<p>To maintain homeostasis, T lymphocytes die through caspase&ndash;dependent apoptosis. However, blockage of caspase activity in T lymphocytes does not increase cell survival. The loss of caspase 8 activity leads to programmed necrosis (necroptosis) upon T cell receptor (TCR) stimulation in T lymphocytes. Necroptosis is correlated with excessive macroautophagy, an intracellular catabolic process characterized by the sequestration of cytoplasmic compartments through double&ndash;membrane vacuoles. Meanwhile, the proper induction of macroautophagy is required for T lymphocyte survival and function. Cellular caspase 8 (FLICE)&ndash;like inhibitory protein (c&ndash;FLIP) promotes survival in T lymphocytes. c&ndash;FLIP suppresses death receptor&ndash;induced apoptosis by modulating caspase 8 activation. Whether this modulation plays a role in the regulation of necroptosis has yet to be studied. Additionally, overexpression of c&ndash;FLIP reduces autophagy induction and promotes cell survival in cell lines. It remains unclear whether c&ndash;FLIP protects primary T lymphocytes by regulating the threshold at which autophagy occurs. In this study, c&ndash;FLIP isoform&ndash;specific conditional deletion models were used to study the role of c&ndash;FLIP in necroptosis and autophagy in primary T lymphocytes.</p><p>Our results showed that the long isoform of c&ndash;FLIP (c&ndash; FLIP_L) regulates necroptosis by inhibiting receptor interacting protein 1 (RIP&ndash;1). Upon TCR stimulation, c&ndash;FLIP_L&ndash;deficient T cells underwent RIP&ndash;1&ndash;dependent necroptosis. Interestingly, though previous studies have generally described necroptosis in the absence of caspase 8 activity and apoptosis, pro&ndash;apoptotic caspase 8 activity and the rate of apoptosis were also increased in c&ndash;FLIPL&ndash;deficient T lymphocytes. Moreover, c&ndash; FLIP_L&ndash;deficient T cells exhibited enhanced autophagy, which served a cytoprotective function. </p><p>Apoptosis can be induced by either death receptors on the plasma membrane (extrinsic pathway), or the damage of the genome and/or cellular organelles (intrinsic pathway). Previous studies in c&ndash;FLIP&ndash;deficient T lymphocytes suggested that c&ndash;FLIP promotes cell survival in the absence of death receptor signals. Independent of death receptor signaling, mitochondria sense apoptotic stimuli and mediate the activation of caspases. Whether c&ndash;FLIP regulates mitochondrion&ndash;dependent apoptotic signaling remains unknown. Here, by deleting the <italic>c&ndash;Flip <italic> gene in mature T lymphocytes, we showed a role for c&ndash;FLIP in the intrinsic apoptosis pathway. In naïve T cells stimulated with the apoptosis inducer, c&ndash;FLIP suppressed cytochrome c release from mitochondria. Bim&ndash;deletion rescued the enhanced apoptosis in c&ndash;FLIP&ndash;deficient T cells, while inhibition of caspase 8 did not. Different from activated T cells, there were no signs of necroptosis in c&ndash;FLIP&ndash;deficient naïve T cells. Together, our findings indicate that c&ndash;FLIP is a key regulator of apoptosis, necroptosis and autophagy in T lymphocytes.</p> / Dissertation

Die Identifizierung von Apoptose-modifizierten Proteinen erfolgte durch Proteomanalyse per 2D-Gelelektrophorese und Massenspektrometrie. Zunächst wurde eine 2DE-Datenbank errichtet, die im Internet zugänglich ist. Die Identifikation von Proteinen durch Peptidmassenfingerabdruck konnte durch Verwendung einer zweiten Matrix verbessert werden. Die Analyse ausgelassener tryptischer Spaltstellen, N-terminaler Pyroglutamatbildung und Oxidation von Tryptophan konnte die Identifikation von Proteinen mittels Peptidmassenfingerabdruck verbessern. Die Apoptose wurde über den Fas-Rezeptor-Signalweg oder durch DNA-Schädigung mittels Cis-Platin eingeleitet und das Totallysat oder die Kompartimente von Jurkat T-Zellen der Proteomanalyse unterworfen. Große Übereinstimmungen der beiden Prozesse wurden festgestellt. 95 Apoptose-modifizierte Proteine wurden identifiziert, wovon 78 Proteine bisher unbekannt für den Apoptoseprozess waren. Auffällig war, dass 40 % der Proteine RNA-Bindungsmotive enthielten und das 21 Onkoprotein oder Onkoprotein-interagierende Proteine identifiziert wurden. Für 39 Proteine konnten bisher proteolytische Spaltungen vorausgesagt werden. Eine Fülle von Informationen wurde über putative Translokationen der Proteine erhalten. Für das Protein p54nrb wurden drei Caspase-3 Spaltstellen durch die Einführung von Mutationen und die Abhängigkeit der Caspase-3 Spaltung von RNA bewiesen. Mitochondriale ribosomale Proteine von Mensch, Maus und Ratte wurden durch Abgleichung von EST-Datenbanken mit partiellen Aminosäuresequenzen aus dem Rind bestimmt. Die Konservierung der Sequenzen der Säugetierproteine der mitochondrialen ribosomalen Proteinen war geringer als von den bekannten cytosolischen ribosomalen Proteinen. Weiterhin wurden unterschiedliche Ergebnisse bzgl. der mitochondrialen Signalsequenzen der Proteine gefunden. RNA-Protein-Wechselwirkungen im Ribosom wurden nach Quervernetzung auf einzelne Aminosäuen bzw. Nukleotide bestimmt. Die Daten wurden zur Verbesserung von ribosomalen Modellen verwendet. Die mittlerweile erhaltenen Kristallstrukturen des Ribosoms zeigten, dass die Ergebnisse der Quervernetzungsexperimente mit den tatsächlichen RNA-Protein-Wechselwirkungen weitgehend übereinstimmen. Die Affinität von verschiedenen Komponenten zu einem Zielmolekül zur Bildung von nicht-kovalenten RNA-Peptid-Wechselwirkungen wurde mit Hilfe von MALDI-MS ermittelt. Die Interaktionen sind stark abhängig von der Anzahl der Arginine. / Apoptosis-modified proteins were identified by proteome analysis via 2D gel electrophoresis and mass spectrometry. First, a internet-accessible 2DE database was rendered. The identification of the proteins by peptide mass fingerprinting was improved using a second matrix. The analysis of missed tryptic cleavage sites, the formation of N-terminal pyroglutamine and oxidation of tryptophan could improve the identification of proteins by peptide mass fingerprinting. Apoptosis was induced via the Fas-receptor signaling pathway or by means of DNA damage by cis-platin. The total lysate and the compartments of Jurkat T cells were analyzed by proteome analysis. High similarities between both processes were observed. 95 apoptosis-modified proteins were identified, 78 of these were until now unknown to be involved in apoptosis. Noticeable, 40% of the proteins include a RNA-binding motif and 21 oncogene or oncogene-interacting proteins were identified. A proteolytic cleavage could be predicted for 39 proteins. Some information was received about the putative translocation of the proteins. Three caspase-3 cleavage sites were shown for the protein p54nrb with the incorporation of mutations. Furthermore, the caspase-3 cleavage was dependent on the occurrence of RNA. Mitochondrial ribosomal proteins of human, mouse and rat were determined by screening of EST-databases with partial amino acid sequences from bovine. The conservation of sequences of mammalian proteins of the mitochondrial ribosomal proteins was less than for known cytosolic ribosomal proteins. Furthermore, different results were obtained considering mitochondrial signal sequences. RNA-protein interaction within the ribosome were determined on single amino acids and nucleotides, respectively, after cross-linking. These data were used to improve models of the ribosome. The in the meantime obtained 3D-structures of the ribosome showed high consistency with the revealed RNA-protein interaction sites after cross-linking. The affinity of different components to a target molecule to form RNA-peptide interactions was determined by MALDI-MS. The interactions were strongly dependent on the number of arginines.

<p>Multiple myeloma (MM) is a virtually incurable B cell malignancy of the bone marrow. One important part of tumor progression and an obstacle for successful therapy is resistance to apoptosis. To combat this resistance, the mechanisms of apoptosis and survival in MM must be better defined. </p><p>In this thesis, we identified Fas up-regulation as a mechanism underlying interferon (IFN)-mediated sensitization to Fas-induced apoptosis in the MM cell line U-266-1970. IFN treatment induced activation of signal transducer and activator of transcription (Stat)1 but, intriguingly, also attenuated activation of MM survival factor Stat3. </p><p>Exploring the role of Stat1 further, we established sub-lines of U-266-1970 with a stable over-expression of Stat1 and of its active mutant Stat1C. These sub-lines displayed a decreased expression and activation of Stat3, and an altered expression of apoptosis-related genes Harakiri, Bcl-2 and Mcl-1. In a drug library screening, Stat1 over-expression was associated with an increased sensitivity to Fas-induced apoptosis and, conversely, an increased resistance to several drugs, including the cyclin dependent kinase (cdk)1 inhibitor CGP74514A. We conclude that Stat1 over-expression does not confer a general resistance or sensitivity to apoptosis in MM, but may strongly affect the response to some specific drugs.</p><p>We also explored the effects of picropodophyllin (PPP), an inhibitor of the insulin-like growth factor I (IGF-I) receptor tyrosine kinase (RTK), in MM. PPP selectively inhibited the IGF-I RTK activity without inhibiting the insulin RTK activity. Furthermore, PPP potently induced cell cycle arrest and apoptosis in all MM cell lines and patient samples tested, also in the presence of survival factors IGF-I and IL-6. We conclude that PPP has great therapeutic potential in MM </p><p>Finally, we examined the expression and regulation of the inhibitors of apoptosis proteins (IAPs) in a panel of MM cell lines and patient samples. The glucocorticoid dexamethasone, which is used in MM therapy, induced a transient up-regulation and a subsequent down-regulation of c-IAP2, as well as a down-regulation of XIAP, possibly influencing the sensitivity to apoptosis induced by this drug. Supporting this notion, abrogation of IGF-IR signaling by PPP, which sensitizes MM cells to dexamethasone-induced apoptosis, enhanced the down-regulation of c-IAP2 and XIAP.</p>

Multiple myeloma (MM) is a virtually incurable B cell malignancy of the bone marrow. One important part of tumor progression and an obstacle for successful therapy is resistance to apoptosis. To combat this resistance, the mechanisms of apoptosis and survival in MM must be better defined. In this thesis, we identified Fas up-regulation as a mechanism underlying interferon (IFN)-mediated sensitization to Fas-induced apoptosis in the MM cell line U-266-1970. IFN treatment induced activation of signal transducer and activator of transcription (Stat)1 but, intriguingly, also attenuated activation of MM survival factor Stat3. Exploring the role of Stat1 further, we established sub-lines of U-266-1970 with a stable over-expression of Stat1 and of its active mutant Stat1C. These sub-lines displayed a decreased expression and activation of Stat3, and an altered expression of apoptosis-related genes Harakiri, Bcl-2 and Mcl-1. In a drug library screening, Stat1 over-expression was associated with an increased sensitivity to Fas-induced apoptosis and, conversely, an increased resistance to several drugs, including the cyclin dependent kinase (cdk)1 inhibitor CGP74514A. We conclude that Stat1 over-expression does not confer a general resistance or sensitivity to apoptosis in MM, but may strongly affect the response to some specific drugs. We also explored the effects of picropodophyllin (PPP), an inhibitor of the insulin-like growth factor I (IGF-I) receptor tyrosine kinase (RTK), in MM. PPP selectively inhibited the IGF-I RTK activity without inhibiting the insulin RTK activity. Furthermore, PPP potently induced cell cycle arrest and apoptosis in all MM cell lines and patient samples tested, also in the presence of survival factors IGF-I and IL-6. We conclude that PPP has great therapeutic potential in MM Finally, we examined the expression and regulation of the inhibitors of apoptosis proteins (IAPs) in a panel of MM cell lines and patient samples. The glucocorticoid dexamethasone, which is used in MM therapy, induced a transient up-regulation and a subsequent down-regulation of c-IAP2, as well as a down-regulation of XIAP, possibly influencing the sensitivity to apoptosis induced by this drug. Supporting this notion, abrogation of IGF-IR signaling by PPP, which sensitizes MM cells to dexamethasone-induced apoptosis, enhanced the down-regulation of c-IAP2 and XIAP.

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