Simulated microgravity induce apoptosis and down-regulation of erythropoietin receptor of UT-7/EPO cells

Hematopoietic progenitor cell proliferation can be alternated on either spaceflight or under simulated microgravity experiments on the ground; however, the underlying mechanism remains largely unknown. In the present study, we have demonstrated that exposure of human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO cells to conditions of simulated microgravity with a rotary culture instrument significantly inhibited the cellular proliferation rate. Adding higher concentrations of EPO to the culture medium failed to improve the inhibitory status. Cell apoptosis was detected by fluorescence staining of cell nuclei and a flow cytometry assay using Annexin V/PI double staining. This microgravity-induced apoptosis in UT-7/EPO cells could be blocked by a pancaspase inhibitor Z-VAD-FMK. Immunoblotting demonstrated that rotary culture resulted in a reduction of the expression of Bcl-xL, an anti-apoptotic protein, and the cleavage of caspase-3. Furthermore, rotary culture reduced surface localization and protein content, as well as the mRNA expression of erythropoietin receptor (EPOR) of UT-7/EPO. Take together, the findings indicated that simulated microgravity may induce mitochondrial related apoptosis of UT-7/EPO cell through depressing the EPO–EPOR pathway.     

Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% (p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO–EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.     

Expression of estrogen receptor α in human breast cancer cells regulates mitochondrial oxidative stress under simulated microgravity

This study investigated intracellular oxidative stress and its underlying mechanisms in a rotary cell culture system used to achieve a simulated microgravity (SMG) environment. Experiments were conducted with human breast cancer cell lines MCF-7 (an estrogen receptor (ER) α positive cell line) and MDA-MB-231 (an ERα negative cell line) encapsulated in alginate/collagen carriers. After 48 h, SMG led to oxidative stress and DNA damage in the MDA-MB-231 cells but a significant increase in mitochondrial activity and minimal DNA damage in the MCF-7 cells. The activity of superoxide dismutase (SOD) significantly increased in the MCF-7 cells and decreased in MDA-MB-231 cells in the SMG environment compared with a standard gravity control. Moreover, SMG promoted expression of ERα and protein kinase C (PKC) epsilon in MCF-7 cells treated with PKC inhibitor Gö6983. Overall, exposure to SMG increased mitochondrial activity in ERα positive cells but induced cellular oxidative damage in ERα negative cells. Thus, ERα may play an important role in protecting cells from oxidative stress damage under simulated microgravity.     

Severe disruption of the cytoskeleton and immunologically relevant surface molecules in a human macrophageal cell line in microgravity—Results of an in vitro experiment on board of the Shenzhou-8 space mission

During spaceflight the immune system is one of the most affected systems of the human body. During the SIMBOX (Science in Microgravity Box) mission on Shenzhou-8, we investigated microgravity-associated long-term alterations in macrophageal cells, the most important effector cells of the immune system. We analyzed the effect of long-term microgravity on the cytoskeleton and immunologically relevant surface molecules. Human U937 cells were differentiated into a macrophageal phenotype and exposed to microgravity or 1g on a reference centrifuge on-orbit for 5 days. After on-orbit fixation, the samples were analyzed with immunocytochemical staining and confocal microscopy after landing. The unmanned Shenzhou-8 spacecraft was launched on board a Long March 2F (CZ-2F) rocket from the Jiuquan Satellite Launch Center (JSLC) and landed after a 17-day-mission. We found a severely disturbed actin cytoskeleton, disorganized tubulin and distinctly reduced expression of CD18, CD36 and MHC-II after the 5 days in microgravity. The disturbed cytoskeleton, the loss of surface receptors for bacteria recognition, the activation of T lymphocytes, the loss of an important scavenger receptor and of antigen-presenting molecules could represent a dysfunctional macrophage phenotype. This phenotype in microgravity would be not capable of migrating or recognizing and attacking pathogens, and it would no longer activate the specific immune system, which could be investigated in functional assays. Obviously, the results have to be interpreted with caution as the model system has some limitations and due to numerous technical and biological restrictions (e.g. 23 °C and no CO₂ supply during in-flight incubation). All parameter were carefully pre-tested on ground. Therefore, the experiment could be adapted to the experimental conditions available on Shenzhou-8.     

T cell regulation in microgravity – The current knowledge from in vitro experiments conducted in space,parabolic flights and ground-based facilities

Dating back to the Apollo and Skylab missions, it has been reported that astronauts suffered from bacterial and viral infections during space flight or after returning to Earth. Blood analyses revealed strongly reduced capability of human lymphocytes to become active upon mitogenic stimulation. Since then, a large number of in vitro studies on human immune cells have been conducted in space, in parabolic flights, and in ground-based facilities. It became obvious that microgravity affects cell morphology and important cellular functions. Observed changes include cell proliferation, the cytoskeleton, signal transduction and gene expression. This review gives an overview of the current knowledge of T cell regulation under altered gravity conditions obtained by in vitro studies with special emphasis on the cell culture conditions used. We propose that future in vitro experiments should follow rigorous standardized cell culture conditions, which allows better comparison of the results obtained in different flight- and ground-based experiment platforms.