The p53-inhibitor pifithrin-alpha inhibits firefly luciferase activity in vivo and in vitro.
BACKGROUND
Pifithrin-alpha is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-alpha in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-alpha provides a useful tool in the laboratory to investigate the function of p53 in model systems.


RESULTS
While investigating the effects of pifithrin-alpha on the transcriptional activity of NF-kappaB, 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-alpha was observed both in vivo and in vitro. Pifithrin-alpha did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-alpha to active extracts inhibited this activity. Furthermore, pifithrin-alpha also inhibited recombinant firefly luciferase protein activity.


CONCLUSIONS
Among its other biological activities, pifithrin-alpha 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.
Rocha
Sonia
S
School of Life Sciences, Division of Gene Regulation and Expression MSI/WTB, Complex Dow Street University of Dundee Dundee, DD1 5EH, Scotland, United Kingdom. s.rocha@dundee.ac.uk
Campbell
Kirsteen J
KJ
Roche
Kevin C
KC
Perkins
Neil D
ND
D000818:Animals; D052160:Benzothiazoles; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics; D001517:Coleoptera / Q000201:enzymology; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D013844:Thiazoles / Q000494:pharmacology*; D014050:Toluene / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors*
D052160:Benzothiazoles; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; D014050:Toluene; C121565:pifithrin; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase