TY - JOUR A1 - Waldholm, Johan A1 - Wang, Zhi A1 - Brodin, David A1 - Tyagi, Anu A1 - Yu, Simei A1 - Theopold, Ulrich A1 - Östlund Farrants, Ann Kristin A1 - Visa, Neus T1 - SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in \(Drosophila\) \(melanogaster\) JF - BMC Molecular Biology N2 - Background: The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA. Results: We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo. Conclusions: We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes. KW - Chromatin-remodeling complexes KW - In-vivo KW - Genes KW - Distinct KW - Brahma KW - Transcription KW - Trithorax KW - Subunit KW - Exons KW - BRM Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142613 VL - 12 IS - 46 ER - TY - JOUR A1 - Wang, Huiqiang A1 - Chen, Nanhai G. A1 - Minev, Boris R. A1 - Szalay, Aladar A. T1 - Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells JF - Journal of Translational Medicine N2 - Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells. Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. KW - tumors KW - therapy KW - metastasis KW - identification KW - lines KW - gene expression KW - in-vitro propagation KW - acute myeloid leukemia KW - epithelial-mesenchymal transition KW - subpopulation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130019 VL - 10 IS - 167 ER - TY - JOUR A1 - Wang, Huiqiang A1 - Chen, Nanhai G. A1 - Minev, Boris R. A1 - Zimmermann, Martina A1 - Aguilar, Richard J. A1 - Zhang, Qian A1 - Sturm, Julia B. A1 - Fend, Falko A1 - Yu, Yong A. A1 - Cappello, Joseph A1 - Lauer, Ulrich M. A1 - Szalay, Aladar A. T1 - Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains JF - PLoS ONE N2 - Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease. KW - lymph nodes KW - cancer treatment KW - metastatic tumors KW - breast cancer KW - blood KW - prostate cancer KW - ascites KW - mouse models Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130059 VL - 8 IS - 9 ER - TY - THES A1 - Wanzek, Katharina T1 - The investigation of the function of repair proteins at G-quadruplex structures in \(Saccharomyces\) \(cerevisiae\) revealed that Mms1 promotes genome stability T1 - Die Untersuchung der Funktion von Reparaturproteinen an G-Quadruplex Strukturen in \(Saccharomyces\) \(cerevisiae\) zeigte, dass Mms1 Genomstabilität fördert N2 - G-quadruplex structures are highly stable alternative DNA structures that can, when not properly regulated, impede replication fork progression and cause genome instability (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu & Spies, 2016; Zimmer et al, 2016). The aim of this thesis was to identify novel G-quadruplex interacting proteins in Saccharomyces cerevisiae and to unravel their regulatory function at these structures to maintain genome integrity. Mms1 and Rtt101 were identified as G-quadruplex binding proteins in vitro via a pull-down experiment with subsequent mass spectrometry analysis. Rtt101, Mms1 and Mms22, which are all components of an ubiquitin ligase (Rtt101Mms1/Mms22), are important for the progression of the replication fork following fork stalling (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). The in vivo binding of endogenously tagged Mms1 to its target regions was analyzed genome-wide using chromatin-immunoprecipitation followed by deep-sequencing. Interestingly, Mms1 bound independently of Mms22 and Rtt101 to G-rich regions that have the potential to form G-quadruplex structures. In vitro, formation of G-quadruplex structures could be shown for the G-rich regions Mms1 bound to. This binding was observed throughout the cell cycle. Furthermore, the deletion of MMS1 caused replication fork stalling as evidenced by increased association of DNA Polymerase 2 at Mms1 dependent sites. A gross chromosomal rearrangement assay revealed that deletion of MMS1 results in a significantly increased genome instability at G-quadruplex motifs compared to G-rich or non-G-rich regions. Additionally, binding of the helicase Pif1, which unwinds G4 structures in vitro (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), to Mms1 binding sites was reduced in mms1 cells. The data presented in this thesis, together with published data, suggests a novel mechanistic model in which Mms1 binds to G-quadruplex structures and enables Pif1 association. This allows for replication fork progression and genome integrity. N2 - Bei G-quadruplex Strukturen handelt es sich um stabile Sekundärstrukturen der DNA, welche das Fortschreiten der Replikationsgabel behindern und Genominstabilität verursachen können, falls sie nicht konsequent reguliert werden (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu & Spies, 2016; Zimmer et al, 2016). Ziel dieser Doktorarbeit war es, neue Proteininteraktionspartner dieser Strukturen in Saccharomyces cerevisiae zu identifizieren und zu untersuchen, wie diese Proteine die Strukturen regulieren um Genomstabilität zu gewährleisten. Mit Hilfe eines Pulldown Assays und anschließender massenspektrometrischer Analyse wurden Mms1 und Rtt101 in vitro als Interaktionspartner von G-quadruplex Strukturen identifiziert. Rtt101, Mms1 und Mms22, Komponenten der Ubiquitinligase Rtt101Mms1/Mms22, spielen eine wichtige Rolle beim Fortschreiten der Replikationsgabel, falls dieses durch Agenzien gehemmt wurde (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). Durch Chromatin-Immunpräzipitation mit anschließender Hochdurchsatzsequenzierung wurden die Bindestellen von Mms1 identifiziert. Interessanterweise hat Mms1 genomweit an G-reiche Sequenzen gebunden. Diese G-reichen Sequenzen bildeten G-quadruplex Strukturen in vitro aus. Die Bindung von Mms1 erfolgte unabhängig von Rtt101 und Mms22 sowie während des gesamten Zellzyklus. Außerdem kam es zu einer Verlangsamung der Replikationsgabel in mms1 Zellen, was durch eine verstärkte Bindung der DNA Polymerase 2 nachgewiesen wurde. Ein gross chromsomal rearrangement assay zeigte, dass die Genominstabilität in mms1 Zellen signifikant erhöht ist, wenn G-quadruplex Motive, im Vergleich zu nicht-G-reichen oder G-reichen Kontrollregionen, vorhanden sind. Zudem war die Bindung der Helikase Pif1, welche G-quadruplex Strukturen in vitro entwindet (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), stark reduziert, wenn Mms1 fehlte. Mit Hilfe der in dieser Doktorarbeit gewonnenen Ergebnisse, sowie mit Hilfe publizierter Daten, lässt sich ein Model postulieren, in welchem Mms1 an G-quadruplexe bindet und somit die Bindung von Pif1 ermöglicht. Dadurch werden das Fortschreiten der Replikationsgabel und die Genomstabilität gewährleistet. KW - Quadruplex-DNS KW - DNS-Reparatur KW - genome stability KW - Bierhefe Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-142547 ER - TY - JOUR A1 - Weibel, Stephanie A1 - Basse-Luesebrink, Thomas Christian A1 - Hess, Michael A1 - Hofmann, Elisabeth A1 - Seubert, Carolin A1 - Langbein-Laugwitz, Johanna A1 - Gentschev, Ivaylo A1 - Sturm, Volker Jörg Friedrich A1 - Ye, Yuxiang A1 - Kampf, Thomas A1 - Jakob, Peter Michael A1 - Szalay, Aladar A. T1 - Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by \(^{19}\)F-Magnetic Resonance Imaging (MRI) JF - PLoS ONE N2 - Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response. KW - inflammation KW - fluorescence microscopy KW - oncolytic viruses KW - fluorescence imaging KW - macrophages KW - magnetic resonance imaging KW - histology KW - in vivo imaging Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130311 VL - 8 IS - 3 ER - TY - THES A1 - Wollny, Claudia T1 - Der p97-Kofaktor UBXD1 ist ein neuer Regulator des NF-kB-Signalweges T1 - The p97-cofactor UBXD1 is a new regulator of NF-kB-signaling N2 - Die essenzielle, Ubiquitin-selektive ATPase p97 reguliert eine Vielzahl unterschiedlicher Prozesse in Eukaryoten. Dazu zählen Proteinqualitätskontrolle, DNA-Reparatur, Signaltransduktion, Zellzykluskontrolle, Autophagie sowie das endolysosomale System. Diese unterschiedlichen Funktionen von p97 werden durch die Bindung von Kofaktoren engmaschig gesteuert und kontrolliert. Die größte und am besten untersuchte Gruppe von p97-Kofaktoren sind die Proteine der UBX Familie. Diese zeichnen sich durch den Besitz einer UBX-Domäne aus, welche die Bindung an p97 vermittelt. Das in höheren Eukaryoten konservierte Familienmitglied UBXD1 besitzt darüber hinaus mit einer PUB-Domäne und einem VIM-Motiv noch mindestens zwei weitere p97-Bindemodule. UBXD1 kann an Vesikel des endolysosomalen Degradationssytems lokalisieren, seine genauen zellulären Funktionen sind jedoch noch weitgehend unbekannt. Ziel dieser Arbeit war die funktionelle Charakterisierung von humanem UBXD1. Dafür wurden Kandidaten eines zuvor durchgeführten Yeast-Two-Hybrid-Screens auf ihre Two Hybrid-Interaktion mit unterschiedlichen UBXD1-Varianten getestet. Darüber hinaus wurde durch Immunpräzipitationsexperimente untersucht, ob die Kandidatenproteine auch in Säugerzellen mit UBXD1 interagieren. Als vielversprechende neue Bindungspartner von UBXD1 wurden so die Ubiquitin-Ligase TRIAD3A und das Ubiquitin-editierende Protein A20 identifiziert. Desweiteren konnte gezeigt werden, dass die Interaktion zwischen UBXD1 und A20 von einer funktionellen PUB Domäne und dem siebten Zinkfinger Motiv von A20 abhängig ist. Da sowohl TRIAD3A als auch A20 negative Regulatoren des NF B Signalweges sind, wurde daraufhin untersucht, ob auch UBXD1 eine Funktion in diesem Signalweg besitzt. Tatsächlich war in UBXD1-depletierten HeLa 57A-Zellen die NF B-abhängige Expression eines Reportgens nach Aktivierung des Signalweges durch TNF, IL-1, Doxorubicin und H2O2 stark reduziert. Dabei spricht die verringerte Aktivierung nach unterschiedlichen Stimuli für eine generelle Rolle von UBXD1 im NF B Signalweg. Durch quantitative Echtzeit-PCR konnte gezeigt werden, dass in HeLa- und HEK293T-Zellen nach UBXD1-Depletion auch die Expression endogener NF B Zielgene verringert ist. Da in UBXD1-depletierten Zellen nach Stimulation mit TNF oder IL-1 bereits die Kerntranslokation des NF B-Transkriptionsfaktor p65 reduziert ist, ist davon auszugehen, dass UBXD1 an einer früheren Phase der Aktivierung des Signalweges beteiligt ist. Möglicherweise ist dies darauf zurückzuführen, dass UBXD1 bekannte Funktionen von A20 reguliert und etwa die Bindung von A20 an Vesikel des endolysosomalen Systems oder an lineare Ubiquitinketten beeinflusst. Diese Arbeit beschreibt somit eine neue Funktion des p97-Kofaktors UBXD1 im NF B-Signalweg. N2 - The essential, ubiquitin-selective ATPase p97 regulates a variety of cellular processes in eukaryotes. Among others, these include protein quality control, DNA repair, signal-transduction, cell cycle control, autophagy and the endolysosomal system. The distinct functions of p97 are tightly controlled by regulatory cofactors. UBX domain-containing proteins are the largest and best studied group of p97 cofactors . They are characterized by a UBX domain, which mediates binding to p97. The family-member UBXD1 is highly conserved in higher eukaryotes and possesses at least two additional p97 binding modules, a PUB domain and a VIM motif. While UBXD1 can localize to vesicles of the endolysosomal degradation system, its exact cellular function is still poorly understood. The aim of this study was the functional characterisation of human UBXD1. To that end, candidates of a previous yeast two-hybrid screen were tested for their two-hybrid interaction with different UBXD1 variants. Immunoprecipitation experiments were used to analyse if the candidates also interact with UBXD1 in mammalian cells. This led to the identification of the ubiquitin-ligase TRIAD3A and the ubiquitin-editing protein A20 as promising new binding partners of UBXD1. Moreover, it could be demonstrated that the interaction between UBXD1 and A20 depends on a functional PUB domain and the seventh zinc finger motif of A20. Because both TRIAD3A and A20 are negative regulators of the NF-B signaling pathway, it was subsequently tested if UBXD1 also has a function in NF-B signaling. Indeed, UBXD1-depleted HeLa 57A cells showed a strongly reduced NF B dependent expression of a reporter gene after activation of the signaling pathway by TNF, IL-1, Doxorubicin and H2O2. The reduced activity observed after various stimuli argues for a general role of UBXD1 in the NF-B signaling pathway. Quantitative real-time PCR demonstrated that the expression of endogenous NF-B target genes in HeLa and HEK293T cells was also reduced upon UBXD1-depletion. Since the nuclear translocation of the NF-B subunit p65 upon stimulation with TNF or IL-1was also reduced in UBXD1-depleted cells, UBXD1 is likely to participate in an earlier phase of NF-B activation. It is possible that UBXD1 regulates a known function of A20 and influences for example the binding of A20 to endocytic vesicles or to linear ubiquitin chains. In summary, this work describes a novel function of the p97 cofactor UBXD1 as a positive regulator of the NF-B signaling pathway. KW - Ubiquitin KW - UBXD1 KW - Signaltransduktion KW - Cofaktor KW - p97 KW - Signalweg KW - Zellbiologie Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132430 ER - TY - THES A1 - Ye, Mingyu T1 - Immunotherapy with Vaccinia virus co-expressing tumor-associated antigens and mouse IL-2 cytokine in mice with mammary cancer T1 - Immuntherapie von Brustkrebs in tumortragenden Mäusen mit genetisch modifizierten Vaccinia Viren, die simultan Interleukin-2 und tumorassoziierte Antigene exprimieren N2 - Interleukin 2 (IL-2) was the first cytokine applied for cancer treatment in human history. It has been approved as monotherapy for renal cell carcinoma and melanoma by the FDA and does mediate the regression of the tumors in patients. One of the possible mechanisms is that the administration of IL-2 led to T lymphocytes expansion, including CD4+ and CD8+ T cells. In addition, a recent study demonstrated that antigen-specific T cells could also be expanded through the induction of IL-2, which plays a crucial role in mediating tumor regression. However, despite the long-term and extensive use of IL-2 in the clinic, the ratio of patients who get a complete response was still low, and only about one-fifth of patients showed objective tumor regression. Therefore, the function of IL-2 in cancer treatment should continue to be optimized and investigated. A study by Franz O. Smith et al. has shown that the combination treatment of IL-2 and tumor-associated antigen vaccine has a strong trend to increased objective responses compared to patients with melanoma receiving IL-2 alone. Peptide vaccines are anti-cancer vaccines able to induce a powerful tumor antigenspecific immune response capable of eradicating the tumors. According to the type of antigens, peptide vaccines can be classified into two distinct categories: Tumor-associated antigens (TAA) vaccine and tumor-specific neoantigens (TSA) vaccine. Currently, Peptide vaccines are mainly investigated in phase I and phase II clinical trials of human cancer patients with various advanced cancers such as lung cancer, gastrointestinal tumors, and breast cancers. Vaccinia virus (VACV) is one of the safest viral vectors, which has been wildly used in cancer treatment and pathogen prevention. As an oncolytic vector, VACV can carry multiple large foreign genes, which enable the virus to introduce diagnostic and therapeutic agents without dramatically reducing the viral replication. Meanwhile, the recombinant vaccinia virus (rVACV) can be easily generated by homologous recombination. Here, we used the vaccinia virus as the therapeutic cancer vector, expressing mouse Interleukin 2 (IL-2) and tumor-associated antigens simultaneously to investigate the combined effect of anti-tumor immune response in the 4T1 mouse tumor model. As expected, the VACV driven mIL-2 expression remarkably increased both CD4+ and CD8+ populations in vivo, and the virus-expressed tumor-associated peptides successfully elicited theantigen-specific T cell response to inhibit the growth of tumors. Furthermore, the experiments with tumor-bearing animals showed that the mIL-2 plus tumor antigens expressing VACV vector gave a better anti-cancer response than the mIL-2 alone expressing vector. The combinations did significantly more inhibit tumor growth than mIL-2 treatment alone. Moreover, the results confirmed our previous unpublished data that the mIL-2 expression driven by synthetic early/late promoter in the Lister strain VACV could enhance the tumor regression in the 4T1 mouse model. N2 - Interleukin 2 (IL-2) war das erste Zytokin in der Geschichte des Menschen, das zur Krebsbehandlung eingesetzt wurde. Es ist von der FDA als Monotherapie für Nierenzellkarzinome und Melanome zugelassen und kann bei Patienten die Rückbildung von Tumorerkrankungen fördern. Einer der möglichen Mechanismen ist, dass die Verabreichung von IL-2 zu einer T-Zell- Expansion führte. Darüber hinaus zeigte eine aktuelle Studie, dass auch antigenspezifische T- Zellen vermehrt werden können, was eine entscheidende Rolle bei der Vermittlung der Tumorregression spielt. Trotz des langjährigen und umfangreichen Einsatzes von IL-2 in der Klinik war der Anteil der Patienten, die eine komplette Antwort Zeigten, jedoch immer noch gering, und nur etwa ein Fünftel der Patienten weist eine objektive Tumorregression auf. Daher sollte die Funktion von IL-2 in der Krebsbehandlung weiter optimiert und untersucht werden. Eine Studie von Franz O. Smith et al. hat gezeigt, dass die Kombinationsbehandlung von IL-2 und tumorassoziiertem Antigenimpfstoff im Vergleich zu Melanomapatienten, die IL-2 allein erhalten, einen starken Trend zu verstärkten objektiven Reaktionen aufweist. Peptidimpfstoff ist ein Anti- Krebs-Impfstoff, der in der Lage ist, eine starke tumorantigenspezifische Immunantwort zu induzieren, die die Tumore ausrotten kann. Je nach Art der Antigene kann es in zwei verschiedene Kategorien eingeteilt werden: Impfstoff gegen tumorassoziierte Antigene (TAA) und Impfstoff gegen tumorspezifische Neoantigene (TSA). Derzeit werden Peptidimpfstoffe hauptsächlich in klinischen Studien in Phasen I und II an Patienten mit verschiedenen fortgeschrittenen Krebsarten wie Lungenkrebs, Magen-Darm-Tumoren und Brustkrebs untersucht ... KW - Immunotherapy KW - Vaccinia virus Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-253095 ER - TY - JOUR A1 - Yin, Jun A1 - Brocher, Jan A1 - Fischer, Utz A1 - Winkler, Christoph T1 - Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for Retinitis pigmentosa JF - Molecular neurodegeneration N2 - Background: Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. Results: We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Conclusion: Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects. KW - Factor gene PRPF31 KW - TRI-SNRNP KW - Transgenic zebrafish KW - Homebox gene KW - Chinese family KW - Mutations KW - RP11 KW - Expression KW - Disease KW - Protein KW - Retinitis pigmentosa (RP) KW - PRPF31 KW - AD5 mutation KW - SP117 mutation KW - haploinsufficiency KW - dominant-negative KW - rod degeneration KW - apoptosis KW - splicing defect Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141090 VL - 6 IS - 56 ER - TY - THES A1 - Zellner, Elisabeth T1 - Wechselwirkungen zwischen Replikationsproteinen und Origin-DNA während Proliferation und terminaler Differenzierung T1 - Interactions between replication proteins and origin DNA during proliferation and terminal differentiation N2 - Ein Teil dieser Arbeit befasste sich mit der Fragestellung, ob beim Übergang von Proliferation zu Teilungsruhe und Differenzierung irreversible Veränderungen in der Zusammensetzung des präreplikativen Komplexes auftreten. Ein dafür geeignetes System ist die murine C2C12-Zell-Linie, die durch Kultivierung in Hungermedium zu Myotuben differenziert werden können. FACS-Analyse und BrdU-Einbau ergaben, dass in den Muskelzellen keine signifikante DNA-Synthese mehr stattfindet. Die Fluktuation von Replikationsproteinen wurde im Verlauf der terminalen Differenzierung untersucht. Gleiche Mengen an Kern- und Cytoplasma-Extrakten von proliferierenden, konfluenten und sich differenzierenden Zellen wurden durch SDS-PAGE aufgetrennt und im Immunblot mit Antikörpern gegen Replikationsproteine untersucht ORC1, CDC6, MCM6 und Geminin konnten nach 132 h nicht mehr detektiert werden, während ORC2, ORC3, MCM3, CDT1 und CDC45 zwar noch vorhanden waren, jedoch in geringerer Menge als in proliferierenden Zellen. Weiterhin wurde die Menge an Replikationsproteinen in durch Serummangel transient aus dem Zellzyklus ausgetretenen G0-Phase-Zellen und Zellen, die durch Serum reaktiviert wurden, untersucht. Die Replikationsproteine waren in quieszenten C2C12- und 3T3-Zellen gleichermaßen wie in den terminal differenzierten Zellen in verringerter Menge vorhanden. Weiterhin konnte gezeigt werden, dass eine Restimulierung von quieszenten, nicht aber terminal differenzierten Zellen, die erneute Expression von Replikationsproteinen zur Folge hat. Im Rahmen dieser Arbeit wurden Chromatin-Immunpräzipitations-Experimente (ChIP) mit proliferierenden und terminal differenzierten C2C12-Zellen durchgeführt. Eine preRC-Assemblierungsstelle befindet sich im OBR-Bereich der murinen rRNA-Gene von -2519 bis -2152 (Fragment B). In proliferierenden C2C12-Zellen konnte die Bindung von ORC1-5, CDC6, CDT1, MCM3, MCM6, CDC45 und HP1 an Fragment B nachgewiesen werden. Während der terminalen Differenzierung werden ORC1, CDC6, CDT1 und CDC45 von der preRC-Bindungsstelle entfernt, ORC2-5, MCM3, MCM6 und HP1 bleiben an Fragment B gebunden. Die Bindung von preRC-Proteinen an Fragment B sollte durch Electrophoretic Mobility Shift Assays (EMSAs) in vitro detailliert untersucht werden. Dazu mussten zunächst preRC-Proteine nativ aus dem Kernextrakt proliferierender FM3A-Zellen durch Ionenaustausch- und Gelfiltrations-Chromatographie angereichert werden. Proteine in den Fraktionen B4 bis B12 bilden einen DNA-Protein-Komplex mit Fragment B. Die ATP-Abhängigkeit der Bildung des DNA-Protein-Komplexes wurde nachgewiesen. Die Ausbildung des DNA-Protein-Komplexes erfolgt sequenzspezifisch an Fragment B. Durch Zugabe spezifischer Antikörper gegen ORC3 und CDT1 zur Bindungsreaktion konnte die Ausbildung des DNA-Protein-Komplexes reduziert werden. Es konnte gezeigt werden, dass die Bildung des DNA-Protein-Komplexes unabhängig von ATP-Hydrolyse erfolgt und dass Diadenosin-Tetraphosphat (Ap4A) die Bindung von preRC-Proteinen an die DNA nicht signifikant stimuliert. Zur Eingrenzung der preRC-Bindungsstelle wurden sowohl am 5´- als auch am 3´-Ende partiell deletierte B-Fragmente eingesetzt. Mit den um 100 bp verkürzten Fragmenten kann der DNA-Protein-Komplex weiterhin gebildet werden. Deletionen von 200 bp entweder vom 5´- oder 3´-Ende verhindern hingegen die Ausbildung des DNA-Protein-Komplexes. Auf einem 119 bp langen Fragment (-2365 bis -2247), das zentral in Fragment B gelegen ist, kann sich der DNA-Protein-Komplex in einer ATP-stimulierten Weise wiederum ausbilden. Die Analyse dieses Bereiches zeigte, dass sich darin zwei auffällige 9 bp-Sequenzen (CTCGGGAGA) befinden, die im Abstand von 63 bp wiederholt werden (-2343 bis -2335; -2280 bis -2272) und die durch die 200 bp-Deletionen ganz oder teilweise eliminiert wurden. Durch ortsgerichtete Mutagenese mittels PCR wurden innerhalb dieser 9 bp-Wiederholungen die Basen C zu A, T zu G und umgekehrt ausgetauscht. In vier sukzessiven Klonierungen wurden je 4 bp ersetzt (S1 bis S4), wobei die erhaltenen Konstrukte als Ausgangs-DNA für die nachfolgende Klonierung dienten. Die Substitutionen S1, S2 und S3 beeinträchtigten die Ausbildung des DNA-Protein-Komplexes im Wesentlichen nicht. Wurden jedoch 8 bp in beiden 9 bp-Wiederholungen ersetzt (S4), war die Ausbildung des DNA-Protein-Komplexes nahezu vollständig inhibiert. S4 hat außerdem eine leichte reduzierte elektrophoretische Mobilität der proteinfreien DNA-Fragmente zur Folge. Vermutlich stellen die 9 bp-Sequenzen jedoch keine Konsensus-Sequenz für die Bindung der preRC-Proteine per se dar, sondern haben vielmehr Effekte auf die Ausbildung spezifischer Sekundär-Strukturen, die wiederum das Binden der preRC-Proteine an diese Region im OBR der murinen rRNA-Gene erlauben könnten. N2 - One task of this work was to analyse the composition of preRCs in proliferating and terminally differentiated cells with the aim to monitor irreversible changes in the nature of preRCs upon transition from proliferation to differentiation. Murine C2C12 cells were used which can be triggered to become terminally differentiated myotubes by exposure to low mitogen medium. Differentiation was assessed by morphological examination. FACS analyses and BrdU incorporation showed the cessation of dna replication. Fluctuation of replication proteins during terminal differentiation was examined. Same amounts of nuclear and cytoplasmatic extracts, respectively, prepared from proliferating, confluent or differentiating C2C12 cells were resolved by SDS-PAGE. Immunoblot analyses were carried out using specific antibodies. After 132 h in DM ORC1, CDC6, MCM6 and geminin could not be detected in terminally differentiated myotubes at all, whereas ORC2, ORC3, MCM3, CDT1 and CDC45 were detectable, albeit at lower levels than in proliferating myoblasts. Levels of replication proteins in serum starved G0 phase cells and cells which were induced to reenter the cell cycle upon serum readdition, were investigated. Levels of replication proteins decreased in quiescent C2C12 and 3T3 cells, although different proteins were reduced to various extends. Reactivation of quiescent cells, but not terminally differentiated myotubes, resulted in reexpression of replication proteins. Chromatin immunoprecipitation analyses (ChIP) were performed comparing chromatin of proliferating cells to that of differentiated cells. A preRC binding site is localized within the OBR of the murine rRNA genes from position -2519 to -2152 (fragment B) upstream of the transcription start site. The in vivo binding of ORC1-5, CDC6, CDT1, MCM3, MCM6, CDC45 and HP1 to fragment B was observed in proliferating C2C12 cells. During terminal differentiation of C2C12 cells ORC1, CDC6, CDT1 and CDC45 are released. ORC2-5, MCM3, MCM6 and HP1, however, remain bound to fragment B. The removal of essential regulatory replication proteins like ORC1, CDC6, CDT1 and CDC45 from chromatin in terminally differentiated cells might contribute to the establishment and maintenance of an “out-of-cycle” state. One aim of the present work was to further characterize the binding of preRC proteins to fragment B in vitro by electrophoretic mobility shift assays (EMSAs). For that purpose, preRC proteins were enriched from nuclear extracts of proliferating FM3A cells by a combination of ion-exchange and gelfiltration chromatography. Purified proteins of fractions B4 to B12 caused a DNA/protein complex with fragment B. Purified preRC proteins bind exclusively to fragment B. When ORC3 and CDT1 antibodies were added to the DNA/protein binding reaction, formation of the DNA/protein complex was reduced, indicating that these proteins may play a role in formation of this DNA/protein complex. It was shown that this DNA/protein complex is formed independently of ATP hydrolysis and that diadenosine tetraphosphate (Ap4A) does not significantly stimulate binding of preRC proteins to DNA. In order to further narrow down the preRC binding site, EMSAs were performed using fragments shortened by 100 bp and 200 bp, respectively, at the 3´- or 5´-end. The DNA/protein complex is formed with both fragments truncated 100 bp at each end. However, if 200 bp are deleted, no shifts were observed, neither with fragment 5.200 nor with 3.200. On a central 119 bp fragment (-2365 to -2247) the DNA/protein complex is formed in an ATP-stimulated manner. Two conspicuous 9 bp sequence elements, CTCGGGAGA, were observed which are repeated at intervals of 63 bp (-2343 to -2335; -2280 to -2272). The sequence of these elements was altered by introducing C to A, T to G and vice versa substitutions. 4 successive substitutions of 4 bp each were constructed and the constructs were used as template DNA for subsequent mutageneses. Substitutions S1, S2 or S3 did not affect formation of the DNA/protein complex. If, however, 8 bp out of 9 bp in both sequence elements were substituted (S4), formation of the DNA/protein complex is abrogated. A naked S4 DNA fragment migrates distinctly slower than WT and S1-S3 suggesting that the secondary structure of the DNA is altered as a result of nucleotide substitutions. Probably, the 9 bp repeats do not constitute a consensus binding site per se, but rather have effects on formation of specific secondary DNA structures which in turn allow binding of preRC proteins to this region of murine rDNA. KW - Replikation KW - Proteine KW - Replikationsursprung KW - Proliferation KW - Zelldifferenzierung KW - DNA-Replikation KW - Proliferation KW - terminale Differenzierung KW - präreplikativer Komplex KW - Origin KW - DNA replication KW - proliferation KW - terminal differentiation KW - prereplicative complex KW - origin Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-15263 ER -