Primary screen for compounds that activate Insulin promoter activity in TRM-6 cells
The assay has been designed to screen for small molecule compounds that modulate insulin promoter activity (1). It is based upon a human pancreatic endocrine cell line, TRM-6 derived from fetal islets (2). These cells have been engineered to express an insulin promoter-fluorescent reporter protein transgene (eGFP) upon stimulation with tamoxifen using a lentiviral vector. In addition, the cell more ..
BioActive Compounds: 1039
Data Source: Columbia University Molecular Screening Center
Source (MLSCN Center Name): Columbia University Molecular Screening Center
Center Affiliation: Columbia University Molecular Screening Center
Assay Provider: Fred Levine and Mark Mercola, The Burnham Institute at UCSD, La Jolla, CA
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: MH077630-01
The assay has been designed to screen for small molecule compounds that modulate insulin promoter activity (1). It is based upon a human pancreatic endocrine cell line, TRM-6 derived from fetal islets (2). These cells have been engineered to express an insulin promoter-fluorescent reporter protein transgene (eGFP) upon stimulation with tamoxifen using a lentiviral vector. In addition, the cell line has been engineered to express a panel of transcription factors that together stimulate insulin gene activity (3, 4); thus, the cells express substantial levels of endogenous insulin mRNA although less than produced by a healthy pancreatic Beta-cell. Discovering compounds that modulate the insulin promoter has the potential to identify signaling pathways that are involved in the establishment and maintenance of mature Beta-cells.
(1) Ohneda K, Ee H, German M. Semin Cell Dev Biol. 2000 Aug;11(4):227-33. Regulation of insulin gene transcription.
(2) Wang S, Beattie GM, Mally MI, Lopez AD, Hayek A, Levine F. Transplant Proc. 1997 Jun;29(4):2219. Analysis of a human fetal pancreatic islet cell line.
(3)Itkin-Ansari P, Marcora E, Geron I, Tyrberg B, Demeterco C, Hao E, Padilla C, Ratineau C, Leiter A, Lee JE, Levine F. Dev Dyn. 2005 Jul;233(3):946-53. NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor.
(4) Itkin-Ansari P, Demeterco C, Bossie S, de la Tour DD, Beattie GM, Movassat J, Mally MI, Hayek A, Levine F. PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line. Mol Endocrinol. 2000 Jun;14(6):814-22.
Keywords: Insulin, Diabetes, reporter construct, GFP, inducible expression, high content, microscopy, Columbia University
1. For the culture and the screen
- RPMI 11mM glucose (Invitrogen, #11875)
- RPMI no glucose (Invitrogen, #11879)
- Fetal Bovine serum (Invitrogen, #16000)
- Phosphate Buffered Saline (PBS), tablets, w/o Ca2+ and Mg2+ (MP Biochemicals, #2810305)
- Tamoxifen (sigma, #T5848)
- Cycloheximide (Sigma, # C-7698)
- Compound plates (2mM in 100% DMSO)
- DMSO (SIGMA, 154938-1l)
- Paraformaldehyde (EMS, #15710)
- Hoechst 33342 (Molecular Probe Invitrogen, #H3570, 10 mg/ml solution in water)
- 384-well Assay plates (Corning, #7109)
- T6PBTEE47mer insGFP cells (TRM6) (frozen stock, Marc Mercola, The Burnham Institute)
3. Working solutions
- Modified RPMI:5.5mM glucose, 10%FBS
- Fixation Solution: 16% Paraformaldehyde
- Hoechst 33342 [1/5,000 (2 uM)] in PBS
1. Cell Culture
- TRM6 cells were obtained from Marc Mercola as frozen stocks
- Cells were grown in modified RPMI medium
- To passage cells, the monolayer was washed first with PBS (10 ml) per T175 and then with a trypsin/EDTA solution (5 ml)
- Cells were detached by incubation with 5 ml of trypsin per T175 flask
- Cells were resuspended in 5 ml of EGM-2 and centrifuged for 5 min at 1000 x g
- Cells were seeded at dilutions of 1:2 or 1:3 and grown until they reached confluency
2. Cell Seeding (using an 8-channel multi-drop, MTX labs)
- Seed 1,000 per well in 60 ViewPlates (per batch) in 50 ul modified RPMI containing 0.75uM Tamoxifen
- Incubate overnight (15 h) at 37 C, 5% CO2 and 100% humidity before stimulation
3. Compounds Addition (using 384-channel pintool)
- Add 50 nl of compounds (2 uM final concentration) from compound daughter plates (2 mM in 100 % DMSO) per well of assay plates
- Columns 1, 2, 23, 24 are used for controls (no compounds addition)
- Add 50 nl of 5mM (5uM final concentration) Tamoxifen (Column 2) per well of assay plate (compound addition control)
- Add 50nl of 1mM Cycloheximide (Column 1) per well of assay plate (inhibitor control)
- Incubate 48h 37 C, 5% CO2 and humidity
- Proceed immediately to fixation
4. Fixation and staining
- Dispense 17 uL of fixation solution per well
- Incubate for 20 min at Room Temperature
- Wash 1x with PBS (1 x 400 uL rolling wash)
- Leave 25 uL residual volume in each well
- Dispense 25 uL of Hoechst solution per well
- Incubate 20 min at Room Temperature
- Wash 3x PBS (3 x 400 uL rolling wash)
- Dispense 50 uL of PBS per well
- Plates (can be stored at 4 C) are ready to be scanned INCell 3000 analyzer (GE, healthcare)
The images acquired on the INCell 3000 were subjected to analysis through the object intensity module (OB1) of the accompanying image analysis software. The percent cells passing a positive intensity threshold in the green channel is used as an index of GFP expressing cells (percentPos).
In this publication we report the primary screening results from the MLSMR 160K library, with a final concentration of 2 uM plus and minus 3 standard deviations.
All calculations were done using Assay Explorer 3.1 from MDL.
For analysis and hit definition percentPos raw results were normalized per plate using high and low controls (non-stimulated and stimulated DMSO /buffer control wells) per plate to give percent activation(PS_Act_2uM). In addition to plate-based statistics we generate control statistics for data and controls wells to evaluate the stability over time and reasonable hit thresholds. For hit selection, a threshold (Hit_Thresh) greater than 75% was applied for each sample of the screened batches. For QC, control wells with a (PS_Nnorm) cell count (normalized cell count per plate) of less than 10% were discarded. For each experiment all images of data wells with percent activation equal or greater than the applied threshold were visually inspected to define the final screening results. The PubChem BioAssay result PUBCHEM_ACTIVITY_SCORE has been calculated based on reported percent activation (PS_Act_2uM) and normalized to 100. Negative percent activation values are reported as PUBCHEM_ACTIVITY_SCORE equal to zero. The result PUBCHEM_ACTIVITY_OUTCOME of this primary screen is reported as active if Hit_Approved is positive (Hit_Thresh > 75% and Hit_Visual are positive), inconclusive if Hit_Approved is negative (Hit_Thresh > 75% and Hit_Visual is negative) and inactive if Hit_Approved is negative (Hit_Thresh < 75% and Hit_Visual are negative).
Possible artifacts can be caused by:
- Fluorescent compounds
- Toxic compounds
- Compounds activating directly trangene transcription in a tamoxifen like fashion
- Additional qualitative comments are reported in the results based on visual inspection
The inactive compounds of this assay have activity score range of 0 to 50 and active compounds range of activity score is 50 to 100
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)