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DNA Repair 10 (2011) 848– 860

Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me pag e: www.elsev ier .com/ locate /dnarepai r

TM protects against oxidative stress induced by oxidized low-densityipoprotein

ichaela Semlitscha, Rodney E. Shackelfordb, Sandra Zirkla, Wolfgang Sattlera, Ernst Mallea,∗

Institute of Molecular Biology and Biochemistry, Center for Molecular Medicine, Medical University of Graz, Harrachgasse 21, A-8010 Graz, AustriaTulane University Health Sciences, Department of Pathology and Laboratory Medicine, New Orleans, LA, USA

r t i c l e i n f o

rticle history:eceived 24 January 2011eceived in revised form 15 April 2011ccepted 10 May 2011vailable online 12 June 2011

eywords:OSodified LDLNA-double-strand breaks

a b s t r a c t

Chronic oxidative stress is involved in the pathogenesis of multiple inflammatory diseases, includingcardiovascular disease and atherosclerosis. The rare autosomal recessive disorder Ataxia-telangiectasia(A-T) is characterized by progressive cerebellar ataxia secondary to Purkinje cell death, immunodefi-ciency, and increased cancer incidence. ATM, the protein mutated in A-T, plays a key role in cellularDNA-damage responses. A-T cells show poor cellular anti-oxidant defences and increased oxidant sensi-tivity compared to normal cells, and ATM functions, in part, as an oxidative stress sensor. The oxidation oflow-density lipoprotein (oxLDL) and its uptake by macrophages is an initiating step in the developmentof atherosclerosis. We demonstrate that oxLDL activates ATM and downstream p21 expression in normalfibroblasts and endothelial cells. In ATM-deficient fibroblasts oxLDL induces DNA double-strand breaks,

therosclerosis21olony forming efficiency assayA.hy926 cellsydrogen peroxide

micronuclei formation and causes chromosome breaks. Furthermore, oxLDL decreases cell viability andinhibits colony formation in A-T fibroblasts more effectively as compared to normal controls. Formationof oxLDL-induced reactive oxygen species is significantly higher in A-T, than normal fibroblasts. Last,pre-treatment of cells with ammonium pyrrolidine dithiocarbamate, a potent antioxidant and inhibitorof transcription factor nuclear factor �B, reduces oxLDL-induced reactive oxygen species formation. Ourdata indicates that ATM functions in the defence against oxLDL-mediated cytotoxicity.

. Introduction

Reactive oxygen species (ROS) are generated constantly as by-roducts of cellular metabolism, particularly by mitochondrialespiration [1,2]. At normal cellular concentrations, ROS play a rolen regulating cell signalling pathways and gene expression [1,2].owever, when the production of ROS exceeds cellular antioxi-ant capacity, damage to cellular macromolecules such as lipids,roteins, and DNA may occur [2,3]. To combat such damage organ-

sms have evolved anti-oxidant protective systems, including thelutathione/glutathione disulfide system, superoxide dismutase,

atalase, metal chelation, and diverse repair systems that maintainedox homeostasis [3,4]. An imbalance between ROS-generatingnd scavenging systems is called “oxidative stress” and plays a

Abbreviations: ATM, A-T mutated protein; ATM-I, ATM-kinase-inhibitor;-T, Ataxia telangiectasia; Carboxy-H2DCFDA, 5-(and-6)-carboxy-2′ ,7′-ichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; DSBs, DNA-doubletrand breaks; LDL, low-density lipoprotein; MTT, 3-[4,5-dimethylthiazol-2-yl]-,5-diphenyltetrazolium bromide; oxLDL, oxidized low-density lipoprotein; pATM,hosphorylated ATM; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygenpecies; �-H2AX, phosphorylated H2AX.∗ Corresponding author. Tel.: +43 316 380 4208; fax: +43 316 380 9615.

E-mail address: ernst.malle@medunigraz.at (E. Malle).

568-7864/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2011.05.004

© 2011 Elsevier B.V. All rights reserved.

crucial role in a variety of pathological disorders, among them car-diovascular and neurodegenerative diseases.

Ataxia telangiectasia (A-T) is a progressive neurodegenerativedisease manifesting in early childhood. The clinical features of A-T include progressive ataxia secondary to cerebellar Purkinje celldeath, premature aging, immunodeficiency, and increased cancerrisk; especially for leukaemia and lymphoma [5]. Patients withA-T lack functioning A-T mutated protein (ATM), a member ofthe phosphatidylinositol 3-kinase like family of serine/threonineprotein kinases [6]. ATM-deficient cells exhibit chromosomalinstability and extreme sensitivity to DNA-double strand break(DSB)-inducing agents, such as ionizing radiation [7]. Hence, themost studied function of ATM is its role in response to DNAdamage. When DNA-DSBs occur, ATM is rapidly activated byautophosphorylation at Ser1981 [8], and in turn rapidly phospho-rylates a number of substrates involved in DNA replication andrepair, cell cycle checkpoint control, and apoptosis [9,10]. How-ever, there is evidence that A-T is not only due to a defect inDNA-DSB response, but also to a diminished control of ROS. Studiesrevealed that ATM-deficient cells are in a constant state of oxida-

tive stress [11]. Reichenbach and co-workers [12] reported that theplasma of A-T patients exhibit a decreased antioxidant capacity.Treatment with antioxidants e.g. N-acetyl-l-cysteine and tem-pol, increased the lifespan of Atm−/− mice and tempol-treatment

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urther decreased levels of ROS and oxidative damage in thymo-ytes of mice [13,14]. Moreover, ATM is activated by oxidantsuch as t-butyl hydroperoxide and H2O2 [15–17]. Additionally,2O2-induced phosphorylation of ATM can be blocked by N-acetyl-

-cysteine, indicating that ATM phosphorylation is responsive toedox imbalance [15].

ROS act as signalling intermediates in many normal cellular pro-esses, and elevated ROS levels are linked to many pathologicalonditions including neurodegenerative diseases, diabetes, cancer,nd atherosclerosis, respectively [18]. The atherosclerotic lesion isharacterized by an accumulation of lipids carried by lipoproteins,uch as low-density lipoprotein (LDL). LDL becomes susceptible tonon)enzymatic oxidative modification when retained in the arteryall [19]. These modifications make the LDL particle a potent affec-

or of cellular functions. In particular, the uptake and degradationf oxidized LDL (oxLDL) by monocyte-derived macrophages is con-idered the leading event in the formation of cholesterol-enrichedoam cells, which are the hallmark of fatty streaks, the earliestecognizable lesion of atherosclerosis [19,20].

Currently, there is no data linking ATM to the cellular responsesollowing oxLDL exposure. However, there is indirect evidencehat ATM may be involved in oxLDL-induced signalling path-ays. Apparently as a consequence of increased levels of plasma

holesterol, heterozygous ATM deficiency may increase the riskf atherosclerosis-related cardiovascular disease in humans [21].polipoprotein E−/− (ApoE−/−) mice heterozygous in Atm developedccelerated atherosclerosis and multiple features of the metabolicyndrome including glucose intolerance, hypertension, obesity andypercholesterolemia [22,23]. Transplantation of ApoE−/−/Atm+/+

ice with bone marrow from ApoE−/−/Atm+/+ or ApoE−/−/Atm−/−

ice revealed 80% increase in lesion severity in animals treatedith Atm null bone marrow [24].

In the present study, we investigated the role of ATM in pro-ection against toxicity of copper-oxLDL [25], a commonly usedxperimental model for oxidative modification of LDL. Here wetudied the effect of oxLDL on ATM activation and downstreamignalling in normal fibroblasts and endothelial cells. We alsonvestigated DNA damage in normal and ATM-deficient fibroblasts.hird, we studied the cytotoxicity of oxLDL on normal and ATM-eficient fibroblasts and last, we examined the effect of ATM statusn oxLDL-induced ROS formation in these cells.

. Materials and methods

.1. Materials

Cell culture dishes, flasks and microtiterplates were fromreiner Bio-One (Frickenhausen, Germany). Dulbecco’s Modifiedagle Medium (DMEM), penicillin/streptomycin and hygromycin

were from Gibco Invitrogen (Lofer, Austria), foetal calf serumFCS) was from PAA (Linz, Austria) and bovine serum albumin (BSA)as from Serva (Heidelberg, Germany). Lysis buffer componentsEPES, EDTA, glycerol, Triton X-100, Na4P2O7 and Na3VO4 were

rom Sigma–Aldrich (Saint Louis, MO, USA) and NaF was from FlukaBuchs, Switzerland). Complete Mini protease inhibitor cocktailablets were from Roche Diagnostics (Mannheim, Germany). Try-an blue stain (0.4%), NuPAGE® 4–12% Bis-Tris Gels, NuPAGE® LDSample buffer, NuPAGE® MOPS running buffer and nitrocelluloseembranes were from InvitrogenTM life technologies (Carlsbad,

A, USA).Bisbenzimide (Hoechst 33258), 3-[4,5-dimethylthiazol-2-

l]-2,5-diphenyltetrazolium bromide (MTT, Thiazolyl blue),mmonium pyrrolidine dithiocarbamate (PDTC), crystal violet andriton X-100 were from Sigma–Aldrich (Saint Louis, MO, USA).arboxy-H2DCFDA (5-(and-6)-carboxy-2′,7′-dichlorodihydro-

air 10 (2011) 848– 860 849

fluorescein diacetate) was from Gibco Invitrogen (Lofer, Austria).Staurosporine and the ATM-kinase-inhibitor (ATM-I, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one) were from Calbiochem(Merck, Darmstadt, Germany). BCATM Protein Assay Kit and SuperSignal West Pico Chemiluminescent substrate were from PierceBiotechnology, Inc. (Rockford, IL, USA). ImmobilonTM WesternChemiluminescent HRP Substrate was from Millipore Corporation(Billerica, MA, USA). H2O2 was from Herba Chemosan (Vienna,Austria); colcemid was from Irvine Scientific (Santa Ana, CA,USA). All other chemicals were from Roth (Vienna, Austria) orSigma–Aldrich (Saint Louis, MO, USA).

The following primary antibodies were used: (i) polyclonalrabbit phospho-ATM antibody (recognizing human ATM phos-phorylated at Ser1981, termed pATM, R&D Systems, Minneapolis,MN, USA); (ii) sequence-specific polyclonal rabbit anti-ATM anti-bodies raised against synthetic peptides corresponding to aminoacids 819–844 (Calbiochem) or 2550–2600 (Abcam, Cambridge,U.K.) of human ATM; (iii) polyclonal rabbit anti-caspase-3 antibody(raised against full-length caspase-3, Santa Cruz Biotechnology,Santa Cruz, CA, USA), (iv) polyclonal anti-�-tubulin (Santa CruzBiotechnology); (v) polyclonal phospho-histone H2AX antibody(recognizing H2AX only when phosphorylated at Ser139, termed�-H2AX, Cell Signaling Technology, Beverly, MA, USA); (vi) rab-bit monoclonal anti-p21 Waf1/Cip1 antibody (12D1, Cell SignalingTechnology, Beverly, MA, USA), (vii) monoclonal anti-�-actinantibody (Santa Cruz Biotechnology); (viii) monoclonal anti-Poly(ADP-ribose) polymerase (PARP) antibody (recognizing the 116 kDaand the 85 kDa apoptosis-related cleaved fragment, Biomol, Ham-burg, Germany). The following secondary antibodies were used: (i)HRP-conjugated goat anti-mouse IgG (Rockland, Gilbertsville, PA,USA) and (ii) HRP-conjugated goat-anti-rabbit IgG (Pierce Biotech-nology, Inc., Rockford, IL, USA).

2.2. Cell culture

WI-38 VA13 (VA13) is a SV-40-immortalized fibroblast cell line(ATCC, Rockville, MD). AT22IJE-T (AT22) is an ATM-deficient SV-40 immortalized fibroblast cell line, originally established fromprimary A-T fibroblasts [26]. VA13 and AT22 cells were grownin DMEM with 1 g/l glucose, 4 mM l-glutamine, 110 mg/l sodiumpyruvate and 25 mM HEPES, supplemented with 5% (v/v) FCSand 100 U/ml penicillin/streptomycin. Human EA.hy926 endothe-lial cells were grown in DMEM with 4.5 g/l glucose, 3.97 mMl-glutamine and 1 mM sodium pyruvate supplemented with 10%(v:v) FCS, 1% penicillin-streptomycin and 1 × HAT supplement [27].All three cell lines were cultured at 37 ◦C in a humidified atmo-sphere of 5% CO2 and 37 ◦C

2.3. Isolation and modification of LDL

LDL was isolated by ultracentrifugation (d = 1.027–1.063 g/ml)from fresh human plasma, obtained from healthy volunteers[28,29]. LDL was sterile-filtered and stored at 4 ◦C. Prior to oxi-dation, LDL was dialyzed overnight against PBS (10 mM; pH 7.4)at 4 ◦C. Oxidation of 500 �g/ml LDL was performed with a finalconcentration of 30 �M Cu2SO4 for 18 h. EDTA (final concentration2.7 mM) terminated the reaction, the samples were saturated withN2 and stored at 4 ◦C. Characterization of oxLDL was performed asdescribed [30]. Before use, oxLDL was sterile-filtered and adjustedto a final protein concentration of 1 mg/ml by dialysis under high

pressure against PBS (10 mM) (to remove EDTA and unreactedCu2SO4). Lipoprotein concentrations are expressed in terms of itsprotein concentration, determined by the Lowry method using BSAas a standard.

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.4. Cell experiments for Western Blot analysis

VA13, AT22, and EA.hy926 cells were seeded in 6-well plates.hen cells reached 70% confluence, they were incubated in

erum-free DMEM overnight. Cells were treated with indicatedoncentrations of lipoproteins for the indicated times. For block-de of the ATM-kinase signalling pathway, cells were pre-incubatedith ATM-I [31] (10 or 30 �M dissolved in DMSO) for 1 h. Cells

reated with PBS and/or DMSO served as controls. DMSO concentra-ion did not exceed 0.01% (v/v). Alternatively, the cells were treatedith 200 �M H2O2 for 15 min and after medium-exchange, the cellsere incubated for further 90 min.

For protein isolation, the cells were washed twice with ice coldBS. Cell lysis was performed on ice in 60 �l lysis buffer (50 mMEPES, 150 mM NaCl, 1 mM EDTA, 10 mM Na4P2O7, 2 mM Na3VO4,0 mM NaF, 1% (v/v) Triton X-100, 10% (v/v) glycerol and Completeini protease inhibitor cocktail tablets; pH 7.4) for 10 min [32]. The

ell lysates were scraped and insoluble cell debris was removed byentrifugation (13,000 rpm, 4 ◦C) for 10 min.

To follow expression of �-H2AX, cleavage of PARP- androcaspase-3, cells were pelleted by centrifugation (1000 rpm,

min) and lysed. Protein content of cell lysates was determinedsing the BCATM Protein Assay Kit, according to the manufac-urer’s instructions. Protein lysates (30–50 �g/ml) were dilutedith NuPAGE® LDS Sample Buffer and NuPAGE® Sample Reduc-

ng Agent (10×) and were boiled for 10 min at 70 ◦C. Proteins wereeparated in NuPAGE® 4–12% Bis-Tris Gels and electrophoreticallyransferred to nitrocellulose membranes [33]. Membranes wererst incubated with Tris-buffered saline Tween 20 (TBS-T, contain-

ng 5% (w/v) non-fat milk) for 2 h, before incubation with polyclonalabbit anti-pATM antibody, polyclonal rabbit anti-ATM antibody,olyclonal rabbit �-H2AX antibody, rabbit monoclonal anti-p21af1/Cip1 antibody, polyclonal rabbit anti-caspase-3 antibody,onoclonal anti-PARP antibody, monoclonal anti-�-actin antibody

r polyclonal anti-�-tubulin antibody (dilution 1:500 to 1:1000 inBS-T containing 5% (w/v) BSA) overnight at 4 ◦C. Immunoreactiveands were visualized using HRP-conjugated goat anti-rabbit (dilu-ion 1:50,000 in TBS-T containing 5% (w/v) non-fat milk) or goatnti-mouse IgG (dilution 1:100,000 in TBS-T containing 5% (w/v)on-fat milk) for 2 h, and subsequently visualized with Super Signalest Pico Chemiluminescent substrate or ImmobilonTM Western

hemiluminescent HRP Substrate.

.5. MTT assay

VA13 and AT22 cells were seeded in 12-well plates in DMEMith 5% (v/v) FCS. When cells reached 50% confluence, the mediumas replaced by serum-free DMEM and the cells were incubated

vernight. Then the cells were treated with lipoproteins for thendicated times and at the indicated concentrations. The cells were

ashed with PBS (10 mM) and incubated with MTT (0.5 mg/ml;issolved in serum-free medium) for 2 h at 37 ◦C [32]. The con-erted dye was solubilised with acidic isopropanol (0.04 M HCl inbsolute isopropanol). MTT reduction was assessed by measuringbsorption at 570 nm on a microplate reader (Victor Multilabelounter, Perkin-Elmer, Waltham, MA, USA) and corrected forackground absorbance at 630 nm. Each treatment was done inriplicate and values are expressed as percentages of untreatedells.

.6. Colony forming efficiency assay

Exponentially growing VA13, AT22, and EA.hy926 cells werelated at a density of 1.5 × 103 cells/100 mm tissue culture dish inhe absence or presence of lipoproteins in normal growth medium.

hen indicated, the cells were preincubated with ATM-I (10 �M

ir 10 (2011) 848– 860

dissolved in DMSO) for 1 h before addition of lipoproteins. After18 h of incubation, the plates were washed 3 times with PBS(10 mM) the medium was replaced, and the cells were culturedfor 12 more days. The cells were fixed for 5 min with methanoland stained with crystal violet (1 g/l; dissolved in aqua dest.) andcell clusters of 50 or more cells were counted as “colonies” under amicroscope.

2.7. Trypan blue exclusion assay

VA13 and AT22 cells were seeded in 6-well plates until 50%confluence was reached. After overnight serum-starvation, cellswere incubated with indicated concentrations of lipoproteins. Atthe indicated times, the cells were washed with PBS (10 mM),trypsinized and resolved in serum-free DMEM. The cell suspen-sion was mixed with 1:1 (v/v) with 0.4% (w/v) Trypan blue stain.Viable cells, identified by a clear cytoplasm, were counted usingCountessTM cell counting chamber slides and the CountessTM Auto-mated Cell Counter (InvitrogenTM life technologies, Carlsbad, CA,USA).

2.8. Micronucleus assay and staining of nuclei with bisbenzimide

VA13 and AT22 cells were seeded in 6-well plates on glasscover slips and cultured in normal growth medium. When cellsreached 50% confluence, cells were serum-starved overnight andincubated with 100 �g/ml lipoprotein for 16 h. Cells were washedwith PBS (10 mM) and fixed with 90% (v/v) methanol for 5 min.Staining of nuclei was performed with 0.5 �g/ml bisbenzimide(Hoechst 33258). Cells on glass cover slips were incubated withthe fluorescence-dye for 30 min in the dark, washed with aquadest., air dried and mounted with glycerol. Micronuclei were scoredand cell images recorded with an FSX100 Box-Type FluorescenceImaging Device (Olympus Corporation, Tokyo, Japan). Before scor-ing the micronuclei, all slides were randomised and coded. Thenumber of micronuclei was determined by counting 500 cells/slide.The criteria for scoring micronuclei were adapted from refer-ences [34,35]; each treatment was done in triplicate. Values areexpressed as percentages of the number of micronuclei in untreatedcells.

2.9. Estimation of chromosome breaks

Logarithmically growing VA13 and AT22 cells were plated in100 mm tissue culture plates. When the cells reached 50% con-fluence, they were treated with 30 �g/ml lipoproteins for 8 h. Toarrest the cells in metaphase, colcemid (100 ng/ml) was addedfor 4 h [36]. The cells were washed with PBS and trypsinized.The reaction was stopped with DMEM and cells were pelleted5 min at 500 × g. Then, the cells were resuspended in 0.075 mMKCl and incubated for 15 min at 37 ◦C. Two hundred microliterof Carnoy’s fixative (methanol:glacial acetic acid, 3:1 [v/v]) wasadded; cells were gently mixed and pelleted (5 min, 500 × g). Thesupernatant was removed, cells were twice gently mixed with5 ml of Carnoy’s fixative and pelleted (5 min at 500 × g) again. Celllysates were dropped on glass slides and dried for 30 min at 90 ◦C.Chromosomes were stained with Giemsa. For scoring chromosomebreaks, 5000 individual chromosomes/treatment were observedunder oil immersion microscopy. Each treatment was done intriplicate.

2.10. Measurement of ROS

The intracellular generation of ROS was measured usingcarboxy-H2DCFDA. H2DCFDA is deacetylated by esterases to non-fluorescent dichlorofluorescein, which is converted to fluorescent

A Repair 10 (2011) 848– 860 851

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Fig. 1. Time-dependent phosphorylation of ATM in normal fibroblasts by oxLDL andinhibition of ATM-activation: (A) VA13 cells were incubated with 100 �g/ml oxLDLat indicated times. (B) VA13 cells were preincubated with 10 �M of the ATM-kinase-inhibitor (ATM-I) for 1 h followed by treatment with 100 �g/ml lipoprotein for90 min. As positive controls, cells were treated with 200 �M H2O2 for 15 min; aftermedium exchange the cells were incubated for another 90 min (105*). Cell lysateswere collected; proteins were subjected to SDS-PAGE and transferred to nitrocel-

M. Semlitsch et al. / DN

ichlorofluorescein (DCF) by ROS. VA13 and AT22 cell were cul-ured in 6 well plates in DMEM containing 5% FCS. Fifty % confluentells were serum-starved overnight and incubated with indicatedoncentrations of lipoproteins for 5, 12 or 24 h. When indicated,ells were pre-treated with PDTC for 30 min (1 mM; dissolved inqua dest.). For inhibition of ATM, cells were preincubated with theTM-I for 1 h before addition of lipoproteins. DMSO concentrationid not exceed 0.01% (v/v). After indicated times, the medium wasspirated and 10 �M carboxy-H2DCFDA, dissolved in PBS (10 mM),as added to the cells [37]. Cells were incubated for another 30 min

t 37 ◦C. To terminate the reaction, cells were kept on ice andashed with ice-cold PBS. Cell lysis was performed with 3% (v/v)

riton X-100 in PBS on a rotary shaker (1350 rpm; Heidolph Instru-ents, Schwabach, Germany) at 4 ◦C (in the dark) for 30 min. To

nsure complete solubilisation of DCF, 50 �l absolute ethanol wasdded and the plates were shaken for a further 15 min [37]. The cellysates were transferred to microfuge tubes and cellular debris wasemoved by centrifugation (13,000 rpm, 4 ◦C, 10 min). One hun-red microliter of the supernatant was transferred into 96-wellicrotiter plates and fluorescence was measured on a Victor Mul-

ilabel Counter (Perkin-Elmer, Waltham, MA, USA) with excitationt 485 nm and emission at 540 nm. All steps concerning carboxy-2DCFDA were performed under light-protected conditions. Therotein content of the cell lysates was determined using an aliquotf the supernatant and the BCATM Protein Assay Kit according tohe manufacturer’s instructions.

.11. Image analysis for generation of intracellular ROS

VA13 and AT22 cells were seeded in 6-well plates, grown to0% confluence, and incubated with serum-free DMEM overnight.here indicated, cells were pre-treated with 1 mM PDTC (dissolved

n aqua dest.) for 30 min. Cells were incubated with 100 �g/mlipoprotein for 5 or 12 h. Carboxy-H2DCFDA (10 �M, dissolved in0 mM PBS) was added to the cells and plates were incubated forurther 30 min at 37 ◦C. To terminate the reaction, dishes were putn ice and cells were washed with PBS. For observation of theells under a fluorescence microscope, 100 �l PBS was added toach well. The cells were observed and photographed using annverted microscope (Nikon Eclipse TE 2000-U; Nikon Instruments,

elville, NY, USA) with a green fluorescent filter and the NIS Ele-ents BR 2.10 software for image acquisition. To allow comparison

etween images, all images were acquired at the same exposureime (600 ms).

.12. Statistical analysis

Data are presented as means ± standard deviation (SD). Two-ay ANOVA or t-test statistical analyses were performed using

rism 5 software (Graphpad Software, La Jolla, CA, USA). In ANOVAnalysis, Bonferroni posttest was used for all pair wise compar-sons of the means of all experimental groups. Values (p < 0.05) wereonsidered significant.

. Results

.1. ATM is activated by oxLDL

Previous reports performed with various cell lines revealed thatependent on the stimulus, activation of ATM occurs between5 and 480 min [38,39]. We here show that VA13 cells (ATM+/+)

xhibited either no (data not shown) or sometimes basal pATMxpression (Fig. 1A). OxLDL increased pATM levels in a time-ependent manner reaching a maximum after 90 min (Fig. 1A).he immunoreactive pATM signal decreased to baseline levels after

lulose membranes. Western Blot experiments were performed using anti-pATMas primary antibody. �-Tubulin was used as loading control. One representativeexperiment out of three is shown.

300 min. H2O2 a known activator of ATM [15,40], resulted in effi-cient phosphorylation of ATM in VA13 cells but not in AT22 cells(termed ATM−/−). Densitometric evaluation of immunoreactivepATM bands revealed that H2O2-mediated induction is approxi-mately 25% higher after 90 min compared with oxLDL-mediatedinduction. Although two different polyclonal antibodies were usedto follow total ATM expression, immunoreactive �-tubulin wasfound to be more precise and reliable as loading control. Fig. 1Bdemonstrates that LDL sometimes tended to phosphorylate ATM inVA13 cells, however, only to levels in between 5 and 10% comparedto oxLDL. Fig. 1B further shows that oxLDL-induced phosphoryla-tion of ATM was completely abrogated by ATM-I (Fig. 1B).

3.2. ATM expression moderates the toxic effect of oxLDL

Cells that fail to repair damaged DNA before entering mitosismay exhibit chromosomal strand breaks, leading to disruption insubsequent cell cycles resulting in a defective colony formation[41]. As ATM plays an important role in the recognition and sig-nalling of DNA damage [42], we studied whether the lack of ATMaffects the clonogenic survival of cells. Fig. 2A shows that oxLDL,but not LDL, caused a dose-dependent inhibition of colony forma-tion in VA13 and AT22 cells. However, at protein concentrationshigher than 3-�g oxLDL/ml, colony formation in AT22 cells wassignificantly (p < 0.001) reduced compared to VA13 cells.

To support our observation, that the presence of ATM affectsthe clonogenic survival, ATM-activation in VA13 cells was inhib-ited before oxLDL treatment. Fig. 2B shows that ATM-I reducedcolony formation in VA13 cells to levels found in AT22 cells whentreated with oxLDL. Again, LDL did not alter colony formation whencompared to untreated control cells.

3.3. ATM and cell viability in the presence of oxLDL

Next, mitochondrial function and cell viability of normal andATM-deficient cells were investigated using two different assaysystems. The MTT-test forms blue formazan crystals that are

reduced by mitochondrial dehydrogenase in living cells [43]. OxLDLdecreased cell viability in VA13 and AT22 cells in a time- (Fig. 3A)and concentration-dependent manner (Fig. 3B). AT22 cells are moresensitive to oxLDL exposure than VA13 cells (Fig. 3). LDL had no

852 M. Semlitsch et al. / DNA Repair 10 (2011) 848– 860

Fig. 2. OxLDL alters colony-forming efficiency in normal and ATM-deficient fibroblasts: (A) Exponentially growing VA13 and AT22 cells were plated at a density of1.5 × 103 cells/100 mm dish in DMEM with increasing lipoprotein concentrations. After 18 h, the medium was replaced and cells were cultured for 12 days. (B) 1.5 × 103

cells of VA13 and AT22 cells, plated on a 100 mm dish, were pre-incubated with 10 �M ATM-I for 1 h before addition of 30 �g/ml lipoprotein. After 18 h the medium wasr ell co5 e com( d AT2

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eplaced by normal growth medium and cells were allowed to grow for 12 days. C0 or more cells were counted. Data are given as number of colonies (as percentaguntreated cells). **p < 0.01 and ***p < 0.001 (significant difference between VA13 an

dverse effect on the viability of either cell type. Next, cell survivalas measured using the Trypan blue exclusion assay. Incubation

f VA13 and AT22 cells with oxLDL up to 24 h decreased the num-er of living cells in a time-dependent manner up to 30% (Fig. 4A).gain, oxLDL was more toxic to AT22 cells at all times, com-ared to VA13 cells. LDL (used as control) had no effect on cellhe survival of both cell lines. To visualize nuclear changes afterreatment with lipoproteins, VA13 and AT22 cells were stainedith Hoechst 33258 and fluorescence intensity was checked. Con-

rol and LDL-treated cells exhibited diffuse chromatin stainingFig. 4B). However, exposure of VA13 cells to oxLDL led to mor-hological changes, such as areas of condensed chromatin andhrunken nuclei. In contrast, AT22 cells treated with oxLDL exhib-ted a decrease in size and number of nuclei, but no chromatinondensation.

.4. OxLDL induces DNA double strand breaks in A-T cells

ATM primarily responds to DSBs [7]. Since phosphorylation ofhe histone H2AX (termed �-H2AX) is a sensitive cellular indicatoror the presence of DNA-DSBs [44,45], the effect of lipoproteins on2AX phosphorylation via ATM was studied. Fig. 5A shows thatxposure of VA13 and AT22 cells to oxLDL led to formation ofmmunoreactive �-H2AX only in AT22 but not in VA13 cells. Also,ime-dependent incubation of both cell lines with oxLDL, but not

DL, confirmed the presence of immunoreactive �-H2AX after 16 hnly in AT22 cells (Fig. 5B). Since the MTT assay demonstrated thatxLDL is toxic to VA13 and AT22 cells (Fig. 4A), PARP cleavage andctivation of procaspase 3 (two hallmarks of apoptosis) were inves-

lonies were fixed, stained with crystal violet and colonies consisting of groups ofpared to untreated cells, set as 100%). Values represent the means ± SD (n = 5). UT2 cells).

tigated. After 16 h of oxLDL exposure neither PARP cleavage norprocaspase 3 processing was observed in either cell type (Fig. 5A).Following time-dependent incubation of cells with lipoproteins upto 24 h, neither LDL nor oxLDL promoted PARP cleavage or activa-tion of caspase 3 (Fig. 5B).

To assess whether the immunoreactive �-H2AX signal corre-lates with micronucleus formation following oxLDL exposure, andto investigate a possible clastogenic effect of oxLDL, the in vitromicronucleus technique was employed. Micronuclei arise duringcell division and contain chromosome breaks lacking centromeresand/or whole chromosomes, and are unable to travel to the spindlepoles during mitosis [34]. Our findings show that oxLDL-treatedAT22 cells displayed a significantly higher micronuclei numbercompared to similarly treated VA13 cells (Fig. 6A). Treatment ofboth cell lines with LDL did not alter the micronuclei number whencompared to untreated controls. Since micronuclei formation is asign of chromosomal damage, the number of chromosomal breakswas further counted in VA13 and AT22 cells in the absence orpresence of lipoproteins. Cells lacking ATM exhibited a slightlyhigher number (although statistically not significant) of chromoso-mal breaks in untreated cells compared to VA13 (Fig. 6B). However,oxLDL significantly (p < 0.001) increased chromosomal breaks inboth cell lines. In VA13 cells, the number of chromosomal breaksafter 8 h increased up to 30. In AT22 cells the number of chro-mosomal breaks increased up to 42. Fig. 6B further shows that

the number of oxLDL-induced chromosomal breaks in AT22 cellsare significantly (p < 0.001) higher when compared to VA13 cells.Treatment of VA13 and AT22 cells with LDL was without effects onchromosomal breaks when compared to untreated cells (Fig. 6B).

M. Semlitsch et al. / DNA Repair 10 (2011) 848– 860 853

Fig. 3. Time- and concentration-dependent effect of lipoproteins on cell viability of in normal and ATM-deficient fibroblasts using the MTT assay: (A) VA13 and AT22 cellswere treated with 100 �g/ml lipoprotein for indicated times. (B) VA13 and AT22 were exposed to 3, 30 and 100 �g/ml lipoprotein for 18 h. MTT reduction was assessedat 570 nm on a microplate reader and values were corrected for background absorbance at 630 nm. Values are expressed as percentages of untreated cells. The data aree p < 0.0

3c

asewtspARwd3tasmhLitc

tm(cR

xpressed as a percentage of untreated cells and represent the means ± SD (n = 6). *

.5. PDTC scavenges oxLDL-induced elevated ROS levels in A-Tells

ATM-deficient cells are in a constant state of oxidative stressnd may exhibit diminished antioxidant capacity [11,46]. Wehow that AT22 cells exhibited approx. 1.5-fold higher ROS lev-ls when compared to VA13 cells (Fig. 7A). Incubation of cellsith oxLDL further increased ROS levels in VA13 and AT22 in a

ime-dependent manner. ROS formation induced by oxLDL wasignificantly (p < 0.001) higher in AT22 cells at 5 and 12 h com-ared to VA13 cells. After 24 h, ROS levels were also higher inT22 cells, although not statistically significant. LDL did not affectOS levels in VA13 or AT22 cells (Fig. 7B). Treatment of cellsith increasing concentrations of oxLDL for 5 h led to a dose-ependent increase of ROS, which is significantly higher (up to-fold compared with untreated cells) in AT22 cells comparedo VA13 cells (Fig. 7C). Findings obtained with the DCFDA/DCFssay (Fig. 7), i.e. incubation of cells with lipoproteins and sub-equent ROS measurements, were confirmed using fluorescenceicroscopy (Fig. 7D). AT22 cells exposed to oxLDL exhibited

igher fluorescence intensity when compared to untreated orDL-treated cells. In line with data shown in Fig. 7B, a slightncrease in fluorescence-intensity could also be observed in oxLDL-reated VA13 cells when compared to untreated or LDL-treatedells.

To confirm, that ATM regulates ROS formation, cells were pre-reated with ATM-I before incubation with oxLDL. DCF fluorescence

easurements revealed that inhibition of ATM led to significantlyp < 0.01) higher levels of basal ROS in VA13 cells but also whenells were treated with oxLDL (Fig. 7E). No significant difference inOS levels were found in oxLDL-treated AT22 cells in the absence

5 and ***p < 0.001 (significant difference between VA13 and AT22 cells).

or presence of ATM-I indicating that the compound per se did notalter ROS formation.

To scavenge ROS, cells were pre-incubated with PDTC, a potentantioxidant and suppressor of transcription factor nuclear factor�B [47], prior to incubation with oxLDL. PDTC effectively reducedoxLDL induced ROS-formation in AT22 and VA13 cells to basal lev-els (Fig. 8A). Also fluorescence microscopy technique showed lessfluorescence intensity in oxLDL-treated cells after preincubationwith PDTC for 1 h (Fig. 8B).

3.6. OxLDL induces p21 via an ATM-dependent pathway

Activation of the ATM-kinase may promote induction of p53[10]; stabilized p53 serves as a transcription factor and stimulatesexpression of the cyclin-dependent kinase inhibitor p21 [48]. Fig. 9shows oxLDL-mediated induction of p21 in VA13 (but not in AT22)cells. Inhibition of the ATM-kinase activity in VA13 cells reducedoxLDL-induced expression of immunoreactive p21 to baseline lev-els.

In the series of experiments, we have tested whether oxLDL-mediated expression of pATM and subsequent induction of p21 isalso operative in cells other than fibroblast. These data indicate thatinduction of pATM by oxLDL in endothelial cells occurs in a time-dependent manner (Fig. 10A) similar as found in VA13 fibroblasts(Fig. 1A); densitometric evaluation of immunoreactive pATM bandsrevealed a 1.7-fold induction after 90 min (Fig. 10A). Furthermore,

pre-incubation of endothelial cells with ATM-I did not only inhibitphosphorylation of the ATM-kinase (Fig. 10B) but also down regu-lated time-dependent expression of p21 (Fig. 10C) as well as colonyformation of oxLDL-treated cells (Fig. 10D).

854 M. Semlitsch et al. / DNA Repair 10 (2011) 848– 860

Fig. 4. Time-dependent effect of lipoproteins on cell viability of normal and ATM-deficient fibroblasts using the Trypan blue exclusion assay: (A) AT22 and VA13 cells werecultured in the presence of 100 �g/ml lipoprotein at indicated times. Cells were stained with Trypan blue and viable cells were counted. Each time point represents the meanvalue ± SD of three experiments performed in triplicates. (B) Fluorescence images of Hoechst 33258-stained VA13 and AT22 cells after treatment with 100 �g/ml lipoproteinfor 16 h. Untreated cells were used as controls. Aggregated and fragmented chromatin appears as bright areas. One representative experiment out of four is shown.

Fig. 5. OxLDL induces phosphorylation of H2AX in ATM-deficient fibroblasts: AT22 and VA13 cells were treated (A) with 100 �g/ml oxLDL for 16 h or (B) with 100 �g/mllipoprotein at indicated times. VA13 cells were treated with 1 �M staurosporine (St) for 4 h to induce phosphorylation of H2AX and apoptosis. Cells were lysed and proteinlysates were subjected to Western Blot analysis following SDS-PAGE. Polyclonal anti-�-H2AX was used as primary antibody. Membranes were then stripped and incubatedwith anti-PARP and anti-caspase-3 antibody as primary antibodies. �-Actin served as a loading control.

M. Semlitsch et al. / DNA Repair 10 (2011) 848– 860 855

Fig. 6. OxLDL promotes formation of micronuclei and chromosomal breaks in AT22 fibroblasts: (A) VA13 and AT22 cells were incubated with 100 �g/ml lipoprotein for 16 h.Cells were fixed with methanol and stained with Hoechst 33285. Numbers of micronuclei/500 cells were counted using a fluorescence microscope. The arrow indicates a typicalmicronucleus formation in AT22 cells. Data are expressed as percentage of untreated cells and represent the means ± SD (n = 6). (B) VA13 and AT22 cells were incubatedw osomo treatc

4

gmaiggmtclotambmcobsaTsamt

ith 30 �g/ml lipoprotein for 8 h. Metaphase preparations were made and chrombservations performed under oil immersion microscopy done in triplicate. UT (unells).

. Discussion

A-T, an autosomal recessive disorder resulting from ATMene mutation, is characterized by a high incidence of lymphoidalignancies, neurodegeneration, immunodeficiency, premature

ging, elevated radiosensitivity, and genomic instability. Genomicnstability is characterized by chromosome breaks, chromosomeaps, translocations, and aneuploidy [42,49]. Recent findings sug-ested that DNA damage links mitochondrial dysfunction to theetabolic syndrome and atherosclerosis, indicating that preven-

ion of mitochondrial dysfunction may represent a novel target ofardiovascular disease [23]. Basically, mitochondrial dysfunction isinked to ATM-heterozygosity [50,51] and results in an imbalancef ROS [23]. As ROS levels are tightly coupled with inflamma-ory diseases e.g. atherosclerosis, increased ROS levels in ATM−/−

nd ATM+/− cells may be due to alterations in cellular defenceechanisms and possibly due to cellular dysfunction induced

y modified/oxidized (lipo)proteins. Among different lipoproteinodifications, the oxidation of LDL by transition metals such as

opper ions represents a suitable experimental approach to mimicxidative modifications of LDL in vivo [19,25,52,53]. OxLDL haseen reported to participate in the development of atherosclero-is primarily by promoting vascular cell growth [54–57]. OxLDL is

potent proinflammatory chemoattractant for macrophages and-lymphocytes. OxLDL is also cytotoxic for endothelial cells and

timulates them to release soluble inflammatory molecules. Inddition, oxLDL has turned out to be highly immunogenic and pro-otes changes in cell cycle protein expression, and subsequent

ranslocation and activation of transcription factors [58,59]. These

al breaks were counted. Each data point represents 5000 individual chromosomeed cells). *p < 0.05 and ***p < 0.001 (significant difference between VA13 and AT22

events help to perpetuate a cycle of vascular inflammation andlipid/(lipo)protein dysregulation within the artery wall and alsomay create a cellular pro-thrombotic state that complicates laterstages of atherosclerosis [59].

In the present study, we demonstrated that oxLDL, known togenerate oxidative stress in the vascular system [19,25], inducedphosphorylation of ATM and downstream activation of p21 infibroblasts and endothelial cells. The immunoreactive pATM sig-nal induced by oxLDL was almost comparable to levels inducedby H2O2. ATM-deficient cells are extremely sensitive to the toxiceffects of H2O2, nitric oxide radical, and t-butyl hydroperoxide,respectively [38,60]. To obtain information on sensitivity of ATM-null fibroblasts to oxLDL, several different cytotoxicity assays wereemployed (Figs. 2–4). All three assays demonstrated that in com-parison to wild-type cells, ATM-deficient fibroblasts are moresensitive to oxLDL treatment – indicating that ATM-expressionlessens oxLDL-mediated toxicity. However, fibroblasts lacking ATMwere more sensitive to oxLDL-treatment in the colony-formingassay, than was observed in the short-term culture assays (MTT-and Trypan blue exclusion assay). This is probably due to defec-tive cell cycle response in A-T cells, as these cells may replicatetheir DNA despite having unrepaired DNA breaks. Both, the MTTand the Trypan blue exclusion assay, and the appearance of con-densed chromatin, demonstrated that oxLDL exhibited mild toxiceffects on VA13 cells, with PARP cleavage and caspase 3 activa-

tion not being detected. We assume that due to the mild toxiceffects of oxLDL in normal fibroblasts, ATM induction triggersan activation of cell cycle checkpoints and not apoptotic cascadeactivation.

856 M. Semlitsch et al. / DNA Repair 10 (2011) 848– 860

Fig. 7. Time- and concentration-dependent intracellular ROS levels in normal and ATM-deficient fibroblasts: (A) To measure basal intracellular ROS levels, AT223 and VA13cells were loaded for 30 min with 10 �M carboxy-H2DCFDA in the absence of agonists. The DCF fluorescence counts detected with VA13 cells were set 100%. (B) Cells wereincubated with 100 �g/ml lipoprotein for indicated times and ROS levels were measured as described above. The DCF fluorescence counts in untreated cells were set 100%.(C) Cells were incubated with indicated concentrations of oxLDL for 5 h before ROS measurements. The DCF fluorescence counts in untreated cells were set 100%. (D) Cellswere incubated with 100 �g/ml lipoprotein for 12 h and loaded with 10 �M carboxy-H2DCFDA for 30 min. DCF fluorescence was observed under fluorescence microscope.(E) Cells were pre-treated with 10 �M ATM-I for 1 h prior to incubation with 100 �g/ml lipoprotein for 5 h. Then ROS levels were measured. The DCF fluorescence counts inuntreated cells were set 100%. (A, B, C, and E) DCF fluorescence values were normalized to cell protein content. Data are expressed as mean ± SD (n = 6). UT (untreated cells).**p < 0.01 and ***p < 0.001 (significant difference between VA13 and AT22 cells).

M. Semlitsch et al. / DNA Repair 10 (2011) 848– 860 857

Fig. 8. PDTC protects against ROS-generation induced by oxLDL in ATM-deficient fibroblasts: (A) AT22 and VA13 cells were incubated with PDTC for 1 h and treated with100 �g/ml oxLDL for 5 h. Levels of endogenous ROS were calculated by measuring DCF fluorescence. Results are expressed as fluorescence intensity as % of control andrepresent the means ± SD (n = 6) ***p < 0.001. (B) Fluorescence microscopy of AT22 and VA13 cells stained with carboxy-H2DCFDA. Cells were pre-treated with 1 mM PDTCfor 1 h and incubated with 100 �g/ml oxLDL for 5 h.

Fig. 9. ATM-kinase-dependent activation of p21 in VA13 fibroblasts by oxLDL: AT22 and VA13 cells were incubated with 100 �g/ml oxLDL at indicated times in the absenceor presence of 10 �M ATM-I (preincubated for 1 h). Cell lysates were subjected to SDS-PAGE, proteins were transferred to nitrocellulose and membranes were incubatedwith anti-p21 Waf1/Cip1 as the primary antibody. �-Actin was used as loading control. One representative experiment out of two is shown.

Fig. 10. ATM-kinase activation and induction of p21 in endothelial cells by oxLDL: EA.hy926 cells were incubated with 100 �g/ml oxLDL (A, B, C) or LDL (B) at indicatedtimes or for 90 min in the absence or presence of 10 �M (C) or 10 and 30 �M (B) ATM-I (preincubated for 1 h). As positive controls, cells were treated with 200 �M H2O2 for15 min; after medium exchange the cells were incubated for another 90 min (105*) (A). Cell lysates were subjected to SDS-PAGE, proteins were transferred to nitrocelluloseand membranes were incubated with anti-pATM (A, B) or anti-p21 (C) as primary antibody. �-Tubulin (A, B) or �-actin (C) was used as loading control. One representativeexperiment out of two is shown. (D) Colony forming efficiency of EA.hy926 cells treated with 30 �g/ml lipoprotein in the absence or presence of 10 �M ATM-I was performedexactly as described in Fig. 2B. Data are given as number of colonies (as percentage compared to untreated cells, set as 100%). Values represent the means ± SD (n = 3). UT(untreated cells). **p < 0.01 and ***p < 0.001.

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OxLDL-mediated toxicity was significantly higher in ATM-eficient fibroblasts. We assume that these cells (defective in G1,

and G2 checkpoint functions [16,61]) are unable to responddequately to oxLDL-induced oxidative stress and/or DNA dam-ge. The result is oxLDL hypersensitivity and eventual cell death.o confirm this hypothesis the effect of oxLDL on DNA damageas investigated. A very early step in the response to DNA-DSBs

s the appearance of immunoreactive �-H2AX [44]. �-H2AX isn essential component for the recruitment and accumulation ofNA repair proteins to sites of DSB damage, including 53BP1,RCA1, RAD51 and MDC1 and the MRE11/RAD50/NBS1 complex62]. In the presence of DNA-DSBs, H2AX is rapidly phospory-ated by ATM [63]. However, H2AX can also be phosphorylated byther members of the phosphatidylinositol 3-kinase family, includ-ng DNA-dependent protein kinase and the ATM-and Rad3-relatedrotein kinase [64,65]. We found that following oxLDL exposure

mmunoreactive �-H2AX was present only in ATM-deficient AT22,ut not in VA13 cells. As oxLDL leads to ATM-phosphorylation inA13 cells, this data indicates that ATM is activated by oxLDL in thebsence of DNA-DSBs. ATM is a key player in DSBs responses, beingctivated by these breaks and phosphorylating key down-streamroteins, leading to cell cycle checkpoint arrest and/or apoptosis66]. However, lack of ATM causes not only a defective responseo DNA-DSBs, but also a defect in regulating cellular responses toxidative stress [7,17]. Our findings are consistent with a recenttudy [17], demonstrating that ATM activation induced by H2O2ccurs in the absence of DNA damage. The observation that oxLDL-ependent H2AX phosphorylation was only observed in ATM−/−

ells suggested that another member of the phosphatidylinositol-kinase family is likely to be involved in this pathway. Further-ore, the appearance of �-H2AX in ATM-deficient cells makes it

easonable to assume that ATM protects against oxLDL-inductionf DNA-DSBs. Increased formation of micronuclei and a higherumber of chromosomal breaks in oxLDL-treated AT22 cells (asompared to VA13 cells) gives further support to this hypothesis.

Accumulating evidence suggests that oxidative stress isnvolved in the pathogenesis of A-T. Loss of ATM leads to increasedxidative damage to proteins and lipids and many cell-types, suchs bone marrow stem cells and thymocytes of mice, exhibit elevatedevels of ROS [7,67–69]. In line with these observations, we detectedncreased basal levels of ROS in ATM-deficient fibroblasts. Treat-

ent with oxLDL further amplified ROS formation in ATM-deficientnd normal fibroblasts. Also, oxLDL-induced ROS formation wasignificantly higher in ATM-deficient AT22 cells and in response toharmacological inhibition of ATM in VA13 cells. This indicates thatTM protects from oxLDL-induced intracellular ROS productionnd that ATM expression may play a critical role in cell function andurvival in atherosclerosis. Most importantly, cellular and molecu-ar responses of fibroblasts from atherosclerosis patients towardsonizing radiation, activating the ATM-stress response, are similaro those observed from cells obtained from A-T patients [70]. ThexLDL-induced elevation of ROS, but no signs of DNA damage, inormal fibroblasts, confirmed the hypothesis, that not DNA-DSBsut ROS triggers oxLDL-induced activation of ATM. These obser-ations parallel recent data [71] where ROS potently and rapidlyctivates ATM in the cytoplasm suggesting that mechanisms otherhan DNA-DSBs in the nucleus are operative to promote activationf ATM.

Administration of antioxidants to Atm−/− mice exhibited aariety of beneficial effects, including extended lifespan, reducedumorigenesis and improvement of motor deficits [13,14,72].re-treatment of ATM-deficient cells with N-acetyl-l-cysteine

ttenuated ROS formation and blocked activation of ATM15,17,67]. Due to redox cycling, N-acetyl-l-cysteine is able toeduce Cu2+- to Cu+-ions that can promote metal-catalyzed lipideroxidation in vitro [73]. However, we here used PDTC to scav-

[

[

ir 10 (2011) 848– 860

enge oxLDL-induced formation of ROS. PDTC induces glutathionesynthesis in endothelial cells and suppresses the activation of tran-scription factor nuclear factor �B [73,74]. Most importantly, PDTCpossesses metal-chelating properties and therefore, generation offree Cu2+-ions, recently reported to activate ATM in murine neu-roblastoma cells and human HeLa cells [75], can be excluded underour experimental conditions. Here we show that PDTC is able todiminish formation of intracellular ROS induced by oxLDL in both,normal and ATM-deficient cells almost to basal levels.

In conclusion, we demonstrated that ATM is involved in oxLDL-mediated signalling. OxLDL-mediated activation of ATM occurs viaintracellular formation of ROS and not via induction of DNA-DSBs.We propose that under conditions of ATM deficiency, oxLDL-dependent ROS production induces DNA damage chromosomalinstability and cell death. As a consequence, H2AX, required for therepair mechanisms of ROS-induced DNA damage in ATM-deficientcells [76], is phosphorylated. Furthermore, we demonstrated thatPDTC acts as a potent antioxidant against oxLDL-induced ROSformation. Our data enforce the role of ATM as a sensor ofoxidative stress that could be important for protection againstoxLDL-mediated cellular toxicity. Therefore, the ability of oxLDLto activate the ATM pathway may represent a critical adaptiveresponse to maintain cell viability at sites of vascular inflammationand atherosclerosis.

Conflict of interest

The authors state that there is no conflict of interest.

Acknowledgments

AT22 cells were kindly provided by Dr. Yosef Shiloh (SacklerSchool of Medicine, Tel Aviv University, Tel Aviv, Israel). The Aus-trian Science Fund (FWF) provided financial support (P19074-B05and F3007). R.S. was supported by a Bank Austria Visiting ScientistsProgram provided to the Medical University of Graz, Austria. Theauthors are grateful to Drs. M. Vadon and S. Sipurzynski (MedicalUniversity of Graz) for providing human plasma.

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