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Raheela Awais, Lyndsay Davies, Luminita Paraoan; Characterizing the role of the p53 effector PERP in cell migration: Insights from uveal melanoma cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5326.
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© ARVO (1962-2015); The Authors (2016-present)
PERP (p53 apoptosis-effector related to PMP-22) is a tetra-span membrane protein specifically expressed during p53-mediated apoptosis. PERP also plays a pivotal role in cell-cell adhesion by enabling desmosome function. Previously, we identified PERP as an important molecular determinant of apoptosis in primary uveal melanoma (UM) tumours. We demonstrated that PERP is significantly down-regulated in the highly metastatic (monosomy 3)-type compared with the less aggressive (disomy 3) tumours. We hypothesize that down-regulation of PERP results in enhanced migratory and invasive properties of UM tumour cells due to decreased cell- cell adhesion in the absence of functional PERP.
PERP expression was characterized in UM cell lines OCM1, MEL202, MEL285 and MEL 290 using qPCR and western blotting. The effect of PERP expression on the migration of GFP-PERP transfected UM cells was assessed using an in vitro scratch assay and time-lapse microscopy, with non-transfected (NT) and GFP-only transfected cells as controls. The kinetic behaviour of cells was analyzed in real time with the Essen Bioscience Cell Player Migration and Kinetic Cell Invasion assay and data analyzed using IncuCyte software. Experiments were done in triplicates and statistical analysis was performed using the Student’s T-test.
Endogenous PERP expression was significantly reduced both at transcriptional and protein level compared to ARPE19 cells. Migration of NT and GFP-only transfected OCM1 cells led to the reduction in the gap created by the scratch. However, GFP-PERP transfected cells migrated significantly less than NT (p=0.005) or GFP-only cells (p=0.02). Kinetic quantification in four UM cell lines also demonstrated a reduction in cell migration capacity following GFP-PERP expression, with highest (71% reduction) in MEL202 and the lowest (13.5% reduction) in MEL285 compared to NT control.
The results are consistent with the hypothesis that reduced levels of PERP lead to enhanced migratory and invasive properties of UM tumour cells resulting in metastasis. Further studies will examine whether PERP’s role in cell adhesion in UM is via direct association with the focal adhesion assembly or by indirect regulation of focal adhesion components.<br /> Acknowledgements - The work has been supported by The Humane Research Trust, UK.
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