Abstract
Purpose :
Downregulation of PERP (p53 apoptosis effector related to PMP22) is a determinant of impaired apoptosis in aggressive monosomy 3 Uveal Melanoma (UM). The functional importance of PERP downregulation in this scenario is further supported by the requirement of chromosome 3-localised p63 for PERP transcription. Given the role of p63-mediated signalling in inflammatory responses, we hypothesised that altered levels of PERP influence the major NFκB inflammatory signalling pathway.
Methods :
The effect of increased PERP expression on NFκB-related proteins was investigated in the human UM cell line Mel202. Changes of specific NFκB proteins and gene expression were assessed in cells transiently transfected with GFP-tagged PERP for 24 hours using Proteome (R&D Systems) and TaqMan (ThermoFisher) NFκB Pathway Arrays. A total of 45 proteins including 4 serine or tyrosine phosphorylation sites and 92 genes, respectively, were analysed. Phosphorylated and non-phosphorylated I Kappa B Alpha (p-IκBα/IκBα) protein expression was determined by immunoblotting.
Results :
Following increased PERP expression, protein levels of the transcription factors cRel, p65, p105/p50 and p100/p52 decreased compared to GFP-only control by 0.63-fold, 0.63-fold, 0.85-fold and 0.73-fold, respectively, 24 hours post-transfection (PT). Decreased protein levels mirrored gene expression changes of cRel, p65 and p105/p50, with the exception of p100/p52 which showed an increase by 4.4-fold. While the increase in PERP level did not lead to changes in non-phosphorylated IκBα protein expression at 24 or 48 hours PT, p-IκBα level showed a significant decrease by 0.61-fold (+/-0.042) followed by a significant increase by 1.4-fold (+/-0.019) after 48 hours (One-Way ANOVA +/-SEM, p<0.05, n=3).
Conclusions :
The results support the hypothesis that increased PERP expression in UM cells affect gene/protein expression related to NFκB signalling pathway. The initial response is characterised by a decrease in transcription factors involved in the translocation and activation of NFκB, corroborated by a decrease in p-IκBα, therefore retaining NFκB in an inactive state, which is conceivably in line with the non-inflammatory characteristics of apoptosis. The subsequent increase of p-IκBα level suggests differences between an early and late response in relation to induction of apoptosis by PERP.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.