Spaceflight altered the gene and protein expression of the membrane transporter proteins in Jurkat cells

We postulated that spaceflight and microgravity may cause changes to cell membrane structure, therefore affecting the expression of drug transporter proteins. We investigated the global gene expression changes and the expression of multidrug resistance proteins in space-flown Jurkat cells. Fluid Processing Apparatus (FPAs) were used in this study. A set of FPAs was prepared as ground controls (F_Ground) and fixated shortly after launching. The flight samples were flown on the Soyuz TMA-11 and fresh medium were added shortly after docking in the ISS. After 30 minutes, another set of FPAs was added fixative (F_T0) meanwhile another set were fixed after 72 hours incubation at 37C (F_T72). The number of cells increased from F_Ground to F_T0 and to F_T72. Post-flight analysis found that only expression of CD3, MRP1(ABCC1) and BCRP(ABCG2) showed significant changes. The flight samples showed a decrease in MRP1(ABCC1) expression at F_T72 compared to F_T0. Expression of BCRP was also significantly decreased in flight samples compared to F_Ground. qRT-PCR results showed that MRP1(ABCC1) mRNA expression complement with the protein expression but mRNA of BCRP(ABCG2) showed different expression from the protein expression. Microarray analysis showed that the voltage-gated K channel gene, Kv.1 was differentially expressed at different time points. We suggest that cellular membrane adapted to spaceflight by modulating the expression of membrane transporter proteins. These findings shed light to the important role of multidrug resistance proteins for drug efflux, biomolecule transport and pharmacokinetics as there is an increasing need to understand how medicine reacts in the human body under microgravity. Sue Mien Then et al. Asia Pac. J. Mol. Med. 2 binding-cassette (ABC) transporters and voltage-gated ion channels which are located at the cellular membrane are postulated to undergo remodeling under microgravity. A recent study on global gene and miRNA expression of proliferating human fibroblast cells after 3 days incubation revealed the upregulation of 2.3-fold of the P-gp gene expression between space flown cells and ground control cells. However, there were no significant difference when the same comparison were made between cells that have been in space for 14 days. The study concluded that spaceflight affect the cells at proliferating stage, however more studies are needed to validate the observation. (7) Another study has shed some clue to this, demonstrating reduced MRP2 (ABCC2) transport activity of estradiol 17-β-glucuronide (E17βG) in microgravity during parabolic flights (8). Studies have shown that microgravity altered the ion channels, such as porin in E. coli, which are located at the membrane layer of the cells (9). P-gp (ABCB1) and cytochrome P450 levels were altered in the kidney of murine model under simulated microgravity studies (10) while gene expression studies in microorganisms (Salmonella sp. and Candida sp.) found upregulation of certain ABC transporter genes during short term spaceflight mission (11, 12). Simulated microgravity has also been shown to cause differential activation of K+ channels in vascular smooth muscle cells (9, 13). However, how microgravity affects these MDR proteins and ion channels in human T-cell acute lymphoblastic leukemia (T-ALL) cell line, i. e. Jurkat cells have yet to be investigated. The Jurkat cells have been used extensively in many microgravity investigations including real space flight and simulated microgravity conditions (14-17). Previous studies have shown that the microtubules of Jurkat cells were altered in microgravity with increased condensation of nuclear DNA, which is one of the characteristics of apoptosis (14). Previous reports also showed that most of the cytoskeletal genes identified such as myosin light polypeptide (MYL5), plectin (PLEC), ankyrin, calponin, tropomodulin and dyactin were up-regulated in space-flown Jurkat (15). Spaceflight has also been shown to alter the human immune system with decreased numbers of Tlymphocytes and natural killer cells in post-flight conditions while there is reduced growth activation and decline in growth rate of the cells total population (16). The activity of natural killer cells were also shown to be reduced at post-flight (18). The intracellular distribution of protein kinase C (PKC) in human leukocytes varied in proportion to the applied g level which showed the cells’ ability for gravisensing (19) and cytoskeleton is sensitive to gravity by responding to the altered gravity conditions (20). Given that the location of the drug transporter proteins and ion channels are at the cell membrane, as well as reported reduction of intracellular ATP in simulated microgravity (21), we postulated that microgravity and any perturbation to the membrane structure during launching might result in changes to the expression of these drug transporter proteins and ion channels. The main objective of this study was to investigate the effects of spaceflight and microgravity on the gene expression, particularly the changes on the plasma membrane such as the MDR proteins and ion channels. The understanding of how spaceflight affects these transmembrane proteins, especially graviperception on the molecular level are crucial as we have yet to understand how cells perceive and adapt to spaceflight and microgravity. We determined the expression of the MDR proteins and their corresponding mRNA expression level as well as gene expression changes of ion channels when the Jurkat cells were exposed to the microgravity condition in the International Space Station (ISS) between different time points compared to the ground controls, namely pre-flight which is 56 hours prior to launching (F_Ground), during docking at ISS which was designated as 0 hour of spaceflight (F_T0) and 72 hours of spaceflight (F_T72). Materials and methods