Counterscreen of A1 inhibitors in HMC1-8 Mcl1 dependent cells, viability readout Measured in Cell-Based System Using Plate Reader - 2045-12_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, but are naturally dependent on a different anti-apoptotic protein (Mcl1) to sequester these anti-apoptotic factors. ..more
BioActive Compounds: 9
Keywords: apoptosis, BH3 domain, Bcl2-A1, BIM, 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, but are naturally dependent on a different anti-apoptotic protein (Mcl1) to sequester these anti-apoptotic factors.
Cells expressing low A1 or with Bax-Bak knocked out should not show cell death after 24 hours if the compound is A1-specific. This cell line, HMC 1-8, is a human cancer cell line with low levels of A1 expression and is immortalized due to factors other than the anti-apoptotic A1.
Expected Outcome: Compounds that cause cell death in this HMC 1-8 low A1 line are most likely not working through A1 inhibition. Compounds significant decrease in viability after 24 hours in this line, indicated by a decrease in luminescence as measured by the CellTiterGlo reagent, are not of further interest.
1. Human cancer cell line HMC 1-8 are cultured in 150mm TC dishes with 30mls 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 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. HMC 1-8 cells 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.
6. Pin transfer 100 nL of compound to the cells and incubate 37 degrees 5% CO2 95% humidity for 24 hours.
7. Remove the plate from the incubator and cool down to room temperature for 30 minutes.
8. Add 30ul of 1:1 diluted CaspaseGlo (Promega) (diluted with 1x PBS) to each well with Combi multidrop (Thermo.) Incubate at room temperature for 1h.
9. Measure luminescence in Envision (Perkin Elmer), US 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 decreasing 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 'Neutral Controls Minus Inhibitors' 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): 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.
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 >= -70% but <= -30%, 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)