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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

A Dual-Color Fluorescence-Based Platform to Identify Selective Inhibitors of Akt Signaling

Background: Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells. Methodology/Principal Findings: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence. Conclusions/Significance: BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of smal

The analysis of Akt phosphorylation in BaFiso cell lines.

<p>Immunoblot analysis of total lysates from the Ba/F3 derived cell lines BY, BC, BYA and BCS. Cells were seeded, grown to 80% confluence and starved for 12 h in IL-3 free medium (−IL-3) or maintained in medium containing 3 ng/ml of the recombinant cytokine (+IL-3). Relevant proteins are indicated by arrows in the blot from a representative experiment.</p

Schematic overview of the BaFiso assay system.

<p>BaFiso consists of paired isogenic cell lines that have been engineered to acquire IL-3 autonomous growth through constitutive activation of Akt or Stat5 signaling. The two cell lines to be compared are individually tagged with either yellow or cyan fluorescent proteins. Equal numbers of yellow and cyan cells were co-cultured, treated with compounds and the change in the relative cell number was calculated on the basis of the distinct fluorescent proteins measured. Our strategy aims to identify lead compounds that specifically kill test cells with activated Akt signaling (yellow cells) and that spare the otherwise isogenic control cells (cyan cells).</p

The viability of BaFiso cell lines in the absence of IL-3.

<p>(A) Parental Ba/F3 derived BY and BC cells, and the BaFiso cell lines BYA and BCS were maintained in the presence or absence of IL-3 (+IL3 and −IL3). Photos were taken 24 h after transferring the cells to medium without IL-3 or in the presence of 3 ng/ml of the recombinant cytokine. (B) Measurement of cell viability using the Alamar blue assay. Alamar blue fluoresces and changes color in response to chemical reduction, and the extent of the conversion is a reflection of cell viability. Metabolic conversion of Alamar blue to its reduced, pink derivative upon cytokine-deprivation (−IL-3) or in its presence (+IL-3). (C), Bar graph showing the results of Alamar Blue cell viability assay. Maximal absorbance of the reduced and oxidized forms of AlamarBlue™, 570 and 600 nm was measured using Victor 1420 multilabel counter 24 h after IL-3 withdrawal. The percentage of cell survival was calculated compared with control cells in the presence of 3 ng/ml of IL3. The data represents three independent experiments performed in triplicate samples. (D) Time course of cell viability upon IL-3 withdrawal. Cells were washed twice in PBS and seeded in media lacking IL-3. Viability was assessed at 12 hour intervals by trypan blue exclusions followed by cell countings. Black rhombs and open squares represent percentage viability of BY and BC cells, respectively. Open triangles and black circles represent percentage viability of BYA and BCS cells, respectively. Data are presented as mean±SD from three independent experiments.</p

Validation of BaFiso assay using a panel of test compounds.

<p>(A) Analysis of the general toxicity of compound treatment. The total cell numbers in each well were determined by nuclear counterstain with the far-red fluorescent DNA probe DRAQ5. The number of DRAQ5-stained nuclei was determined after exposure to 30 µM Cisplatin (Cis), 100 µM Minerval (Min), 500 nM Akt Inhibitor X (AIX), 20 µM Genistein (Gen), 1 nM Leptomycin B (LMB), 20 nM Staurosporine (Stau), 1 µM UCN-01, 20 nM Raf1 Kinase Inhibitor (RKI), 20 µM LY294002 (LY), 10 µM Etoposide (Eto) and 1 mM lithium chloride (LiCl) for 12 hours and compared to vehicle treatment. (B) Equal numbers of BCS and BYA cells were co-cultured in IL-3-free medium. We exposed the paired BaFiso cell lines to 3 µM, 30 µM and 300 µM Cisplatin (Cis), 25 µM, 100 µM, 200 µM Minerval (Min), 50 nM, 500 nM, 5 µM Akt Inhibitor X (AIX), 200 nM, 20 µM, 50 µM Genistein (Gen), 0.5 nM, 1 nM, 4 nM Leptomycin B (LMB), 2 nM, 20 nM, 10 µM Staurosporine (Stau), 200 nM, 1 µM, 10 µM UCN-01; 5 nM, 20 nM, 200 nM Raf1 Kinase Inhibitor (RKI), 1 µM, 20 µM, 50 µM LY294002 (LY), 100 nM, 10 µM, 100 µM Etoposide (Eto) and 100 µM, 1 mM, 10 mM lithium chloride (LiCl), and Dimethyl sulfoxide (DMSO) as a negative control (striped bar). Three images specific for ECFP, EYFP or DRAQ5 from each well were acquired using BD Pathway Bioimager. The ECFP/EYFP ratio was determined by dividing the number of ECFP positive cells by the number of EYFP positive cells. Light, dark grey and black bars represent low, medium and high concentrations of the corresponding compounds, respectively. The data shown here represents three independent experiments. The average Z' value for BaFiso was 0.53. (C) Untreated, co-cultured BaFiso cells imaged before exposure to Akt Inhibitor X and (D) 12 h after treatment with 5 µM AIX.</p

The generation of BaFiso cell lines.

<p>Ba/F3 cells were transduced with retroviral supernatant carrying pBabePuro-EYFP or pBabePuro-ECFP. Cell clones were established and sorted in a fluorescence activated cell sorter (FACS) to generate lines homogeneously expressing the corresponding fluorescent tags. (A) and (C), viable Ba/F3 cells show robust and homogeneous expression of the respective fluorescent protein. (B) and (D), corresponding light field views. (E), Generation of stable BaFiso cell lines. Clonal Ba/F3 cells stably expressing EYFP (BY) or ECFP (BC) were used to generate stable BaFiso cell lines that co-express yellow fluorescence and myr-Akt (BYA), or cyan fluorescence and STAT5A1*6 (BCS). Cell clones were established and analyzed. (F), Analysis of transgene expression and downstream activation of the corresponding signaling pathways by western blotting. Antibodies against total Akt, Stat5a, Flag phospho-Akt (Ser473), phospho-p70 S6 (Thr389) and Pim-1 were used and the signals normalized to the respective α-tubulin levels.</p