Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

^a University of Alabama in Huntsville, Microgravity Biotechnology Laboratory, Huntsville, Alabama 35899, USA
^b INSERM U311, Establissement de Transfusion Sanguine de Strasbourg, 67065 Strasbourg Cedex, France
^c The Rockefeller University, Steinman-Cohn Laboratory New York, New York 10021, USA
^d Department of Pharmaceutics, University of Texas, Austin, Texas 78235, USA

	ABSTRACT

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Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post- flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.—Lewis, M. L., Reynolds, J. L., Cubano, L. A., Hatton, J. P., Lawless, B. D., Piepmeier, E. H. Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat). FASEB J. 12, 1007–1018 (1998)

Key Words: cytoskeleton • Fas/APO-1 • glucose metabolism • MTOC

	INTRODUCTION

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THE EFFECT OF spaceflight on cells is not clearly understood, although changes at the cellular level are well documented and reviewed (1). Some of these effects include reduced secretion of growth hormone (2, 3), blunted response to mitogen activation in T lymphocytes (4), and retarded osteoblast growth and an abnormal actin cytoskeleton (5). We hypothesized that spaceflight-induced alterations in the cytoskeleton may underlie some of these effects and that apoptosis may contribute to the blunted growth response of certain cell types during spaceflight. We assessed these hypotheses by flying cells of an actively growing human lymphoblastoid cell line, Jurkat, on the Space Shuttle.

The cytoskeleton is highly sensitive to changes in the gravity environment. Simulated low gravity (g)² in a fast-rotating clinostat enhanced EGF-induced, cytoskeleton-related cell rounding (6), rotation of Xenopus myocytes on a slow clinostat caused disorganization, condensation, and irregular arrangement of filaments (7), microtubule assembly was significantly different in the low g (30 s) and 2 g phases during parabolic flight on the KC-135 aircraft (8), and actin stress fibers in mouse osteoblasts flown on the Space Shuttle became coalesced (5). Our objective in examining cell growth responsiveness and cytoskeletal morphology in lymphocytes was to advance understanding of the effect of the spaceflight environment on cell growth and aging, manifested as programmed cell death.

This report is the first of which we are aware to document time-related changes in microtubules and the apparent return to normal morphology during spaceflight. A novel finding is the microgravity environment-associated, time-dependent release of the apoptosis-related Fas/APO-1 protein into the medium of space-flown cells. This information contributes significantly to the understanding of growth responsiveness and cell death during spaceflight and may help in predicting potential compromise to immune function in humans during missions of long duration. Results also contribute to the understanding of gravity effects and cellular response to environmental factors.

In most cells, shape is determined and maintained by cytoskeleton polymerization forces, weaker in some cell types than in others (9), and extension of microtubules to the cell membrane (10) to maintain mostly uniform cell surface tension. Interruption of membrane–cytoskeletal association induces shedding of cell surface receptors and blebbing of the membrane (11). In the absence of gravity, very subtle cell volume changes may result from hydrostatic pressure shifts (9), potentially causing disjunction between the membrane and critical cytoskeletal elements. Mechanical changes may be transduced into biochemical responses through the cytoskeletal scaffolding within the cell (12). If microtubules are disrupted, molecular transport by cytoskeletal elements would be affected and cell surface, receptor-dependent signal transduction reactions could not occur.

A number of reports describe the effect of altered gravity on signal transduction and growth regulation. Simulated microgravity in a fast rotating clinostat reduced the rate of transcription of the fos proto-oncogene in the human epidermoid carcinoma cell line, A431 (13). On the Soviet satellite Cosmos 2044, cellular interactions between T lymphocytes and monocytes resulted in normal interleukin 1 and 2 production. However, the activation of lymphocytes was dramatically inhibited when a phorbol ester, which directly activates protein kinase C (PKC), was used (14). This inhibition did not result from poor phorbol ester binding, but instead from blunted activation after binding. Also, the distribution of PKC isoforms in the cytosolic fraction of Jurkat and U937 cells was altered in microgravity compared to the 1 g onboard centrifuge and ground control (15).

A direct relationship between cytoskeletal integrity and apoptosis, programmed cell death, has been demonstrated. Inhibition of actin filament assembly (16) and disruption of microtubules (17) increase apoptosis whereas stabilization of the microtubule cytoskeleton inhibits the apoptotic process (18). Apoptosis is genetically controlled and is characterized by condensation and segmentation of the nucleus, condensation and fragmentation of the cytoplasm, membrane blebbing, cytoskeletal disruption, loss of mitochondrial function, and activation of endogenous proteases, leading to cell death (19). One of the most thoroughly studied programmed cell death factors is Fas. Survival of activated lymphocytes is mediated through cell surface receptors involving Fas and Fas ligand (Fas-L) (20). Human Fas belongs to the tumor necrosis factor receptor family and consists of 325 amino acids with a membrane-spanning region in the middle of the molecule. A cytoplasmic `death domain' of about 70 amino acids extending into the cytoplasm is required for transduction of the apoptotic signal (21). Fas is highly expressed in activated lymphocytes and lymphoblastoid cells. The Fas and APO-1 cell surface proteins, first characterized in 1989, are designated as Fas/APO-1 because molecular cloning studies have shown them to be identical (22). Fas and Fas-L-induced death in activated lymphocytes may be mediated by different mechanisms. Fas and Fas-L may be expressed on the same or different cells, or soluble Fas-L may be released into the medium and cause death of the same cell or another cell bearing a Fas receptor (21). Regulation of cell survival in activated lymphocytes is dynamic and not completely understood. Activated lymphocytes constitutively express Fas and intermittently express Fas-L on their surface (20). Together on the same cell, they may be able to induce auto-apoptosis by membrane folding. We selected Fas/APO-1 for evaluation of apoptosis in Jurkat cells because Fas is one of the most studied and best-defined factors promoting apoptosis.

To characterize the effects of spaceflight on the integrity of microtubules and to evaluate cell growth responsiveness and apoptosis, we exposed Jurkat cells to conditions of spaceflight on the Space Shuttle. Because our previous experiments showed that osteoblast growth is reduced in microgravity (23) and because of the well-documented findings that T lymphocyte response to mitogen stimulation is blunted during spaceflight (4), we investigated apoptosis as a potential contributor to the lack of increase in population size in space-flown cells. Jurkat cells, a human T lymphoblastoid cell line, were chosen for this investigation because they can be consistently growth stimulated by increasing serum concentration; they are similar to cells that are known to be affected by spaceflight (4), and have been used in previous spaceflight experiments (14, 15). Cells were growth stimulated and fixed with formalin while in microgravity. The onboard centrifuge provided an in-flight 1 g control in addition to the ground controls. Filtration of cells from medium at 4, 24, and 48 h after activation allowed us to evaluate time-related changes in the cells as well as the cell-free medium. We found that zero population growth was concomitant with collapsed microtubules, disorganized MTOCs, and DNA condensation characteristic of apoptosis. We conclude that the blunted response of certain cell types to growth stimulation during spaceflight is a result of cytoskeletal anomalies and increased apoptosis.

	MATERIALS AND METHODS

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Hardware
The Biorack hardware, manufactured by COMAT (Toulouse, France) and made available by the French Centre National d'Etudes Spatiales, consisted of polycarbonate cassettes with three sets of six wells machined into each to allow sample replication. The cassettes were housed in metal containers and were designed to withstand freezing at -20°C. Each sample set consisted of: 1) a cell culture chamber containing 500 µl of cell suspension; 2) a growth activator chamber with 125 µl of medium containing a high concentration of fetal bovine serum (FBS); 3) a 500 µl reservoir to receive conditioned medium filtered from cells at the fixation step. Chambers were interconnected by vanes that permitted transfer of solutions by a crew member who manually turned the vanes and pushed the manifold plunger. Fixative was injected manually into cell chambers with a modified commercial pipetor (Wheaton, Millville, N.J.). The Biorack facility provided an in-flight 1 g centrifuge control. Ground control hardware, which operated simultaneously, was identical except that the ground control centrifuge provided 1.4 g (the sum of 1 g and lowest centrifugation). The Biorack facility maintained temperature at 37°C during the experiment. All experiment handling was conducted in the Biorack glovebox. A more detailed description of the Biorack facility is presented elsewhere (24).

Cell culture preparations
The human lymphoblastoid cell line, Jurkat clone E6–1, was obtained from the American Type Culture Collection (Rockville, Md.) and certified free of mycoplasma contamination by polymerase chain reaction testing. Culture medium consisted of RPMI 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 10% heat-inactivated FBS (Summit Biotechnology, Santa Ana, Calif.), 2 mM glutamine, l mM sodium pyruvate, 1 ml/100 of 100X nonessential amino acids, and penicillin and streptomycin, 100 units and 100 µg/ml, respectively (Life Technologies/GIBCO BRL, Grand Island, N.Y.), and 12.5 mM HEPES Buffer (Sigma, St. Louis, Mo.). Cells were transported to the Launch Site Support Facility laboratory at Kennedy Space Center and maintained under optimal growth conditions prior to loading into the Biorack hardware.

Experiment details
In preparation for flight, the cells were suspended in medium containing 2% FBS. The Jurkat cells, medium containing FBS calculated to give a 10% final concentration when injected into cell wells, and 3% formalin (Tousimis, Rockville, Md.) in phosphate-buffered saline (PBS) were loaded into their respective chambers in the Biorack cassettes approximately 20 h before launch. Identical cassettes were set up for the concurrent ground experiment. Loaded cassettes were maintained in the laboratory until installed at launch minus 16 h in the Shuttle middeck or Biorack ground control facility. Twenty hours after launch, the experiment was activated and one set of cassettes was maintained static while an identical set was placed in the onboard 1 g in-flight centrifuge. Identical sets for the ground control were held static or placed in the 1.4 g centrifuge. Cell growth was stimulated at launch plus 20 h by increasing serum concentration to 10% and temperature to 37°C. Upon experiment initiation, each cell culture chamber contained approximately 770,000 cells suspended in 625 µl of medium. At 4 and 48 h after activation, cells were filtered from culture medium, fixed with formalin, and stored at 5°C. Cell-free medium in reservoir wells from a second set of cassettes was stored at -20°C for analysis of glucose and Fas/APO-1. Samples remained at these temperatures for the duration of the 10-day mission and until processed in the laboratory after landing.

Because of concerns for crew safety, crew time limitations, and launch schedules, unique constraints (which must be taken into account when interpreting results) are imposed on Space Shuttle flight experiments. The fact that the experiment had to be installed in the Shuttle 16 h before launch required that cells be loaded into the hardware well in advance of that time. Once loaded, cells were held for about 20 h before launch in 2% serum medium at 20°C. Crew time limitations prevented early attention to the experiment in space, and so the test was initiated 20 h after launch. Thus, the cells remained in the hardware under growth-limiting conditions for approximately 40 h before activation. All flown cells were subjected to the same launch stresses of vibration, acceleration, and 20 h in microgravity before the 1 g in-flight controls were placed on the centrifuge. Ground controls replicated the flight experiment, except for launch stresses and the 20 h in microgravity, before initiation of the experiment. Comparisons between static and the 1 g in-flight centrifuge provide information on microgravity-related effects; comparisons between flight and ground are valid because they provide insight into understanding the responses of cells to the combined conditions of spaceflight, including launch stresses, and the microgravity environment.

Postflight analyses
Cell count and glucose metabolism
Formalin-fixed cells from each of the six replicate wells of a cassette fixed at 4 and 48 h were counted with a hemacytometer. Glucose concentration in cell-free medium was evaluated with a Glucose Analyzer 2 (Beckman Instruments, Brea, Calif.), using a Beckman glucose analysis kit. Concentrations were assessed from the mean of three glucose analyzer readings of each of the six replicate wells for each cassette.

Cytoskeletal evaluations
The morphology of the microtubule cytoskeleton was evaluated by indirect immunofluorescence. Formalin-fixed cells were washed twice in PBS, centrifuged onto air-dried albumin-coated coverslips, and permeabilized with a solution of 0.5% Triton-X 100 (Boehringer-Mannheim, Indianapolis, Ind.) in PBS. Coverslips were dipped in polyethylene glycol-stabilizing buffer and incubated with anti-tubulin antibody (Amersham, Arlington Heights, Ill.) for 1 h at 37°C. They were then rinsed and incubated for 1 h with anti-mouse FITC-conjugated immunoglobulin G whole antibody (Amersham). Coverslips were rinsed again in PBS, mounted on a glass slide, and sealed with nail polish. Microtubule morphology was visualized using a Zeiss laser scanning confocal microscopy system (excitation at 488 nm, emission at 515–540 nm) kindly provided by Dr. B. R. Brinkley, Baylor University, Houston, Texas. Propidium iodide staining was observed concurrently (excitation at 488 nm, and emission at 590–610 nm).

Flow cytometry
Formalin-fixed cells were washed in PBS and treated with 70% ethanol prior to staining with propidium iodide and analysis by flow cytometry. Cells were incubated with RNase (1 mg/ml in PBS pH 7.0) for 30 min at room temperature. The cells were then incubated with 0.1 mg/ml propidium iodide in 10 mM Tris pH 7.6, 150 mM NaCl for 30 min at room temperature in the dark. The cells were washed three times with PBS. Fluorescence and light scattering were measured on a FACSCALIBER flow cytometer (Becton-Dickinson, San Jose, Calif.). The excitation wavelength band was 488 nm and fluorescence was detected at 585 nm (bandwidth 42 nm). Low- and high-angle light scattering were measured by separate detectors. The measurements were gated by both low- and high-angle light scattering. The thresholds were set to eliminate cellular debris and doublets. For standardization of measurements, Calibrite (Becton-Dickinson, San Jose, Calif.) fluorescent beads were run at the beginning and end of each experiment. DNA content was determined for each cell as measured by fluorescence of propidium iodide. Differences in cell cycle between the treatment groups were detected by analysis of cell population DNA content using Modfit software.

Evaluation of apoptosis
For visualization of apoptotic nuclei, formalin-fixed cells were spun onto bovine serum albumin-coated coverslips and permeabilized with Triton X 100 (Boehringer, Indianapolis, Ind.) for 3 min. Cells were then incubated at room temperature with 25 µg/ml of Hoechst 33258 (Molecular Probes, Eugene, Oreg.) for 5 min and the bright blue-stained nuclei were counted using a Nikon Labophot microscope equipped with epifluorescence and a 364 nm filter. The percent of cells with condensed nuclear DNA characteristic of apoptosis was determined from counts of 300 cells dispersed in different fields on two coverslip preparations for each gravity condition.

Fas/APO-1 protein analysis
Cell-free culture medium, collected at 4, 24, and 48 h after activation of the cells, was tested for soluble Fas/APO-1 (sFas/APO-1) protein by an enzyme-linked immunosorbent assay (ELISA) according to methodology and reagents supplied with kits purchased from Oncogene Sciences (Uniondale, N.Y.). The concentration (in units/ml) was determined by comparison of the optical density of samples and the kit standard diluted and read in wells of a 96-well plate on a BioTek EL 340 microplate reader.

Statistics
Statistical analyses were performed using an InStat 1.14 program for the Macintosh computer. One-way analysis of variance and unpaired two-tailed t tests were applied to obtain the mean, standard error of the mean, and P values for replicate samples for each gravity condition. Statistical significance was considered to be P 0.05.

	RESULTS

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Effects of spaceflight on microtubule morphology
The microtubule cytoskeleton of Jurkat cells fixed 4 h after activation in microgravity was significantly altered in both static and in-flight 1 g controls. This spaceflight effect is not unexpected since all flown cassettes were subjected to the same launch vibration and acceleration forces before being placed on the in-flight 1 g centrifuge 20 h after launch. Filaments were shortened, coalesced, lacked normal branching at the cell membrane, and MTOCs were disrupted ( Fig. 1B). In the ground control, discrete microtubules radiated from well-organized MTOCs and branched at the cell membrane ( Fig. 1A). Forty-eight hours later, MTOCs and microtubules in flown 1 g centrifuged ( Fig. 2E, F) and static ( Fig. 2C, D) cells appeared to be reorganized. Appearance of the cytoskeletal complex was not markedly different from that of cells grown in the laboratory and fixed and stained immediately ( Fig. 2A, B), except that the immunofluorescence images were less sharp in flown cells due to the length of time the cells remained in fixative before processing for microscopy after the mission.

View larger version (72K): [in this window] [in a new window]   Figure 1. Laser scanning confocal microscopy of Jurkat cells stained to show the microtubule cytoskeleton in cells fixed 4 h after growth stimulation in ground control (A) and microgravity (B). In flown cells, the microtubule filaments extended from poorly defined organizing centers (MTOCs) and were coalesced and shortened. In comparable ground controls, microtubules radiated in discrete filaments from organized MTOCs and branched toward the cell membrane. Bars represent 5 µm. (Laser scanning confocal microscopy was kindly provided by Dr. B. R. Brinkley, Integrated Microscopy Laboratory, Baylor College of Medicine, Houston, Texas.)

View larger version (58K): [in this window] [in a new window]   Figure 2. Laser scanning confocal microscopy of cells showing two confocal planes 1 µm apart for ground control cells grown in the laboratory (A, B) and flown cells fixed 48 h after growth stimulation in microgravity, and either held static in microgravity (C, D) or maintained on the 1 g in-flight centrifuge (E, F). Microtubules and MTOCs in flown cells fixed at 48 h appeared to be well organized compared to flown cells fixed at 4 h (Fig. 1B). At 48 h, cells appeared to recover cytoskeletal integrity, and filament extension and branching was visible. In comparison to nonflown cells grown in the laboratory (A, B), the organization of the cytoskeletal complex in flown cells was less distinct. This is attributed to the time that the cells remained in the fixative after fixation in space and before processing for microscopy in the laboratory after landing. Bars represent 5 µm. (Laser scanning confocal microscopy was kindly provided by Dr. B. R. Brinkley, Integrated Microscopy Laboratory, Baylor College of Medicine, Houston, Texas).

Apoptosis
Morphological detection
DNA condensation characteristic of apoptosis was evaluated in Hoechst-stained cells fixed 4 ( Fig. 3) and 48 ( Fig. 4) h after growth stimulation. The number of cells with apoptotic nuclear morphology, expressed as percent of approximately 300 cells counted for each gravity condition, is shown in Table 1. Centrifuged 4 h cultures appeared to have more apoptotic cells in both flight (5%) and ground (10%) compared to static cultures. We attribute this to handling during placement of the cultures on the centrifuges. We found a significant difference between flown and ground cultures at the 4 h time point. By averaging the number of apoptotic cells in the static and centrifuged samples, we show that flown cultures had significantly more apoptotic cells (30.5%) as ground cultures (17%). By 48 h, the frequency of morphologically detectable apoptosis in flight static cells was essentially the same as the 1 g centrifuge and ground controls.

View larger version (66K): [in this window] [in a new window]   Figure 3. Appearance of nuclei in cells fixed 4 h after growth stimulation in microgravity. Although there was no increase in the number of cells during spaceflight, mitotic figures (m) in Hoechst-stained nuclei preparations indicate that some cells were actively dividing. Nuclear fragmentation characteristic of apoptosis (a) was also evident.

View larger version (68K): [in this window] [in a new window]   Figure 4. Appearance of nuclei in space-flown Jurkat cells. Laser scanning confocal microscopy, showing two planes 1 µm apart, of nuclei in cells cultured in the laboratory (A, B) and in microgravity 48 h after growth stimulation: in-flight static culture (C, D) and 1 g centrifuged in-flight control (E, F). Nuclei in static flown and 1 g centrifuged cells were smaller (P=0.0110 and 0.0569, respectively) than in ground cultured cells. Apoptotic nuclei (a) were evident in flown cells.

Fas/APO-1 protein
To further characterize spaceflight-related apoptosis, we tested cell culture medium for Fas/APO-1 protein at 4, 24, and 48 h ( Fig. 5). We found a dramatic effect on the dynamics of Fas/APO-1 release by static (but not 1 g centrifuged) cultures in microgravity. At 4 h, flown cultures had approximately 0.1 unit/ml. Between 4 and 24 h, the concentration in static flown cultures increased by approximately 15-fold; by 48 h, the concentration was almost 65 times greater than at 4 h. This time-dependent increase in Fas/APO-1 concentration occurred only in the cultures held static in microgravity. Centrifugation in microgravity significantly reduced Fas/APO-1 levels. Centrifuged cultures had approximately threefold less Fas/APO-1 at 24 h (P=0.0005) and 13-fold less at 48 h (P=0.0011) than static microgravity cultures. Fas/APO-1 increased between 24 and 48 h in the 1 g centrifuged microgravity samples, but levels were less than l unit/ml and differences were not statistically significant (P=0.6660). The standard errors of the means of the in-flight 1 g centrifuged cells at 48 h and the 1 g ground controls at 4 h overlapped; thus, no statistically significant difference (P=0.8188) was demonstrated. Clearly, expression of Fas/APO-1 was up-regulated in the microgravity environment. All ground samples were negative for Fas/APO-1.

View larger version (18K): [in this window] [in a new window]   Figure 5. Time-dependent increase in Fas/APO-1 protein during spaceflight. Cell-free medium samples were tested for Fas/APO-1 protein using an ELISA kit, as described in Materials and Methods. Bars represent the mean and SEM of four to six wells for each gravity condition (µg = flight static; 1gC = in-flight 1 g centrifuge; 1g = ground static; 1.4gC = ground centrifuge). No Fas/APO-1 was found in space-flown samples at 4 h. A time-dependent increase in Fas/APO-1 concentration in static microgravity samples, but not the centrifuged in-flight 1 g controls, shows that production of this cell death-related factor was up-regulated in the microgravity environment. The Fas/APO-1 concentration in 1 g in-flight centrifuged cultures was dramatically reduced (by approximately 16-fold) at 24 (P=0.0005) and 48 h (P=0.0011) compared to static flown cultures. Ground controls were consistently negative, and statistical analyses (In-Stat 1.14 Macintosh computer program) showed no statistically significant difference between in-flight 1 g centrifuged and the ground-based 1 g cultures (P=0.8188).

Cell growth
Centrifugation at 1 g during spaceflight had no significant effect at the 95% confidence level (P=0.2345) on cell growth by 48 h ( Fig. 6). The low cell count for the 4 h microgravity static cassette resulted from a hardware anomaly during the fixation step. This had no effect on the morphology or growth potential of the cells before this step. The number of cells in flown populations at 48 h after growth stimulation in microgravity did not increase over input number (P>0.05), yet comparable ground controls increased by 1.5-fold during the same period (P=0.0001). Although the flight populations did not increase in number, mitotic cells were present ( Fig. 3), indicating that some cells in the population were actively dividing in microgravity. There was no difference in cell count between the number of cells loaded into the cassettes and the counts 4 h after activation. Thus, flight and ground cells both maintained the desired static growth state before initiation of the experiment 40 h after loading of the cassettes.

View larger version (32K): [in this window] [in a new window]   Figure 6. Cell growth. A 0.5 ml volume of cell suspension containing 1.5 million cells/ml was loaded into Biorack cassette wells (770,000 cells/well) at approximately 20 h before launch (0 h). Forty hours later, the experiment was activated as described in Materials and Methods. One set of cassettes was held static; an identical set was placed on the in-flight 1 g control centrifuge in microgravity and the 1.4 g centrifuge for the ground test. Bars show the mean and SEM of hemacytometer counts of cells recovered from six replicate wells of each cassette under the four gravity conditions (µg = flight static; 1gC = in-flight 1 g centrifuge; 1g = ground static; 1.4gC = ground centrifuge) at 4 and 48 h after activation of the experiment. There was no significant (P>0.05) increase in Jurkat cell populations between 0 and 48 h in microgravity. Ground control cells increased by approximately 1.5-fold during the same 48 h period (P=0.0001). Centrifugation had no effect on cell number in either flown or ground-based cultures. (All flown cultures experienced the same launch stresses and 20 h in microgravity before the control cassettes were placed on the in-flight 1 g centrifuge). *Because of a hardware anomaly during fixation, some cells were lost.

Glucose metabolism
Four hours after activation, there was no significant difference in glucose use regardless of gravity condition (data not shown). During prelaunch and before activation of the experiment 20 h after launch, cells under all gravity conditions used approximately 40 mg/dl of the available 187 mg/dl glucose in the medium, indicating the desired retardation in metabolism under conditions of low serum and low temperature before launch and growth stimulation of the cells in orbit. Although flown cells did not increase in number, they were metabolically active. By 48 h, both in-flight 1 g controls and static cultures had used approximately 60% of the glucose available in the medium. Expressed as glucose used per 100,000 cells, there was no difference between static and 1 g controls in microgravity ( Fig. 7). When compared to ground controls, however, we found a very significant increase in the amount of glucose used by flown cultures. By 48 h, flown cells used approximately 1.6-fold more glucose than the ground controls in both static (P=0.0046) and centrifuged (P=0.0003) cultures.

View larger version (24K): [in this window] [in a new window]   Figure 7. Glucose consumption 48 h after growth stimulation. Glucose in cell-free medium was assayed as described in Materials and Methods. The amount of glucose consumed was calculated by subtracting the glucose measured in cell-free medium for each well from the glucose concentration in unused culture medium, divided by the number of cells counted in the corresponding well, and expressed as milligrams of glucose used per 100,000 cells. Bars represent the mean and SEM of six replicate cultures for each gravity condition (µg = flight static; 1gC = in-flight 1 g centrifuge; 1g = ground static; 1.4gC = ground centrifuge). Cells in microgravity used approximately 1.6 times more glucose (P=0.0046 for static and P=0.0003 for centrifuged) than comparable ground controls. Centrifugation had no apparent effect on glucose use in either flight or ground cultures.

Cell cycle
Because flown cells did not increase in number, we used flow cytometry to evaluate the possibility of cell cycle arrest. Flown cells appeared to be initially arrested in G2M based on the significantly higher number at 4 h compared to ground static cultures (P=0.04) and in-flight 1 g centrifuged compared to ground static (P=0.0011) ( Fig. 8A). The difference in G2M between centrifuged compared to static microgravity cultures was not significant (P=0.07). Though more in-flight centrifuged cells appeared to be in G2M than ground centrifuged cells, the difference was not significant (P=0.07). At 4 h, centrifugation appeared to promote both flight and ground cells into G2M ( Fig. 8A) and S ( Fig. 8B), with a corresponding decrease in G1 ( Fig. 8C). The differences between static and centrifuged cultures were significant for ground controls in all phases, but were significant for flown cells only in G1 (P=0.04). There were no significant differences in G1 between flight and ground controls ( Fig. 6C). Together, these findings indicate that DNA replication was occurring in the flight cells but that cells at 4 h were not progressing through G2M. By 48 h, there were no significant differences in cell cycle phase between static and centrifuged cultures for either flown or ground controls, and cells were distributed in all cell cycle phases.

View larger version (36K): [in this window] [in a new window]   Figure 8. Cell cycle distribution. Formalin-fixed cells were stained with propidium iodide and evaluated by flow cytometry, as described in Materials and Methods. For each sample, approximately 10,000 individual cells were analyzed in three separate flow cytometry runs. The percentage of cells in the G2M phase (A), S phase (B), and G1 phase (C) is represented. At 4 h after growth stimulation in microgravity, cells appeared to be blocked in G2M as indicated by comparing cells in G2M in flight static with ground static cultures (P=0.04), and flown centrifuged with ground static cultures (P=0.0011). There was no significant difference (P=0.07) between flight static and the 1 g centrifuged cells in G2M, though there appeared to be more cells in G2M in centrifuged flown populations. Centrifugation appeared to promote active cycling (increase in G2M and S and corresponding decrease in G1) in both flight and ground cultures. Differences at 4 h between static vs. centrifuged ground cultures was significant in all phases. However, the difference between static and in-flight centrifuged cells for the flown cells was significant only for G1. The presence of mitotic figures (Fig. 3) and cell cycle data indicate that spindle formation and DNA synthesis in some cells may not be altered during spaceflight, but that cytoskeletal changes associated with cell division are affected, as the flown populations did not increase in number. By 48 h, there were no significant differences in cell cycle phase among the gravity conditions, and cells were found in all phases.

	DISCUSSION

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We flew cells of the Jurkat human lymphoblastoid cell line on the Space Shuttle to characterize spaceflight effects on the microtubule cytoskeleton, cell growth, metabolism, and apoptosis. A significant and novel finding was the time-dependent increase in the apoptosis-related factor, Fas/APO-1, in culture medium of flown cells ( Fig. 5). This appears to be a microgravity environment-related response, since in-flight static cultures had significantly more Fas/APO-1 than the in-flight 1 g centrifuged controls. The comparable ground controls were negative for Fas/APO-1 and not statistically different from 1 g centrifuged flown cells (P>0.8). The number of cells with DNA condensation characteristic of apoptosis was increased in flown cells early in the experiment. These data support our hypothesis that apoptosis is a mechanism limiting lymphocyte population increase in the microgravity environment.

In interpreting results of spaceflight experiments, it is important to distinguish between the stresses associated with spaceflight, including launch vibration, experiment-induced stresses, and effects of the microgravity environment per se. We address these factors as they relate to the responses of the Jurkat cells described in this report.

All flown cells were handled identically preflight. They all experienced the same launch stresses and the 20 h period in microgravity before the 1 g inflight control cassettes were placed in the Biorack centrifuge and the experiment was activated. This may clarify why no dramatic differences were seen in microtubule morphology, number of apoptotic cells, cell count, and glucose use between the in-flight static and the in-flight 1 g centrifuged cells. If responses to the microgravity environment occur immediately, then placing the cultures on the in-flight 1 g centrifuge 20 h later would not prevent, and may not reverse, these effects. Microtubule disorganization and apoptotic nuclear morphology at 4 h are a likely result of the stresses associated with launch. We are currently investigating the effects of simulated launch stresses on Jurkat cells. The number of apoptotic cells at 4 h was greater in the in-flight 1 g centrifuge and 1.4 g ground controls than in static cultures ( Table 1). This is attributed to handling during placement of the cells on the centrifuge and the start/stops of the centrifuge when cassettes were removed for sampling.

Experiment-related stresses include length of time the cells were maintained in the hardware after loading and before initiation of the experiment 20 h into the mission, preactivation temperature, and low serum content (2%) in the medium. We loaded cells into the Biorack cassettes under growth-limiting conditions to prevent cell overgrowth and nutrient and oxygen depletion before activation of the experiment in orbit. We demonstrated in our previous ground-based studies that cells maintained at 20°C in medium containing 2% serum for as long as 48 h resumed growth after serum concentration was increased to 10% and the temperature was raised to 37°C. We selected the optimal concentration of cells (~770,000 cells/well) to allow one to two doublings over 48 h and to provide enough cells for postflight evaluations. In ground-based studies comparing cells in flight hardware to control cultures in flasks, we found no differences in cytoskeletal morphology or glucose use rates, and the culture medium tested negative for Fas/APO-1 protein. Ground-based testing also confirmed good preservation of microtubules with 3% formalin in PBS.

No generalized quantitative trends were observed for the gravity treatment groups, but there were trends specific to the effect measured. For example, for Fas/APO-1 levels in culture medium at 24 h (48 h samples showed the same trend): microgravity static > microgravity 1 g centrifuge (P=0.0005) > ground. All ground samples were negative. With respect to morphologically detected apoptotic cells at 4 h ( Table 1), the trend was: microgravity 1 g centrifuge > microgravity static > ground 1.4 g centrifuge > ground static. At 48 h, the number of apoptotic cells was about the same among treatments. There was no trend for cell growth and glucose consumption, and centrifugation made no difference in either case. Cells in microgravity used approximately 1.6-fold more glucose than ground cultures (P=0.0046 static and P=0.0003 centrifuged) and did not increase in number. For cell cycle distribution, expressed as the percentage of cells in S plus G2M, the trend, though not statistically significant (significance is defined as P<0.05), at 4 h was: microgravity 1 g centrifuge > ground 1.4 g centrifuge (P>0.5) > microgravity static (P>0.5) > ground static (P=0.04). There was a significant difference between microgravity static and ground static (P=0.04), but differences were not significant for other gravity conditions. Flown cells appeared to be blocked in G2M at 4 h, but by 48 h there was no evidence of cell cycle arrest.

Disruption of cytoskeletal integrity has long been suggested as a gravity-sensing mechanism in single cells (7, 25). Flow cytometry data showed that DNA replication was occurring in-flight, but cells sampled 4 h after activation were not progressing through G2M ( Fig. 8), possibly due to the anomalies observed in the cytoskeleton. The presence of mitotic figures at 4 h ( Fig. 3) indicates that spindle formation in some cells may not be altered, but rather that cytoskeletal changes associated with cell division are altered as a result of spaceflight. This adds credence to our hypothesis that the blunted growth response observed in space-flown cells results, at least in part, from cytoskeletal anomalies. By 48 h there were no statistical differences in cell cycle phase between flight and ground controls. Our finding that cytoskeletal filaments in the flown Jurkat cells were coalesced and shortened ( Fig. 1B) is consistent with abnormalities in actin stress fibers described by Hughes-Fulford and Lewis (23) for static cultures of anchorage-dependent mouse osteoblasts flown on the Shuttle. Piepmeier et al. (26) also report lack of cytoskeletal polymerization and amorphic shape of microtubules in space-flown HL60 cells. We also found that MTOCs were significantly degraded. These anomalies were apparent in cells fixed 4 h after activation and appear to result from launch vibration stresses. However, the microgravity environment may also play a role and cannot be ruled out.

After 48 h in microgravity, cells appeared to reorganize their cytoskeleton; cell number did not increase and the level of Fas/APO-1 in the culture medium and glucose use increased significantly. Disruption of the cytoskeleton can have a profound effect on signal transduction and cell growth since PKC or one of its downstream targets (13) appears to be involved in sensing gravity changes in mammalian cells (14, 15). During signal transduction, PKC is associated with the cytoskeleton (27–29). In cardiac myocytes, an isozyme of PKC is translocated on actin stress fibers, and molecules required for binding and activation of PKC are associated with these elements (28). If we assume that other structural components such as actin, in addition to the microtubules we report, would also be deranged in our flown Jurkat cells, then it is reasonable to expect that any downstream function in the signal transduction cascade would be compromised and lead to altered growth response. An intact cytoskeleton is necessary for signal transduction, and membrane–cytoskeletal interactions are involved in transduction of second messengers by signal amplification (30).

We do not believe that the observed zero population growth resulted from the indirect effects of nutrient depletion or spent metabolite build-up near the cells due to lack of convective mixing in microgravity. Indirect effects of microgravity, in contrast to direct effects due to weight of the cell, result from external fluid environment and include diffusion of nutrients into and metabolites away from the cell's microenvironment (31). Metabolite mixing in the culture medium was facilitated in-flight when the 1 g centrifuge was stopped and started a number of times during sampling, yet cells did not increase in number and glucose use was the same in flown static and the 1 g in-flight controls. In addition, Brownian motion and diffusion, unaffected in microgravity (31), would induce some mixing.

There was a significant 1.6-fold greater use of glucose (P<0.005) by the flown vs. ground cultures at 48 h. This effect was not due to experimental conditions because the terrestrial controls were subjected to the same hardware, temperature, medium serum content, and time line as the flight experiment. Hatton et al. also found that glucose utilization in Jurkat cells flown on STS-56 was approximately 1.6-fold higher than the 1 g controls (J. P. Hatton, unpublished results). Conversely, Hughes-Fulford and Lewis (23) reported reduced glucose use attributable to decreased cell number in flown osteoblasts; thus, metabolic responsiveness appears to be cell type specific.

The design of the experiment, based on space available to accommodate multiple cassettes, did not allow us to directly measure cell viability by incorporation of tritiated thymidine or BrdUr. It is reasonable to assume that a substantial segment of the cell population was viable based on significant glucose consumption, presence of mitotic cells, absence of necrotic or swollen cells and debris, and cell cycle traverse shown by flow cytometry. This led us to evaluate apoptosis as a mechanism for control of population density during spaceflight. Necrotic cell death caused by mechanical damage is characterized by cell swelling and lysis, whereas apoptotic cell death is programmed and under genetic control and is typified by cell shrinking, membrane blebbing, nuclear condensation, and breakdown of the cells into smaller membrane-bound fragments or apoptotic bodies (19). Microscopic examination did not reveal swollen cells nor did we see cellular fragments characteristic of necrotic cells. Instead, we observed apoptotic bodies ( Fig. 3and Fig. 4C–F) and a time-dependent increase in Fas/APO-1 protein in the culture medium ( Fig. 5). The stresses associated with launch and maintenance of cells in low serum medium at 20°C for 20 h prelaunch and 20 h postlaunch before activation of the experiment in microgravity could have predisposed flown cells to apoptosis. These prelaunch and launch conditions may be more important than microgravity in inducing apoptosis early in the flight. After 48 h, the number of apoptotic cells in the flight groups decreased to 12% (static) and 15% (1g centrifuge), values comparable to the ground controls. This and the apparent return of normal cytoskeletal morphology (Fig 2C–F), significant glucose metabolism ( Fig. 7), the presence of mitotic figures ( Fig. 3), and flow cytometry data ( Fig. 8) suggest that a part of the population was surviving and some cells were actively dividing in the microgravity environment. Yet there was no increase in cell number in the flown populations by 48 h, indicating that microgravity per se or a preoccurring event influenced cell growth and death dynamics. We observed a significant time-dependent increase in cell death-related Fas/APO-1 protein in the medium of flown cells at 24 and 48 h, adding credence to the hypothesis that apoptosis is a mechanism by which cells regulate response to spaceflight.

Whether the concomitant increase in Fas/APO-1 protein in medium of flown Jurkat cells and the observed cytoskeletal disorganization are related is not clear; however, this combination may contribute to the limited cell growth during spaceflight previously reported by others (4, 23). In our experiments, Fas/APO-1 protein in culture medium increased in a time-dependent manner. This was not due to overcrowding, since the ground control had 1.5-fold more cells and reached growth-limiting density, yet without release of Fas/APO protein. Nor was an increase in Fas/APO-1 in flown cells a consequence of nutrient depletion, since glucose concentrations in the medium at 48 h were adequate to support cell growth.

The time-dependent increase in Fas/APO-1 appears to be a response to the microgravity environment per se. Whether this is directly microgravity related or a residual effect of launch stresses in unclear. Fas/APO-1 is a likely inducer of apoptosis in the flown Jurkat cells because actively growing lymphocytes are growth regulated via the Fas and Fas-L mechanism. Under normal conditions in the body, the immune system must balance proliferation with programmed cell death, and in lymphocytes there is a relationship between apoptosis and cell cycle that involves PKC (32). The survival of actively growing lymphocytes is controlled by cell surface receptors that specifically induce apoptosis. Interruption of membrane–cytoskeletal association causes shedding of cell surface receptors and blebbing of certain areas on the membrane (11). A member of the tumor necrosis factor family, Fas interacts with Fas ligand to specifically induce cell death (19). Jurkat cells are actively proliferating (malignant) lymphocytes and, as such, would be sensitive to Fas-dependent cell death regulation. This is the first report, to our knowledge, of a time-dependent increase in the Fas death factor in space-flown lymphocytes.

The role of soluble Fas (sFas), as measured in our samples, has not been clearly defined. Cheng et al. (33) reported an elevated level of sFas in the serum of some patients with systemic lupus erythematosus. He suggests that sFas is produced by a Fas mRNA generated by alternative splicing and that the encoded sFas lacks the membrane domain. Thus, sFas theoretically could be an inhibitor of Fas-mediated apoptosis by competitive binding to Fas-L. If sFas blocks apoptosis, then our flown Jurkat cells may have begun to recover at 24 and 48 h from the level of apoptosis observed in the 4 h samples. This apparent adjustment to the microgravity environment is supported by glucose use ( Fig. 7), DNA synthesis ( Fig. 8), cytoskeletal reorganization ( Fig. 2C–F), and the decrease in number of apoptotic cells found in the flight samples at 48 h ( Table 1). If this model is valid and sFas inhibits Fas-mediated apoptosis, then given time in microgravity to overcome the initial lag in growth in the first few days and with hardware designed for perfusion to resupply spent nutrients, some populations may resume growth in microgravity.

Regulation of programmed cell death in T lymphocytes is mediated by a number of interacting factors. In preliminary tests, we found no increase in the apoptosis inhibiting factor, Bcl-2, in medium of flown cells (data not shown). Bcl-2 does not appear to be associated with apoptosis in the Jurkat cells in this study. Apoptosis as a mechanism regulating population dynamics during spaceflight is not understood, and unraveling its complexity will provide direction to future spaceflight research.

	ACKNOWLEDGMENTS

 
The authors express appreciation to the following institutions and individuals: the European Space Agency Biorack Team for overall management of the STS-76 flight experiment; Lockheed Martin Engineering and Sciences, Moffett Field, California, for mission management support; COMAT, Inc., Toulouse, France, and the French Centre National de'Etudes Spatiales for use of the Biorack hardware; the crew of STS-76 for in-flight support; and C. A. Yancey and K. L. Murphy for technical and administrative assistance thoughout the experiment. This research was supported by NASA Grant NAG2–985.

	FOOTNOTES

 
¹ Correspondence: Wilson Hall, Rm. 360, University of Alabama in Huntsville, Huntsville, AL 35899, USA. E-mail: lewisml{at}email.uah.edu

² Abbreviations: MTOC, microtubule organizing centers; PBS, phosphate-buffered saline; PKC, protein kinase C; FITC, fluorescein isothiocyanate; BrdUr, 5-bromo-2-deoxyuridine; ELISA, enzyme-linked immunosorbent assay; sFas, soluble Fas; FBS, fetal bovine serum; Fas-L, Fas ligand; g, gravity.

Received for publication September 19, 1997. Accepted for publication February 25, 1998.

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