open access

Vol 49, No 3 (2011)
Original paper
Submitted: 2012-01-05
Published online: 2011-10-28
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p21WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

Dan Wang, Yu-Zhen Zhang, Bing Yang, Feng-Xiang Zhang, Ming-Yong Cao, Cheng Wang, Ming-Long Chen
DOI: 10.5603/FHC.2011.0063
·
Folia Histochem Cytobiol 2011;49(3):445-451.

open access

Vol 49, No 3 (2011)
ORIGINAL PAPERS
Submitted: 2012-01-05
Published online: 2011-10-28

Abstract

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21WAF1 (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21WAF1 and a plasmid expressing small hairpin RNA (shRNA) targeting p21WAF1 were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO2, balancing the incubator chamber with N2 for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16INK4a, and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16INK4a expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451)

Abstract

We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21WAF1 (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21WAF1 and a plasmid expressing small hairpin RNA (shRNA) targeting p21WAF1 were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO2, balancing the incubator chamber with N2 for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16INK4a, and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16INK4a expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451)
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Keywords

H9c2; hypoxia reoxygenation; p21WAF1; β-galactosidase; senescence

About this article
Title

p21WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 49, No 3 (2011)

Article type

Original paper

Pages

445-451

Published online

2011-10-28

Page views

2474

Article views/downloads

2872

DOI

10.5603/FHC.2011.0063

Bibliographic record

Folia Histochem Cytobiol 2011;49(3):445-451.

Keywords

H9c2
hypoxia reoxygenation
p21WAF1
β-galactosidase
senescence

Authors

Dan Wang
Yu-Zhen Zhang
Bing Yang
Feng-Xiang Zhang
Ming-Yong Cao
Cheng Wang
Ming-Long Chen

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