Dose response cell-based assay to measure STAT3 activation
Members of the signal transducer and activator of transcription (STAT) family of transcription factors mediate inflammation, cell survival, differentiation, and proliferation (1, 2). In response to stimuli such as growth factors and cytokines (1-3), cytosolic STATs are activated by phosphorylation by the Janus-activated kinases (Jaks), inducing STAT dimerization, nuclear translocation, and more ..
BioActive Compounds: 60
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frank, Dana Farber Cancer Institute
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 X01 MH079826-01
Grant Proposal PI: David Frank, Dana Farber Cancer Institute
External Assay ID: STAT3_ACT_LUMI_1536_EC50
Name: Dose response cell-based assay to measure STAT3 activation
Members of the signal transducer and activator of transcription (STAT) family of transcription factors mediate inflammation, cell survival, differentiation, and proliferation (1, 2). In response to stimuli such as growth factors and cytokines (1-3), cytosolic STATs are activated by phosphorylation by the Janus-activated kinases (Jaks), inducing STAT dimerization, nuclear translocation, and binding to specific enhancer elements in target genes (2). Although structurally similar, STAT proteins possess diverse biological roles (2). For example, STAT1 activity is pro-inflammatory, anti-proliferative and mediates the effects of IFN-gamma, while STAT3 activity is anti-inflammatory, pro-apoptotic, and mediates IL-6 signaling (2, 4). Studies showing that STAT3 is activated in breast and prostate cancers, that genetic inhibition of STAT3 reduces cell proliferation, survival, and wound healing (1, 4, 5), and that disrupting STAT3-EGFR interactions reduces tumor growth (6), suggest that STAT3 activation has broad cellular effects. As a result, the identification of selective STAT3 modulators may provide useful tools for exploring STAT3 biology.
1. Alvarez JV, Febbo PG, Ramaswamy S, Loda M, Richardson A, Frank DA. Identification of a genetic signature of activated signal transducer and activator of transcription 3 in human tumors. Cancer Res. 2005 Jun 15;65(12):5054-62.
2. Schindler C, Levy DE, Decker T. JAK-STAT signaling: from interferons to cytokines. J Biol Chem. 2007 Jul 13;282(28):20059-63.
3. Germain D, Frank DA. Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin Cancer Res. 2007 Oct 1;13(19):5665-9.
4. Levy DE, Darnell JE Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002 Sep;3(9):651-62.
5. Takeda K, Kaisho T, Yoshida N, Takeda J, Kishimoto T, Akira S.1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell- specific Stat3-deficient mice. J. Immunol. 161:4652-4660.
6. Buerger C, Nagel-Wolfrum K, Kunz C, Wittig I, Butz K, Hoppe-Seyler F, Groner B. Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells. J Biol Chem. 2003 Sep 26;278(39):37610-21.
STAT3, acute-phase response factor, APRF, transcription factor, HTS, assay, activation, activator, dose response, luciferase, luminescence, reporter, 1536 Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT3 activation" (PubChem AID 871), and that confirmed activity in a set of experiments entitled, "Confirmation cell-based high throughput screening assay to measure STAT3 activation" (AID 1267). The compounds selected for testing in this AID met the following criteria: 1) they were declared active in AID 871 and AID 1267; 2) they were declared inactive in a set of experiments entitled, "Primary cell-based high throughput screening assay to measure STAT1 activation"(AID 932); 3) they were inactive in a set of experiments entitled, "Counterscreen assay for STAT3 activators: cell-based high throughput assay to measure NF-kappaB activation" (AID 1309); and 4) they were inactive in a set of experiments entitled, "Counterscreen assay for STAT3 activators: cell-based high throughput assay to measure STAT1 activation" (AID 1318).
In this assay STAT3 activation was measured using a human U3A fibrosarcoma cell line that stably expresses a human STAT3::luciferase construct. This cell line is deficient in STAT1. Test compounds were screened for their ability to increase IL-6-mediated STAT3::luciferase reporter activity. Cells were exposed to test compounds, followed by treatment with IL-6 to activate STAT3 transcription. Changes in STAT3::luciferase activity were monitored by measuring well luminescence. As designed, a STAT3 activator will increase IL-6-mediated STAT3 transcription, thus increasing transcription of the luciferase reporter gene, leading to increased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal test concentration of 55.7 uM.
The activator and inhibitor dose response assays using STAT3::luciferase cells were run simultaneously. U3A cells were grown in T-175 flasks in Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v fetal bovine serum and antibiotics (50 ug/ml each of penicillin and streptomycin, and 100 ug/mL neomycin) at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).
Prior to the start of the assay, cells were resuspended at a density of 1.88 million cells/mL in phenol red-free growth medium, and filtered through a 0.7 micron filter. Next, 4 ul of cell suspension (7,520 cells per well) were dispensed into each well of 1536-well plates. The assay was started by immediately dispensing 28 nL of test compound in DMSO, or DMSO alone (0.6% final concentration) to the appropriate wells. Next, the plates were incubated for 1 hour at 37 degrees C (5% CO2, 95% RH). Then, 1 ul of human recombinant IL-6 (33 ng/mL final nominal EC80 concentration, set as 100% activation) was dispensed into all wells. The plates were then incubated for 6 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of SteadyLite HTS luciferase substrate at room temperature to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the ViewLux plate reader.
The percent activation was defined using the following mathematical formula:
% Activation = 100*(Test_Compound/Median_Low_Control)
Test_Compound is defined as the luminescence value of a well containing IL-6 and test compound.
Median_Low_Control is defined as the median luminescence value of wells containing IL-6 and DMSO.
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 55.7 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 55.7 uM. Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 micromolar were considered active.
Any compound with a percent activation value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activation value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
List of Reagents:
Dulbecco's Modified Eagle's Media I (Invitrogen, part 11965-092)
Dulbecco's Modified Eagle's Media, no Phenol Red (Invitrogen, part 21063-029)
Fetal Bovine Serum (Hyclone, part SH30088-03)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
Recombinant human IL-6 (Peprotech, part 200-06)
SteadyLite HTS Assay Kit (PerkinElmer, part 6016989)
T175 Flasks (Corning, part 431080)
1536-well plates (Greiner, part 789173)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the plate, compounds that non-specifically modulate STAT3 or luciferase activity, and compounds that quench or enhance luminescence within the well. All test compound concentrations reported are nominal; the specific test concentration for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)