Accumulation of DNA Damage and Cell Death after Fractionated Irradiation

Martina Rezacova,a Gabriela Rudolfova,a Ales Tichy,a,b Alena Bacıkova,c Darina Mutna,a Radim Havelek,a

Jirina Vavrova,b Karel Odrazka,d Emilie Lukasovac,1 and Stanislav Kozubeka

a Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Czech Republic; b Department ofRadiobiology, Faculty of Military Health Sciences Hradec Kralove, University of Defence Brno, Czech Republic; c Department of Molecular

Cytology and Cytometry, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic; and d Department of RadiationOncology, Multiscan and Pardubice Regional Hospital, Pardubice, Czech Republic

Rezacova, M., Rudolfova, G., Tichy, A., Bacıkova, A.,Mutna, D., Havelek, R., Vavrova, J., Odrazka, K., Lukasova,E. and Kozubek, S. Accumulation of DNA Damage and CellDeath after Fractionated Irradiation. Radiat. Res. 175, 000–000(2011).

The purpose of this work was to find how fractionatedradiation used in the treatment of tumors affects the ability ofcancer as well as normal cells to repair induced DNA double-strand breaks (DSBs) and how cells that have lost this abilitydie. Lymphocytic leukemia cells (MOLT4) were used as anexperimental model, and the results were compared to those fornormal cell types. The results show that cancer and normal cellswere mostly unable to repair all DSBs before the next radiationdose induced new DNA damage. Accumulation of DSBs wasobserved in normal human fibroblasts and healthy lymphocytesirradiated in vitro after the second radiation dose. Thelymphocytic leukemia cells irradiated with 4 3 1 Gy and asingle dose of 4 Gy had very similar survival; however, there wasa big difference between human fibroblasts irradiated with 4 3

1.5 Gy and a single dose of 6 Gy. These results suggest thatexponentially growing lymphocytic leukemia cells, similar torapidly proliferating tumors, are not very sensitive to fractionsize, in contrast to the more slowly growing fibroblasts and mostlate-responding (radiation therapy dose-limiting) normal tissues,which have a low proliferation index. g 2011 by Radiation Research Society

INTRODUCTION

Ionizing radiation is one of the tools used fortreatment of cancer. The aim of this treatment is toinduce very high levels of DNA damage in cancer cellsso that they are unable to repair the damage, stopdividing, and finally die. It was found empirically that tominimize the secondary effects of radiation in normaltissues, it is appropriate to divide the radiation doserequired for elimination of cancer cells into a number ofsmall fractions delivered at equal intervals (1, 2).

Conventionally, doses of 1–2 Gy are delivered once aday for several weeks. Most studies dealing with theeffect of exposure to ionizing radiation use single-doseirradiation. Some studies performed with fractionatedirradiation have focused on the effect of splitting thedose on the relationship between DNA damage repairand cell recovery [e.g. (3, 4)]. Other studies haveinvestigated apoptosis (5), radiation sensitivity of cells(6, 7), and the influence of dose size on the rate ofsublethal damage repair (8). While a few studies haveshown accumulation of DNA DSBs after exposure ofcells to fractionated radiation (9–11), the question ofhow the cells die after fractionated irradiation has notyet been completely answered.

The most dangerous DNA damage induced byionizing radiation is the interruption of genome integrityby DNA double-strand breaks (DSBs), which representspotentially lethal damage (12–15). Discovery of histoneH2AX phosphorylation on serine 139 by ATM kinase atthe site of DSBs after irradiation enabled the use of thisepigenetic modification for DSB detection (16). Manyproteins involved in various stages of DSB repair (17–20) and related modification of chromatin structure (21–23) accumulate in the region of phosphorylated H2AX(c-H2AX). Their mutual interaction at the site of DSBsis crucial for correct repair (24). In response to DSBinduction, cells activate DNA damage checkpointpathways to stop cell division until completion of therepair of damage to protect genomic integrity andpromote survival of the cell (15, 25, 26). Proteinsimportant in cell cycle arrest, such as p53 andcheckpoint kinases 1 and 2, are activated by ATR andATM kinases immediately after DSB induction (26, 28).

It was shown recently that human cells (29), similar tocells of other organisms (30), have the ability to divideafter a checkpoint-imposed cell cycle arrest despite thepresence of persistent DNA damage. This overriding ofthe checkpoint (checkpoint adaptation) may facilitateelimination of cells with excessive DNA damage byallowing progression through mitosis (29, 31). Cellular

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1 Address for correspondence: Institute of Biophysics, Academy ofSciences of the Czech Republic, Kralovopolska 135, 612 65 Brno; e-mail: lukasova@ibp.cz.

RADIATION RESEARCH 175, 000–000 (2011)0033-7587/11 $15.00g 2011 by Radiation Research Society.All rights of reproduction in any form reserved.DOI: 10.1667/RR2478.1

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senescence has been identified as another cellularresponse induced by genotoxic stress that leads to theultimate and irreversible loss of replicative capacity ofsomatic cell cultures (32). The senescence is used by cellsas a safeguard mechanism that can prevent aged ordamaged cells from dividing (33).

In this paper we present results showing the effects oflong-lasting genotoxic stress induced by repeated irra-diation of MOLT4 human lymphocytic leukemia cells.MOLT4 cells were used as a model because of their easyacquisition and manipulation. For comparison, normalskin fibroblasts, which represent one of the mostcommon types of stromal cells forming the connectivetissue of all organs, were used. The repair of DSBs wasalso followed after two radiation doses of 2 Gy inlymphocytes isolated from the blood of healthy donors.Fibroblasts, similar to lymphocytes, are exposed toradiation damage during radiotherapy of most cancers.

MATERIAL AND METHODS

Cell Culture

Blood was obtained from two healthy donors with informedconsent. The blood samples were anonymized. Peripheral bloodmononuclear cells (PBMC) were isolated from heparinized blood bycentrifugation on a Histopaque-1077 (Sigma) cushion according tothe manufacturer’s instructions. The cells were cultured in Iscove’smodified Dulbecco’s medium (Sigma) supplemented with 20% fetalbovine serum (FBS) (PAN Laboratories GmbH, Austria), 2 mMglutamine (Sigma), 100 U/ml penicillin (Sigma) and 0.1 mg/mlstreptomycin (Sigma), starting at a density of 5 3 105 cells/ml.MOLT4 cells were obtained from the American Type CultureCollection (Manassas, VA) and were cultured in the same mediumas lymphocytes. Cell numbers were assessed before irradiation and 1 hand 24 h after each radiation dose. Human skin fibroblasts fromjuvenile foreskin were from PromoCell (Heidelberg, Germany). Theywere cultured in DMEM (Gibco) containing 100 U/ml penicillin(Sigma), 0.1 mg/ml streptomycin (Sigma) and 10% FBS (PAN,Austria). They were subcultured when they reached about 80%

confluence to a starting density of 1 3 105 cells/ml. Cells from the 5thto the 10th subculture were used for all experiments.

Irradiation

MOLT4 cells and lymphocytes were transferred in 10-ml aliquotsinto 25-cm2 flasks (Nunc) and irradiated at room temperature using a60Co c-ray source. Doses of 1–4 Gy were delivered at a dose rate of0.4 Gy/min. Immediately after irradiation, the flasks were placed intoa 37uC incubator with 95%air/5% CO2, and aliquots of cells werecollected at various times after irradiation for analysis. Lymphocytesfrom healthy donors were irradiated with two doses of 2 Gy at aninterval of 24 h, and MOLT4 cells were irradiated with four doses of1 Gy at intervals of 24 h. The lower dose was used for MOLT4 cellsbecause of the higher radiosensitivity of these cells compared tolymphocytes. Fibroblasts were synchronized in confluence by serumdeprivation for 3 days in DMEM without serum. They weretransferred into the medium with serum 24 h before irradiation at astarting density of 1 3 105 cells/ml. Cells were irradiated with fourdoses of 1.5 Gy at intervals of 24 h and with a single dose of 6 Gy at adose rate of 1 Gy/min using a 60Co c-ray source. The dose of 1.5 Gywas used because the DSB repair after this dose in fibroblasts iscomparable to that after a dose of 1 Gy in MOLT4 cells. To separatedividing cells, Nocodazol (Sigma) 40 ng/ml was added to the medium

2 h after irradiation, dividing cells were shaken off 18 h later andfixed, and DSBs together with cyclin B1 were immunodetected.

Colony-Forming Units (CFU)

Exponentially growing fibroblasts were plated at 100 and 1000 cellsin 100-mm petri dishes containing 12 ml of DMEM with 10% FBS in4 replicates. After 14 days of incubation, the cells were fixed andstained with 1% crystal violet in methanol for 30 min at roomtemperature. Colonies with about 50 cells were counted. For detectionof MOLT4 CFU, the MethoCult H4230 methylcellulose mediumwithout cytokines (Stem Cell) was used. Suspensions of 100 and 1000cells in the methylcellulose medium were plated in 35-mm dishes intriplicate according to the recommendations of the manufacturer.Colonies that were 3 mm in diameter were counted after 14 days ofincubation.

Statistics

All experiments were repeated two or three times. Evaluation ofdata and statistical analyses were performed using the Sigma Plotstatistical package (Jandel Scientific).

Immunocytochemistry

Immunodetection of c-H2AX and other proteins was performed asdescribed (34). Cells containing DSBs detected by means of antibodyagainst c-H2AX and forming IRIF (ionizing radiation-induced foci)and or cells with a high level of cyclin B1 were counted by eye afterfixation with 4% paraformaldehyde among at least 500 cells persample on microscope slides in the visual field of the confocalmicroscope at a magnification of 10003. Cells containing high levelsof cyclin B1 were large and easily distinguishable from those withlower levels of the protein (see Fig. 2A). Blind counting of bothparameters was also used. Integrated optical density (IOD) was usedfor the detection of DSBs in lymphocytes isolated from the peripheralblood of healthy donors; this method is rapid, and the resultscorrespond well to the numbers of c-H2AX foci as defined earlier(35).

Senescence-associated b-galactosidase fluorescence detection (SA-

bgal) was done according to the protocol described by Debacq-Chainiaux et al. (36). SA-bgal-positive cells were viewed using aconfocal microscope.

Antibodies and Reagents

Anti-phospho-H2AX-ser139, anti-trimethyl histone H3K9 andanti-HP1b were from Upstate Biotechnology (Lake Placid, NY);anti-phospho-p53-ser15 and anti-53BP1 and 5-dodecanoylamino-fluorescein di-b-D-galactopyranoside (C12FDG) were from CellSignaling, Danvers, MA; anti-p16 and anti-p21were from MilliporeCorporation; anti-Chk1, anti-phospho-Chk1-ser317, anti-cyclinB1and anti-actin were from Abcam, Cambridge UK; anti p53 was fromSanta Cruz Biotechnology, Santa Cruz, CA; bafilomycin A1 wasfrom Sigma-Aldrich Co. Secondary antibodies donkey anti-mouse-FITC and anti-rabbit-Cy3 were from Jackson Laboratory, BarHarbor, ME. TOPRO-3 was from Molecular Probes, Eugene, OR.

Confocal Microscopy

Images were obtained using a high-resolution confocal cytometerbased on an automated Leica DM RXA fluorescence microscopeequipped with a CSU-10a confocal unit (Yokogawa, Japan), aCoolSnap HQ CCD camera (Photometrics, Tucson, AZ), an Ar/Krlaser (Innova 70C, Coherent, Palo Alto, CA), and an oil immersionPlan Fluotar objective (1003/NA1.3). The pinhole size of the NipkowCSU-10a spinning disc corresponds to about 1 Airy unit for theemission maximum of 510 nm.

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Automated exposure, image quality control and other procedureswere performed using FISH 2.0 software (37, 38). Forty opticalsections were captured at 0.2-mm intervals along the z-axis at aconstant temperature of 26uC. Integrated optical density (IOD) wasmeasured using the image analysis software ImagePro 4.11 (Media-Cybernetics).

Flow Cytometry Analysis and Detection of Apoptosis

The Apoptest-FITC kit (DakoCytomation, Brno, Czech Republic)was used for the detection of early apoptosis. Cells with permeablecell membranes (late apoptotic or necrotic) were detected bypropidium iodide (PI) staining on a Coulter Epics XL flow cytometerequipped with a 15 mW argon-ion laser with excitation at 488 nm(Coulter Electronics, Hialeah, FL). List mode data were analyzedusing Epics XL System II software (Coulter Electronics). Thedistribution of cells in the cell cycle was determined in cells fixed in70% ethanol and stained with 0.1 mg/ml PI.

Western Blotting

Cellular lysates were prepared as described previously (39), andtotal protein concentration was determined by a protein assay (Bio-Rad). Extracts containing equal amounts of proteins (5 mg to 10 mg)were loaded on 9% or 15% SDS-PAGE gels and after separation weretransferred onto PVDF membranes (Bio-Rad). The membranes wereblocked in Tris-buffered saline with 0.1% Tween 20 and 5% nonfatmilk or 5% BSA. After incubation with primary antibodies,appropriate secondary antibodies conjugated with horseradishperoxidase were used for detection of protein bands on the membraneusing SuperSignal West Pico Substrate (Pierce). The signal wasobtained using a Fujifilm LAS-3000 Imager.

RESULTS

Effect of Dose Fractionation on the Induction and Repairof DSBs, Cell Proliferation, Adaptation and Senescencein MOLT4 Lymphocytic Leukemia Cells

Examples of cells containing IRIF 1 h and 24 h afterexposure to fractionated doses of c rays are shown inFig. 1A and B. Each radiation dose resulted in a delay ofcell proliferation (Fig. 1C). Many cells were unable torepair all the DSBs before the next radiation dose inducednew DNA damage. The fractions of cells with unrepairedIRIF increased with the number of radiation doses 24 hafter irradiation (Fig. 1D) and decreased during the next24 h (i.e., 48 h after each irradiation). Some cellscontaining unrepaired DSBs 24 h after the secondirradiation had a very high level of cyclin B1 in thecytoplasm (Fig. 2A), indicating that these cells hadarrested at the end of G2 phase [the onset of cyclin B1synthesis is in S phase, and it accumulates during G2

phase and reaches maximum levels just prior to mitosis(40, 41)]. Therefore, despite the presence of unrepairedDSBs, they appear to be preparing for cell division. Suchcells comprised 20% of the 500 cells counted, whichagreed with the cell fraction arrested in G2 detected byflow cytometry (about 26%) (Figs. 2C, 1E). Cells with ahigh level of cyclin B1 were not present in the cellsuspensions fixed 48 h after the second irradiation,indicating that they had indeed completed cell division.

If the cells were irradiated with the third radiation dose(24 h after the second dose), then cells with high levels ofcyclin B1 persisted longer and disappeared between 24 hand 25 h after the third irradiation (Fig. 2C). In somecells, the higher level of cyclin B1 had already shifted fromthe cytoplasm into the nuclei at 24 h after the second dosebut especially after the third irradiation, and in somenuclei chromosome condensation and mitoses containingc-H2AX on some chromosomes were observed, indicat-ing that cell division was progressing (Fig. 2A). Thefraction of cells arrested in G2 phase increased to 55% 24 hafter the third radiation dose (Fig. 1E); however, a highlevel of cyclin B1 was observed in only 22% of the cells(Fig. 2C). A higher level of cyclin B1 in cells arrested inG2 24 h after the second and third radiation dose was alsoshown by Western blotting (Fig. 2B). Cyclin B1 was notexpressed in cells irradiated with four doses; these cellssynthesized an increased level of p21, indicating that theycould enter senescence. All cells maintained elevatedphosphorylation of Chk1(ser317) and p53(ser15)throughout the fractionated irradiation until 24 h afterthe last dose (Fig. 2B, D). The level of proteinscharacteristic for senescence (p16 and p21) had notincreased at 96 h after the single dose of 4 Gy (Fig. 2B).

Effect of Dose Fractionation on the Induction ofApoptosis in MOLT-4 Cells

The percentage of apoptotic cells detected by AnnexinV positivity 24 h after the last irradiation was highest(60%) after a single dose of 3 Gy, but only 34% of cellsirradiated with 3 3 1 Gy were apoptotic (Fig. 3A). Thelevel of Annexin V-positive cells increased with time afterthe individual fractionated doses and a single dose of 3 Gy72 h irradiation (Fig. 3B). The greatest increase of thenumber of apoptotic cells was after 1 Gy and 2 3 1 Gy(about 100% and 62%, respectively) and the smallest wasafter a single dose of 3 Gy (26%). The increase in thenumber of apoptotic cells 72 h after 3 3 1 Gy (54%)resulted in a similar proportion of Annexin V-positivecells (75%) as found after a single dose of 3 Gy (80%).

Effect of Radiation Dose Fractionation on DSB Inductionand Repair in Human Lymphocytes Isolated fromPeripheral Blood In Vitro

PBMC were irradiated in vitro with 2 Gy (Fig. 4A).One hour after irradiation, an increase in the IOD for c-H2AX per cell was seen. Twenty-four hours later, DSBswere repaired, which was seen as the disappearance ofalmost all c-H2AX signal and a significant decrease inIOD (Fig. 4A, B). The cells were then irradiated with asecond dose of 2 Gy. The signal of c-H2AX 1 h after thesecond irradiation was comparable with the cellularresponse to the first radiation dose. However, the cellswere unable to repair all DSBs within the next 24 h, anda significant number of c-H2AX signals persisted in 52%

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of PBMC (Fig. 4C). We cannot exclude the possibilitythat apoptotic cells might contribute to the PBMC withincreased c-H2AX fluorescence shown in Fig. 4B and C.

Effect of Dose Fractionation on the Induction and Repairof DSBs, Cell Proliferation, Adaptation and Senescencein Normal Skin Fibroblasts

The number of cells containing unrepaired DSBs(IRIF) 24 h after irradiation with 1.5 Gy increased as the

number of doses increased (Fig. 5A). IRIF were inalmost all cells at 24 h and 48 h after a single dose of6 Gy. We did not observe an increased number of DSBsin cells synchronized with serum deprivation comparedto nonsynchronized cells. The number of cells contain-ing IRIF decreased only slightly at 48 h after the lastfractionated dose. An increasing number of radiationdoses resulted not only in an increase of cells containingIRIF 24 h after irradiation but also in an increasednumber of IRIF per cell (Fig. 5B). The number of cells

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FIG. 1. Induction and repair of DSBs after irradiation of MOLT4 lymphocytic leukemia cells with 3 Gy of c rays delivered in three 1-Gyfractions with an interval of 24 h or as a single dose. Panel A: Examples of cells 1 h after the last irradiation (IR). DSBs are detected as c-H2AX(green) colocalizing with 53BP (red). Panel B: Cells fixed 24 h after the last irradiation. Unrepaired DSBs are detected as c-H2AX (green)colocalizing with 53BP (red). Panel C: Growth of cells during fractionated irradiation. The cells were irradiated with the first dose of 1 Gy and3 Gy (two vials per dose) after cell counting. Living cells were counted at 24 h, i.e. just before the next irradiation, in all vials. Flashes indicate thetime of irradiation. Error bars represent SEM from four measurements. Panel D: The size of cell fractions with unrepaired DSBs at 24 h and 48 hafter the last irradiation. Error bars represent SEM from three experiments. Panel E: Cell distribution in the cell cycle 24 h after the lastfractionated dose or a single dose of 3 Gy. The distribution is shown as DNA histograms obtained by flow cytometry in one experiment intriplicate. Fractionated irradiation with a dose of 1 Gy with an interval of 24 h resulted in an increase of the cell fraction arrested in G2.

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containing two and more IRIF per cell decreased withinthe next 24 h (i.e. 48 h after irradiation), but the totalnumber of cells containing IRIF did not changesignificantly, indicating that cells repaired DSBs slowlywithout dying during this time.

Cell division was observed 24 h after the first and thesecond irradiation and in control nonirradiated cells.Only about 8% of mitoses (2 of 25) contained IRIF afterthe first irradiation, unlike the second radiation dose,when c-H2AX foci were observed in 76% of mitoses (23

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FIG. 2. Cyclin B1 expression in MOLT4 cells arrested in G2 phase after fractionated irradiation (IR) with doses of 1 Gy at intervals of 24 h.Panel A: Representative projections of MOLT4 cells expressing high levels of cyclin B1 (red) at 24 h after doses of 2 3 1 Gy and 1 h and 24 hafter 3 3 1 Gy. After single doses of 1 Gy and 3 Gy and fractionated doses of 4 3 1 Gy, cells with a high level of cyclin B1 were not observed ateither 24 h or 48 h irradiation. One hour after irradiation with the third dose, the cells contained a high number of DSBs, represented by greenfoci of c-H2AX; however, 23 h later, the number of these foci decreased, indicating that the cells had repaired DNA damage in spite of being atthe boundary of mitosis. Panel B: Western blotting of proteins detected in cell extracts prepared 20 h after the last irradiation. At 96 h after thesingle radiation dose of 4 Gy, the level of p16 and p21 is shown. Panel C: The size of the cell fraction synthesizing high levels of cyclin B1. Thesecells were observed only 24 h after the second irradiation and 1 h and 24 h after the third irradiation. Flashes indicate the time of irradiation.Error bars represent SEM from three experiments. Panel D: Examples of cells that did not express a high level of cyclin B1 24 h after the lastirradiation and thus did not adapt to the G2 checkpoint. Representative nuclei with immunodetected phosphorylated p53-ser15 and Chk1-ser317(both red) and c-H2AX foci (green) 24 h after the last irradiation are also shown.

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of 30). After the third and the fourth radiation doses,dividing cells were sporadic. Cells preparing for divisionafter the first and second irradiations had an increasedlevel of cyclin B1 (Fig. 5C-II). The highest level of cyclinB1 was 12 h after the second irradiation (Fig. 5D). Thecessation of cell division after the third and fourthirradiations was also demonstrated by the absence ofany S phase in the flow cytometry analysis of DNAlevels (Fig. 6A). Unlike the MOLT4 cells, the dividingfibroblasts were not retained at the end of G2 byfractionated irradiation. Fibroblasts remained in the G1

phase during the whole process of irradiation and didnot die by apoptosis (Fig. 6A, B). The number ofapoptotic cells 24 h and 48 h after each fractionated doseof c rays was almost the same as in control nonirradi-ated cells. We tested the SA-bgal activity to determinewhether the long-term presence of unrepaired DSBstriggers senescence in these cells. However, only a smallnumber of SA-bgal-positive cells were observed 72 hafter the single dose of 6 Gy and the fourth irradiation inregions where the cells were confluent.

Fractionated irradiation considerably reduced thecapacity of exponentially growing fibroblasts to formcolonies 24 h and 48 h after irradiation. This capacitywas significantly lower than in MOLT4 cells (Fig. 6C).It decreased to about 20% after the single dose of 1.5 Gy,to about 2% after the fourth irradiation with the samedose, and to about 0.17% after a single dose of 6 Gy.

DISCUSSION

Repair Capacity and Cell Death of LymphocyticLeukemia Cells Induced by Long Persistence of DSBsduring Fractionated Irradiation

We found that during the fractionated irradiation theMOLT4 cells cannot repair all DSBs induced by an

individual fraction before the administration of the nextradiation dose. The persistence of a certain amount ofunrepaired DSBs was found not only in lymphocyticleukemia cells but also in peripheral lymphocytesirradiated in vitro and in normal skin fibroblasts. Thenormal skin fibroblasts irradiated with four dosesaccumulated unrepaired DSBs for several days withoutdying. Important differences were observed in the abilityof lymphocytic leukemia cells to repair DSBs induced byfractionated radiation and those induced by a singledose that corresponded to the cumulative dose from fourfractions. The lower numbers of DSBs induced by lowerdoses of fractionated radiation were repaired more easilybetween fractions than the large number of DSBsinduced by the single dose. After the high single dose(3 Gy), a large number of unrepaired IRIF persisted inalmost all cells 24 h after irradiation (Fig. 1D). In thesecells, the activity of p53 increased significantly (Fig. 2B),

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FIG. 3. Apoptosis induction in MOLT4 cells exposed to fraction-ated doses of 1 Gy or a single dose of 3 Gy of c rays. Panel A:Fractions of Annexin V-positive cells detected by flow cytometry 24 hafter the last irradiation (Post-IR) with fractionated doses of 1 Gy ora single dose of 3 Gy. Panel B: Fraction of Annexin V-positive cellsdetected 72 h after the last irradiation with fractionated doses of 1 Gyor a single dose of 3 Gy. Error bars represent SD from threeexperiments in both panels.

FIG. 4. DSB induction and repair in peripheral blood mononu-clear cells (PBMC) during fractionated irradiation in vitro. DSBinduction and repair during fractionated irradiation of lymphocytesin vitro with a dose of 2 Gy repeated two times in the interval of 24 h;samples were analyzed 1 h and 24 h after the first and the secondirradiation. Panel A: Representative photomicrographs of the cellsduring fractionated irradiation after detection of c-H2AX (green) and53BP1 (red). Cell nuclei counterstained with TOPRO-3 are outlined:flashes denote irradiation and arrows the time of irradiation. Panel B:Quantification of the intensity of green (c-H2AX) fluorescence,measured as integrated optical density of each cell. At least 50 cellswere analyzed in each sample. The bars represent median ± 1st and3rd quartile. *Significantly different from both control cells and cells24 h after the first irradiation, P , 0.001 by Kolomogorov’s test.Panel C: Frequency of incidence of cells with increasing amounts of c-H2AX 24 h after the first irradiation (left) and 24 h after the secondirradiation (right). Fifty-two percent of the cells retained increasedamounts of c-H2AX 24 h after the second irradiation.

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which probably led to p53-dependent apoptosis of 60%and 80% of cells blocked in G1 at 24 h and 72 h afterirradiation, respectively (Fig. 3A, B). The distribution ofcells within the cell cycle changed when the radiationwas delivered in small fractions. The cells with a lownumber of unrepaired DSBs after fractionated irradia-tion were progressively arrested in G2 (Fig. 1E).Induction of new DSBs in cells containing as yetunrepaired damage probably impaired repair capacity,as indicated by the increased proportion of cells withunrepaired IRIF for a larger number of doses 24 h and

48 h after irradiation (Fig. 1D). In addition, these cellspersisted for a longer time, especially after the fourthradiation dose. Successive accumulation of incompletelyrepaired DSBs in cells after repeated irradiation led to apermanent DNA damage response. The cells remainedin a state with an activated G2 cell cycle checkpoint andp53 induced by the damage, and some of these cells wereprobably shifted to senescence as indicated by anincreased level of p21 protein. The majority of cellssurviving with DNA damage after fractionated dosesdied 72 h after the last radiation dose, as seen in the

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FIG. 5. Induction and repair of DSBs in human skin fibroblasts and cells dividing with unrepaired DSBs after irradiation with a single dose of6 Gy or fractionated doses of 1.5 Gy delivered at interval of 24 h. Panel A: The size of cell fractions with unrepaired DSBs at 24 h and 48 h afterthe last irradiation. Panel B: Cells containing DSBs at 24 h and 48 h after the last irradiation (shown in panel A) are composed of cell fractionscontaining different numbers of DSBs per cell. The size of these fractions is expressed as a percentage of the total number of cells with DSBs at24 h or 48 h after the last irradiation. There is an increase of cell fractions containing more DSBs per cell with an increased number offractionated doses 24 h after irradiation. The fractions of cells with more DSBs per cell decrease and those containing less DSBs per cell increase48 h after irradiation. The decrease of DSBs per cell is also evident at 48 h after irradiation with a single dose of 6 Gy. After this dose, almost allcells contained .3 DSBs per cell 24 h after irradiation; however, a small fraction of cells containing one, two and three DSBs per cell appeared48 h after irradiation. Error bars represent SEM from three experiments. Panel C: Examples of mitoses containing DSBs (green spots of c-H2AX) at 20 h after the second irradiation. (I) Upper row represents the total image composed of 40 slices of 0.2 mm, the lower row representsthe central slices. (II) Examples of fibroblasts expressing a higher amount of cyclin B1 (red) indicating their presence at G2 phase, (c-H2AX,green spots). The fibroblasts that started to express a high amount of cyclin B1 entered mitosis rapidly and were not detained in this state, unlikeMOLT4 cells. Panel D: The level of cyclin B1 in human skin fibroblasts during fractionated irradiation detected by Western blotting.

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increase of Annexin V-positive cells (Fig. 3B). The cellsdetected by Annexin V or PI positivity could comprisenot only cells dying by the apoptosis but also thosedying during metaphase as a result of caspase activationand subsequent mitochondrial damage (42, 43). Thoseauthors explained that mitotic catastrophe is a specialcase of apoptosis linked to the abnormal activation ofcyclin B1/Cdk1 in cells containing DNA damages. It ispossible that some MOLT4 cells arrested in G2 andexpressing high levels of cyclin B1 and entering mitosiswith unrepaired DSBs died during this process. This

adaptation of cells with damaged DNA to an active G2

checkpoint was recently demonstrated in human cells(29).

In our experiments, the checkpoint adaptation startedby massive synthesis of cyclin B1 in cells containingunrepaired DSBs arrested at G2/M phase 24 h after thesecond and third irradiations. These cells did not divideimmediately after the increase in cyclin B1 levels butrather during the next 24 h, and when the cells wereirradiated with the third dose, their persistence on theborder of prophase was prolonged for another 24 h.

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FIG. 6. Cell cycle distribution, level of apoptosis and viability of human skin fibroblasts during fractionatedirradiation with 1.5 Gy. Viability of MOLT4 lymphocytic leukemia cells during fractionated irradiation with1 Gy. Panel A: Distribution of fibroblasts in the cell cycle according to DNA histograms obtained by flowcytometry 24 h (upper graph) and 48 h (lower graph) after the last fractionated dose. Panel B: Level ofapoptosis in human fibroblasts detected by flow cytometry 24 h (upper graph) and 48 h (lower graph) after thelast fractionated dose. The values represent the mean from three measurements. Panel C: Cell viability ofhuman skin fibroblasts (lower graph) and MOLT4 lymphocytic leukemia cells (upper graph) 24 h and 48 h afterthe last dose. Cells used for detection of CFU were not submitted to synchronization or to nocodazoletreatment. All plating experiments were repeated three times in triplicate for MOLT4 and in four replicates forfibroblasts. Error bars represent SEM from three experiments.

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Then the cells entered mitosis, and some of them mayhave died. The fourth irradiation did not prolongpersistence of cells on the border of prophase. Weassume that the extensive DNA damage that accumu-lated progressively in cells irradiated with three and fourdoses increased the genotoxic stress that pushed cells toapoptosis and that a lower level of DNA damage couldinitiate senescence. The results of some authors (44, 45)show that cellular responses may depend on the extentof genotoxic damage produced by chemotherapeuticagents; while high DNA damage induces massiveapoptosis, a low level of this damage initiates senescencein cells containing functional p53 and p21.

Response of Normal Skin Fibroblasts toFractionated Irradiation

The process of adaptation occurring in cells withpersistently unrepaired DSBs was also found in normalskin fibroblasts, although to a lesser extent than inlymphocytic leukemia cells. These cells preserving 1 to10 c-H2AX foci per cell were retained in the G0/G1 phaseuntil subculture (Fig. 6A). Only a small part of thesecells divided 24 h after the first and second radiationdoses. Many mitoses observed after the second radiationdose contained nonrepaired IRIF, indicating that theyescaped from cell cycle arrest imposed by DNA damagecheckpoints (Fig. 5A). The long-term arrest of normalhuman fibroblasts in the G0/G1 phase was also observedafter a single radiation dose (46) and has been referred toas being ‘‘senescence-like’’ (47). However, we detectedSA-bgal activity in only a small number of cells after asingle dose of 6 Gy, which was similar to that after thefractionated radiation doses, and we did not observechanges in the chromatin structure accompanyingsenescence during 72 h after irradiation. The generalopinion that the cell cycle delay occurs primarily toenable repair of DSBs seems not to be the only reason,and other potential functions during radiation-inducedgenotoxic stress response in a tissue environment mustbe taken into consideration to explain this cell behavior.According to some authors (14, 48), fibroblasts providesignals to epithelial cells during growth, development,differentiation and apoptosis. Therefore, the DNAdamage induced by radiation may cause fibroblasts toundergo cell arrest and then differentiation to providespecific stress signals to neighboring cells (49).

We presume that the long persistence of fibroblastswith unrepaired DSBs in the G0/G1 phase might berelated to their natural capacity to stay in quiescenceafter reaching confluence in vitro. Fibroblasts that havenot yet reached the end of their limited life span in vitroare able to restart proliferation after subculture;however, those cells exposed to genotoxic stress obvi-ously lose this capacity. The progressive accumulation ofDSBs and the inability to repair this damage trigger

premature quiescence in non-confluent cells that prob-ably precedes premature senescence. These cells, similarto the cells in premature senescence, are able to remainalive for months (50); however, contrary to cells inpremature senescence, they do not display features ofreplicative senescence (morphology, SA-bgal activity)and die during subculture. This is a favorable responsebecause it prevents eventual proliferation and theneoplastic transformation of cells with damaged DNA.In the tissue, the DNA damage-induced quiescent andsenescent cells could be removed by immune cells orundergo apoptosis (51).

Conclusion

Our results show that the lymphocytic leukemiaMOLT4 cells and normal skin fibroblasts are mostlyunable to repair all DSBs before the application of thenext radiation dose that induces new DNA damage.Similarly, human lymphocytes irradiated in vitro alreadyaccumulate DNA DSBs after the second dose. Exposureof lymphocytic MOLT4 leukemia cells to a singlerelatively high dose induces apoptosis in high numbersof cells 24 h after irradiation, in contrast to lowerfractionated doses. After these doses, apoptosis pro-gresses more slowly and reaches almost the same level asa cumulative dose 72 h after irradiation. In contrast tolymphocytic leukemia cells, normal skin fibroblast withunrepaired DSBs do not die by apoptosis after eithersmall fractionated doses or a high single dose. Theypersist for a long time in quiescence without signs ofsenescence. These cells exposed to low doses offractionated radiation lose their proliferation capacityduring subculture. While the lymphocytic leukemia cellsirradiated with 4 3 1 Gy and a single dose of 4 Gy havevery similar survival, there is a big difference betweenhuman fibroblasts irradiated with 4 3 1.5 Gy and asingle dose of 6 Gy, to the advantage of the fractionateddose.

These results suggest that exponentially growinglymphocytic leukemia cells, like rapidly proliferatingtumors, are not very sensitive to a fraction size, incontrast to the more slowly growing fibroblasts andmost late-responding (radiation therapy dose-limiting)normal tissue, which have a low proliferation index. Thisresult is important in light of recent developments inoptimizing fractionation regimens in radiotherapy.

The sensitivity of cells to DNA-damaging agents isdetermined by the status of principal tumor suppressorgenes (ATM, p53, DNA-PK, Chk1 and Chk2) that arefrequently mutated in cancer cells, influencing theirsensitivity to DNA damage (52). Therefore, it would bebeneficial to determine the response of individual typesof cancers to different schedules of fractionated irradi-ation in vitro to determine the optimal radiation dosesneeded for cancer eradication.

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ACKNOWLEDGMENTS

This work was supported by the Ministry of Defence of the CzechRepublic (Project No. MO0FVZ0000501), the Grant Agency of theCzech Republic (Project No. P302/10/1022), and Ministry of

Education of the Czech Republic (Projects No. MSM 0021620820and No. LC535).

Received: October 19, 2010; accepted: January 21, 2011; publishedonline:

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