Weightlessness Induced Apoptosis in Normal Thyroid Cells and ...

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Endocrinology 144(9):4172– 4179 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-0171

Weightlessness Induced Apoptosis in Normal Thyroid Cells and Papillary Thyroid Carcinoma Cells via Extrinsic and Intrinsic Pathways PETER KOSSMEHL, MEHDI SHAKIBAEI, AUGUSTO COGOLI, MANFRED INFANGER, ¨ NBERGER, CHRISTOPH EILLES, JOHANN BAUER, FRANCESCO CURCIO, JOHANN SCHO HOLGER PICKENHAHN, GUNDULA SCHULZE-TANZIL, MARTIN PAUL, AND DANIELA GRIMM Institute of Clinical Pharmacology and Toxicology (P.K., H.P., M.P., D.G.), Benjamin Franklin Medical Center, Institute of Anatomy (M.S., G.S.-T.), and Klinik fu¨r Unfall und Wiederherstellungschirurgie (M.I.), Benjamin Franklin Medical Center, Freie Universita¨t Berlin, 14195 Berlin, Germany; Space Biology Group (A.C.), ETH Zurich, 8005 Zurich, Switzerland; Dipartmento di Patologia e Medicina Sperimentale e Clinica, University of Udine Medical School (F.C.), 33100 Udine, Italy; Clinic of Nuclear Medicine, University of Regensburg (J.S., C.E.), 93042 Regensburg, Germany; and Max Planck Institute of Biochemistry (J.B.), 82152 Martinsried, Germany Apoptosis plays a pivotal role in development, tissue homeostasis, cancer, immune defense, and response to weightlessness. It can be initiated by external signals via death receptors, but may also emerge from mitochondria. We exposed mitochondria-rich thyroid carcinoma cells (ONCO-DG1 cell line) and normal thyroid cells (HTU-5) to conditions of simulated microgravity. After 24 h, 10% of the cancer cells had entered a Fas-dependent apoptotic pathway, but destruction and redistribution of mitochondria, microtubuli disruption, and caspase-3 activation were also detected, demonstrating the activation of extrinsic as well as intrinsic pathways. Furthermore, ONCO-DG1 cells grown on the clinostat showed elevated amounts of Bax, but reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed

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XPERIMENTS PERFORMED DURING real and simulated microgravity have shown substantial changes in molecular or biochemical cell regulation (1–5). One of the alterations observed under these conditions is the downregulation of thyroid function, resulting in mild hypothyroidism (6, 7). Understanding this microgravity-induced hypothyroidism is very important, because many organ systems are affected by thyroid function, such as bone mineralization, muscle trophism, fat deposition, and even normal function of the left ventricle of the heart. Recently, we described an increase in programmed cell death in cells of the follicular thyroid carcinoma cell line ML-1 (8) cultured in a three-dimensional clinostat simulating microgravity (5). Programmed cell death observed was accompanied by spontaneous formation of multicellular tumor spheroids (MCTSs); up-regulation of various cytoskeletal and extracellular matrix proteins, Fas protein, and the TSH receptor; as well as simultaneous down-regulation of thyroglobulin, free T3 (fT3), and fT4 hormones and of Bcl-2. The finding of low thyroglobulin, fT3, and fT4 secretion during clinorotation was explained by the increase in apoptosis and was suggested to Abbreviations: DAPI, 4⬘,6-Diamidino-2-phenylindole; fT3, free T3; MCTS, multicellular tumor spheroid; PARP, poly(ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4ⴕ,6-diamidino-2-phenylindole staining, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADPribose)polymerase activity and apoptosis. Apoptosis was also detected in normal HTU-5 cells, as demonstrated by electron microscopy, activation of caspase-3, increases in Fas and Bax, and elevation of 85-kDa apoptosis-related cleavage fragments resulting from enhanced poly(ADP-ribose) polymerase activity. Gravitational unloading affects the mitochondria and thereby may trigger apoptosis in thyroid cells subjected to weightlessness by clinorotation. (Endocrinology 144: 4172– 4179, 2003)

mirror the mild hypothyroidism found in astronauts as well as in an animal model (rat) returning from a space mission (6, 7). Various cellular mechanisms lead to apoptosis (9, 10). Recent work has provided evidence that mitochondria play a central role in both caspase-dependent and -independent forms of apoptosis. DNA damage, kinase inhibition, trophic factor deprivation, ischemia, UV radiation, and oxidative stress are important apoptotic signals channeled through mitochondria (11). To see a possible role for mitochondria in weightlessness-induced apoptosis, we used the mitochondria-rich permanent papillary thyroid cell line ONCO-DG1 of epithelial origin to characterize the stimulation of apoptosis in thyroid cells under microgravity. This cell line is tumorigenic in nude mice and also capable of forming MCTSs (12). The study of ONCO-DG1 cells has been carried out in parallel with HTU-5 thyroid cells to compare the behaviors of normal and tumor cells. Materials and Methods Cell culture Cells of the human oxyphilic papillary thyroid carcinoma cell line ONCO-DG1 (passage 40) were cultured in RPMI 1640 medium containing 100 ␮m sodium pyruvate and 2 mm l-glutamine and supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Invitrogen, Eggenstein, Germany) until subconfluence. A primary culture of human thyroid cells (HTU-5) was developed

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from normal human thyroid tissue and grown using methods previously reported (13). The basic culture medium for these cells was Coon’s F-12 medium modified by the addition of MgCl to a final concentration of 0.5 mm, CaCl2 to 1.1 mm, and glucose to 3.6 mm. The medium (humed) was also supplemented with 5% fetal bovine serum, a five-hormone mixture (insulin, somatostatin, hydrocortisone, transferrin, and glyclcylhistidyl-lysine), and bovine hypothalamus and bovine pituitary (Pel Freez Biologicals, Rogers, AK) extract as previously described (14). In the present study HTU-5 cells were used at passage 9. The subconfluent monolayers (12 ⫻ 106 cells/dish) of ONCO-DG1 cells (n ⫽ 6) as well as HTU-5 cells (n ⫽ 6) were either clinorotated for 24 h or further cultured at 1 ⫻ g under identical conditions in a 37 C room. To start a culture, we used a syringe to fill culture flasks with complete medium, taking care to avoid air bubbles. Randomly, 12 filled culture flasks (of each cell type, n ⫽ 6) were screwed onto the threedimensional clinostat developed by Hoson and colleagues (15) in Japan and manufactured by Fokker Space (Leiden, The Netherlands). Another six samples of each cell type were placed at the bottom next to the machine in a room at a temperature of 37 C. Rotation was 60°/sec.

Spinning culture of papillary thyroid carcinoma cells for spheroid formation (16) A suspension (400 ml) of ONCO-DG1 cells was adjusted to a cell density of 5 ⫻ 105 cell/ml in 37 C complete medium. One hundred milliliters of the cell suspension were transferred to a 250-ml Spinner flask (12). Four Spinner flasks were placed in a 5% CO2/95% air humidified incubator at 37 C and immediately stirred with a magnetic stirrer at 120 rpm. Three- and 6-d-old multicellular spheroids were investigated under conditions of simulated microgravity for 24 h in the three-dimensional clinostat and under 1 ⫻ g conditions at the bottom in the clinostat room.

Evaluation of apoptosis: acridine orange/ethidium bromide, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, and 4⬘,6-diamidino-2-phenylindole (DAPI) staining The monolayers remaining under 1 ⫻ g conditions at the bottom of the plastic culture flasks (BD Biosciences, Heidelberg, Germany) and the MCTSs formed during 24 h of clinorotating were examined by phase contrast microscopy. After culture, control and microgravity-exposed cells were stained with acridine orange/ethidium bromide according to the method of Zhou et al. (17). Clinorotation (for 24 h) induced DNA fragments of ONCO-DG1 cells, which were labeled by immunohistochemical staining with the Apopdetect Peroxidase Kit (Qbiogene, Heidelberg, Germany) according to the manufacturer’s protocol. The cells were fixed with 4% paraformaldehyde and postfixed with ethanol/ acetic acid (2:1). They were then incubated with terminal deoxynucleotidyl transferase enzyme. After reacting with antidigoxigenin conjugate, a peroxidase substrate was added to develop color. Cells were counterstained with methylene green. TUNEL-positive cells were counted using quantitative image analysis. The percentage of apoptotic cells was determined from counts of 300 cells dispersed in different fields on five coverslip preparations for each gravity condition. The cells for DAPI staining were fixed with 4% formaldehyde and incubated in the DAPI medium containing 4⬘,6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR). Stained nuclei were investigated using fluorescence microscopy (5).

Western blot analysis Western blot analyses of various components of cells exposed to microgravity and of control cells were carried out following routine protocols. Antibodies against the following antigens were used for this study: activated caspase-3, ␤1 integrin, Fas, Bax, and Bcl-2 (Chemicon, Hofheim, Germany); laminin, vinculin, and vimentin (Sigma Chemie, Deisenhofen, Germany); and poly(ADP-ribose) polymerase (PARP; Coulter, Heidelberg, Germany). SDS-PAGE and immunoblotting were carried out following routine protocols (5). The samples were homogenized by shearing forces in lysis buffer (50 mm Tris-HCl, pH 7.2; 150 mm NaCl; 1% Triton X-100; 1 mm sodium orthovanadate; 50 mm sodium

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pyrophosphate; 100 mm sodium fluoride; 0.01% aprotinin; 4 ␮g/ml pepstatin A; 10 ␮g/ml leupeptin; and 1 mm phenylmethylsulfonylfluoride) on ice for 30 min. For immunoblotting, equal amounts of total proteins (50 ␮g total protein, each fraction was loaded per lane) were separated on 10% SDS-PAGE polyacrylamide gels under reducing conditions. Subsequently, the homogenates were transferred onto a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using a Transblot electrophoresis apparatus (Mini Trans Blot, Bio-Rad Laboratories, Richmond, CA) for 1 h at 120 V. Membranes were blocked with 5% (wt/vol) skim milk powder in PBS/0.1% Tween 20 overnight at 4 C and incubated with primary antibodies diluted in blocking buffer for 1 h at room temperature. After five washes in blocking buffer, membranes were incubated with alkaline phosphatase-conjugated secondary antibody diluted in blocking buffer for 30 min at room temperature. Membranes were finally washed five times in blocking buffer and three times in 0.1 m Tris (pH 9.5) containing 0.05 m MgCl2 and 0.1 m NaCl; specific binding was detected using nitro blue tetrazolium and 5-bromo-4chloro-3-indoyl-phosphate (p-toluidine salt; Pierce, Rockford, IL) as substrates and was quantitated by densitometry (Personal Densitometer 50301, Molecular Dynamics, Krefeld, Germany). Protein determination was performed with the bicinchoninic acid system, using BSA as a standard.

Laser scan and electron microscopy Cells stained by rhodamine 123 as described previously (12) were investigated by confocal laser scanning microscopy (Leitz, Wetzlar, Germany). Analyses of mitochondria and nuclei were performed by transmission electron microscopy exactly as described previously (18).

Immunofluorescence staining For immunofluorescence staining, cells and MCTSs were seeded out into one of a four-chamber Supercell chamber slide (BD Biosciences) and were incubated for 30 min (adhesion time). Subsequently, the adherent cells were washed twice, and the monoclonal antibodies were added. Antibodies against the following antigens were used for this study: laminin, collagen I (both from Sigma Chemie), fibronectin (Chemicon, Hofheim, Germany), Fas (APO-1, CD 95; Biozol, Eching, Germany), Bax, Bcl-2, caspase-3-CPP32 (Coulter, Heidelberg, Germany), and osteopontin (Developmental Studies Hybridoma Bank, Department of Biological Sciences University of Iowa, Iowa City, IA). Antigen-antibody complexes were visualized with the indirect immunofluorescence technique and examined by fluorescence microscopy and image analysis (19).

Automatic image analysis Morphometry, including automatic image analysis, was applied to quantitatively assess positive cells and extracellular matrix proteins using a computer-assisted image analysis system (Olympus Optical, Hamburg, Germany). Automatic image analysis was applied to quantitatively assess changes in the expression of cellular antigens. Supercell chamber slides (BD Biosciences) were fixed with acetone (⫺20 C) for 10 min. Slides were selected to visualize antigen-antibody complexes with indirect immunofluorescence. All slides were visualized by fluorescence microscopy using an oil immersion objective with a calibrated magnification of ⫻400. Visual fields had 757 ⫻ 506 square pixels with a resolution of 0.2053 ␮m/pixel (area ⫽ 0.0161 mm2). Automatic image analysis used an 8-bit color system that translates colors to 256 gray levels for automatic border detection. These measurements were performed by two independent investigators, who were blinded for modality of treatment. Variability was assessed by performing repeated analyses and was calculated as 1% (intraobserver) and 3% (interobserver). Areas positive for an antigen demonstrated a yellow-green color, which is translated by an 8-bit color depth system to 256 gray levels. Differences are used to identify borders. Areas were calculated as the sum of all positive areas related to the area of the entire visual field times 100. Twenty randomly selected visual fields were analyzed to calculate the average of respective volume fractions (variance, ⬍2%) (19).

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Statistics Statistical analysis was performed using SPSS 10.0 (SPSS, Inc., Chicago, IL). Results are expressed as the mean ⫾ se. Comparisons between multiple groups were assessed by one-way ANOVA, including a modified least significant difference (Bonferroni) multiple range test to detect significant differences between two distinct groups, which were further analyzed using the Mann-Whitney U test. The strength of the relationship between two variables was assessed by calculation of the productmoment correlation coefficient (r). Statistical significance was accepted at the level of P ⬍ 0.05.

Results Effect of clinorotation on morphology

After 24-h culture of subconfluent starting cultures, confluent viable monolayers were found in control cultures (Fig. 1A), but most clinorotated ONCO-DG1 cells had detached

from the bottom of the culture dishes, and several spheroids had formed and were floating in the medium under simulated low gravity conditions (Fig. 1B). The maximum size of the MCTSs was 0.3 mm in diameter. Moreover, smaller cell aggregates were detected. Within the spheroids, the cells were randomly distributed over the total volume of the spheroids, which showed irregular cell arrangements, but no concentric cell layers. Initially, after 12 h of clinorotation, we observed normal thyroid cells forming regular round aggregates with a maximum diameter of 0.3 mm that were nonadherent after 24 h and formed no necrotic centers. Preformed Spinner flask spheroids cultured for 24 h under simulated microgravity did not change their shape compared with 1 ⫻ g controls as investigated by phase contrast microscopy. Clinorotation induces programmed cell death

Acridine orange/ethidium bromide staining of ONCODG1 control cells and MCTSs revealed according to Zhou et al. (17) 1) that the 1 ⫻ g control cells had remained impermeable for the dyes (Fig. 1C), whereas 2) MCTS cells exerted nuclear condensation (phase II) or took up the dyes (phase III; Fig. 1D), indicating dead cells. In addition, only the clinorotated papillary thyroid carcinoma cells showed chromatin condensation, membrane blebbing, loss of nuclear envelope, and cellular fragmentation into apoptotic bodies. Similarly, the control cells remained TUNEL negative (Fig. 1E), whereas in clinostat cultures 10% of the cells were TUNEL positive (Fig. 1F). DAPI labeling of HTU-5 monolayer cells (Fig. 1G) revealed normal viable cells, whereas DAPI staining of clinorotated HTU-5 cells (Fig. 1H) and already formed Spinner flask MCTSs investigated after 24 h of clinorotation (Fig. 1I, 3-dold spheroids; Fig. 1J, 6-d-old spheroids) demonstrated cells showing chromatin condensation, membrane blebbing, loss of nuclear envelope, and cellular fragmentation into apoptotic bodies, indicating a significant increase in programmed cell death under simulated microgravity. Electron microscopy of clinorotated HTU-5 cells revealed characteristic signs of programmed cell death (Fig. 5, C and D). Clinorotation changes the expression of cellular antigens

Immunohistochemical characterization of ONCO-DG 1 MCTSs proved the microscopic observations (Fig. 1, D and F), as staining with anti-Fas and anti-caspase-3 antibodies

FIG. 1. Phase contrast microscopy of the ONCO-DG1 monolayers remaining under 1 ⫻ g conditions at the bottom of the plastic culture flasks (A), MCTSs formed during 24-h clinorotation (B), ONCO-DG1 control cells (C and E), and simulated microgravity-exposed cells (D and F) stained with acridine orange/ethidium bromide (C and D) or according to the TUNEL assay (E and F). The arrow indicates brown TUNEL-positive nuclei of ONCO-DG1 cells. G and H, DAPI staining of HTU-5 control cells (G) and DAPI-stained clinorotated HTU-5 cells (H) exerting chromatin condensation and membrane blebbing. Signs of apoptosis are also evident after 24 h of simulated microgravity in 3-d-old (I) and 6-d-old (J) spheroids of the ONCO-DG1 cell line already established by Spinner flask technique before exposure to microgravity (original magnification, ⫻200).

TABLE 1. Evaluation of cellular antigens detected by immunofluorescence and measured by quantitative image analysis Antigens

1 g Control cells area %

Simulated 0 g area %

Laminin Collagen I Fibronectin Osteopontin Fas Caspase-3

n.d. n.d. 0.8 ⫾ 0.05 n.d. n.d. 0.2 ⫾ 0.1

1.7 ⫾ 0.16a 3 ⫾ 0.01a 4.8 ⫾ 0.1a 23 ⫾ 0.5a 10 ⫾ 0.01a 10 ⫾ 00.1a

All values are mean control; n ⫽ 5.0.

SEM;

n.d., not detectable;

a

P ⬍ 0.05 vs. 1 g



FIG. 2. Western blot analyses of various components of ONCO DG1 cells exposed to microgravity and of control cells (A, PARP; B, laminin, vinculin, ␤1 integrin, vimentin; D, Bcl-2, Bax, Fas; E, activated caspase-3). C, MCTS stained by fluorescence-labeled antilaminin antibodies. F, Densitometric data.

FIG. 3. Western blot analyses of HTU-5 cells exposed to conditions of simulated microgravity (zero gravity) and of control cells (1 ⫻ g). A, PARP; B, Bcl-2, Bax, and Fas; C, activated caspase-3; D, laminin. E, Densitometric data.

resulted in clearly positive areas if the cells had been exposed to simulated microgravity, but not in control cells (Table 1). Furthermore, in three-dimensional ONCO-DG1 MCTSs formed under low gravity conditions, the extracellular matrix proteins collagen I, laminin, fibronectin, and osteopontin were significantly up-regulated (Table 1), as quantified by image analysis.

Investigation of extracellular matrix proteins and PARP by Western blot analysis

The observation that clinorotation induced vinculin protein synthesis (1.8-fold) in MCTSs, enhanced vimentin (1.3fold) as well as ␤1 integrin expression (1.4-fold) compared with ground control cells, was confirmed by Western blot analyses (Fig. 2B). Laminin was 1.9-fold up-regulated under

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FIG. 4. Western blot analyses of ONCO-DG1 Spinner flask spheroids (3 and 6 d old) exposed to conditions of simulated microgravity (zero gravity) and of control cells (1 ⫻ g). A, PARP; B, Bcl-2, Bax, and Fas; C, activated caspase-3; D, laminin. Densitometric data of 3-d-old spheroids (E) and 6-d-old MCTSs (F) are shown.

zero gravity as shown by multicellular spheroids stained by fluorescein-labeled antilaminin antibodies (Fig. 2C). In addition, laminin was increased in clinorotated HTU-5 cells (Fig. 3D) and clinorotated 3- and 6-d-old Spinner flask MCTSs (4.4-fold/3.3-fold) of the ONCO-DG1 cell line (Fig. 4D). Western blots also proved that effectors of apoptosis, such as Fas (1.3-fold) and Bax proteins were increased, whereas bcl-2 was decreased in ONCO-DG1 cells cultured under simulated zero gravity conditions (Fig. 2D). Bcl-2 (4.2-fold), Bax (1.4-fold), and Fas protein (2-fold) were increased in clinorotated HTU-5 cells cultured for 24 h at simulated zero gravity (Fig. 3B). Interestingly, Spinner flask spheroids (3 and 6 d old) exerted an increase in bcl-2 (2.6-/2.3-fold), Bax (2.7-/2.1-fold), and Fas (2.3-/1.7-fold) under conditions of simulated microgravity (Fig. 4B). Activated caspase-3 remained undetectable in 1 ⫻ g cultures of ONCO-DG1 and HTU-5 cells as well as in 3- and 6-d-old spheroids of the ONCO-DG1 cell line and was upregulated in all zero gravity cultures (Figs. 2E, 3C, and 4C). Finally, the cleavage of 116-kDa PARP into 85-kDa apoptosis-related cleavage fragments (Fig. 2A) was increased 2.7-fold in microgravity-exposed ONCO-DG1 cells compared with control ONCO-DG1 cells. In 3- and 6-d-old ONCO-DG1-MCTSs clinorotation induced a 2.6-fold increase in the 85-kDa cleavage fragment of PARP compared with the corresponding control spheroids (Fig. 4A). Interestingly, 85-kDa apoptosis-related cleavage fragments were also 1.7-fold elevated in normal thyroid HTU-5 cells grown under conditions of simulated microgravity compared with controls (Fig. 3A). These features indicated a significant increase in apoptosis under conditions of simulated microgravity. Investigation of mitochondria

Finally, ONCO-DG1 cells cultured at 1 ⫻ g always showed a clear abundance of mitochondria. This could best be dem-

onstrated by laser scan microscopy of rhodamine 123-stained control cells (Fig. 5A). In cells that had been exposed to zero gravity for 24 h, chromatin condensation was visible as well as blebbing of the cell. Mitochondria had accumulated toward one side of the cell, indicating disruption of the microtubule network. They had become swollen; inner cristae were disorganized and contained larger inner spaces of matrix material (Fig. 5B). Discussion

Among the observations described in this manuscript, the most significant and novel findings were, first, the detection of programmed cell death in papillary thyroid cancer cells as well as in normal thyroid cells under conditions of simulated microgravity, second, the increase in caspase-3 activation in both cell types, and third, the disorganization of mitochondria in ONCO-DG1 papillary thyroid cancer cells. In addition, we detected characteristic morphological signs of programmed cell death, such as an elevated cleavage of 116-kDa PARP to the 85-kDa apoptosis-related cleavage fragment, TUNEL positivity and dye permeability, as well as morphological changes. Moreover, the amount of Fas antigen was increased compared with 1 ⫻ g controls in thyroid cancer cells as well as in normal thyroid cells. Recently, it was reported that Fas antigen is expressed on papillary thyroid cancer cells (20). The Fas/Fas ligand system has been proposed as a mechanism of tumor cell defense, although there is controversy regarding the relevance of Fas ligand in such a role (21, 22). The specific role of the Fas receptor in thyroid cancer is not known. Fas expressed on tumor cells may play a role in the immune control of tumor growth and may provide a possible target for treatment. Death of tumor cells has been demonstrated after inducing sensitivity to Fas-mediated apoptosis with anticancer drugs, such as doxorubicin (23). In parallel, we found a higher expression of Bax and a


FIG. 5. The confocal laser scanning microscopic investigation of rhodamine 123-stained control cells (A) revealed the characteristic abundance of mitochondria of the ONCO-DG1 cell line. Electron microraphs of microgravity-exposed ONCO-DG1 cells (B) exert characteristic features of apoptosis, such as chromatin condensation of the ONCO-DG1 cell. Mitochondria had become swollen, and inner cristae were disorganized and contained larger inner spaces of matrix material (arrows). Electron micrographs of normal HTU-5 cells cultured under normal 1 ⫻ g conditions (C) and of clinorotation-exposed HTU-5 cells (D), demonstrating chromatin condensation as well as blebbing of the cell.

decrease in Bcl-2 in ONCO-DG 1 cancer cells. Elevated Bax and p53 levels were found in patients with thyroid carcinomas (24). HTU-5 cells exerted a low level of Bax expression, a finding in agreement with data published for adenomas and goiters (24). The observed responses of clinorotated cells are related to the microgravity environment, because static controls were negative for morphological signs of apoptosis as well as activated caspase-3 and Bax proteins, but were strongly positive for Bcl-2. Bcl-2 as a prosurvival member of the Bcl-2 family can act by preventing the release of apoptogenic molecules from organelles such as mitochondria (25). These data are in agreement with our earlier results (5) and with observations made on other cells exposed to real or simulated microgravity (4, 26). For these experiments, we used the ONCO-DG1 cell line, because it exhibited an abundance of mitochondria (12). The cell line is able to form multicellular spheroids, without changes in viability, when the liquid overlay method and the Spinner flask technique are used (27). We investigated already formed Spinner flask spheroids under conditions of simulated microgravity. Western blot analysis revealed an increase in Fas, PARP, and activated caspase-3 in MCTSs grown at zero gravity for 24 h compared with control spheroids. In contrast to monolayer ONCO-DG 1 cells exposed to microgravity, Bcl-2 was increased, and Bax was elevated in clinorotated Spinner flask spheroids as well as in HTU-5 cells. The increase in Fas in all cell types may trigger apoptosis in simulated weightlessness. DAPI staining of viable cells immediately investigated after clinorotation revealed nuclei of the papillary thyroid carcinoma-MCTS showing chromatin condensation, membrane blebbing, loss of nuclear envelope, and cellular fragmentation into apoptotic bodies, indicating a significant increase in programmed cell death under microgravity. It is concluded that MCTS formation as such can be excluded as the cause of apoptosis in these cells. As observed in ML-1 cells (5), multicellular ONCO-DG1


spheroids grown in the clinostat exerted an increase in the cytoskeletal intermediate filament vimentin as well as in extracellular matrix components, such as laminin, osteopontin, fibronectin, collagen I, and ␤1 integrin, compared with corresponding static control cells. Secreted extracellular matrix components have been demonstrated in MCTSs established at 1 ⫻ g conditions (28), but are also detected in elevated amounts in spheroids formed under clinorotation. The fact that laminin increases in 3- and 6-d-old MCTSs that had been first formed by the Spinner flask method and then grown for 24 h under conditions of microgravity seems to be due to spheroid formation per se and the effect of zero gravity on the cells in the three-dimensional cell complex. Our data support the hypothesis that simulated microgravity induces programmed cell death and demonstrates for the first time that apoptosis of papillary thyroid carcinoma cells is triggered by microgravity via a mechanism that includes, in addition to the Fas-dependent pathway, the destruction of mitochondria. As mitochondria disorganization is reported to be an early event upstream of the caspase-3 activation step, the mitochondrial system might be an early target affected by weightlessness (29). Earlier space studies suggested that weightlessness affects the correct assembly of microtubuli and filamentous actin (30, 31) and induces intracellular redistribution of mitochondria (4). Hence, impairment of the assembly of microtubuli and F-actin might be a reason for the redistribution of mitochondria and induction of apoptosis in thyroid cells. In cancer research, chemotherapeutically induced models of programmed cell death are usually applied to identify targets for treatment of the disease (32). The effect of zero gravity is not specific for thyroid cells. Apoptosis of different cell types after spaceflight and under conditions of simulated microgravity has been described in the literature. In 1998, Lewis et al. (26) showed for the first time that real microgravity alters microtubuli in human Jurkat lymphocytes. They demonstrated that cytoskeletal alterations, growth retardation, and metabolic changes are accompanied by increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We obtained similar results investigating papillary thyroid carcinoma cells. After 24 h of microgravity, the number of Fas-positive cells increased compared with that of 1 g controls. Using a clinostat, Sarkar et al. (33) showed that clinorotation of osteoblastic rat osteosarcoma 17/2.8 cells resulted in apoptosis. Apoptotic death was associated with alterations of the cytoskeleton of osteoblasts. Onishi et al. (34) reported on postflight accumulations of tumor suppressor p53 protein in rat muscle. Moreover, it has been shown that microgravity induced apoptosis in cultured glial cells (35), and clinorotation induced apoptosis in luteal cells of pregnant rats (36). The principal aim of our study was to learn how to induce selective apoptosis in all malignant cells of a thyroid tumor so that frequently observed progression and metastases can be prevented. Several researchers have shown that simulated zero gravity conditions induce three-dimensional growth of normal cells and tumor cells (37–39). Three-dimensional aggregates function in the same way as their corresponding tissues in vivo; however, this is not the case in a cellular monolayer. Using various earth-based zero gravity simula-

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tion techniques, scientists have successfully developed experimental models for the understanding and development of treatments for cancer, osteoporosis, and disorders of the immune system. Moreover, zero gravity may become an important tool in tissue engineering. In this context a threedimensional clinostat can provide a convenient experimental system that allows the culture of many thyroid cancer spheroids, consisting of cells progressing through apoptosis and others resistant to apoptosis. Therefore, clinorotation, which is an important terrestrial model system for studying the effects of reduced gravity on cells to develop experimental systems and hypotheses concerning gravitational cell biology, may additionally become an important tool to trigger apoptosis under controlled physical conditions. Thyroid hormones control many cells in our organism and, as indicated by a variety of postspaceflight hormonal changes, its complex regulation may be influenced by gravity. One of the hormonal alterations observed under the conditions of real and simulated microgravity is the downregulation of thyroid function resulting in mild hypothyroidism (5–7). Molecular modifications observed on thyroid cells under microgravity conditions could explain the hormonal in vivo changes found in space. It has been shown that FRTL-5 cells, normal rat follicular thyroid cells in continuous culture, respond to TSH in a dose-dependent manner in terms of cAMP production. At each hormonal dosage, the cellular response was increased in hypergravity (40). In addition, a significant inhibition of cAMP production was demonstrated in microgravity (41). Moreover, after 24 and 48 h of simulated microgravity, human follicular thyroid carcinoma ML1 cells revealed significantly decreased fT3 and fT4 secretion and up-regulated TSH receptor expression (5). Our results provide new information on papillary thyroid cancer cells and normal thyroid cells grown under simulated microgravity. Apoptosis accompanied by redistribution and destruction of mitochondria is initiated after formation of MCTSs within a short time, whereas the expression of extracellular matrix proteins and apoptosis via extrinsic and intrinsic pathways (42) increased. This information may help us to determine the roots of the negative physiological changes that humans and animals face during a long stay in orbit (6, 7). These data strongly support that the action of weightlessness on thyroid mitochondria might be a major reason for the development of hypothyroidism in space and that diminished thyroid function may be the cause of the health problems suffered in space by men and animals. Acknowledgments We thank Mrs. Ursula Schwikowski for producing the figures for the Western blot analyses. Received December 19, 2002. Accepted May 22, 2003. Address all correspondence and requests for reprints to: Daniela Grimm, M.D., Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universita¨ t Berlin, Garystrasse 5, D-14195 Berlin, Germany. E-mail: [email protected]. This work was supported by Eidgenössische Technische Hochschule Zurich (A.C.).

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