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(Journal of Leukocyte Biology. 2001;69:755-761.)
© 2001 by Society for Leukocyte Biology

Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

Luis A. Cubano* and Marian L. Lewis{dagger}

* Nephrology Section, Department of Medicine, Tulane University Medical Center, and Tulane Environmental Astrobiology Center, New Orleans, Louisiana, and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

Key Words: microgravity • gene expression • molecular chaperones • vibration • lymphocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells flown on the space shuttle respond in a number of ways not typical of ground-based cultures. For instance, human lymphocytes are growth retarded or arrested [1 2 3 ] and undergo apoptosis at a higher rate than ground-based controls [3 ]. To determine the mechanisms causing the observed arrest of growth in space, it is important to understand whether mechanical stress from shuttle launch vibration is a factor. In previous studies, hsp70 gene expression was found to be up-regulated in Jurkat cells cultured under conditions of serum starvation [4 ]. In contrast, cultures in rotating bioreactors, which mimic some aspect of microgravity, and centrifugation in excess of the typical 3 g during shuttle launch did not up-regulate hsp70 mRNA [4 ].

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The human cell line Jurkat clone E6-1 was obtained from the American Type Culture Collection (Manassas, VA). The cells were certified free of mycoplasma contamination by the American Type Culture Collection. The culture medium consisted of RPMI 1640 (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Summit Biotechnology, Santa Ana, CA), 2 mM glutamine, 1 mM sodium pyruvate, 1 mL/100 of 100x nonessential amino acids, 100 U of penicillin, 100 µm/mL of streptomycin (Life Technologies, Grand Island, NY), and 12.5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (Sigma, St. Louis, MO). To evaluate cell growth and metabolism, cells were counted with a hemacytometer, and viability was determined by trypan blue exclusion. The glucose concentration in cell culture medium samples was determined by use of Beckman glucose analysis kits and a Glucose Analyzer 2 (Beckman Instruments, Brea, CA). Jurkat cells were selected for this study because they have been used in previous space shuttle missions [3 , 20 , 21 ].

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.



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  		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.

 
Flight experimental details
In preparation for the shuttle flight, the cells were suspended at 1.5 million per mL in medium containing 2% FBS and held at 20°C to retard growth prior to initiation of the experiment in microgravity. Two (A and C) of the four BPM syringes were loaded with the cells, thus providing two independent samples at each sampling time. Syringe B was loaded with a growth activator consisting of a medium containing enough FBS to give a final concentration of 10% FBS when this activator was injected into the syringes containing the cells. Syringe D of each BPM was loaded with a guanidinium-based mRNA extraction solution. Identical BPMs were set up as ground controls. All BPMs were maintained at 20°C for ~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.

 
Vibration test details
For vibration tests, Jurkat cells at 1.5 million cells/mL in 3 mL of medium containing either 10% or 2% FBS were loaded into duplicate sets of 10-mL syringes. All air space and bubbles were carefully removed. After 24 h at room temperature to mimic the conditions of previous spaceflight experiments, the syringes were placed in StyrofoamTM containers and transferred at ambient temperature (~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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of vibration on cell growth, viability, and metabolism
To assess the effect of shuttle launch vibration on gene expression, cells in syringes handled in the same manner as those flown during the STS-95 shuttle flight were subjected to simulated shuttle launch vibration as described in Materials and Methods. Some cells were lost initially to vibration, and counts at 24 h were lower than for nonvibrated controls (Fig. 3 ). However, between 24 and 48 h, active growth resumed, although the cell number remained ~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.



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  		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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  		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.

 
Heat shock protein gene expression in vibrated and nonvibrated cells
The mRNA from vibrated and nonvibrated cells was extracted and evaluated by RT-PCR using primers for hsp70a (constitutive), hsp70b (inducible), and hsp27 as described in Materials and Methods. Representative PCR gels are shown in Figure 5 for nonvibrated controls and vibrated cells. mRNA was transcribed for both heat shock protein genes in vibrated and nonvibrated cells at 24 h. Both vibrated and nonvibrated cells that were maintained in medium with 2% serum at 20°C for 24 h before vibration failed to transcribe mRNA for hsp27 (lane 2) and hsp70b (lane 6) at 30 min.



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  		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.

 
Figure 6 shows that mRNA was transcribed for hsp70a (constitutive) by cells maintained in medium with 10% serum throughout the test as well as those maintained in medium with 2% serum for 24 h and then switched to 10% serum at the time of activation after vibration to simulate the shuttle flight experiment.



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  		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.

 
Effect of simulated shuttle launch vibration on heat shock protein gene expression
The PCR gels shown in Figures 5 and 6 were scanned as described in Materials and Methods, and the PCR gel band density values and ratios are shown in Table 1 . For all heat shock protein mRNAs except hsp70b and hsp27, which were not transcribed at 30 min by cells in low-temperature, low-serum culture, all band density ratios were <2 for vibrated versus nonvibrated samples. Thus, the vibrated cells, handled by the same procedures and subjected to the same vibrational stress as those in the flight experiment, did not up-regulate mRNA for the heat shock proteins, including hsp27.


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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

 
Effect of time in culture on expression of heat shock protein genes
The PCR band densities from Figures 5 and 6 , shown in Table 1 , allow comparison of heat shock protein mRNAs in cells cultured for 30 min and 24 h after vibration. There were no twofold or greater differences in heat shock protein mRNA expressed in cultures maintained in medium with 10% serum throughout the test, and expression was higher at 24 h than at 30 min. Between 30 min and 24 h, in cells that were held in low-serum medium at low temperature for 24 h before vibration, mRNA for the constitutive hsp70a increased by a factor of 3.2 (P =0.0001) (in nonvibrated cells) or 2.2 (P =0.0013) (in vibrated cells).

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.



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  		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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  		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

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To evaluate the expression of genes during spaceflight and define which genes are stimulated by microgravity, it is necessary to establish the contribution of flight-related perturbations. One such perturbation is vibrational stress during launch of the spacecraft. Understanding how vibration affects the expression of heat shock proteins can allow the characterization of the effects of spaceflight on stress-related genes. Previous reports have shown that the expression of the hsp70 gene is not increased in flown rats [24 ] and cells cultured in rotating bioreactors [4 ]. Here we present the effects of vibrational stress on the expression of hsp70 and hsp27 in human lymphoblastoid cells. Our results showed that response to vibrational stress does not significantly alter the expression of the heat shock proteins that we tested.

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 ].


   ACKNOWLEDGEMENTS
 
Financial support was provided by NASA grant NAG2-985 and NASA Graduate Student Research Program grant 97-GSRP-076.

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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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