Life Sciences 89 (2011) 241–249

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Increased expression of the coxsackie and adenovirus receptor downregulates αvβ3and αvβ5 integrin expression and reduces cell adhesion and migration

Dragomira Majhen, Nikolina Stojanović, Tea Špeljko 1, Anamaria Brozovic, Tihana De Zan,Maja Osmak, Andreja Ambriović-Ristov ⁎Laboratory for Genotoxic Agents, Division of Molecular Biology, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia

⁎ Corresponding author. Tel.: +385 1 4571 240; fax:E-mail address: andrea@irb.hr (A. Ambriović-Ristov)

1 Present address: Department of Neuroscience, CroatiUniversity of Zagreb Medical School, Zagreb, Croatia.

0024-3205/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.lfs.2011.06.009

a b s t r a c t

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Article history:

Received 11 March 2011Accepted 4 June 2011

Keywords:Coxsackie and adenovirus receptorαvβ3 integrinαvβ5 integrinAdhesionMigrationAdenovirus type 5 transductionRhabdomyosarcoma

Aims: Coxsackie and adenovirus receptor (CAR) is a tumor suppressor and a primary receptor for adenovirustype 5 (Ad5). Our study aims to examine the influence of forced expression of CAR in rhabdomyosarcoma cells(RD) on expression levels of integrins implicated in Ad5 entry, and the effect of CAR on cell–extracellularmatrix adhesion and migration.Main methods: CAR expressing clones were established from RD cells by stable transfection. Flow cytometrywas used to evaluate the expression of CAR and integrins. Adhesion was measured in plates previously coatedwith vitronectin or fibronectin. Boyden chambers were used to investigate migration. Transfection ofcells with siRNAwas used to achieve integrin silencing. Ad5-mediated transgene expression wasmeasured byβ-gal staining.Key findings: Increased expression of CAR in RD cells reduces the expression ofαvβ3 and αvβ5 integrins. Cellsoverexpressing CAR exhibit significantly reduced adhesion to vitronectin and fibronectin, and reduced cell

migration. Specifically silencing αvβ3 integrin in RD cells reduced cell migration indicating that reducedmigration could be the consequence of αvβ3 integrin downregulation. This study also demonstrates thenegative effect of reduced levels ofαvβ3 andαvβ5 integrins on Ad5-mediated transgene expression with Ad5retargeted to αv integrins.Significance: The pharmacological upregulation of CAR aimed to increase Ad5-mediated transgene expressionmay actually downregulate αvβ3 and αvβ5 integrins and thus alter Ad5-mediated gene transfer. Themechanism of decreased cell migration, a prerequisite for metastasis and invasion, due to increased CARexpression may be explained by reduced αvβ3 integrin expression.

© 2011 Elsevier Inc. All rights reserved.

Introduction

The coxsackie and adenovirus receptor (CAR) is a 46 kDa class Imembrane glycoprotein with a C-terminal cytoplasmic domain, atransmembrane domain and an extracellular region (Philipson andPettersson, 2004). CAR is the high affinity receptor for adenovirus type5 (Ad5) (Bergelson et al., 1997) and plays a vital role in intercellularcell adhesion (Bruning and Runnebaum, 2003; Cohen et al., 2001;Excoffon et al., 2004). The intracellular C-terminal tail of CAR plays norole in the Ad5 infection (Walters et al., 2001), but is important for theendogenous function of CAR as a cell adhesion protein and a growthmodulatory protein (Excoffon et al., 2004). Whereas many normaltissues express readily detectable levels of CAR, low or absentexpression of CAR is often observed in corresponding primarytumor tissues and cell lines (Buscarini et al., 2007; Jee et al., 2002;

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Korn et al., 2006; Li et al., 1999; Matsumoto et al., 2005). IncreasedCAR expression in prostate and bladder cancer cell lines can inhibitthe in vitro and in vivo growth of tumor cells (Okegawa et al., 2000,2001), and in vitro growth of malignant glioma cells (Kim et al., 2003).CAR overexpression in ovarian, cervical and gastric cancer cellsreduces migration, suggesting that CAR shows features of a tumorsuppressor (Anders et al., 2009b; Bruning and Runnebaum, 2004).

Adenovirus type 5 (Ad5) infection initiates with high affinitybinding to CAR (Bergelson et al., 1997; Fuxe et al., 2003; Tomko et al.,1997), followed by interaction with αvβ1, αvβ3, αvβ5, α5β1 andα3β1 integrins on the cell surface, promoting Ad5 internalization(Sharma et al., 2009; Wickham et al., 1993, 1994). Rhabdomyosarco-ma (RMS) is the most common pediatric soft-tissue sarcoma, arisingfrommuscle progenitor cells. There remains a need for more effectivetherapies such as Ad5-mediated gene therapy. RMS cells express theAd5 co-receptors, αv integrins, but have low or undetectableexpression of CAR; they are less sensitive or even resistant tooncolysis by wild type Ad5 (Cripe et al., 2001). There are two possibleways to increase Ad5-mediated gene transfer into RMS: (i) to use Ad5vectors targeted to cell surface receptors other than CAR (Kanerva and

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Hemminki, 2004) or (ii) to increase endogenous CAR expression(Bruning and Runnebaum, 2003; Hemminki et al., 2003; Okegawaet al., 2007; Pong et al., 2006; Shieh et al., 2006; Vincent et al., 2004). Apromising retargeted Ad5 vector is Ad5RGD4C, which contains the αvintegrin binding motif CDCRGDCFC incorporated into the HI-loop ofthe Ad5 fiber protein (Dmitriev et al., 1998; Majhen et al., 2009).

There are only two reports that have explored the influence ofCAR-overexpression on integrins. Yamashita et al. (2007) describedthat stable and transient CAR expression in mouse B16BL6 melanomacells induced the suppression of αv, α4, β3 and β1 integrin mRNAexpression levels. In contrast, Farmer et al. (2009) described that inhuman breast cancer MCF-7 cells CAR overexpression promotes β1and β3 integrin activation resulting in increased cell–extracellularmatrix adhesion. It is critical to improve our understanding of thecrosstalk between CAR and integrins, since altered integrin expressionor activity and Ad5 susceptibility could significantly impact our viewof cancer progression in diverse tissues and the mechanism behindthe success or failure of novel treatments. In this study, we evaluatedthe influence of forced expression of CAR in rhabdomyosarcoma cellson expression of integrins implicated in Ad5 entry, and analyzed theeffect of CAR on cell–extracellular matrix adhesion, migration, andAd5 infection.

Materials and methods

Cell lines

The human rhabdomyosarcoma (RD) cell line was obtained fromthe American Type Culture Collection (CCL-136, ATCC; USA). Cellswere grown as a monolayer culture in DMEM (Invitrogen, USA)supplemented with 10% FCS (Invitrogen) at 37 °C with 5% CO2.

RD cells expressing CAR

CAR expressing cell clones RD-G7 and RD-D6 were establishedfrom RD cells by stable transfection with the plasmid pCDNA3-hCAR(Kim et al., 2003) (kindly provided by Dr JT Douglas, Gene TherapyCenter, University of Alabama at Birmingham, Birmingham, AL, USA).The plasmid was transfected into RD cells using Lipofectamine(Invitrogen). Cell clones were selected in the presence of 600 μg ofgeneticin (Invitrogen) per milliliter and screened by flow cytometryfor CAR expression.

Expression of CAR and integrins

Flow cytometry was used to analyze expression of CAR, integrinsubunit αv and integrin heterodimers αvβ3 and αvβ5 on the surfaceof studied cells. Briefly, adherent cells were grown in tissue culturedishes (until 80% confluence), detached with Versene (Invitrogen)andwashed twicewith PBS. To analyze the expression of CAR, integrinsubunit αv and integrin heterodimers αvβ3 and αvβ5, cells wereincubated with murine mAb anti-CAR, clone RmcB (Upstate CellSignaling Solutions, USA), anti αv 272-17E6 (Calbiochem, Germany),anti-αvβ3 23 C6 (Pharmingen, USA) or anti-αvβ5 MAB1961 (Chemi-con, USA), respectively. The binding of unlabelled primary antibodieswas revealed by using PE-conjugated anti-mouse antibody (DAKO,USA). Isotype control samples were incubated with mouse IgG1(Sigma, Germany) followed by PE-conjugated anti-mouse antibody(DAKO).

Confocal microscopy of CAR and integrins αvβ3 and αvβ5

For confocal microscopy analysis cells were seeded on cover slips.The next day cells were incubatedwith the primarymurine antibodiesmAb anti-CAR, clone RmcB (Upstate Cell Signaling Solutions),anti-αvβ3 23 C6 (Pharmingen) and anti-αvβ5 MAB1961 (Chemicon)

for 1 hour on ice, washed, incubated with Alexa-Fluor 594 secondaryantibody (Molecular Probes) for 1 hour on ice, fixed with 2%paraformaldehyde at room temperature and mounted in Vectashield(Abcys, USA). Nuclei were counterstained with DAPI. Analysis wasperformed using a Leica TSC SP2 (Lasertechnichk GmbH, Germany)microscope.

Measurement of Ad5-mediated transgene expression

We used three replication deficient adenoviral vectors: Ad5wt (wtdenotes wild type fiber), and recombinants Ad5FbΔ639 andAd5FbΔ639RGD4C (Ambriović-Ristov et al., 2003; Majhen et al.,2009). Ad5FbΔ639 and Ad5FbΔ639RGD4C have been constructedfrom Ad5wt and both have short fiber shafts (8β repeats in the fibershaft instead of the 22β repeats), while Ad5FbΔ639RGD4C has anadditional RGD4C motif inserted in HI loop of fiber knob. All threeadenoviral vectors possess a β-gal reporter gene, placed under thecontrol of the Rous Sarcoma Virus (RSV) promoter. They wereamplified in HEK-293 cells, purified by two rounds of CsCl densitycentrifugation, dialyzed and stored in PBS with 10% glycerol at−80 °C. The virus particle concentration was measured by opticaldensity according to the protocol described by Mittereder et al.(1996).

Ad5-mediated transgene expression was measured in 96-wellplates. Twenty-four hours after plating of 2×104 RD, RD-G7 and RD-D6 cells per well they were infected for 1 hour in a total volume of50 μl (in triplicate) with serial twofold dilutions of viruses normalizedfor physical particle number. Viruses were then removed and freshmedium was added. Twenty-four hours after infection, cells werefixed with 0.5% glutaraldehyde and stained for β-Gal expression. Thenumber of stained cells was determined by light microscopy.

Adhesion

For adhesion experiments, RD, RD-G7 and RD-D6 cells (4×104 cellsin 0.1 ml of DMEM containing 10% FCS) were seeded in 96-well platespreviously coated with 0.04 mg/ml vitronectin or fibronectin, andincubated for 90 minutes. The nonadherent cells were removed by twowashes with PBS. Attached cells were fixed with 4% paraformaldehydefor 10 minutes at 37 °C, stained for 90 minutes with 1% crystal violet(Sigma) in PBS, extensively washed with distilled water, and dried atroom temperature. The dye was resuspended with 100 μl of 0.5% TritonX-100 in PBS perwell, and color yieldwasmeasured using 96-well platereader at 590 nm.

Migration

Boyden chambers (pore size, 8 μm) (BD Biosciences, USA) wereused to investigate migration. Serum starved (24 hours) RD cells andCAR-expressing cell clones RD-G7 and RD-D6 (7.5×104 cells in 0.5 mlof DMEM containing 0.1% BSA)were placed in the upper chamber, and1 ml of DMEM containing 10% FCS was placed in the lower chamber of24-well plate. After 22 h in culture, cells on the upper side of the filterswere removed with cotton-tipped swabs, the filters were fixed inparaformaldehyde for 15 min and stainedwith 1% crystal violet in PBSfor 90 min. Cells on the underside of the filters were photographedunder a microscope and counted.

Silencing

To achieve integrin αvβ3 or αvβ5 silencing, cells were transientlytransfected with 20 nM Silencer Select Predesigned siRNA (Ambion,USA) for integrin β3 (Cat# 4392421) or 10 nM for integrin β5 (Cat#4390817) using Lipofectamine RNAiMAX reagent (Invitrogen) accord-ing to the manufacturer's instructions. As a negative control, cells weretransfected with equivalent concentration of a nontargeting control

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siRNA Silencer Select Predesigned siRNA Negative control #1 (Cat#4390844). Silencing of integrin αvβ3 or αvβ5 was verified by flowcytometry as described above.

Statistical analysis

Each experiment was repeated at least three times. All data wereanalyzed by unpaired Student's t-test and expressed as means±standard deviation. Only p-valuesb0.05 were considered statisticallysignificant.

Fig. 1. Stable CAR expression reduces the expression of αvβ3 and αvβ5 integrins. Surface expcells and RD-derived stable CAR transfectants RD-G7 and RD-D6. RD, RD-G7 and RD-D6 cellsclone RmcB, anti-αvβ3 23 C6 or anti-αvβ5 MAB1961, followed by rabbit PE-conjugated-ayielded similar results are shown. The statistical analysis of data for mean fluorescencetransfectants RD-G7 and RD-D6 are presented as fold means of RD±standard deviation. **

Results

Forced expression of CAR in RD cells reduces expression of αvβ3 andαvβ5 integrins

In order to study the interplay of CAR and integrins in RD cells thathave very low endogenous CAR, we generated RD cell clones RD-G7and RD-D6 stably expressing full length human CAR. The expressionof CAR was measured by flow cytometry and showed a 140 (RD-G7)and 400 (RD-D6) fold increase in cell surface CAR mean fluorescentintensity (MFI), respectively, as compared to RD cells (Fig. 1A). We

ression of (A) CAR, (B) αvβ3 integrin, (C) αvβ5 integrin, (D) αv integrin subunit in RDwere detached by Versene and analyzed by flow cytometry using murine mAb anti-CAR,ntimouse antibody. The representative data of three independent experiments whichintensities (MFI) from three independent experiments for RD-derived stable CAR

* denotes pb0.0005, ** denotes pb0.005, * denotes pb0.05.

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additionally analyzed CAR expression in RD-G7 and RD-D6 cell clonesby semiquantitative RT-PCR and found approximately 50 (RD-G7) and80 (RD-D6) fold increase in CAR-specific mRNA, respectively, relativeto parental RD cells (data not shown).

In order to study the influence of CAR expression on the expressionof integrin subunits αv, α4, α5, β1, β3 and β5, we examinedexpression by semiquantitative RT-PCR and found that the mRNAlevels of the analyzed integrin subunits were not influenced by CARexpression (data not shown). Then we analyzed the cell surfaceexpression of integrin subunits αv, α4, α5, β1 and integrinheterodimers αvβ3 and αvβ5 by flow cytometry. We did not finddifferences in expression of α4, α5 and β1 integrin subunits betweenparental RD, and CAR overexpressing cell clones RD-G7 and RD-D6(data not shown). However, significantly decreased expression ofintegrin heterodimers αvβ3 (Fig. 1B) and αvβ5 (Fig. 1C) wasobserved in RD-CAR clones relative to parental RD cells. It is verylikely that αvβ3 and αvβ5 integrins are not the only αv integrinsexpressed by RD cells but they are the most important for Ad5transduction (Sharma et al., 2009; Wickham et al., 1993, 1994). Toinvestigate whether the downregulation of αvβ3 and αvβ5 integrinswas a reflection of a decrease in total αv expression, we specificallymeasured αv expression by flow cytometry. As can be seen inFig. 1D we observed a significant decrease in total αv integrin on thesurface of RD-G7 and RD-D6 cells, respectively, indicating thatαvβ3 and αvβ5 integrins comprise a significant proportion of totalαv heterodimers.

To confirm that decreased expression of αvβ3 and αvβ5 integrinsis indeed the consequence of CAR expression we analyzed theexpression of αvβ3 and αvβ5 integrins and αv integrin subunit intwo additional cell clones stably expressing CAR. In both of the cellclones we observed not only decreased expression of αvβ3 and αvβ5integrins but also decreased expression of αv integrin subunit (datanot shown).

Membrane expression of CAR, αvβ3 and αvβ5 integrins in RD andRD-derived CAR stable transfectants

To examine the membrane expression and localization of CAR,αvβ3 and αvβ5 integrins on the surface of RD cells and CAR-expressing cell clones RD-G7 and RD-D6, we performed immunola-beling of live, unpermeabilized cells at 4 °C followed by confocalmicroscopy. RD cells do not grow as a typical monolayer culture.Therefore, we analyzed CAR localization at several depths. As shownin Fig. 2A, RD cells were not labeled by anti-CAR antibody. In contrast,in RD-G7 and RD-D6 cells CAR is present on both the cell surface andat sites of cell–cell adhesion. When analyzed on cells at similar depth(as judged by the intensity of DAPI staining), CAR can be identified incell–cell contacts (Fig. 2B). Analysis of αvβ3 and αvβ5 integrinexpression in RD, RD-G7 and RD-D6 cells by confocal microscopydemonstrated staining in all three cell types (Fig. 2C). As confocalmicroscopy is not a quantitative method we were unable to see thedifference in αvβ3 and αvβ5 integrin expression between RD cellsand their CAR overexpressing clones.

Functional involvement of decreased αvβ3 and αvβ5 integrinsexpression in Ad5-mediated transgene expression

To investigate how changes in cell surface CAR expression arerelated to Ad5 infection we measured Ad5wt (replication defective

Fig. 2. Membrane staining of CAR, αvβ3 and αvβ5 integrins in RD and RD-derived CARimmunostained withmurinemAb anti-CAR, clone RmcB or anti-αvβ3 23 C6 or anti-αvβ5MAperformed using a TCS SP Leica (Lasertechnichk GmbH) microscope. (A) Membrane stainingx–y optical sections taken along the z axis of CAR staining in RD-G7 or RD-D6 cells. Nuclei weRD-G7 or RD-D6 cells. Scale bar in (A) and (B) is 105 μm. Scale bar in (C) is 55 μm.

Ad5 bearing wild type Ad5 fiber)-mediated transgene expression inCAR expressing clones RD-G7 and RD-D6 in comparison to parentalRD cells. CAR expressing cell clones RD-G7 and RD-D6 expressapproximately 100 (RD-G7) to 180-fold (RD-D6) more Ad5-deliveredtransgene than parental RD cells, respectively (Fig. 3A).

Ad5 retargeting toαv integrins through incorporation of an RGD4Ctargeting motif into HI-loop of Ad5 fiber protein represents onestrategy to overcome the frequent downregulation of CAR in tumorcells (Dmitriev et al., 1998; Kanerva and Hemminki, 2004). To studythe importance of αvβ3 and αvβ5 integrin expression level in RD-derived CAR-overexpressing cell clones, we measured transgeneexpression with two replication defective adenoviruses Ad5FbΔ639and Ad5FbΔ639RGD4C. Entry of both of these recombinant adenovi-ruses is less dependent of CAR expression owing to a shortened andrigid fiber (Ambriović-Ristov et al., 2003; Majhen et al., 2009). This isconfirmed by measurement of Ad5FbΔ639-mediated transgeneexpression that is only 8-fold and 11-fold higher in cell clones RD-G7 and RD-D6 than in RD cells, respectively (Fig. 3B). Ad5FbΔ639RGDis essentially the same as Ad5FbΔ639 but possesses an RGD4Cretargeting motif incorporated into the HI-loop of fiber protein. RDcells express both, αvβ3 and αvβ5 integrins, and the ratio oftransgene expressions of Ad5FbΔ639RGD4C/Ad5FbΔ639 reflects theavailability of these integrins on the cell surface to mediate efficienttransgene expression. Ad5FbΔ639RGD4C-mediated transgene ex-pression in RD cells is approximately 10-fold higher than thatobserved for Ad5FbΔ639 (denoted as ratio 10, Fig. 3B). In both CARexpressing cell clones RD-G7 and RD-D6,αvβ3 andαvβ5 integrins aredownregulated as compared to RD cells (Fig. 1). While the ratio oftransgene expressions of Ad5FbΔ639RGD4C / Ad5FbΔ639 in RD cellsis 10, for RD-G7 is 6 and for cell clone RD-D6 is 5, confirmingdecreased availability of αvβ3 and αvβ5 integrins for transductionand the importance of these co-receptors for Ad5-mediated genetransfer in cell clones RD-G7 and RD-D6.

CAR overexpression in RD cells decreases adhesion to vitronectin andfibronectin

To determine whether the decreased expression of αvβ3 andαvβ5 integrins would influence adhesion to extracellular matrixproteins we performed an adhesion assay to vitronectin andfibronectin. The αvβ5 integrin binds preferentially to vitronectinwhile αvβ3 integrin, in addition to vitronectin, binds to fibrinogen,von Willebrand factor, and fibronectin. RD derived cell clonesexpressing CAR, RD-G7 and RD-D6, showed a significant decrease inadhesion to both ligands as compared to parental cells. Namely, RD-G7 and RD-D6 cells demonstrated 60 and 50% reduction in adhesion tovitronectin (Fig. 4A), and 30 and 35% reduction in adhesion tofibronectin (Fig. 4B), respectively.

Reduced migration of RD cells overexpressing CAR: a possible role ofintegrin αvβ3

We next examined the possibility that CAR expression mightinfluence cell migration. We compared the ability of RD cells and CARexpressing clones RD-G7 and RD-D6 tomigrate using Boyden chamberswith 8-μm pore size membrane. Twenty two hours after cells wereplaced into the upper well, the migrated cells at the bottom of themembranewere fixed, stained and counted. As can be seen in Fig. 5 RD-G7 and RD-D6 cells expressing CAR showed reduced cell migration.

stable transfectants. RD, RD-G7 and RD-D6 cells were grown on glass coverslips andB1961, followed by Alexa-Fluor 594-conjugated goat-antimouse antibody. Analysis wasof CAR in RD, RD-G7 or RD-D6 cells. Nuclei were counterstained with DAPI. (B) Stack ofre counterstained with DAPI. (C) Membrane staining of αvβ3 and αvβ5 integrins in RD,

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Fig. 3. Adenovirus-mediated transgene expression in RD and RD-derived CAR stable transfectants. (A) Ad5wt, (B) Ad5Δ639 and Ad5Δ639RGD4C. Cells (2×104/well) were plated in96-well plates. Twenty-four hours later, theywere infected for 1 h at 37 °Cwith two-fold serial dilutions of Ad5wt, Ad5Δ639 or Ad5Δ639RGD4C and stained for β-gal expression 24 hafter infection. Blue cells were counted under light microscopy for each well in which the number of stained cells fell between 50 and 150. Results are presented as transgeneexpression relative to RD cells. Ratios of absolute numbers of stained cells for Ad5Δ639RGD4C and Ad5Δ639: pb0.01. The data presented are representative of three independentexperiments, which yielded similar results.

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To specifically investigate whether decreased expression of αvβ3integrin is responsible for this effect we performed specific β3silencing in RD cells using siRNA that led to decreased expression ofαvβ3 integrin (to 40% of the RD value; Fig. 6A) and measured cellmigration. Silencing of αvβ3 integrin, as confirmed by flowcytometry, led to a significant inhibition of cell migration i.e. to 60%of the RD value (Fig. 6B). In contrast, silencing of β5 in RD cells led todecreased expression of integrin αvβ5 but significantly impaired cellproliferation, preventing us to conclude about influence on cellmigration (data not shown). Therefore, the inhibition of cell migrationin RD cells with silenced integrin αvβ3 expression indicates thatdownregulation of this integrin could be the mechanism of CAR-induced reduced migration.

Discussion

Tumor cells often show a decrease in cell–cell and cell–matrixadhesion. An increasing body of evidence now indicates that aberrantcell adhesion is causally involved in tumor progression and metastasis,rather than merely being a consequence of it (Nollet et al., 1999). CARwas identified as a primary Ad5 receptor (Bergelson et al., 1997) but hasalso been classified as an adhesion molecule as it can form homophilicinteractions between neighboring cells (Bruning and Runnebaum,2003; Cohen et al., 2001; Excoffon et al., 2004). The previously proposedinhibitory effects of CAR on cancer cell growth and motility have beensuggested to result from increased intercellular adhesion (Bruningand Runnebaum, 2004; Okegawa et al., 2000; Wang et al., 2005). Bothαvβ3 and αvβ5 integrins, but particularly αvβ3 integrin, have been

Fig. 4. Adhesion assays of RD, RD-G7 and RD-D6 cells to vitronectin (A) or fibronectin (B) cowith vitronectin or fibronectin, and incubated for 90 minutes. Nonadherent cells were respectrofotometry. Data are presented as adhesion relative to RD cells. The representative datapb0.001, * denotes pb0.05.

extensively studied for their involvement in tumor cell adhesion andmotility. It is known that expression of αvβ3 and αvβ5 integrins ontumor cells is correlatedwithdisease progression invarious tumor typeslike melanomas and carcinomas of the breast, prostate, pancreas andcervix (reviewed in Desgrosellier and Cheresh, 2010). Integrin αvβ3 isalso involved in the resistance of tumor cells to antitumor drugs.Namely, we have previously shown in human laryngeal carcinoma cellsHEp2, that increased expression of αvβ3 integrin confers resistance tocisplatin, mitomycin C, and doxorubicin through a mechanism ofglutathione-dependent increased ability of αvβ3-expressing cells toeliminate drug-induced reactive oxygen species (Brozovic et al., 2008).

RD cells express very low levels of endogenous CAR while RD cellclones transfectedwith plasmid containing a CAR-specific cDNA, RD-G7and RD-D6, express CAR in large quantities, as shown by flow cytometry(Fig. 1A). It should be mentioned that CAR overexpression did notsignificantly change cell morphology, as seen by light microscopy (datanot shown) and immunocytochemistry (Fig. 2A,B). In RD-G7 and RD-D6cells, CAR is present on the cell surface and can be seen at cell–celljunctions as observed for CAR-expressing CHO cells (Excoffon et al.,2005).

The first data concerning the mechanism of crosstalk between CARand integrins are observed in B16BL6 mouse melanoma cell model(Yamashita et al., 2007). CAR is not detected in endothelial cells in vivo(Raschperger et al., 2006) suggesting that B16BL6 cells expressingCAR are not attached to endothelial cells by interaction with CAR,while integrins play a prominent role in the binding of tumor toendothelial cells (Rice and Bevilacqua, 1989) and in tumor metastasis(Seftor et al., 1999). That is the reason why Yamashita et al. (2007)

ated plates. RD, RD-D7 and RD-G6 cells were seeded in 96-well plates previously coatedmoved by washing and attached cells were quantitated by crystal violet staining andof three independent experiments which yielded similar results are shown. *** denotes

Fig. 5. Enforced expression of CAR in RD cells reduces cell migration. The cell migration was performed with a total of 7.5×104 of 24 h serum starved cells in 0.5 ml of DMEM+0.1%BSA that were placed in the Boyden chamber (pore size, 8 μm) and 1 ml of DMEM containing 10% FBS was added in the lower chamber. After 22 h in culture, cells on the upper side ofthe filters were removedwith cotton-tipped swabs; the filters were fixed in paraformaldehyde and stainedwith crystal violet. Cells on the underside of the filters were photographedunder a microscope and counted. Averages of four microscope fields±standard deviation. The representative data of three independent experiments which yielded similar resultsare shown. *** denotes pb0.001.

Fig. 6. Decreased expression of integrin αvβ3 in RD cells reduces cell migration. (A) Silencing of integrin subunit β3 in RD cells led to decreased expression of integrin αvβ3. RD cellswere transfected with negative control (si-nc) or β3 (si-β3)-specific si RNA. The success of silencing wasmeasured by flow cytometry as described in Fig. 1, 96 h after transfection, i.e.in the moment of seeding cells in Boyden chambers for migration. (B) The cell migration of RD cells transfected with negative control (si-) or β3 si(β3)-specific si RNA. The cellmigration was performed same as in Fig. 5. Averages of four microscope fields±standard deviation. The representative data of three independent experiments which yielded similarresults are shown. ** denotes pb0.01.

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examined the mRNA levels of integrins by RT-PCR analysis in B16BL6cells overexpressing CAR. They found lower amounts of αv, α4, β1and β3 integrins that reduced migration in vitro and reduced lungmetastatic potential, suggesting that decreased integrin expression byCAR may contribute, at least in part, to reduced metastasis, possiblybecause of reduced attachment (binding) and invasion (migration) ofB16BL6 cells to lung endothelial cells. We show here that forcedexpression of CAR in RD cells: (i) decreases αvβ3 and αvβ5 integrinexpression at the protein level (Fig. 1), (ii) decreases adhesion tovitronectin and fibronectin (Fig. 4) and (iii) reduces cell migration(Fig. 5). However, in our cell system, we did not observe changes ofintegrins on the transcription level. In addition, no changes in α4 andβ1 integrin expression were observed (data not shown). Somewhatdifferent result concerning the mechanism of crosstalk between CARand integrins has been recently demonstrated by Farmer et al. (2009)in MCF-7 human breast carcinoma cells. They found that forcedexpression of CAR drove p44/42 MAPK activation and this in turnpromoted β1 and β3 integrin activation resulting in increased celladhesion, possibly through talin, an integrin binding protein whichbinds to the integrin cyto domain and leads to inside-out receptorsignaling. However, differently from our findings, they didn't observechanges of the integrin expression on the protein level.

The overexpression of CAR in ovarian, cervical and gastric cancer celllines inhibits cancer cell migration (Anders et al., 2009a; Bruning andRunnebaum, 2004). Integrins α4β1 and αvβ3 are reported to beimportant for cell migration and metastasis. Yamashita et al. (2007)postulated that their reducedexpressionmay contribute, at least in part,to reducedmigration andmetastasis. Our results showthat the silencingof either β3 or β5 integrin subunit in RD cells led to decreasedexpression of αvβ3 or αvβ5 integrin heterodimers. However, onlydecreased expression of αvβ3 integrin significantly inhibited cellmigration (Fig. 6). Therefore in this study we propose that forcedexpression of CAR reduces migration through down regulation of αvβ3integrin. We are aware that increasing the expression of αvβ3 integrinin CAR-overexpressing cell clonesmay provide direct evidence to provethis hypothesis. However, this is very difficult to achieve experimentallydue to the heterodimeric nature of integrins. Namely, the transfectedβ3subunit could compete for availableαv in a cell anddecrease anotherαvheterodimer likeαvβ5, aswe described previously in amodel of humanlaryngeal carcinoma cell line (Ambriović-Ristov et al., 2004).

Adenovirus-mediated transgeneexpressionhas thepotential to alterthe clinical course of cancer. However, CAR expression has beenreported to be low or sometimes absent in many advanced clinicalcancers (Korn et al., 2006;Matsumoto et al., 2005) limiting the utility ofthis intervention. Several studies have investigated the feasibility ofincreasing adenovirus-mediated transgene expression by incubatingcancer cells with various agents (Hemminki et al., 2003; Pong et al.,2006). For example, the data presented by Hemminki et al. (2003)identify depsipeptide (FR901228 or FK228), trichostatin A, topotecan,and etoposide as promising agents, alone or in combination, forincreasing adenoviral transgene expression in ovarian cancer cells.These treatments have increased both CAR expression and adenoviraltransgene expression in several cell lines. Similar effects have beenobservedbyPonget al. (2006); epigeneticmodifier FK228 increases CARexpression as well as the uptake of adenovirus in bladder cancer in vivoand in vitro. Our data demonstrate that overexpression of CAR in RDcells increases Ad5-mediated transgene expression. In addition, weshow that CAR induced downregulation of αvβ3 and αvβ5 integrinsdecreased their availability for Ad5RGD4C transgene expression incomparison to parental RD cells. Therefore, in cells with limited amountof αv integrins, CAR induced downregulation of αvβ3 and αvβ5integrins may influence the efficacy of adenovirus internalization ofboth, Ad5wt and Ad5RGD4C. The expression of αvβ5 integrin is evenmore important than expression of αvβ3, since it has been shown thatαvβ5 integrin is a limiting factor for adenovirus endosomal release(Majhen et al., 2009; Wickham et al., 1994).

Ultimately, the effect of improving Ad5-mediated transgene expres-sion via pharmacological manipulation of CAR could have a significanteffect on biological properties of treated tumor cells (Bruning andRunnebaum, 2003; Hemminki et al., 2003; Okegawa et al., 2007; Ponget al., 2006; Shieh et al., 2006; Vincent et al., 2004). We show here thatforced expression of CAR in RD cells reduces adhesion tofibronectin andvitronectin, as well as cell migration. For both, invasion and metastaticspread, an impaired adhesion of cancer cells is considered as a crucialprerequisite. Cell migration is also one necessary requirement forefficient cell invasion and metastasis. Thus, we postulate that pharma-cological upregulation of CAR in certain types of tumor cells like RD,could also slowdown tumorprogression. Inaddition, themechanismsofaction of compounds used to increase CAR expression in order toenhance Ad5 infectivity are completely unknown and it is very likelythat they influence the transcription of an unknown number of geneswith a cumulative effect that is difficult to predict. Therefore weunderline the need for analyzing not only Ad5 transgene expression incells after pharmacological upregulation of CAR, but also biologicaleffects of this combined treatment.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgements

This work was supported by funds of the Ministry of Science,Education and Sports of the Republic of Croatia (Project Nos. 098-0982913-2850 and 098-0982913-2748). We thank Katherine J. D. A.Excoffon, Department of Biological Sciences, Wright State University,Dayton, OH, USA, for many helpful suggestions regarding this work.

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