Androgen responsiveness of Renilla luciferase reporter vectors is promoter, transgene, and cell line dependent.
Renilla based reporters are frequently used as transfection controls for luciferase transcriptional reporter assays. However, recent evidence suggests that a commonly used reporter (HSV-thymidine kinase driven Renilla) is responsive to androgen receptor (AR) and glucocorticoid receptors in the presence of the cognate ligands, dihydrotestosterone (DHT) and dexamethasone (DEX), respectively [1]. We further validate this important technical difficulty by illustrating that in LNCaP prostate cancer cells, spurious Renilla luciferase activity is a function of (a) the promoter driving Renilla expression, (b) the presence of co-transfected transgenes, and (c) the androgen responsiveness of the cell line used. Using inhibitors of transcription and translation we showed that transcript interference or translational modulation is not a major means by which androgens affect Renilla luciferase activity. As luciferase reporter assays are a frequent means of studying transcriptional co-regulation in the highly androgen dependent LNCaP cell line, our data serves as a cautionary note that alternative normalization techniques should be employed to avoid misinterpretation of data.
Mulholland
David J
DJ
The Prostate Centre, Jack Bell Research, Vancouver, BC, Canada. djm@interchange.ubc.ca
Cox
Michael
M
Read
Jason
J
Rennie
Paul
P
Nelson
Colleen
C
D000728:Androgens / Q000494:pharmacology*; D000818:Animals; D037421:Anthozoa / Q000201:enzymology / Q000235:genetics; D001681:Biological Assay; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D008297:Male; D011471:Prostatic Neoplasms / Q000201:enzymology* / Q000473:pathology; D015203:Reproducibility of Results; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D000728:Androgens; D008156:Luciferases