Johann Bauer

Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells

This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML‐1. Cultured on a three‐dimensional clinostat, ML‐1 cells formed three‐dimensional MCTSs (MCTS diameter: 0.3±0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up‐regulated the thyroid‐stimulating hormone‐receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML‐1 cells grown on the clinostat showed elevated amounts of the apoptosis‐associated Fas protein, of p53, and of bax but showed reduced quantities of bcl‐2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase‐mediated dUTP digoxigenin nick end labeling, 4′, 6‐diamidino‐2‐phenylindole staining, DNA laddering, and 85‐kDa apoptosis‐related cleavage fragments. These fragments resulted from enhanced 116‐kDa poly(ADP‐ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

Electrophoresis of cells and the biological relevance of surface charge

Recent developments in electrophoresis of cells are reviewed. Problems and progress in automation and miniaturization of analytical electrophoresis instruments as well as in the interpretation of experimentally determined electrophoretic mobility (EPM) data are summarized: In recent times, the EPM determination techniques not only became more reliable and faster, but also more knowledge could be gained about the cell surface electrical properties, the structure of the glycocalyx as well as its influence on the cell peripheral regions and microenvironment by applying cell electrophoresis. In addition, ways are shown to solve discrepancies between physical requirements of a preparative cell electrophoresis procedure and the quantities of ions, which have to be dissolved in cell suspension media. As the modern machines allow the purification of untagged cells suspended in more cell friendly and physiological media, they are likely to be valuable tools in several useful practical applications in clinical transplantation, gene therapy and treatment of disease states.

Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation

Cell separation by counterflow centrifugal elutriation (CCE) or free flow electrophoresis (FFE) is performed at lower frequency than cell cloning and antibody-dependent, magnetic or fluorescence-activated cell sorting. Nevertheless, numerous recent publications confirmed that these physical cell separation methods that do not include cell labeling or cell transformation steps, may be most useful for some applications. CCE and FFE have proved to be valuable tools, if homogeneous populations of normal healthy untransformed cells are required for answering scientific questions or for clinical transplantation and cells cannot be labeled by antibodies, because suitable antibodies are not available or because antibody binding to a cell surface would induce the cell reaction which should be investigated on purified cells or because antibodies bound to the surface hamper the use of the isolated cells. In addition, the methods are helpful for studying the biological reasons for, or effects of, changes in cell size and cellular negative surface charge density. Although the value of the methods was confirmed in recent years by a considerable number of important scientific results, activities to further develop and improve the instruments have, unfortunately, declined.

The Effects of Weightlessness on the Human Organism and Mammalian Cells

It has always been a desire of mankind to conquest Space. A major step in realizing this dream was the completion of the International Space Station (ISS). Living there for several months confirmed early observations of short-term spaceflights that a loss of gravity affects the health of astronauts. Space medicine tries to understand the mechanism of microgravity-induced health problems and to conceive potent countermeasures. There are four different aspects which make space medicine appealing: i) finding better strategies for adapting astronauts to weightlessness; ii) identification of microgravity-induced diseases (e.g. osteoporosis, muscle atrophy, cardiac problems and others); iii) defining new therapies to conquer these diseases which will benefit astronauts as well as people on Earth in the end; and iv) on top of that, unveiling the mechanisms of weightlessness-dependent molecular and cellular changes is a requirement for improving space medicine. In mammalian cells, microgravity induces apoptosis and alters the cytoskeleton and affects signal transduction pathways, cell differentiation, growth, proliferation, migration and adhesion. This review focused on gravi-sensitive signal transduction elements and pathways as well as molecular mechanisms in human cells, aiming to understand the cellular changes in altered gravity. Moreover, the latest information on how these changes lead to clinically relevant health problems and current strategies of countermeasures are reviewed.

Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells.

Many space missions have shown that prolonged space flights may increase the risk of cardiovascular problems. Using a three‐dimensional clinostat, we investigated human endothelial EA.hy926 cells up to 10 days under conditions of simulated microgravity (µg) to distinguish transient from long‐term effects of µg and 1g. Maximum expression of all selected genes occurred after 10 min of clinorotation. Gene expression (osteopontin, Fas, TGF‐β1) declined to slightly upregulated levels or rose again (caspase‐3) after the fourth day of clinorotation. Caspase‐3, Bax, and Bcl‐2 protein content was enhanced for 10 days of microgravity. In addition, long‐term accumulation of collagen type I and III and alterations of the cytoskeletal alpha‐ and beta‐tubulins and F‐actin were detectable. A significantly reduced release of soluble factors in simulated microgravity was measured for brain‐derived neurotrophic factor, tissue factor, vascular endothelial growth factor (VEGF), and interestingly for endothelin‐1, which is important in keeping cardiovascular balances. The gene expression of endothelin‐1 was suppressed under µg conditions at days 7 and 10. Alterations of the vascular endothelium together with a decreased release of endothelin‐1 may entail post‐flight health hazards for astronauts. J. Cell. Biochem. J. Cell. Biochem. 101: 1439–1455, 2007.

Establishment and characterization of the follicular thyroid carcinoma cell line ML-1

Abstract.The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.

A Delayed Type of Three-Dimensional Growth of Human Endothelial Cells Under Simulated Weightlessness

Endothelial cells (ECs) form three-dimensional (3D) aggregates without any scaffold when they are exposed to microgravity simulated by a random positioning machine (RPM) but not under static conditions at gravity. Here we describe a delayed type of formation of 3D structures of ECs that was initiated when ECs cultured on a desktop RPM remained adherent for the first 5 days but spread over neighboring adherent cells, forming little colonies. After 2 weeks, tube-like structures (TSs) became visible in these cultures. They included a lumen, and they elongated during another 2 weeks of culturing. The walls of these TSs consisted mainly of single-layered ECs, which had produced significantly more beta(1)-integrin, laminin, fibronectin, and alpha-tubulin than ECs simultaneously grown adhering to the culture dishes under microgravity or normal gravity. The amount of actin protein was similar in ECs incorporated in TSs and in ECs growing at gravity. The ratio of tissue inhibitor of metalloproteinases-1 to matrix metalloproteinase-2 found in the supernatants was lower at the seventh than at the 28th day of culturing. These results suggest that culturing ECs under conditions of modeled gravitational unloading represents a new technique for studying the formation of tubes that resemble vascular intimas.

Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space

This study focuses on the effects of short‐term [22 s, parabolic flight campaign (PFC)] and long‐term (10 d, Shenzhou 8 space mission) real microgravity on changes in cytokine secretion and gene expression patterns in poorly differentiated thyroid cancer cells. FTC‐133 cells were cultured in space and on a random positioning machine (RPM) for 10 d, to evaluate differences between real and simulated microgravity. Multianalyte profiling was used to evaluate 128 secreted cytokines. Microarray analysis revealed 63 significantly regulated transcripts after 22 s of microgravity during a PFC and 2881 after 10 d on the RPM or in space. Genes in several biological processes, including apoptosis (n=182), cytoskeleton (n=80), adhesion/extracellular matrix (n=98), proliferation (n=184), stress response (n=268), migration (n=63), angiogenesis (n=39), and signal transduction (n=429), were differentially expressed. Genes and proteins involved in the regulation of cancer cell proliferation and metastasis, such as IL6, IL8, IL15, OPN, VEGFA, VEGFD, FGF17, MMP2, MMP3, TIMP1, PRKAA, and PRKACA, were similarly regulated under RPM and spaceflight conditions. The resulting effect was mostly antiproliferative. Gene expression during the PFC was often regulated in the opposite direction. In summary, microgravity is an invaluable tool for exploring new targets in anticancer therapy and can be simulated in some aspects in ground‐based facilities.—Ma, X., Pietsch, J., Wehland, M., Schulz, H., Saar, K., Hübner, N., Bauer, J., Braun, M., Schwarzwälder, A., Segerer, J., Birlem, M., Horn, A., Hemmersbach, R., Waβer, K., Grosse, J., Infanger, M., Grimm, D. Differential gene expression profile and altered cytokine secretion of thyroid cancer cells in space. FASEB J. 28, 813–835 (2014). www.fasebj.org

Gravity-sensitive signaling drives 3-dimensional formation of multicellular thyroid cancer spheroids

This study focused on the effects induced by a random positioning machine (RPM) on FTC‐133 thyroid cancer cells and evaluated signaling elements involved in 3‐dimensional multicellular tumor spheroid (MCTS) formation. The cells were cultured on the RPM, a device developed to simulate microgravity, and under static 1‐g conditions. After 24 h on the RPM, MCTSs swimming in culture supernatants were found, in addition to growth of adherent (AD) cells. Cells grown on the RPM showed higher levels of NF‐κB p65 protein and apoptosis than 1‐g controls, a result also found earlier in endothelial cells. Employing microarray analysis, we found 487 significantly regulated transcripts belonging not only to the apoptosis pathway but also to other biological processes. Selected transcripts were analyzed with quantitative real‐time PCR using the same samples. Compared with 1‐g IL‐6, IL‐8, CD44, and OPN were significantly up‐regulated in AD cells but not in MCTSs, while ERK1/2, CAV2, TLN1, and CTGF were significantly down‐regulated in AD cells. Simultaneously, the expression of ERK2, IL‐6, CAV2, TLN1, and CTGF was reduced in MCTSs. IL‐6 protein expression and secretion mirrored its gene expression. Thus, we concluded that the signaling elements IL‐6, IL‐8, OPN, TLN1, and CTGF are involved with NF‐κB p65 in RPM‐dependent thyroid carcinoma cell spheroid formation.—Grosse, J., Wehland, M., Pietsch, J., Schulz, H., Saar, K., Hübner, N., Eilles, C., Bauer, J., Abou‐El‐Ardat, K., Baatout, S., Ma, X., Infanger, M., Hemmersbach, R., Grimm, D. Gravity‐sensitive signaling drives 3‐dimensional formation of multicellular thyroid cancer spheroids. FASEB J. 26, 5124–5140 (2012). www.fasebj.org

Spheroid formation of human thyroid cancer cells in an automated culturing system during the Shenzhou-8 Space mission

Human follicular thyroid cancer cells were cultured in Space to investigate the impact of microgravity on 3D growth. For this purpose, we designed and constructed a cell container that can endure enhanced physical forces, is connected to fluid storage chambers, performs media changes and cell harvesting automatically and supports cell viability. The container consists of a cell suspension chamber, two reserve tanks for medium and fixative and a pump for fluid exchange. The selected materials proved durable, non-cytotoxic, and did not inactivate RNAlater. This container was operated automatically during the unmanned Shenzhou-8 Space mission. FTC-133 human follicular thyroid cancer cells were cultured in Space for 10 days. Culture medium was exchanged after 5 days in Space and the cells were fixed after 10 days. The experiment revealed a scaffold-free formation of extraordinary large three-dimensional aggregates by thyroid cancer cells with altered expression of EGF and CTGF genes under real microgravity.