Inhibitors of Bcl2-A1 in Bax/Bak -/- Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-02_Inhibitor_Dose_DryPowder_Activity
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 BIM. The primed cells still maintain a balance between anti and pro-apoptotic proteins, but rely on A1 to sequester BIM. ..more
BioActive Compounds: 4
Keywords: apoptosis, BH3 domain, Bcl2-A1, BIM, caspase, cancer
Primary Collaborator: Todd Golub, Broad Institute, email@example.com
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 BIM. The primed cells still maintain a balance between anti and pro-apoptotic proteins, but rely on A1 to sequester BIM.
Cells expressing low A1 or with Bax-Bak knocked out should not show caspase activation if the compound is A1-specific. An A1 inhibitor causes the release of A1-bound BIM, which activates BAX/BAK, and will not score in this counterscreen cell line because the downstream Bax/Bak element has been knocked out.
Expected Outcome: Compounds that cause caspase activation in this cell line are likely due to off-target effects or general toxicity and are not of further interest.
1. MEF cells with a Bax/Bak double deletion mutation are cultured in 150 mm TC dishes with 30 mls of growth media supplemented with 0.5-1 ug/ml blasticidin in a 37 deg C incubator (5% CO2). Use 30 ml media for a 150 mm dish. Do not 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 grown on T200 mm cell culture flasks are washed once with 1X PBS (Gibco), and digested with 1 ml (or 3 ml) 1X trypsin (CellGro Mediatech) for 1-2 minutes.
3. Add 10 ml complete growth media (RPMI-1640 (Cellgrow Mediatech), 10% heat inactivated FBS (Thermo), 1X pen/strep/glutamine (Gibco)) to the plate, mix cells and break clumps, then transfer the cells to a 50 ml 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), 30 ul/well (2500 cells/well), with Combi (Thermo) while gently stirring the media.
6. Pin transfer 100 nL of compound to the cells and incubate at 37 deg C, 5% CO2 and 95% humidity for 3 hours.
7. Remove the plate from the incubator and cool down to room temperature for 30 minutes.
8. Add 10 ul of 1:1 diluted CaspaseGlo (Promega) (diluted with 50 mM 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), ultrasensitive luminescence aperture, 0.1 s/well
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) 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 (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
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 (Clofoctol only) was set to a normalized activity value of 100.
Experimental wells values were scaled to this range. Due to the high activity of Clofoctol, small values of the resulting normalized compound wells can still indicate significant activity.
Due to the toxic nature of high levels of caspase activation, several compounds result in bell-shaped curves in which caspases rise at moderate concentration but fall at toxic higher concentrations. For curves of this shape, the decreasing portion of the curve was masked and sigmoidal curves were fit to the increasing data range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION(AC): AC50
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.
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -5% and < 5% at all tested concentrations, or
b) AC > AC_limit, or
c) 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
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 not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
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)