ANALYTICAL

Analytical Biochemistry 320 (2003) 66–74

www.elsevier.com/locate/yabio

BIOCHEMISTRY

High-throughput screening for the identification ofsmall-molecule inhibitors of retinoblastoma protein

phosphorylation in cells

S. Elaine Barrie,a Ebun Eno-Amooquaye,b Anthea Hardcastle,a Georgina Platt,b

Juliet Richards,a David Bedford,a Paul Workman,a Wynne Aherne,a

Sibylle Mittnacht,b and Michelle D. Garretta,*

a Cancer Research U.K. Centre for Cancer Therapeutics at the Institute of Cancer Research,

Brookes Lawley Building, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UKb Cancer Research U.K. Centre for Cell and Molecular Biology at the Institute of Cancer Research,

Chester Beatty Laboratories, 237 Fulham Road, Sutton, Surrey, SW3 6JB, UK

Received 10 January 2003

Abstract

The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and

regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-

dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK in-

hibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor

suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to

development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the

level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a

fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on

pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is re-

producible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules

that will block pRb phosphorylation.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Screen; Inhibitors; Retinoblastoma protein; Phosphorylation; CDK; Cancer

1 Abbreviations used: PP1, protein phosphatase 1; CDK, cyclin-

Tumour progression often involves the genetic alter-

ation of a class of genes known as tumor suppressors [1].The classic example of a tumor suppressor is the reti-

noblastoma gene, RB, which was originally identified in

1986 as a genetic locus associated with the development

of inherited retinal cancer [2]. Since then it has become

clear that RB plays an important role in the develop-

ment of many other tumor types. The RB gene itself

encodes a nuclear protein of 105 kDa (pRb) that func-

tions as a negative regulator of cell cycle progression.This function is mediated through its ability to bind to

* Corresponding author. Fax: +44-208-770-7899.

E-mail address: Michelle.Garrett@icr.ac.uk (M.D. Garrett).

0003-2697/$ - see front matter � 2003 Elsevier Science (USA). All rights res

doi:10.1016/S0003-2697(03)00349-X

and regulate the activity of a variety of transcriptionally

active proteins, including the E2F family of transcrip-tion factors [3]. Phosphorylation of pRb blocks the

binding of these proteins, thereby obliterating its ability

to act as a suppressor of proliferation.

The phosphorylation state of pRb is tightly regulated

in cells via opposing enzymatic reactions; activating

dephosphorylation carried out by members of the pro-

tein phosphatase 1 (PP1)1 superfamily and inactivating

dependent kinase; CKIs, CDK inhibitors; INK4, inhibitors of CDK4;

CIP/KIP, CDK interacting protein/kinase inhibitory protein; TRF-

Cellisa; time-resolved fluorescence-based cellular immunosorbant

assay.

erved.

S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74 67

phosphorylation performed by members of the cyclin-dependent kinase (CDK) family of serine/threonine

kinases [4,5]. Dephosphorylation arises as a matter of

routine concurrent with the completion of mitosis, thus

providing active pRb, which restrains unlicensed transit

from G1 into the next S phase [6].

Phosphorylation and deactivation of pRb during G1

involves sequential modification by the cyclin D-de-

pendent kinases CDK4 and CDK6 in early to mid G1and the cyclin E-dependent kinase CDK2 toward late

G1 [7]. The overall cellular activity of the CDKs is

controlled through a number of mechanisms including

CDK phosphorylation, cyclin synthesis, cyclin degra-

dation, and the action of two families of CDK inhibitors

(CKIs) [8]. The first family comprises the inhibitors of

CDK4 (INK4) proteins that bind to and specifically

inhibit the cyclin D-dependent kinases, while the secondfamily, known as the CDK interacting protein/kinase

inhibitory protein (CIP/KIP) family, can bind to either

the cyclin D-dependent kinases or the cyclin E/CDK2

during G1 [9].

Pathways involved in the regulation of G1 CDK ac-

tivity are frequently affected in tumor cells. For example,

cyclin D1 expression (and thus cyclin D-dependent ki-

nase activity) can be activated by both Ras and Wntsignaling pathways, which in turn are known to be de-

regulated in a large fraction of different cancers [10,11].

CDK4 and CDK6 are themselves found overexpressed

or rendered resistant to the action of INK4 CKIs in a

number of tumor types. The INK4 CKIs are also subject

to mutational inactivation or gene silencing in other

instances [12]. Finally, the function of Archipelago, the F

box protein critically involved in degradation of cyclinE, is lost in a subset of human tumors [13,14].

Thus in many types of tumor, pRb phosphorylation

may arise inappropriately and its inhibition may there-

fore have therapeutic value as an anticancer treatment.

With this in mind, we have developed a high-throughput

cell-based assay for detection of changes in phosphory-

lation on pRb. The aim here is to use the assay to screen

for small molecules that will block pRb phosphorylationin human tumor cells, although this type of assay could

be used to study the effect of any agent, of known or

unknown function, on pRb phosphorylation. We have

taken this approach rather than that of using bio-

chemical screens for inhibitors of specific gene products

on the pRb pathway, as compounds identified using this

cell-based assay format may have superior pharmaco-

logical properties including cell permeability and intra-cellular stability. This strategy also allows the targeting

of more than one particular protein or pathway and

indeed may allow for the identification of previously

unknown but superior targets upstream of pRb. It

should be pointed out that a number of small-molecule

inhibitors have been identified for several targets known

to be upstream of pRb, mostly using biochemical assays.

These include inhibitors of receptor tyrosine kinases,compounds that inhibit different stages of the ras sig-

naling pathway, and a number of small molecules that

block the activity of a range of CDKs [15]. Nevertheless,

there is a continuing need for additional inhibitors of

existing and new targets that have structural novelty and

could form the basis of new drug optimization program

[16]. One key issue here is the fact that, although many

cancer drug development programs are initiated everyyear in countless organizations, only a very few com-

pounds make it to a Phase I clinical trial and so initia-

tion of new drug screens and drug development

programs is of critical importance.

In this paper we describe the development of a high-

throughput immunoassay to detect changes in the cel-

lular level of pRb phosphorylation using a time-resolved

fluorescence-based cellular immunosorbant assay (TRF-Cellisa) format. Application of the TRF-Cellisa to a

screen of 2000 compounds has led to the identification of

a small molecule that blocks the cellular phosphorylation

of pRb, a result confirmed by Western blot analysis of

lysates prepared from cells treated with this compound.

This assay could also serve as a readout to assess the

ability of any agent to affect the cellular phosphorylation

of pRb, for example a known chemotherapeutic drug ora small molecule identified in a biochemical screen.

Materials and methods

Materials

The human HT29 and HCT116 colon carcinoma andC33A cervical cell lines were obtained from ATCC and

maintained at 37 �C, 5% CO2 in Dulbecco�s modified

Eagle�s medium containing 10% fetal calf serum. The

mouse monoclonal antibody 14001A (Catalogue No.

554136, clone G3-245) to measure total expression of

pRb and the mouse monoclonal antibody 14441A

(Catalogue No. 554164, clone G99-549) to measure the

nonphosphorylated form of serine 608 of pRb were bothfrom PharMingen. The rabbit polyclonal antibody for

detection of phosphorylated serine 807/811 on pRb

(Catalogue No. 9308S) was from Cell Signalling Tech-

nology-NEB (UK) Ltd. Rabbit antimouse IgG labeled

with Europium (Catalogue No. AD0124) and proprie-

tary Assay and Enhancement solutions were obtained

from Perkin–Elmer Life Sciences. The 96-well micro-

plates were Microtest tissue culture plates from Falcon(Catalogue No. 3072). Roscovitine was purchased from

Sigma.

Generation of phospho-specific monoclonal antibodies

Monoclonal antibodies with specificity for phos-

phorylated Serine 608 of pRb were raised using the

68 S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74

synthetic peptide COOH-C-ADMYLSPPLRSPK-NH2(where SP is the phosphorylated serine residue) coupled

to keyhole limpet hemocyanin for an immunogen. Hy-

bridoma 51B7 was selected from a series of candidates

with suitable specificity based on its performance in

single-cell staining.2 Hybridoma 7F10, with specificity

for threonine 356 of pRb, was raised against synthetic

peptide COOH-C-ERERTPRKNN-NH2 (where TP

is the phosphorylated threonine residue) coupled tomycobacterium tuberculosis PPD. Hybridoma fusions

were conducted essentially as described by Harlow and

Lane [17].

Cell culture and compound treatment

Cells were plated in 96-well microplates at 8000 cells

per well (unless stated otherwise), in a volume of 160 llof growth medium and left for 48 h before treatment was

carried out. For compound treatment, 40 ll of growth

media containing various concentrations of compound

was added to each well, with medium containing the

compound vehicle, dimethyl sulfoxide (DMSO), added

to the control wells.

Cell-based immunoassay for the detection of phospho-pRb

After the desired treatment time, the medium was

removed and the cells were fixed by exposure to 200 llper well of 4% paraformaldehyde, 0.3% Triton X-100 in

phosphate-buffered saline (PBS) for 15min at room

temperature. The plates were washed once with PBS and

either probed immediately or stored at 4 �C after filling

with Tris-buffered saline (TBS), pH 7.6, containing 0.1%sodium azide. The plates were emptied and treated with

100 ll per well of blocking solution composed of 0.1%

Tween 20, 5% milk in TBS for 1 h before probing with

primary antibody. The primary antibodies were diluted

into the same blocking solution, and the plates were left

exposed to the antibody at 4 �C overnight. Three washes

with 0.1% Tween 20 in water (Tween 20 wash) were then

carried out. Signal detection used a rabbit antimouseIgG labeled with Europium (secondary antibody) di-

luted in proprietary Assay buffer to 0.3 lg/ml. After 2 h,

the plates were washed again three times with Tween 20

wash, and 100 ll per well of proprietary Enhancement

solution was added. The plates were shaken for at least

10min before reading in a Victor plate reader, with

excitation at 340 nm and emission at 615 nm (Perkin–

Elmer Life Sciences). Protein determinations were car-ried out after this using the sulforhodamine blue [18] or

bicinchoninic acid assays (Pierce).

2 This monoclonal antibody with specificity for phosphorylated

Serine 608 of pRb is now available from Serotech (MCA2104/

MCA2105).

Western blot analysis

This was performed as described previously [19].

Quantitation was performed using the public domain

program Image J developed at the US National Insti-

tutes of Health and available at http://rsb.info.nih.gov/

nih-image.

Results

Detection of pRb phosphorylation in cell monolayers: site

and phospho-specificity of the 51b7 monoclonal antibody

The aim of this work was to develop a high-

throughput immunoassay to detect cellular changes in

pRb phosphorylation that could be used for screeningpurposes. To monitor changes in pRb phosphorylation,

we generated a mouse monoclonal antibody, 51B7,

which specifically detects serine 608 when this residue

is phosphorylated. Serine 608 represents one of the 15

known CDK phosphorylation sites of pRb [5]. Good

evidence is also available for the cell cycle-dependent

regulation of this site. In vitro serine 608 is effectively

phosphorylated by CDK4/cyclin D1 and in cells is seento become modified in mid G1 phase [20]. Furthermore,

loss of modification in response to growth factor with-

drawal and treatment of cells with antiproliferative do-

ses of TGF beta and following M phase exit have all

been demonstrated [6,21]. Thus phosphorylation chan-

ges at this site are likely to provide an adequate surro-

gate assay for activity changes in a variety of pathways

affecting pRb phosphorylation and activity.Site specificity of this antibody has been demon-

strated in C33A cervical carcinoma cells, which lack

pRb and thus are not recognized by the antibody

(Fig. 1A and data not shown). C33A cells were trans-

fected with plasmid constructs encoding wild-type or

mutant (serine 608 to alanine) pRb, along with cyclin

D1 and CDK4 to promote pRb phosphorylation and

green fluorescent protein (GFP) to identify transfectedcells. Immunostaining of these cells with 51B7 shows

that while this monoclonal antibody recognizes wild-

type pRb, it cannot detect the mutant protein, suggest-

ing that the signal generated with 51B7 is specific to the

serine 608 site (Fig. 1A).

Phospho-specificity of 51B7 was tested in the

HCT116 colon carcinoma cell line by treating fixed and

permeabilized cells with lambda phosphatase to removethe phosphate, in the absence or presence of the phos-

phatase inhibitors sodium fluoride and beta glycero-

phosphate (Fig. 1B). HCT116 cells were used for this

analysis as they harbor wild-type pRb and were one of

the cell lines used for assay development (see below).

Upon phosphatase treatment of the fixed and permea-

bilized HCT116 cells, the signal detected using 51B7 is

Fig. 1. Site- and phospho-specific recognition of pRb by the 51B7 monoclonal antibody. (A) cells were transfected with a combination of expression

plasmids encoding GFP, human cyclin D1, human CDK4, and either wild-type pRb (Rb-WT) or a pRb mutant in which the serine at position 608 is

replaced by alanine (S608A). Following transfection, cells were seeded onto glass cover slips and stained using either the 51B7 monoclonal antibody,

which is specific for phosphorylated S608 (51B7), or the 14001A monoclonal antibody, which recognizes total pRb expression (Total Rb). (B) Fixed

HCT116 colon carcinoma cells were left untreated (Mock) or treated with lambda phosphatase in the absence (PPT) or presence of phosphatase

inhibitors (PPT/INH). Cells were subsequently stained using 51B7 or 14001A antibodies for detection of phosphorylation of S608 on pRb (51B7) or

total pRb expression (Total Rb).

S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74 69

lost. In contrast the signal obtained with the 14001A

monoclonal antibody, which detects total expression of

pRb, is still present. We therefore conclude that the

51B7 signal is phospho-specific. The site and phospho-

specificity of 51B7 were also confirmed by Western blotanalysis (data not shown).

Development of the TRF-Cellisa: a high-throughput

immunoassay for detection of pRb phosphorylation in cells

Two human colon carcinoma cell lines, HCT116 and

HT29, were chosen for the assay development. Both

cell lines express functional pRb but harbor differingmutations on cellular pathways known to impinge on

the regulation of Rb phosphorylation. In particular,

HCT116 has wild-type p53 but possesses a mutation in

Kirsten RAS (K-ras). In contrast, the HT29 cell line

expresses mutated p53, but has no known mutations in

K-ras. This information can be found on the Molecular

Targets section of the web site for the DevelopmentalTherapeutics Program of the National Cancer Institute,

at http://dtp.nci.nih.gov/mtargets/mt_index.html.

To validate the assay, we selected the CDK inhibitor

roscovitine as our positive control. In addition to being

a CDK inhibitor in vitro, roscovitine is also reported to

block the cellular phosphorylation of pRB [21]. To

verify that roscovitine would inhibit pRb phosphoryla-

tion in our test cell lines, both were treated with 10, 20,30, or 50 lM compound, and loss of pRb phosphory-

lation was assayed by Western blot analysis using 51B7

70 S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74

and 14001A. Inhibition of pRb phosphorylation onserine 608 (P-Ser608) of pRb in both HT29 and HCT116

colon carcinoma lines was confirmed by a decrease in

the 51B7 phospho-specific antibody signal and as a band

shift from the hyperphosphorylated state to the hypo-

phosphorylated state using 14001A to detect total ex-

pression of pRb. This was seen in both cell lines after

treatment with 30 and 50 lM of roscovitine (Fig. 2).

Quantitation of the Western blots (as described underMaterials and methods) confirmed a marked loss of the

phospho-serine 608 signal in both cell lines after treat-

ment with roscovitine.

The cell-based immunoassay for detection of pRb

phosphorylation was set up using a TRF-Cellisa format

on a fixed monolayer of cells. This assay utilizes the

51B7 mouse monoclonal antibody for detection of the

P-Ser608 signal and a Europium-labeled rabbit anti-mouse secondary antibody as the readout. The condi-

tions for fixing and blocking were selected to give a good

signal versus background response, which was routinely

on the order of 8 for this assay (data not shown).

Critical features of any biological assay are the linear

relationship between the signal observed and the num-

ber of cells or the amount of protein present and whe-

ther this relationship will hold after treatment withmodulators of the signal being detected. To test this

linear relationship for the P-Ser608 signal, cells were

plated in 96-well microplates at seeding densities of be-

tween 1000 and 16,000 per well and treated with either

Fig. 2. Western blot analysis of total and P-Ser608 pRb levels in cells treated

HCT116 cell lines were treated with medium alone (C1), medium containing

harvested. Samples were subjected to SDS–polyacrylamide gel electrophoresi

(Total Rb) using the 14001A monoclonal antibody and phosphorylation at se

of the Western blots was carried out and the values obtained are given belo

the vehicle control, DMSO, or 30 lM roscovitine for24 h. The net signal was then plotted against the optical

density measurement from SRB assays performed on

each well, which give a measure of the amount of pro-

tein present per well. The 51B7 antibody gave signals in

both DMSO- and roscovitine-treated cells that were

directly proportional to the amount of protein in each

well and this linear relationship was maintained in both

cell lines (Fig. 3A). It was also noted that the proteinlevel in wells treated with roscovitine was lower than

that in DMSO-treated wells seeded with an equivalent

cell number. This is because the growth of the roscovi-

tine-treated cells is inhibited compared to that of the

vehicle control. It can also be seen in this experiment,

however, that at any particular protein concentration,

there is a smaller 51B7 signal in roscovitine-treated wells

versus DMSO-treated wells. Thus, there is a specific lossof phospho-pRb signal per unit of protein in wells in-

cubated with the positive control compound. This ob-

servation is important as it means that comparing the

phospho-pRb signal/protein ratios will distinguish

compounds that specifically cause a loss of phospho-

pRb signal per unit protein from those that just cause a

reduction in cell density without affecting the pRb

phosphorylation.This study was repeated using the mouse monoclonal

antibody 14441A, which specifically recognizes the

nonphosphorylated form of the serine 608 site (non-P-

Ser608) on pRb [20]. Using this antibody for detection,

with the CDK inhibitor roscovitine. Exponentially growing HT29 and

the drug vehicle DMSO (C2), or 10–50lM roscovitine for 24 h, and

s on 6% gels and Western blotted for detection of total pRb expression

rine 608 (P-Ser608) using the 51B7 monoclonal antibody. Quantitation

w each blot as a percentage (%) of the DMSO control (C2).

Fig. 3. Linear relationship between the Europium signal and the protein amount for the TRF-Cellisa. Exponentially growing HT29 and HCT116 cells

were plated at seeding densities of between 1000 and 16,000 per well in 96-well microplates and treated with either DMSO (r) or 30lM roscovitine

(�) for 24 h, and the TRF-Cellisa assay was performed using the 51B7 monoclonal antibody for the P-Ser608 pRb signal (A) and the 14441A

monoclonal antibody for the non-P-Ser608 signal (B). Once the TRF-Cellisa had been completed, all wells were subjected to the SRB protein assay.

S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74 71

it would be envisaged that the Europium signal would

increase in those wells treated with roscovitine as

phosphorylation on pRb was lost. Like 51B7, 14441A

gave signals in both cell lines that were directly pro-

portional to the amount of protein in each well and thislinear relationship was maintained with roscovitine

(Fig. 3B). As predicted, wells treated for 24 h with

roscovitine exhibited a strong increase in Europium

signal, compared to DMSO-treated wells at the same

protein concentration, suggesting that there is a rosco-

vitine-induced specific increase in the non-P-Ser608

signal, per unit protein.

The next step was to determine whether the sensitivityof signal detection using the TRF-Cellisa was equivalent

or better than detection by Western blot and to deter-

mine the versatility of the assay format, by testing it on

two cell lines using two different antibodies (Fig. 4). For

this experiment, both colon carcinoma cell lines were

treated with 10, 20, or 30 lM roscovitine for 24 h. The

TRF-Cellisa was performed on the plates using the 51B7

antibody to detect a loss of pRb phosphorylation. A lossof 51B7 signal was easily detectable with 20 or 30 lMroscovitine (Fig. 4A, filled bars) and was similar to the

signal loss detected by Western blot analysis (Fig. 2),

suggesting that the TRF-Cellisa has comparable sensi-

tivity. The experiment was repeated using the 14441A

antibody for detection of non-P-Ser608 pRb. At 20 and

30 lM roscovitine, which causes a decrease in P-Ser608

signal, there is a concomitant increase in the non-P-Ser608 signal (Fig. 4A, hatched bars). Thus the TRF-

Cellisa was shown to be functional in two cell lines with

two antibodies that detect different forms of pRb.

To assess the effect of treatment time on signal de-

tection, both colon carcinoma cell lines were grown in

the presence of 10, 20, or 30 lM roscovitine for 18, 24,or 30 h and the TRF-Cellisa was performed with the

51B7 monoclonal antibody. The results show that the

assay could be performed at any of these times after

roscovitine treatment and so a time point of 24 h was

selected for all future experiments.

Performance of the TRF-Cellisa in a high-throughput

screen to identify inhibitors of pRb phosphorylation

The TRF-Cellisa was next used to screen 2000 com-

pounds at a concentration of 10 lM on the HCT116 cell

line, using the 51B7 monoclonal antibody for the de-

tection of P-Ser608 pRb. Roscovitine (30 lM) was in-

cluded as a positive control on each plate. The mean of

the response to this concentration of roscovitine was

54% with standard deviations of 6.9% between platesand 10% within plates. The assay reproducibility is

shown in Fig. 5, which depicts the roscovitine versus

DMSO signals for each of the 28 plates screened. A hit

from this screen was defined as a compound that gave a

decrease of P 50% in P-Ser608 signal/protein level in

the assay. From this screen we identified five hits,

hereafter referred to as compounds A, B, C, D, and E.

Only compound A reconfirmed repeatedly at 10 lM inthe TRF-Cellisa and by Western blot, lowering the

Fig. 4. Detection of the effects of roscovitine using the TRF-Cellisa. Exponentially growing HT29 and HCT116 cell line were treated with medium

alone (Con), medium containing the drug vehicle DMSO (DMSO), or 10–30lM CDK inhibitor roscovitine. (A) After 24 h treatment the TRF-

Cellisa was performed with either the 51B7 monoclonal antibody for detection of P-Ser608 pRb (black bars) or the 14441A monoclonal antibody for

detection of non-P-Ser608 pRb (hatched bars). (B) After 18, 24, or 30 h (hatched, black, and checkered bars, respectively) of treatment, the TRF-

Cellisa was performed with the 51B7 monoclonal antibody for detection of P-Ser608 pRb. Once the TRF-Cellisa had been completed, all wells were

subjected to the SRB protein assay. The results (Europium signal/protein) are shown as a percentage of the DMSO control value and are the mean

plus one standard error of three replicate wells in a representive experiment.

Fig. 5. Reproducibility of the TRF-Cellisa in a screen of 2000 com-

pounds for inhibitors of pRb phosphorylation. HCT116 cells were

plated at 8000 per well and compounds (10 lM) added for 24 h, after

which the TRF-Cellisa was performed using the 51B7 monoclonal

antibody for detection of P-Ser608 signal on pRb. Once the TRF-

Cellisa had been performed, all wells were subjected to protein assay.

The results (Europium signal/protein) are shown for each plate. The

closed and open circles each represent the mean value�one standard

deviation for eight replicate wells treated with DMSO and 30 lM of

the CDK inhibitor roscovitine, respectively.

72 S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74

confirmed hit rate to 0.05% (data not shown). The

ability of compounds A and B to block the cellular

phosphorylation of pRb at several different sites

(including phospho-serine 608) was investigated using

Western blot analysis and antibodies that each recognizea different phosphorylated residue on pRb. Compound

A, but not compound B, caused a decrease in the P-

Ser608 signal to 28% of the control, which is in agree-

ment with the TRF-Cellisa, where a hit was defined as a

compound that gave a decrease of P50% in P-Ser608

signal/protein level (Fig. 6). A concomitant increase in

the non-P-Ser608 signal was detected in the compound

A-treated sample using the 14441A antibody (Fig. 6).Loss of phosphorylation on pRb was also detected at

amino acids 807, 811, and 356 (all known sites of CDK

phosphorylation) in samples treated with compound A

(Fig. 6). From these results we conclude that compound

A can block the cellular phosphorylation of pRb.

Discussion

Our objective was to develop a high-throughput im-

munoassay for the detection of pRb phosphorylation in

cells and to use this assay in a screen to identify cell-

permeable small molecules that will block pRb phos-

phorylation in human tumor cells. This type of assay

could also be used to study the effect of any agent, of

Fig. 6. Western blot analysis of samples prepared from cells treated

with compounds A and B. Cells were plated in 90-mm dishes, treated

with either DMSO (Con) or 10 lM of either compound A (A) or

compound B (B) for 24 h, and harvested. Samples were subjected to

SDS–polyacrylamide gel electrophoresis on 6% gels and Western

blotted for detection of total pRb expression (Total Rb) using

the 14001A monoclonal antibody, non-phospho-Ser608 on pRb (Non-

P-Ser608) using the 14441A monoclonal antibody, P-Ser608 on pRb

(P-Ser608) using the 51B7 monoclonal antibody, and P-Ser807/811 (P-

Ser807/811) and P-Thr356 (P-Thr356) signals on pRb using the anti-

bodies described under Materials and methods. Quantitation of the

Western blots was carried out and the values obtained are given below

each blot as a percentage (%) of the control for all blots except the

non-phospho-Ser608 where values are given as a percentage of

compound A.

S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74 73

known or unknown function, on pRb phosphorylation.

A high-throughput assay of any type needs to be rapid

and reproducible, but must not compromise selectivity

or sensitivity of detection. The techniques of Western

blotting and enzyme-linked immunosorbant assay weretherefore inappropriate for our purposes, since the for-

mer has a much lower throughput, while the latter, al-

though in 96-well format, is less sensitive than

Europium-based assays [22,23].

The assay was developed using both HCT116 and

HT29 cells, to construct a robust format that could

potentially be used on multiple cell lines. The choice of

antibody is pivotal to the success of the immunoassay,as it needs to be selective for its target in the context of a

fixed cell and available in sufficient quantities for high-throughput purposes. The monoclonal antibody 51B7

fulfilled these criteria as it was both site- and phospho-

specific against fixed cells (Fig. 1), and could be used for

direct comparisons between results generated in the

TRF-Cellisa versus Western blot (Figs. 2–4 and 6). As a

monoclonal antibody, it could also be produced in large

quantities. The choice of a Europium-labeled secondary

antibody for quantifying the amount of primary anti-body bound in the assay was based on the sensitivity of

detection (1 fmol of Europium gave a signal of 30,000

units; data not shown) and the linearity of the signal

over a wide protein range (Fig. 3).

For any biological assay, the linear relationship be-

tween the signal observed and the number of cells or the

amount of protein present and whether this relationship

will hold after treatment with modulators of the signalbeing detected are crucial. Using the 51B7 monoclonal

antibody, the TRF-Cellisa gave signals that were di-

rectly proportional to the amount of protein in each well

and detected a specific loss of phosphorylation on serine

608 of pRb in wells treated with the CDK inhibitor

roscovitine (Fig. 3A and B). This observation is im-

portant as it means that the assay will distinguish

compounds that specifically cause a loss of phospho-pRb signal in cells from those that just cause a reduction

in cell density without affecting the pRb phosphoryla-

tion. At this point, the 14441A monoclonal antibody

was also tested in the TRF-Cellisa to ascertain whether

an antibody other than 51B7 could be utilized to mon-

itor pRb phosphorylation in this assay format. This

antibody appeared to perform well in the assay (Figs. 3

and 4), but was not used further, due to cost issues.Screening of 2000 compounds with the TRF-Cellisa

was performed using the 51B7 monoclonal antibody for

signal detection. The screen had acceptable reproduc-

ibility, the mean of the response to the positive control

of 30 lM roscovitine being 54% with standard devia-

tions of 6.9 and 10% between and within plates, re-

spectively. Cell-based assays do tend to have higher

coefficients of variation than biochemical assays, due tothe length of the assay, which is often the doubling time

of the cells. Moreover, compound-associated cytotox-

icity or antiproliferative effects may alter the cell num-

ber. It is for this reason that it is so important that

protein levels in each well are measured after the TRF-

Cellisa is completed.

The initial 0.25% ‘‘hit’’ rate from the screen (com-

pounds A–E) and the confirmed ‘‘hit’’ rate of 0.05%(compound A) is not unreasonable. The lack of confir-

mation of all the initial ‘‘hits’’ was disappointing but is

common in high-throughput screens. Western blot

analysis of lysates prepared from cells treated with

compounds A and B confirmed the TRF-Cellisa result

that compound A is an inhibitor of pRb phosphoryla-

tion in cells.

74 S.E. Barrie et al. / Analytical Biochemistry 320 (2003) 66–74

To conclude, a high-throughput immunoassay toidentify small-molecule inhibitors of pRb phosphoryla-

tion in cells has been developed. This assay, known as

the TRF-Cellisa, has been used to test 2000 compounds

for their ability to block the cellular phosphorylation of

pRb. From this pilot screen, it is clear that the assay is

rapid and reproducible and has allowed the identifica-

tion of one active compound, compound A. We now

intend to use this assay for a more comprehensive high-throughput screen.

Acknowledgments

This work was supported by Cancer Research U.K.

and the Institute of Cancer Research. We also thank

David Mason for his help with formatting the figures.

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