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

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

DNA DOUBLE STRAND BREAK REPAIR DURING HEAD-DOWN-TILT BEDREST: AGBRESA MEETS RADIATION

BACKGROUND Radiation and reduced gravity impose a major burden on health and performance during human spaceflight. While radiation increases cancer risk and limits tissue regeneration, reduced gravity predisposes to musculoskeletal and cardiovascular deconditioning. Deconditioning could conceivably limit the recovery from radiation damage. Our aim was to develop a terrestrial ex vivo model that could be utilized to study the effect of simulated reduced gravity using head-down-tilt bed-rest on repair of ionizing-radiation-induced DNA damage

Intercellular Communication of Tumor Cells and Immune Cells after Exposure to Different Ionizing Radiation Qualities

Ionizing radiation can affect the immune system in many ways. Depending on the situation, the whole body or parts of the body can be acutely or chronically exposed to different radiation qualities. In tumor radiotherapy, a fractionated exposure of the tumor (and surrounding tissues) is applied to kill the tumor cells. Currently, mostly photons, and also electrons, neutrons, protons, and heavier particles such as carbon ions, are used in radiotherapy. Tumor elimination can be supported by an effective immune response. In recent years, much progress has been achieved in the understanding of basic interactions between the irradiated tumor and the immune system. Here, direct and indirect effects of radiation on immune cells have to be considered. Lymphocytes for example are known to be highly radiosensitive. One important factor in indirect interactions is the radiation-induced bystander effect which can be initiated in unexposed cells by expression of cytokines of the irradiated cells and by direct exchange of molecules via gap junctions. In this review, we summarize the current knowledge about the indirect effects observed after exposure to different radiation qualities. The different immune cell populations important for the tumor immune response are natural killer cells, dendritic cells, and CD8+ cytotoxic T-cells. In vitro and in vivo studies have revealed the modulation of their functions due to ionizing radiation exposure of tumor cells. After radiation exposure, cytokines are produced by exposed tumor and immune cells and a modulated expression profile has also been observed in bystander immune cells. Release of damage-associated molecular patterns by irradiated tumor cells is another factor in immune activation. In conclusion, both immune-activating and -suppressing effects can occur. Enhancing or inhibiting these effects, respectively, could contribute to modified tumor cell killing after radiotherapy

The role of nuclear factor kappa B in the radiation-induced bystander response

Radiation-induced bystander effects play a special role in the cellular response to ionizing radiation. Besides direct consequences of radiation exposure such as cell death, cell cycle arrest and deoxyribonucleic acid (DNA) repair, signaling pathways are activated that result in the secretion of different factors for intercellular communication (cytokines, radicals, damage markers and extracellular vesicles). These factors incite multiple effects in nearby non-irradiated target cells (bystander cells), among those are induction of DNA damage as well as further signal transduction. DNA damage in bystander cells can lead to cell death or activation of repair, similar to direct irradiation responses. Continuing activation of signaling pathways leads to further secretion of signaling factors thereby amplifying and promoting the damage signal originating from the irradiated cell. A key molecule in intra- and intercellular signal transduction is the transcription factor nuclear factor kappa B (NF-kappa B). Target genes of the transcription factor encode proteins that affect intracellular processes like repair and cell cycle progression as well as cytokines that are secreted for intercellular communication. In this work the role of NF-kappa B in the radiation-induced bystander response was investigated. To this end, embryonic fibroblasts from wildtype (wt) and NF-kappa B essential modulator (NEMO) knock-out (ko) mice were used. In these NEMO ko murine embryonic fibroblasts (MEF), the NF-kappa B response is dysfunctional. Direct X-ray exposure of MEF wt cells resulted in reduced survival, induction of premature senescence at high doses, cell cycle arrest in G2/M phase and DNA double strand breaks (DSB) that were partially repaired with time. Furthermore, X-irradiation of MEF wt cells led to nuclear translocation of the NF-kappa B subunit p65, indicating activation of NF-kappa B. MEF NEMO ko cells show a similarly reduced survival upon X-irradiation, a more sensitive response regarding senescence induction, cell cycle arrest in G2/M phase and DNA DSB induction compared to MEF wt. Bystander MEF cells were incubated with culture medium conditioned by irradiated cells. MEF wt bystander cells show NF-kappa B activation, a dose threshold-dependent reduction of cellular survival, induction of premature senescence and induction of DNA DSB, but no changes in cell cycle progression. MEF NEMO ko bystander cells show an increased survival fraction after treatment with conditioned medium and a more sensitive response regarding senescence induction, but no changes in cell cycle progression similar to MEF wt cells. The amount of DNA DSB in MEF NEMO ko bystander cells depends on incubation time and conditioning dose. The survival response of bystander cells has been found to depend on the NF-kappa B status of the recipient cells, indicating involvement of NF-kappa B in the amplification and transmission of the bystander signal.Strahlen-induzierte Bystander Effekte spielen bei der zellulären Reaktion auf ionisierende Strahlung eine besondere Rolle. Neben den direkten Folgen von Strahlenexposition wie Zelltod, Zyklusarrest und Reparatur werden zusätzlich Signalwege aktiviert, an deren Ende verschiedene Faktoren für die interzelluläre Kommunikation ausgeschüttet werden (Zytokine, Radikale, Schadensmarker und extrazelluläre Vesikel). Diese Faktoren lösen in naheliegenden nicht-bestrahlten Zielzellen (Bystander Zellen) diverse Effekte aus, unter anderem DNA-Schäden und weitere Signaltransduktions-Prozesse. DNA-Schäden in Bystander Zellen können, ähnlich den direkten Strahlenfolgen, zu Zelltod oder Reparatur führen. Anhaltende Aktivierung von Signalwegen führt zu erhöhter Ausschüttung von Signalfaktoren. Dadurch kommt es zu einer Verstärkung und Weiterleitung des Schadenssignals, welches von der bestrahlten Zelle ausgeht. Ein Schlüsselmolekül in intra- und interzellulärer Signaltransduktion ist der Transkriptionsfaktor nuclear factor kappa B (NF-kappa B). Die Zielgene des Transkriptionsfaktors kodieren Proteine, welche intrazelluläre Vorgänge wie Reparatur und Zellzyklusverlauf beeinflussen, sowie Zytokine, die ausgeschüttet werden, um interzelluläre Kommunikation zu ermöglichen. In dieser Arbeit wurde die Rolle von NF-kappa B in der strahleninduzierten Bystander Antwort untersucht. Dazu wurden embryonale Fibroblasten von wildtyp (wt) und NF-kappa B essential modulator (NEMO) knock-out (ko) Mäusen verwendet. In diesen NEMO-ko murinen embryonalen Fibroblasten (MEF) ist die NF-kappa B-Antwort dysfunktional. Exposition mit Röntgenstrahlung bewirkte in MEF-wt-Zellen eine reduzierte Überlebensfähigkeit, sowie Induktion von früher Seneszenz bei hohen Dosen, Zellzyklusarrest in der G2/M Phase und DNA Doppelstrangbrüche (DSB), welche mit der Zeit teilweise repariert wurden. Des Weiteren führte Röntgenbestrahlung von MEF-wt-Zellen zur einer nukleären Translokation der NF-kappa B Untereinheit p65, was eine Aktivierung von NF-kappa B erkennen lässt. MEF-NEMO-ko-Zellen zeigten ein ähnlich reduziertes Überleben und eine sensitivere Reaktion bezüglich der Seneszenz-Induktion, des Zellzyklusarrestes und des DNA-DSB-Aufkommens verglichen mit MEF-wt-Zellen. Bystander-MEFZellen wurden mit Kulturmedium inkubiert, welches von bestrahlten Zellen konditioniert wurde. MEF-wt-Bystander-Zellen zeigten NF-kappa B Aktivierung, eine Dosis-Schwellenwert-abhängige Reduzierung des Überlebens, das Auftreten von früher Seneszenz und von DNA-DSB, allerdings keine Veränderung der Zellzyklusprogression. MEF-NEMO-ko-Bystander-Zellen wiesen eine erhöhte Überlebensfraktion auf, nachdem sie mit konditioniertem Medium behandelt wurden, sowie ein sensitiveres Seneszenz-Auftreten, jedoch – ähnlich den wt-Zellen – keine Veränderung der Zellzyklusprogression. Die Anzahl von DNA-DSB in MEF-NEMO-ko-Bystander-Zellen war abhängig von der Inkubationszeit und der Konditionierungsdosis. Die Überlebensantwort von Bystander-Zellen hing vom NF-kappa B Status der Empfängerzellen ab, was impliziert, dass NF-kappa B an der Verstärkung und Übertragung des Bystander-Signales beteiligt ist.Scholarship of the Helmholtz Space Life Sciences Research School (SpaceLife), German Aerospace Center (DLR; Cologne, Germany), which was funded by the Helmholtz Association during a period of 6 years (grant VH-KO-300) and received additional funds from the DLR, including the Aerospace Executive Board and the Institute of Aerospace Medicine. The work was supported by the DLR grant FuE-Projekt ‘‘ISS LIFE’’ (Program RF-FuW, program part 475)

The role of nuclear factor κB in the radiation-induced bystander response

Radiation-induced bystander effects play a special role in the cellular response to ionizing radiation. Besides direct consequences of radiation exposure such as cell death, cell cycle arrest and deoxyribonucleic acid (DNA) repair, signaling pathways are activated that result in the secretion of different factors for intercellular communication (cytokines, radicals, damage markers and extracellular vesicles). These factors incite multiple effects in nearby non-irradiated target cells (bystander cells), among those are induction of DNA damage as well as further signal transduction. DNA damage in bystander cells can lead to cell death or activation of repair, similar to direct irradiation responses. Continuing activation of signaling pathways leads to further secretion of signaling factors thereby amplifying and promoting the damage signal originating from the irradiated cell. A key molecule in intra- and intercellular signal transduction is the transcription factor nuclear factor κB (NF-κB). Target genes of the transcription factor encode proteins that affect intracellular processes like repair and cell cycle progression as well as cytokines that are secreted for intercellular communication. In this work the role of NF-κB in the radiation-induced bystander response was investigated. To this end, embryonic fibroblasts from wildtype (wt) and NF-κB essential modulator (NEMO) knock-out (ko) mice were used. In these NEMO ko murine embryonic fibroblasts (MEF), the NF-κB response is dysfunctional. Direct X-ray exposure of MEF wt cells resulted in reduced survival, induction of premature senescence at high doses, cell cycle arrest in G2/M phase and DNA double strand breaks (DSB) that were partially repaired with time. Furthermore, X-irradiation of MEF wt cells led to nuclear translocation of the NF-κB subunit p65, indicating activation of NF-κB. MEF NEMO ko cells show a similarly reduced survival upon X-irradiation, a more sensitive response regarding senescence induction, cell cycle arrest in G2/M phase and DNA DSB induction compared to MEF wt. Bystander MEF cells were incubated with culture medium conditioned by irradiated cells. MEF wt bystander cells show NF-κB activation, a dose threshold-dependent reduction of cellular survival, induction of premature senescence and induction of DNA DSB, but no changes in cell cycle progression. MEF NEMO ko bystander cells show an increased survival fraction after treatment with conditioned medium and a more sensitive response regarding senescence induction, but no changes in cell cycle progression similar to MEF wt cells. The amount of DNA DSB in MEF NEMO ko bystander cells depends on incubation time and conditioning dose. The survival response of bystander cells has been found to depend on the NF-κB status of the recipient cells, indicating involvement of NF-κB in the amplification and transmission of the bystander signal

Auswirkung von ionisierender Strahlung auf intestinale Epithelzelllinien H-4 und Caco-2

Kosmische Strahlung ist ein großer Risikofaktor für die bemannte Raumfahrt. Bei Langzeitaufenthalten im All tragen die verschiedenen Komponenten und Qualitäten der kosmischen Strahlung, sowie unvorhersehbare Sonnenstürme, zur erhöhten Strahlenexposition von Astronauten bei. Die gesundheitlichen Folgen reichen vom akuten und chronischen Strahlensyndrom bis zu der Entwicklung von Tumoren, abhängig von der Strahlendosis. Hohe Dosen ionisierender Strahlung können die Funktion des Knochenmarks beeinträchtigen und die Epithelzellen des Dünndarms schädigen. Die daraus resultierende Ablösung der Epithelzellen kann den Verlust der intestinalen Mukosa zur Folge haben. Voraussetzung für die Untersuchung gastrointestinaler Strahlenschäden in vitro ist die Auswahl eines geeigneten Modellsystems. Hierzu wurden in dieser Arbeit zwei Zelllinien mit zellbiologischen und biochemischen Methoden auf folgende Eigenschaften untersucht: (i) zelluläre Morphologie, (ii) Chromosomenanzahl, (iii) Wachstumsverhalten, (iv) Zellzyklusverteilung, (v) Zellalterung in Kultur und (vi) Anwesenheit von darmspezifischen Enzymen. Es konnte nachgewiesen werden, dass die tumorassoziierte Zelllinie Caco2 und die nicht-maligne Zelllinie H4 intestinale Epithelzelllinien sind. Nach Exposition mit Röntgenstrahlung wurden die Zelllinien auf klonoges Überleben und Zellzyklusprogression untersucht. Die Ergebnisse zeigen eine Ansammlung von Zellen in der G2/M-Phase des Zellzyklus und vergleichbare Überlebenskurven

Improved cellular survival of bystander cells via NF-κB associated recovery after X-irradiation

Radiation-induced bystander effects (RIBE) are an acknowledged issue of radiation therapy. Radiation of tumor tissue has been shown to affect non-irradiated neighboring cells in a paracrine and endocrine manner. Transduction of bystander signaling though remains to be investigated in detail. A part of the transduction is the receptor-initiated activation of signaling pathways by secreted factors of the irradiated cell during irradiation damage response. This work focusses on the activation of the transcription factor Nuclear Factor kappaB (NF-kappaB) in bystander cells after irradiation. NF-kappaB is a well-known contributor to inflammatory processes like cyto- / chemokine production as well as to stress reactions such as the DNA damage response and cell cycle regulation. Using a mouse embryonic fibroblasts (MEF) in vitro model with a genetic knock-out of an NF-kappaB regulator (NEMO, NF-kappaB essential modulator), clonogenic survival and cell cycle distribution was determined in directly irradiated cells and in cells incubated with conditioned medium from X-irradiated cells (bystander treatment). Directly irradiated NEMO ko cells, plated for clonogenic survival immediately after X-irradiation, display the same dose-effect curve as the wildtype (wt) (a/b NEMO ko = 13.92 ± 2.4 vs. a/b wt = 12.37 ± 2.6). But when allowed to recover for 24 h, the wt cells show a broader shoulder in the curve (a/b = 3.5 ± 2.9), indicating a role of NF-kappaB in the repair of radiation induced DNA damages. Looking into the survival of bystander cells, the survival curves show a statistically different slope, with NEMO ko cells surviving better than wt cells (S16 Gy: NEMO ko = 1.66 vs wt = 0.83). The different behavior may correlate with NF-kappaB dependent DNA repair in bystander cells for NEMO ko and wt cells. Cell cycle analysis revealed a 6 hour delayed arrest in G2/M phase in directly irradiated NEMO ko cells compared to wt cells. This indicates that NF-kappaB regulated DNA repair pathways are important for recovery of radiation induced damages. Bystander NEMO ko show an even further delayed arrest at 48 h, while wt bystander cells show no G2/M arrest at all. This supports the assumption that damages have to overcome a certain threshold to be recognized as repair-worthy. As NF-kappaB has been reported to be involved in homologous recombination; cells with impairment in NF-kappaB pathways, such as NEMO ko, register damages caused by bystander treatment differently from wt cells. This leads to G2/M arrest extending time for repair in NEMO ko bystander cells

Transcription Factors in the Cellular Response to Charged Particle Exposure

Charged particles, such as carbon ions, bear the promise of a more effective cancer therapy. In human spaceflight, exposure to charged particles represents an important risk factor for chronic and late effects such as cancer. Biological effects elicited by charged particle exposure depend on their characteristics, e.g., on linear energy transfer (LET). For diverse outcomes (cell death, mutation, transformation, and cell-cycle arrest), an LET dependency of the effect size was observed. These outcomes result from activation of a complex network of signaling pathways in the DNA damage response, which result in cell-protective (DNA repair and cell-cycle arrest) or cell-destructive (cell death) reactions. Triggering of these pathways converges among others in the activation of transcription factors, such as p53, nuclear factor κB (NF-κB), activated protein 1 (AP-1), nuclear erythroid-derived 2-related factor 2 (Nrf2), and cAMP responsive element binding protein (CREB). Depending on dose, radiation quality, and tissue, p53 induces apoptosis or cell-cycle arrest. In low LET radiation therapy, p53 mutations are often associated with therapy resistance, while the outcome of carbon ion therapy seems to be independent of the tumor’s p53 status. NF-κB is a central transcription factor in the immune system and exhibits pro-survival effects. Both p53 and NF-κB are activated after ionizing radiation exposure in an ataxia telangiectasia mutated (ATM)-dependent manner. The NF-κB activation was shown to strongly depend on charged particles’ LET, with a maximal activation in the LET range of 90–300 keV/μm. AP-1 controls proliferation, senescence, differentiation, and apoptosis. Nrf2 can induce cellular antioxidant defense systems, CREB might also be involved in survival responses. The extent of activation of these transcription factors by charged particles and their interaction in the cellular radiation response greatly influences the destiny of the irradiated and also neighboring cells in the bystander effect

Bystander Effects in the Cellular Radiation Response

The overall response to tumor radiation therapy results from direct radiation damage and indirect bystander effects (RIBE) mediated by secreted molecules and paracrine transfer of short-lived mediators. RIBE can have both detrimental and protective actions on cancerous as well as healthy tissues. Nuclear Factor κB (NF-κB), which governs immune responses, is a prime candidate for mediating RIBE. NF-κB also modulates DNA repair while promoting cellular survival as part of the DNA damage response. After exposure to ionizing radiation NF-κB is activated in directly targeted and non-targeted bystander cells. We tested the hypothesis that the NF-κB status is relevant for induction of RIBE, using a mouse embryonal fibroblasts (MEF) knock-out variant that is unable to activate NF-κB (NF-κB essential modulator knock-out (NEMO ko))