Cytotoxicity counterscreen assay for inhibitors of Wee1 degradation
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 more ..
BioActive Compounds: 2
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Franck Madoux, SRIMSC
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R21NS056991-01
Grant Proposal PI: Nagi Ayad, TSRI
External Assay ID: CytoxHeLa_INH_LUMI_1536_CC50
Name: Cytotoxicity counterscreen assay for inhibitors of Wee1 degradation
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.
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. PMID: 7880537.
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 phospho-dependent 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.
Wee1, WEE1hu, FLJ16446, DKFZp686I18166, cyclin B1, CCNB, CCNB1, cell cycle, cancer, HeLa, degradation, inhibitor, cytotoxicity, viability, dose response, counterscreen, luminescence, luciferase, 1536-well, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine the cytotoxicity of compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput screening assay for inhibitors of Wee1 degradation" (PubChem AID 1321), and that confirmed activity in a set of experiments entitled, "Confirmation cell-based high throughput screening assay for inhibitors of Wee1 degradation" (PubChem AID 1410). The assay employs the CellTiter-Glo luminescent reagent, which contains luciferase to catalyze the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. As designed, cytotoxic compounds will reduce viable cell numbers and ATP levels, resulting in decreased well luminescence. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 50 uM.
HeLa cells were routinely cultured in T175 flasks in growth media consisting of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% FBS and 1% pen-strep-neo antibiotic mix. Prior to the start of the assay, HeLa cells were plated at 500 cells per well in 1536-well plates in 5 microliters of growth media. Plates were incubated for 4 hours at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity. Next, 25 nL of test compound in DMSO, or DMSO alone were added to the appropriate wells. Plates were then placed back in the incubator for 20 hours. After incubation, 5 ul of CellTiter-Glo reagent were added to each well, and the plates were allowed to incubate for 15 minutes at room temperature. Next, well luminescence was measured for 30 seconds per well using the ViewLux reader. The percent cytotoxicity was expressed relative to wells containing media only (100%) and wells containing cells treated with DMSO only (0%).
The percent cytotoxicity was defined according to the following mathematical formula:
% Cytotoxicity = 100* (Median_Negative_Control - Test_Compound) / (Median_Negative_Control - Median_Positive_Control)
Negative_Control is defined as wells containing cells treated with DMSO,
Test_Compound is defined as wells containing cells treated with test compound,
Positive_Control is defined as wells containing media only.
For each test compound, percent cytotoxicity was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported CC50 values were generated from fitted curves by solving for the X-intercept at the 50% inhibition level of the Y-intercept. In cases where the highest concentration tested did not result in > 50% cytotoxicity or where no curve fit was achieved, the CC50 was determined manually depending on the observed cytotoxicity at the individual concentrations. Compounds with CC50 values greater than 10 uM were considered inactive. Compounds with CC50 values equal to or less than 10 uM were considered active.
Any compound with a percent cytotoxicity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent cytotoxicity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by potency, with the most potent compounds assigned the highest activity scores.
The inactive compounds of this assay have activity score range of 0 to 71 and active compounds range of activity score is 97 to 100.
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)
CellTiter-Glo Reagent (Promega, part G7572)
T175 flasks (Corning, part 430828)
1536-well plates (Greiner, part 789173)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: presence of lint or dust located in or on wells of the microtiter plate, and compounds that nonspecifically modulate 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Categorized Comment - additional comments and annotations
Assay Type: Toxicity
Assay Cell Type: HeLa
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)