Correlation of Hypoxia and Pro-senescence Protein Expression in Green Sea Turtle (Chelonia mydas) Lung Epithelial and Dermal Fibroblast Cell Culture

Anggraini Barlian, Yemima Dani Riani


Recent studies have shown hypoxia-induced gene expression correlated with cellular senescence. HIF-1α (hypoxia-inducible factor 1-alpha), p53, and pRB were induced under hypoxia and correlated with cellular senescence. The localization and expression of HIF-1α, p53, and pRB in Chelonia mydas lung epithelial and dermal fibroblast cell cultures were analyzed under normoxic and hypoxic conditions (at 4 and 24 hours). Human dermal fibroblast was used for comparison purposes. Protein localization was analyzed with immunocytochemistry, while protein expression was analyzed with the Western blot and enhanced chemiluminescence (ECL) method. HIF-1α, p53, and pRB were localized in the nuclei of the C. mydas cell cultures treated with hypoxia. The C. mydas lung epithelial cell cultures had a higher increase of HIF-1α expression than the human dermal fibroblast cell culture. The hypoxic conditions did not affect p53 expression significantly in C. mydas lung epithelial and dermal fibroblast cell cultures. Meanwhile, pRB expression changed significantly under hypoxia in the C. mydas dermal fibroblast cells. Expression of p53 and pRB in the human cell cultures was higher than in the C. mydas cell cultures. This research suggests that C. mydas and human cell cultures have different pro-senescence protein expression responses under hypoxic conditions.


Green sea turtle; hypoxia; immunocytochemistry; pro-senescence protein; Western blotting

Full Text:



Finch, C.E., Update on Slow Aging and Negligible Senescence, Gerontology, 55(3), pp. 307-313, 2009. DOI:10.1159/000215589

Barlian, A., Riani, Y.D., Christina, C., Lestari, A. & Fajriah, N., Molecular Study of Aging in Green Sea Turtle: Telomere, Telomerase and Antioxidant Enzyme, In New Hope in Aging Medicine, 2013.

Krivoruchko, A. & Storey, K.B., Forever Young: Mechanism of Natural Anoxia Tolerance and Potential Links to Longevity, Oxid. Med. Cell Longev., 3(3), pp. 186-198, 2010. DOI:10.4161/oxim.3.3.12356

Koh, M.Y., Spivak-Kroizman, T.R. & Powis, G., HIF-1 Regulation: Not So Easy Come Easy Go, Trends Biochemical Sci., 33(11), pp. 526-534, 2008. DOI:10.1016/j.tibs.2008.08.002

Campisi, J., Senescent Cells, Tumor Supression, and Organismal Aging: Good Citizens, Bad Neighbors, Cell., 120(4), pp. 513-522, 2005. DOI:10.1016/j.cell.2005.02.003

Sermeus, A. & Michiels, C., Reciprocal Influence of the p53 and the Hypoxic Pathways, Cell Death Dis., 2(5), e164, 2011. DOI:10.1038/ cddis.2011.48

Budde, A., Marra, N.M., Petersen, G. & Brune, B., Retinoblastoma Susceptibility Gene Product pRB Activates Hypoxia-inducible Factor-1 (HIF-1), Oncogene, 24(10), pp. 1082-1088, 2005. DOI:10.1038/ sj.onc.1208369

Wang, R., Jin, F., & Zhong, H., A Novel Experimental Hypoxia Chamber for Cell Culture, Am. J. Cancer Res., 4(1), pp.53-60, 2014. PMCID: PMC3902232

Jain, M. & Sznajder, J.I., Effects of Hypoxia on the Alveolar Epithelium, Proc. Am. Thorac. Soc., 2(3), pp. 202-205, 2005. DOI: 10.1513/pats. 200501-006AC

Pak, O., Aldashev, A., Welsh, D. & Peacock, A., The Effects of Hypoxia on the Cells of the Pulmonary Vasculature, Eur. Respir. J., 30(2), pp. 364-372, 2007. DOI:10.1183/09031936.00128706

Hayflick, L. & Moorhead, P.S., The Serial Cultivation of Human Diploid Cell Strains, Exp. Cell. Res., 25(3), pp. 585-621, 1961. DOI:10.1016/ 0014-4827(61)90192-6

Moroz, E., Carlin, S., Dyomina, K., Burke, S., Thaler, H.T. & Blasberg, R., Real Time Imaging of HIF-1α Stabilization and Degradation, 4(4), e5077, 2009. doi: 10.1371/journal.pone.0005077

Semenza, G. & Wang, G.A., Nucleur Factor Induced by Hypoxia via de Novo Protein Synthesis to the Human Erythropoietin Gene Enhancer at A Site Required for Transcriptional Activation, Mol. Cell. Biol., 12(12), pp. 5447-5454, 1992.

Uchida. T., Rossignol, F., Matthay, M.A., Mounier, R., Couette, S. & Clottes, E., Prolonged Hypoxia Differentially Regulates HIF1α and HIF2α Expression in Lung Epithelial Cells, J. Biol. Chem., 279, pp. 14871-14878, 2004. DOI:10.1074/jbc.M400461200

Rufini, A., Tucci, P., Celardo, I. & Melino, G., Senescence and Aging : the Critical Roles of p53, Nature, 32, pp. 5129-5143, 2013. DOI:10.1038/ onc.2012.640

Gianciti, C. & Giordano, A., RB and Cell Cycle Progression, Nature, 25, pp. 5220-5227, 2005. doi:10.1038/sj.onc.1209615

Graeber, T.G., Peterson, J.F., Tsai, M., Monica, K., Fornace, A.J. & Giaccia, A.J., Hypoxia Induces Accumulation of p53 Protein, but Activation of A G1-phase Checkpoint by Low Oxygen Conditions is Independent of p53 Status, Cell. Mol. Biol., 14(9), pp. 6264-6277, 1994.

Mizuno, S., Bogaard, H.J., Voelkel, N.F., Umeda, Y., Kadowaki, M. & Ameshima, S., Hypoxia Regulates Human Lung Fibroblast Proliferation via p53 Dependent and Independent Pathways, Respiratory Res., 10(17), pp. 14870-14878, 2009. DOI:10.1186/1465-9921-10-17

Danielsen, T., Hvidsten, M., Stokke, T. & Rofwstad, E.K., Hypoxia Induces p53 Accumulation in the S-phase and Accumulation of Hypophosphorylated Restinoblastoma Protein in all Cell Cycle Phase of Human Melanoma Cells, British Journal of Cancer, 78(12), pp. 1547-1558, 1998.

Amellem, O., Sandvik, J.A., Stokke, T. & Pettersen, E.O., The Retinoblastoma Protein-associated Cell Cycle Arrest in S-phase under Moderate Hypoxia is Disrupted in Cells Expressing HPV18 E7 Oncoprotein, British Journal of Cancer, 77(6), pp. 862-872, 1998.

Amellem, O. & Pettersen, E.O., Cell Inactivation and Cell Cycle Inhibition as Induced by Extreme Hypoxia, The Possible Role of Cell Cycle Arrest as A Protection Against Hypoxia-induced Lethal Damage, Cell Prolif., 24(2), pp. 127–141, 1991. DOI:10.1111/j.1365-2184.1991. tb01144.x

Chandel, N.S., McClintock, D.S., Feliciano, C.E., Wood, T.M., Melendez, J.A. & Rodriguez, A.M., Reactive Oxygen Species Generated at Mitochondrial Complex III Stabilize Hypoxia-inducible Factor-1 alpha during Hypoxia: A Mechanism of O2 Sensing, J. Biol. Chem., 275, pp. 25130-25138, 2005. DOI: 10.1074/jbc.M001914200

Guzy, R.D., Hoyos, B., Robin, E., Chen, H., Liu, L. & Mansfield, K.D., Mitochondrial Complex III is Required for Hypoxia-induced ROS Production and Cellular Oxygen Sensing, Cell Metabol, 1(6), pp. 401-408, 2005. DOI:10.1016/j.cmet.2005.05.001

Wu, C., Miloslavskaya, I., Demontis, S., Maestro, R. & Galaktionov, K., Regulation of Cellular Response to Oncogenic and Oxidative Stress by Seladin-1, Nature, 432, pp. 640-645, 2004. DOI:10.1038/nature03173

Feng, Z. , Lin, M. & Wu, R., The Regulation of Aging and Longevity: A New and Complex Network, Genes. & Cancer, 2(4), pp. 443-452, 2011. DOI:10.1177/1947601911410223



  • There are currently no refbacks.

View my Stats

Creative Commons License
This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License.