Development of a high-throughput Gaussia luciferase reporter assay for the activation of the GADD45a gene by mutagens, promutagens, clastogens, and aneugens.
Exposure to genotoxic carcinogens leads to increased expression of the GADD45a gene in mammalian cells. This signature of genotoxic hazard has previously been exploited in the GreenScreen HC assay, in which GADD45a expression is linked to green fluorescent protein (GFP) expression in the human TK6 lymphoblastoid cell line. This article describes the development and validation of an alternative assay ("BlueScreen HC"), in which expression is linked to Gaussia luciferase (GLuc) expression, yielding a luminescent reporter, the preferred optical output in high-throughput screening. The coelentrazine substrate of GLuc is relatively unstable, and a new buffer is reported that improves its stability. A more sensitive method is demonstrated for the measurement of cell densities in the assay, using the fluorescent cyanine dye thiazole orange. A protocol amendment also allows the assessment of pro-genotoxicity using S9 liver extracts. Compounds from the European Centre for the Validation of Alternative Methods (ECVAM) recommended list for the assessment of new or improved genotoxicity assays were evaluated with and without S9 in the new assay. The new GLuc assay was as effective as the GFP assay in producing positive results for all classes of genotoxic carcinogen and negative results for all nongenotoxins tested.
Gentronix Ltd., CTF Building, Manchester, UK.
Chris
C
Hughes
Adam
A
Rabinowitz
Matthew
M
Tate
Louise
L
Birrell
Jodie
J
Allsup
Nicholas
N
Billinton
Richard M
RM
Walmsley
D052160:Benzothiazoles / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008156:Luciferases / Q000235:genetics; D049449:Luminescence; D009152:Mutagenicity Tests; D009153:Mutagens / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000235:genetics*; D011804:Quinolines / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D015533:Transcriptional Activation / Q000187:drug effects*; D000097642:GADD45 Proteins
D052160:Benzothiazoles; D018797:Cell Cycle Proteins; D008156:Luciferases; D009153:Mutagens; D009687:Nuclear Proteins; D011804:Quinolines; D054852:Small Molecule Libraries; C439070:GADD45A protein, human; D000097642:GADD45 Proteins; C051000:thiazole orange