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BioAssay: AID 463177

Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: fluorescence activated cell sorting (FACS)-based cell-based assay to identify inducers of Hela cell apoptosis

Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: fluorescence activated cell sorting (FACS)-based cell-based assay to identify inducers of Hela cell apoptosis. ..more
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 Tested Compounds
 Tested Compounds
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Active(2)
 
 
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 Tested Substances
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Active(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 463177
Data Source: The Scripps Research Institute Molecular Screening Center (HELA-APOPTOSIS_ACT_FLUOR_FACS_%ACT MCSRUN ROUND 1 ASSAY 9)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-08-31
Hold-until Date: 2011-08-30
Modify Date: 2011-08-31

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
1321Primary Cell-based High Throughput Screening Assay for Inhibitors of Wee1 DegradationScreening depositor-specified cross reference: Primary (WEE1 inhibitors in singlicate)
1410Confirmation cell-based high throughput screening assay for inhibitors of Wee1 degradationScreening depositor-specified cross reference: Confirmation (WEE1 inhibitors in triplicate)
1412Dose Response Cell-based Assay for Inhibitors of Wee1 DegradationConfirmatory depositor-specified cross reference: Dose response (WEE1 inhibitors in triplicate)
1413Cytotoxicity counterscreen assay for inhibitors of Wee1 degradationConfirmatory depositor-specified cross reference: Counterscreen (cytotoxicity in triplicate)
1414Counterscreen assay for inhibitors of Wee1 degradation: dose response cell-based assay to identify inhibitors of cyclin B degradationConfirmatory depositor-specified cross reference: Dose response counterscreen (cyclin B inhibitors in triplicate)
1807Summary of probe development efforts to identify inhibitors of Wee1 degradationSummary1 depositor-specified cross reference: Summary (WEE1 inhibitors)
2088Late stage results from the probe development effort to identify inhibitors of Wee1 degradation.Screening depositor-specified cross reference: Late stage (WEE1 inhibitors in triplicate)
434972Late stage results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to determine inhibition of Wee1 degradation by kinase inhibitorsOther depositor-specified cross reference: Dose response (WEE1 inhibitors in triplicate)
463076Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay for inhibitors of fms-related tyrosine kinase 3 (FLT3)Confirmatory depositor-specified cross reference: Late stage (FLT3 inhibitors)
463077Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay to identify CK1d inhibitorsConfirmatory depositor-specified cross reference: Late stage (CK1d inhibitors)
463080Late stage assay provider results from the probe development effort to identify inhibitors of WEE1 degradation: luminescence-based dose response assay to identify stabilizers of WEE1Confirmatory depositor-specified cross reference: Late stage dose response (WEE1 stabilizers in quadruplicate)
504929Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: Amplified proximity luminescence-based biochemical assay to identify inhibitors of residues 1-40 of amyloid betaOther1 depositor-specified cross reference
504930Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: radioactivity-based cell-based assay to identify inhibitors of granule cell progenitor (GCP) proliferationConfirmatory1 depositor-specified cross reference
504935Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based dose response assay for cytotoxic compounds using neuroblastoma cell lineOther1 depositor-specified cross reference
463169Late stage assay provider results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of Wee1 degradationScreening same project related to Summary assay
463170Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of p21 (CDKN1A) degradationScreening same project related to Summary assay
463171Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of p27 (CDKN1B) degradationOther same project related to Summary assay
463186Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of cyclin B degradationOther same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Nagi Ayad, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R21NS056991-01
Grant Proposal PI: Nagi Ayad, TSRI
External Assay ID: HELA-APOPTOSIS_ACT_FLUOR_FACS_%ACT MCSRUN ROUND 1 ASSAY 9

Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: fluorescence activated cell sorting (FACS)-based cell-based assay to identify inducers of Hela cell apoptosis.

Description:

Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling (1-4). The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclinB protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur (1-4). Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome (5, 6). Studies showing that Wee1 expression is reduced in colon carcinoma cells (7) and that Wee1 overexpression can block cell division (8), suggest that Wee1 may act as a tumor suppressor. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis.

References:

1. Lee MH, Yang HY. Negative regulators of cyclin-dependent kinases and their roles in cancers. Cell Mol Life Sci 2001; 58: 1907-1922.
2. Heald R, McLoughlin M, McKeon F. Human Wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell 1993; 74: 463-474.
3. Coleman, TR & Dunphy, WG. Cdc2 regulatory factors. Curr Opin Cell Biol. 1994 Dec;6(6):877-82.
4. Kellogg, DR. Wee1-dependent mechanisms required for coordination of cell growth and cell division. J Cell Sci. 2003 Dec 15;116(Pt 24):4883-90.
5. Smith A, Simanski S, Fallahi M, Ayad NG. Redundant ubiquitin ligase activities regulate wee1 degradation and mitotic entry. Cell Cycle. 2007 Aug;6(22):2795-9.
6. Watanabe N, Arai H, Nishihara Y, Taniguchi M, Watanabe N, Hunter T, and Osada H. M-phase kinases induce phosphodependent ubiquitination of somatic Wee1 by SCFbeta-TrCP. PNAS 2004 101: 4419-4424.
7. Backert S, Gelos M, Kobalz U, Hanski ML, Bohm C, Mann B, Lovin N, Gratchev A, Mansmann U, Moyer MP, Riecken EO, Hanski C. Differential gene expression in colon carcinoma cells and tissues detected with a cDNA array. Int J Cancer. 1999 Sep 9;82(6):868-74.
8. McGowan, C. H.; Russell, P. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J. 1993. 12: 75-85.
9. Madoux F, Simanski S, Chase P, Mishra JK, Roush WR, Ayad NG, Hodder P. An Ultra-High Throughput Cell-Based Screen for Wee1 Degradation Inhibitors. J Biomol Screen. 2010 Aug 13. [Epub ahead of print]

Keywords:

Late stage, late stage AID, powders, SAR, purchased, synthesized, apoptosis, viability, cytotoxicity, HeLa, hela, subG1, mitosis, counterscreen, Wee1, WEE1hu, FLJ16446, DKFZp686I18166, cell cycle, cancer, HeLa, degradation, inhibitor, inhibition, luminescence, luciferase, stabilize, fold change, stabilization assay, stabilization, dose response, quadruplicate, 96, assay, assay provider, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, B7SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates can induce apoptosis in parental HeLa cells, as measured by the percentage of cells in the subG1 phase of the cell cycle (9). The prediction is that a small-molecule inhibitor of Wee1 degradation would inhibit cell cycle progression because overexpression of nondegradable Wee1 induces a G2-M arrest. In this assay, compounds were tested using a 7-point dose response starting at a nominal high concentration of 5 uM, followed by determination of the percentage of cells in subG1, G1, S, and G2 phases of the cell cycle.

Protocol Summary:

HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 C in an atmosphere of 10% CO2 and 95% relative humidity (RH). HeLa cells were treated for 20 hours with different concentrations of test compounds. Next, compound-treated HeLa cells were harvested by collecting culture media, washing once in phosphate-buffered saline (PBS), and trypsinizing. Cells were centrifuged at 400 g for 5 min at 4 C. Cells were then washed with cold PBS, centrifuged at 400 g for 5 min at 4 C, and rapidly resuspended with 7.5 mL 70% ethanol and incubated at -20 C overnight. Cells were then centrifuged at 400 g for 10 min at 4 C and washed with 10 mL cold PBS. The supernatant was removed and the cell pellet resuspended in 38 mM sodium citrate containing 69 mM of propidium iodide and 19 mg/mL of Rnase A. Fluorescence-activated cell sorting (FACS) analysis was performed on a BD Biosciences (San Jose, CA) LSR II system and analyzed using Flowjo 8.7.3 software.

The fold-change inhibition for each compound was calculated as follows:

[Cells_treated_with_Test_Compound] / [Cells_treated_with_Vehicle (DMSO)]

The percentage of cells in each stage of the cell cycle was determined directly from FACS.

PubChem Activity Outcome and Score:

Compounds that induce >= 20% of the HeLa cell population to enter the "subG1" phase of the cell cycle at concentrations greater than 100 nM are considered Active. Compounds that induce < 20% of the HeLa cell population to enter the "subG1" phase of the cell cycle at all concentrations are considered inactive.

The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

List of Reagents:

Dulbecco's Modified Eagle's Media (Invitrogen, part 11965-092)
Fetal Bovine Serum (Hyclone, part SH 30088.03)
Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)
Propidium iodide (Invitrogen, Carlsbad, CA)
T75 flasks (BD Falcon 353136)
96-well plates (Corning part 3917)
Comment
This assay was performed by the assay provider. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. Possible artifacts of this assay can include, but are not limited to: presence of lint or dust in the test well; compounds that nonspecifically modulate the proteasome or well luminescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Cells in SubG1, no treatmentIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
2Cells in SubG1, DMSOIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
3Cells in SubG1 at 0.01 uM (0.01μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
4Cells in SubG1 at 0.02 uM (0.02μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
5Cells in SubG1 at 0.05 uM (0.05μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
6Cells in SubG1 at 0.1 uM (0.1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
7Cells in SubG1 at 0.5 uM (0.5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
8Cells in SubG1 at 1 uM (1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
9Cells in SubG1 at 5 uM (5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
10Cells in G1, no treatmentIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
11Cells in G1, DMSOIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
12Cells in G1 at 0.01 uM (0.01μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
13Cells in G1 at 0.02 uM (0.02μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
14Cells in G1 at 0.05 uM (0.05μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
15Cells in G1 at 0.1 uM (0.1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
16Cells in G1 at 0.5 uM (0.5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
17Cells in G1 at 1 uM (1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
18Cells in G1 at 5 uM (5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
19Cells in S, no treatmentIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
20Cells in S, DMSOIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
21Cells in S at 0.01 uM (0.01μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
22Cells in S at 0.02 uM (0.02μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
23Cells in S at 0.05 uM (0.05μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
24Cells in S at 0.1 uM (0.1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
25Cells in S at 0.5 uM (0.5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
26Cells in S at 1 uM (1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
27Cells in S at 5 uM (5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
28Cells in G2, no treatmentIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
29Cells in G2, DMSOIndicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
30Cells in G2 at 0.01 uM (0.01μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
31Cells in G2 at 0.02 uM (0.02μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
32Cells in G2 at 0.05 uM (0.05μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
33Cells in G2 at 0.1 uM (0.1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
34Cells in G2 at 0.5 uM (0.5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
35Cells in G2 at 1 uM (1μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%
36Cells in G2 at 5 uM (5μM**)Indicates the percentage of cells in the indicated phase of the cycle at the indicated text compound concentration.Float%

** Test Concentration.
Additional Information
Grant Number: 1R21NS056991-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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