Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators.
BACKGROUND
Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.


RESULTS
The U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers. The U2transLUC assay measures nuclear-cytoplasmic FOXO shuttling and FOXO-driven transcription, providing a means to analyze these two key features of FOXO regulation in the same experiment. We challenged the U2transLUC system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity.


CONCLUSIONS
Combining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. The U2transLUC assay facilitates the maintenance and monitoring of homogeneous FOXO transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.
Zanella
Fabian
F
Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncologicas, Melchor Fernandez Almagro 3, 28029 Madrid, Spain. fzanella@cnio.es
Rosado
Aranzazú
A
Garcia
Beatriz
B
Carnero
Amancio
A
Link
Wolfgang
W
D001483:Base Sequence; D001693:Biological Transport, Active; D002460:Cell Line; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical / Q000379:methods; D051858:Forkhead Transcription Factors / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic; D014162:Transfection
D051858:Forkhead Transcription Factors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases