Luminescence Cell-Based Counter Screen to Identify Inhibitors of Cytokine Induced Apoptosis
The goal of this project is to identify probes that inhibit apoptosis induced by cytokines in the rat insulinoma cell line, INS-1E. The primary assay measures cell viability via ATP levels as a surrogate for apoptosis but does not measure apoptosis directly. A more selective assay is to measure the activity of a direct mediator of apoptosis, such as Caspase 3. INS1-E rat insulinoma cells are more ..
BioActive Compounds: 167
Keywords: Type I diabetes, INS-1E cells, Caspase, cytokine, apoptosis
The goal of this project is to identify probes that inhibit apoptosis induced by cytokines in the rat insulinoma cell line, INS-1E. The primary assay measures cell viability via ATP levels as a surrogate for apoptosis but does not measure apoptosis directly. A more selective assay is to measure the activity of a direct mediator of apoptosis, such as Caspase 3. INS1-E rat insulinoma cells are treated with 50 ng/mL interferon-gamma, 25 ng/mL Tumor necrosis factor-alpha and 10 ng/mL Interleukin-1-beta for 48 hours in the presence of 100 nL compounds that were leads from the primary screen. A range of concentrations of compound are used from 0.2 uM to 25 uM and IC50 values will be determined. Cells are assayed with Promega's Caspase Glo 3/7, a luciferase-based reagent for measurement of caspase activity.
A compound that prevents the induction of or reduces Caspase-3 activity will decrease the luminescence signal in a dose-dependent manner. These are the type of compounds that will be interesting for further studies. Some compounds will not prevent Caspase activation and will no longer be considered for the project. Compounds should have an IC50 of less than 10 uM.
The cytokines induce apoptosis in primary islet beta cells and in the model cell line, INS-1E cells. The primary assay measures cellular viability but not actvity of any components of the apoptosis pathway. This assay measures the levels of Caspase 3 and 7 activity in cells with a luminescence-based assay from Promega called Caspase Glo 3/7. It is known that cytokine treatment leads to a 3 to 4 fold increase in Caspase activity after 48 hours of treatment. INS-1E cells will be treated with cytokines and various concentrations of compounds for 48 hours. The compounds that decrease Caspase activity will be considered for additional studies. In addition, any compound that increases the luminescence signal due to structural properties will be eliminated because these tend to increase the level of luminescence in a dose-dependent manner. The positive control is suberoylanilide hydroxamic acid (SAHA).
Day 0: Collect cells and seed 8,000 cells/well of INS-1E rat beta-cell line in 30 uL using white-walled, bar coded, 384-well plates Corning 8867; incubate at 37 degrees C overnight
Day 1: Add 10 uL medium with cytokine cocktail to each well using the Combi (Cocktail is 250 mL INS1E media, 92.5 uL IFN-gamma, 32.5 uL TNF-alpha and 20 uL IL-1-beta).
Day 3: Add 20 uL Caspase-Glo 3/7 reagent to each well.
Agitate with 'Big Bear' for 15 seconds to maximize cell lysis. Incubate 1 hour at room temperature.
Use Perkin Elmer Envision to read plate luminescence with ultra sensitive settings.
RPMI 1640 (Phenol red free), 5% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50 uM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 ug/ml streptomycin
Cytokines: 10 ng/mL IL-1 beta (R&D Systems, 501-RL-050), 25 ng/mL TNF-alpha (R&D Systems, 410-MT-050), 50 ng/mL IFN-gamma (Sigma, I4777-.1MG)
Dose Data Analysis
Presence of controls: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
Expected outcome: Active compounds result in decreasing readout signal.
Highest concentration tested: 26 uM
Active concentration limit: 260 uM
In cases where the data points at the highest concentration tested were deemed invalid, the next-highest concentration values were used to determine the active concentration limit.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
Active Concentration: AC50
Active concentration values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
Active concentration values were calculated up to the active concentration limit.
Curves with constant fits near 0% activity are assumed to have active concentration values outside the active concentration limit.
Activity_Outcome = 1 (inactive) when
a) active concentration > active concentration limit, or
b) compound shows activity but in a direction opposite to the expected outcome
Activity_Outcome = 2 (active) when active concentration <= active concentration limit
Activity_Outcome = 3 (inconclusive) when
a) Curve fitting strategy resulted in a constant fit with activity >30% but <70%, or
b) The fit was not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE is set to 0 when compound is described as 'inactive' in this experiment; that is, curve fit is:
a) constant fit showing near 0% activity at all tested concentrations, or
b) active concentration is calculated to be greater than the active concentration limit, or
c) active but in the opposite direction of the expected outcome in these cases, values describing curve fitting parameters (Sinf, So, Hill Slope, log_AC50, log_AC50_SE) are set to null
PUBCHEM_ACTIVITY_SCORE is set to -10*Log(AC50), where AC50 is in molar, when compound is described as 'active' in this experiment.
Scores relate to active concentrations in this manner:
1 pM 120
1 nM 90
1 uM 60
1 mM 30
1 M 0
PUBCHEM_ACTIVITY_SCORE is set to 100 when compound is described as 'superactive' in this experiment, that is, curve fit is:
a) constant fit showing full inhibition at all tested concentrations, or
b) active concentration is calculated to be less then lowest tested concentration
* Activity Concentration. ** Test Concentration.
Data Table (Concise)