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Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators.

Zanella F, Rosado A, Garcia B, Carnero A, Link W - BMC Cell Biol. (2009)

Bottom Line: 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.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.U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncologicas, Melchor Fernandez Almagro 3, 28029 Madrid, Spain. fzanella@cnio.es

ABSTRACT

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.

Conclusion: 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.

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Related in: MedlinePlus

Validation of U2transLUC assay using a panel of test compounds. A. The nuclear accumulation of the GFP-FOXO reporter protein and FOXO driven luciferase activity induced by the test compounds. We exposed U2transLUC cells to three different concentrations of the test compounds. The results shown here are the values obtained from the treatment of U2transLUC cells with 4 nM Ratjadone A, 20 μM LY294002, 1 ng/ml PDGF, 100 μM Minerval, 1 μM Forskolin, 4 nM Leptomycin B, 30 μM Cisplatin, 100 nM PI-103, 0.5 nM Rapamycin, 20 ng/ml IGF, 5 ng/ml EGF and Dimethyl sulfoxide (DMSO) as a negative control. The grey bar graphs and the corresponding left hand y-axis depict the values in relation to the control values normalized to the corresponding measure of viability values. The relative luciferase activity was calculated dividing the value obtained for Firefly luciferase activity for each well by the average GFP intensity from the same well. The right hand y-axis and the hatched bars indicate the percentage of cells in each well exhibiting nuclear/cytoplasmic (Nuc/Cyt) ratios of fluorescence intensity greater than 1.8 normalized to the percentage in DMSO-treated wells. B. Representative images of treated cells using high throughput format of the U2transLUC system. Images of cells expressing GFP-FOXO and stained with DRAQ5 were obtained by automated microscopy 2 hour after drug exposure. The images correspond to U2transLUC cells exposed to 4 nM Ratjadone A, 4 nM Leptomycin B, 20 μM LY294002, 100 nM PI-103 and DMSO. The images are shown from the GFP and DRAQ5 channels, as well as the corresponding merged image in the case of the DMSO treated control cells.
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Figure 4: Validation of U2transLUC assay using a panel of test compounds. A. The nuclear accumulation of the GFP-FOXO reporter protein and FOXO driven luciferase activity induced by the test compounds. We exposed U2transLUC cells to three different concentrations of the test compounds. The results shown here are the values obtained from the treatment of U2transLUC cells with 4 nM Ratjadone A, 20 μM LY294002, 1 ng/ml PDGF, 100 μM Minerval, 1 μM Forskolin, 4 nM Leptomycin B, 30 μM Cisplatin, 100 nM PI-103, 0.5 nM Rapamycin, 20 ng/ml IGF, 5 ng/ml EGF and Dimethyl sulfoxide (DMSO) as a negative control. The grey bar graphs and the corresponding left hand y-axis depict the values in relation to the control values normalized to the corresponding measure of viability values. The relative luciferase activity was calculated dividing the value obtained for Firefly luciferase activity for each well by the average GFP intensity from the same well. The right hand y-axis and the hatched bars indicate the percentage of cells in each well exhibiting nuclear/cytoplasmic (Nuc/Cyt) ratios of fluorescence intensity greater than 1.8 normalized to the percentage in DMSO-treated wells. B. Representative images of treated cells using high throughput format of the U2transLUC system. Images of cells expressing GFP-FOXO and stained with DRAQ5 were obtained by automated microscopy 2 hour after drug exposure. The images correspond to U2transLUC cells exposed to 4 nM Ratjadone A, 4 nM Leptomycin B, 20 μM LY294002, 100 nM PI-103 and DMSO. The images are shown from the GFP and DRAQ5 channels, as well as the corresponding merged image in the case of the DMSO treated control cells.

Mentions: The U2transLUC-based assay was formatted for 96-well plates and the workflow has been automated. To assess the specificity of the transcriptional and the translocational read-out, we challenged the U2transLUC system with small molecule inhibitors under living cell conditions in order to observe the translocation of the fluorescent reporter protein and luciferase activity as the transcriptional end point. We exposed U2transLUC cells to a panel of test compounds with known biological activity. Three different doses were used that resulted in a range of final concentrations greater than two orders of magnitude around the IC50 value for each individual compound. The live cells were stained with the fluorescent nuclear dye DRAQ5 and images were obtained on an automatic microscope. The nuclear/cytoplasmic (Nuc/Cyt) fluorescence intensity ratios were determined and the percentage of cells per well displaying nuclear translocation or the inhibition of nuclear export was calculated. Compounds that induced the nuclear accumulation of the fluorescent signal above 60% of that obtained from wells treated with LY294002 were considered as hits. Several compounds known to inhibit PI3K activity or the nuclear export machinery fulfilled these criteria (Fig 4A). As expected from the results of a previous study in U2foxRELOC cells [9,15], the PI3K inhibitors PI-103 and LY294002 were capable of inducing FOXO translocation into the nucleus of U2transLUC cells. By contrast, known activators of the PI3K/Akt pathway produced little nuclear localization of GFP-FOXO, including EGF, IGF and PDGF. The exposure of U2transLUC cells to the nuclear export inhibitors leptomycin B or ratjadone A provoked the nuclear accumulation of fluorescent signal, as shown previously for U2foxRELOC cells [9,15]. Conversely, through the measurement of the FOXO-dependent production of firefly luciferase, leptomycin B and ratjadone A exerted no significant effect on the transcriptional activity of FOXO (Fig 4B). Finally, exposure to PI-103 and LY294002 triggered a dramatic increase in FOXO-dependent luciferase activity. Together, these data show that the U2transLUC assay serves as an automated high throughput assay that can identify inhibitors of PI3K/Akt signaling through the high content analysis of protein translocation in conjunction with an independent analysis of the transcriptional activity of FOXO transcriptions factors.


Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators.

Zanella F, Rosado A, Garcia B, Carnero A, Link W - BMC Cell Biol. (2009)

Validation of U2transLUC assay using a panel of test compounds. A. The nuclear accumulation of the GFP-FOXO reporter protein and FOXO driven luciferase activity induced by the test compounds. We exposed U2transLUC cells to three different concentrations of the test compounds. The results shown here are the values obtained from the treatment of U2transLUC cells with 4 nM Ratjadone A, 20 μM LY294002, 1 ng/ml PDGF, 100 μM Minerval, 1 μM Forskolin, 4 nM Leptomycin B, 30 μM Cisplatin, 100 nM PI-103, 0.5 nM Rapamycin, 20 ng/ml IGF, 5 ng/ml EGF and Dimethyl sulfoxide (DMSO) as a negative control. The grey bar graphs and the corresponding left hand y-axis depict the values in relation to the control values normalized to the corresponding measure of viability values. The relative luciferase activity was calculated dividing the value obtained for Firefly luciferase activity for each well by the average GFP intensity from the same well. The right hand y-axis and the hatched bars indicate the percentage of cells in each well exhibiting nuclear/cytoplasmic (Nuc/Cyt) ratios of fluorescence intensity greater than 1.8 normalized to the percentage in DMSO-treated wells. B. Representative images of treated cells using high throughput format of the U2transLUC system. Images of cells expressing GFP-FOXO and stained with DRAQ5 were obtained by automated microscopy 2 hour after drug exposure. The images correspond to U2transLUC cells exposed to 4 nM Ratjadone A, 4 nM Leptomycin B, 20 μM LY294002, 100 nM PI-103 and DMSO. The images are shown from the GFP and DRAQ5 channels, as well as the corresponding merged image in the case of the DMSO treated control cells.
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Figure 4: Validation of U2transLUC assay using a panel of test compounds. A. The nuclear accumulation of the GFP-FOXO reporter protein and FOXO driven luciferase activity induced by the test compounds. We exposed U2transLUC cells to three different concentrations of the test compounds. The results shown here are the values obtained from the treatment of U2transLUC cells with 4 nM Ratjadone A, 20 μM LY294002, 1 ng/ml PDGF, 100 μM Minerval, 1 μM Forskolin, 4 nM Leptomycin B, 30 μM Cisplatin, 100 nM PI-103, 0.5 nM Rapamycin, 20 ng/ml IGF, 5 ng/ml EGF and Dimethyl sulfoxide (DMSO) as a negative control. The grey bar graphs and the corresponding left hand y-axis depict the values in relation to the control values normalized to the corresponding measure of viability values. The relative luciferase activity was calculated dividing the value obtained for Firefly luciferase activity for each well by the average GFP intensity from the same well. The right hand y-axis and the hatched bars indicate the percentage of cells in each well exhibiting nuclear/cytoplasmic (Nuc/Cyt) ratios of fluorescence intensity greater than 1.8 normalized to the percentage in DMSO-treated wells. B. Representative images of treated cells using high throughput format of the U2transLUC system. Images of cells expressing GFP-FOXO and stained with DRAQ5 were obtained by automated microscopy 2 hour after drug exposure. The images correspond to U2transLUC cells exposed to 4 nM Ratjadone A, 4 nM Leptomycin B, 20 μM LY294002, 100 nM PI-103 and DMSO. The images are shown from the GFP and DRAQ5 channels, as well as the corresponding merged image in the case of the DMSO treated control cells.
Mentions: The U2transLUC-based assay was formatted for 96-well plates and the workflow has been automated. To assess the specificity of the transcriptional and the translocational read-out, we challenged the U2transLUC system with small molecule inhibitors under living cell conditions in order to observe the translocation of the fluorescent reporter protein and luciferase activity as the transcriptional end point. We exposed U2transLUC cells to a panel of test compounds with known biological activity. Three different doses were used that resulted in a range of final concentrations greater than two orders of magnitude around the IC50 value for each individual compound. The live cells were stained with the fluorescent nuclear dye DRAQ5 and images were obtained on an automatic microscope. The nuclear/cytoplasmic (Nuc/Cyt) fluorescence intensity ratios were determined and the percentage of cells per well displaying nuclear translocation or the inhibition of nuclear export was calculated. Compounds that induced the nuclear accumulation of the fluorescent signal above 60% of that obtained from wells treated with LY294002 were considered as hits. Several compounds known to inhibit PI3K activity or the nuclear export machinery fulfilled these criteria (Fig 4A). As expected from the results of a previous study in U2foxRELOC cells [9,15], the PI3K inhibitors PI-103 and LY294002 were capable of inducing FOXO translocation into the nucleus of U2transLUC cells. By contrast, known activators of the PI3K/Akt pathway produced little nuclear localization of GFP-FOXO, including EGF, IGF and PDGF. The exposure of U2transLUC cells to the nuclear export inhibitors leptomycin B or ratjadone A provoked the nuclear accumulation of fluorescent signal, as shown previously for U2foxRELOC cells [9,15]. Conversely, through the measurement of the FOXO-dependent production of firefly luciferase, leptomycin B and ratjadone A exerted no significant effect on the transcriptional activity of FOXO (Fig 4B). Finally, exposure to PI-103 and LY294002 triggered a dramatic increase in FOXO-dependent luciferase activity. Together, these data show that the U2transLUC assay serves as an automated high throughput assay that can identify inhibitors of PI3K/Akt signaling through the high content analysis of protein translocation in conjunction with an independent analysis of the transcriptional activity of FOXO transcriptions factors.

Bottom Line: 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.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.U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncologicas, Melchor Fernandez Almagro 3, 28029 Madrid, Spain. fzanella@cnio.es

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus