Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Ovarian Carcinoma (BG1Luc4E2) Cell-based assay to identify inhibitors of Estrogen Receptor-Dependent Gene Expression, Set 2
Name: Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Ovarian Carcinoma (BG1Luc4E2) Cell-based assay to identify inhibitors of Estrogen Receptor-Dependent Gene Expression, Set 2 ..more
BioActive Compounds: 12
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Michael Denison, University of California, Davis
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1-X01-DA026558-01
Grant Proposal PI: Michael Denison
External Assay ID: ESTROGEN-RECEPTOR_INH_LUMI_0096_3X%INH_SET2 MCSRUN
Name: Late stage assay provider counterscreen for the probe development effort to identify activators of the Aryl Hydrocarbon Receptor (AHR): Luminescence-based Human Ovarian Carcinoma (BG1Luc4E2) Cell-based assay to identify inhibitors of Estrogen Receptor-Dependent Gene Expression, Set 2
Transcription factors are critical regulators of gene expression (1). Under conditions such as environmental stress and exposure to endogenous toxins, transcription factors can rapidly modulate the transcription of genes whose products regulate cell proliferation and metabolism. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor of the basic helix-loop-helix protein superfamily involved in the biological response to aromatic hydrocarbons, and regulates the expression of xenobiotic-metabolizing enzymes such as cytochrome P450, aldehyde dehydrogenase, quinone reductase, and other phase I and phase II detoxification genes (2, 3). In response to various compounds, including the environmental pollutants dioxins, benzo(a)pyrene, dietary contaminants, grapefruit juice, endogenous toxins, and plant products such as carotinoids, nicotine and caffeine (2, 4-6), cytosolic AHR complexes with chaperones hsp90, p23, and XAP2, translocates to the nucleus where it dimerizes with the AHR nuclear translocator (ARNT) to influence target gene transcription (7, 8). Gain-of-function studies in mice reveal the oncogenic potential of AHR (9), while other reports show roles for AHR in diverse biologic events such as organ development (10, 11), immune function and allergy (12), and estrogen responsiveness (13). The identification of agonists of AHR will provide useful tools to elucidate the roles of this receptor in cell metabolism, transcriptional control, and tumor formation (14-16).
1. Ptashne, M., Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci, 2005. 30(6): p. 275-9.
2. McMillan, B.J. and Bradfield, C.A., The aryl hydrocarbon receptor sans xenobiotics: endogenous function in genetic model systems. Mol Pharmacol, 2007. 72(3): p. 487-98.
3. Puga, A., Tomlinson, C.R., and Xia, Y., Ah receptor signals cross-talk with multiple developmental pathways. Biochem Pharmacol, 2005. 69(2): p. 199-207.
4. Bock, K.W. and Kohle, C., Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions. Biochem Pharmacol, 2006. 72(4): p. 393-404.
5. Denison, M.S. and Nagy, S.R., Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev Pharmacol Toxicol, 2003. 43: p. 309-34.
6. de Waard, P.W., Peijnenburg, A.A., Baykus, H., Aarts, J.M., Hoogenboom, R.L., van Schooten, F.J., and de Kok, T.M., A human intervention study with foods containing natural Ah-receptor agonists does not significantly show AhR-mediated effects as measured in blood cells and urine. Chem Biol Interact, 2008.
7. Hankinson, O., The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol, 1995. 35: p. 307-40.
8. Petrulis, J.R. and Perdew, G.H., The role of chaperone proteins in the aryl hydrocarbon receptor core complex. Chem Biol Interact, 2002. 141(1-2): p. 25-40.
9. Andersson, P., McGuire, J., Rubio, C., Gradin, K., Whitelaw, M.L., Pettersson, S., Hanberg, A., and Poellinger, L., A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors. Proc Natl Acad Sci U S A, 2002. 99(15): p. 9990-5.
10. Ramos, K.S., Transcriptional profiling and functional genomics reveal a role for AHR transcription factor in nephrogenesis. Ann N Y Acad Sci, 2006. 1076: p. 728-35.
11. Walisser, J.A., Glover, E., Pande, K., Liss, A.L., and Bradfield, C.A., Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types. Proc Natl Acad Sci U S A, 2005. 102(49): p. 17858-63.
12. Lawrence, B.P., Denison, M.S., Novak, H., Vorderstrasse, B.A., Harrer, N., Neruda, W., Reichel, C., and Woisetschlager, M., Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel low-molecular-weight compound. Blood, 2008. 112(4): p. 1158-65.
13. Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K., Tohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama, Y., and Kato, S., Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature, 2003. 423(6939): p. 545-50.
14. Zhao, B., Baston, D.S., Hammock, B., and Denison, M.S., Interaction of diuron and related substituted phenylureas with the Ah receptor pathway. J Biochem Mol Toxicol, 2006. 20(3): p. 103-13.
15. Garrison, P.M., Tullis, K., Aarts, J.M., Brouwer, A., Giesy, J.P., and Denison, M.S., Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam Appl Toxicol, 1996. 30(2): p. 194-203.
16. Han, D., Nagy, S.R., and Denison, M.S., Comparison of recombinant cell bioassays for the detection of Ah receptor agonists. Biofactors, 2004. 20(1): p. 11-22.
17. Rogers JM, Denison MS., Recombinant cell bioassays for endocrine disruptors: development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals. In Vitr Mol Toxicol. 2000 Spring;13(1):67-82.
late stage, powders, purchased, synthesized, AHR, bHLHe76, aryl hydrocarbon receptor, receptor, transcription factor, triplicate, dose response, counterscreen, estrogen, estrogen receptor, ER, hormone, stably transfected, stable, estradiol, BG1, BG1Luc4E(2), E2, assay provider, 96, well, cell, ovarian, ovary, reporter, plasmid, activator, agonist, activation, luciferase, luminescence, reporter, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine the ability of powder samples identified as possible AhR activator probe candidates to inhibit estrogen-dependent luciferase reporter gene expression, in an AHR-dependent manner, in stably transfected ovarian (BG1) carcinoma cells in vitro. The assay employs human ovarian carcinoma (BG1) cells that have been stably transfected with the estrogen receptor-responsive luciferase reporter plasmid pGucLuc6.1ERE (contains 2 synthetic estrogen response elements) and the resulting cells (BG1Luc4E2) respond to estrogenic chemicals with the induction of firefly luciferase gene expression, which is easily measured in a microplate luminometer. Cells are incubated with phenol red-free media containing charcoal-stripped serum for 5 days (to reduce endogenous estrogen levels) and then incubated with estradiol 17b in the absence or presence of test compounds for 24 hours (TCDD is used as a positive control), followed by cell lysis and detection of well luminescence using Promega Steady-Glo Luciferase reagent. As designed, compounds that act as AHR agonists will not only stimulate the AHR signaling pathway, but they will also inhibit estrogen receptor-dependent luciferase reporter gene expression in these cells (17), with the extent of inhibition directly related to the level of AHR activation. Compounds are tested in triplicate at 10 uM and in a dose response up to a maximum nominal concentration of 100 uM.
General maintenance, BG1Luc4E2 cells: BG1Luc4E2 cells should be maintained in alpha-MEM (Invitrogen, 12000-063) containing 10% premium fetal bovine serum (Atlanta Biologicals, Cat S11150) and 400 mg G418 per liter media. Cells should not exceed 90% confluency before passaging.
Estrogen-stripping period, BG1Luc4E2 cells: Five days prior to plating of the BG1Luc4E2 cells into clear-bottomed while 96-well plates, the media that the cells are grown in should be changed from alpha-MEM containing 10% FBS serum into phenol red-free Dulbecco's Modified Eagle's Medium (DMEM) (low glucose; with 1 g/L glucose and L-glutamine; without phenol red, NaHCO3 (Sigma, D2902-10L)) containing 10% charcoal-stripped fetal bovine serum (Atlanta Biologicals, Cat S11650). This provides sufficient time for background estrogenic activities to drop to very low levels.
Protocol for plating BG1Luc4E2 cells into 96-well plate:
On the fifth day of DMEM media exposure
1) Double rinse BG1Luc4E2 cells with PBS (5 mL per plate per rinse), trypsinize and transfer into 50 mL sterile tubes.
2) Fill all tubes to 50 mL mark with media (only phenol red-free DMEM, 10% charcoal-stripped serum should be during the plating procedure).
3) Centrifuge at room temperature for 5 minutes at 1,100 rpm.
4) In tissue culture hood, carefully aspirate media from centrifuged tubes. Add 10 mL media to tube and gently resuspend the cells.
5) Remove a 10 uL aliquot of resuspended cells for cell counting. For the bioassay, the optimal cell density for BG1Luc4E2 cells in the 96-well plate format is 750,000 cells/mL. A 100 uL aliquot of cells is added per well using a cell trough and multichannel pipette. Cells are incubated at 37 C for 12-24 hours before use.
Protocol for treating BG1Luc4E2 cells:
1) In tissue culture hood, prepare treatments with 1000 uL pipette and sterilized tips in 7 mL glass tubes using a 1:100 ratio of chemical (or sample) to phenol red-free DMEM/FBS media (i.e. 10 uL chemical diluted in 990 uL media); a maximal inducing concentration of 17-beta-estradio1 (1 nM) is included as the positive control. Vortex all treatments for several seconds. Treatment volumes should account for triplicate wells.
2) Dump media from plated 96-well plate(s) into appropriate biological waste container, taking care not to contaminate the cells during this procedure but to remove as much media as possible.
3) Carefully fill the appropriate wells of a 96-well microtiter plate with 100 uL chemical suspension prepared in the above step and incubate at 37 C for 24 hours. All chemicals were examined in at least triplicate incubations.
Protocol for lysing BG1Luc4E2 cells and measurement of luciferase activity:
1) Microscopically examine the health of cells in every treated well in the 96-well plate.
2) Dump media from 96-well plate(s) into appropriate biological waste container.
3) Wash wells twice with 100 uL PBS per well.
4) Check cell health and confluency under the microscope after the PBS rinses to ensure that cells were not lost during washing. Remove any remaining PBS.
5) Add 50 uL of room temperature Promega lysis buffer (1X) to each well (1X lysis buffer is prepared by adding 30 mL 5X lysis buffer to 120 mL MilliQ water; store in glass bottles).
6) Shake the plate at a moderate speed for at least 20 minutes to ensure cell lysis.
7) Prepare the luminometer. Add 1 bottle of room temperature luciferase buffer to 1 bottle substrate (buffer and substrate from Luciferase Assay System). Apply white backing tape to plate containing lysed cells. Read luminescence of treated wells after automatic injection of Promega stabilized luciferase reagent.
IC50 values from sigmoidal concentration-response curves were determined using the four-parameter Hill equation (SigmaPlot (Systat)).
PubChem Activity Outcome and Score:
Compounds that reduced estrogen-induced reporter activity less than 20% were considered inactive. Compounds that reduced estrogen induced reporter activity equal to or greater than 20% were considered active.
The reported PubChem Activity Score has been normalized to 100% observed % max response. Negative % max response values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-27, and for inactive compounds 20-0.
List of Reagents:
BG14E2 cell line
Minimum Essential Medium a (Invitrogen, part 12561072)
G418 sulfate powder (Gemini Bio-Products, part 400-11P)
Trypsin-EDTA solution (Invitrogen, part 15400054)
Fetal Bovine Serum, Premium (Atlanta Biologicals, part S11150)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
P100 tissue culture plates (Corning)
Dulbecco's Modified Eagle's Medium - phenol red-free (Sigma, product D2902)
charcoal-treated fetal bovine serum (Atlanta Biologicals, product S11650)
96-well white, clear-bottomed culture plates (Corning)
Steady-Glo Luciferase Assay System (Promega, part E2650)
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Accustandard, product D404N)
This assay was run by the assay provider. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)