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Research Article
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Received: 15 May 2010, Revised: 24 September 2010, Accepted: 4 October 2010, Published online in Wiley Online Library: 00 Month 2010
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(wileyonlinelibrary.com) DOI:10.1002/jmr.110664
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SLabel-free sensing and atomic forcespectroscopy for the characterizationof protein–DNA and protein–proteininteractions: application to estrogen receptorsA. Berthiera,b*, C. Elie-Cailleb, E. Lesniewskac, R. Delage-Mourrouxa
and W. Boireaub
7677
In this paper we describe a new surface plasmonJ. Mol. Rec
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resonance (SPR) biosensor dedicated to potential estrogeniccompounds prescreening, by developing an estrogen receptor (ER) specific DNA chip. Through the covalent binding ofa DNA strain wearing the estrogen response element (ERE) to an activated 6-mercapto-1-hexadecanoic acid and11-mercapto-1-undecanol self-assembled monolayer on gold surface, the SPR biosensor allows to detect specifically,quickly, and without any labeling the binding of ER in the presence of estrogen. In parallel, we investigated the ERinteraction with itself, in order to study the formation of ER dimer apparently needed to activate the gene expressionthrough ERE interaction. For that, we engaged force spectroscopy experiments that allowed us to prove that ER needsestrogen for its dimerization. Moreover, these ER/ER intermolecular measurements enabled to propose an innovativescreening tool for anti-estrogenic compounds, molecules of interest for hormono-dependant cancer therapy. Copy-right � 2010 John Wiley & Sons, Ltd.
Keywords: estrogen receptor; force spectroscopy; DNA sensor
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E T* Correspondence to: A. Berthier, Estrogenes, Expression Genique et Pathologiesdu Systeme Nerveux Central, EA3922, IFR 133, Universite de Franche-Comte,Besancon cedex, France.
a A. Berthier, R. Delage-Mourroux
Q2EstrogenesQ2, Expression Genique et Pathologies du Systeme Nerveux Central,
EA3922, IFR 133, Universite de Franche-Comte, Besancon cedex, France
b A. Berthier, C. Elie-Caille, W. Boireau
Institut FEMTO-ST, UMR 6174 CNRS, Clinical and Innovation Proteomic
Platform (CLIPP), Universite de Franche-Comte, Besancon cedex, France
c E. Lesniewska
Laboratoire Interdisciplinaire Carnot Bourgogne UMR CNRS 5209, Nanos-
ciences Department, University of Bourgogne, Dijon Cedex, France 1
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INTRODUCTION
Estrogens, and particularly estradiol-17-b (E2), are femalehormones involved in development, growth, and maintenanceof reproductive tissues (Diel, 2002). These hormones interact withestrogen receptors (ER) which are transcription factors activatedby ligand binding (O’Malley, 2005). These activated nuclearreceptors modulate expression of estrogen-responsive genesafter the interaction with palindromic DNA sequences calledestrogen response element (ERE; Klinge, 2001). Many moleculescould interact with these ER, as well as plant derived compoundscalled phytoestrogens (genistein, glucopyranosylchrysin, etc.;Maggiolini et al., 2001; Berthier et al., 2007), synthetic drugs (ICI182,780, Tamoxifen, etc.; Wakeling et al., 1991; Ahn and Sheen,1997), or endocrine disturbers (polychlorobiphenyls, parabens,etc.; Okubo et al., 2001; Ho et al., 2008). Interactions of thesecompounds have an impact on the conformational state of thereceptors. Thus, the agonist or antagonist nature of the bondedcompounds allows or blocks receptor dimerization, DNAinteraction, and coregulators recruitment (Arbuckle et al., 1992;Brzozowski et al., 1997; Shiau et al., 1998; Klinge, 2000; Gruberet al., 2002). All estrogenic or anti-estrogenic compounds couldact on the animal and human health and treatments: (i)phytoestogens as lignans could be used to prevent thehormonally dependent cancer arrival or to limit climactericcomplaints (Thompson, 1998; Power and Thompson, 2003;Bergman Jungestrom et al., 2007; Feng et al., 2008); (ii) drugs likeTamoxifen or fulverstran1 (ICI 182,780) were applied to cancersand menopause treatments (Love et al., 1994; Landgren et al.,2002; Martino et al., 2004a,b; Jacobsen et al., 2008; McCormackand Sapunar, 2008; Saji and Kuroi, 2008). At present, public and
ognit. 2010; 9999: 1–7 Copyright � 2010
politic attentions are focussed on a new class of molecules calledendocrine disturbers. These compounds, resulting from anenvironmental pollution (drugs, pesticides, and industrial waste)or from our lifestyle (plastics, cosmetics, etc.), affect fertility andenhance risk of cancer (Inadera, 2006; Vanparys et al., 2006; Hoet al., 2008; Soto et al., 2008; Tiemann, 2008).All these data incite to identify some new potential estrogenic
or anti-estrogenic compounds essential to protect human health(therapy, prevention) and environment.Classically, the identification of new potential estrogenic
compounds was based on cellular or animal models. However,the ER dimerization and its interaction with DNA being the initialsteps of the estrogenic genomic mechanism, a prescreening ofmolecules of interest could be processed upstream, in order toreduce time and money consumptions.Surface plasmon resonance (SPR) biosensors and/or on-chip
force spectroscopy analysis, which allow real time biomolecular
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Table 1. 50amine modified oligonucleotides (D1 and D4) and complementary single strand (C3 and C6) sequences
Names SequencesDouble strandsequences
D1 50-H2N-(CH2)12-ATATATAGTTCTTTGATCAGGTCACTGTGACCTGAACTTGCT-30 ERE
C3 AGCAAGTTCAGGTCACAGTGACCTGATCAAAGAACTD4 50-H2N-(CH2)12-ATATATGTCCAAAGTCAATCGCCAGCACGATGATCAAAGTCC-3
0 ControlC6 GGACTTTGATCATCGTGCTGGCGATTGACTTTGGAC
The ERE (bold and underlined) and control sequences were created by hybridization of D1/C3 and D4/C6 oligonucleotides, respectively.
A. BERTHIER ET AL.
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RRECinteraction detections and quantification, can be applied tothese prescreening steps. These biosensors are based on theimmobilization of biomolecules on a gold chip directly or indirectlyby physical or chemical adsorption (Boireau et al., 2002; Aoyagiet al., 2008). In order to get free of non-specific protein adsorption, apassivation step of the metallic film is essential (Rella et al., 2004).This process implies the development of indirect grafting strategies.Actually, many kinds of surfaces were provided to SPR biosensor’susers, for instance 3D hydrogels like commercial carboxymethyldextran (CMD) or 2D self-assembled monolayers (SAMs).For the ER/ERE interaction studies, several authors have oriented
their DNAgrafting strategies on the CMDbased chips (Cheskis et al.,1997; Kostelac et al., 2003; Asano et al., 2004; Habauzit et al., 2008).However, the CMD based surfaces were known to create somekinetic disturbing as mass transport and rebinding (Schuck, 1996;Yarmush et al., 1996; Fong et al., 2002). Thus, to get free of theseartifacts, one of the grafting alternative was based on a mixtureof 16- mercapto-1-hexadecanoic acid (MHA) and 11- mercap-to-1-undecanol (MUO). Previous works have shown that carboxylicfunctions of the MHA allowed molecular grafting and that thehydroxyl groups of the MUO reduced the non-specific adsorptionphenomenon (Frederix et al., 2003; Huang et al., 2005). The graftingof molecules like modified DNA and proteins on such SAMspresenting a primary amine group was enabled by this latestsurface choice after carboxylic function activation.To address the question of ER dimerization, we analyzed
interactions between an ER functionalized gold surface and an ERmodified AFM tip by force spectroscopy. Indeed, AFM force
UNCO
Figure 1. (A) Detection of ER/DNA specific interaction. The ERawas incubatemin) on ERE (black) or control DNA sequences (gray). (B) Ligand impact. The ER
of E2, or with an excess of ICI 182 780 supplemented or not by E2 (10 nM). The s
the ERE signal. Data presented as histograms were the means of three inde
wileyonlinelibrary.com/journal/jmr Copyright � 2010 John
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spectroscopy is able to monitor intermolecular interactions indifferent medium conditions. Recording the applied force over thestretching distance revealed a characteristic sawtooth-like patternof force peaks. The appearance of a ‘‘repetitive’’ pattern of forcepeaks while retracting ER tip to the ER surface confirmed (1) that ERinteracts with itself and (2) that this interaction, estrogen specificand repetitive, described a specific pathway. These observations fitwith a dimerization of ER in the presence of estrogen.In this paper we have described a new SPR biosensor
dedicated to potential estrogenic compounds prescreening, bydeveloping an ER specific DNA chip, the ERE chip. In parallel, forcespectroscopy experiments allowed us to (1) prove that ER needsestrogen for its dimerization and (2) propose an innovativescreening tool for anti-estrogenic compounds.
MATERIALS AND METHODS
Materials
DNA immobilization was allowed by a primary amine grafted viaa 12 carbons alkyl chain on 50 extremities of D1 and D4 singlestrand oligonucleotides. Modified oligonucleotides (D1 and D4)were able to create ERE or double strand control sequenceby hybridization with complementary sequence C3 and C4. AllDNA sequences were presented in Table 1 (Eurogentec, Liege,Belgium).Human recombinant estrogen receptor-a (ERa; PanVera,
Invitrogen Corporation, Carlsbad, USA) was conserved at
dwith 10 nM E2 (30min at room temperature) and injected (90ml at 30ml/awas incubated with ligand solvent (ethanol), with equimolar proportion
pecific interaction signals were the control interaction signal subtracted to
pendent experiments.
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Q1LABELQ1-FREE SENSING AND AFS FOR THE CHARACTERIZATION OF PROTEIN–DNA AND PROTEIN–PROTEIN INTERACTIONS
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�808C into 10ml aliquots to limit the number of freeze-thawcycles. Estradiol-17-b (E2; Sigma–Aldrich, St. Louis, USA) and ICI182 780 provided by AstraZeneca (Reims, France) was prepared inethanol at 1 nM and 1mM, respectively.The MUO and MHA (Sigma–Aldrich, St. Louis, USA)
were prepared in ethanol. MHA carboxylic functionswere activated with amine coupling kit consisted of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N -hydroxysuccinimide (NHS) 1:1 mix (GE Healthcare Life Sciences,Pittsburgh, USA).
Sensor chips
Gold chips were processed as previously described (Mangeatet al., 2009). Briefly a 2 nm thick chromium layer is deposited on aglass wafer (diameter: 13mm and thickness: 0.17mm) withplasma sputtering technology to improve the adherence of goldto the substrate. Then a 40 nm thick gold layer is sputtered on thetop of the Cr layer. After cleaning of the surface (ethanol/water),the chip was functionalized with a mixed SAM. This SAM wasprocessed by an overnight chip immersion in a 1mM MUO/MHA(90:10 by mol) bath. Such functionalized gold surfaces were
UNCORRECT
Figure 2. Characterization of ER bio-functionalized surfaces. Thiols function
images and their corresponding section analysis. Pieces of APTES/glutaraldehy
The image presented in G is the zoom of one red delimited area in image
J. Mol. Recognit. 2010; 9999: 1–7 Copyright � 2010 John Wiley
OFS
either used for DNA or ER immobilization, in order to study DNA/ER sensing by SPR or ER/ER dimerization by force spectroscopy,respectively.
SPR experiments
All SPR experiments were run on BIAcore 2000 apparatus at 258Cusing BIACORE 2000 Control Software version 3.2 (GE HealthcareLife Sciences).
DNA immobilization
Modified DNA immobilizations were processed with HBS-Nrunning buffer (10mM Hepes pH 7.4, NaCl 150mM). First,the surface was washed with 15ml of 40mM Octyl-Glucopyranoside (OG, Sigma–Aldrich, St. Louis, USA) at 30ml/min. Then, the MHA carboxylic functions were activated with twoinjections (70ml at 10ml/min) of EDC/NHS v/v mix. In orderto reduce the surface charge repulsion, 10mM of amine-modifiedoligonucleotides were diluted in a 10mM Hepes buffer pH8.4 supplemented by 0.4mM hexadecyltrimethylammoniumbromide (CTAB). Oligonucleotides were then injected on the
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alized gold chip before (A and C) and after (B and D) ER graftings. AFM
de functionalized silicon nitride, before (E) and after (F and G) ER graftings.
F. Z range is 10 (E and F) or 5 (G) nm.
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TED P
ROQ3
A. BERTHIER ET AL.
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ORRECchip at 10ml/min for 15min. The excess of NHS-ester functionwas inactivated with 1M ethanolamine (140ml at 10ml/min).For the hybridization process, the running buffer was replaced
by HBS-EP buffer (GE healthcare Life Sciences). Complementaryoligonucleotides (2.5mM) were injected at 20ml/min for 2min. Atthe end of injection, the surface was cleaned by a 15ml pulse ofOG at 30ml/min.
Force spectroscopy
ER was immobilized on MUO/MHA (90:10 by mol) functionalizedgold surface. For that, the surface was washed with 40mMOctyl-Glucopyranoside. Then, the MHA carboxylic functions wereactivated with two injections of EDC/NHS v/v, and the goldsurface was incubated during 30min in the presence of ERsolution (20 nM). The surface was then washed with ER buffer.The AFM used was a Nanoscope III (Veeco, Santa Barbara, CA).
Imaging was performed in contact mode using NPS-oxidesharpened silicon nitride probes (Veeco) exhibiting springconstants of 0.3 N/m. For the force spectroscopy measurements,the AFM tip, made of silicon nitride, was functionalized by 1%APTES in toluene during 2 h, washed extensively with toluenethen with ethanol. The second step consisted in an incubation in0.2% glutaraldehyde solution during 10min, followed byextensive washing with water. The modified tips were thenincubated in 20 nM ER solution during 30min, then washed withER buffer. DNP-S AFM probes (from Veeco) were employed,presenting a spring constant around 0.3 N/m. For all the curves, a200 nm ramp size and an interaction time of 1msQ3 were used.The loading rate was varied, from 30 000 to 520 000 pN/s, in orderto study the dependency of the forces necessary to rupturespecific bonds on the loading rate.
ER/DNA interactions
For the protein/DNA interaction studies the running buffer wasreplaced by the interaction buffer (40mM Hepes pH 7.4, 100mMNaCl, 10mM MgCl2, 1mM DTT, and 0.2% Triton X-100). First, 10nMERa was incubated with 1nM E2 or 1mM ICI 182 780 for 30min atroom temperature in interaction buffer. Then 90ml of activatedreceptor were injected at 30ml/min on ERE or control channels.Finally, the DNA surface was regenerated by 10min injection of0.1% SDS, 100mM EDTA denaturizing solution (at 5ml/min).
Figure 3. Force curves measured between ER tip and ER gold chip. (A)Scattered spectra represent superimposition of force curves, that high-
light common features. A typical force curve is highlighted in red. Blue
circles and arrows indicate the average peak position and the rupture
distance. Loading rate: 150 000 pN/s, in the presence of 1 nM E2. (B)Histogram presenting the repartition of rupture forces measured in the
absence (ER/ER; in gray) or presence (in black) of 1 nM E2 (ER/ERþ E2) in
the medium.
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RESULTS AND DISCUSSION
ERE chip processing
The 50 amine modified single strand oligonucleotides D1 and D4(10mM) were immobilized on activated mixed SAM. Thencomplementary oligonucleotides (C1 and C4) were injected tocreate double strand ERE specific sequence or control sequence,respectively. After 2min of injection the D1/C3 (ERE) and D4/C6(control) hybridization degrees were 69.3� 6.9 and 78.8� 6.5%,respectively (data not shown). Double strand SPR signals were150� 30 RU (Response Unit) for ERE and 244� 55 RU for controlsequence (data not shown).
ER/ERE interaction validation
The specificity of the ER/DNA interaction was determined by aninjection of E2 activated ER on control and ERE sequences. The ER
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(10 nM) interaction signal, in presence of 10 nM E2, was 3.63� 0.8fold higher on ERE than on control sequence (for example seeFigure 1A). Thus, we can conclude that our ERE chip constitutes avalid tool for specific ER/DNA interaction detection.In order to determine the ligand impacts on ER/DNA interactions,
the receptor has been injected with or without ligand. In presenceof E2, the specific interaction signal was 1.7 fold higher than inabsence of ligand (157RU vs. 91RU, respectively). When ER has beenincubated with 1mM ICI 182 780, supplemented or not by 10nM E2,the specific interaction was 1.8 fold reduced compared to ligandfree condition (Figure 1B).
ER dimerization characterization by force spectroscopy
(a) Characterization of ER biofunctionalized surfaces
The quality of the working surfaces, the ER modified siliconnitride AFM tip and the ERmodified gold chip, was assessed usingAFM to prove the presence of ER on both surfaces (Figure 2).The surface of the MHA/MUO functionalized gold chip was
compared before and after ER graftings. The surface presented
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LABELQ1-FREE SENSING AND AFS FOR THE CHARACTERIZATION OF PROTEIN–DNA AND PROTEIN–PROTEIN INTERACTIONS
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an increase in rms value (on 400 nm� 400 nm surface area), from0.2 nm (rmax¼ 0.9 nm) to 0.9 nm (rmax¼ 3.4 nm) before and afterprotein grafting, respectively, that reveals the presence of proteinmaterial on the gold surface (Figure 2A–D).We also wanted to prove the presence of ER on AFM tip, made
in silicon nitride. For that, we prepared pieces of silicon nitrideand functionalized them with APTES and glutaraldehyde andcontrolled the surface by AFM before and after ER grafting.After incubation of the functionalized surface in ER and awashingstep, we distinguished motifs on the surface and also somebigger aggregates. While zooming on such aggregates, itappeared that these bigger complexes were composed of motifsof 12–15 nm in diameter (Figure 2E–G). Then, the majority ofthe surface is covered by proteins, with a good homogeneityand only few aggregates of the protein are present. Thus, we canevaluate that the APTESþ glutaraldehyde surface enables thegrafting of ER protein on the silicon nitride surface, meaningthat the ER is effectively bound to the silicon nitride tip when ERtip is employed in force curves experiments.
UNCORRECT
Figure 4. Dynamics of ER/ER interaction. (A) Typical force curves regis-
tered in the presence of 1 nM E2, at three different loading rates (30 000,150 000, and 520 000 pN/s in black, dark gray, and light gray, respectively).
J. Mol. Recognit. 2010; 9999: 1–7 Copyright � 2010 John Wiley
(b) Force spectroscopy measurements
Here we used single molecule force spectroscopy to investigatethe molecular forces driving ER/ER interactions. If an ER dimer isformed, under conditions favorable for interaction between thetwo molecules, single molecule force spectroscopy should detectit while retracting the tip from the surface. In the case ofdimerization, when an ER modified AFM tip interacts with an ERmodified surface, the dimer is ruptured at the retraction stepupon removal of the tip from the surface. Such an event producesa specific rupture signature on the force curve. We observedthat when the ER modified AFM tip is used on the ERbiofunctionalized gold chip surface after a preliminary 10min
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Figure 5. Inhibition and reversibility of ER/ER interaction. (A) In buffer
and in the presence of 1 nM E2, forces curves were registered before
inhibitor addition (in black), in the presence of 1mM ICI 182 780 (in red
and gray) and after an extensive rinsing and again in buffer containing1 nM E2 (in blue). (B) Histogram presenting the proportion of curves
presenting peaks or not in the different tested conditions.
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incubation in the presence of 1 nM E2, interaction events canbe recorded. We collected then several force curves (50) in thiscondition (loading rate 150 000 pN/s), i.e., in the presence of 1 nME2. We obtained the scattered spectra presented in Figure 3A.Scattered spectra represent superimpositions of force curvesobtained in the same conditions. Superimpositions highlightcommon features (a ‘‘five peaks’’ typical curve) of the unfoldingevents and reduce deviations, which may occur in individualspectra. More than 90% of force curves presented interactionpeaks in the presence of 1 nM E2. The peak intensity was around10 pN. The results are different in the absence of 1 nM E2, sincethe majority of force curve present no peak. Moreover, whena peak appeared in these conditions, its intensity was really low,close to zero (Figure 3B, the single gray bar).Moreover, the forces necessary to rupture specific bonds are
known to depend on the loading rate (Merkel et al., 1999).Consistent with this study, 100 force curves were registered at fourdifferent loading rate, from 30 000 to 520 000pN/s. Typical forcecurves obtained for three different loading rates are represented inFigure 4A. We found that the binding force between the ER tip andthe ER surface, in the presence of 1nM E2 in the solution, increasedlinearly with the logarithm of the loading rate (Figure 4B).In our force spectroscopy experiment, we also tested the effect
of ICI 182 780, a molecule known as an inhibitor of ER/ERdimerization. While the buffer contained 1 nM E2 and 1mM of ICI182 780, we registered 50 curves and from them, very few curveswere presenting peaks, and in this case only a single peak atshort rupture distance was observed (Figure 5A, in red).Nevertheless, the majority of curves even presented no moreinteraction peaks (Figure 5A, in gray). Then, the presence of ICI182 780 seemed to have inhibited the interaction between the ERtip and the ER surface. After this, we rinsed our surface, tip andAFM fluidic cell extensively with buffer, added again 1 nM E2 andproceeded again in force curves experiments. We observed thatforce curves presented again several peaks (3–5 peaks), andthat the stretching distance fit with the stretching distanceregistered before addition of the inhibitor (Figure 5A, in blue).Nevertheless, after recording 50 force curves in these
conditions (‘‘after ICI incubation and rinsing’’), we noticed thatonly 50% of curves roughly presented this pattern of peaks(Figure 5B). This observation could mean that the ER/ERinteraction is ‘‘quasi-reversible’’ after ICI 182 780 incubation,as if the inhibitor remained partially fixed on ER after washing.This is probably the case. Indeed, to ensure the inhibition of ER/ERinteraction, we used a 1mM inhibitor concentration, comparedto 1 nM concentration of E2. But, it is known that the binding
wileyonlinelibrary.com/journal/jmr Copyright � 2010 John
affinity of ICI 182 780 to the ER is 89% that of oestradiol (Howell,2006). Then, such a large excess of inhibitor, having an affinitybinding comparable to the ligand E2, could induce adsorbedmolecules left on ER. This could explain that the interactionbetween the tip and the surface was reduced after ICI incubation.Moreover, this result indicates that the absence of peaks oncurves obtained in the presence of 1mM ICI 182 780 waseffectively due to an inhibition of the tip/surface interaction, andnot to an artifact of experiments.Then these unbinding events between ER tip and ER gold chip
seem to be specific since:
(1) N
Wile
o interaction is detected between a naked tip and the ERsurface (data not shown).
(2) O
FS
nly few and weak interactions appeared between the ERmodified tip and ER modified gold surface in the absence ofE2.
In the presence of 1 nM E2:
(1) A
Orecurrent pattern of 3–5 peaks appeared, attesting that theER/ER interaction follows a highly controlled and specificmultiple-steps mechanism.(2) T
ROhe forces needed to rupture the ER/ER interaction is loadingrate dependent, meaning that these interactions are ERspecific.(3) T
Phe addition of the ICI 182 780 inhibitor prevents the ER/ERinteraction.EDCONCLUSION
This SPR biosensor, consisting in ERE presenting DNA sequence,allows the selection of molecules which induce ER/EREinteraction. These preselected compounds could be enough toisolate compounds which are able to induce gene transactiva-tion. The estrogenic activity should then be studied with ananimal or cellular model. These xeno-estrogenes could be usedfor example to menopause treatment. However, hormono-dependant cancer therapy needs molecules which reduce targetgene expressions by ER dimerization and/or its interaction withDNA inhibition. The differentiation of anti-estrogenic andinefficient compounds will be impossible using the SPR EREbiosensor. The force spectroscopy assays represent then arelevant and complementary tool since this strategy allowsto preselect quickly anti-estrogenic compounds which inhibitER dimerization (initial step of gene transactivation).
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REFERENCES
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AhnMR, Sheen YY. 1997. Antiestrogen interaction with estrogen receptorsand additional antiestrogen binding sites in human breast cancerMCF-7 cells. Arch. Pharm. Res. 20(6): 579–585.
Aoyagi S, Rouleau A, BoireauW. 2008. TOF-SIMS structural characterizationof self-assembly monolayer of cytochrome b5 onto gold substrate.Appl. Surf. Sci. 255(4): 1071–1074.
Arbuckle ND, Dauvois S, Parker MG. 1992. Effects of antioestrogens on theDNA binding activity of oestrogen receptors in vitro. Nucleic Acids Res.20(15): 3839–3844.
Asano K, Ono A, Hashimoto S, Inoue T, Kanno J. 2004. Screening ofendocrine disrupting chemicals using a surface plasmon resonancesensor. Anal. Sci. 20(4): 611–616.
Bergman JungestromM, Thompson LU, Dabrosin C. 2007. Flaxseed and itslignans inhibit estradiol-induced growth, angiogenesis, and secretionof vascular endothelial growth factor in human breast cancer xeno-grafts in vivo. Clin. Cancer Res. 13(3): 1061–1067.
Berthier A, Girard C, Grandvuillemin A, Muyard F, Skaltsounis AL, JouvenotM, Delage-Mourroux R. 2007. Effect of 7-O-beta -D- glucopyranosyl-chrysin and its aglycone chrysin isolated from Podocytisus carama-nicus on estrogen receptor alpha transcriptional activity. Planta Med.73(14): 1447–1451.
Boireau W, Bombard S, Sari MA, Pompon D. 2002. Bioengineering andcharacterization of DNA–protein assemblies floating on supportedmembranes. Biotechnol. Bioeng. 77(2): 225–231.
y & Sons, Ltd. J. Mol. Recognit. 2010; 9999: 1–7
117
118
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106
UNCORREC
Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O,Ohman L, Greene GL, Gustafsson JA, Carlquist M. 1997. Molecularbasis of agonism and antagonism in the oestrogen receptor. Nature389(6652): 753–758.
Cheskis BJ, Karathanasis S, Lyttle CR. 1997. Estrogen receptor ligandsmodulate its interaction with DNA. J. Biol. Chem. 272(17): 11384–11391.
Diel P. 2002. Tissue-specific estrogenic response and molecular mechan-isms. Toxicol. Lett. 127(1–3): 217–224.
Feng J, Shi Z, Ye Z. 2008. Effects of metabolites of the lignans enter-olactone and enterodiol on osteoblastic differentiation of MG-63cells. Biol. Pharm. Bull. 31(6): 1067–1070.
Fong CC, Wong MS, Fong WF, Yang M. 2002. Effect of hydrogel matrix onbinding kinetics of protein–protein interactions on sensor surface.Anal. Chim. Acta 456(2): 201–208.
Frederix F, Bonroy K, Laureyn W, Reekmans G, Campitelli A, Dehaen W,Maes G. 2003. Enhanced performance of an affinity biosensor inter-face based on mixed self-assembled monolayers of thiols on gold.Langmuir 19(10): 4351–4357.
Gruber CJ, Tschugguel W, Schneeberger C, Huber JC. 2002. Production andactions of estrogens. N. Engl. J. Med. 346(5): 340–352.
Habauzit D, Armengaud J, Roig B, Chopineau J. 2008. Determination ofestrogen presence in water by SPR using estrogen receptor dimer-ization. Anal. Bioanal. Chem. 390(3): 873–883.
Ho PW, Garner CE, Ho JW, Leung KC, Chu AC, Kwok KH, Kung MH, Burka LT,Ramsden DB, Ho SL. 2008. Estrogenic phenol and catechol metab-olites of PCBs modulate catechol-O-methyltransferase expression viathe estrogen receptor: potential contribution to cancer risk. Curr.Drug Metab. 9(4): 304–309.
Howell A. 2006. Pure oestrogen antagonists for the treatment of advancedbreast cancer. Endocrinol. Relat. Cancer 13(3): 689–706.
Huang L, Reekmans G, Saerens D, Friedt JM, Frederix F, Francis L, Muylder-mans S, Campitelli A, Van Hoof C. 2005. Prostate-specific antigenimmunosensing based on mixed self-assembled monolayers, camelantibodies and colloidal gold enhanced sandwich assays. Biosens.Bioelectron. 21(3): 483–490.
Inadera H. 2006. The immune system as a target for environmentalchemicals: xenoestrogens and other compounds. Toxicol. Lett.164(3): 191–206.
Jacobsen DE, Samson MM, Schouw YT, Grobbee DE, Verhaar HJ. 2008.Efficacy of tibolone and raloxifene for the maintenance of skeletalmuscle strength, bone mineral density, balance, body composition,cognitive function, mood/depression, anxiety and quality of life/well-being in late postmenopausal women �70 years: study designof a randomized, double-blind, double-dummy, placebo-controlled,single-center trial. TrialsQ4 9: 32.
Klinge CM. 2000. Estrogen receptor interaction with co-activators andco-repressors. Steroids 65(5): 227–251.
Klinge CM. 2001. Estrogen receptor interaction with estrogen responseelements. Nucleic Acids Res. 29(14): 2905–2919.
Kostelac D, Rechkemmer G, Briviba K. 2003. Phytoestrogens modulatebinding response of estrogen receptors alpha and beta to theestrogen response element. J. Agric. Food Chem. 51(26): 7632–7635.
Landgren MB, Bennink HJ, Helmond FA, Engelen S. 2002. Dose-responseanalysis of effects of tibolone on climacteric symptoms. BjogQ5
109(10): 1109–1114.Love RR, Wiebe DA, Feyzi JM, Newcomb PA, Chappell RJ. 1994. Effects of
tamoxifen on cardiovascular risk factors in postmenopausal womenafter 5 years of treatment. J. Natl. Cancer Inst. 86(20): 1534–1539.
Maggiolini M, Bonofiglio D, Marsico S, Panno ML, Cenni B, Picard D, AndoS. 2001. Estrogen receptor alpha mediates the proliferative but notthe cytotoxic dose-dependent effects of two major phytoestrogenson human breast cancer cells. Mol. Pharmacol. 60(3): 595–602.
J. Mol. Recognit. 2010; 9999: 1–7 Copyright � 2010 John Wiley
ED PRO
OFS
Mangeat T, Berthier A, Elie-Caille C, Perrin M, Boireau W, Pieralli C,Wacogne B. 2009. Gold/silica biochips: applications to surface plas-mon resonnace and fluorescence quenching. Laser Phys. 19(2):252–258.
Martino S, Costantino J, McNabb M, Mershon J, Bryant K, Powles T, SecrestRJ. 2004a. The role of selective estrogen receptor modulators in theprevention of breast cancer: comparison of the clinical trials. Oncol-ogist 9(2): 116–125.
Martino S, Cauley JA, Barrett-Connor E, Powles TJ, Mershon J, Disch D,Secrest RJ, Cummings SR. 2004b. Continuing outcomes relevant toEvista: breast cancer incidence in postmenopausal osteoporoticwomen in a randomized trial of raloxifene. J. Natl. Cancer Inst.96(23): 1751–1761.
McCormack P, Sapunar F. 2008. Pharmacokinetic profile of the fulvestrantloading dose regimen in postmenopausal women with hormonereceptor-positive advanced breast cancer. Clin. Breast Cancer 8(4):347–351.
Merkel R, Nassoy P, Leung A, Ritchie K, Evans E. 1999. Energy landscapes ofreceptor-ligand bonds explored with dynamic force spectroscopy.Nature 397(6714): 50–53.
Okubo T, Yokoyama Y, Kano K, Kano I. 2001. ER-dependent estrogenicactivity of parabens assessed by proliferation of human breast cancerMCF-7 cells and expression of ERalpha and PR. Food Chem. Toxicol.39(12): 1225–1232.
O’Malley BW. 2005. A life-long search for the molecular pathways ofsteroid hormone action. Mol. Endocrinol. 19(6): 1402–1411.
Power KA, Thompson LU. 2003. Ligand-induced regulation of ERalpha andERbeta is indicative of human breast cancer cell proliferation. BreastCancer Res. Treat. 81(3): 209–221.
Rella R, Spadavecchia J, Manera MG, Siciliano P, Santino A, Mita G. 2004.Liquid phase SPR imaging experiments for biosensors applications.Biosens. Bioelectron. 20(6): 1140–1148.
Saji S, Kuroi K. 2008. Application of selective estrogen receptormodulatorsfor breast cancer treatment according to their intrinsic nature. BreastCancer
Schuck P. 1996. Kinetics of ligand binding to receptor immobilized in apolymer matrix, as detected with an evanescent wave biosensor. I. Acomputer simulation of the influence of mass transport. Biophys. J.70(3): 1230–1249.
Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL.1998. The structural basis of estrogen receptor/coactivator recog-nition and the antagonism of this interaction by tamoxifen. Cell 95(7):927–937.
Soto AM, Vandenberg LN, Maffini MV, Sonnenschein C. 2008. Does breastcancer start in the womb? Basic Clin. Pharmacol. Toxicol. 102(2):125–133.
Thompson LU. 1998. Experimental studies on lignans and cancer. BaillieresClin. Endocrinol. Metab. 12(4): 691–705.
Tiemann U. 2008. In vivo and in vitro effects of the organochlorinepesticides DDT, TCPM, methoxychlor, and lindane on the femalereproductive tract of mammals: a review. Reprod. Toxicol. 25(3):316–326.
Vanparys C, Maras M, Lenjou M, Robbens J, Van Bockstaele D, Blust R, DeCoen W. 2006. Flow cytometric cell cycle analysis allows for rapidscreening of estrogenicity in MCF-7 breast cancer cells. Toxicol. inVitro 20(7): 1238–1248.
Wakeling AE, Dukes M, Bowler J. 1991. A potent specific pure antiestrogenwith clinical potential. Cancer Res. 51(15): 3867–3873.
Yarmush ML, Patankar DB, Yarmush DM. 1996. An analysis oftransport resistances in the operation of BIAcore; implicationsfor kinetic studies of biospecific interactions. Mol. Immunol.33(15): 1203–1214.
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