Vol 49, No 3 (2011)
Original paper
Submitted: 2012-01-05
Published online: 2011-10-28
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.
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)
Keywords
H9c2; hypoxia reoxygenation; p21WAF1; β-galactosidase; senescence
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