Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate pro-apoptotic inducer Measured in Cell-Based System Using Plate Reader - 2045-08_Inhibitor_Dose_DryPowder_Activity_Set2
The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID. In this assay, the parental control cells do not depend on A1 for survival. However, they can be primed to depend on A1 by co-expressing A1 and tBid. The primed cells still maintain a balance between anti and pro-apoptotic proteins, but rely on A1 to sequester tBid. ..more
BioActive Compounds: 5
Keywords: apoptosis, BH3 domain, Bcl2-A1, BIM, tBid, caspase, cancer
Primary Collaborator: Todd Golub, Broad Institute, firstname.lastname@example.org
The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID. In this assay, the parental control cells do not depend on A1 for survival. However, they can be primed to depend on A1 by co-expressing A1 and tBid. The primed cells still maintain a balance between anti and pro-apoptotic proteins, but rely on A1 to sequester tBid.
An A1 inhibitor causes the release of A1-bound tBid, which activates BAX/BAK, and leads to caspase activation that can be quantitatively measured using a luciferin-linked caspase substrate (peptide sequence DEVD) available commercially as Promega's Caspase Glo 3/7 reagent.
Expected Outcome: Compounds that cause caspase activation will show an increase in luminescence signal as measured by the caspase glo reagent. Additional assays will determine whether this caspase activation is being caused by general toxicity or off target effects (as is the case for the nonspecific positive control, clofoctol) or due to the on-target disruption of the A1-tBid interaction (as is the case for the specific but weaker positive control, ABT-263, which mimics the BH3 homology domain of tBid.) Compounds active in both this assay and the primary assay using BIM overexpression are particularly interesting since they are likely working through A1 inhibition rather than through activation of one or the other pro-apoptotic proteins BIM or tBid.
1.MEF cells expressing A1-2A-tBid are cultured in 150mm TC dishes with 30mls of growth media supplemented with 0.5-1 ug/ml blasticidin in a 37oC incubator (5% CO2). Use 30 ml media for a 150 mm dish. Do let cells go beyond 95% confluency (about 30X10^6 cells per 150mm dish). Split cells 1 to 6-10 (3-4X10^6 cells) for subsequent passage every other day.
DAY 1 MORNING
2.MEF cells expressing A1-2A-tBid grown on T200 mm cell culture flasks are washed once with 1XPBS (Gibco), and digested with 1ml (or 3ml) 1X trypsin (CellGro Mediatech) for 1-2 minutes.
3.Add 10ml complete growth media (RPMI-1640 (Cellgrow Mediatech), 10% heat inactivated FBS (Thermo), 1X penn/strep/glutamine (Gibco)) to the plate, mix cells and break clumps, then transfer the cells to a 50ml centrifuge tube through a cell strainer (BD Falcon # 352340) to get rid of any clumps. Count the cells, and centrifuge cells at 1000 rpm for 4 minutes.
4.Aspirate off the supernatant, and resuspend the cells in complete media at density of 1X10^5 cells/ml.
5.Plate cells in white 384 well plates (Corning 3570), 30ul/well (2500 cells/well), with Combi (Thermo) while gently stirring the media. Add 2 uL TSA.
6.Pin transfer 100 nL of compound to the cells and incubate 37 degrees 5% CO2 95% humidity for 3 hours.
7.Remove the plate from the incubator and cool down to room temperature for 30 minutes.
8.Add 10ul of 1:1 diluted CaspaseGlo (Promega) (diluted with 50mM HEPES) to each well with Combi multidrop (Thermo.) Shake the plate on the combi nest for 1 minute. Incubate at room temperature for 1h.
9.Measure luminescence in Envision (Perkin Elmer), std lum, 0.1s/well
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=9) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal Max_Concentration.
This assay measures caspase activation. At high levels of caspase activation, cell death is expected to occur, decreasing the readout and resulting in bell shaped curves for potent compounds. For this reason, higher concentration data points that showed a decrease following an increase were masked and curves were fit to the increasing portion of the curve only.
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 10.0. 10% was used because the positive control in these experiments, Clofoctol, causes very high levels of caspase activation (15-20x) while as little as 2 fold activation is biologically relevant. Therefore, 10% of the Clofoctol signal was considered a threshold for significant activation. It was necessary to use such a potent control to ensure activity across all cell lines and allow normalization in all experiments.
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_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 deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)