vilasová_et_al

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Regular paper

Changes in phosphorylation of histone H2A.X and p53 in response ofperipheral blood lymphocytes to gamma irradiation

Zdeka Vilasov1, Martina ezov2, Jiina Vvrov1, Ale Tich1,Doris Vokurkov2,Friedo Zoelzer3, Zuzana ehkov1, Jan Osterreicher1 and Emilie Lukov4

1University of Defence, Faculty of Military Health Sciences, Department of Radiobiology, Hradec Krlov, CzechRepublic; 2Charles University in Prague, Faculty of Medicine in Hradec Krlov, Department of Biochemistry,Hradec Krlov, Czech Republic; 3South Bohemian University, Department of Radiology and Toxicology, esk

Budjovice, Czech Republic; 4Czech Academy of Sciences, Institute of Biophysics, Brno, Czech Republic

Received: 08 January, 2008; revised: 27 May, 2008; accepted: 12 June, 2008available on-line: 15 June, 2008

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phy-hmagguinin (PHA)-simuad, human priphra bd mnnucar cs (PBMCs) -ra -diation, and analyse changes of proteins related to repair of DNA damage and apoptosis, suchas H2A.X, p53, p53 phsphryain a srins-15 and -392, and p21 and hir ds dpndnc.Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G 0 phase, and withvry w amuns f prins p53 and p21. Using cnfca micrscpy w dcd ds dpnd-n (0.55 Gy) inducin f fci cnaining H2A.X (1 h ar -ray xpsur), which ar frmdarund radiain-inducd dub srand braks f DNA. Appsis was dcd frm 24 h arirradiain by h ds f 4 Gy nwards by Annxin V binding and amin B cavag. Svnyw hurs ar irradiain 70% f CD3+ lymphocytes were A+. Neither increase in p53 nor itsphsphryain n srin-392 ar irradiain was dcd in hs cs. Hwvr, massiv in-cras in p21 (cycin-dpndn kinas inhibir 1A) was dcd ar irradiain, which can brspnsib fr a ccurrnc f appsis in hs quiscn cs. PHA-simuain isf (72 h)caused an increase in early apoptosis (A+PI) in cmparisn nn-simuad PBMCs (38% A+rsp. 13.4%). Ar PHA-simuain as h amun f H2A.X, p53, and p21 incrasd, bu nphsphryain f p53 n srin-392 r -15 was dcd. Racin -radiain was dirnin PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated onsrins-15 and -392 4 h ar irradiain by h ds f 4 Gy. Phsphryain f p53 a srin-15incrasd in a ds-dpndn mannr in h sudid ds rang 0.27.5 Gy. As h amunf p21 incrasd ar irradiain. Svny w hurs ar irradiain f PHA-simuad CD3+ T

ymphcys by h ds f 4 Gy 65% f cs wr A+.

Keywords:lymphocyte, ionizing radiation, p53, phytohemagglutinin (PHA), apoptosis, DNA damage

INtRoDUCtIoN

In vitro and in vivo irradiation of peripheralblood mononuclear cells (PBMC) by -irradiation in-duces programmed cell death apoptosis (Hertveldet al., 1997; Philipp et al., 1997; Cornelissen et al.,2002; Vokurkov et al., 2006). This process is timeand dose-dependent. It was demonstrated that in

PBMC isolated from blood of healthy donors and ir-radiated by the dose of 7 Gy apoptosis is not detect-

able within the rst 6 h, but 16 h aer irradiation50% of the cells are Annexin V positive (A+), and 48h aer irradiation almost all cells are A+ (Vokurkovet al., 2006).

The molecular mechanisms of ionizing radia-tion (IR)-induced apoptosis in cells of lymphocytic

Corresponding author: Martina Rezacova, Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove,Simkova 870, 500 01 Hradec Kralove, Czech Republic; tel: (420) 4958 16166; fax: (420) 4955 12715; e-mail: [email protected]: AT, ataxia telangiectasia; ATM, ataxia telangiectasia mutated; A, Annexin V; IR, ionizing radiation; IRIF, ion-izing radiation-induced foci; OD, optical density; IOD, integral optical density; NBS, Nmegen breakage syndrome; PHA,

phytohemagglutinin; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buered saline; TPA, phorbol ester.

Vol. 55 No. 2/2008, 381390

on-line at: www.actabp.pl


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382 2008Z. Vilasov and others

origin were studied in some detail on MOLT-4 cells(T-lymphocytic leukemia) (Szkanderov et al., 2003;2005; Vvrov et al., 2004). Immediately aer DNADSB damage, histone H2A.X is phosphorylated atserine-139 H2A.X (Rogakou et al., 1998; ezovet al., 2008). Bekker-Jensen et al. (2006) described thereaction of U-2-OS cells to IR-induced double strand

breaks (DSB) of DNA, during which accumulation

of many proteins (53BP1, H2A.X, phosphorylatedATM kinase, Nbs1) in IR-induced foci (IRIF) wasdetected. These proteins assemble around DSBs andform optically discernible foci.

One of the initial processes in the response ofeukaryotic cells to IR is the activation of ATM kinase(Bakkenist & Kastan, 2003) by auto-phosphorylationat serine-1981 within minutes aer irradiation, whichwas also detected in MOLT-4 cells (Tich et al.,2007). The product of the ATM gene plays a pivotalrole in sensing DNA damage and consequentially inmodulating cell cycle check points. Activated ATM

kinase phosphorylates a broad range of substratesrelated to radiation damage repair and/or apoptosis.One of its substrates is protein p53, which is post-tranlationally modied in response to IR, includingphosphorylation at serine-15 and -392, within hoursaer irradiation (Szkanderov et al., 2005). Thesemodications prevent Mdm2-mediated degradationof p53 and are probably the main reason for the in-crease in the total amount of p53. Protein p53 acts asa transcriptional factor, which aer interaction withDNA activates transcription of other genes, whichregulate the cell cycle and apoptosis, including p21,a cyclin-dependent kinase inhibitor (Lakin & Jack-son, 1999). In MOLT-4 cells p21 levels increased 4 haer irradiation by the dose of 7.5 Gy (Szkanderovet al., 2003). It is generally accepted that p53 is a keymolecule responsible for cell cycle arrest in the G1phase as a response to DNA damage. Human mono-nuclear cells in peripheral blood are predominantlyquiescent, in the G0 phase of the cell cycle. Fukao etal. (1999) demonstrated that ATM protein is presentat low levels in freshly isolated PBMCs and increas-es approx. 6- to 10-fold in response to a mitogenicstimulus, reaching a maximum aer 3 to 4 days.Increase in ATM in PBMCs is therefore related to

proliferation. The increase in ATM protein was ac-companied by an increase in ATM kinase activity.Freshly isolated PBMCs were also shown to expresslow levels of p53 mRNA that is rapidly degraded(Voelkerding et al., 1995). Progressive stabilizationof the p53 mRNA occurred during growth induction

by incubation in the presence of phytohemagglutinin(PHA) and phorbol ester (TPA), whereby p53 mRNAsteady state levels were increased 4.5 to 5-fold. Ac-cordingly, protein p53 was not detected in quiescentlymphocytes, but was detected in lymphocytes stim-ulated with PHA (Voelkerding et al., 1995).

It remains to be claried whether H2A.X ismainly involved in ATM mediated DNA DSB dam-age response (Fernandes-Capetillo et al., 2002) orwhether it is also involved in recruitment of 53BP1to sites of DNA DSB damage. Kang et al. (2005) stud-ied interactions among H2A.X, Nbs1 and p53. Theydemonstrated that Nbs1, and not H2A.X is responsi-

ble for ATM-dependent suppression of interchromo-

somal rearrangement, which implies dierent rolesof Nbs1 and H2A.X in DNA damage response. H2A.X functions mainly as a downstream mediator ofATM kinase, in a pathway parallel to the p53 path-way. Nbs1 seems to have two major roles: ATM ac-tivator and an adapter to mediate ATM activity aerDNA DSB damage.

The main aim of this study was to comparethe reaction of quiescent and proliferating PHA-stimulated PBMCs to -radiation and analyse chang-es of proteins related to repair of DNA damage andapoptosis, such as H2A.X, p53 and phosphoryla-

tion of p53 at serine-15 and -392, and of p21. In thispaper we demonstrate that even aer irradiation thelevel of protein p53 (and its phosphorylation) are

below detection limits of western blot analysis inquiescent PBMCs, but p53 responds to -radiationin proliferating PHA-stimulated lymphocytes. PHA-stimulation alone causes an increase in H2A.X andp21. These proteins increase aer IR in both quies-cent and PHA-stimulated PBMCs, but with dierentdynamics. We also assess possible biodosimetric ap-plications of these parameters.

MAteRIAl AND MetHoDS

PBMCs and culture conditions. Peripheralblood mononuclear cells were isolated from volun-teers heparinized blood by centrifugation on theHistopaque-1077 (Sigma) cushion according to themanufacturers instructions. Aer washing twicewith cold PBS the cells were resuspended in Iscovesmodied Dulbeccos medium (Sigma) supplementedwith 20% fetal bovine serum (PAA LaboratoriesGmbH, Austria), 2 mmol/l glutamine (Sigma), 100UI/ml penicillin (Sigma) and 0.1 mg/ml streptomy-

cin (Sigma) at the density of 5 105 cells/ml. Thecell suspension was maintained in an incubator(37oC, humidied atmosphere with 5% CO

2) till ir-

radiation (23 h). For preparation of PHA-stimulatedlymphoblasts, PHA (PHA-P, Sigma) was added at aconcentration of 10 g/ml and PBMCs were incubat-ed 72 h in an incubator under the same conditionsas unstimulated PBMCs.

In vitro irradiation. Cells in 10 ml aliquotswere transferred into 25 cm2 asks (Nunc) and ir-radiated at room temp. using a 60Co gamma X-raysource. Doses below 10 Gy were delivered with a


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Vol. 55 383p53 pathway in irradiated quiescent and proliferating lymphocytes

dose-rate of 0.4 Gy/min at a distance of 1 m fromthe source, the dose of 10 Gy with 1.5 Gy/min dose-rate and a distance of 0.5 m from the source. Im-mediately aer irradiation the asks were placed ina 37oC incubator with 5% CO

2and aliquots of cells

were collected at various time intervals aer irradia-tion for analysis.

Cell cycle analysis. Aer dierent times of

incubation, PBMCs were washed with cold PBSand xed with 70% ethanol. For the detection oflow molecular-weight fragments of DNA, the cellswere incubated for 5 min at room temp. in buer(192 ml 0.2 mol/l Na

2HPO

4+ 8 ml 0.1 mol/l citric

acid, pH 7.8) and then stained with propidiumiodide (PI) in Vindelovs solution for 60 min at37oC. The DNA content was determined on owcytometer Cytomics FC500 (Beckman Coulter, Hi-aleah, FL, USA) using a 15 mW argon-ion laser for488 nm excitation; the total emission above 560nm was recorded. List mode data were analyzed

using Multicycle AV soware (Phoenix Flow Sys-tems, Inc., San Diego, CA, USA). A minimum ofthree independent measurements were performed.

CD25 xprssin. Expression of CD25 wasdetermined by anti-CD25-PE (clone 33B3.1, Beck-man Coulter). Flow cytometric analysis was per-formed on a Coulter Epics XL ow cytometerequipped with a 15 mW argon-ion laser for 488nm excitation (Coulter Electronic, Hialeah, FL,USA). A minimum of 10 000 cells were collectedfor each 2-colour sample in a list mode le format.List mode data was analyzed using Epics XL Sys-tem II soware (Coulter Electronic, Hialeah, FL,USA).

thr-cr w cymry. PBMCs werewashed in ice-cold washing and staining buer(WSB, PBS containing 0.2% gelatin and 0.1% NaN

3)

and kept in the cold during subsequent processingto prevent further apoptosis development. For vis-ualization of the T lymphocyte subset surface im-munostaining using the mouse anti-human CD3/PB (clone UCHT1, DakoCytomation) was chosen.Aer 15 min incubation and a single wash cycle (2ml WSB, centrifugation, vortexing) the cells weretreated with uorescein isothiocyanate-conjugated

Annexin V (A-FITC) and propidium iodide (PI)from the Apoptest kit (DakoCytomation) accordingto the manufacturers instructions. Flow cytom-etry (FCM) was performed on a nine-color CyAn(DakoCytomation) ow cytometer. A minimum of50 000 events were analyzed in each sample andthe results were evaluated using the Summit v.4.3soware. Too small objects including cell debrisand the majority of non-adherent monocytes wereexcluded from analysis by a standard approachusing the lymphogate region in the forward andside (right angle) scaer characteristics (FSC and

SSC, respectively). Lymphogate was carefully setup to include smaller cells in the later stages ofapoptosis into the analyzed population.

Wsrn bing. PBMCs were washed witha PBS at various times aer irradiation. The lysatescontaining an equal amount of protein (30 g) wereloaded onto dierent lanes of a polyacrylamide gel.Aer electrophoresis, the separated proteins were

transferred to a PVDF membrane. The membraneswere blocked in Tris-buered saline containing0.05% Tween 20 and 5% non-fat dry milk and thenincubated with a primary antibody (p53 and phos-phorylated p53 (serine-392) from Exbio, p21 and

H2AX from Cell Signaling) at 4C overnight. Aerwashing, the blots were incubated with a second-ary antibody (DakoCytomation) and the signal wasdeveloped with a chemiluminiscence detection kit(Boehringer).

elISA. For quantitative assay of p53 andphosphorylated p53 (serine-15) PathScan Sandwich

ELISA kits by Cell Signaling Technology were used.The optical density (OD) measured at 450 nm wasploed against dose D (in Gy) and the resultingcurve turned out to be of sigmoidal shape compat-ible with MichaelisMenten kinetics

OD = a + b D / (c + D)

This was checked with the help of a Lineweav-erBurk plot, showing direct proportionality between1/D and 1/OD (Lineweaver & Burk, 1934).

Immunocytochemistry. The cells were xedwith 4% freshly prepared paraformaldehyde for

10 min at room temp., rinsed quickly in PBS, thenwashed 3 5 min in PBS, permeabilized in 0.2% Tri-ton X-100/PBS for 15 min at room temp., rinsed inPBS and washed 2 5 min. Before incubation withprimary antibodies (overnight at 4oC), the cells were

blocked with 7% inactivated FCS + 2% bovine se-rum albumin in PBS for 30 min at room temp. Fordetection of H2A.X mouse monoclonal anti-phos-pho-Histone H2A.X (Upstate) was used. Secondaryantibody: Anity pure donkey anti-mouse-FITC-conjugated and anity pure donkey anti-rabbit-Cy3-conjugated were purchased from Jackson Laboratory

(Bar Harbor, ME, USA). Antibodies were applied toeach slide (aer their pre-incubation with 5.5% don-key serum in PBS for 30 min at room temp.) andincubation for 1 h in the dark followed by washing(3 5 min) in PBS. Counterstaining was performed

by TOPRO-3 (Molecular Probes, Eugene, OR, USA)in saline sodium citrate prepared fresh from thestock solution.

Images were obtained on a high-resolutionconfocal cytometer based on a completely automatedLeica DM RXA uorescence microscope equippedwith CSU-10a confocal unit (Yokogawa, Japan) and


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a CoolSnap HQ charged-coupled device camera(Photometrix, Melbourne, Australia). Forty opticalsections of 0.3 m were acquired for each nucleusand stored in the computer memory. The expositiontime and dynamic range of camera in the red, greenand blue channels were adjusted to the same valuesfor all slides to obtain quantitatively comparable im-ages. Integral optical density (IOD) was measured

using image analysis soware ImagePro 4.11 (Me-diaCybernetics, USA).

ReSUltS

Simuain by PHA

The reaction of PBMCs to ionizing radiationwas evaluated using non-stimulated and PHA-stim-ulated cells. PBMCs circulating in peripheral blood

are predominantly present in G0 phase of cell cycle,in our case only 0.7% of the cells were in S phase(Fig. 1A, B). The cells did not express CD25 antigen,i.e. the alpha chain of the receptor for interleukin-2(Fig. 1C). Long stimulation (72 h) by 10 g/ml PHAcaused activation of T-lymphocytes and inducedCD25 on almost all cells (Fig. 1C). DNA contentanalysis revealed an increase in the percentage of

cells in S phase, with a maximum 48 h aer the be-ginning of stimulation, when 12.2% of the cells werein S phase (Fig. 1C).

Apoptosis induction

Apoptosis induction in T-lymphocytes (CD3+)was analysed by FCM 24 and 72 h aer the irradia-

tion by the dose of 4 Gy. The eect of irradiationon non-stimulated and PHA-stimulated PBMC wascompared. Separated PBMC were cultivated for 24h and subsequently irradiated by the dose of 4 Gy.PHA-stimulated lymphocytes were incubated withPHA for 72 h and subsequently irradiated by thedose of 4 Gy. In both groups apoptosis inductionwas evaluated 24 and 72 h aer irradiation. Figure 2shows representative results of FCM analysis of ra-diation-induced apoptosis in CD3+ lymphocytes us-ing dual staining by Annexin V (A) and propidiumiodide (PI). This method allows detection of live

cells (A

PI

), early apoptotic cells (A+

PI

), and lateapoptotic cells (A+PI+).Table 1 summarizes mean values of radia-

tion-induced apoptosis in PBMC samples obtainedfrom three dierent blood donors. It is apparent that72 h-long stimulation causes increase in early ap-optotic cells (37.94% A+PI versus 16.71% in control).Irradiation by the dose of 4 Gy increases A+ cells

Figur 1. Simuain f ymphcys byPHA.A, B. Dynamics of cell cycle changes in-duced in PBMCs by in vitro stimulationby 10 g/ml PHA. The amount of DNAin each cell was determined aer PIstaining by ow-cytometric analysis. ThePBMCs entered the cell cycle and thenumber of cells in the S and G

2phases

increased aer PHA-stimulation. C. Ex-pression of CD25 antigen and forwardscaer (corresponding to cell size) analy-sis of T-lymphocytes (CD3+) aer 72 hof stimulation by PHA. While only 3.5%of control T-lymphocytes are CD25+,this fraction increased to 95.6% aer thestimulation by PHA.


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to 37.51% 24 h aer the irradiation and further to69.68% 72 h aer the irradiation. The majority of the

apoptotic cells were A

+

PI

cells, i.e. early apoptotic.Also in the PHA-stimulated group irradiation by thedose of 4 Gy caused an increase in apoptotic cells to70.64% (24 h aer irradiation) and 65.22% (72 h aerirradiation)

Detection of ionizing radiation induced foci

At the site of DSB in the DNA, protein com-plexes are assembled, which form optically discern-ible foci IRIF. These sites can be identied bydetection of the phosphorylated form of histoneH2A.X by an immunocytochemical method on

confocal microscope. We identied H2A.X foci inquiescent PBMCs 1 h aer irradiation by the doses

0.510 Gy (Fig. 3A). The intensity of the phospho-rylation in irradiated PMBCs is such that it seemsto have a potential as a biodosimetric marker: theintegral optical density was measured using imageanalysis (Fig. 3B). In the dose range 0.55 Gy a lin-ear dose-dependence was observed.

Prin changs inducd by radiain ar dirn

in quiescent and stimulated PBMCs

We analyzed changes in proteins related toDNA damage repair and apoptosis using the west-ern blot method in quiescent and stimulated PBMCs.The cells were irradiated by a dose of 4 Gy and thedynamics of the process were studied.

In quiescent lymphocytes, H2A.X was notdetected early aer irradiation (1 and 4 h), but in-creased at 2472 h. This increase in H2A.X detect-able by western blot occurred during apoptosis,which was conrmed by caspase cleavage of lam-in B. We also detected an increase in cell cycle regu-

lator p21 2472 h aer irradiation. This increase wasnot accompanied by a detectable increase in p53 or

by p53 phosphorylation (Fig. 4A).In PHA stimulated cells slight

phosphorylation of H2A.X was de-tected even without irradiation.The phosphorylation increasedfrom 1 h aer the irradiation on-wards, with a maximum at 24 h.PHA stimulation caused a signi-cant increase in the p53 amount incomparison with quiescent PBMCs,

Figur 2. Appsis inducin 24 h ar irradiain by adose of 4 Gy in quiescent and PHA-stimulated PBMCs.Representative results of multiparameter ow cytomet-ric analysis of in vitro irradiated (4 Gy) and subsequentlycultivated (24 h) human PBMC stained with anti-CD3/PBmonoclonal antibody and counterstained with ApoptestTM.A. control CD3+ lymphocytes; B. irradiated CD3+ lym-phocytes; C. CD3+ lymphocytes aer 96 h stimulation withPHA; D. CD3+ lymphocytes aer 96 h stimulation withPHA and 24 h aer irradiation.

tab 1. Appsis inducin 24 h ar irradiain by h ds f 4 Gy inquiescent and PHA-stimulated PBMCs

Nonstimulated ly PHA stimulated lyA+ A+ PI A+ A+ PI

24 h C 16.71 4.53 13.44 4.55 42.99 4.97 37.94 6.574Gy 37.51 3.20 27.24 2.32 70.64 11.57 54.24 4.42

72 h C 22.97 7.76 15.25 5.72 51.46 5.61 41.34 0.954Gy 69.68 7.95 40.28 2.39 65.22 12.66 49.13 10.41

Figure 3. Phosphorylation of histone H2A.X on serine-139 in quiscn PBMCs 1 h ar irradiain.A. Examples of nuclei of control (C) and irradiated PB-MCs (dose 1, 2, 5 and 10 Gy). One hour aer irradiationthe cells were xed and immunostained with antibodyagainst H2A.X. Cell nuclei were counterstained by TO-PRO3, and are marked by solid line. B. This graph sum-marizes quantication of the intensity of green (H2A.X-FITC) uorescence measured as integral optical density ofeach cell. At least 50 cells were analyzed in each sample.Median values and 1st and 3rd quartile are indicated.


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but this p53 was not phosphorylated. Further upreg-ulation of p53 was detected 172 h aer irradiation.From 4 h aer irradiation onwards, phosphorylationof p53 at serine-392 was detected. Furthermore, pro-tein p21 was induced in non-irradiated PHA stimu-lated cells and its amount increased aer irradiation,with a maximum increase at 48 h (Fig. 4B).

Dose-dependence of radiation-induced changes inPHA-stimulated cells

Dose-dependence of changes in H2A.X, p53,p53 phosphorylated at serine-392, and p21 in PHA-

stimulated lymphocytes were evaluated 4 h aerirradiation by western blot analysis. Both H2A.X and p21, but not p53 and p53 phosphorylation atserine-392 showed a dose-dependent increase overthe dose range studied (0.54 Gy) (Fig. 5).

A very sensitive method for quantitativemeasurements of p53 and p53 phosphorylated atserine-15 is the so-called sandwich ELISA. Figure 5

shows a dose-dependent increase of p53 phospho-rylation at serine-15 4 h aer irradiation over thedose range 0.57.5 Gy by using western blot andELISA methods. We also measured the amount oftotal p53, but similar to our ndings with the west-ern blot analysis, no dose-dependence was observed(Fig. 6).

DISCUSSIoN

Many studies (Schultz et al., 2000; Sti et al.,2004; Bekker-Jensen et al., 2006) have shown that

one of the earliest responses of eukaryotic cells toDSB in the DNA caused by IR is the phosphoryla-tion of histone H2A.X at serine-139. This modica-tion occurs in DSB anking chromatin within min-utes aer irradiation. At the same time various oth-er proteins are recruited to these sites, forming in-tranuclear ionizing radiation induced foci (IRIF).The most important function of IRIF is repair ofradiation-induced DNA damage. A signicant de-crease in the number of initially formed IRIF detect-ed by immunocytochemical evaluation of live cellsis apparent during 24 haer irradiation. The initial

Figur 4. Cmparisn f changs in H2A.X, p53, p53 phsphryad a srin-392, p21 vs and appsis during72 h ar irradiain by a ds f 4 Gy in quiscn and PHA-simuad PBMCs.A. Freshly isolated quiescent PBMCs. The cells were isolated from peripheral blood, irradiated by the dose of 4 Gy andthen incubated for 172 h. MOLT-4 cells (4 h aer the irradiation by the dose of 4 Gy) were used as positive control forp53 detection. B. PHA-stimulated proliferating PBMCs. Isolated PBMCs were stimulated with PHA (72 h, 10 g/ml),irradiated by the dose of 4 Gy and then incubated for 172 h. Representative western blots are shown. The cells werelysed and analysed at the time intervals indicated. Aer irradiation, the level of protein p53 (and its phosphorylation)was below detection limits of western blot analysis at quiescent PBMCs, but p53 responded to -radiation in proliferat-ing PHA-stimulated PBMCs. PHA-stimulation also caused an increase in H2A.X and p21. These proteins increase aerIR in both quiescent and PHA-stimulated PBMCs, but with dierent dynamic. Apoptosis was detected by analysis oflamin B cleavage a 68-kDa nuclear protein is cleaved into a 48-kDa fragment by active caspases when apoptosis isinduced. -Actin was used as a loading control.

Figur 5. Ds dpndnc f changs in H2A.X, p53,p53 phsphryad a srin-392, and p21 4 h ar irra -diation of PHA-stimulated PBMCs.Isolated PBMCs were stimulated with PHA (72 h, 10 g/ml), irradiated with the indicated dose, incubated for 4 h,lysed and analyzed by western blot. Representative west-ern blots are shown. A dose-dependent response was ob-served in the dose range 0.54 Gy for the phosphorylationof H2A.X at serine-139 and for the increase in p21. Nei-ther p53 nor p53 phosphorylated at serine-392 exhibited adose-dependent response. -Actin was used as a loadingcontrol.


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increase in H2A.X phosphorylation is dependentupon ATM kinase activation (Banth et al., 2004).ATM kinase is activated quickly aer DNA damageand triggers down-stream pathways of DNA repairand/or cell death. Among the proteins detected inIRIF are 53BP1, Nbs1, Mre11, Rad50, BRCA1, Mdc1and ATM (Bekker-Jensen et al., 2006). In this paperwe have shown that the increase in H2A.X focidetected by confocal microscopy and quantied byimage analysis 1 h aer in vitro irradiation of qui-escent lymphocytes is dose-dependent in the doserange 0.55 Gy. However, using western blot anal-ysis this early increase in H2A.X is not detectablein quiescent lymphocytes. Western blot techniquecan detect H2A.X increase only at later times,when the phosphorylation of H2A.X is related tothe onset of apoptosis (2472 h aer irradiation bythe dose of 4 Gy). These data are in good agree-ment with the recent suggestions by Mukherjee etal. (2006) that H2A.X is phosphorylated also during

DNA fragmentation occurring in the course of ap-optosis. Mukherjee et al. (2006) found that duringapoptosis, H2A.X phosphorylation is catalyzed byDNA-PKcs, while ATM is dispensable for the proc-ess. In our experiments immunocytochemical de-tection of H2A.X by confocal microscopy is moresensitive than western blot analysis, reveals initial

H2A.X related to IRIF formation and is more suit-able for quantication by image analysis.

The level of H2A.X phosphorylation increasedaer stimulation of PBMC by PHA (72 h, 10 g/ml)and as shown here it was detectable by western blotanalysis. The increase in H2A.X that we detected

by western blot 4 h aer irradiation of stimulatedlymphocytes was dose dependent. These data are ingood agreement with those of Hamasaki et al. (2007),who used a ow cytometry system for measurementof IR-induced H2A.X in quiescent and PHA stimu-lated lymphocytes 4 h aer the irradiation. It can beconcluded that measurement of H2A.X during the

Figur 6. Ds dpndnc f changs in p53 and p53 phsphryad a srin-15 vr h ds rang 0.57.5 Gy, 4 har irradiain f PHA-simuad PBMCs.A. Isolated PBMCs were irradiated with the indicated dose, incubated for 4 h, and analysed by ELISA. The optical den-sity OD at 450 nm was ploed against dose D (in Gy) and the resulting curve turned out to be compatible with Michae-lisMenten kinetics. The equation of the ed curve was OD = a + b D/(c + D). The calculated parameters ( + standarddeviation from 6 experiments) were as follows:

a(OD at 0 Gy)

b(maximum OD)

c(D for half-maximum OD)

p53 0.050 + 0.007 0.164 + 0.057 6.4 + 4.6 Gyp53_ser 15 0.034 + 0.006 4.0 + 1.2 75 + 23 Gy

While p53 did not exhibit a dose-dependent increase (as also demonstrated in Fig. 4), the phosphorylation of p53 at ser-ine-15 exhibited a dose-dependence over the whole dose range studied (0.57.5 Gy).B. Isolated PBMCs were irradiated with the indicated dose, incubated for 4 h, lysed and analyzed by western blot. Rep-resentative western blots are shown. A dose-dependent response was observed in the dose range 0.57.5 Gy for thephosphorylation of p53 at serine-15.


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rst hours aer the irradiation is a good marker ofthe received dose of radiation.

The eect of two dierent genotoxic stres-sors actinomycin (DNA-intercalating agent) andirradiation on T cells in the G0/G1 phase wascompared by Porcedda et al. (2006). They showedthat while actinomycin induces DSB in normalquiescent T lymphocytes and targets them to ap-

optosis, T cells from AT and NBS patients resistactinomycin-induced apoptosis and survive. Fol-lowing irradiation by the dose of 2 Gy quiescentT cells from normal donors form 30 IRIF per cellon the average, but normal T cells resolve virtu-ally all damage within 72 h (average number ofIRIF decreases to 0.5 per cell) and survive. T cellsfrom AT and NBS patients also survive, despitethe maintenance of 4 H2A.X foci per cell aer72 h. They also compared irradiation dose 0.5 Gyand actinomycin in concentration 0.05 g/ml, bothof which induce 8 foci per cell aer 1 h. Twenty

four hours aer exposure to actinomycin the re-sidual damage was 72100%, while aer irradia-tion it was lower, 920%. Although both agentsinduce the same number of foci in the cells, it isquestionable whether the same molecular path-way is triggered by actinomycin and irradiation.Phosphorylation of kinase ATM was signicantlyhigher aer irradiation than aer exposure to ac-tinomycin. On the other hand, actinomycin causeda more intensive and longer increase in p53 andits phosphorylation on serine-15.

When p53 receives information about DNAdamage within the cell, it may arrest the cells ad-vance through its growth- and division-cycle and,at the same time, orchestrate localized responsesto facilitate the repair of damage. However, undercertain conditions, p53 can also emit signals thatawaken the normally latent suicide program ap-optosis (Weinberg, 2007). Increase in p53 was de-tected in many cell types aer exposure to IR, UVradiation, and chemotherapeuticals which damageDNA. Measurement of p53 protein levels indicatedthat they could vary drastically from one cell typeto another (Weinberg, 2007). Generally, increasein p53 is predominantly regulated by blocking its

degradation in proteasomes (Weinberg, 2007). Ir-radiation of leukemic MOLT-4 cells by IR inducesphosphorylation of ATM, which in turn phospho-rylates p53 (which prevents its degradation) andits negative regulator Mdm2 (a ubiquitin ligase)(Tich et al., 2007). While MOLT-4 cells derivedfrom T-lymphocytic leukemia have high levels ofp53 (Szkanderov et al., 2003), we did not detectp53 nor its phosphorylation on serine-392 in qui-escent lymphocytes, not even aer irradiation.This does correspond with ndings of Jones et al.(2004), who did not detect signicant levels of p53

protein nor its phosphorylation in PBMCs irradi-ated by UVC and concluded that UVC-inducedATR-p53 pathway is downregulated in non-cy-cling lymphocytes. Voelkerding et al. (1995) ob-served very low expression of p53 mRNA in G0quiescent lymphocytes, and its rapid degradationwith a half life of 1 h. They also demonstrated thatPHA and TPA stimulation leads to stabilization of

p53 mRNA in the nucleus, half-life increased to 6h. Fukao et al. (1999) showed that in PBMCs in astate of quiescence ATM kinase is present at lowlevels and increases approx. 610-fold in responseto mitogenic stimuli (PHA 10 g/ml) reaching amaximum aer 34 days. Two hours aer irra-diation by the dose of 6 Gy of stimulated PBMCsthey detect p53 and p21 by western blot analy-sis. Our ndings correspond with their data. Wecompared the dynamics of p53 induction aer ir-radiation by IR in both quiescent and stimulatedlymphocytes. p53 increase was observed only in

stimulated lymphocytes, as was p53 phosphor-ylation at serines-392 and -15. The increase in theamount of p53 was not dose-dependent 4 h aerthe irradiation. On the other hand, phosphoryla-tion of p53 at serine-15 analyzed 4 h aer the irra-diation is dose-dependent over the studied dose-range (0.57.5 Gy).

Despite the fact that p53 was not detectedin quiescent lymphocytes and a reaction to irradi-ation was not observed either, p21 levels increasedaer irradiation in both quiescent and stimulatedlymphocytes in a dose-dependent manner. p21(also known as cyclin-dependent kinase inhibitor1A CDKN1A) inhibits activity of cyclin-CDK2and cyclin-CDK4 complexes, which causes cellcycle arrest in G1/S phase and allows DNA-dam-age repair. It was shown that histone deacetylaseinhibitors induce increased expression of p21 by

both p53-dependent and p53 independent mecha-nisms (Ocker & Schineider-Stock, 2007) and thatthis activation includes many other factors suchas ATM (Ju & Muller, 2003) or c-myc (Zhao et al.,2006). It seems that the main pathway involved inp21 induction by histone deacetylase inhibitors istriggered through Sp1/Sp3 pathway activation. On

the other hand, Pei et al. (2001) studied radio-sensitivity of B-cell chronic lymphocytic leukemia.They detected increase in p53 and p21 and highsensitivity to radiation-induced apoptosis only inp53 wild type leukemias. When p53 was absent,no expression of p21 was found aer irradiationand the cells were more radioresistant. In cells ofhuman leukemia HL-60 and U937 exit from cellcycle is a prerequisite for terminal dierentiationand p21 is induced during terminal dierentiationof cells (Gartel & Tyner, 1999). In some cell typesp21 expression protects the cells from p53-depend-


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ent apoptosis induction (Gorospe et al., 1997). Lowexpression of p53 in T lymphocytes irradiated bythe dose of 2 Gy in contrast to high expression af-ter actinomycin treatment (Porcedda et al., 2006),and low p53 and high expression of p21 in quies-cent lymphocytes aer irradiation by the dose of4 Gy detected in our study indicate that increasedp21 in lymphocytes enhances repair of radiation-

induced damage and prevents apoptosis.Apoptosis induction in quiescent lym-

phocytes was described in the study of Vokurkovet al. (2006). It proves that even aer exposure to ahigh lethal dose of 7 Gy apoptosis is not inducedwithin 6 h aer irradiation. Apoptotic cells detect-ed by Annexin V binding were found only 16 haer the irradiation and 48 h aer the irradiation80% of lymphocytes were A+. When sensitivity ofdierent T lymphocyte subpopulations (CD3+CD4+,CD3+CD8+ and CD3CD8+) was studied, the mostradiosensitive cells were natural killer cells CD3

CD8+

. In present study we compare apoptosis in-duction in quiescent CD3+ and PHA-stimulated Tlymphocytes. We prove that 72 h-long stimulationof T lymphocytes by PHA induces apoptosis (de-tected by Annexin V binding and lamin B cleav-age). Apoptotic cells were mostly A+/PI, which in-dicates an early stage of apoptosis. Also apoptosiswas detected in unstimulated and PHA-stimulatedcells from 24 h aer the irradiation by the doseof 4 Gy onwards. In both, unstimulated and PHA-stimulated cells 6570% of cells were apoptotic 72h aer the irradiation.

Phosphorylation of p53 in response to DNAdamage is one mechanism by which its activitycan be modulated. Serine-15 was identied as asite on p53 phosphorylated in response to DNAdamage induced by UV or IR (Siliciano et al.,1997; Shieh et al., 1997) UV and IR action is dier-ent with respect to phosphorylation of p53 at ser-ine-392 in colon cancer cells RKO (Kapoor & Lo-zano, 1998). However, using T-cell leukemic cellsMOLT-4 phosphorylation of p53 at serine-392 wasshown aer IR as well (Szkanderov et al., 2003).As we demonstrate in this paper, IR induces phos-

phorylation of p53 at both serines-15 and -392 inPHA stimulated human lymphocytes. However,phosphorylation at serine-15 was dose-dependent,while phosphorylation at serine-392 was not.

In this paper we have shown that irradia-tion of stimulated lymphocytes causes an increasein phosphorylation of histone H2A.X, an increasein p53 and its phosphorylation, and an increasein p21. Immunocytochemical detection of H2A.X and ELISA measurement of p53 phosphorylatedon serine-15 can be used as indicator of receiveddose of IR.

Acknowledgements

The authors would like to thank the Min-istry of Defence of the Czech Republic for -nancial support. (projects MO0FVZ0000501 andOBUKHK205001).

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