* Nephrology Section, Department of Medicine, Tulane University Medical Center, and Tulane Environmental Astrobiology Center, New Orleans, Louisiana, and
Department of Biological Sciences and Microgravity Biotechnology Laboratory, University of Alabama, Huntsville
Correspondence: Marian L. Lewis, Ph.D., Wilson Hall, Room 360, University of Alabama at Huntsville, Huntsville, AL 35899. E-mail: lewisml{at}email.uah.edu
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Key Words: microgravity • gene expression • molecular chaperones • vibration • lymphocytes.
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We undertook the present ground-based study to determine whether a stress response is triggered by space shuttle launch vibration. Cells, when confronted with environmental stresses, exhibit a highly conserved [5 ] protection mechanism that results in increased tolerance to the given stress [6 ]. One of the best-characterized components of stress responses is heat shock proteins, also know as molecular chaperones, which assist in cell survival [7 , 8 ]. The production of heat shock proteins is associated with changes in gene expression, including the induction of genes that express the heat shock proteins and the repression of genes being expressed prior to the response [9 ]. Protein synthesis is also altered in the cells as features of heat shock protein mRNAs are recognized and given priority for the synthesis of the heat shock proteins [10 ]. Heat shock protein transcription is under the control of heat-inducible promoters [11 ] which contain heat shock elements at which heat shock factors bind [12 ]. Heat shock promoters are activated not only by heat stress but also by other events not limited to exposure to cytotoxic chemicals, glucose starvation, and radiation exposure. These stresses have the common result of increasing the number of partially denatured or improperly folded proteins. This triggers the production of heat shock proteins, which the cells use to degrade [13 ] or capture [14 ] the misfolded proteins and correctly fold them, which in turn provides the increased tolerance to the stress and, most importantly, promotes cell survival.
Although considerable work has gone into determining the mechanisms for the space-related immune cell changes, these mechanisms are still largely unknown. Among the most significant changes seen in space-flown mammalian cells are reduced growth activation and a decline in the growth rate in the total population [1 3 , 15 , 16 ]. Other changes include chromosomal aberrations [17 ], changes in cytokine production [2 ], and increased apoptosis [3 ]. In this study, we evaluated the heat shock protein mRNA in flown lymphocytes and compared the results with those for cells exposed to simulated shuttle launch vibration as a means to understand the contribution of launch stress on cell growth and expression of stress genes.
During the launch phase of flight on the space shuttle, cells are subjected to considerable vibrational stress. Therefore, we wanted to determine whether this stress is sufficient to induce a measurable response. We evaluated heat shock protein mRNA in Jurkat cells grown in a reduced-serum medium and cells exposed to vibration characteristic of a typical space shuttle launch. The results of these experiments provided a baseline for comparison of cells flown in space with those subjected to some of the stresses of spaceflight in ground-based tests. Prior reports have indicated an increase or decrease in expression of certain genes in cells exposed to vibration [18 ] and acceleration [4 , 19 ] simulating space shuttle launches. Our results expand the database that can be used as a baseline in differentiating between the effects due to stresses of spaceflight and the effects of microgravity per se on cells.
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Flight hardware
Bioprocessing modules (BPMs) were used for the STS-95
spaceflight experiment (Fig. 1
). This hardware consists of four syringes interconnected by tubing
via a four-way valve with filters (not shown) placed between the valve
and the syringe to separate cells and culture medium at the extraction
step. This simple, manually operated hardware allows for large volumes
of cells (5 mL) to be flown. Duplicate cultures of cells in syringes A
and C (Fig. 1)
were growth stimulated in microgravity when an astronaut
turned the valve to connect syringes A and B or syringes C and B. When
the B syringe plunger was pressed, the activator (medium with 22% FBS)
was injected into syringes A and C. After 24 h, the cells in
syringes A and C, which were equipped with a 0.40-µm-pore-size HTTP
Isopore membrane (Millipore, Bedford, MA), were fixed by pushing the
plunger, which delivered the medium from syringes A and C into B and
left the cells in A and C. By turning the valve to connect D with A and
D with C, the guanidinium isothiocyanate (GITC)-containing mRNA
extraction solution was added to the cells. The whole BPM was
immediately placed in a -80°C freezer for the duration of the
flight. All ground controls were handled using identical procedures.
 View larger version (24K): [in a new window] |
Figure 1. Bioprocessing modules. The hardware used for the STS-95
experiment consisted of simple, manually operated bioprocessing
modules. Duplicate cultures of cells in syringes A and C were
growth stimulated in microgravity when an astronaut turned the valve to
connect syringes A and B or C and B. By pressing the B syringe plunger,
the activator (medium with 22% FBS) was injected into syringes A and
C. After 24 h, the cells in both A and C [equipped with a
0.45-µm membrane filter (not shown)] were fixed by pushing plungers
to deliver the medium from A and C into B, leaving the cells in A and
C. By turning the valve to connect D with A and to connect D with C,
the GITC-containing mRNA extraction solution was added to the
cells. The complete BPM was immediately placed in a -80°C freezer
for the duration of the flight. All ground controls were handled in the
same manner.
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30 h before launch. At 4.5 h after launch and accessing
microgravity, the cells were growth stimulated by increasing the serum
concentration to 10% and raising the temperature to 37°C.
Twenty-four hours after activation, the cells in both cell-containing
syringes of a BPM were filtered from the medium, and the guanidinium
solution was added. The BPMs were immediately frozen and maintained at
-80°C until the mRNA was extracted in the laboratory after shuttle
landing. Ground controls were handled according to the same procedures
and the same timeline as the flight experiment.
Vibration parameters
Vibration tests were conducted at the vibration facility at the
Marshall Space Flight Center in Huntsville, AL. Syringes containing
cells suspended in culture medium were placed in a frame and bolted to
the vibration table (Fig. 2
). The vibration intensity and duration profile was set to mimic
that of the space shuttle STS-95 launch and included vibration in the
x, y, and z axes. Vibration parameters were as
follows: 20 Hz at 0.00054 g2/Hz, 20–150 Hz at
+6.0 dB/oct, 150–1,000 Hz at 0.03 g2/Hz,
1,000–2,000 Hz at -6.0 dB/oct, 2,000 Hz at 0.0075
g2/Hz, and composite = 6.5 g
root mean square. Environment exposure duration = 60 s each
in x-, y-, and z-axes.
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Figure 2. Simulated launch vibration test equipment. Ten-milliliter syringes
containing Jurkat cells suspended in culture medium were bolted onto
tables, like the one shown, and vibrated in the x-, y-, and
z-axes. Vibration profiles were designed to simulate the
time and intensity of those aspects of the STS-95 shuttle mission. All
tests were conducted in the vibration laboratory at Marshall Space
Flight Center, Huntsville, AL.
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20–22°C) to the Marshall Space Flight Center vibration
laboratory. The syringes were subjected to vibration stress as
described above. Identical sets of syringes were held under static
conditions as controls. After vibration, all syringes were transported
at ambient temperature back to the laboratory, where the concentration
of serum in the medium was increased to 10% FBS in the syringes
initially set up with 2% serum. Syringes were placed at 37°C, and
mRNA was extracted after 30 min and after 24 h. Vibration tests
were performed twice.
mRNA extraction procedure
For all mRNA procedures, plasticware was autoclaved or washed
with diethyl pyrocarbonate-treated water to prevent degradation by
ribonuclease (RNase). After the vibration tests, the cells in each
syringe were transferred to a 15-mL centrifuge tube and separated from
the medium by centrifugation for 5 min at 800 rpm using a table top
centrifuge (International Equipment Co. model HN-S). The medium, except
for 1 mL, was transferred to a new tube and stored at -20°C for
later glucose analysis. The cell pellet was resuspended in the 1 mL of
medium remaining in the tube, and the suspension was transferred to a
2-mL microcentrifuge tube (Applied Scientific, San Francisco, CA). The
cells were centrifuged for 5 min at 800 rpm, the supernatant was
removed, 500 µL of guanidinium solution were added to the cell
pellet, and then the samples were incubated at 60°C for 25 min.
For the flight experiment, mRNA was extracted as follows. After the shuttle landing, the frozen mRNA samples were returned to the laboratory and stored frozen. The extraction procedure for flown cells was continued by transferring the guanidinium-treated samples from syringes to 15-mL centrifuge tubes and placing them directly in a water bath at 60°C for 25 min.
The subsequent procedures were common to both vibrated and flown samples. Following the 25-min incubation at 60°C, the mRNA was extracted by a modification of the procedure described by Chomczynski and Sacchi [22 , 23 ]. One-tenth volume of sodium acetate was added to the mixture, after which 1.2 volumes of phenol-chloroform-isoamyl alcohol (125:24:1) were added (Sigma). The cells were vortexed for 20 s and placed on ice for 15 min, after which they were centrifuged at 3,000 rpm (Eppendorf centrifuge 5415c) for 20 min at 4°C. The top phase was transferred to a new tube, and 1 volume of isopropanol (Sigma) was added. The samples were centrifuged again for 20 min at 4°C. The isopropanol was decanted, and 1 mL of ice-cold ethanol was added. At this point, the flight samples were transferred to 2-mL tubes. The tubes were incubated on ice for 5 min and centrifuged for 12 min at 7500 4°C. The ethanol was decanted, and the samples were allowed to air dry. The samples were then resuspended in 20 µL of water, and the mRNA concentration was determined using a GeneQuant IITM spectrophotometer (Pharmacia Biotech, Cambridge, England). To check the quality of the mRNA, a sample was electrophoresed on a 1%; agarose–formaldehyde gel at 120 V for 25 min.
Primers
Heat shock protein primer sequences for constitutive and
inducible hsp70 were designed by M. Hughes-Fulford, Veterans Affairs
Medical Center, San Francisco, CA [4
]. Primers for hsp27
were designed by one of us (L. C.) from a sequence obtained from
GenBank using the computer program MacVectorTM 6.0. The primers’
predicted product sizes were 283, 284, and 400 nucleotides. Primers for
ß actin were run in all tests as a housekeeping gene. Primer sets
were as follows: for hsp70a (constitutive):
forward-CCATGGTGCTGACCAAGATGAAG and reverse-TCGTCGATCGTCAGGATGGACAC;
hsp70b (inducible): forward-CCATGGTGCTGACCAAGATGAAG and
reverse-CACCAG-CGTCAATGGAGAGAACC; hsp27:
forward-TGTCCCTGGATGTCAACCACTTC and
reverse-AAAAGAACACACAGGTGGCGG; actin: forward-CCGCAAATGCTTCTAGGC and
reverse-GGTCTCACGTCAGTGTACGG.
Reverse transcription-polymerase chain reaction (RT-PCR) procedure
Complementary DNA was synthesized using 1.5 µg of RNA and
reagents included in the SuperScriptTM reverse transcriptase kits (Life
Technologies). One microliter of oligo(dT) was added to each tube. The
volume was brought to 12 µL with diethyl pyrocarbonate-treated water.
Tubes were incubated at 70°C for 10 min, then placed on ice for 2
min. Two microliters of 10x PCR buffer, 25 mM MgCl2, 0.1 M
dithiothreitol, and 1 µL of a 10 mM deoxynucleoside triphosphate
solution were added to each tube, followed by incubation at 42°C for
5 min and addition of 1 µL of SuperScriptTM. The tubes were incubated
for 50 min at 42°C, placed at 70°C for 15 min, and then chilled on
ice. One microliter of RNase H was added, and tubes were incubated for
20 min at 37°C. One microliter of complementary DNA template, 45 µL
of PCR SupermixTM (Life Technologies), 1 µL each of forward primer
(hsp70; Operon, Alameda, CA) and reverse primer (hsp27; Research
Genetics, Huntsville, AL), 2 µL of RediloadTM (Research Genetics),
and 2 drops of mineral oil (Sigma) were placed in the 200-µL tubes
for the PCR. The RobocyclerTM thermocycler (Stratagene, La Jolla, CA)
program was set as follows: 3 min of initial denaturation at 95°C,
followed by 30–32 cycles of 1.5 min at 95°C, 1 min at the annealing
temperature required for each specific primer used, and 2 min at 72°C
and then one cycle of 5 min at 72°C; the reaction was stopped at
6°C. A 2% agarose gel containing ethidium bromide was run to view
PCR products, and photographs of gels were taken using a FCR-10TM
camera (Fotodyne, Hartland, WI) and PolaroidTM 667 film.
Densitometry of PCR product bands
Photographs of the PCR gels were scanned into a digitized image,
and grey scale band densities were determined using a MacintoshTM
computer and NIH ImageTM 1.61 software. The cells were either
maintained in a medium with 10% serum throughout the test or held in a
medium with 2% serum for 24 h before vibration and then switched
to 10% serum for 48 h after vibration to mimic the conditions of
the STS-95 shuttle flight experiment. The band density ratios of flown
versus ground or vibrated versus nonvibrated control cells were
determined, and a difference of twofold or greater was arbitrarily set
as an acceptable value for differences
Statistical analyses
The experimental design and hardware for the flight and ground
control experiments allowed us to make evaluations from two replicate
cell syringes at each time point. Two independent vibration tests were
performed, and duplicate syringes were set up for each sample time,
thus providing four independent samples for evaluation. For glucose
analyses, the mean and SD were determined from three
glucose analyzer readings for each of the replicate samples for each
time point. The ratio values for the PCR gels are shown as the mean and
SD of a minimum of two scan values for each gel. For
comparing flight to ground and vibrated to nonvibrated cultures,
statistical analyses were performed using an InStatTM 1.14 program for
the Macintosh computer. One-way analysis of variance and unpaired
two-tailed t-tests were applied to obtain the mean,
SE, SD, and P values for replicate
samples for each condition. Statistical significance was considered to
be P 
0.05.
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30% less than controls
(P =0.0007). There were no differences in viability
(data not shown) of vibrated and nonvibrated cells, and cultures were
>80% viable at 48 h. The viability of both vibrated and
nonvibrated cells dropped from 96% to 91% during the previbration
period when cells were maintained at 20°C in low-serum medium to
retard cell growth. Both the vibrated and nonvibrated cells remained
metabolically active throughout the 48-h test and used glucose as they
continued to grow (Fig. 4
). On a per-cell basis, considering cells per milliliter in
culture, the vibrated cells at 24 and 48 h used 1.4
(P =0.0001) and 1.5 (P =0.0005) times,
respectively, more glucose per cell than controls.
 View larger version (17K): [in a new window] |
Figure 3. Representative growth curves for vibrated and nonvibrated cells. Cells,
suspended in medium with 2% serum, were loaded into the syringes
24 h before vibration to mimic the STS-95 shuttle flight
experiment, and the vibration tests were conducted as described in
Materials and Methods. Cells were counted at 30 min after vibration,
when the serum concentration was increased to 10%, and syringes were
placed in a standard CO2 incubator to activate the
experiment (0 h). Subsequent counts were made 24 and 48 h later.
Each data point represents the mean and SE of cells counted
in five hemacytometer squares for duplicate samples at each time point.
There was an initial decrease in the number of cells after vibration,
and there were fewer cells at 24 h in vibrated cultures than in
nonvibrated controls. By 24 h, vibrated cells had resumed active
growth, but counts were about 30% less than for controls at 48 h
(asterisk, P =0.0007), corresponding to initial cell
loss during vibration. No cell growth occurred during the
prestimulation period, when cells were maintained in medium with 2%
serum at 20°C.
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 View larger version (18K): [in a new window] |
Figure 4. Effect of vibration on glucose metabolism. Cells were suspended in
10-mL syringes in medium containing 2% or 10% FBS and remained at
room temperature for 24 h before vibration test initiation to
mimic conditions of the shuttle flight experiment as previously
described. Glucose use was measured 24 h before the vibration test
when the cells were placed in the syringes, then 30 min and 24 and
48 h after vibration as described in Materials and Methods.
Measurements were done in triplicate at each time point, and the data
represent the mean and SD of three evaluations (absence of
error bars indicates a very small SD). At 24 h on a
per-cell basis, the vibrated cells had used 1.4;tm more glucose per
cell than controls (asterisk, P =0.0001). By 48 h,
vibrated cells metabolized about 1.5;tm more glucose per cell than
controls did (asterisk, P =0.0005). Both the vibrated
and nonvibrated cells remained metabolically active throughout the 48-h
test.
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 View larger version (35K): [in a new window] |
Figure 5. Effect of simulated shuttle launch vibration on expression of heat
shock protein mRNA. Cells were maintained at 20°C in medium
containing 2% serum for 24 h before vibration to mimic the time
profile and conditions of the STS-95 shuttle flight experiment. After
vibration, serum was increased to 10% and cells were placed at 37°C
to initiate growth. mRNA was extracted and RT-PCR was conducted as
described in Materials and Methods. Representative PCR gels are shown
for nonvibrated control and vibrated cells. Lanes for each panel are as
follows: lane 1, DNA ladder; lane 2, hsp27, 30 min; lane 3, hsp27,
24 h; lane 4, hsp70a, 30 min; lane 5, hsp70a, 24 h; lane 6,
hsp70b, 30 min; and lane 7, hsp70b, 24 h. All three heat shock
protein genes transcribed mRNA in vibrated and nonvibrated cells at 24
h. Vibrated and nonvibrated cells maintained in medium with 2% serum
at 20°C for 24 h before vibration did not transcribe mRNA for
hsp27 (lane 2) or hsp70b (lane 6) and only faint bands are visible for
hsp70a at 30 min. Numbers on left are base pairs and correspond to base
pair bands on the DNA ladder.
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 View larger version (54K): [in a new window] |
Figure 6. A representative PCR gel comparing expression of hsp70a in cells
maintained in medium with 10% or 2% FBS. mRNA extraction and RT-PCR
were conducted as described in Materials and Methods. Lanes 2–5 show
bands for samples from cells maintained on medium with 10% FBS, and
lanes 6–9 show bands for samples from cells maintained on medium with
2% FBS for 24 h before the vibration test. Lane 1, DNA ladder;
lane 2, control, 30 min; lane 3, control, 24 h; lane 4, vibration,
30 min; lane 5, vibration, 24 h; lane 6, control, 30 min; lane 7,
control, 24 h; lane 8, vibration, 30 min; lane 9, vibration,
24 h. The constitutive hsp70a was transcribed at each sample time
and in cells maintained at both serum concentrations. Numbers on left
are base pairs and correspond to base pair bands on the DNA ladder.
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Table 1. PCR Gel Densitometry Values and Ratios Showing Effect of Time and Serum
Concentration on Heat Shock Protein Gene Expression in Vibrated Cells
and Nonvibrated Controls
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Effect of serum concentration on heat shock protein mRNA
The effect of maintaining cells in medium with 2% or 10% serum
for the 24 h prior to the vibration test may also be determined
from the scans of PCR gels shown in Figures 5
and 6
. As shown in Figure 5
and Table 1
, cells maintained in low-serum medium at low temperature
for 24 h before vibration did not transcribe mRNA for hsp70b or
hsp27 at 30 min in either the vibrated or nonvibrated controls. Thus,
low-serum culture markedly reduced (more than twofold) transcription of
these genes. By 24 h, after increasing the serum to 10% and
raising the temperature to 37°C, there were no twofold or greater
differences in expression of hsp70b or hsp27 between cells grown on
either the 2 or 10% serum medium. mRNA for the constitutive hsp70a as
well as ß-actin was transcribed under all test conditions.
Effect of spaceflight on heat shock protein gene expression
A representative PCR gel (Fig. 7
) showing expression of heat shock protein genes in Jurkat cells
flown on the STS-95 shuttle flight indicated that mRNA for all three
heat shock protein genes was transcribed in microgravity. The density
of the band in lane 2 (flight cells) compared to lane 3 (ground
controls) represents up-regulation of hsp27 in flown cells after
24 h in microgravity. Genes for both the constitutive and
inducible forms of hsp70 were expressed at 24 h in both flight and
ground samples. The PCR gel band densities of flown cells versus ground
control cells are shown in Table 2
. Both hsp70a and hsp70b were expressed in flown and ground control
cells, and there was no notable difference in expression between flight
and ground cells harvested 24 h after activation for hsp70a
(P =0.1031) and hsp70b (P =0.0781). For
hsp27, the band density in flown cells was 2.2 times greater than that
of the ground control (P =0.0007). The up-regulation of
hsp27 in microgravity is a new finding.
 View larger version (64K): [in a new window] |
Figure 7. Effect of spaceflight on expression of heat shock protein mRNA. Cells
were loaded into BPMs and maintained at 20°C for 24 h in medium
containing 2% FBS before launch of STS-95. On orbit, serum was
increased to 10% as described in Materials and Methods. Cells were
filtered from medium 24 h after activation, and a GITC solution
was added to the cells. Each BPM was immediately frozen at -80°C.
mRNA was extracted post-flight as described in Materials and Methods.
The PCR gel lanes are as follows: lane 1, DNA ladder; lane 2, hsp27,
flight; lane 3, hsp27, ground; lane 4, hsp70a, flight; lane 5, hsp70a,
ground; lane 6, hsp70b, flight; and lane 7. hsp70b, ground. mRNA for
both hsp70a and hsp70b was transcribed at 24 h in all samples.
Up-regulation of mRNA for hsp27 in microgravity is shown by comparing
lane 2 (flight) and lane 3 (ground control). Numbers on left are base
pairs and correspond to base pair bands on the DNA ladder.
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View this table: [in a new window] |
Table 2. Density of RT-PCR Gel Bands with Heat Shock Protein Message from
Space-Flown Cells and Cells Subjected to Simulated Shuttle Launch
Vibration
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A possible reason for the increased expression of heat shock proteins over time in culture may involve the stress levels and protein synthesis in the cells [25 ]. The magnitude of the stress and how much the stress affects the ability of the cell to synthesize new proteins could affect the time required for elevation of protein synthesis. Serum stimulation can also result in production of heat shock protein mRNA, with greater differences noted by 12–18 h after the stimulation [26 ]. For this reason, we evaluated the expression of the genes in cells maintained in medium with 10% FBS throughout the experiment compared with cells maintained for 24 h in medium with 2% serum, as for shuttle flight experiments. Genes for hsp70b and hsp27 were not transcribed at 30 min in cells cultured in medium with 2% serum. This was reflected by the significant increase in expression of hsp70b and hsp27 at 24 h (more than twofold).
The reasons for the increase of hsp27 in the flight samples remain to be determined. The increase could be related to microgravity or other factors related to conditions of spaceflight. The increase of hsp27 in flight samples (Table 2) may be due to the involvement of hsp27 in the protection against Fas-induced apoptosis [27 ] or regulatory activities in actin dynamics [28 ]. We also found increased consumption of glucose by flight samples [3 ], which could result in oxidative stress leading to hsp27 expression [29 ].
The future of space exploration will involve long-duration spaceflights to Mars and beyond, and crewmembers will be subjected to a number of stressors, including radiation. The stimulation of heat shock proteins and their ability to protect thymocytes from radiation-induced apoptosis [30 ] in crewmembers may protect against increased death of lymphocytes in space. Research to find ways to counteract the effects of microgravity on crewmembers is now focused on exercise and addition of artificial gravity (centrifugation) to reduce bone demineralization and prevent muscle atrophy. Little has been done to evaluate other possible ways to counteract the effects of space travel. The next generation of countermeasures could include ways to safely stimulate the production of heat shock proteins in cells and experimental animals to determine whether this stimulation benefits the system being studied. Further understanding of heat shock protein stimulation and function in microgravity could lead to the development of Earth-based therapies against a variety of conditions, including myocardial infarctions, cancer, and aging [31 ].
The authors express their appreciation to the astronauts of shuttle flight STS-95, especially to Senator John Glenn and Scott Parazynski, who conducted the experiments while in orbit, and to the Marshall Space Flight Center vibration laboratory. We are grateful to C. A. Yancey for technical assistance and K. L. Murphy for administrative support throughout the experiments.
Received May 18, 2000; revised October 23, 2000; accepted December 15, 2000.
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