The role of G-protein-coupled receptors in the biological activity of γδ T lymphocytes

The data collected in this work represent the first evidence that G-protein-coupled signalling exist in γδ T cells via histamine and fMLP receptors. The bigenic amine histamine and the bacterial peptide fMLP have been demonstrated to be novel chemoattractant factors for circulating human γδ T cells, which are critical members of the immunological tumor surveillance machinery. Here, we analyzed the influence of histamine on the interaction of human γδ T cells with tumor cells such as the A2058 human melanoma cell line, the human Burkitt's non-Hodgkins lymphoma cell line Raji, the T-lymphoblastic lymphoma cell line Jurkat, and the NK cell-sensitive erythroleukaemia line K562. We found that histamine inhibits the spontaneous cytolytic activity of γδ T cells in response to these cell lines. The downregulation of γδ T cell mediated cytotoxicity involves the histamine receptor subtype 2 (H2R), the activation of Gs proteins and increased cAMP intracellular levels. On the other hand, histamine activates the common signalling pathways of chemotaxins such as Gi-protein-dependent actin reorganization, the increase of intracellular Ca2+ and the induction of migratory response in γδ T lymphocytes. Our data indicate that histamine contributes to the mechanism by which tumor cells escape immunological surveillance. The bacterial-cell-wall-derived peptide N-formyl-Met-Leu-Phe (fMLP) is a well characterized chemotactic factor for phagocytes such as neutrophils, monocytes and dendritic cells. Here, we analyzed the influence of fMLP on isolated human peripheral blood γδ T cells. We found that fMLP induces intracellular calcium transients, actin reorganization, CD11b upregulation and the migration of γδ T cells. Pretreating γδ T cells with pertussis toxin inhibited all fMLP-stimulated cell responses, implicating the involvement of Gi proteins in the induced signalling cascade. The present data suggest that, in addition to phagocytes, N-formyl peptides also regulate the trafficking and activation of γδ T cells

A TTRAP adaptor molekula szerepének vizsgálata a TGF-beta ligand család szignalizációs mechanizmusában = Role of the TTRAP adaptor molecule in the signaling mechanisms of TGF-beta family ligands

A TGF-β fehérje egy esszenciális növekedési faktor, amely fontos szabályzó szerepet tölt be a sejtek életének szinte minden mozzanatában. Mindezek mellett kiemelkedő szereppel bír a daganatos betegségek elleni védelemben is, gátolva a szervezetben a sejtek kontrollálatlan burjánzását. Tumor fejlődés során a TGF-β jelátviteli útvonal komponenseiben mutációk és epigenetikai változások halmozódnak fel, amelyek nem csak a citokin sejtosztódást gátló hatásaival szembeni rezisztencia kialakulását eredményezik, hanem gyakran paradox módon azt a tumorok progresszióját elősegítő tényezővé változtatják. Kutatásaink során ennek a fontos biológiai jelenségnek a jobb megértését tűztük célul. Figyelmüket döntően egy új adaptor fehérjére, TTRAP-re összpontosítottuk. Megállapítottuk, hogy TTRAP fontos szerepet tölt be mind a Smad függő mind a Smad független TGF-β jelátviteli folyamatokban. Kifejlesztettünk egy egér emlő epiteliáls sejtvonalon (NMuMG) alapuló modell rendszert amelynek felhasználásával bizonyítottuk, hogy a molekula fontos komponense a TGF-β indukálta programozott sejthalál (apoptózis) folyamatának. Kutatásaink a rákellenes szerek egy olyan új osztályának a kifejlesztéséhez teremthetik meg az alapot, amelyek specifikusan gátolják TGF-β tumor fejlődést elősegítő hatásait előrehaladott rák betegségekben. | TGF-β is a pleiotropic cytokine that regulates mammalian development, differentiation, and homeostasis. It is also a potent anticancer agent that prohibits the uncontrolled proliferation of epithelial, endothelial, and hematopoietic cells. Aberrations in the TGF-β pathway bring about resistance to TGF-β-mediated growth arrest and thus give rise to human malignances. Paradoxically, these genetic and epigenetic aberrations also conspire to convert TGF-β from a suppressor of tumor formation to a promoter of their growth, survival and metastasis. Our main objective was to better understand the mechanisms that underlie the ability of TGF-β to mediate tumor suppression in normal cells, and conversely, to facilitate cancer progression in malignant cells. We focused our attention on elucidating the role of the TTRAP adaptor molecule in TGF-β signaling. We have shown that TTRAP is an important component of both Smad-dependent and Smad-independent branches of TGF-β signaling. In addition, we have developed a murine mammary epithelial cell line (NMuMG) based model system for studying TTRAP's role in various TGF-β dependent biological responses. Using this model, we have demonstrated that TTRAP is a critical component of TGF-β induced apoptosis. In summary, our results may open up new avenues for developing drugs capable of selectively negating TGF-β's pro-oncogenic effects in late stage malignances

Validation of biological recognition elements for signal transduction as first step in the development of whole cell biosensors

Choosing the proper combination of receptor element, cell type and measurable signal requires major consideration for developing cell-based biosensors. In order to use physiologically relevant cellular responses towards (geno)toxic conditions, information on the mechanism of action and of the expected outcome of exposure needs to be considered

Radiation Response of Murine Embryonic Stem Cells

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approache

Hypergravity attenuates Reactivity in Primary Murine Astrocytes

Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning and memory as well as motion. Alterations or even disruptions of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we use increased gravitational (mechanical) loading induced by hypergravity to identify a potential method to modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the reactivity phenotype in vivo on custom-built centrifuges for cultivation as well as for livecell imaging under hypergravity conditions in a physiological range (2g and 10g). This resulted in significant changes to astrocyte morphology, behavior and reactivity phenotypes, with the ultimate goal being to enhance neuronal regeneration for novel therapeutic approaches

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5

The Use of ProteoTuner Technology to Study Nuclear Factor κB Activation by Heavy Ions

Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects

Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies

The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y