Purchase this article with an account.
L. Davies, D. Spiller, D. Gray, M. White, I. Grierson, L. Paraoan; Investigation of Apoptosis by p53 Effector PERP in Uveal Melanoma Cell Lines. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5553.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
PERP (p53 apoptosis effector related to PMP-22) is an apoptosis-specific effector of p53. We have previously reported that down-regulation of PERP correlates with the aggressive (monosomy 3) type of uveal melanoma. This study evaluated the ability of PERP to induce apoptosis in uveal melanoma and control cell lines.
Green-fluorescent protein (GFP)-PERP fusion constructs were engineered and used to transfect human uveal melanoma cells (92-1, MEL202 and OCM-1) and control (HeLa and ARPE19) cells. Expression of GFP-PERP and processing of initiator caspases-8, -9 and Bid protein were assessed by Western blotting. Confocal fluorescence microscopy was used to determine the subcellular localization of GFP-PERP in transfected cells. Cell viability and apoptosis were monitored by flow-cytometry and time-lapse microscopy in transfected and control (non-transfected and GFP-only-transfected) cells, using Alexa Fluor 647-annexin-V conjugate and propidium iodide in the presence and absence of the a pan-caspase inhibitor Z-VAD-FMK.
GFP-PERP fusion protein was targeted to the plasma membrane of living uveal melanoma and control cells, consistent with the predicted subcellular target for the PERP protein based on the putative topology of the gene product. Elevated levels of PERP expression caused an increase in susceptibility of uveal melanoma cells to apoptosis. Uveal melanoma cells expressing GFP-PERP showed typical morphological features of apoptosis, including membrane blebbing and packaging of DNA into apoptotic bodies, whereas GFP-PERP expressing cells exposed to Z-VAD-FMK exhibited normal cell morphology. Processing of caspase-8 and reduction of full-length Bid was detected in GFP-PERP expressing cells.
PERP expression triggers the death of uveal melanoma cells by apoptosis, which is mediated via the caspase-dependent apoptosis pathway. We have shown that elevated PERP expression is accompanied by processing of caspase-8 and Bid, suggesting activation of the extrinsic apoptotic pathway with possible engagement of the intrinsic pathway. 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 under further investigation by us.
This PDF is available to Subscribers Only