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Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity.

Thiel CS, Paulsen K, Bradacs G, Lust K, Tauber S, Dumrese C, Hilliger A, Schoppmann K, Biskup J, Gölz N, Sang C, Ziegler U, Grote KH, Zipp F, Zhuang F, Engelmann F, Hemmersbach R, Cogoli A, Ullrich O - Cell Commun. Signal (2012)

Bottom Line: In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response.Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls.The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy, Faculty of Medicine, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. oliver.ullrich@anatom.uzh.ch.

ABSTRACT
In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

No MeSH data available.


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Expression of p21 Waf1/Cip1 mRNA in real microgravity provided by parabolic flights in non-stimulated (con) or stimulated (PMA or CD3/CD28) Jurkat T cells or primary CD4+ T cells. p21 Waf1/Cip1 mRNA expression is demonstrated as ratio between expression in microgravity (μg) and 1 g in fig.5ab. In fig. 5c, p21 Waf1/Cip1 mRNA expression is demonstrated as the ratio between expression in microgravity (μg) with a pharmacological inhibitor (VP, CU or AIQ) and without. Three independent experiments were analysed. VP = valproic acid, CU = curcumin, AIQ = 5-aminoisoquinoline. Results are expressed as mean ± SEM. Data were analysed by Wilcoxon test. p < 0.1 = *, p < 0.05 = **. In fig 5d, phosphorylation of cdc2 in Jurkat T cells after 20s real microgravity provided by parabolic flight manouvres in non-stimulated (con) or stimulated (PMA or CD3/CD28) cells is shown. PMA or CD3/CD28 was added at the onset of altered gravity. Loading control: corresponding gel stained by Coomassie after blotting. Five independent experiments during different flight days were performed and pooled protein was analysed.
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Figure 5: Expression of p21 Waf1/Cip1 mRNA in real microgravity provided by parabolic flights in non-stimulated (con) or stimulated (PMA or CD3/CD28) Jurkat T cells or primary CD4+ T cells. p21 Waf1/Cip1 mRNA expression is demonstrated as ratio between expression in microgravity (μg) and 1 g in fig.5ab. In fig. 5c, p21 Waf1/Cip1 mRNA expression is demonstrated as the ratio between expression in microgravity (μg) with a pharmacological inhibitor (VP, CU or AIQ) and without. Three independent experiments were analysed. VP = valproic acid, CU = curcumin, AIQ = 5-aminoisoquinoline. Results are expressed as mean ± SEM. Data were analysed by Wilcoxon test. p < 0.1 = *, p < 0.05 = **. In fig 5d, phosphorylation of cdc2 in Jurkat T cells after 20s real microgravity provided by parabolic flight manouvres in non-stimulated (con) or stimulated (PMA or CD3/CD28) cells is shown. PMA or CD3/CD28 was added at the onset of altered gravity. Loading control: corresponding gel stained by Coomassie after blotting. Five independent experiments during different flight days were performed and pooled protein was analysed.

Mentions: Jurkat T cells and primary T cells were exposed to 20s of microgravity during parabolic flights and analysed for their differential gene expression of p21 Waf1/Cip1 (cyclin-dependent kinase inhibitor 1) which functions as a regulator of cell cycle progression at the G1 phase by directly inhibiting the activity of cyclinE/CDK2 and cyclinD/CDK4 complexes (Figure 5). Three different conditions were tested: 1.) medium was injected as a control solution to identify effects of microgravity on gene expression without stimulation (con), 2.) PMA was used to activate directly the signal transduction enzyme protein kinase C (PKC) and 3.) CD3/CD28 antibodies were applied to stimulate the cells via their T cell receptor (TCR) and CD28 receptor (CD3/CD28). Comparison of 1 g and μg showed that even for non-stimulated conditions, an increased p21 Waf1/Cip1 gene expression (1.9 fold in Jurkat T cells and 2.3 in primary CD4+ T cells, Figure 5ab) is detectable. In CD3/CD28-stimulated Jurkat T cells and primary CD4+ T cells, increased p21 Waf1/Cip1 gene expression was upregulated 2.9-fold and 4.1-fold, respectively (Figure 5ab).


Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity.

Thiel CS, Paulsen K, Bradacs G, Lust K, Tauber S, Dumrese C, Hilliger A, Schoppmann K, Biskup J, Gölz N, Sang C, Ziegler U, Grote KH, Zipp F, Zhuang F, Engelmann F, Hemmersbach R, Cogoli A, Ullrich O - Cell Commun. Signal (2012)

Expression of p21 Waf1/Cip1 mRNA in real microgravity provided by parabolic flights in non-stimulated (con) or stimulated (PMA or CD3/CD28) Jurkat T cells or primary CD4+ T cells. p21 Waf1/Cip1 mRNA expression is demonstrated as ratio between expression in microgravity (μg) and 1 g in fig.5ab. In fig. 5c, p21 Waf1/Cip1 mRNA expression is demonstrated as the ratio between expression in microgravity (μg) with a pharmacological inhibitor (VP, CU or AIQ) and without. Three independent experiments were analysed. VP = valproic acid, CU = curcumin, AIQ = 5-aminoisoquinoline. Results are expressed as mean ± SEM. Data were analysed by Wilcoxon test. p < 0.1 = *, p < 0.05 = **. In fig 5d, phosphorylation of cdc2 in Jurkat T cells after 20s real microgravity provided by parabolic flight manouvres in non-stimulated (con) or stimulated (PMA or CD3/CD28) cells is shown. PMA or CD3/CD28 was added at the onset of altered gravity. Loading control: corresponding gel stained by Coomassie after blotting. Five independent experiments during different flight days were performed and pooled protein was analysed.
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Related In: Results  -  Collection

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Figure 5: Expression of p21 Waf1/Cip1 mRNA in real microgravity provided by parabolic flights in non-stimulated (con) or stimulated (PMA or CD3/CD28) Jurkat T cells or primary CD4+ T cells. p21 Waf1/Cip1 mRNA expression is demonstrated as ratio between expression in microgravity (μg) and 1 g in fig.5ab. In fig. 5c, p21 Waf1/Cip1 mRNA expression is demonstrated as the ratio between expression in microgravity (μg) with a pharmacological inhibitor (VP, CU or AIQ) and without. Three independent experiments were analysed. VP = valproic acid, CU = curcumin, AIQ = 5-aminoisoquinoline. Results are expressed as mean ± SEM. Data were analysed by Wilcoxon test. p < 0.1 = *, p < 0.05 = **. In fig 5d, phosphorylation of cdc2 in Jurkat T cells after 20s real microgravity provided by parabolic flight manouvres in non-stimulated (con) or stimulated (PMA or CD3/CD28) cells is shown. PMA or CD3/CD28 was added at the onset of altered gravity. Loading control: corresponding gel stained by Coomassie after blotting. Five independent experiments during different flight days were performed and pooled protein was analysed.
Mentions: Jurkat T cells and primary T cells were exposed to 20s of microgravity during parabolic flights and analysed for their differential gene expression of p21 Waf1/Cip1 (cyclin-dependent kinase inhibitor 1) which functions as a regulator of cell cycle progression at the G1 phase by directly inhibiting the activity of cyclinE/CDK2 and cyclinD/CDK4 complexes (Figure 5). Three different conditions were tested: 1.) medium was injected as a control solution to identify effects of microgravity on gene expression without stimulation (con), 2.) PMA was used to activate directly the signal transduction enzyme protein kinase C (PKC) and 3.) CD3/CD28 antibodies were applied to stimulate the cells via their T cell receptor (TCR) and CD28 receptor (CD3/CD28). Comparison of 1 g and μg showed that even for non-stimulated conditions, an increased p21 Waf1/Cip1 gene expression (1.9 fold in Jurkat T cells and 2.3 in primary CD4+ T cells, Figure 5ab) is detectable. In CD3/CD28-stimulated Jurkat T cells and primary CD4+ T cells, increased p21 Waf1/Cip1 gene expression was upregulated 2.9-fold and 4.1-fold, respectively (Figure 5ab).

Bottom Line: In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response.Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls.The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Anatomy, Faculty of Medicine, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. oliver.ullrich@anatom.uzh.ch.

ABSTRACT
In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

No MeSH data available.


Related in: MedlinePlus