A375 melanoma cells and CHO cells were obtained from ATCC (Manassas, VA) and propagated as recommended. The CellSensor® p53RE-bla HCT-116 cell line was purchased from Invitrogen (Carlsbad, CA) and cultured under continuous selection with Blasticidin. Cells were maintained at 37 °C, with 5% CO2 and 90–95% humidity in a tissue culture incubator.
The Maybridge HitFinder library
The Maybridge HitFinder library was purchased from Thermo-Fisher Scientific (Waltham, MA). It consists of 14,400 premier compounds representing drug-like diversity. All screening compounds fit Lipinski guidelines for “drug-likeness”, and all have purity greater than 90%. The library is arrayed in 45 384-well microtiter plates and is available for screening at 1 mM and 0.1 mM stock concentrations. These are individually designed compounds, produced by innovative synthetic techniques and have the potential for structural alterations to increase in efficacy and decrease potential toxicity.
High throughput screening
We screened Maybridge HitFinder library, a collection of 14,400 compounds, for their ability to (1) cause selective death of MAGE positive cells, (2) activate p53 in MAGE positive cells, and (3) cause death with low or no activation of caspase. High throughput screening was performed at the Keck Small Molecule screening Facility at the University of Wisconsin Comprehensive Carbone Center (UWCCC), Madison, WI.
p53 activation screen
We used a p53 responsive assay with a quantitative β-lactamase read out that employs CellSensor® p53RE-bla HCT-116 cell line (Invitrogen, Carlsbad, CA). CellSensor® p53RE-bla HCT-116 cell line has been modified from parental HCT-116 human colon cancer cell line by stable integration of a beta-lactamase reporter gene under control of the p53 Response Element (p53RE). Modulation of p53 pathway can be measured selectively and quantitatively as β-lactamase reporter activity. Cells were plated in 384 well plates and next day were treated with compounds at 10 μM concentration. 24 h later, β-lactamase assay was performed according to manufacturer’s directions. Briefly, cells were loaded with Live-BLAzer™-FRET B/G Substrate (Invitrogen, Carlsbad, CA) for 2.5 h and fluorescence emission values at 460 nm and 530 nm were obtained using a fluorescence plate reader. Live-BLAzer™-FRET B/G Substrate is an engineered fluorescent substrate containing two fluorophores, coumarin and fluorescein. In the absence of β-lactamase activity, the substrate molecule remains intact and excitation of coumarin results in fluorescence resonance energy transfer to the fluorescein moiety and emission of green light. However, in presence of β-lactamase activity, the substrate is cleaved, separating the fluorophores and disrupting energy transfer. Excitation of the coumarin in the presence of beta-lactamase enzyme activity results in a blue fluorescence signal. The resulting coumarin-to-fluorescein ratio provides a normalized reporter response represented as Relative Fluorescence Units (RFU).
Cytotoxicity of the compounds was estimated by CytoTox-Glo Cytotoxicity Assay (Promega Corporation, Madison, WI), a luminescent assay that estimates dead cells ina population. The assay isbased on the activity of intra-cellular proteases that are released when cell membranes become porous in dying or dead cells. In this assay, the peptide substrate used for these proteases, AAF-aminoluciferin (alanyl-alanylphenylalanyl-aminoluciferin), is impermeable and hence can be utilized only by extracellular proteases. The lysed aminoluciferin is luminescent and can be estimated by a luminometer. Cytotoxicity results are represented as Relative Luciferase Units (RLU).
We have previously shown that apoptosis induced by MAGE knockdown is caspase independent. In order to eliminate compounds that cause cell death by caspase dependent mechanisms, therefore, probably not acting via MAGE-KAP-1 interactions, we measured caspase activity by Caspase-Glo® 3/7 Assay (Promega Corporation, Madison, WI). We chose caspase 3/7 assay because caspase 3 is a key mediator of caspase dependent apoptosis in mammalian cells. This is a luminescent assay that utilizes the caspase-3/7 DEVD-aminoluciferin substrate. Degradation of this substrate by active caspase 3/7 decreases luminescence. Results are represented as Relative Luciferase Units (RLU).
Mammalian two hybrid assay
We used a modified mammalian two hybrid assay to directly measure binding of MAGE-A3 or C2 to KAP-1 and if the compounds interfere with this interaction. We modified a commercially available mammalian two hybrid assay (Matchmaker™ Mammalian Two Hybrid Assay Kit, Clontech, Mountain View, CA) by replacing the promoter of the bait plasmids with a stronger CMV promoter, to drive expression of the bait fusion proteins, resulting in increased sensitivity and reproducibility. pM plasmid (bait), empty or expressing MAGE-C2, MAGE-A3, or the MHD regions of MAGEC2 and MAGE-A3, were cloned under CMV promoter in p3xflag-CMV plasmid (Sigma–Aldrich, St Louis, MO). Similarly, KAP-1 or RBCC region of KAP-1 were cloned in the pVP-16 plasmid (prey). To quantify the interaction of MAGE-A3 or C2 with KAP-1, and to determine if the compounds interfere with this interaction, MHD region of MAGE-A3 or C2 was used as bait and the RBCC region of KAP-1 was used as prey. The interaction was measured by the secreted alkaline phosphatase (SEAP) assay using the Phospha-Light™ Secreted Alkaline Phosphatase Reporter Gene Assay System (Applied Biosystems/Life Technologies Corporation, Carlsbad, CA) performed according to manufacturer’s directions. Briefly, CHO cells were co-transfected with pM-3XFLAG-CMV, pVP16 and pG5SEAP vectors with Lipofectamine reagent (Invitrogen, Carlsbad, CA). The next day, cells were treated with various concentrations of compounds and 48 h later, SEAP activity was estimated. The output was measured as secreted alkaline phosphatase (SEAP) and analyzed by a luminescent substrate.
A375 melanoma cells were treated with compounds at 10 μM concentration and collected after 24 h. Cells were lysed in RIPA buffer and subjected to SDS–PAGE. Membranes were probed with MAGE-C2 and Actin antibodies (SantaCruz Biotechnology, Santa Cruz, CA).
Protein lysates (500 μg) were immunoprecipitated with MAGE-C2 antibody (SantaCruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were subjected to SDS–PAGE and immunoblotted with antibodies for KAP-1 (Novus Biologicals, Littleton, CO).