Apoptosis and cell survival are two seemingly opposing fate-determining processes that are regulated by distinct and complex signaling pathways. Caspase-8, an apical caspase, plays a pivotal regulatory role in initiating apoptosis. c-Src, a prototypical member of the Src family kinases (SFKs), regulates a myriad of cellular processes including cell mitogenesis, proliferation, growth and migration. Although the regulation of caspase-8 by c-Src has been suggested, the reciprocal regulation of these two seemingly opposing signaling molecules, caspase-8 and c-Src, has never been explored. To study this reciprocal regulation, we asked three questions. (1) Can active caspase-8 negatively regulate c-Src activity to allow the propagation of apoptosis? (2) Can c-Src negatively regulate caspase-8 activity to prevent the propagation of apoptosis? (3) Can caspase-8, when its enzymatic activity is inhibited, further promote c-Src activity to allow the propagation of cell survival? To address these questions, we first investigated the effect of active caspase-8 on the activation and activity of c-Src. We discovered that active caspase-8 inhibited c-Src activation and some of its downstream effectors. Next, we investigated whether c-Src could tyrosine phosphorylate caspase-8. We discovered that c-Src could phosphorylate caspase-8 at multiple tyrosine sites. We then examined whether tyrosine phosphorylated caspase-8 prevents apoptosis. We found that phosphorylation of caspase-8 at Y465 prevented its cleavage, and activity towards activating caspase-3 and towards causing cell morphological changes associated with apoptosis. Finally, we studied whether tyrosine phosphorylation of caspase-8 could further promote the activation of c-Src. We showed that phosphorylation of caspase-8 at both Y465 and Y397 resulted in the activation of c-Src and extracellular signal-regulated kinase 1/2 (Erk1/2). In conclusion, this work demonstrated the reciprocal regulation of two opposing signaling molecules, caspase-8 and c-Src. These results also suggest an elegant mechanism for a cell to commit efficiently and rapidly to a fate-determining process, either apoptosis or survival, by further suppression of the opposing signaling pathway.

A bactéria Legionella pneumophila é um bacilo Gram-negativo, flagelado causador da doença dos legionários e febre de Pontiac. O inflamassoma mais importante no controle da replicação desta bactéria é o composto por Naip5/NLRC4, que é responsável pelo reconhecimento de flagelina. A ativação do inflamassoma de Naip5/NLRC4 pela flagelina induz a ativação de caspase-1, induzindo a formação de poros na membrana, piroptose e controle da replicação desta bactéria. A participação da proteína adaptadora ASC é essencial para a nucleação deste complexo e secreção de citocinas inflamatórias como IL-1? e IL-18 por esta via. Além do controle da replicação de L. pneumophila pelo inflamassoma NLRC4 dependente de caspase-1, foi demonstrado que existe uma via induzida por NLRC4 independente de caspase- 1/11. Dessa forma, camundongos e células Nlrc4-/- são mais susceptíveis à infecção por esta bactéria do que as células Casp1/11-/-. Neste trabalho, nós identificamos que a via independente de caspase-1/11 é composta por Naip5/NLRC4/ASC/Caspase-8 e é essencial para o controle da replicação de Legionella spp. flageladas em macrófagos e in vivo. Através da utilização de BMDMs Casp1/11-/- e Asc/Casp1/11-/- transduzidos com NLRC4-GFP ou ASC-GFP, identificamos que a formação de punctas de NLRC4 e ASC dependem do reconhecimento de flagelina e que ASC é essencial para a formação desses punctas. Também foi identificado que a infecção com L. pneumophila que expressa flagelina leva à ativação de caspase-8 de maneira dependente de ASC e Naip5, mas independente de caspase-1/11. De acordo com esses dados, o silenciamento de caspase-8 em macrófagos Casp1/11-/- aumentou a susceptibilidade dessas células à infecção com L. pneumophila flagelada. Além disso, macrófagos e camundongos Asc/Casp1/11-/- foram tão susceptíveis quanto os Nlrc4- /- e mais susceptíveis que os Casp1/11-/-. Nós observamos que o inflamassoma de NLRC4/ASC/Caspase-8 induz formação de poros e morte celular independente de gasdermina-D (GSDMD). Por meio da utilização de células de camundongos C57BL/6, foi observado que caspase-8 é recrutada para o inflamassoma de Naip5/NLRC4/ASC/Caspase-1. Entretanto, a ativação de caspase-8 só ocorre na 10 ausência de caspase-1 ou GSDMD. Nossos dados sugerem que a ativação de caspase-8 no inflamassoma composto por NLRC4/ASC/Caspase-8 representa uma via alternativa que opera para garantir o controle da replicação de bactérias flageladas em situações nas quais ou caspase-1 ou GSDMD estão inibidas. / Legionella pneumophila is a flagellated Gram-negative bacillus that is the causative agent of the legionnaire\'s disease and Pontiac fever. The most important inflammasome for the control of L. pneumophila replication is the Naip5/NLRC4, responsible for the flagellin recognition. The activation of the Naip5/NLRC4 inflammasome leads to caspase-1 activation, consequently pore formation, pyroptosis and control of bacterial replication. The participation of the adaptor molecule ASC is essential for this complex nucleation and the secretion of inflammatory cytokines like IL-1? and IL-18 by this pathway. Besides the control of L. pneumophila replication by Naip5/NLRC4/Caspase-1 inflammasome, it was demonstrated there are NLRC4 responses independent of caspase-1/11. These explain why mice and macrophages Nlrc4-/- are more susceptible than Casp1/11-/-. In this work, we identified that the caspase-1/11-independent pathway is composed of Naip5/NLRC4/ASC/Caspase-8 and it is essential for the control of flagellated Legionella spp. replication in macrophages and in vivo. Infection of Casp1/11-/- and Asc/Casp1/11-/- macrophages, transduced with NLRC4-GFP or ASC-GFP, showed that flagellin-positive bacteria triggered puncta formation that is ASC-dependent. Accordingly, Naip5 and ASC, but not caspase-1/11, were required for caspase-8 activation in response to flagellated bacteria. Silencing caspase-8 in Casp1/11-/- BMDMs increased the susceptibility to L. pneumophila infection. Furthermore, the macrophages and mice Asc/Casp1/11-/- are as susceptible as Nlrc4-/-, but more susceptible than Casp1/11-/-. We also found that the NLRC4/ASC/Caspase-8 inflammasome induces GSDMD-independent pore formation and cell death. Using C57BL/6 cells, we observed that caspase-8 is recruited to Naip5/NLRC4/ASC/Caspase-1 inflammasome. However, caspase-8 is just activated in the absence of caspase-1 or GSDMD. Our data suggest that caspase-8 activation in the NLRC4/ASC/Caspase-8 inflammasome represents an alternative pathway that operates to ensure the control of flagellated bacteria replication in situations which either caspase-1 or GSDMD are inhibited.

A barreira placentária é constituída por duas camadas de células sinciciais, uma camada de células trofoblásticas gigantes e o endotélio fetal. Apesar da importância das camadas sinciciais no transporte molecular entre mãe e feto, o exato mecanismo de formação dessa barreira não está completamente elucidado em camundongos. Em humanos, estudos sugerem que a formação do sinciciotrofoblasto ocorre por um processo de fusão celular dependente de Caspase- 8, uma proteína iniciadora da cascata de apoptose. Desta forma, este estudo teve como proposta analisar o processo de formação das camadas sinciciais do labirinto em placentas de camundongos e o possível envolvimento da caspase-8 neste processo. Sítios de implantação foram coletados de camundongos fêmeas nos dias 8,5 a 11,5 de gestação e caracterizados morfologicamente através de marcadores de células precursoras sinciciais (EpCAM) e de células sinciciais maduras (Slc16A3) por meio de reações imunohitoquímicas. A expressão gênica dos marcadores diferenciais de células sinciciais também foi analisada por RT-PCR na região labiríntica dissecada nos diferentes dias de gestação. A expressão de Caspase-8 total e clivada também foi avaliada por Western blot e a relação entre a presença de Caspase-8 clivada e a indução de apoptose, avaliada por TUNEL e pela imunolocalização da Citoqueratina 18 clivada. Tambem foram realizadas análises com células labirínticas cultivadas, isoladas nos dias 8,5 a 10,5 de gestação. As células cultivadas foram caracterizadas morfologicamente e avaliadas quanto a expressão gênica de marcadores sinciciais e proteica de Caspase-8. Nossos resultados mostraram que os primeiros sinais morfológicos de formação da barreira placentária ocorream no dia 9,5 de gestação. O marcador EpCAM foi encontrado na base da placenta nos dias 8,5 e 9,5. No dia 11,5 de gestação, o labirinto já se encontra estruturado e funcional, o que foi indicado pela expressão de Slc16A3, nos dias 10,5 e 11,5 de gestação. A expressão gênica dos fatores de transcrição associados ao desenvolvimento das camadas sinciciais mostraram expressões crescentes ao longo do período estudado. Caspase-8 total e clivada mostrou intensa expressão no dia 9,5 de gestação, e aparentemente não estava associada à morte celular por apoptose, uma vez que não se detectou reatividade pela reação de TUNEL ou imunomarcação de Citoqueratina 18 clivada nas células labirínticas em formação em nenhum dos dias estudados. Células labirínticas obtidas aos 9,5 dias de gestação e cultivadas formaram ninhos celulares ao longo das 48 horas de cultura, com indícios morfológicos de sincicialização. A imunolocalização do marcador de células progenitoras do labirinto, EpCAM foi mais intensa nas culturas de 6 horas e se limitou a áreas ao redor dos ninhos celulares após 48 horas. Inversamente, a imunolocalização do transportador sincicial Slc16A3 não foi observada após 6 horas de cultura, mas foi bastante intensa no centro dos ninhos celulares após 48 horas. As culturas de labirinto de 9,5 das de gestação, também mostraram aumento de expressão das Sincitinas A e B ao longo do tempo de cultivo. A análise da expressão proteica de Caspase-8 mostrou expressão mais alta após 6 horas de cultivo do que a observada nos demais tempos experimentais. Por outro lado, a forma ativa (clivada) da caspase aumentou gradativamente após 24 e 48 horas de cultivo. Culturas submetidas ao tratamento com o inibidor farmacológico de Caspase-8 z-IEDT-fmk, mostraram perfis morfológicos alterados com redução da formação dos ninhos celulares e diminuição da reatividade ao Slc16A3. A expressão dos marcadores de diferenciação Sincitina A e B também foi significativamente diminuída (p<0.05) nestes experimentos em que a inibição da Caspase-8 clivada foi comprovada por Western blot. Estes achados mostraram a expressão de Caspase- 8, principalmente no dia 9,5 de gestação, nas células trofoblásticas labirínticas da placenta de camundongos e sugerem sua participação na formação das camadas sinciciais do labirinto. / Two layers of syncytial cells, a layer of trophoblastic giant cells and the fetal endothelium form the placental barrier. Despite the importance of the syncytial layers in molecular transport between mother and fetus, its exact developmental mechanism is still not completely elucidated in rodents. In humans, studies suggest that the formation of the syncytiotrophoblast occurs through a cell fusion process dependent on Caspase-8, an apoptosis cascade-initiating protein. In this way, this study had the proposal to analyze the process of formation of the syncytial layers of the labyrinth in placentas of mice and the possible involvement of Caspase-8 in this process. Implantation sites were collected from female mice on days 8.5 to 11.5 of gestation and morphologically characterized by the labyrinthine precursor cell marker EpCAM, and the mature syncytial cell marker - Slc16A3, through immunohistochemical reactions. The gene expression of the differential markers of syncytial cells was analyzed by RT-PCR in the labyrinthine region dissected on the different days of gestation. Total and cleaved Caspase-8 expression was also evaluated by Western blot and the relationship between the presence of cleaved Caspase-8 and the induction of apoptosis as assessed by TUNEL and the immunolocalization of the cleaved Cytokeratin 18. Analyzes were also performed with cultured labyrinth cells, isolated on days 8.5 to 10.5 of gestation. The cultured cells were characterized morphologically and evaluated for the gene expression of syncytial markers and Caspase-8 protein. Our results showed that the first morphological signs of placental barrier formation occurred on day 9.5 of gestation. The EpCAM marker was found at the base of the placenta on days 8.5 and 9.5. At day 11.5 of gestation, the labyrinth is already structured and functional, which was indicated by the expression of Slc16A3, on days 10.5 and 11.5 of gestation. The gene expression of the transcription factors associated with the development of the syncytial layers showed increased throughout the studied period. Total and cleaved Caspase-8 showed intense expression at day 9.5 of gestation, and apparently was not associated with cell death by apoptosis, since no reactivity was detected by the TUNEL reaction or cleaved Cytokeratin 18 immunolabeling in the labyrinthine zone. Labyrinthine cells obtained at 9.5 days of gestation formed nests during the 48 hours of culture, with morphological signs of syncytialization. Immunolocalization of the progenitor cell marker EpCAM was more intense in the 6-hour cultures and was limited to areas around the cell nests after 48 hours. Conversely, immunolocalization of the syncytial transporter Slc16A3 was not observed after 6 hours of culture but was quite intense at the center of the cell nests after 48 hours. Labyrinthine cell cultures of 9.5 gestation days also showed increased expression of A and B syncytins throughout the culture time. Analysis of the protein expression of Caspase- 8 showed higher expression after 6 hours of culture than that observed in the other experimental times. On the other hand, the active (cleaved) form of Caspase gradually increased after 24 and 48 hours of culture. Cultures submitted to the pharmacological inhibitor of Caspase-8 - z-IEDT-fmk showed altered morphological 18 profiles with reduction of cell nests formation and a decrease of reactivity to Slc16A3. The expression of the differentiation markers A and B Syncytins was also significantly decreased (p <0.05) in the experiments, in which the inhibition of the cleaved Caspase-8 was confirmed by Western blot. These findings show the expression of Caspase-8, mainly on day 9.5 of gestation, in the labyrinthine cells of the mice placenta and suggest its participation in the formation of the labyrinthine syncytial layers.

A bactéria Legionella pneumophila é um bacilo Gram-negativo, flagelado causador da doença dos legionários e febre de Pontiac. O inflamassoma mais importante no controle da replicação desta bactéria é o composto por Naip5/NLRC4, que é responsável pelo reconhecimento de flagelina. A ativação do inflamassoma de Naip5/NLRC4 pela flagelina induz a ativação de caspase-1, induzindo a formação de poros na membrana, piroptose e controle da replicação desta bactéria. A participação da proteína adaptadora ASC é essencial para a nucleação deste complexo e secreção de citocinas inflamatórias como IL-1? e IL-18 por esta via. Além do controle da replicação de L. pneumophila pelo inflamassoma NLRC4 dependente de caspase-1, foi demonstrado que existe uma via induzida por NLRC4 independente de caspase- 1/11. Dessa forma, camundongos e células Nlrc4-/- são mais susceptíveis à infecção por esta bactéria do que as células Casp1/11-/-. Neste trabalho, nós identificamos que a via independente de caspase-1/11 é composta por Naip5/NLRC4/ASC/Caspase-8 e é essencial para o controle da replicação de Legionella spp. flageladas em macrófagos e in vivo. Através da utilização de BMDMs Casp1/11-/- e Asc/Casp1/11-/- transduzidos com NLRC4-GFP ou ASC-GFP, identificamos que a formação de punctas de NLRC4 e ASC dependem do reconhecimento de flagelina e que ASC é essencial para a formação desses punctas. Também foi identificado que a infecção com L. pneumophila que expressa flagelina leva à ativação de caspase-8 de maneira dependente de ASC e Naip5, mas independente de caspase-1/11. De acordo com esses dados, o silenciamento de caspase-8 em macrófagos Casp1/11-/- aumentou a susceptibilidade dessas células à infecção com L. pneumophila flagelada. Além disso, macrófagos e camundongos Asc/Casp1/11-/- foram tão susceptíveis quanto os Nlrc4- /- e mais susceptíveis que os Casp1/11-/-. Nós observamos que o inflamassoma de NLRC4/ASC/Caspase-8 induz formação de poros e morte celular independente de gasdermina-D (GSDMD). Por meio da utilização de células de camundongos C57BL/6, foi observado que caspase-8 é recrutada para o inflamassoma de Naip5/NLRC4/ASC/Caspase-1. Entretanto, a ativação de caspase-8 só ocorre na 10 ausência de caspase-1 ou GSDMD. Nossos dados sugerem que a ativação de caspase-8 no inflamassoma composto por NLRC4/ASC/Caspase-8 representa uma via alternativa que opera para garantir o controle da replicação de bactérias flageladas em situações nas quais ou caspase-1 ou GSDMD estão inibidas. / Legionella pneumophila is a flagellated Gram-negative bacillus that is the causative agent of the legionnaire\'s disease and Pontiac fever. The most important inflammasome for the control of L. pneumophila replication is the Naip5/NLRC4, responsible for the flagellin recognition. The activation of the Naip5/NLRC4 inflammasome leads to caspase-1 activation, consequently pore formation, pyroptosis and control of bacterial replication. The participation of the adaptor molecule ASC is essential for this complex nucleation and the secretion of inflammatory cytokines like IL-1? and IL-18 by this pathway. Besides the control of L. pneumophila replication by Naip5/NLRC4/Caspase-1 inflammasome, it was demonstrated there are NLRC4 responses independent of caspase-1/11. These explain why mice and macrophages Nlrc4-/- are more susceptible than Casp1/11-/-. In this work, we identified that the caspase-1/11-independent pathway is composed of Naip5/NLRC4/ASC/Caspase-8 and it is essential for the control of flagellated Legionella spp. replication in macrophages and in vivo. Infection of Casp1/11-/- and Asc/Casp1/11-/- macrophages, transduced with NLRC4-GFP or ASC-GFP, showed that flagellin-positive bacteria triggered puncta formation that is ASC-dependent. Accordingly, Naip5 and ASC, but not caspase-1/11, were required for caspase-8 activation in response to flagellated bacteria. Silencing caspase-8 in Casp1/11-/- BMDMs increased the susceptibility to L. pneumophila infection. Furthermore, the macrophages and mice Asc/Casp1/11-/- are as susceptible as Nlrc4-/-, but more susceptible than Casp1/11-/-. We also found that the NLRC4/ASC/Caspase-8 inflammasome induces GSDMD-independent pore formation and cell death. Using C57BL/6 cells, we observed that caspase-8 is recruited to Naip5/NLRC4/ASC/Caspase-1 inflammasome. However, caspase-8 is just activated in the absence of caspase-1 or GSDMD. Our data suggest that caspase-8 activation in the NLRC4/ASC/Caspase-8 inflammasome represents an alternative pathway that operates to ensure the control of flagellated bacteria replication in situations which either caspase-1 or GSDMD are inhibited.

Thesis (Ph.D. in Physiology and Biophysics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 89-98). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Stimulation of β-adrenergic receptor (β-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of β1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of β-AR induces β1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of β1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of β1A integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-β1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-β1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-β1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-β1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-β1-induced apoptosis. TAC-β1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of β1 integrin may be a mechanism of induction of apoptosis during β-AR-stimulated cardiac remodeling.

No description available.

No description available.

A eliminação de células-alvo por linfócitos T citotóxicos (CTLs) exerce um papel importante na imunidade protetora contra patógenos e células tumorais, o que pode ser desencadeada pela ação de perforina e granzima, ou pelas interações Fas-FasL. A interação Fas-FasL promove a apoptose pela formação de DISC, que é composto pela cauda citoplasmática de Fas, a proteína adaptadora FADD e a pro-caspase-8. Nesse complexo, a caspase-8 é ativada e induz a clivagem das caspases efetoras -3, -6 e -7, e/ou BID - membro da família Bcl-2, desencadeando o sinal apoptótico. Devido à sua relevância na sinalização apoptótica, a caspase-8 é alvo de inúmeros estudos. Uma correlação entre mutações no gene CASP8 de células tumorais e alta atividade citolítica foi demonstrada, sugerindo que a deficiência em caspase-8 pode representar um mecanismo de evasão imune. Nesse projeto, nosso objetivo foi avaliar o efeito da deficiência em caspase-8 na eliminação de células-alvo por linfócitos T CD8&#43. Para isso, nós estabelecemos uma técnica de citotoxicidade in vitro utilizando blastos de camundongos OTI como células efetoras e células RMA como alvo. Nesse ensaio, as células-alvo foram marcadas e pulsadas com diferentes concentrações de peptídeo OVA e colocadas em co-cultura a diferentes razões efetora: alvo (E:A) com linfócitos OTI, estimulados por 7 dias com ConA e rIL-2. A eliminação específica foi avaliada após 16 horas de co-cultura. Como controle para avaliar a especificidade ao MHC de classe I, utilizamos as células RMA-S, uma vez que expressam níveis reduzidos desta molécula na superfície. Os resultados demonstraram que o ensaio estabelecido é eficiente e antígeno-específico. Foi também observado que a eliminação das células RMA-S é menor em comparação com as células RMA, sendo essa diferença melhor discriminada em condições com números limitados de células efetoras. Para avaliar o efeito da caspase-8, células RMA caspase-8-suficientes e -deficientes, obtidas pela técnica CRISPR-Cas9, foram utilizadas como alvo nesse ensaio. Os resultados demonstram que, em condições com menor número de células efetoras, as células caspase-8-deficientes são significativamente mais resistentes à eliminação por linfócitos T CD8&#43, em comparação com as células caspase-8-suficientes. Portanto, nossos dados corroboram com a ideia de que a deficiência em caspase-8 poderia representar um mecanismo de evasão imune, principalmente na eliminação por linfócitos T CD8&#43. / The killing of target cells by cytotoxic T lymphocytes (CTLs) plays a major role in protective immunity to pathogens and tumor cells, which can be triggered by either the action of perforin and granzymes or FAS-FASL interactions. The Fas-FasL interaction leads to apoptosis through the formation of DISC, which is composed by the cytoplasmic tail of Fas, the adaptor protein FADD and the pro-caspase-8. At the complex, caspase-8 become activated and transduce the apoptotic signal by cleaving the effector caspases-3, -6 and -7 and/or the Bcl-2 family member Bid. Due to its role on apoptotic signaling, caspase-8 is the target of several studies. Recently, it has been demonstrated a correlation between mutations in CASP8 in tumors from patients with high cytolytic activity, suggesting that deficiency in caspase-8 may represent an immune evasion mechanism. Our project aimed to evaluate the effect of caspase-8 deficiency on the elimination of target cells by CD8&#43 T lymphocytes. In order to assess this elimination, we have established an in vitro cytotoxic assay using as effector, blasts from OTI mice, and as target, RMA cells. In this assay, the target cells were labeled, pulsed with OVA peptide at different concentrations and then co-cultured with OTI lymphocytes, which were stimulated with ConA and rIL-2, at different effector: target (E: T) ratios. The specific elimination was evaluated after 16 hours of co-culture. As a control to assess the specificity to MHCI of our assay, RMA-S cells were used because they have reduced levels of MHCI at surface. The results have shown that OTI blasts have effector capacity and that our established assay is antigen-specific. In addition, we observed that RMA-S elimination is reduced in comparison to RMA cells. The difference between RMA and RMA-S is better detected in conditions with limited number of effector cells. In order to assess the effect of caspase-8 in the resistance to elimination by CD8&#43 T cells, caspase-8-sufficient and deficient RMA cells (obtained by CRISPR-Cas9 technique) were used as targets in this assay. Our results demonstrate that caspase-8-deficient cells are more resistant to elimination than wild-type counterparts. This difference is better detected at conditions with reduced number of effector cells. Thus, our data suggests the deficiency in caspase-8 results in lower elimination by CD8&#43 T lymphocytes, suggesting this might represent an immune evasion mechanism.

Das maligne Melanom ist für 90 % der Sterbefälle bei Hautkrebs verantwortlich (Garbe et al., 2022). Verschiedene Mutationen wirken sich auf die Signalwege aus und fördern die Karzinogenese, was zu einem ständigen Wandel der Melanomtherapie führt. Zusätzlich zu der, in frühen Stadien kurativen, chirurgischen Therapie, erzielt die zielgerichtete Therapie mit BRAF- und MEK-Inhibitoren in den letzten Jahren durchschlagende Erfolge für das Gesamtüberleben der Patienten, bei nicht-resezierbaren Metastasen wird weiterhin die Chemotherapie empfohlen. Zunehmende Therapieresistenzen schränken die Melanomtherapie ein. Müller et al. (2020) weisen auf Wildtyp p53 exprimierende Tumorzellen hin, die mithilfe der Expression von nukleärer Caspase-8 den G2/M-Zellzyklusarrest umgehen und so die Proliferation und Expansion entarteter Tumorzellen fördern. Bei der Untersuchung von 16 Melanomzellproben, die sich in ihrem BRAF-/NRAS- und p53 Mutationsstatus, sowie in ihren klinischen Vortherapien unterscheiden, konnte die potenziell klinische Relevanz der nukleären Caspase-8 herausgearbeitet werden. Wir konnten bestätigen, dass nukleär lokalisierte Caspase-8 bei Wildtyp p53 exprimierenden Tumorzellen zu Chemoresistenz und auch in geringem Maß zur Resistenz gegenüber zielgerichteten Therapien führt. Zunächst wurde die subzelluläre Lokalisation von Caspase-8 in den vorhandenen 16 Melanomzellproben mithilfe immunzytochemischer Färbung bestimmt, wobei sich herausstellte, dass die Melanomzellproben mit nukleärer Caspase-8, alle von Metastasen stammen. Das hohe Metastasierungspotenzial dieser Zellproben wurde weiterhin dadurch betont, dass die Melanomzellproben mit nukleär lokalisierter Caspase-8 die stärkste Migration aufwiesen. Dem gegenüberstehend präsentierten sie die geringste Proliferation, was den direkten Zusammenhang zwischen migrativen und proliferativen Eigenschaften bei Tumorzellen unterstreicht. Ebenso zeigten BRAF-mutierte Zellproben die stärkste Migration und bekräftigen, dass Melanompatienten mit BRAF-Mutation früher Metastasen entwickeln. Das stärkste Wachstum zeigten hingegen die Zellproben mit diffus lokalisierter Caspase-8, was die protektive Relevanz der diffusen Caspase-8 für Tumorzellen verdeutlicht. Ein Einfluss des p53-Mutationsstatus auf die Proliferation und Migration konnte ebenfalls nachgewiesen werden. Die Zellproben mit Wildtyp p53 migrierten stärker, wohingegen die Zellprobe mit mutp53(E285K) stark proliferierte. Bei Korrelation der subzellulären Caspase-8-Lokalisation mit der Caspase-8- und p53-Expression der Melanomzellproben mithilfe Westernblotanalysen zeigte sich, dass metastatische Zellproben verstärkt Caspase-8 exprimieren. Dies betraf besonders die metastatischen Zellproben mit nukleär lokalisierter Caspase-8, die kein p53 oder wenig Wildtyp p53 exprimieren. Diese inverse Korrelation der Caspase-8- und p53 Expression konnte ebenfalls bei den unstimulierten Zellproben mit zytoplasmatischer Caspase-8 festgestellt werden. Zellprobe M31 mit mutp53(E285K) exprimiert konstant stark p53 und wies Chemoresistenz auf. Bei Untersuchung des Zelltods 24 h und 48 h nach Cisplatinstimulation, sowie 48 h nach Stimulation mit Dabrafenib, Trametinib und deren Kombination mithilfe des IncuCyte® Readers, wurde deutlich, dass die Zellproben mit nukleär lokalisierter Caspase-8 chemoresistenter sind als die restlichen Zellproben, wobei die Zellproben mit diffus lokalisierter Caspase-8 am resistentesten gegenüber den zielgerichteten Therapeutika sind. Die protektive Rolle der diffusen Caspase-8 für die Tumorzellen konnte unterstützend durch die Zunahme der Apoptose bei einigen Zellproben nach Herunterregulierung von Caspase-8 nachgewiesen werden. Hervorzuheben ist, dass die Zellproben mit zytoplasmatischer Caspase-8 am sensitivsten, sowohl auf zielgerichtete Therapeutika als auch auf Chemotherapie reagierten. Insgesamt induzierte Cisplatin deutlich mehr Zelltod als die zielgerichteten Therapien, weshalb die potenzielle Überlegenheit der Chemotherapie, besonders bei rezidivierten Melanomen bedacht werden muss. Es bestätigte sich ebenfalls die Relevanz des BRAF-/NRAS Mutationsstatus für die Therapiesensitivität, wobei BRAF-mutierte Zellproben die größte Chemosensitivität präsentierten und NRAS-mutierte Zellproben vor allem nach der Stimulation mit Trametinib und der Kombination Dabrafenib + Trametinib Zelltod aufwiesen. Überraschenderweise zeigten sich die Zellproben vortherapierter Melanompatienten sensitiver gegenüber Dabrafenib und Cisplatin als die Zellproben therapienaiver Melanompatienten, was die Bedeutung von Chemotherapie und Dabrafenib für vortherapierte, rezidivierte Tumore bzw. Metastasen hervorhebt. Weiterhin konnte der Impact des p53-Mutationsstatus herausgearbeitet werden. Die Zellproben mit mutiertem oder ohne p53 wiesen die größte Cisplatinresistenz auf, wohingegen die Zellproben mit Wildtyp p53 am meisten Zelltod zeigten, gefolgt von den Zellproben mit dem Einzelnukleotidpolymorphismus (wt(P72R)). Nach Herunterregulierung des mutierten p53 nahm die Apoptose nach Cisplatinstimulation hoch signifikant zu. Dahingegen zeigten besonders die Zellproben mit zytoplasmatischer Caspase-8 und Wildtyp p53 eine Hochregulierung von p53 nach Cisplatinstimulation im Westernblot. Wir konnten nachweisen, dass die Wildtyp p53 exprimierende Zellprobe M40 nukleär lokalisierte Caspase-8 aufweist und die stärkste Chemoresistenz zeigte, wohingegen die Zellproben mit diffus oder zytoplasmatisch lokalisierter Caspase-8, die Wildtyp p53 oder den Einzelnukleotidpolymorphismus (wt(P72R)) exprimieren, am chemosensitivsten waren. Die Therapieresponsivität von Melanomen wird durch eine Vielzahl an Faktoren beeinflusst, die für die optimale Behandlung der Patienten bei Beginn einer Therapie möglichst genau bestimmt werden sollten. Wir empfehlen für die Risikostratefizierung und Prognose des Krankheitsverlaufes, sowie für die Entscheidung des Therapiewegs, die Bestimmung der subzellulären Caspase-8-Lokalisation in Kombination mit dem p53-Mutationsstatus. Weiterhin weisen wir auf die Bedeutung des BRAF-/NRAS-Mutationsstatus sowie mögliche Vortherapien in Bezug auf Therapieresistenz hin und stellen die Bedeutung der Chemotherapie, besonders für rezidivierte, BRAF-mutierte Melanompatienten bei Resistenzentwicklung gegen BRAF- und/oder MEK-Inhibitoren heraus. / Malignant melanoma is responsible for 90 % of skin cancer deaths (Garbe et al., 2022). Various mutations affect the signaling pathways and promote carcinogenesis, which leads to a constant change in melanoma therapy. In addition to surgical therapy, which is curative in the early stages, targeted therapy with BRAF and MEK inhibitors has achieved resounding successes for the overall survival of patients in recent years while chemotherapy is still recommended for non-resectable metastases. Increasing therapy resistance limits melanoma therapy, with Müller et al. (2020) pointing to wild-type p53-expressing tumor cells that use the expression of nuclear caspase-8 to bypass the G2/M cell cycle arrest and thus promote the proliferation and expansion of degenerated tumor cells. By investigating 16 melanoma cell samples differing in their BRAF/NRAS and p53 mutation status, as well as in their prior clinical therapies, the potential clinical relevance of nuclear caspase-8 could be elaborated. We were able to confirm that nuclear localized caspase-8 in wild-type p53-expressing tumor cells leads to chemoresistance and, to a lesser extent, to resistance to targeted therapies. First, the subcellular localization of caspase-8 in the existing 16 melanoma cell samples was determined using immunocytochemical staining, which revealed that the melanoma cell samples with nuclear caspase-8 all originated from metastases. The high metastatic potential of these cell samples was further emphasized by the fact that the melanoma cell samples with nuclear localized caspase-8 showed the strongest migration. In contrast, they presented the lowest proliferation, underlining the direct correlation between migratory and proliferative properties in tumor cells. Likewise, BRAF-mutated cell samples showed the strongest migration and verified that melanoma patients with BRAF mutations develop metastases earlier. In contrast, the cell samples with diffusely localized caspase-8 showed the strongest growth, which illustrates the relevance of diffusely localized caspase-8 in its protective function in tumor cells. The impact of the p53 mutation status on proliferation and migration could also be demonstrated. The cell samples with wild-type p53 migrated stronger, whereas the cell samples with mutp53(E285K) showed stronger proliferation. Correlation of subcellular caspase-8 localization with caspase-8 and p53 expression of melanoma cell samples using western blot analysis showed that metastatic cell samples express more caspase-8. This was particularly true for the metastatic cell samples with nuclearly localized caspase-8, which express little wild-type p53 or no p53. This inverse correlation of caspase-8 and p53 expression was also observed in the unstimulated cell samples with cytoplasmically localized caspase-8, respectively. Cell sample M31 with mutp53(E285K) consistently expressed high levels of p53 and exhibited chemoresistance. When examining cell death 24 h and 48 h after cisplatin stimulation, as well as 48 h after stimulation with dabrafenib, trametinib and their combination using the IncuCyte® Reader, it became clear that the cell samples with nuclearly localized caspase-8 are the most chemoresistant, whereby the cell samples with diffusely localized caspase-8 are the most resistant to the targeted therapeutics. The protective role of diffusely localized caspase-8 for the tumor cells was supportively demonstrated by the increase in apoptosis in some cell samples after downregulation of caspase-8. It should be emphasized that the cell samples with cytoplasmically localized caspase-8 responded most sensitively to both targeted therapies and chemotherapy. Overall, cisplatin induced significantly more cell death than the targeted therapies, which is why the potential superiority of chemotherapy, especially in recurrent melanoma, must be considered. The relevance of BRAF/NRAS mutation status for therapy sensitivity was also confirmed, with BRAF-mutated cell samples presenting the greatest chemosensitivity and NRAS-mutated cell samples showing cell death particularly after stimulation with trametinib and the combination dabrafenib + trametinib. Surprisingly, the cell samples of pre-treated melanoma patients were more sensitive to dabrafenib and cisplatin than the cell samples of treatment-naive melanoma patients, which highlights the importance of chemotherapy and dabrafenib for pre-treated, recurrent tumors and metastases. Furthermore, the impact of the p53 mutation status could be worked out. The cell samples with mutated or no p53 showed the highest cisplatin resistance, whereas the cell samples with wild-type p53 showed the highest cell death, followed by the cell samples with the single nucleotide polymorphism (wt(P72R)). After downregulation of mutant p53, apoptosis increased highly significantly after cisplatin stimulation. In contrast, especially the cell samples with cytoplasmically localized caspase-8 and wild-type p53 showed an upregulation of p53 in the Western blot after cisplatin stimulation. We demonstrated that wild-type p53 and nuclear caspase-8 expressing cell sample M40 exhibited the strongest chemoresistance, whereas the cell samples with diffusely or cytoplasmically localized caspase-8 expressing wild-type p53 or the single nucleotide polymorphism (wt(P72R)) were the most chemosensitive. In summary, it is clear that the treatment response of melanoma is influenced by a variety of factors that should be determined as precisely as possible for the optimal treatment of patients at the start of therapy. We recommend the determination of subcellular caspase-8 localization in combination with the p53 mutation status for risk stratification and prognosis of disease progression, as well as for the optimal therapy. Furthermore, we point out the importance of BRAF/NRAS mutation status and possible prior therapies with regard to therapy resistance and emphasize the importance of chemotherapy, especially for relapsed, BRAF-mutated melanoma patients who developed resistance to BRAF and/or MEK inhibitors.