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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 18, 2001 as doi:10.1096/fj.00-0820fje. |
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University of Alabama in Huntsville, Department of Biological Sciences, Huntsville, Alabama 35899, USA;
* Tulane University Medical Center, Department of Medicine, New Orleans, Louisiana, USA;
University of Texas, College of Pharmacy, Austin, Texas, USA;
University of Texas Medical Branch, Galveston, Texas, USA; and
U.S. Army Institute of Surgical Research, San Antonio, Texas, USA
2Correspondence: Department of Biological Sciences, University of Alabama in Huntsville, Wilson Hall 360, Huntsville, AL 35899, USA. E-mail: lewisml{at}email.uah.edu
SPECIFIC AIMS
This study addresses the hypothesis that in addition to previously reported microtubule disruption and growth arrest, spaceflight may alter expression of genes to fundamentally affect cytoskeletal and cellular function. The effect of the space shuttle environment while in orbit and simulated shuttle launch vibration on gene expression have been studied and compared to ground and nonvibrated controls using cDNA microarray to evaluate RNA message for 4324 genes at 24 h as well as more than 20,000 genes and expressed sequence tags (EST) at 48 h in leukemic T lymphocytes (Jurkat) flown on Space Transportation System (STS) 95.
PRINCIPAL FINDINGS
1. Cytoskeletal gene expression in space-flown Jurkat cells differs
from ground controls
The cDNA microarray identified 11 cytoskeletal genes that were
regulated differently in space-flown vs. ground control cells. Among
these were up-regulated messages for tropomodulin, plectin (human
plectin, PLEC1 mRNA), C-NAP1 (Homo sapiens centrosomal
Nek2-associated protein 1), calponin (human mRNA for calponin), myosin
(human 20 kDa myosin light chain, MLC-2 mRNA), an EST weakly similar to
ankyrin D. melanogaster and dynactin H. sapiens
mRNA. Gelsolin precursor message was down-regulated. Eight of these
genes evaluated qualitatively by RT-PCR (Fig. 1
) showed up- or down-regulation in agreement with the cDNA microarray.
Exceptions were dynactin and ankyrin (EST similar) for which different
primer sequences specific for ankyrin and dynactin were used in the
RT-PCR.
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2. Microgravity promotes up-regulation of growth control, cellular
function, and tumor suppresser genes
Of the more than 20,000 genes examined, 
98% were similarly
expressed in flown and ground control cells. In addition to
cytoskeletal genes, we found differences between flight and controls
for genes that regulate metabolism, signal transduction, adhesion,
transcription, apoptosis, and tumor suppression. Especially noteworthy
is the Tsc2 gene, which encodes the tumor suppressor
tuberin. This gene was expressed 11.7-fold more in space at 24 h
than in corresponding ground controls. Other cell cycle-related genes
up-regulated on orbit included cyclin-dependent kinase 6 (CDK6), which
was expressed fivefold more than in controls at 24 h, and the G1/S
boundary cell cycle inhibitor prohibitin, which was double that of
ground controls at 48 h. A list of genes expressed in space-flown
and vibrated cells is available at
http://www.uah.edu/biology/faculty/lewis/spacegenes. The identity of
the cDNA incorporated into the microarray GeneFiltersTM (Research
Genetics, Huntsville, AL) is available at http://www.resgen.com.
3. Vibration disrupts microtubules and microtubule organizing
centers (MTOCs) and up-regulates two cytoskeletal genes also
up-regulated in space-flown cells
We subjected Jurkat cells to vibration simulating the STS-95
shuttle launch to determine whether gene expression and growth arrest
result from launch rather than the shuttle’s orbital environment. We
found that vibration severely disrupts microtubules and MTOCs. This was
most evident in cells sampled immediately (10 min) and 4 h after
vibration (Fig. 2
). By 24 h, damage was no longer apparent and the cells continued
to grow. This is in contrast to our previous studies showing that
space-flown Jurkat cells reorganize the microtubule cytoskeleton by
24 h yet remain growth-arrested. Hence, growth arrest in
space-flown Jurkat cells does not appear to be a direct outcome of
launch vibration.
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Messages for two cytoskeletal genes—plectin (a cytoskeletal element linker) and C-NAP1 (a centriole-associated protein)—were up-regulated in vibrated and space-flown cells. This identifies a common response to vibration and suggests a major role for these genes in reorganizing cytoskeletal elements disrupted by shuttle launch vibration. Messages for eight cytoskeleton-related genes increased only in the orbital phase of the mission and not in vibrated cells, indicating an orbit-related up-regulation.
4. The time course of expression of cytoskeletal genes in
space-flown and vibrated cells is different
Most genes expressed in vibrated vs. nonvibrated cells were
up-regulated soon (4 h) after vibration. Seven cytoskeletal messages,
including plectin and C-NAP1, were increased and five were
down-regulated. Due to crew time and hardware constraints, no flight
samples were obtained at 4 h. By 24 h, when the cytoskeleton
was reorganized and cells were actively growing, the vibrated cells no
longer expressed cytoskeletal genes with the exception of keratin type
1 (cytoskeletal 20). At 24 h, the space-flown cells up-regulated
the message for a myosin light polypeptide, which was expressed
11.3-fold more in flown than in ground control cells. Messages for
plectin and a tau protein were down-regulated to approximately half
that of nonvibrated controls.
Forty-eight hours after vibration, only the messages for keratin type 1 (cytoskeletal 14) and plectin were expressed more in vibrated than in nonvibrated controls. Compared with ground controls, the space-flown cells up-regulated 10 cytoskeletal genes at 48 h. This suggests that some property of the space environment (microgravity per se, radiation, electromagnetic fields, or other orbital attributes) influences cytoskeletal gene expression.
CONCLUSIONS AND SIGNIFICANCE
Cytoskeletal organization depends on proteins that link and interconnect filaments to each other, to cell membranes, and to other cellular components. Disruption of cytoskeletal structure leads to cellular dysfunction, growth arrest, and apoptosis. Previous reports describe anomalies in the actin and microtubule cytoskeleton in several types of space-flown cells. This is the first report to describe the use of cDNA microarray to screen genes (>20,000) in T cells (Jurkat) after 24 and 48 h on orbit and the first to identify the cytoskeletal genes expressed in space-flown human leukemic T cells. Based on differential expression of the genes encoding cytoskeletal proteins in flown vs. vibrated and static ground controls, we conclude that membrane-cytoskeletal, centriole-centriole, and cytoskeletal filament associations are altered on orbit.
The observation that the message for plectin, an important cytoskeletal scaffolding protein, is up-regulated by both spaceflight and vibration is a novel finding. This suggests a role for plectin in reorganization of the cytoskeleton after damage by vibration. Plectin is a versatile cytoskeletal linker that provides mechanical strength and structure by interconnecting intermediate filaments, microtubules, and actin to each other and to the cell membrane.
In contrast to vibrated cells, which resume growth after apparent
re-reorganization of the cytoskeleton, space-flown cells up-regulated
cytoskeletal genes at 48 h but remained growth arrested. Although
cytoskeletal anomalies may not be the only factors that cause T cell
growth arrest in microgravity, our hypothesis that cytoskeletal gene
expression is affected in space is validated. This strengthens the
concept that cytoskeletal anomalies fundamentally influence functional
processes (such as growth) of cells in microgravity (Fig. 3
). Growth inhibition of Jurkat cells in space does not appear to be a
primary result of shuttle launch vibration.
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The increase in plectin and other cytoskeletal messages at 48 h in space may imply that cytoskeletal elements, made de novo, are not properly reassociated. Recent reports that microtubule polymerization is inhibited in HL-60 cells cultured with microtubule stabilizing drugs during spaceflight and gravity dependence in microtubule self-organization in vitro in low-gravity support this hypothesis. The increased gene expression in microgravity at 48 h may also indicate anomalies in translation downstream of transcription. The limited numbers of cells from the STS-95 flight experiment were used for RNA extraction; thus, the question of whether translational activity occurs normally remains unresolved.
We previously reported disruption of MTOCs in space-flown Jurkat cells fixed 4 h after shuttle launch. We now find that the message for Nek2-associated protein 1 (C-NAP1) is up-regulated at 48 h. The C-NAP1 protein plays a role in regulation of centriole-centriole cohesion during the cell cycle; disruption of MTOC function thus could affect cell growth. Our gene expression results provide indirect evidence to corroborate reports of Schatten et al., who found abnormalities in the centriole-centrosome region in dividing cells during spaceflight.
Although cytoskeletal anomalies may not be the only factors that cause T cell growth arrest in space, our data provide the molecular basis to strongly infer that cytoskeleton organizing processes as well as normal cellular functions are altered. We cannot rule out the possible role of other growth regulatory genes on T cell growth arrest in space. Indeed, our results provide the first evidence to show that expression of the tumor suppresser gene tuberin is increased in space-flown Jurkat cells. We also found expression differences in flown vs. ground controls in other genes regulating proteins associated with signal transduction, cell cycle, and apoptosis.
In summary, our results provide new information on gene expression in response to spaceflight and vibration, an environmental health hazard in some occupations. For almost 3 decades, spaceflight research has consistently demonstrated reduction in response of human T lymphocytes to mitogen stimulation in microgravity, yet the causal mechanisms remain largely unresolved. The differential expression of cytoskeletal, growth regulatory and tumor suppressor genes in space-flown vs. ground control and vibrated cells provides a gene-level basis to support our previous findings, and those of others, indicating that growth arrest is a characteristic of space-flown T cells. The question of exactly how the cytoskeleton reorganizes in cells in microgravity remains unanswered and whether the observed cytoskeletal anomalies directly affect cell growth is yet to be determined. However, the difference in expression of cytoskeleton-related genes in space-flown cells compared to ground control and vibrated cells validates our hypothesis that spaceflight alters cytoskeletal gene expression and fundamentally affects functional processes of the cytoskeleton and cells in space. With the International Space Station now operable and long-term space habitation more common, it is increasingly important to understand effects of the spaceflight environment on both normal and abnormal cells of the human immune system.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0820fje ; to cite this
article, use FASEB J. (June 18, 2001)
10.1096/fj.00-0820fje 
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