelltod (Apoptose). Jedoch arretieren nur Kontroll-Lymphome unter Therapie in vitro wenn Apoptose blockiert ist, nicht aber Suv39h1 oder p53-defiziente Lymphomzellen. Diese Resultate identifizieren Ras-induzierte Seneszenz als einen neuen, H3K9me-abhängigen Tumorsuppressor-Mechanismus, wobei dessen Inaktivierung die Entwicklung von aggressiven, aber dennoch Apoptose-kompetenten Lymphomen herbeiführt. / Cellular “failsafe” programs like apoptosis or senescence are genetically encoded, stress-responsive mechanisms that ultimately counteract malignant transformation. Acute induction of oncogenic Ras provokes cellular senescence that involves the p16/Retinoblastoma (Rb) pathway to induce a permanent arrest, but the tumor suppressive mechanism in vivo still remains questionable. Senescent cells display heterochromatic features on S-phase relevant genes involving methylation of histone H3 on lysine 9 (H3K9me), which may depend on the Rb-associated histone methyltransferase Suv39h1. In the present thesis it was shown that Eµ-N-Ras transgenic mice harboring targeted heterozygous lesions at the Suv39h1, or the p53 locus for comparison, succumb to invasive T cell lymphomas that lack expression of Suv39h1 or p53, respectively. By contrast, most N-Ras-transgenic wildtype (“control”) animals develop a non-lymphoid neoplasia significantly later. Proliferation of primary lymphocytes is directly stalled by a Suv39h1-dependent, H3K9me-related senescent growth arrest in response to oncogenic Ras, thereby cancelling lymphomagenesis at an initial step. Suv39h1-deficient lymphoma cells grow rapidly but, unlike p53-deficient cells, remain highly susceptible to adriamycin-induced apoptosis. In contrast, only control, but not Suv39h1-deficient or p53-deficient lymphomas senesce after drug therapy when apoptosis is blocked. These results identify H3K9me-mediated senescence as a novel Suv39h1-dependent tumour suppressor mechanism whose inactivation permits the formation of aggressive but apoptosis-competent lymphomas in response to oncogenic Ras.

Die Aufrechterhaltung der sogenannten failsafe Programme ist ein wichtiges Kennzeichen des zellulären Lebens, da das genaue Gleichgewicht zwischen Proliferation und Wachstumsarrest es sicherstellt, dass die Zelle sich selber vor potentiell gefährlichen Mutationen schützen kann. Apoptose und Seneszenz bewahren die Zellhomeostase und sind von aüsserster Bedeutung dafür, die Zelle vor maligner Transformation zu bewahren. Die Seneszens zeichnet sich dabei durch Veränderungen des Heterochromatins bei Genen aus, welche für den Eintritt in die DNA-Synthese-Phase des Zellzyklus verantwortlich sind. Insgesamt wurde die Bedeutung epigenetischer Modifikationen bei malignen Erkrankungen in den letzten Jahren immer deutlicher. So konnte gezeigt werden, dass die Rb-assoziierte Histon-Methyltransferase Suv39h1 ein entscheidender Vermittler der Seneszenz in Ras-induziertem Maus Lymphom-Modell ist. In dieser Arbeit wurde gezeigt, dass die transgenen Eµ-myc Maüse (ähnlich dem Burkitt''s Lymphom im Menschen), wenn sie mit Suv39h1-defizienten Maüsen gekreuzt werden. Die Überexpression von Myc, wie sie in humanen und murinen Tumoren zu beobachten ist, induziert primär ein apoptotische Antwort mit dem zentralen Vermittler p53. Allerdings bewirkte die Suv39h1 Defizienz, obwohl sie die Lebenserwartung signifikant verkürzte, keine Veränderung der Apoptose-Rate in diesem Modell. Dieses Ergebnis demonstriert die Tumor-suprimierende Wirkung des Suv39h1 in Myc-getriebener Lymphomagenese. Weiterhin wurde in dieser Arbeit ein zusätzlicher Mechanismus zur Vermittlung Myc-induzierter Seneszenz unter Einbeziehung des Zytokin-Signaling identifiziert. Im Falle der Expression von intaktem Suv39h1 in der Tumorzelle kann TGF-beta die Myc-induzierte Seneszenz in vivo beeinflussen. Schliesslich ergaben die Untersuchungen, dass die Suv39h1-defizienten Lymphome über eine beeinträchtigte Therapie-Anwort verfügen, da sie keine Therapeutika-induzierte Seneszenz ausführen können. / Maintenance of cellular failsafe pathways (apoptosis and senescence) is one of the hallmarks of life, as fine equilibrium between proliferation and growth arrest ensures that the cell can protect itself from the potentially dangerous mutations. Senescence is a mechanism distinguished by heterochromatin modifications leading to silencing of the genes responsible for the entry to the DNA synthesis [S] phase of the cell cycle. Involvement of epigenetic modifications in cancer development has been subject of a more intense research in the past few years. It was shown that the Rb-associated histone methyltransferase Suv39h1 is a critical mediator of senescence in a Ras-induced mouse lymphoma model. Additional mechanisms of the senescence regulation are currently being under investigation. In this thesis Eµ-myc transgenic mice, crossed to mice deficient for Suv39h1, were shown to succumb to the same type of B-cell lymphoma (similar to Burkitt''s lymphoma in humans). Overexpression of Myc, as seen in human and mouse tumors will primarily induce an apoptotic response using p53 as a mediator of this response. In Eµ-myc, Suv39h1-/- mouse model deficiency in Suv39h1 has significantly shortened life expectancy, but it did not affect spontaneous apoptosis rates. This finding established Suv39h1 as a tumor suppressor in Myc-lymphomagenesis. Further, an additional mechanism of mediating oncogene-induced senescence involving cytokine signaling was identified and reported here. When intact Suv39h1 is present in the tumor, TGF-beta is able to mediate oncogene-induced senescence in vivo. Finally, in the treatment studies it was shown that Suv39h1 deficient lymphomas have an impaired response to chemotherapy, caused by their inability to execute drug-induced senescence. This finding can be of use for the design of novel cancer therapies.