The p53-inhibitor Pifithrin-α inhibits Firefly Luciferase activity in vivo and in vitro

While investigating the effects of pifithrin-α on the transcriptional activity of NF-κB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-α was observed both in vivo and in vitro. Pifithrin-α did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-α to active extracts inhibited this activity. Furthermore, pifithrin-α also inhibited recombinant firefly luciferase protein activity.Among its other biological activities, pifithrin-α is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.The tumour suppressor protein, p53 is one of the most intensively studied proteins throughout biomedical research. Due to its central role in genome surveillance, cell cycle arrest and apoptosis induction, compounds affecting this protein, either re-activating it or inactivating it, are of exceptional interest and use in the field of cancer, Alzheimer's disease, Parkinson's disease, stroke and brain trauma [1-3].In recent years, a chemical inhibitor of p53, Pifithrin-α(PFT-α), has been identified and used both in vitro and in vivo to investigate p53 function [4]. PFT-α reversibly inhibits p53-transcriptional activity, inhibiting p53-induced apoptosis, cell cycle arrest and DNA-synth