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Keywords:

  • clinostat;
  • osteoblasts;
  • apoptosis;
  • cytoskeleton;
  • gravity

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity environment. We then evaluated the responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6–24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell-surface integrin β1 and disorganization of actin cytoskeleton. These results suggest that vector-averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Microgravity, in space flights or in altered gravitational environments, has been shown to exert profound effects on important cellular processes such as proliferation, differentiation, and gene expression.(1–3) Osteoblasts or osteoblast-like cells were shown to be highly sensitive to microgravity both by morphological and functional studies.(4–7) Mouse MC3T3-E1 osteoblasts, cultured in STS-56 shuttle flight for 4 days, showed reduced growth rate, decrease in the size of the nucleus, and abnormal actin cytoskeleton with reduced stress fibers.(6) Human osteosarcoma cell line MG-63 cells, cultured for 9 days in the unmanned Foton 10 space flight, showed reduced induction in collagen Iα1, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) expression in response to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or transforming growth factor β2 (TGF-β2) treatment.(7) These studies concluded that microgravity adversely affects growth and differentiation of osteoblasts.

One of the important determinants of cellular growth and differentiation is apoptosis or programmed cell death. Apoptosis is a cell suicide pathway that is involved in a variety of physiological and pathological events such as tissue morphogenesis, development, cancer, and neurodegenerative disorders.(8,9) Transmission electronmicroscopic analysis of lymphocytes, cultured in Spacelab D-1, showed the presence of a large number of vacuoles indicating that apoptosis may be enhanced on exposure of cells to the microgravity of space.(2) One of the hallmarks of apoptosis is depolymerization of actin cytoskeleton.(10) Apoptosis is increased by inhibition of actin filament assembly and disruption of microtubules.(11,12) Arecent study with human T lymphoblastoid cells (Jurkat) flown on the space shuttle showed that these cells undergo apoptotic death in association with anomalies of cytoskeleton.(13) Therefore, it is possible that the reduction in growth and differentiation of osteoblasts, in actual space flight experiments, may result from apoptotic cell death.

Cell-surface integrins play an important role in regulation of cell adhesion and proliferation.(14) Integrin-mediated signals are required for the survival of anchorage-dependent cells including endothelial and epithelial cells. Integrins link the components of the extracellular matrix (ECM), for example, collagen, fibronectin, and vitronectin, to the intracellular actin cytoskeleton and associated proteins.(15) Interaction of integrins with ECM generates cell survival signals that are transduced to the interior via the actin cytoskeleton. Deprivation of these signals results in apoptosis of the cells.(16) Integrins are heterodimers composed of α-subunits and β-subunits.(17) Both subunits bind to ECM whereas only the β-subunit binds to cytoskeletal proteins. In osteoblasts, the major subunit of integrin appears to be β1, which serves as receptors for collagen, fibronectin, laminin, and vitronectin.(18–21)

In the present study, we found that, after exposure to vector-averaged gravity environment in a clinostat, ROS 17/2.8 cells showed evidence of apoptotic death in association with perinuclear localization of the cell-surface integrin β1 and disorganization of actin cytoskeleton. However, vector-averaged gravity did not affect the proliferation of the cells. We suggest that apoptotic cell death might be one of the mechanisms for reduced bone formation observed in actual space flights.(22–24)

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cell culture

ROS 17/2.8 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), 50 U/ml of penicillin G and 50 μg/ml of streptomycin at 37°C in 5% CO2 and 100% relative humidity. To determine the appropriate serum concentration needed to maintain cell viability, we cultured ROS 17/2.8 cells in the medium containing 1, 2, 5, and 10% FBS. It was demonstrated that cells grow well in 2, 5 and 10% FBS (data not shown). In addition, the changes, observed in clinorotated cells, were evident in all three serum concentrations (data not shown). Therefore, 2% FBS concentration was used in all experiments. The day before clinostat rotation, 1 × 105 cells were plated either on Falcon 3018 12.5-cm2 flasks (Becton Dickinson Labware, Franklin Lakes, NJ, U.S.A.) or on Lab-Tek chamber slides (Nunc, Naperville, IL, U.S.A.). After 24 h, the medium was removed and the flask was filled completely with fresh medium (approximately 40 ml) to eliminate the presence of air bubbles, thus diminishing the likelihood of turbulence and shear forces during rotation of cultures. The flasks were divided into three groups: stationary (control condition), horizontal rotation (motion control condition) in which the cultures were rotated around the vertical axis, and clinorotation in which the cultures were rotated around the horizontal axis. All cultures were placed in an incubator at 37°C. Rotation was carried out at 50 revolutions per minute (rpm) for 6, 12, and 24 h. This speed of rotation was selected after experiments with 10, 40, and 50 rpm in which the last clinorotation resulted in reproducible, detectable changes from the stationary and motion controls. The same speed was used by Guignandon et al., taking into account theoretical considerations defined by Block and Briegleb.(25,26) Brown also described the feasibility of 50 rpm for the fast rotating clinostat.(27) The pH of the medium did not change in any of the cultures during the time period of the experiment.

Measurement of total cellular DNA content

Total cellular DNA content was measured using fluorochrome Hoechst 33258 dye (Molecular Probes, Eugene, OR, U.S.A.) according to the method described by Rago et al.(28) Briefly, attached and floating cells were harvested separately and washed in phosphate-buffered saline (PBS). The cells were lysed in distilled water for 1 h followed by freezing at −80°C and thawing. Fluorochrome Hoechst 33258 dye was added to a final concentration of 10 μg/ml and fluorescence was determined by excitation at 350 nm and measuring emission at 460 nm using a fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Herring sperm DNA was used as standards.

Cell viability assay

Cell viability was assessed by trypan blue exclusion test.(29) At 24 h, the adherent and detached cells were collected separately, stained with trypan blue and counted under a microscope using a hemocytometer at ×40 magnification. Cells from at least three flasks were counted per experiment. To determine whether clinorotation affects the growth of the cells, viable cells were counted by trypan blue exclusion test after 24 h rotation and 2 × 103 cells were plated per well in a Falcon 96-well plate (Becton Dickinson Labware, Franklin Lakes, NJ, U.S.A.) at a density similar to that plated before clinorotation. The cells were cultured for 12, 24, and 48 h under normal conditions in 10% FBS and cell viability was determined by WST assay kit (Dojindo, Kumamoto, Japan) according to the manufacturer's protocol. The data represent mean ± SD (n = 8). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Fisher's protected least significant difference (PLSD) analysis.

DNA fragmentation assay

Both adherent and detached cells from eight 12.5-cm2 flasks were pooled and washed in PBS. The cells were lysed in 100 μl of cell lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM EDTA, and 0.5% Triton X-100) for 10 minutes on ice and centrifuged at 12,000 rpm for 20 minutes to separate intact from fragmented DNA. The resulting supernatant containing the fragmented DNA was treated with RNase A (0.4 mg/ml) for 1 h at 37°C followed by proteinase K (0.4 mg/ml) for 1 h at 37°C. After precipitation with 5 M NaCl and isopropanol, DNA was collected and resuspended in 20 μl of TE buffer (10 mM Tris-HCl; 1 mM EDTA, pH 8.0) and electrophoresed in a 2% agarose gel. DNA bands were stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR, U.S.A.). DNA fragmentation also was analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) method using ApoAlert DNA Fragmentation Assay Kit (Clontech, Palo Alto, CA, U.S.A.) according to the manufacturer's instructions.

Quantitative analysis of apoptosis was performed using the Cell Death Detection ELISA kit (Boehringer Mannheim GmbH, Mannheim, Germany) according to the manufacturer's protocol. This assay is based on the quantitative sandwich-enzyme immunoassay using mouse monoclonal antibodies directed against DNA and histones, respectively. It allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. At first, antihistone antibody was fixed absorptively on a microtiter plate. The nucleosomes contained in the sample then bound via their histone components to the immobilized antihistone antibody. Then anti-DNA antibody coupled with peroxidase reacted with the DNA of the nucleosome. The amount of peroxidase was determined photometrically with 2, 2′-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) as a substrate using a microplate reader (BIO-RAD Laboratories, Hercules, CA, U.S.A.) at 405 nmper 3 × 105 cells. An increased absorbancy indicates a higher percentage of cells undergoing apoptosis.

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Figure FIG. 1.. Decrease in the number of adherent ROS 17/2.8 cells after 12–24 h of culture in the clinostat. ROS 17/2.8 cells were cultured in DMEM containing 2% FBS for 6, 12, and 24 h in three groups: stationary, horizontal rotation, and clinostat at 50 rpm. Cells were photographed with a Polaroid camera at ×40 magnification. The panels in the figures represent the best estimate of a typical field of cells under each condition.

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Flow cytometry

Flow cytometry was performed to detect Annexin V binding and to perform cell cycle analysis. Annexin V binding was detected using the Annexin-V-FLUOS staining kit (Boehringer Mannheim GmbH, Mannheim, Germany) according to the manufacturer's protocol. After 12 h and 24 h of culture, ROS 17/2.8 cells were harvested, resuspended in 100 μl staining solution containing Annexin-V-fluorescein and propidium iodide (PI) and incubated for 30 minutes at room temperature. For cell cycle analysis, the cells were harvested and fixed with ice-cold 70% methanol. After washing twice with PBS, the cells were incubated in RNase A in PBS (40 U/ml) at 37°C for 30 minutes and then were stained with PI (50 μg/ml) for 1 minute. For both analyses, after passing through a nylon-mesh filter, the cells were analyzed by flow cytometry using a flow cytometer (Coulter Epics XL, Coulter Corp., Miami, FL, U.S.A.) equipped with a 500-mW argon laser and a helium-neon laser. Appropriate settings of forward and side scatter gates were used to examine 10,000 cells per experiment. The percentage of cells in G0 + G1, S, and G2 + M of the cell cycle were analyzed and quantitated with the Multicycle software (Coulter Electronics, Coulter Corp., Miami FL, U.S.A.).

Immunocytochemistry

For staining of actin, the cells were fixed, permeabilized with 0.2% Triton X-100 in PBS for 5 minutes, and incubated with rhodamine phalloidin (Molecular Probes, Eugene OR, U.S.A.) for 20 minutes. The cells were visualized with a confocal laser scanning microscope (Zeiss LSM 510; Carl Zeiss Jena GmbH, Jena, Germany).

To visualize integrin β1, cells were fixed in 4% paraformaldehyde in PBS for 10 minutes, incubated with anti-integrin β1 antibody overnight at 4°C (1:50) (Transduction Laboratories, Lexington, KY, U.S.A.), recognizing amino acids 76–256 of the extracellular domain, washed in PBS, and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 2 h. The cells were visualized with a confocal laser scanning microscope (Zeiss LSM 510, Carl Zeiss Jena GmbH, Jena, Germany). Two hundred cells were counted for each group to determine the percentage of cells showing perinuclear distribution of integrin β1. Three-dimensional images from the confocal laser scanning microscope were converted into binary format and total pixel number of integrin β1 per cell was determined using the National Institutes of Health (NIH) image software (version 1.62) for 25 cells in each group. Statistical analysis was carried out using one-way ANOVA followed by Fisher's PLSD analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

ROS 17/2.8 cells were cultured in DMEM containing 2% FBS for 6, 12, and 24 h in three groups, namely, stationary, horizontal rotation (motion control), and clinorotation (experimental group). Figure 1 shows that the number of adherent cells at the two early time points was similar in all three test groups. However, at 24 h after initiation of the experiment experimental cultures (rotated in the vector-averaged condition in clinostat) appeared to contain fewer attached cells and many detached cells. To determine the extent of cell detachment in the three groups at 24 h, we collected adherent and detached cells separately, stained with trypan blue and counted at ×40 magnification using a hemocytometer. Figure 2 shows that almost all the adherent cells were viable in stationary and motion control cultures. Moreover, in both control conditions, very few detached cells were observed (2.5% and 0%, respectively). However, in vector-averaged (clinorotated) cultures, there was a significant (p < 0.01 as analyzed by one-way ANOVA) decrease (35%) in viable adherent cells. In addition, a significant (p < 0.01) proportion of adherent cells from these cultures were stained with trypan blue. The detached cells also were dead. The total number of cells (adherent and detached) under clinorotation was similar to the total number of cells in stationary and horizontal rotation. When the total cellular DNA content was determined in all of the three groups, the combined DNA content of adherent and detached cells under clinorotation was similar to those in stationary cultures and under horizontal rotation (Table 1). These results indicate that the decrease in the number of adherent cells under clinorotation is caused by cell death rather than inhibition of cell proliferation. To determine whether 24-h clinorotation affects the growth of the viable cells, they were counted and plated in a 96-well plate at a density similar to that plated before clinorotation. The growth of the cells was assessed at 12, 24, and 48 h of culture under normal conditions. As shown in Fig. 3, viable cells from all of the three groups grew at a similar rate, indicating that clinorotation did not affect the proliferation rate of the viable cells.

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Figure FIG. 2.. Total number of viable ROS 17/2.8 cells are decreased by clinorotation. ROS 17/2.8 cells were cultured in three groups (as in Fig. 1). After 24 h, the adherent and detached cells were collected separately, stained with trypan blue, and counted using a hemocytometer. The data represent mean ± SD of three independent experiments. The asterisk represents a significant decrease (p < 0.01) versus viable cell number in stationary culture. The # represents a significant increase (p < 0.01) versus trypan blue stained cells in stationary cultures.

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The major biochemical hallmark of apoptotic cell death is cleavage of chromosomal DNA at internucleosomal sites into 180 base pairs and its multiples. To identify whether the cells in clinostat underwent apoptosis, the presence of fragmented DNA in the cells was analyzed. As shown in Fig. 4, no DNA fragmentation could be observed among the three groups at 12 h. However, at 24 h, DNA ladder pattern was evident in the cells cultured under clinorotation but not in those in stationary cultures and under horizontal rotation. DNA fragmentation was analyzed further by TUNEL method. As shown in Fig. 5, no TUNEL-positive cells were present in stationary cultures and under horizontal rotation, whereas TUNEL-positive cells were seen in the clinorotated cultures (arrows in Fig. 5). To quantify the extent of apoptosis, we performed sandwich-enzyme immunoassay as described (in the Methods section). Figure 6 shows that apoptosis was more than 3-fold higher in clinorotated cultures when compared with the control groups.

An additional characteristic feature of apoptosis is the translocation of phosphotidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca+2-dependent phospholipid-binding protein with high affinity for PS.(30) To detect Annexin V binding and to establish whether there is any change in the cell cycle of ROS 17/2.8 cells under clinorotation, the cells were incubated with Annexin-V-fluorescein and PI and analyzed by flow cytometry (Fig. 7). As shown in Fig. 7A, Annexin V staining showed a single peak of membrane-unbound molecule at 12 h in all three groups, indicating the absence of apoptotic changes. However, at 24 h, a distinct peak for Annexin V bound to cell membrane was detected only in the cells cultured under clinorotation (Fig. 7A, black arrow). To differentiate necrotic and apoptotic cell population in Annexin V–positive cells, dual parameter Annexin V and PI analysis was performed. As shown in Fig. 7B, an increase in apoptotic cells and the absence of necrotic cells was observed at 24 h of clinorotation but not in control groups.

Table Table 1.. Total DNA Content of ROS 17/2.8 Cells Does Not Change in Clinorotation
   Clinostat
 StationaryHorizontal rotationAdherentDetachedTotal
  1. ROS 17/2.8 cells were cultured as in Fig. 1. After 24 h, the adherent and detached cells were collected separately and incubated with Fluorochrome Hoechst 33258 dye. Total DNA was measured using a fluorescent spectrophotometer. The data represents mean ± SD of three independent experiments.

Total DNA (μg)23.13 ± 2.3324.85 ± 0.4912.18 ± 0.149.74 ± 0.6321.9 ± 0.47
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Figure FIG. 3.. Clinorotation does not affect the proliferation rate of viable cells. ROS 17/2.8 cells were cultured as in Fig. 1. After 24 h, viable cells were counted by trypan blue exclusion test and 2 × 103 viable cells were plated per well in a 96-well plate. The cells were cultured under normal condition and cell viability was assessed by WST assay at 12, 24, and 48 h. The data represent mean ± SD (n = 8).

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Figure FIG. 4.. Clinorotation results in fragmentation of DNA in ROS 17/2.8 cells after 24 h. ROS 17/2.8 cells were cultured as in Fig. 1. After 12 h and 24 h, the adherent and detached cells were collected together; fragmented DNA was isolated and analyzed as described in the Materials and Methods section. Note the increased presence of DNA fragmentation in clinorotated cells at 24 h compared with controls. MWM, molecular weight marker.

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Figure 7C shows data of forward and side scatters and PI staining for analysis of cell cycle. At 12 h, a comparable and low number of apoptotic cells (ranging from 4.9% to 9.3%) were observed in all groups. At this time, the cell cycle was normal with no growth arrest as exemplified by cell cycle analysis. At 24 h, a limited number of apoptotic cells were shown in the stationary and horizontal rotated cultures (Fig. 7C). However, under clinorotation at 24 h, 27.4% of the cells exhibited apoptotic characteristics (Fig. 7C), in agreement with the findings of Annexin V staining. However, the cell cycle was normal without any growth arrest. Growth arrest was not observed by cell cycle analysis employing PI alone (data not shown).

Because apoptosis is associated with cytoskeletal depolymerization, we examined actin staining patterns to determine if clinorotation affects the cytoskeleton. In stationary and horizontal rotation cultures, we found predominantly linear actin filaments (Fig. 8). The cells in these two groups were marked by the presence of lamellipodia and microspikes (Fig. 8, arrows). However, in the clinorotated cells the actin cytoskeleton was disorganized, lost its filamentous distribution, and became condensed. The cells became more rounded, having lost their processes.

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Figure FIG. 5.. TUNEL-positive cells are present in clinorotation after 24 h. ROS 17/2.8 cells were cultured as in Fig. 1. After 24 h, the cells were fixed and stained by the TUNEL method. The upper panel represents the cells in bright field while the lower panel shows the cells viewed with a fluorescence microscope at ×400 magnification. The arrows in the clinorotated cultures indicate TUNEL-positive cells. The panels in the figures represent the best estimate of a typical field of cells under each condition. Note the increased number of TUNEL-positive cells in clinorotated cells in comparison with those in the controls.

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Figure FIG. 6.. Clinorotation results in apoptosis of ROS 17/2.8 cells. ROS 17/2.8 cells were cultured as in Fig. 1. After 24 h, the adherent cells were harvested. Apoptotic change in three culture groups was determined by cell death detection ELISA. The data represents mean ± SD from three independent experiments. The asterisk represents a significant increase (p < 0.01) versus O.D. units in stationary culture.

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Immunocytochemistry was performed to determine the distribution of integrin β1. In stationary and horizontal rotation groups, ROS 17/2.8 cells showed a diffuse staining pattern for integrin β1, which also was evenly distributed in cell processes (Fig. 9). Clinorotation was associated with a decrease in the diffuse distribution of integrin β1 staining of the cells, especially in the periphery of the cells. In addition, integrin β1 staining was detected predominantly in the perinuclear region. Among the clinorotated cells, 30.5% showed the perinuclear distribution of integrin β1 (1.5% and 2% in stationary and horizontal rotated cultures, respectively) (Table 2) and in these cells the total pixel number of integrin β1 per cell was significantly less than that in the typical cells in stationary and horizontal rotated cultures (Table 2).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The clinostat is an effective, ground-based tool that can be used to verify data from space flight (where experimental duplication is very difficult) and to test hypotheses and experimental conditions for eventual space flights in which the effects of microgravity on mammalian cells have to be tested.(2) Rotation in clinostats appears to mimic the microgravity environment by nulling the gravitational vector through continuous averaging.(2,31) In the present study, we rotated osteoblast-like ROS 17/2.8 cells in a clinostat at 50 rpm and observed that this vector-averaged gravity environment resulted in apoptotic cell death in association with disorganization of actin cytoskeleton and redistribution of cell-surface integrin β1. These effects could be attributed to vector-averaged gravity because the cells in stationary culture and those under horizontal rotation, designed to control for turbulence, shear forces against the cells, and general vibrations as stressors, did not show any significant differences. Hughes-Fulford et al. have shown that the total number of MC3T3-E1 osteoblast cells, flown in the STS-56 shuttle flight, was significantly reduced.(6) These findings are consistent with the results reported here using clinorotation. We extended these findings by showing more specifically that the reduction in cell number is caused by apoptosis and not by growth arrest. This conclusion is further strengthened by the observation that 24 h clinorotation did not affect the proliferation rate of viable cells after subculturing under normal conditions (Fig. 3). Guignandon et al. reported that the growth curves of ROS 17/2.8 cells in ground controls and in space-flown cultures are roughly similar.(4) The differences between our respective results and theirs may be attributable, in part, to differences in detection methods, in the cell number at plating, and to the longer time points (up to 6 days) that they used.

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Figure FIG. 7.. Clinorotation does not result in cell cycle arrest in ROS 17/2.8 cells. ROS 17/2.8 cells were cultured as in Fig. 1. After 12 h and 24 h, the cells were stained with Annexin-V-fluorescein and PI and analyzed by flow cytometry. (A) Annexin-V-fluorescein. Fluorescent peak of Annexin V not bound to the cell membrane was indicated by white arrow. The peak with Annexin V bound to cell membrane was indicated by black arrow. (B) Dual parameter analysis with Annexin V (FL1 LOG) and PI (FL3) at 24 h. R1, R2, and R3 represent living, apoptotic, and necrotic cells, respectively. Note the increase in apoptotic cells (R2) and the absence of necrotic cells (R3) in clinorotated cells. (C) Data of forward and side scatters and PI for cell cycle analysis; X axis represents DNA content while Y axis represents number of cells. The percentage of cells that underwent apoptosis was indicated in each panel.

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One of the important causative factors of apoptosis is loss of cellular adhesion to the ECM. Epithelial and endothelial cells undergo apoptosis on disruption of cell-ECM interactions by either maintaining cells in suspension or treating cells with arginine-glycine-aspartate-containing peptides to block functionally integrin signals.(14,16) Similar integrin-mediated suppression of apoptosis also was shown in mammary and bronchial epithelial cells.(32,33) In this study, we observed that the distribution of integrin β1 was confined essentially to the perinuclear region in clinorotated cells in association with a quantitative decrease in amount of staining. This redistribution of integrin β1 may be responsible for cell detachment described in this article (Fig. 9). Gronowicz et al. reported that glucocorticoids induce similar changes in the distribution and the amount of integrin β1 as well as detachment of osteoblasts from ECM.(34) Recently, it was shown that glucocorticoids also induce apoptotic death of osteoblasts.(35)

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Figure FIG. 8.. Clinorotation results in disorganization of actin cytoskeleton in ROS 17/2.8 cells. ROS 17/2.8 cells were cultured as in Fig. 1. After 24 h, the cells were fixed and stained with rhodamine phalloidin. The cells were viewed with a confocal laser scanning microscope at ×400 magnification. The arrows indicate lamellipodia with microspikes. The panels in the figures represent the best estimate of a typical field of cells under each condition. Scale bar = 20 μm.

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Figure FIG. 9.. Clinorotation results in perinuclear distribution of integrin β1 in ROS 17/2.8 cells. ROS 17/2.8 cells were cultured as in Fig. 1. After 24 h, the cells were fixed and stained with antibody against integrin β1. The cells were viewed with a confocal laser scanning microscope at ×400 magnification. The panels in the figures represent the best estimate of a typical field of cells under each condition. Scale bar = 20 μm.

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Another precipitating factor for apoptosis might be the depolymerization of actin cytoskeleton. Agents inhibiting actin polymerization have been shown to result in fragmentation of DNA.(11) In addition, in several cell lines, it has been shown that microgravity leads to the disorganization of actin cytoskeleton. Actin stress fibers became coalesced in mouse osteoblastic MC3T3-E1 cells in the STS-56 shuttle flight.(6) Interestingly, similar findings were reported for a different cell type (myocytes) after rotation in a clinostat.(36) Also, microtubule filaments of Jurkat cells flown on the space shuttle were shortened, coalesced, lacked normal branching at the cell membrane, and microtubule organization centers were disrupted.(13) Our study is consistent with these findings. Thus, ROS 17/2.8 cells cultured under clinorotation became more rounded and actin cytoskeleton was disorganized. Furthermore, integrin β1 showed perinuclear distribution, suggesting that these cells were in the process of detachment. The combined findings of actin disorganization and integrin β1 redistribution are likely to be important steps in the induction of apoptosis.

It is intriguing to speculate why altered gravity, including microgravity, might produce apoptosis of ROS 17/2.8 cells. It is well known that bone tissue is sensitive to mechanical stress and loading.(37) Subnormal mechanical stress as a result of bed rest or immobilization results in decreased bone mass and disuse osteoporosis.(38) Microgravity, either in space or in simulated environments, also produces mechanical unloading. From the perspective of an isolated cell, microgravity might be considered to cause “unloading” of the cytoskeleton, resulting in altered orientation and organization of cytoskeletal filaments.(36) Under normal gravity conditions, Kaiser et al. have shown that lack of hemodynamic forces results in apoptosis of vascular endothelial cells.(39) Therefore, they speculated that mechanical forces might be essential stimuli for the maintenance of blood vessels. Similarly, in case of osteoblasts, mechanical loading might be necessary for survival and sustenance of the cells. Lack of mechanical stimuli in clinostat might be one of the factors to cause apoptosis of ROS 17/2.8 cells. In clinostat, the gravity vector is nulled by continuous averaging and, in this manner, it simulates the near zero gravity force encountered in space. However, during rotation other physical forces may contribute to results obtained by using the clinorotation paradigm. Of these, shear forces, turbulence, and vibrations are the most important. By comparing the results to motion controls (which mimic these forces), we may conclude that effects seen in vector-averaged clinorotation are attributable to the perturbed gravity environment experienced by the cells. We may rule out the effect of hydrostatic pressure, which is minimal because the cells were placed near the axis of rotation as described by Albrecht-Buehler.(40) Therefore, vector-averaged gravity environment alone is most likely to cause mechanical unloading of the cells. It is also possible that in the microgravity of space (and to a lesser extent in the clinostat), the lack of convection forces may be responsible for alternations to cell function including those we report here for osteoblasts.

Table Table 2.. Total Pixel Number of Integrin b1 in ROS 17/2.8 Cells
 StationaryHorizontal rotationClinostat
  1. Two hundred cells were counted for each group to determine the percentage of cells showing perinuclear distribution of integrin β1. Pixel number of integrin β1 per cell was determined for typical cells in stationary and horizontal rotation groups and for cells showing perinuclear distribution of integrin β1 in clinorotated group using NIH image software (version 1.62). The data represent mean ± SD (n = 25).

  2. *Represents significant difference from stationary and horizontal rotation groups (p < 0.001).

Percent of cells showing perinuclear distribution of integrin β11.52.030.5
Pixel number of integrin β1/cell981.3 ± 148.9977.1 ± 175.8703.1 ± 108.0*

Space flight is known to be associated with bone loss in astronauts and cosmonauts. Several studies have attributed this bone loss to the inhibition of bone formation.(22–24) One of the mechanisms of reduced bone formation may be the increased death of osteoblasts. In our study, we used osteosarcoma-derived ROS 17/2.8 cells instead of primary culture of osteoblasts. This may somewhat limit our ability to extrapolate the death of ROS 17/2.8 cells to space flight–induced bone loss. However, ROS 17/2.8 cells resemble the osteoblastic cells very closely in the expression of differentiation markers.(41) Therefore, we suggest that ROS 17/2.8 cells and osteoblasts are very likely to be affected similarly by altered gravity. Our finding that osteoblasts undergo apoptosis under clinorotation might lead to a better understanding of bone loss in space.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We express our gratitude to Dr. David A. Walsh, School of Anatomy, The University of New South Wales, Sydney, Australia for his valuable suggestions and help with flow cytometry; to Dr. Yoshiko Takagishi, Department of Teratology and Genetics, Research Institute of Environmental Medicine, Nagoya University for providing rhodamine phalloidin and giving suggestions about immunocytochemistry; and to Mr. Yoshiyuki Kawamoto, Nagoya University School of Agriculture for his excellent assistance with flow cytometry. This study was carried out as a part of the “Ground Research Announcement for the Space Utilization” promoted by National Space Development Agency of Japan and the Japan Space Forum (to H.S.). Additional support was obtained from Grants-in-Aid for Science Research (A) no. 10301005 in 1998 (to K.K.) and (C) no. 09671045 in 1998 (to T.N.).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Cogoli A, Tschopp A, Fuchs BP 1984 Cell sensitivity to gravity. Science 225: 228230.
  • 2
    Cogoli A, Cogoli GM 1997 Activation and proliferation of lymphocytes and other mammalian cells in microgravity. Adv Space Biol Med 6: 3379.
  • 3
    Bikle DD, Harris J, Halloran BP, Morey HE 1994 Altered skeletal pattern of gene expression in response to spaceflight and hindlimb elevation. Am J Physiol 267: E822E827.
  • 4
    Guignandon A, Genty C, Vico L, Lafage-Proust MH, Palle S, Alexandre C 1997 Demonstration of feasibility of automated osteoblastic line culture in space flight. Bone 20: 109116.
  • 5
    Kumei Y, Shimokawa H, Katano H, Hara E, Akiyama H, Hirano M, Mukai C, Nagaoka S, Whitson PA, Sams CF 1996 Microgravity induces prostaglandin E2 and interleukin-6 production in normal rat osteoblasts: role in bone demineralization. J Biotech 47: 313324.
  • 6
    Hughes-Fulford M, Lewis ML 1996 Effects of microgravity on osteoblast growth activation. Exp Cell Res 224: 103109.
  • 7
    Carmeliet G, Nys G, Stockmans I, Bouillon R 1998 Gene expression related to the differentiation of osteoblastic cells is altered by microgravity. Bone 22: 139S143S.
  • 8
    Steller H 1995 Mechanisms and genes of cellular suicide. Science 267: 14451449.
  • 9
    Thompson CB 1995 Apoptosis in the pathogenesis and treatment of disease. Science 267: 14561462.
  • 10
    Levee MG, Dabrowska MI, Lelli JJ, Hinshaw DB 1996 Actin polymerization and depolymerization during apoptosis in HL-60 cells. Am J Physiol 271: C1981C1992.
  • 11
    Kolber MA, Broschat KO, Landa GB 1990 Cytochalasin B induces cellular DNA fragmentation. FASEB J 4: 30213027.
  • 12
    Martin SJ, Cotter TG 1990 Disruption of microtubules induces an endogenous suicide pathway in human leukaemia HL-60 cells. Cell Tissue Kinetics 23: 545559.
  • 13
    Lewis ML, Reynolds JL, Cubano LA, Hatton JP, Lawless BD, Piepmeier EH 1998 Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat). FASEB J 12: 10071018.
  • 14
    Meredith JJ, Fazeli B, Schwartz MA 1993 The extracellular matrix as a cell survival factor. Mol Biol Cell 4: 953961.
  • 15
    Hynes RO 1992 Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69: 1125.
  • 16
    Frisch SM, Francis H 1994 Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124: 619626.
  • 17
    Turner CE, Burridge K 1991 Transmembrane molecular assemblies in cell-extracellular matrix interactions. Curr Opin Cell Biol 3: 849853.
  • 18
    Clover J, Dodds RA, Gowen M 1992 Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture. J Cell Sci 103: 267271.
  • 19
    Hughes DE, Salter DM, Dedhar S, Simpson R 1993 Integrin expression in human bone. J Bone Miner Res 8: 527533.
  • 20
    Grzesik WJ, Robey PG 1994 Bone matrix RGD glycoproteins: Immunolocalization and interaction with human primary osteoblastic bone cells in vitro. J Bone Miner Res 9: 487496.
  • 21
    Saito T, Albelda SM, Brighton CT 1994 Identification of integrin receptors on cultured human bone cells. J Orthopaed Res 12: 384394.
  • 22
    Morey ER, Baylink DJ 1978 Inhibition of bone formation during space flight. Science 201: 11381141.
  • 23
    Wronski TJ, Morey ER 1983 Effect of spaceflight on periosteal bone formation in rats. Am J Physiol 244: R305R309.
  • 24
    Vico L, Chappard D, Palle S, Bakulin AV, Novikov VE, Alexandre C 1988 Trabecular bone remodeling after seven days of weightlessness exposure (BIOCOSMOS 1667). Am J Physiol 255: R243R247.
  • 25
    Guignandon A, Usson Y, Laroche N, Lafage-Proust M, Sabido O, Alexandre C, Vico L 1997 Effects on intermittent or continuous gravitational stresses on cell-matrix adhesion: Quantitative analysis of focal contacts in osteoblastic ROS 17/2.8 cells. Exp Cell Res 236: 6675.
  • 26
    Block I, Briegleb W 1986 Gravisensitivity of the acellular slime mold Physarum polycephalum demonstrated on the fast-rotating clinostat. Eur J Cell Biol 41: 4450.
  • 27
    Brown AH 1992 Centrifuges: Evolution of their uses in plant gravitational biology and new directions for research on the ground and in spaceflight. ASGSB Bulletin 5: 4357.
  • 28
    Rago R, Mitchen J, Wilding G 1990 DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Biochem 191: 3134.
  • 29
    Oosterhuis JW, Verschueren RC, Eibergen R, Oldhoff J 1982 The viability of cells in the waste products of CO2-laser evaporation of Cloudman mouse melanomas. Cancer 49: 6167.
  • 30
    Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C 1995 A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 184: 3951.
  • 31
    Gruener R 1998 Neuronal responses to vector-averaged gravity: A search for gravisensing and adaptation mechanisms—a preliminary report. Jpn J Aerospace Env Med 35: 6383.
  • 32
    Howlett AR, Bailey N, Damsky C, Petersen OW, Bissell MJ 1995 Cellular growth and survival are mediated by β1 integrins in normal human breast epithelium but not in breast carcinoma. J Cell Sci 108: 19451957.
  • 33
    Aoshiba K, Rennard SI, Spurzem JR 1997 Cell-matrix and cell-cell interactions modulate apoptosis of bronchial epithelial cells. Am J Physiol 272: L28L37.
  • 34
    Gronowicz GA, McCarthy MB 1995 Glucocorticoids inhibit the attachment of osteoblasts to bone extracellular matrix proteins and decrease β-1 integrin levels. Endocrinology 136: 598608.
  • 35
    Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC 1998 Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest 102: 274282.
  • 36
    Gruener R, Roberts R, Reitstetter R 1994 Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight. Biol Sci Space 8: 7993.
  • 37
    Burger EH, Klein NJ 1998 Microgravity and bone cell mechanosensitivity. Bone 22: 127S130S.
  • 38
    Houde JP, Schulz LA, Morgan WJ, Breen T, Warhold L, Crane GK, Baran DT 1995 Bone mineral density changes in the forearm after immobilization. Clin Orthoped Relat Res 317: 199205.
  • 39
    Kaiser D, Freyberg MA, Friedl P 1997 Lack of hemodynamic forces triggers apoptosis in vascular endothelial cells. Biochem Biophys Res Commun 231: 586590.
  • 40
    Albrecht-Buehler G 1992 The simulation of microgravity conditions on the ground. ASGSB Bulletin 5: 310.
  • 41
    Majeska RJ, Nair BC, Rodan GA 1985 Glucocorticoid regulation of alkaline phosphatase in the osteoblastic osteosarcoma cell line ROS 17/2.8. Endocrinology 116: 170179.