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L. I. Paraoan, L. Davies, D. G. Spiller, D. Gray, B. Damato, M. R. H. White, I. Grierson; Cell Death Effector PERP in Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2007;48(13):558.
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We have previously reported that down-regulation of PERP (p53 apoptosis effector related to PMP-22) correlates with the aggressive (monosomy 3) type of uveal melanoma. This study assessed the susceptibility of uveal melanoma cells to apoptosis induced by overexpression of PERP.
Green-fluorescent protein (GFP)-PERP fusion constructs were engineered and used to transfect uveal melanoma cell lines and control Hela cells. Western blot analysis was used to confirm the expression and stability of the GFP-PERP fusion proteins. Confocal fluorescence microscopy was used to determine the subcellular localization of the protein and to monitor the fate of newly synthesized GFP-PERP in transfected cells. Time-lapse microscopy and flow cytometry using Alexa Fluor 568-annexin-V conjugate and propidium iodide were employed to monitor apoptosis and cell viability in transfected and control (non-transfected and GFP-only-transfected) cells.
The GFP-PERP fusion protein (48kDa) was detected by both anti-PERP antibody and anti-GFP antibody in lysates of transfected cells. Expression of GFP-PERP fusion protein was observed on the plasma membrane of live uveal melanoma and HeLa cells, consistent with the predicted subcellular target for the PERP protein, based on the putative topology of the gene product. Overexpression of PERP caused a reduction in cell viability compared with control cells. Cells expressing GFP-PERP showed typical morphological features of apoptosis, including membrane blebbing and packaging of DNA into apoptotic bodies.
Overexpression of PERP has a pro-apoptotic effect on uveal melanoma cells and HeLa cells. Reduced expression and/or loss of function of PERP may, therefore, be part of a mechanism employed by uveal melanomas to evade p53-dependent apoptosis. The mechanism of action and effects of restoration of PERP function in uveal melanoma cells are currently further investigated by us.
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