107 research outputs found
Zellzyklus- und Dichteabhängigkeit todesrezeptorvermittelter Apoptose und Untersuchungen des von RKO-Zellen konditionierten Mediums
No full textIm Rahmen dieser Arbeit sollte untersucht werden, ob eine Zellzyklusabhängigkeit der CD95- vermittelten Apoptose besteht. Dazu wurde ein ecdysoninduzierbares Genexpressionsystem für die induzierte Überexpression der CDK-Inhibitoren p21 und p27 in RKO-Zellen (Kolonkarzinomzellen) zur Herbeiführung eines Zellzyklusarrests in der G1-Phase benutzt. Nach Induktion mit dem Ecdysonhomolog Muristeron wurde durch Zugabe von rekombinanten hCD95-Liganden Apoptose ausgelöst und anschließend untersucht. Die erzielten Ergebnisse zeigen, dass der Induktor Muristeron an sich und nicht die p21- bzw. p27-Überexpression die anti-apoptotische Akt-Kinase aktiviert, die Expression des anti-apoptotischen Bcl-xL erhöht, die Caspase-8-Aktivierung (entweder am CD95-DISC oder durch "Feedback"-Aktivierung durch Caspase-3) und die darauf folgenden Ereignisse verhindert und somit die hCD95L-induzierte Apoptose blockiert. Zusätzlich beeinflusst der Induktor auch das Genexpressionsmuster der behandelten Zellen, was ebenfalls für die Hemmung der Apoptose mit verantwortlich sein könnte. Somit ist das ecdysoninduzierbare Genexpressionsystem zur Apoptoseuntersuchung in RKO-Zellen nicht verwendbar. Mit der Untersuchung des Apoptoseverhaltens proliferierender RKO-Zellen konnte gezeigt werden, dass überlebende Zellen nach hCD95L-Behandlung vermehrt in der G0/G1-Zellzyklusphase nachweisbar sind, während apoptotische (Caspase-3-positive) Zellen aus der G2/M-Phase heraus sterben. Allerdings weisen die apoptotischen Zellen kaum Cyclin B1 auf, ein für die G2-Phase wichtiges und typisches Cyclin. Somit bleibt die genaue Verknüpfung von Zellzyklusregulation und Apoptose auch nach diesen Analysen ungeklärt. In einem dritten Ansatz - Zellzyklusarrest durch Dichtearretierung - konnte eine Hemmung der CD95- vermittelten Apoptose in der arretierten Zellpopulation nachgewiesen werden. Allerdings sekretieren RKO-Zellen einen anti-apoptotischen Faktor in ihr Medium, dessen Konzentration und Wirkung mit größerer Zelldichte zunimmt und somit für die Protektion, unabhängig von Zellzyklusarrest oder Proliferation, verantwortlich ist. Konfluente und auch mit konditioniertem Medium behandelte RKO-Zellen zeigen im Vergleich zu dünn ausgesäten RKO-Zellen Veränderungen, die denen sehr ähnlich sind die beim Übergang einer epithelverankerten Zelle zu einer migrierenden Einzelzelle (EMT) auftreten. Beispielsweise verändert sich die Zusammensetzung des Zytoskeletts, die Zellen verlieren den Zell-Zell-Kontakt und lösen sich ab, bleiben aber am Leben. Zusätzlich steigt die Sekretion von Zytokinen an, die Angiogenese, Migration und Invasion positiv beeinflussen. Sowohl konfluente als auch mit konditioniertem Medium behandelte sub-konfluente Zellen sind apoptoseresistent (hCD95L, TRAIL, UV, Staurosporin), woran u.a. die Kinasen PKC und PI3K, aber auch das anti-apoptotische Bcl-xL beteiligt sind. Die Zellen sterben interessanterweise, wenn ein agonistischer anti-CD95-Antikörper statt des rekombinanten CD95-Liganden verwendet wird, was vermuten lässt, dass eine mangelhafte Vernetzung der einzelnen DISC-Komplexe zur Apoptosehemmung führt, welche durch den Antikörper dann aber erzwungen wird. Zwar handelt es sich hierbei um ein reines Zellkulturmodell, dennoch könnte es bedeuten, dass die Umgebung in einer dichten RKO-Zellkultur vergleichbar ist mit der in größeren soliden Tumoren. Die Zellen brauchen Nährstoffe, versuchen über eine Neovaskularisierung Anschluss an ein Blutsystem zu finden und sekretieren Lockstoffe, Wachstumsfaktoren sowie Proteasen, um die Metastasierung zu erleichtern. PI3K, cPKCs und Bcl-xL tragen dabei zu einer Apoptoseresistenz bei, welche die Zellen zum einen resistent gegenüber Anoikis, Nährstoffmangel, aber auch gegen angreifende zytotoxische T-Zellen macht. Eine weitere Aufklärung der hier ablaufenden Prozesse würde es erleichtern, Möglichkeiten zu finden, in diese Signalwege einzugreifen, um die Apoptosesensitivität wieder herzustellen und die Metastasierung zu verhindern. Insbesondere ist die Identifizierung des für die Apoptoseprotektion verantwortlichen Zytokins das nächste wichtige Ziel bei der Fortsetzung dieser Arbeiten.The aim of this study was to investigate whether a cell cycle dependency in CD95 mediated apoptosis exists. Therefore an ecdysone-inducible expression system was used to induce overexpression of the CDK-inhibitors p21 and p27 in RKO cells (colon carcinoma cell line) to arrest then in the G1 cell cycle phase. Upon transgene induction with the ecdysone homologue muristerone, apoptosis was induced via addition of recombinant hCD95 ligand and then further analyzed. The results demonstrate that muristerone itself and not the overexpression of p21 or p27 activates the anti-apoptotic Akt kinase, increases expression levels of anti-apoptotic Bcl-xL, inhibits caspase-8 activation (possibly at the CD95 DISC or alternatively at the level of feedback activation by caspase-3) as well as downstream events, and therefore inhibits hCD95L-induced apoptosis. Additionally the inductor influences the gene expression profile of treated cells, which could also be involved in the inhibition of apoptosis. In conclusion, these data show that the Ecdysone-inducible gene expression system is not useful to study apoptosis in RKO cells. By investigating apoptotic behavior of proliferating RKO cells the observation was made that, upon hCD95L treatment, surviving cells are more likely to be found in the G0/G1-cell cycle phase. Apoptotic (caspase-3 positive) cells preferentially die from G2/M-phase as judged by the DNA profile, although they do not express the G2/M-typical cyclin B. Therefore, the question of a direct link between cell cycle and apoptosis still remains open. In a third approach - cell cycle arrest via contact inhibition of dense cells - an inhibition of CD95- mediated apoptosis in cell cycle arrested cells could be proven. Further analysis revealed that RKO cells secret an anti-apoptotic factor into the medium, with increasing concentration (and effectiveness) with higher cell density. This factor is responsible for apoptosis protection, independently of cell cycle arrest or proliferation. Dense RKO cells and cells plated at low density before treatment with conditioned medium show changes similar to those in epithelial-mesenchymal transition (EMT) if compared to sparsely seeded RKO cells: cytoskeleton reorganization, loss of cell-cell-contact, loss of adherence and anoikis resistance. In addition, secretion of angiogenesis-, migration- and invasion-inducing cytokines increases. Dense, as well as sparse cells treated with conditioned medium are apoptosis-resistant (hCD95L, TRAIL, UV, staurosporine). The kinases PKC and PI3K and the anti-apoptotic Bcl-xL contribute to the resistance phenotype. The cells die however if treated with anti-CD95-antibody, suggesting an insufficient clustering of single DISC-complexes and inhibition of apoptosis when the cells are treated with recombinant hCD95 ligand. The agonistic CD95-antibody on the other hand forces DISC clustering. This cell culture model may be comparable to the situation in large solid tumors. The cells need nutrients, try to connect to the vascular system through angiogenesis and secrete chemoattractants, growth factors and proteases to facilitate metastasis formation. PI3K, cPKC and BclxL induce apoptosis resistance, which protects cells against anoikis, serum deprivation and cytotoxic T cells. Further investigations of underlying mechanisms could provide new targets to manipulate the signaling pathways in order to restore apoptosis sensitivity in tumor cells and to prevent metastasis formation. In this context, it will be highly interesting to identify the anti-apoptotic factor secreted by RKO cells
Advanced 3D cell culturing and monitoring system
No full textDie vorgelegte Doktorarbeit stellt ein 3D-Zellkultursystem mit einem vollautomatisierten analytischen, biochemischen Assay und vollautomatisierter Kultivierung mit Mediumwechsel vor. Dieses integrierte Kultivierungs- und Analysesystem wurde in dieser Arbeit mit 3D-Hepatozytenkulturen in Polycarbonat-MatriGrid®-Gerüsten für das 3D-Wachstum von Zellkulturen prototypisiert. Das System perfundiert MatriGrid- Kulturen kontinuierlich mit Wirkstoff-ergänztem Medium und führt bei Bedarf eine Bewertung der Wirkstofftoxizität durch Beobachtung und Messung der Konzentration eines Indikators, des Biomarkers Albumin, durch. Das System kann die MatriGrid-Kultur mit unterschiedlichen Flussraten perfundieren, automatisierte Medienwechsel durchführen und mit dem mitgelieferten ELISA-Modul Proben des zu analysierenden Kulturmediums nach Bedarf untersuchen. Das System unterstützt die parallele Kultivierung von Zellen in mehreren Bioreaktoren. Das Fluidnetzwerk wurde aus Materialien konstruiert, die wenig Proteine und kleine Moleküle binden, absorbieren oder adsorbieren, um seine Anwendung für niedrige Biomarkerkonzentrationen und Langzeitexperimente zu erweitern. Die Doktorarbeit beschreibt das Systemdesign, den Aufbau, das Testen und die Verifikation unter Verwendung von 3D-gewachsenen HepaRG-Zellkulturen. Die zeitabhängige Wirkung von APAP auf die Albuminsekretion wurde über 96 h untersucht, wobei sowohl mit dem neu entwickelten System als auch konventionell in Mikrotiterplatten, gemessen wurde. Es zeigte sich, dass die Ergebnisse vergleichbar sind. Dieses Resultat belegt die Verwendung des Systems als eigenständiges Gerät, das in Echtzeit arbeitet und in der Lage ist, gleichzeitig Zellkultur- und Mediumanalyse in mehreren Bioreaktoren durchzuführen, mit erhöhter Zuverlässigkeit der 3D-Kultivierung, in einfacher Handhabung und Messung. Auf diese Weise soll das neu entwickelte 3D-Zellkultivierungs- und Analysesystem 3DZellkultivierungstechniken und -experimente für weitere Forschungsgruppen bekannt machen.This thesis presents a 3D cell culturing system with a fully automated analytic biochemistry assay and fully automated culturing with medium change. This integrated culturing and analytic system was prototyped in this work with 3D hepatocyte cultures in polycarbonate MatriGrid® scaffolds for 3D growth of cell cultures. The system continuously perfuses MatriGrid cultures with drug supplemented medium and performs, on demand, drug toxicity evaluation by observing and measuring the amount of an indicator, the biomarker albumin. The system can perfuse the MatriGrid cultures using different flow rates, performs automated medium changes and can make on-demand samples of the culture medium to be analyzed with the included ELISA module. The system supports parallel culturing of cells in multiple bioreactors. The fluidic network was constructed from low protein and small molecules binding, absorbing or adsorbing materials to extend its application for low biomarker concentration and long-term experiments. The thesis describes the system design, construction, testing, and verification using 3D-grown HepaRG cell cultures. The time-dependent effect of APAP on albumin secretion over 96 h, measured with newly developed system and conventional microtitre plates was measured and the results are comparable. These results confirm the use the system as a standalone device that works in real time and is capable of simultaneous cell culture and medium analysis in multiple bioreactors, with increased reliability of 3D-culturing, ease of handling and measurement. This way the newly developed 3D cell culturing and analysis system is aimed to promote 3D cell culturing techniques and experimentation to more research groups
Drug sensitivity profiling of 3D tumor tissue cultures in the pediatric precision oncology program INFORM
No full textAbstract The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75–78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs
OTHR-04. DEVELOPMENT OF A FUNCTIONAL PLATFORM FOR REAL-TIME PERSONALIZED DRUG SENSITIVITY PROFILING OF PATIENT-DERIVED 3D FRESH TUMOR TISSUE CULTURES IN THE PEDIATRIC PRECISION ONCOLOGY PROGRAM INFORM
No full textAbstract The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75-78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs
Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the Gatekeeper Residue is Implicated
No full textThe discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as drug targets. The isozyme HDAC10 contributes to chemotherapy resistance and has recently been described to be a polyamine deacetylase, but no studies toward selective HDAC10 inhibitors have been published. Using two complementary assays, we found tubastatin A, an HDAC6 inhibitor, to potently bind HDAC10. We synthesized tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10 binding. HDAC10 inhibitors mimicked knockdown by causing dose-dependent accumulation of acidic vesicles in a neuroblastoma cell line. Furthermore, docking into human HDAC10 homology models indicated that a hydrogen-bond between a cap group nitrogen and the gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, our data provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the tubastatin A scaffold
HDAC Family Members Intertwined in the Regulation of Autophagy: A Druggable Vulnerability in Aggressive Tumor Entities
No full textThe exploitation of autophagy by some cancer entities to support survival and dodge death has been well-described. Though its role as a constitutive process is important in normal, healthy cells, in the milieu of malignantly transformed and highly proliferative cells, autophagy is critical for escaping metabolic and genetic stressors. In recent years, the importance of histone deacetylases (HDACs) in cancer biology has been heavily investigated, and the enzyme family has been shown to play a role in autophagy, too. HDAC inhibitors (HDACi) are being integrated into cancer therapy and clinical trials are ongoing. The effect of HDACi on autophagy and, conversely, the effect of autophagy on HDACi efficacy are currently under investigation. With the development of HDACi that are able to selectively target individual HDAC isozymes, there is great potential for specific therapy that has more well-defined effects on cancer biology and also minimizes toxicity. Here, the role of autophagy in the context of cancer and the interplay of this process with HDACs will be summarized. Identification of key HDAC isozymes involved in autophagy and the ability to target specific isozymes yields the potential to cripple and ultimately eliminate malignant cells depending on autophagy as a survival mechanism
Inhibition of histone deacetylases permits lipopolysaccharide-mediated secretion of bioactive IL-1b via a caspase-1-independent mechanism
No full textHistone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immunemodulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1 beta processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1 beta maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1 beta secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1 beta, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1 beta cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1 beta by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1 beta, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications
Differences in protein structural regions that impact functional specificity in GT2 family β- Glucan synthases
No full text© 2019 Oehme, Shafee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Most cell wall and secreted β-glucans are synthesised by the CAZy Glycosyltransferase 2 family (www.cazy.org), with different members catalysing the formation of (1,4)-β-, (1,3)-β-, or both (1,4)- and (1,3)-β-glucosidic linkages. Given the distinct physicochemical properties of each of the resultant β-glucans (cellulose, curdlan, and mixed linkage glucan, respectively) are crucial to their biological and biotechnological functions, there is a desire to understand the molecular evolution of synthesis and how linkage specificity is determined. With structural studies hamstrung by the instability of these proteins to solubilisation, we have utilised in silico techniques and the crystal structure for a bacterial cellulose synthase to further understand how these enzymes have evolved distinct functions. Sequence and phylogenetic analyses were performed to determine amino acid conservation, both family-wide and within each sub-family. Further structural analysis centred on comparison of a bacterial curdlan synthase homology model with the bacterial cellulose synthase crystal structure, with molecular dynamics simulations performed with their respective β-glucan products bound in the trans-membrane channel. Key residues that differentially interact with the different β-glucan chains and have sub-family-specific conservation were found to reside at the entrance of the trans-membrane channel. The linkage-specific catalytic activity of these enzymes and hence the type of β-glucan chain built is thus likely determined by the different interactions between the proteins and the first few glucose residues in the channel, which in turn dictates the position of the acceptor glucose. The sequence-function relationships for the bacterial β- glucan synthases pave the way for extending this understanding to other kingdoms, such as plants
leads to impaired T cell proliferation and an increased memory T cell pool after staphylococcal enterotoxin B injection
No full textAuthor Correction: The influence of different sample preparation on mechanical properties of human iliotibial tract
No full textArt. 10404, 2 S.This Article contains errors. The determined values of the Young's moduli deviate from the literature by about two orders of magnitude. Due to the recalculation, the values of the determined Young's moduli in Figure 7B are incorrect. The correct Figure 7B appears below as Figure 1. In addition, a new table is included with each calculated Young's modulus for better transparency. The calculated Young's moduli are listed in Table 1 below. As a result of this error, Table 2 is incorrect. The correct Table 2 appears below. In the Results section, ""A slight increase in the Young's Modulus of FDF0 samples (0.91 (SD 0.29) MPa) and FDF1 samples (0.83 (SD 0.28) MPa), compared to F samples (0.73 (SD 0.44) MPa) has been observed (Fig. 7)."" should read: "A slight increase in the Young's Moduli of FDF0 samples (274.46 (SD 115.60) MPa) and FDF1 samples (252.26 (SD 83.16) MPa), compared to F samples (247.73 (SD 167.91) MPa) has been observed (Fig. 7)." Finally, the Acknowledgements section is incomplete. "We acknowledge support from the German Research Foundation (DFG) and Leipzig University within the program of Open Access Publishing." should read: "We acknowledge support from the German Research Foundation (DFG) and Leipzig University within the program of Open Access Publishing. Additionally, the authors would like to thank Matthias Oehme and especially PD Dr. Hanno Steinke for their support in managing the cadaver acquisition."1
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