| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 25, 2002 as doi:10.1096/fj.01-0673fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universität Berlin, Germany;
*
Max-Planck Institute of Biochemistry, Martinsried, Germany;
Institute of Anatomy, Freie Universität Berlin, Germany;
Department of Nuclear Medicine, University of Regensburg, Germany; and
Space Biology Group, ETH Zurich, Switzerland
2Correspondence: Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, FU Berlin, Garystr. 5, D-14195 Berlin, Germany. E-mail. dgrimm{at}zedat.fu-berlin.de
SPECIFIC AIMS
The principal aim of this study was to investigate the effects of appropriate simulation of microgravity with a 3-dimensional random positioning machine (clinostat) on thyroid hormone-producing human follicular thyroid carcinoma cells. The specific objective was to investigate the effects of simulated microgravity on the cytoskeleton, extracellular matrix protein production, and endocrine activity, and to address the hypothesis that simulated microgravity induces programmed cell death and that apoptosis is a mechanism for the mild hypothyroidism after spaceflight.
PRINCIPAL FINDINGS
1. Effect of clinorotation on morphology
After 12 h of clinorotation of a subconfluent ML-1 monolayer, formation of multicellular spheroids (MCTS) was visible in all culture dishes on a 3-dimensional random positioning machine. After 24 h, most ML-1 cells had detached from the bottom of the culture dishes and had formed spheroids. After 48 h, nearly all cells were growing in the form of 3-dimensional spheroids of up to 0.3 ± 0.01 mm in diameter. Cells were distributed randomly over the total volume of the spheroids and arranged irregularly, but not in concentric cell layers. Necrotic centers were not observed even after 3 days of culture under simulated 0 g conditions. After 24 h of 0 g exposure, cells of MCTS and monolayers stained by propidium iodide, DAPI, and TUNEL labeling revealed nuclei of the follicular thyroid carcinoma cells showing chromatin condensation, membrane blebbing, loss of nuclear envelope, and cellular fragmentation into apoptotic bodies indicating a significant increase of programmed cell death under microgravity (Fig. 1
); 1 g control cells did not show typical morphological signs of apoptosis. Transmission electron microscopy confirmed these data. When the cells were stained with fluorescence-labeled anti-vimentin antibodies, 100% were vimentin positive whereas the control population exerted only 30% positive cells. The filaments had rearranged, were radiating and branching toward the cell membrane, and were thicker than control filaments. Western blot analysis confirmed these findings.
|
2. Simulated microgravity alters the expression of extracellular matrix proteins and adhesion molecules
After 24 h low gravity incubation, spheroids exhibited an increase of the extracellular matrix proteins collagen I, III, laminin, fibronectin, and chondroitin sulfate. This was confirmed by Western blot analysis, which indicated (Fig. 2
) that collagen I and III, fibronectin, chondroitin sulfate, laminin, and well as vinculin were produced at a lower rate by control ML-1 cells grown in dishes in the incubator and ML-1 cells grown in flasks on the ground in the clinostat room. There was a clear significant increase of collagen I (3-fold), collagen III (4.5-fold), fibronectin (2.3-fold), laminin (6.6-fold), chondroitin sulfate (2.2-fold), and vinculin (2-fold) vs. 1 g control cells.
|
3. Clinorotation induced apoptosis
Morphological parameters (i.e., chromatin condensation) and biochemical markers (i.e., DNA laddering) demonstrated apoptosis of thyroid cancer cells cultured in simulated microgravity. Normally, 2% of the ML-1 cells showed expression of Fas protein as measured by flow cytometry of 10 000 single cells. After 24 and 72 h of simulated microgravity, the amount of Fas-positive cells was increased up to 20% and 15%. Tumor suppressor antigen p53, normally expressed only in 1.5% of ML-1 cells, was significantly elevated up to 19% after 24 h and 25% after 72 h by clinorotation; 17.5% of the ML-1 cells showed a positivity for Bcl-2 on ground. Microgravity reduced the expression of Bcl-2 (24 h: 8.1% and 72 h: 5.9%). The cleavage of 116 kDa poly(ADP-ribose)polymerase resulted in the 85 kDa apoptosis-related cleavage fragment and was associated with an increase of bax (4-fold). This was more pronounced in cells cultured under simulated 0 g conditions.
4. Hormone release of ML-1 cells
Cultures of ML-1 cells constitutively released thyroid hormones such as fT3, fT4, and thyroglobulin into the supernatant. After 24 and 48 h of clinorotation, fT3 and fT4 secretion of the ML-1 cells was significantly reduced. To investigate the thyroglobulin content of the ML-1 cells, we performed flow cytometry. 10,000 control ML-1 cells showed a 99% positivity for thyroglobulin. Clinorotation reduced the percentage of thyroglobulin-positive cells to 63% after 24 h and 59% after 72 h. Expression of the TSH receptor was up-regulated under conditions of simulated microgravity.
CONCLUSIONS AND SIGNIFICANCE
These experiments could indicate a regulatory basis why astronauts show low thyroid hormone levels after spaceflight, which may be explained by an increase of apoptosis in thyrocytes because of microgravity (Fig. 3
).
|
A significant and novel finding was the time-dependent increase in promotors of apoptosis in thyroid carcinoma cells. Our data support the hypothesis that microgravity induces programmed cell death and that apoptosis is a mechanism limiting survival of thyroid carcinoma cells under conditions of simulated microgravity.
It is important to distinguish between the stress-associated changes by clinorotation (including experiment-induced stress) and effects of the clinostat room. We addressed these factors by applying ground controls and incubator controls. All samples were treated identically before the experiment. They were randomized for clinorotation (24, 48 or 72 h), ground control, or incubator control groups.
Three-dimensional growth of normal cells and tumor cells in vitro is an established cell culture model with advantages in many research applications because cultures of tumor cells, often referred to as MCTS, resemble small microtumors in vivo rather than cells grown in 2-dimensional monolayers. After 12 h of clinorotation, we observed spheroid formation of ML-1 cells. They formed regular round aggregates with a diameter of 0.3 mm that were nonadherent after 24 h. After 48 h, only a few cells were still adherent on the bottom of the culture flasks. We proved again that clinostat and bioreactor systems are advantageous in a variety of experiments limited to the biological effects of real microgravity and exclude effects of cosmic radiation. Applications include tissue engineering, maintenance of normal prostate tissue, and studies of normal and cancer cells.
Our finding that the cytoskeletal intermediate filament protein vimentin is increased under conditions of simulated microgravity is consistent with abnormalities in actin stress fibers and microtubulin in Jurkat cells flown on the space shuttle. These results were found in cells fixed 24 h after the start of clinoration and can also be detected at 48 and 72 h of clinorotation.
ML-1 MCTS harvested after clinorotation revealed an impressive increase of extracellular matrix components compared with corresponding 1 g control cells. We also examined the thyroid-specific metabolic capability of the cell line grown under conditions of simulated microgravity. In controls, the ML-1 cell line secretes fT3 and fT4 permanently into the supernatant. After 24 and 48 h of clinorotation, this metabolic capability of fT3 and fT4 production is reduced and is accompanied by an up-regulation of the thyroid stimulating hormone (TSH) receptor. This result agrees with the finding that a mild hypothyroidism was induced in rats and monkeys in space. The hypothalamic-pituitary-thyroid axis involves the thyroid hormones thyroxine and triiodothyronine, which are lowered in space, suggesting a mild hypothyroidism. A similar trend was reported for humans during the D2 mission, with slightly higher TSH levels in space suggestive of mild hypothyroidism with compensatory pituitary hyperfunction. This is an important result, because all organ systems are affected by thyroid function: normal function of the left ventricle of the heart, for instance, requires a normal thyroid function. This question has received little attention and is why additional studies are necessary.
Further, >95% of ML-1 1 g control cells had intracellularly accumulated thyroglobulin antigens, but only 59% of the ML-1 cells under clinorotation expressed thyroglobulin. This was accompanied by a decrease of thyroglobulin secretion into the supernatant. Our hypothesis is that the increase of apoptotic cells in clinostat cultures may cause the changes in cell metabolism. Clinorotation induced apoptotic cell death. There are two major pathways leading to apoptosis: a 1) positive induction by a ligand binding to a plasma membrane receptor and 2) negative induction by loss of a trophic signal. The apoptotic mechanism of a cell involves the death receptors, adaptor molecules, caspases, mitochondria, and the Bcl-2 proteins. Exposure to low gravity induced an elevated cleavage of 116 kDa poly(ADP-ribose)polymerase (PARP) to the 85 kDa apoptosis-related cleavage fragment of ML-1 cells, which is also present in lower amounts in control cells. PARP plays a pivotal role in DNA damage repair, but its cytoprotective function is changed when enormous DNA damages cannot be repaired. These damaged cells will be eliminated by apoptosis. After 24 h of microgravity, the level of Fas-positive cells increased compared with ground controls. We found higher expression of p53 and bax and a decrease in Bcl-2. These data agree with postflight accumulations of tumor suppressor p53 protein in rat muscle. Up-regulation of promotors of apoptosis, down-regulation of inhibitors in thyroid cancer cells, and the appearance of programmed cell death in thyroid cancer may provide a possible target for treatment in the future.
Our results provide the following: 1) new information on follicular thyroid cancer cells grown under low gravity conditions; 2) conditions of simulated microgravity induce formation of multicellular tumor spheroids within a short time and increase the expression of extracellular matrix proteins; 3) ML-1 cells grown under simulated weightlessness show characteristic signs of apoptosis; 4) The finding of low fT3 and fT4 secretion of this cell line during the first 48 h of clinorotation can be explained by the increase of programmed cell death. Mild hypothyroidism in space may affect bone mineralization, muscle trophism, fat deposition—in fact, all organs. Future studies under conditions of real microgravity during short and longer term missions are needed to investigate these important findings in space. 5) Our study demonstrates that the 3-dimensional clinostat can provide a convenient experimental system that allows us to culture large numbers of thyroid cancer spheroids for pharmacological studies.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0673fje; to cite this article, use FASEB J. (February 25, 2002) 10.1096/fj.01-0673fje 
This article has been cited by other articles:
|
|
 |
|
  J. B. Laoag-Fernandez, H. Matsuo, H. Murakoshi, A. L. Hamada, B. K. Tsang, and T. Maruo 3,5,3'-Triiodothyronine Down-Regulates Fas and Fas Ligand Expression and Suppresses Caspase-3 and Poly (Adenosine 5'-Diphosphate-Ribose) Polymerase Cleavage and Apoptosis in Early Placental Extravillous Trophoblasts in Vitro J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4069 - 4077. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Graebe, E. L. Schuck, P. Lensing, L. Putcha, and H. Derendorf Physiological, Pharmacokinetic, and Pharmacodynamic Changes in Space J. Clin. Pharmacol., August 1, 2004; 44(8): 837 - 853. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Maccarrone, N. Battista, M. Meloni, M. Bari, G. Galleri, P. Pippia, A. Cogoli, and A. Finazzi-Agro Creating conditions similar to those that occur during exposure of cells to microgravity induces apoptosis in human lymphocytes by 5-lipoxygenase-mediated mitochondrial uncoupling and cytochrome c release J. Leukoc. Biol., April 1, 2003; 73(4): 472 - 481. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
