Bookmark and Share
BioAssay: AID 1447

Identification of compounds which are cytotoxic to PPC-1 cells.

This assay is a counter screen for AID 1443, "uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cells". ..more
_
   
 Tested Compounds
 Tested Compounds
All(1248)
 
 
Active(618)
 
 
Inactive(630)
 
 
 Tested Substances
 Tested Substances
All(1248)
 
 
Active(618)
 
 
Inactive(630)
 
 
 Related BioAssays
 Related BioAssays
AID: 1447
Data Source: Burnham Center for Chemical Genomics (BCCG-A125-TRAIL-Cytotox-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2008-12-10
Modify Date: 2010-12-29

Data Table ( Complete ):           Active    All
BioActive Compounds: 618
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1443uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cellsscreening
1756SAR analysis of compounds cytotoxic to MDA-MB-435 cells.confirmatory
1765Mechanism of action of compounds that potentiate TRAIL-induced apoptosis in PPC-1 cellsother
1787Isobologram analysis of chemical compounds and TRAIL to sensitize TRAIL-resistant tumor cells to TRAILother
1773Comparison of the effects of compounds on TRAIL-mediated extrinsic apoptotic pathway in TRAIL-resistant MDA-MB-435 cells.other
1774Comparison of the effects of compounds on apoptotic pathways (intrinsic and ER stress pathway) in TRAIL-resistant MDA-MB-435 cells.other
2619SAR analysis of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435.other
1745Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in normal 184B5 cellsother
1752SAR analysis of compounds that potentiate TRAIL-induced apoptosis in PPC-1 cells.confirmatory
1755SAR analysis of compounds that potentiate TRAIL-induced apoptosis in MDA-MB-435 cells.confirmatory
1748Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MCF7other
1754SAR analysis of compounds cytotoxic to PPC-1 cells.confirmatory
1624Confirmation screening of compounds that potentiate TRAIL-induced apoptosis of cancer cells.confirmatory
1640Summary of compounds that potentiate TRAIL-induced apoptosis of cancer cells.summary2
1746Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in primary human hepatocytes.other
1747Confirmation screening of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435other
2647SAR analysis of compounds that potentiate TRAIL-induced apoptosis in TRAIL-resistant cancer cells, MDA-MB-435/MDR.other
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Production Centers Network (MLPCN)
Grant Number: X01 MH083230-01
Assay Provider: Dr. Dmitri Rosenoff, Sanford-Burnham Medical Research Institute, San Diego CA

This assay is a counter screen for AID 1443, "uHTS for the identification of compounds that potentiate TRAIL-induced apoptosis of cancer cells".

The goal of this assay is to determine if compounds are cytotoxic to PPC-1 cells. The assay uses a luminescence readout via ATPlite (Perkin Elmer) and is based on the ATP content within cells. The more viable cells there are in a well the more ATP is available, and subsequently the higher the signal in the well. Compounds which are cytotoxic will cause a drop in the luminescent signal.

References:
Bodmer JL, Meier P, Tschopp J, Schneider P. Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J Biol Chem 2000, 275:20632-7.

Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D, Mounho B, Hillan K, Totpal K, DeForge L, Schow P, Hooley J, Sherwood S, Pai R, Leung S, Khan L, Gliniak B, Bussiere J, Smith CA, Strom SS, Kelley S, Fox JA, Thomas D, Ashkenazi A. Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. Nat Med 2001, 7:383-5.

Singh TR, Shankar S, Chen X, Asim M, Srivastava RK. Synergistic interactions of chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand on apoptosis and on regression of breast carcinoma in vivo. Cancer Res 2003, 63:5390-400

Greil R, Anether G, Johrer K, Tinhofer I. Tracking death dealing by Fas and TRAIL in lymphatic neoplastic disorders: pathways, targets, and therapeutic tools. J Leukoc Biol 2003, 74:311-30.

Smyth MJ, Takeda K, Hayakawa Y, Peschon JJ, van den Brink MR, Yagita H. Nature's TRAIL-on a path to cancer immunotherapy. Immunity 2003, 18:1-6.
Protocol
Assay materials:

1) PPC-1 cells were provided by the assay provider
2) ATPLite (Perkin Elmer)

uHTS protocol:

Day 1
1) PPC-1 cells are grown in DMEM without phenol red, 10% FBS, 2mM L-glutamine, 1mM Na-pyruvate, 15ug/ml gentamycin and are harvested at 100% confluency.
2) Using a Biomek FX cells are seeded into a 1536 well plate (Corning #3727) at a concentration of 250 cells/well in 5ul. Cells are seeded in columns 3 through 48. Media is added to wells in columns 1 and 2 (Positive control).
3) Plates are spun in an Eppendorf model 5810 centrifuge @ 500 RPM for 5min.
4) Plates are placed in stacks of 6 (with a dummy plate filed on the top and bottom of each stack) and wrapped in Saran Wrap. This helps to eliminate any edge effects.
5) Incubate overnight in 37 oC 5% CO2 incubator

Day 2
1) Unwrap plates
2) Add 2.5 nl of 10mM compound to wells (columns 5-48).
3) Add 2.5 nl of 100% DMSO to wells in columns 1 through 4.
4) Plates are spun in an Eppendorf model 5810 centrifuge @ 500 RPM for 5min
5) Plates are placed in stacks of 6 (with a dummy plate filed on the top and bottom of each stack) and re-wrapped in Saran Wrap.
6) Incubate overnight in 37 oC 5% CO2 incubator

Day 3
1) Unwrap plates
2) Add 3ul of ATPlite to each well
3) Spin plate in a Velocity11 Vspin
4) Shake plate
5) After 20 min read luminescence using a Perkin Elmer Viewlux (read time= 10sec, filter=clear, gain=high, binning=2x)
Comment
Compounds that reconfirmed with an average of >=50% activity are defined as actives in this assay, thus are considered cytotoxic.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay demonstrated by a compound at 5 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
Active compounds will have activity scores between 20 and 40. While inactive compounds will have activity scores between 0 and 20.
2) Second tier (41-80 range) is reserved for liquid resupply single concentration confirmation and or dose-response confirmation data
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_QualifierThis qualifier is to be used with the next TID, IC50. If qualifier is "=", IC50 result equals to the value in that column;if qualifier is ">", IC50 result is greater than that value. if qualifier is "<", IC50 result is smaller than that value.String
2IC50IC50 value determined using sigmoidal dose response equationFloat
3Std.Err(IC50)Standard Error of IC50 valueFloat
4nHHill coefficient determined using sigmoidal dose response equationFloat
5Mean %Activity at 5 uM (5μM**)Mean % activity of reconfirmation replicatesFloat
6STD ErrorStandard error (n=2) of reconfirmation replicatesFloat
7Mean HighMean fluorescence ratio of the negative controlsFloat
8STD Deviation HighAverage of the standard deviation of negative controls (n=64) in the corresponding plateFloat
9Mean LowMean fluorescence ratio of the positive controlsFloat
10STD Deviation LowAverage of the standard deviation of positive controls (n=64) in the corresponding plateFloat

** Test Concentration.
Additional Information
Grant Number: X01 MH083230-01

Data Table (Concise)
PageFrom: