ASSAY and Drug Development TechnologiesVolume 4, Number 4, 2006© Mary Ann Liebert, Inc.

A Cellular Assay for Measuring the Inhibition of GlycogenSynthase Kinase-3 via the Accumulation of �-Catenin in

Chinese Hamster Ovary Clone K1 Cells

Karen Yeow, Laurence Novo-Perez, Pascale Gaillard, Patrick Page, Jean-Pierre Gotteland,Alexander Scheer, and Paul Lang

Abstract: Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that exists as twoisozymes, GSK3� and GSK3�. It plays important roles in regulating cell structure, function, andsurvival, and dysregulation of its function is linked to disorders such as Alzheimer’s disease and typeII diabetes. In resting cells, GSK3 is active and regulates the function of many downstream targets,including �-catenin. We describe the development of a cell-based assay designed to measure theactivity of GSK3 by directly measuring the accumulation of �-catenin in Chinese hamster ovaryclone K1 (CHOK1) cells. �-Catenin levels were assessed using an antibody-based staining protocolwith a luminometric readout. The assay is set up in a 96-well format. The use of GSK3 inhibitorsdemonstrated that this assay could be used to compare the effects of various small molecules onGSK3 inhibition in CHOK1 cells.

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Introduction

GLYCOGEN SYNTHASE KINASE-3 (GSK3) is a serine-threonine protein kinase that exists as two isozymes,

GSK3� and GSK3�, encoded by two highly homologousand ubiquitously expressed genes.1 GSK3 was originallyidentified through its ability to phosphorylate and inhibitglycogen synthase.2 It plays important roles in regulat-ing cellular structure, function, and survival, and dys-regulation of its function is linked to diseases such asAlzheimer’s disease and type II diabetes.3,4 Recent workhas shown that GSK3 increases �-amyloid productionand that LiCl and small molecule inhibitors can reduceA� levels.5,6 In addition, it has been reported that the useof GSK3 inhibitors can lower blood glucose levels in ro-dent models of type II diabetes. These effects are thoughtto occur primarily through an increase in hepatic glyco-gen synthesis and a decrease in hepatic gluconeogene-sis.3 Much effort is currently directed at understanding

the regulation and function of GSK3, and inhibiting itsactivity in these diseases.

In resting cells, GSK3 is constitutively active and canbe inhibited by a variety of extracellular stimuli, includinginsulin and growth factors such as platelet-derived growthfactor. GSK3 activity is significantly decreased by phos-phorylation of Ser21 and Ser9 in the � and � isoforms, re-spectively. Phosphorylation of these serine residues can beaccomplished through the action of several kinases, in-cluding Akt, protein kinase A, and protein kinase C.3 In-sulin signaling can also deactivate GSK3, via the actionsof the phosphatidylinositol 3-kinase–Akt pathway.7 GSK3can also be inhibited in response to the Wnt group of cellspecification factors. Secreted Wnt ligands are involved inthe control of gene expression, cell behavior, cell adhe-sion, and polarity, in embryonic development and cancer.8

It is thought that Wnt signaling inactivates GSK3 by tar-geting the subcellular pool of GSK3 in complex with axin,�-catenin, and adenomatous polyposis coli.9

Serono Pharmaceutical Research Institute, Plan-les-Ouates, Switzerland.

ABBREVIATIONS: BSA, bovine serum albumin; CHOK1, Chinese hamster ovary clone K1; DMSO, dimethyl sulfoxide; EC50, 50% effec-tive concentration; eIF2B, eukaryotic initiation factor 2B; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; GSK3, glycogensynthase kinase-3; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; PBS, phosphate-buffered saline; S/B, signal to background; TBS,Tris-buffered saline; Tcf, ternary complex factor.

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The numerous downstream targets of GSK3 reflect itsbroad regulatory influence on cellular function: over 40proteins have been reported to be phosphorylated byGSK3.10 Among the numerous downstream targets ofGSK3 are the neuronal microtubule-associated proteinsMAP-1B and Tau, members of the insulin-signaling path-way such as insulin receptor substrate-1, the translationinitiation factor eukaryotic initiation factor 2B (eIF2B),and transcription factors such as ternary complex factor(Tcf)-1 and �-catenin.10

One of the most well-studied downstream targets ofGSK3 is �-catenin. �-Catenin is a 92-kDa cytoplasmicprotein that binds to the cytoplasmic tail of E-cadherin,connecting cadherins to the actin cytoskeleton. Cytoso-lic �-catenin functions as a transcription factor, inducingthe transcription of downstream target genes such as cy-clin D1, c-myc, and Tcf-1.11

In the absence of Wnt stimulation in the resting cell,�-catenin is destabilized via phosphorylation by GSK3.12

This phosphorylated form of �-catenin is targeted for de-struction via the proteasome. Activation of the Wnt sig-naling pathway results in functional inhibition of GSK3.Alternatively, as a result of GSK3 inactivation by Wntsignaling, �-catenin dissociates from the destructioncomplex and is no longer subject to degradation. Free �-catenin accumulates and enters the nucleus, where itlinks Tcf and other transcription factors to activate tran-scription of Wnt target genes.

Activation of the Wnt/�-catenin signaling pathway hasbeen linked to diverse human cancers, including mela-nomas and colorectal cancers.8 Cytosolic �-catenin lev-els are increased in many tumors, resulting in uncheckedproliferative activity.12,13 A large proportion of coloncancers arise from mutations in �-catenin that render itresistant to degradation.14 Mice expressing a stable mu-tant �-catenin in the colon develop intestinal polypswithin a few weeks after birth.15 Therefore, the long-termuse of GSK3 inhibitors should be done with caution. In-deed, LiCl and the small molecule GSK3 inhibitorSB216763 are able to increase �-catenin levels in trans-formed cell lines.16,17 Therefore, assessing �-catenin lev-els is important for two reasons: to determine the effi-cacy of compounds to inhibit GSK3 and to determinetheir potential oncogenicity in elevating �-catenin levels.

We describe a cellular assay that measures the accu-mulation of endogenous �-catenin in Chinese hamsterovary clone K1 (CHOK1) cells in response to GSK3 in-hibition, such as through the use of LiCl or small mole-cule inhibitors. The assay uses a �-catenin polyclonal an-tibody to detect the levels of �-catenin that accumulatesin the cells. The assay is performed in 96–well format,using a simple protocol amenable to automation. In ad-dition, detection of �-catenin is performed in intact cells,thereby maintaining the complexity of the biological mi-lieu. This assay is well adapted for medium throughput

testing of compounds that affect �-catenin accumulation,such as for hits-to-leads compound progression.

This �-catenin assay could be used alongside addi-tional cellular assays, such as those measuring glucoseproduction,18 for the progression of a GSK3 inhibitor pro-gram. This strategy would enable the design and selec-tion of inhibitors with desired effects (such as on lower-ing glucose levels) while eliminating those that promotethe accumulation of �-catenin.

Materials and Methods

Reagents

The CHOK1 cell line19 (CCL61) was obtained fromthe American Type Culture Collection (Molsheim,France), and all tissue culture materials were obtainedfrom Gibco-Invitrogen (Basel, Switzerland), unlessstated otherwise. Fetal calf serum (FCS) was obtainedfrom Hyclone (Logan, UT). White-walled, 96-well View-plates® were purchased from Perkin-Elmer (Wellesley,MA). For the cell staining, 37% formaldehyde, Tween20, Triton X-100, sodium azide, and hydrogen peroxidesolution were obtained from Sigma (Lausanne, Switzer-land), and dimethyl sulfoxide (DMSO) from Fluka(Buchs, Swizerland). Cell lysis buffer (catalog number9803) and �-catenin antibody (catalog number 9562)were obtained from Cell Signaling Technology (Danvers,MA), and secondary antibody [horseradish peroxidase(HRP)-conjugated goat anti-rabbit, P0448] from Dako(Copenhagen, Denmark). The eIF2B antibody (catalognumber 44-530) was obtained from Biosource Interna-tional (Nivelles, Belgium), and the anti-actin antibody(catalog number 4968) was obtained from Cell SignalingTechnology. LiCl was obtained from Merck (Darmstadt,Germany). The alsterpaullone Compound 2 (catalognumber 270 275) was purchased from Alexis Biochem-icals (Lausen, Switzerland). CHIR98023 (Chiron Corp.,Emeryville, CA) was synthesized as previously de-scribed.20,21 SB216763 and SB415286 were purchasedfrom Sigma (product numbers S3442 and S3567, re-spectively). The in-house-synthesized GSK3 inhibitorCompound 1 and the c-Jun N-terminal kinase (JNK) in-hibitor Compound 4 were synthesized at the ChemistryDepartment, Serono Pharmaceutical Research Institute,Plan-les-Ouates, Switzerland.22–24

Cell culture

CHOK1 cells were cultured routinely in Dulbecco’sModified Eagle’s Medium/F12 medium supplementedwith 10% decomplemented FCS, 2 mM L-glutamine, and1% penicillin-streptomycin solution. Cells were main-tained in a tissue culture incubator at 37°C in 5% CO2

and passaged twice a week when they reached conflu-

Yeow et al.452

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ence. Cells were seeded as described below for each ex-periment.

Cell treatments

All small molecule inhibitors were solubilized inDMSO at an initial concentration of 10 mM. Before be-ing added to cell cultures, intermediate dilutions of stocksolutions were made in DMSO and cell culture mediumto obtain final concentrations in the micromolar range. Inall experiments, DMSO concentrations were kept con-stant for each cell treatment. A final concentration of0.4% DMSO was used.

Western blotting

Cells were plated in six-well dishes (250,000 cells perwell) and left to adhere overnight in a tissue culture in-cubator. The following day, the cells were treated witheither LiCl or small molecule inhibitors of GSK3, as in-dicated in Fig. 1. Control cells were left untreated ortreated with DMSO only. After 1 h of treatment, cellswere rinsed in 1 ml of ice-cold phosphate-buffered saline(PBS), and extracts were made by scraping the cells witha sterile plastic cell scraper into 300 �l of cell lysis buffer.Cell debris was removed by centrifuging the lysates to8050 g for 20 min, and lysates were stored at �80°C.Equal amounts of protein were separated by sodium do-decyl sulfate-polyacrylamide gel electrophoresis on a10% acrylamide gel. Proteins were then transferred toHybond-C Extra membranes (Amersham, Bucking-hamshire, UK), and the membranes were blocked for 1 h at room temperature in Tris-buffered saline (TBS)containing 0.1% Tween 20 and 5% bovine serum albu-min (BSA). The membranes were washed in wash buffer(TBS containing 0.1% Tween 20) for 30 min and thenprobed with primary antibody followed by secondary an-tibody for 1 h each. The antibodies were diluted in TBScontaining 0.1% Tween and 1% BSA. After each incu-bation, membranes were washed for 30 min with threechanges of wash buffer. Protein quantification was per-formed using the MicroBCA® Protein Assa kit (Pierce,Rockford, IL). Proteins were visualized using the Super-signal® enzyme-linked immunosorbent assay (ELISA)Pico Chemiluminescent substrate (Pierce).

Final assay protocol

The day before cell treatment, a total of 10,000 cellsper well were distributed in Packard White 96-wellViewPlates, in a final volume of 200 �l of growthmedium per well. The following day, cells were treatedwith different dilutions of compounds (as described inthe figure legends) for 3 h in a tissue culture incubator(37°C in 5% CO2). Controls were treated with eitherDMSO alone or with 10 �M GSK3 inhibitor Compound

1. The plates were then processed at room temperature(unless stated otherwise) using the following protocol.Unless otherwise stated, final volumes of 150 �l perwell were used for all steps of the protocol. Cells werefixed by the direct addition of 25 �l per well of 37%formaldehyde solution for 20 min. Plates were thenwashed in wash buffer (PBS containing 0.1% Triton X-100) for 3 � 5 min. Background peroxidase activity wasblocked by incubation in a solution of PBS containing0.1% Triton X-100, 1% H2O2, and 0.1% sodium azide,followed by 3 � 5 min washes in wash buffer. The cellswere incubated for 1 h at room temperature in PBS con-taining 0.1% Triton X-100 and 10% FCS, followed bya 5-min wash in wash buffer. Incubation in primary an-tibody was performed overnight at 4°C, with gentle ag-itation. All subsequent steps were performed at roomtemperature. The next day, the plates were washed 3 �5 min in wash buffer, followed by an incubation for 1h in secondary antibody and 3 � 5 min washes in washbuffer. Two final washes were performed in PBS with-out Triton X-100. Plates were incubated in SupersignalELISA reagents (at a volume of 100 �l per well) ac-cording to the manufacturer’s directions, before readingon a Luminoskan Ascent luminometer (Thermo Elec-tron, Waltham, MA). The primary and secondary anti-bodies were diluted in PBS containing 1% Triton X-100and 1% BSA, at final dilutions of 1:250 and 1:400, re-spectively.

A Cellular Assay for �-Catenin Accumulation 453

FIG. 1. Inhibition of GSK3 results in accumulation of �-catenin in CHOK1 cells. CHOK1 cells were treated with vari-ous GSK3 inhibitors, including LiCl, and two small moleculeGSK3 inhibitors, CHIR98023 and Compound 1. Cell proteinextracts were harvested after 1 h of treatment, and western blotswere performed using an anti-�-catenin antibody. As furtherevidence for GSK3 inhibition, blots were probed with eIF2B,another downstream target of GSK3. Blots were probed withan anti-actin antibody as a control.

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Assay automation

The assay was successfully automated using theTitertek (Huntsville, AL) MAP C®. With the exceptionof the overnight incubation step in primary antibody, allsteps in the protocol were performed using the roboticstation. For the incubation in primary antibody, the plateswere removed from the station and placed on a rocker at4°C overnight. The next day, they were replaced onto therobotic station, and the following steps of the assay pro-tocol were performed as described using the TitertekMAP C. All solutions containing BSA or FCS were fil-tered using 0.22-�m (pore size) Millipore® (Billerica,MA) filters, to prevent clogging of the dispensing nee-dles. No other modifications were made to the assay pro-tocol.

Data analysis

Data were analyzed and plotted using XLFit® (IDBusiness Solutions, Guildford, Surrey, UK), and calcu-lations of Z� factors were performed according to the fol-lowing formula:

Z� � 1 � {[(3 � standard deviation of positive control)� (3 � standard deviation of negative control)]/

(mean of positive control � mean of negative control)}

Signal to background (S/B) ratios were calculated asfollows:

S/B � mean signal of wells treated with compound/mean of control wells

Results

Proof-of-concept experiments

We first tested whether or not �-catenin did indeed ac-cumulate in CHOK1 cells upon inhibition of GSK3. Thecells were treated with known inhibitors of GSK3 in or-der to determine whether this had an effect on �-cateninaccumulation. Preliminary experiments were performedusing CHOK1 cells plated in six-well dishes and treatedfor 1 h with LiCl, CHIR98023 (an aminopyrimidineGSK3 inhibitor), or Compound 1 (an in-house-synthe-sized benzimidazole GSK3 inhibitor). Compound 1showed in vitro activities against GSK3� and GSK3�with 50% inhibitory concentration values of 20 nM and8 nM, respectively (data not shown). �-Catenin accumu-lation was assessed by western blotting. As shown in Fig.1, treatment of the cells with 20 mM LiCl or small mol-ecule GSK3 inhibitors resulted in increased �-cateninlevels. The �-catenin band was negligible in untreatedcontrol cell lysates or in lysates from cells treated withDMSO alone. To provide additional evidence that treat-

ment of the CHOK1 cells with LiCl or GSK3 inhibitordid indeed affect GSK3, we investigated another knowndownstream target of GSK3, the transcription factoreIF2B.25,26 Western blotting with an eIF2B antibody oncell lysates from cells treated with either LiCl or GSK3inhibitor showed a decrease in the eIF2B band, confirm-ing data from the literature. Immunoblots were strippedand reprobed using an anti-actin antibody as a loadingcontrol.

Assay development and optimization

Results from the western blotting experiments sug-gested that it would be possible to engineer a cellular assay measuring �-catenin as a readout, under these con-ditions. We chose to use a protocol based on immuno-fluorescence staining (see Materials and Methods).27 Theassay was set up in 96-well plates, in order to maintain

Yeow et al.454

FIG. 2. (A) Quantification of �-catenin accumulation inCHOK1 cells using a luminescence-based readout. Treatmentof CHOK1 cells with GSK3 inhibitors CHIR98023 and Com-pound 1 resulted in �-catenin accumulation in CHOK1 cells.Cells were treated with inhibitors for 1 h and then stained fol-lowing the cell assay protocol as described in Materials andMethods. Hatched bars, control; dark gray bars, CHIR98023;white bars, Compound 1. Data presented are means � standarderror of the mean of triplicate wells. (B) Effect of DMSO onbackground levels of �-catenin in CHOK1 cells. Cells weretreated for 3 h with varying concentrations of DMSO, beforetreatment according to the cell ELISA protocol. Data presentedare means � standard error of the mean of triplicate wells.

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a reasonable throughput and be amenable for screeningand automation purposes. The objective of these initialexperiments was to determine whether the increase in �-catenin seen by western blot could be measured usinga luminescence-based readout. CHOK1 cells were plated,and the following day treated with GSK3 inhibitors. Cellswere then fixed, permeabilized, and stained using a poly-clonal anti-�-catenin antibody. The secondary antibodyused was an HRP-conjugated goat anti-rabbit antibody.Staining of the cells was visualized using a luminol-based, chemiluminescent substrate developed for readingon luminometers. Figure 2A shows the results of thestaining using �-catenin antibody, visualized by the luminescent substrate. Background levels of �-cateninwere present in untreated cells, but these levels increasedin a concentration-dependent manner upon treatment withsmall molecule GSK3 inhibitors CHIR98023 and Com-pound 1 (Fig. 2A). The slight reduction in signal seenwith the lowest concentration of inhibitor falls within therange of values obtained for the “0” controls and doesnot represent a bona fide reduction in signal. Cells weretreated for 1 h with GSK3 inhibitors in this initial ex-periment.

An important condition to define is the concentrationof DMSO that the assay is able to tolerate without af-fecting the signal or background levels. To this end, wedetermined the effect of various concentrations of DMSOon the background �-catenin levels in our assay. Figure2B illustrates the effects of DMSO on background lumi-nescence on the CHOK1 cells treated using the cell stain-ing protocol. At 2% DMSO and higher, background lu-minescence levels decreased in comparison to theuntreated control. However, the use of 0.5% and 0.1%DMSO had no effect on background levels in the CHOK1cells. Therefore, we did not exceed 0.5% DMSO whenperforming the cell treatments.

We next optimized various parameters for the assay,namely, antibody concentrations and the length of timethat cells were treated with compound. Different con-centrations of primary and secondary antibodies weretested in various combinations, with the objective of max-imizing the S/B ratio. Cells were treated with the smallmolecule GSK3 inhibitor Compound 1 as previously de-scribed, but the final staining was performed using thedilutions of primary and secondary antibodies as shownin Fig. 3A. Results were expressed as signal-to-noise ra-tios. The signal-to-noise ratio was calculated by dividingthe maximum signal (obtained with treatment using 10�M compound) with the value from control (DMSO-treated) cells. Figure 3A shows that the optimal signal-to-noise ratios obtained under these conditions were witha 1:250 dilution of primary antibody in combination witha 1:400 dilution of the secondary antibody.

Upon determining the optimal antibody concentra-tions, we optimized the length of time the cells weretreated with the small molecule GSK3 inhibitor. In Fig.3B, cells were treated with 10 �M compound for 1 h, 2 h, and 3 h. The signal-to-noise ratios were used to de-pict the accumulation of �-catenin in the cells from eachtreatment set. The S/B ratios increased from 1 h to 3 h oftreatment with compound, with a maximum of 3.4 after3 h of treatment. Overnight treatment of the cells resultedin significant cell death (data not shown). Therefore, wechose a 3-h incubation time with compounds because thelarger signal-to-noise window would permit more sensi-tive detection of any changes in �-catenin accumulationin the cells, as compared with the 1-h treatment time.

Assay robustness criteria

In order to ensure the robustness of the cell responseand the reliability of the results obtained using this as-

A Cellular Assay for �-Catenin Accumulation 455

FIG. 3. Optimization of assay parameters. (A) Testing of optimal antibody concentrations. Cells were treated with compoundfor 1 h and then treated according to the cell-based ELISA protocol. Various dilutions of primary (1°) and secondary (2°) anti-body were tested to determine the conditions with the best signal-to-noise ratios. (B) Determination of optimal cell treatmenttimes with compound. Cells were treated with compound for between 1 and 3 h, and the ratio of luminometric units betweencontrol and treated cells was used to determine S/B ratios for the different incubation times.

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say, we assessed various criteria in a series of independentexperiments. The first parameter we assessed was in-traplate variation in the readings between control andtreated cells (Fig. 4A). Because this assay was set up in a96-well format for the purposes of screening throughput,we wished to test whether there was any variation in sig-nal from different rows on the plate. Cells were treatedwith GSK3 inhibitor for 3 h, and the signal (as measuredin luminometric units in cells treated with 10 �M com-pound) was compared with the background from controlcells. Figure 4A shows that there is a uniform response inrows 2 and 11 of a 96-well plate. The S/B ratios remainconsistent (3.2–3.3), with Z� factors of around 0.5.

Figure 4B depicts the results from testing the variationin signal between different plates, in a series of three in-dependent experiments performed on different dates.Again, the signal and background readings remained con-sistent, as did the signal-to-noise ratios and Z� factors.These results demonstrate that the signal obtained remainsconsistent across independent experiments. Figure 4C il-lustrates that consistent results were obtained between dif-ferent operators, in experiments performed independently.

Profiling of the assay

Upon finalizing the cell staining protocol and ensur-ing that the assay performed in a robust and reliable man-

ner, we chose to profile the assay using various smallmolecule inhibitors (Fig. 5). CHOK1 cells were treatedwith three concentrations of each inhibitor: 1 �M, 3 �M,and 10 �M. In addition to CHIR98023 and Compound1, we treated the cells with three other GSK3 inhibitors,SB216763, SB415286, and Compound 2. We also usedan in-house-synthesized JNK inhibitor, Compound 4.Cells were treated with these compounds for 3 h ac-cording to the protocol, and results were expressed rela-tive to those obtained using the positive control, 10 �MCompound 1 (which was designated as the “100% stim-ulus”). Results were expressed as percentages relative tothe 100% control. The maleimide inhibitor SB216763 at10 �M resulted in an increase of �-catenin to 70% of thepositive control, followed by 17% and 8% at 3 �M and1 �M, respectively. Interestingly, treatment of the cellswith the related maleimide SB415286 resulted in onlyvery low levels of �-catenin increases (10%, 5%, and 3%at 10 �M, 3 �M, and 1 �M, respectively). The alster-paullone Compound 2 resulted in the highest accumula-tion of �-catenin, with a maximum value of 107% at 10�M, relative to the positive control. Treatment of the cellswith the JNK inhibitor did not result in increased �-catenin levels (Fig. 5).

The 50% effective concentration (EC50) levels weredetermined for CHIR98023 and Compound 1 (Fig. 6).Both of these inhibitors had a concentration-dependent

Yeow et al.456

FIG. 4. Assay optimization. Cells weretreated with GSK3 inhibitor Compound 1 andprocessed according to the final assay proto-col. All data points were performed in tripli-cate, in three independent experiments. Alldata presented are means � standard error ofthe mean of a total of nine wells. (A) Varia-tion of signal in rows 2 and 11 of a 96-wellplate. (B) Variation in signal among three sep-arate plates. (C) Variation in signal betweentwo different operators. Hatched bars, control;white bars, 10 �M Compound 1.

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effect on �-catenin accumulation in the CHOK1 cells,with EC50 values of 4.5 � 1.0 �M for CHIR98023 and2.0 � 0.2 �M for Compound 1. Representative EC50

graphs are depicted in Fig. 6. Consistent EC50 valueswere obtained using this assay, for both the GSK3 in-hibitors tested. Identical EC50 values were obtained forall compounds tested using the automated assay proto-col (data not shown).

Figure 7 depicts the S/B ratios and Z� factors from20 individual plates, performed over the course of a 2-month interval. The results shown in Fig. 7 were ob-tained using the automated assay protocol. The S/B ra-tios varied between 2 and 4.5, and the Z� factors didnot fall below 0.5. The Z� factor satisfies the require-ments for assessing both specific signal and uniformityof the response for an accurate evaluation of assay per-formance.28 In general, a Z� value greater than 0.5 sug-gests that an assay is suitable for automation andscreening.

Discussion

In light of the importance of GSK3 as a therapeutictarget, a cellular assay measuring its endogenous ac-tivity would be a valuable tool for research and com-pound progression. This study describes the develop-ment of a cell-based assay to detect and measureendogenous �-catenin accumulation in intact CHOK1

cells, in response to treatment with GSK3 inhibitors.The focus was on the development of a robust and re-producible assay with a reasonably high throughput, ina 96-well format. We chose to develop a cellular assaywith a view towards maintaining the complexity of thecellular environment, for testing small molecule in-hibitors for their ability to inhibit GSK3 and therebyinfluence �-catenin accumulation within the cell. Al-though there is much prior literature on the accumula-tion of �-catenin in response to various signals, thiscell-based assay is one of the first that enables the de-tection and quantification of �-catenin in 96-well for-mat, using intact cells.

Our initial proof-of-concept experiments using LiCland a known small molecule GSK3 inhibitor29 revealedthat accumulation of �-catenin did occur in CHOK1 cellsunder these conditions. To confirm that the increase in�-catenin levels under these conditions was indeed due

A Cellular Assay for �-Catenin Accumulation 457

FIG. 5. Profiling of the assay using a selection of small mol-ecule inhibitors. CHOK1 cells were treated with compounds at10 �M (gray bars), 3 �M (black bars), and 1 �M (white bars).Each data point was performed in triplicate, in three independentexperiments. Data presented are means � standard error of themean of triplicate wells. CDK5, cyclin-dependent kinase 5.

FIG. 6. EC50 values (in �M) for GSK3 inhibitors on �-catenin accumulation in CHOK1 cells: (A) CHIR98023 and(B) Compound 1. Each data point was performed in triplicate.Data presented are means � standard error of the mean of trip-licate wells.

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to GSK3 inhibition, we performed western blots to de-tect levels of eIF2B, another known downstream targetof GSK3.26,30 In keeping with previously published data,inhibition of GSK3 through the use of LiCl or a smallmolecule inhibitor resulted in a decrease in eIF2B levels.This suggests that the increase in �-catenin observed un-der these conditions was likely due to GSK3 inhibition.

These results prompted us to test whether such an in-crease could be directly measured in CHOK1 cells vialuminescence. Further work on optimizing cell treatmentconditions and assay parameters resulted in a robust andreliable protocol for detection of �-catenin in these cells.Our assay measures endogenous �-catenin production infixed, adherent CHOK1 cells using a polyclonal antibodyagainst total �-catenin. Live cells were first treated for a3-h period with small molecule inhibitors of GSK3, andthen fixed, processed, and stained using the �-catenin an-tibody, followed by staining with a secondary antibodycoupled to HRP. Levels of �-catenin are quantified us-ing a luminescence system, generating a stable signal thatcan be read using a luminometer. The relatively simplesteps in the assay protocol are amenable to automation;indeed, the assay has been fully automated and run on aTitertek MAP C station. Identical results were obtainedfor EC50 values in comparison to the manually run pro-tocol (data not shown).

In order to further profile this assay, we tested severalcompounds, including three GSK3 inhibitors and oneJNK inhibitor. Among the three GSK3 inhibitors tested,the alsterpaullone Compound 2 resulted in the highest accumulation of �-catenin levels, followed by the

maleimide SB216763. The related maleimideSB415286 was not as potent as SB216763 in inducing�-catenin accumulation in our assay. The results withthe maleimide inhibitors reflect previously publisheddata.16 The authors showed that at 10 �M, SB415286did not induce transcription of a �-catenin-LEF/Tcf re-porter gene in human embryonic kidney 293 cells. Bycomparison, SB216763 yielded a 2.5-fold stimulationof reporter activity above basal levels.16 Our results withboth these inhibitors reflected these published results incells treated according to our assay protocol. The useof JNK inhibitor Compound 3 resulted in no augmen-tation of �-catenin levels. Taken together, these resultssuggest that our assay can be used to compare the effi-cacy and cell permeability of small molecule inhibitorsof GSK3, by measuring their effect on �-catenin accu-mulation in the cells.

Our final assay has distinct advantages over other meth-ods currently used to measure �-catenin production. Theuse of native CHOK1 cells eliminates the necessity for re-porter genes and the generation of stably transfected celllines. Our assay is performed in a 96-well format, allow-ing for higher throughput for compound screening, as com-pared with reporter assays or immunohistochemical analy-sis. �-Catenin is an adherens junction protein that canaccumulate in the cytoplasm and translocate to the nucleusin response to the appropriate signals.31 Although our as-say does not discriminate between nuclear and cytoplasmic�-catenin, it enables the direct measurement of total cellu-lar �-catenin levels in response to treatment with small mol-ecules. The cell treatments, processing, staining, and finalreadings can be completed within 1 working day, enablingthe rapid selection of molecules for further testing, possi-bly using a cellular imaging-based approach.32

It should be noted that the long-term use of lithiumions has not been associated with increased cancer risk.33

No increases in cytosolic �-catenin levels have been seenin tissues from Zucker diabetic rats upon treatment witha GSK3 inhibitor.21 GSK3 inhibition in primary cells maybe insufficient to cause an elevation in �-catenin levels;this may be a characteristic of transformed cell lines.3

Nevertheless, the accumulation of �-catenin is also awell-documented oncogenic event,31 and our assay couldbe used as a screen to eliminate inhibitors that have thiseffect. Data from the �-catenin assay could complementdata from other functional assays, such as those measur-ing glucose production, in order to progress compoundsthat target GSK3 without triggering the accumulation of�-catenin.

Conclusions

There is a current trend towards the development ofcell assays that reflect the complexity of biological sys-

Yeow et al.458

FIG. 7. Representative S/B ratios (gray bars) and Z� factors(dots) from 20 individual plates. Cells were treated with theGSK3 inhibitor Compound 1, and S/B ratios were calculatedas described in the text. Z� factors were calculated according toZhang et al.28

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tems. Although in vitro assays remain indispensabletools in drug discovery, there is an ever-increasing de-mand for taking into account the functioning of targetswithin a more “natural” context, with emphasis on bio-logically relevant cellular assays.34 We have developeda robust cellular assay for measuring �-catenin accu-mulation in CHOK1 cells, in a 96-well format. This as-say is suitable for automation and can be used to screenfor modulators of GSK3. Using a fast-growing cell lineand a rapid and convenient luminometric readout, thisprotocol is suitable for compound progression and smallmolecule screening.

Acknowledgments

The authors wish to thank Drs. Dominique Perrin andAnthony Nichols for critical reading of the manuscriptand Ms. Céline Hattrait for assistance with compoundscreening.

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Address reprint requests to:Karen Yeow. Ph.D.

Serono Pharmaceutical Research Institute14 Chemin des Aulx

1228 Plan-les-Ouates, Switzerland

E-mail: Karen.Yeow@phocus.com

or

Paul Lang, Ph.D.Serono Pharmaceutical Research Institute

4 Chemin des Aulx1228 Plan-les-Ouates, Switzerland

E-mail: Paul.Lang@serono.com

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