Cell-Based Assay for Identifying the Modulators of Antioxidant Response Element Signaling Pathway

Abstract

The antioxidant response element (ARE) signaling pathway plays an important role in the amelioration of cellular oxidative stress. Thus, assays that detect this pathway can be useful for identifying chemicals that induce or inhibit oxidative stress signaling. The focus of this chapter is to describe a cell-based ARE assay in a quantitative high throughput screening (qHTS) format to test a large collection of chemicals for oxidative stress induction ability. The assay is described through cell handling, assay preparation, and instrument usage.

1. Introduction

Oxidative stress, an imbalance of reactive oxygen species (ROS), and antioxidant defenses plays roles in chemical-induced toxicity, cancer, and age-related diseases [1–3]. ROS can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. ROS also acts as cellular messengers in redox signaling that can cause disruptions in normal cellular signaling mechanisms [4]. The induction of many cytoprotective enzymes in response to ROS is mediated by antioxidant response elements (ARE), which activate the nuclear factor erythroid 2-related factor (Nrf2).

ARE is a cis-acting enhancer locating in the 5’ flanking region of many phase II detoxification genes. Nrf2 is a member of the Cap ‘n’ Collar family of transcription factors. Under oxidative stress, Nrf2 is released from keap1 and quickly translocated to the nucleus where it binds to the ARE (Fig. 1). The Nrf2/ARE transcriptional pathway plays an important role in the regulation of genes that control the expression of proteins critical to the detoxification and elimination of ROS and electrophiles [5,6]. Several studies suggest the protective role of Nrf2/ARE activation against environmentallyinduced oxidative stress and the Nrf2/ARE pathway as a potential therapeutic target [7,8]. Here we describe a cell-based ARE β-lactamase (bla) reporter gene assay (ARE-bla assay) using Fluorescence Resonance Energy Transfer (FRET) technology to identify the chemical compounds that modulate the ARE signaling pathway. The ARE-bla cell line contains a β-lactamase reporter gene under control of ARE stably integrated into HepG2 cells. The assay uses a FRET-based fluorescent substrate, CCF4-AM, for detection. Once inside the cells, CCF4-AM is hydrolyzed by cytoplasmic esterases to form a polar molecule (CCF4). Upon excitation at 405 nm, the energy is transferred to the fluorescein moiety by FRET resulting in emission at 530 nm. In the presence of β-lactamase expression, the CCF4 substrate is cleaved at β-lactam ring by β-lactamase, which leads to excitation at 405 nm and emission at 460 nm [9] (Fig. 1). Thus, the fluorescence measured in these cells quantitatively corresponds to the activity change of ARE signaling. The ARE-bla assay has been optimized in a quantitative high-throughput screening (qHTS) platform to detect the compounds that activate this pathway. This chapter describes a protocol for this assay in a 1536-well plate format.

Fig. 1.

Schematics of ARE-bla reporter gene assays.

2. Materials

2.1. Cell line and cell culture condition

1. All of the cell-culture-related medium and components (Table 1) are purchased from Life Technologies.

2. Cell line: A CellSensor^® ARE-bla HepG2 cell line (Life Technologies) that contains three stably integrated copies of the ARE derived from the reduced form of human nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase 1 gene (NQO1) driving the expression of a downstream beta-lactamase reporter gene [7]. The cells were maintained in culture medium at 37°C under a humidified atmosphere and 5% CO2.

3. Culture media: DMEM medium supplement with 10% dialyzed fetal bovine serum, 0.1mM non-essential amino acids, 25mM HEPES, 100U/mL penicillin, 100μg/mL streptomycin, and 5μg/mL blasticidin.

4. Thaw media: same as culture medium but without blasticidin.

5. Assay media: DMEM medium supplement with 1% dialyzed fetal bovine serum, 0.1mM non-essential amino acids, 25mM HEPES, 100U/mL penicillin and 100μg/mL streptomycin.

6. Freezing Medium: Recovery^™ Cell Culture Freezing Medium from Life Technologies.

7. 0.25% Trypsin-EDTA.

8. Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium.

Table 1:

Cell culture and assay medium

Components	Culture Medium	Assay Medium	Thaw Medium
DMEM	90%	99%	90%
Dialyzed FBS	10%	1%	10%
NEAA	0.1 mM	0.1 mM	0.1 mM
HEPES	25 mM	25 mM	25 mM
Penicillin	100 U/mL	100 U/mL	100 U/mL
Streptomycin	100 μg/mL	100 μg/mL	100 μg/mL
Blasticidin	5 μg/mL	-	-

2.2. Assay Reagents and Chemicals

1. Reagents for fluorescence detection: LiveBLAzer^™ FRET B/G Loading Kit (Life Technologies) including Solution A (CCF4-AM), Solution B and Solution C.

2. Solution D (Life Technologies) containing an anion transport inhibitor that is used in conjunction with CCF4-AM-based assay to prevent active cellular export of the FRET-based substrates after loading. It was used in combination with Solution A, B, and C to create CCF4-AM substrate mixture.

3. Dimethyl sulfoxide (DMSO) was used to resolve compounds and vehicle control for basal signal.

4. β-Naphthoflavone was used as positive control for ARE-bla Assay.

2.3. Supplies and Equipment

1. T225 cell culture flasks.

2. Disposable, sterile centrifuge tubes.

3. 1536-well assay plates: black well/clear bottom, cell culture treated and with low fluorescence background plate.

4. Cell strainer: a receptacle with a 40 μm nylon filter that is used to remove clumped cells from cell suspensions.

5. Lids for assay and compound plate: these reusable lids are made from stainless steel and contain a rubber gasket that sits around the top outer edge. The cellular assay lid contains small evenly placed holes that allow air exchange necessary for cell-based assays. The weight of the lid allows the gasket to form a strong barrier around the plate, virtually eliminating edge effects.

6. PinTool workstation (Wako Automation): the PinTool performs transfer of 23 nL of compound from a 1536-well compound plate to a 1536-well assay plate (see Note 1).

7. BioRAPTR FRD workstation (Beckman Coulter): a liquid handling system that can transfer of 0.2–10 μL of up to four different reagents or cells simultaneously into a 1536 well plate.

8. Multidrop Combi Dispenser: a high speed dispenser capable of one reagent or cells using eight-channel detachable dispensing cassettes.

9. CyBi-well Vario pipettor, a 96, 384 and 1536 channel simultaneous pipettor: It requires the use of disposable tips, which is used for the preparation of positive control plate in 1536- well plate format.

10. Cellometer Auto Cell Counter (Nexcelom Bioscience) is used to count viable cells.

11. EnVision Multilabel plate reader (PerkinElmer) covers a wide range of fluorescence, absorbance, and luminescence readouts commonly used in high throughput assays. The EnVision reader includes two detectors enabling simultaneous dual wavelength reading that is well suited for β-lactamase reporter assays. For an ARE-bla assay, reading requires dual bottom mirror and compatible filter sets (Table 2).

Table 2:

Filter selection for ARE-bla assay

Filters	Excitation	Emission
Channel 1 (Green)	409/20 nm	530/30 nm
Channel 2 (Blue)	409/20 nm	460/40 nm
Ratio (Ch2/Ch1)	460 nm/530 nm

3. Methods

3.1. Cell Culture

1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath with gentle agitation for 1–2 min.

2. Place the vial into a laminar flow hood. Before opening the vial, wipe the outside of the vial with 70% ethanol.

3. Transfer the thawed cells into a 50 mL conical sterile centrifuge tube with 30 mL prewarm thaw medium.

4. Centrifuge the cell supernatant for 4 minutes at 200 × g.

5. Carefully aspirate supernatant without disturbing the cell pellet.

6. Gently re-suspend cell pellet in thaw medium.

7. Transfer the desired amount of the cells to a T225 tissue culture flask.

8. Place the flask in an incubator with a humidified atmosphere of 5% CO₂ until passage. Maintain the cells at 30% to 90% confluence prior to passage. During the first passage, switch to culture medium.

9. After 48–72 hours or cells with 80 – 90% confluence, aspirate medium and rinse once with 10 mL DPBS, followed by the addition of 10 mL of 0.25% Trypsin/EDTA and swirl to coat the cells evenly around flask.

10. Place the flask in incubator at 37°C for 2–3 min or until the cells detach.

11. Add 10 mL of culture medium to deactivate Trypsin.

12. Transfer the cells to a 50 mL conical tube and centrifuge at 200 × g for 4 min.

13. Carefully aspirate supernatant and re-suspend cell pellet in the culture medium.

14. Count cells using a Cellometer auto cell counter.

15. Transfer the cell suspension to a T225 tissue culture flask.

16. Incubate until next passage or assay. Cell should be passaged at least twice a week. Please not let cells grow over 90% confluence.

3.2. Preparation of Positive Control Plate:

1. Assay-specific controls (positive and negative) are located on an additional 1536-well compound plate in columns 1–4. These controls will transfer simultaneously with the test compounds to the assay plate. The final concentration of DMSO in the assay is less than 0.5%.

2. To make a positive control plate for ARE-bla assay, 5 uL of β-naphthoflavone stock solution in DMSO is dispensed into each well in columns 1, 2 and 3 (Fig. 2). CyBi-well Vario pipettor can be used to prepare the control plate.

Fig. 2.

Control plate map in 1536-well format. Column 1 contains a sixteen-concentration titration ranging from 92 μM to 2.8 nM in duplicates. Columns 2 and 3 contain replicates for the β-naphthoflavone: 46 μM in column 2 and 23 μM in column 3 in 32 replicates for each concentration. Additionally, DMSO, as a negative control, is dispensed into column 4.

3.3. ARE-bla Assay

1. Harvest the ARE-bla HepG2 cells and resuspend the cell pellet in assay medium (see Note 2).

2. Place the cells on a cell strainer to remove clumped cells before counting.

3. Count cell number and determine cell viability. Cell viability of 95% or greater will have a better window of signal to basal.

4. Prepare cell stock in assay medium at density of 0.4×10⁶ cells/mL.

5. Dispense 5 μL of cells prepared at step 4 into each well of a 1536-well black, clear-bottom, tissue culture-treated assay plates using a Multidrop Combi dispenser or BioRAPTR FRD.

6. Place a pre-cleaned plate lid over the plate and incubate assay plates at 37°C under a humidified atmosphere and 5% CO₂ for 5 hours to allow cells to attach.

7. Transfer 23 nL of compounds and controls to the assay plate by a PinTool.

8. Incubate the assay plates at 37°C, 5% CO₂ for 16 h.

9. Freshly prepare 6X CCF4-AM substrate mixture prior to assay termination: add 6 μL of 1 mM of CCF4-AM substrate to 60 μL of Solution B and mix, and then add 874 μL of Solution C and 60 μL of Solution D to the combined solution and vortex.

10. Dispense 1 μL of 6X CCF4-AM substrate mixture to each well using a BioRAPTR dispenser after 16 hours compound treatment.

11. Incubate the plates in the dark at room temperature for 2 h for fluorescence development.

12. Measure fluorescence intensity at 460 and 530 nm emission and 405 nm excitation using an EnVision plate reader. Data is expressed as the ratio of 460nm/530nm emissions (see Note 3).

13. Percentage of activity of the compounds is calculated by normalizing the raw data to DMSO wells (0% activity) and maximal β-napthoflavone wells (100% activity).

14. The compound half maximum effective concentration (EC50) and maximum response (efficacy) values were calculated using a four-parameter Hill equation in GraphPad Prism software (Fig. 3).

Fig. 3.

β-Naphthoflavone stimulated β-lactamase activity in ARE-bla HepG2 cells in 1536-well plate format.

4. Notes

1. To avoid cross contamination, it is important to wash pins with appropriate reagents (e.g. DMSO and methanol) before and after use.

2. For screening, the cells that have passed one passage after thawing are recommended.

3. The ratiometric readouts from dual emissions (460 and 530 nm) can minimize well-to-well and plate-to-plate variation caused by differences in plating cell density.