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Luminita I. Paraoan, David Spiller, Michael R. White, Ian Grierson, Lyndsay Davies; Enhancement Of P53-dependent Pro-Apoptotic Response By Perp In Uveal Melanoma Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5118.
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We have previously shown that PERP (p53 apoptosis effector related to PMP-22) protein levels influence the protein levels of its own transcriptional regulator p53 in uveal melanoma. Additionally, PERP expression causes nuclear localization of p53 that is critical for its transcriptional function. The purpose of this study was to investigate the functional status of the elevated p53 following PERP expression and to identify the potential pro-apoptotic genes activated.
Fluorescent fusion constructs of PERP fused to green fluorescent protein under a CMV promoter (GFP-PERP), and of MDM2 fused to yellow fluorescent protein under its human native promoter (MDM2-YFP) were used to transfect the human uveal melanoma cell line MEL202. The subcellular localization of PERP and MDM2 fluorescent fusion proteins in transfected cells was analysed by confocal microscopy and the expression level of MDM2 was analysed using ImageJ. Western blotting assessed the phosphorylation status of p53. Expression of p53-target genes in response to elevated PERP was evaluated by real-time quantitative PCR. Pifithrin-α was used to inhibit p53-dependent gene transcription.
The expression of MDM2-YFP was significantly higher in cells co-expressing GFP-PERP, and it was significantly reduced by pifithrin-α treatment. Phosphorylation of p53 at Ser20 and Ser46 was significantly increased in cells expressing elevated PERP protein. The p53 pro-apoptotic target genes DR4 and LRDD were significantly upregulated in response to PERP expression, but levels of the cell cycle arrest-related protein p21 remained unchanged.
Elevated PERP protein levels increase levels of transcriptionally active p53. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative regulator MDM2 and enhance specific pro-apoptotic gene transcription (DR4, LRDD) also occurs subsequent to PERP expression. Together, these results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.
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