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The role of the p53 protein in nitrosative stress-induced apoptosis of PC12 rat pheochromocytoma cells

Abstract

PC12 rat pheochromocytoma cells are widely used to investigate signaling pathways. The p143p53PC12 cell line expresses a Val143Ala mutant p53 protein that is less capable of binding to the p53 consensus site in DNA than its wild-type counterpart. Nitric oxide (NO), depending on its concentration, is able to activate several signal transduction pathways. We used sodium nitroprusside (SNP), an NO donor compound, to analyze NO-induced cellular stress in order to clarify the mechanism and role of nitrosative stress in pathological processes, including inflammation and cancer. SNP caused cell death when applied at a concentration of 400 μM, p143p53PC12 cells showing higher sensitivity than wild-type PC12 cells. The mechanisms leading to the increased SNP-sensitivity of p143p53PC12 cells were then investigated. The 400-μM SNP treatment caused stress kinase activation, phosphorylation of the eukaryotic initiation factor eIF2α and p53 protein, proteolytic activation of protein kinase R, caspase-9, and caspase-3, p53 stabilization, CHOP induction, cytochrome c release from mitochondria, and a decline in the level of the Bcl-2 protein in both cell lines. All these SNP-induced changes were more robust and/or permanent in cells with the mutant p53 protein. We thus conclude that (1) the main cause of the SNP-induced apoptosis of PC12 cells is the repression of the bcl-2 gene, evoked through p53 stabilization, stress kinase activation, and CHOP induction; (2) the higher SNP sensitivity of p143p53PC12 cells is the consequence of the stronger and earlier activation of the intrinsic apoptotic pathway.

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Acknowledgments

The authors are grateful to András Balogh, Mária Németh, and Ibolya Koloszár for their help throughout the experiments.

Author information

Correspondence to József Szeberényi.

Additional information

This work was supported by grants from the International Association for the Promotion of Cooperation with Scientists from the Independent States of the former Soviet Union (INTAS 51022) and the Science, Please! Research Team on Innovation (SROP-4.2.2/B-10/1-2010-0029). The purchase of the Olympus FluoView 1000 laser scanning confocal microscope was supported by a grant from the National Development Agency (GVOP-3.2.1-2004-04-0172/3.0).

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Varga, J., Bátor, J., Péter, M. et al. The role of the p53 protein in nitrosative stress-induced apoptosis of PC12 rat pheochromocytoma cells. Cell Tissue Res 358, 65–74 (2014). https://doi.org/10.1007/s00441-014-1932-7

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Keywords

  • PC12 cell line
  • Nitric oxide
  • Sodium nitroprusside
  • Apoptosis
  • p53 protein