Brachytherapy 11 (2012) 149e156

Comparison of cellular damage response to low-dose-rate 125I seedirradiation and high-dose-rate gamma irradiation in human lung

cancer cells

Honghong Chen*, Yizhong Bao, Lei Yu, Rongfei Jia, Wenying Cheng, Chunlin ShaoDepartment of Radiation Biology, Institute of Radiation Medicine, Fudan University, Shanghai, P. R. China

125

ABSTRACT PURPOSE: To investigate the difference of c

Received 23 Sept

accepted 13 May 201

* Corresponding a

Radiation Medicine, F

200032, P. R. China.

E-mail address: h

1538-4721/$ - see fro

doi:10.1016/j.brach

ellular response between low-dose-rate (LDR) Iseed irradiation and high-dose-rate (HDR) g-irradiation in human lung cancer cells.METHODS AND MATERIALS: A549 and NCI-H446 cells with or without wortmannin (WM)treatment were exposed to 125I seeds and g-rays, respectively. Cell survival, micronuclei (MN)formation, and the expressions of Ku70/Ku80 proteins were measured.RESULTS: There was a strong negative correlation between survival and MN formation for bothirradiations, and the MN inductions of NCI-H446 were about twofolds of those of A549, and thesurvival of NCI-H446 was lower than that of A549, indicating the radiosensitivity of NCI-H446cells was greater than that of A549 cells. Interestingly, at 4-Gy radiation, NCI-H446 cells weremore sensitive to LDR irradiation than HDR irradiation. WM treatment enhanced theradiosensitivity of A549 cells evenly to 125I seed and g-irradiation, but this treatment led NCI-H446cells to be more sensitive to LDR 125I. Further results revealed that the expression ofphosphorylated Ku80 protein was enhanced in irradiated A549, but in contrast, it was markedlydecreased in NCI-H446 cells after 4-Gy LDR 125I irradiation as that compared with g-irradiatedand nonirradiated cells.CONCLUSION: NCI-H446 cells were more sensitive to LDR 125I irradiation than HDRirradiation, and this sensitivity could be further enhanced by WM treatment. But no obviousdifferences of cellular response to both irradiations were observed in A549. Ku as molecularmarkers together with cell proliferation rate can be used to predict the radiosensitivity of tumorcells to LDR 125I seed irradiation. � 2012 American Brachytherapy Society. Published by ElsevierInc. All rights reserved.

Keywords: LDR 125I seeds; HDR g-irradiation; Radiosensitivity; Ku protein; Lung cancer cells

Introduction

125I seed interstitial brachytherapy is an alternative orcomplementary modality to external radiotherapy of solidtumors (1, 2). The therapeutic effect of externalradiotherapy has been well understood, while the currentknowledge of the effect of 125I seed brachytherapy is verylimited. The main difference of these two kinds of therapiesis that the dose rate of 125I seed brachytherapy is at least

ember 2010; received in revised form 8 May 2011;

1.

uthor. Department of Radiation Biology, Institute of

udan University, No. 2094, Xie-Tu Road, Shanghai

Tel.: þ86-021-64437130; fax: þ86-021-64048043.

hchen@shmu.edu.cn (H. Chen).

nt matter � 2012 American Brachytherapy Society. Publis

y.2011.05.002

two orders of magnitude lower than that of externalradiotherapy, and it also varies with time and spatialdistribution of 125I seeds although the total dosagemight be high. Previous studies (3, 4) showed that in thepresence of continuous low-dose-rate (LDR) irradiation(0.05 ~ 0.1 Gy/h) and with the lapse of time, DNA repairplays an important role in the dose-rate effect of irradiation.To obtain the desired therapeutic dose, the LDR irradiationtime has to be prolonged, during which cell damage repair,cell cycle redistribution and cell reproliferation may occurso that the cell radiosensitivity may undergo changesaccordingly. Clinical trial demonstrated that 125I seedbrachytherapy is a feasible adjuvant procedure to reducethe incidence of recurrence and prolong survival aftersublobar resection in medically compromised patients withearly stage non-small-cell lung cancer (5, 6). Few studies

hed by Elsevier Inc. All rights reserved.

150 H. Chen et al. / Brachytherapy 11 (2012) 149e156

reported the difference of tumor cells’ response betweenLDR 125I seed irradiation and high-dose-rate (HDR)g-ray irradiation, and it is not clear whether a sensitizerof HDR radiation such as phosphatidylinositol-3 (PI-3)kinase inhibitor can enhance the tumor killing effect ofLDR 125I seed irradiation.

Radiosensitivity of tumor cells is a comprehensivereflection of multiple mechanisms where radiation-inducedDNA double-strand breaks (DSBs) and DNA repair playextremely important roles in determining the intrinsicradiosensitivity of tumor cells and thus is a key determinantfor the outcome of radiotherapy. Nonhomologous endjoining (NHEJ) is the main repair mechanism of DSB inmammalian cells, where DNA-dependent protein kinase(DNA-PK) is a key element during NHEJ repair (7).DNA-PK is a serine/threonine kinase consisting of a cata-lytic subunit (DNA-PKcs) and a DNA-binding Ku subunit.Ku, a heterodimer composed of Ku70 and Ku80 proteins, isthe major DSB-sensing protein and is regarded as thecornerstone of NHEJ. Binding of Ku to DNA is a prerequi-site for interaction with other NHEJ proteins by recruitingother NHEJ proteins to the DSB sites. DNA-PKcs can beactivated by interaction with Ku in response to DSBs andpromote the phosphorylation in trans across DSB andtrigger the repair of DNA damage (8). It has been reportedthat there was a high degree of correlation between theexpression of Ku protein and the disease-free survival inpatients with rectal and cervix carcinoma after radiotherapy(9, 10). Tumors with a high percentage of Ku70 and/or K80positive cells tend to be radioresistant, which suggests thattumor cells with the ability to repair damaged DNA aremore likely to survive and proliferate after irradiation.Conversely, tumors with low level of Ku70 and/or Ku80tend to be radiosensitive, which is supported by the signif-icantly higher survival of clinical patients. Moreover, accu-mulating evidence suggests that defection or inhibition ofeither component of DNA-PK can enhance radiosensitivityof tumor cells because of a decreased ability of DSB repair(11e18). For example, shRNA-mediated Ku80 genesilence inhibits cell proliferation and sensitizes radiation-induced apoptosis in cancer cells (11, 12). The dominantnegative Ku70 overexpression results in decreasedKu-DNA end-binding activity and increases radiosensi-tivity of human glioma U-87 MG cells and human colo-rectal tumor HCT-8 cells under both aerobic and hypoxicconditions (13). Wortmannin (WM), a potent and selectiveinhibitor of DNA DSB repair proteins, can boost lethaleffect induced by different doses of X-rays in radioresistantbladder tumor cell lines (15). But, all these studies con-cerned with HDR irradiation. Up to our knowledge, therehas no literature to confirm whether radiosensitivity oftumor cells during continuous LDR irradiation undergoesany change or not, or whether this change (if any) is asso-ciated with DNA-PK. Tomita et al. (19) reported that theNHEJ-defective mammalian cells were more sensitive toLDR radiation than HDR radiation, suggesting that

NHEJ-related factors may be useful as molecular markersto predict the sensitivity of human cancer to LDR radiation.

In the present study, we compared the effects ofcontinuous LDR 125I seed irradiationwithHDRg-irradiationon cell proliferation, DNA damage, and the expression of Kuprotein in two human lung cancer cell lines of A549 andNCI-H446 with different radiosensitivity to HDR radiation,and WM was applied to explore the efficacy of a sensitizerto LDR 125I seed irradiation, in an attempt to provideexperimental clues for enhancing and predicting thetherapeutic effect of 125I seed brachytherapy on the treatmentof malignant tumors.

Methods and materials

125I seed irradiation mold

Type-6711 sealed 125I seeds with single-source apparentradioactivity of 34.3e37.0 MBq (0.93e1.0 mCi) wereprovided by Ningbo Jun-An Pharmaceutical TechnologyCo. Ltd. (Ningbo, Zhejiang, China). In vitro cell irradiationwith 125I seeds was performed as that described in ourprevious literature (20). Briefly, eight 125I seeds wereequally placed within recesses around the circumferenceof a 30 mm diameter� 1 mm thick polystyrene dish. Theninth seed was placed in the center of the dish. The distancebetween the cell plane and the source plane was 5 mm(using water as the medium between). This irradiator waslocated in a steel cabinet inside a CO2 incubator at anaccess-control area, which was restricted to authorizedusers. Manipulations of the cell dishes were undertakenusing tongs and wearing lead-lined gloves. To achieveoptimum uniformity dose to cells, the dish was turned by22.5 degrees at the middle time of irradiation. Dosedistribution was calculated using the brachytherapyplanning system Fudan TPS2.0 based on the AmericanAssociation of Physicists in Medicine (AAPM) Task Group(TG) No. 43 brachytherapy formalism (Fig. 1) (21).

Cell culture and WM treatment

Non-small-cell lung cancer cell line of A549 and onevariant of small-cell lung cancer of NCI-H446 were obtainedfrom Shanghai Cell Bank, Chinese Academy of Sciences(Shanghai, China). Cells were cultured in RPMI1640medium (Invitrogen by Life Technologies, Carlsbad, CA)containing 10% bovine calf serum (Hangzhou ShijiqingBiological Engineering Co., Ltd., Hangzhou, Zhejiang,China), 100 mg/mL streptomycin and 100 U/mL penicillinat 37 �C, 5% CO2, and saturated humidity. A total of5� 104e2� 105 cells in log phase were seeded intoa 35-mm Greiner dish for 125I seed irradiation and a totalof 2� 105e106 cells in log phase were seeded intoa 60-mm Greiner dish for 137Cs irradiation 24 h beforeirradiation so that cells were grown to 50e70% confluenceat the time of irradiation. Cells in some dishes were treated

Fig. 1. Typical dose distribution on the 125I seed plane and cell plane. Plot a, arrangement of nine 125I seeds and dose distribution on the plaque. Plot b, dose

distribution on the cell plane. The white circle represents the cell dish. 1, 2, and 3 curve represent 1, 2, 3 Gy isodose curve, respectively.

151H. Chen et al. / Brachytherapy 11 (2012) 149e156

with 20 mM WM (Tocris Cookson Inc., Ellisville, MO) 3 hbefore irradiation to inhibit PI-3 kinase irreversibly (22).

Cell irradiation

For 125I seed irradiation, the 35-mm dish that were seededwith cells 24 h before irradiation was placed at 5 mmdirectlyabove the 125I seed plane and the cells were continuallycultured for different times until the dose minima of thevarying doses along the outer perimeter of the dish reachedto 2 and 4 Gy, respectively. The average dose rate whenthe first cells were irradiated was 4.56e6.08 cGy/h, anddecayed over the time of the experiments to4.32e5.83 cGy/h based on different radioactivity of 125Iseed and absorbed dose of irradiated cells. When each patchof cells was started on irradiation, the mean dose rate on thecell plane was calculated again to correct the dose rate fordecay. The irradiation time varied from 46 to 60 h for 2 Gyand from 92 to 120 h for 4 Gy of 125I seed irradiation.137Cs g-ray irradiation at 0.86 Gy/min (Gammacell-40,MDS Nordion Inc., Toronto, Ontario, Canada) was usedfor the HDR irradiation. The dose rate of 137Cs irradiatorwas calibrated by the secondly standard dosimetrylaboratory in Shanghai Institute of Measurement and TestingTechnology. On the basis of the irradiation condition (theheight of cell culture media is 5 mm), and calculated withthe Monte Carlo simulation code of MCNP5X, the ratio ofabsorbed dose to air Kerma was estimated to be about 0.8by considering only the 662 kV of photon emitted from137Cs. As soon as possible after irradiation, the cells werewashed thoroughly with phosphate-buffered saline,trypsinized and reseeded for further assay of colonyformation and micronucleus (MN) induction. Ku70/80protein expression and phosphorylated Ku80 proteinexpression were assayed by Western Blot.

Colony-forming assay

After 125I seed and 137Cs irradiation, cells weretrypsinized and washed with phosphate-buffered salinetwice and counted. According to irradiation dose,100e500 of A549 and NCI-H446 cells were seeded on60-mm dishes to grow for 10e14 days. Then the colonies

were fixed, stained with Gimesa (Sigma-Aldrich, St. Louis,MO), and counted. The percentage of colony formation wascalculated as the ratio of the numbers of colonies to thenumbers of seeded cells. The surviving fraction wasnormalized to the nonirradiated control. The clonogenicsurvival assay was first repeated three times for each cellline with three replicates each time. If significant differencebetween cell survival exposed to 125I seed and 137Csirradiation was observed, the experiment was repeatedanother two times to further ensure the data reproducibility.

MN assay

MN were evaluated by the cytokinesis-block technique.Briefly, 2 mg/mL cytochalasin B (Sigma-Aldrich) wasadded to the medium allowing cells to be cultured for24 h. The cells growing on the dish were then fixed in situwith methanol/acetic acid (9:1 v/v) and stained withGiemsa. At least 500 binucleated cells were randomlycounted using a microscope for each dish, and the MNformation rate (MFR) was calculated as the ratio of thenumber of MN in binucleated cells to the number ofcounted binucleated cells. Three independent experimentswith three replicates were first performed for each cell line.Like the clonogenic survival assay, the experiment wasrepeated another two times if significant difference betweenMN formation after exposure to 125I seed and 137Csirradiation was observed.

Protein extraction and western blotting assay

As soon as possible after 125I seed irradiation or 3 h afterHDR irradiation, cells were harvested, washed, and stored at�80 �C. For protein extraction, 100 mL of the cell lysis(Pierce Biotechnology, Rockford, IL) containing proteinaseinhibitor (1:100) (Sigma-Aldrich), phosphatase inhibitor(1:100) (Sigma-Aldrich), phenylmethanesulfonyl fluoride(PMSF) (1 mM) (Sigma-Aldrich), and Na3VO4 (2 mM)(Sigma-Aldrich) was added to 1� 106 cells for 30-min treat-ment with occasional mixing every 10 min. Cell lysate wasclarified at 12,000 rpm at 4 �C for 15 min. The supernatantwas collected and used immediately or stored at �80 �C.Total protein concentration was measured using the protein

Fig. 2. Clonogenic survival of A549 and NCI-H446 cells irradiated with

low-dose-rate 125I seeds and high-dose-rate g-ray with and without wort-

mannin (MW) treatment. For A549 cells, results were from three indepen-

dent experiments with three replicates each experiment; for NCI-H446 cells,

results were from five independent experiments with three replicates each

experiment to confirm the significant difference between exposure to 125I

seed and 137Cs g-ray irradiation except in the case of 2-Gy irradiation alone;

bars, standard error (SE).)p!0.05,))p!0.01,)))p!0.001 compared

between the two cell lines under the same treatment and between 125I seed

152 H. Chen et al. / Brachytherapy 11 (2012) 149e156

assay kit (BCA Protein Assay Kit, Pierce Biotechnology)with bovine serum albumin (Sigma-Aldrich) as the standard.Aliquot of 30 mg protein of each sample was separated with10% sodium dodecyl sulfate polyacrylamide gel electropho-resis and then electrotransferred onto polyvinylidene fluoridemembrane (Millipore, Bedford, MA). The Ku proteins weredetected by adding anti-Ku(p70)Ab-4(Clone N3H10),Ku(p80)Ab-2 (Clone 111) (Lab Vision, Fremont, CA),Ku80 (phospho T714) (Novus Biologicals, Littleton, CO),and anti-mouse immunoglobulin G (IgG)-horseradish perox-idase-linked antibodies or anti-rabbit IgG-horseradish perox-idase antibody (Santa Cruz Biotechnology, Santa Cruz, CA)sequentially and then detected by enhanced chemilumines-cence (ECL) detection kit (Pierce Biotechnology). Thecommercial monoclonal antibody of b-actin (Santa CruzBiotechnology) was used as an internal control.

Statistical analysis

The data were obtained from at least three independentexperiments with three replicates in each case, and all dataare presented as means� standard error (SE). Significantdifferences in data of different groups were testified byStudent’s t test and one-way ANOVA at p ! 0.05 usingthe SPSS 12.0 software (SPSS Inc., Chicago, IL).

and 137Cs g-ray irradiation. #p!0.05, ###p!0.001 compared between

2 Gy and MWþ2 Gy for the same kind of cell line under the same kind

of irradiation.

Results

Clonogenic survival

As seen in Fig. 2, after 125I seed irradiation and HDR g-rayirradiation, the surviving fraction of A549 and NCI-H446cells were significantly decreased in a dose-dependentmanner( p!0.001), and the surviving fraction of NCI-H446 cellswas significantly lower than that of A549 cells at 4 Gy. ForA549 cells, the surviving fraction between 125I seedirradiation and HDR irradiation was similar; whereas forNCI-H446 cells, no obvious difference of cell survivalbetween 125I seed irradiation and HDR irradiation wasobserved at 2 Gy, and the surviving fraction exposed to4 Gy 125I seed irradiation was half of that exposed to the samedose of HDR irradiation. The WM treatment resulted insignificant decrease of survivals of A549 ( p!0.05) andNCI-H446 cells ( p!0.001) for both 125I seed and HDR irra-diation at 2 Gy, but the surviving fraction of NCI-H446 cellswas still lower than that of A549 cells ( p!0.001 and p!0.01). Moreover, there was no significant difference on thesurviving fraction of WM-treated A549 cells between 2 Gy125I seed irradiation and HDR irradiation. In contrast, thesurviving fraction of WM-treated NCI-H446 cells exposedto 125I seed irradiation was lower than that exposed to HDRirradiation ( p!0.05).

Micronucleus formation induction

Figure 3 illustrates that the spontaneous MFR of A549and NCI-H446 cells had no significant difference

( pO0.05). After 125I seed irradiation and HDR irradiation,MFR of A549 and NCI-H446 cells was obviously increasedwith radiation dose ( p!0.001), and MFR values ofNCI-H446 cells were 2.6e2.8-folds of A549 cells for alldoses. For A549 cells, MFR between 125I seed irradiationand HDR irradiation was similar at 2 and 4 Gy. While forNCI-H446 cells, MFR value at 4 Gy 125I seed irradiationwas obviously higher than that exposed to HDR irradiation( p!0.01) although they were similar at 2 Gy. The WMtreatment increased the MFR values by about 1.5 timesfor both A549 cells and NCI-H446 cells that were exposedto 2 Gy 125I and HDR irradiations, and the MFR ofWM-treated NCI-H446 cells exposed to 125I seedirradiation was significantly higher than that exposed toHDR radiation ( p!0.05).

It is well known that, as chromosome fragments,radiation-induced MN are resulted from the unrepairedDNA DSBs. Because DSB is a critical lesion leading to celldeath, some type of correlation should exist between theinduction of MN and the decreased cell survival. It wasfound that the MN frequencies of A549 and NCI-H446cells were negatively correlated with the cell survival forboth irradiations with a linear relationship (Fig. 4). ForA549 cells, the values of the correlation coefficient werer25 0.94 and 0.96 for LDR and HDR irradiation,respectively. For NCI-H446 cells, the values of thecorrelation coefficient were r25 0.99 and 0.97 for LDRand HDR irradiation, respectively.

Fig. 3. Micronucleus formation in A549 and NCI-H446 cells irradiated

with low-dose-rate 125I seeds and high-dose-rate g-ray with and without

wortmannin (MW) treatment. For A549 cells, results were obtained in

three independent experiments with three replicates each experiment; for

NCI-H446 cells, results were obtained in five independent experiments

with three replicates each experiment to confirm the significant difference

between exposure to 125I seeds and 137Cs g-ray irradiation except in

the case of 2 Gy irradiation alone; bars, standard error (SE). )p!0.05,

))p!0.01, )))p!0.001 compared between the two cell lines under

the same treatment and between 125I seed and 137Cs g-ray irradiation.

#p!0.05, ##p!0.01 compared between 2 and MWþ2 Gy for the same

kind of cell line under the same kind of irradiation.

153H. Chen et al. / Brachytherapy 11 (2012) 149e156

Expression of Ku80 and Ku70 proteins

As shown in Fig. 5, the cells of A549 and NCI-H446have similar level of Ku80 protein, but the expression ofKu70 protein in A549 was significantly higher than thatin NCI-H446. Exposure to 2 and 4 Gy 125I seed or HDRirradiation had no influence on the total Ku80 and Ku70protein expressions in both cell lines. The WM treatmentdid not change the total Ku80 and Ku70 protein expressionsin A549 and NCI-H446 cells with or without LDR andHDR irradiation.

Ku80 phosphorylation in A549 and NCI-H446 cells

Because the Ku-DNA-binding activity correlates with Kuprotein phosphorylation level (23), the phosphorylated Ku80protein level in A549 andNCI-H446 cells was assayed by theWestern blot assay (Fig. 6). For A549 cells, a major increaseof Ku80 phosphorylation was detected after 2 or 4 Gy of 125Iseed and HDR irradiation, whereas WM treatment couldinhibit Ku80 phosphorylation of irradiated and nonirradiatedA549 cells. The phosphorylated Ku80 protein expression ofNCI-H446 cells increased slightly after 2 Gy 125I seed irradi-ation and 2 and 4 Gy HDR irradiation. However, exposure to4 Gy 125I seed irradiation resulted in obvious decrease of thephosphorylated Ku80 protein level in NCI-H446 cells. WMtreatment inhibitedKu80 phosphorylation inNCI-H446 cellswith 125I seed and HDR irradiation or even withoutirradiation.

Discussion

The present results show that both continuous LDR 125Iseed irradiation and HDR g-ray irradiation led tosignificant decrease of cell survival and obvious increaseof MN in both A549 and NCI-H446 cells, and there wasstrong negative correlation between survival and MNformation. This is in agreement with other report of a strongnegative relationship between clonogenic radiosensitivityand residual DNA DSBs (24). A previous study has demon-strated that NCI-H446 cells was more sensitive to HDRirradiation than A549 cells (25). We found that NCI-H446 cells sensitive to HDR irradiation remained moresensitive to LDR 125I seed irradiation than A549 cells. Itis interesting to note that a change in sensitivity wasobserved at 4 Gy when NCI-H446 cells were irradiatedwith LDR and HDR sources. For NCI-H446 cells, thedecrease of cell survival and increase of MN formationinduced by 4 Gy of 125I seed irradiation were obviouslygreater than those of 4 Gy HDR irradiation, indicating thatNCI-H446 cells were more sensitive to LDR irradiation ata higher dose. But this phenomenon was not observed inA549, that is to say, with respect to a high-dose irradiatedNCI-H446 cells but not A549 cells, LDR irradiation caninduce more lethal damage than HDR irradiation.

It has been reported that tumor growth rate is animportant factor in determining the therapeutic effects of125I seed brachytherapy. Some investigations showed that125I seed brachytherapy was more effective for the cancerwith a long tumor potential doubling time than that witha short tumor potential doubling time (26). We alsoobserved that the doubling time of NCI-H446 cells is about34 h, nearly two times of that of A549 cells with a doublingtime of 18 h. Our results that the NCI-H446 cells havea higher radiosensitive than A549 cells further supportedthe previous finding that cell killing effect of 125I seedbrachytherapy was higher on the slow proliferating tumors.Moreover, the correlation between cell survival and MNinduction suggests that, compared with irradiated A549cells, NCI-H446 cells were much poor in repairing DNAdamage, especially, its efficacy of DNA damage repairduring LDR irradiation may be much lower than that afterHDR irradiation.

To understand the relationship between the difference ofcellular response and DSB repair, we examined theexpressions of total Ku70/80 protein and Ku80phosphorylation for both LDR 125I seed irradiation andHDR irradiation. Importantly, we found that, although theconstitutive Ku80 protein expression was similar in bothcell lines, Ku70 protein expression had higher intrinsiclevel in A549 cells than in NCI-H446 cells. We speculatedthat the lower basal level of Ku70 and the imbalance ofKu70 and Ku80 protein expression in NCI-H446 maycontribute to a higher spontaneous MFR and lowercolony-forming efficiency compared with A549 cells,which can be represented by the fact that the MN formation

Fig. 4. Relationship between clonogenic surviving fraction and micronucleus formation frequency. a. A549 cells exposed to 125I seed irradiation. b. A549

cells exposed to high-dose-rate (HDR) 137Cs g-ray irradiation. c. NCI-H446 cells exposed to 125I seed irradiation. d. NCI-H446 cells exposed to HDR 137Cs

g-ray irradiation.

154 H. Chen et al. / Brachytherapy 11 (2012) 149e156

and colony formation inhibition in NCI-H446 cellsirradiated by 4 Gy continuous LDR 125I seed and HDRirradiation were two- to threefold of those in A549 cells.

In addition, although the total protein levels of Ku70 andKu80 in the two cell lines did not change (Fig. 5), the level ofphosphorylated Ku80 protein in A549 cells increased after 2and 4 Gy 125I seed irradiation and HDR g-irradiation. ForNCI-H446 cells, there was slight increase in phosphorylatedKu80 protein expression after 2 Gy 125I seed irradiation and2 and 4 Gy HDR g-irradiation. However, interestingly, whenthe dosage of continuous LDR irradiation accumulates to4 Gy, the phosphorylated Ku80 protein expression of NCI-H446 cells markedly decreased, suggesting that the DNAdamage/repair system of NCI-H446 cells has changedduring 4 Gy continuous LDR 125I seed irradiation. Negroniet al. (23) showed that the increase of Ku-end-bindingactivity was coincident with the increase of phosphorylatedKu80 protein level, and upregulation of Ku-end-bindingactivity without any marked change in Ku expressionunderlie radiation resistance in a bladder tumor cell line.Therefore, it is very likely that, as a consequence of the low-ered Ku-end-binding activity, the decreased Ku80 proteinphosphorylation result in a decrease of DSB repair efficiency

and then increase the radiosensitivity of NCI-H446 tocontinuous LDR 125I seed irradiation. But the reasons why125I seed irradiation can reduce Ku80 proteinphosphorylation in NCI-H446 cells still need furtherinvestigation (Fig. 6).

To know the effects of DSB repair inhibition on the cellresponse to LDR 125I seed irradiation, we treated bothcell lines with WM and found this drug increased cellsensitivity to both LDR 125I seed irradiation and HDR g-irra-diation. The fungal metabolite WM is an irreversible andnonselective inhibitor of PI-3 kinases including DNA-PK,ataxia telangiectasiamutated (ATM), and ataxia telangiectasiaand Rad3 related (ATR) involved in DNA damage signalingand repair. The IC50 for WM-mediated inhibition of DNA-PK kinase activity was at 5e10 mM and maximal radiosensi-tization was observed in human lung cancer cells treated with20 mM WM (22, 27). We found that treatment with 20 mMWM significantly enhanced radiation-induced MN formationand decreased cell survival in A549 and NCI-H446 cells andincreased their radiosensitivity to both 2 Gy LDR 125I seedand HDR irradiation (Figs. 2 and 3). This agreed with thedecrease of phosphorylatedKu80 protein level inWM-treatedA549 and NCI-H446 cells (Fig. 5). However, treatment of

Fig. 5. Western blot assay of total Ku70 and Ku80 in A549 and

NCI-H446 cells irradiated with low-dose-rate 125I seeds and high-dose-rate

g-ray with and without wortmannin (WM) treatment. No changes of total

Ku70 and Ku80 were observed 3 h after exposure to 125I seed and 137Cs

g-ray radiation with and without WM.

155H. Chen et al. / Brachytherapy 11 (2012) 149e156

NCI-H446 cells with WM allowed cells to be more sensitiveto 125I seed irradiation than to HDR irradiation, whereas thereis no such phenomenon inWM-treatedA549 cells, suggestingthat the WM-dependent radiosensitization of NCI-H446 cellsis partly because of the inhibition of DNA-PK, but otherunknown factors may also be involved.

Fig. 6. Western blot assay of phosphorylated Ku80 (pKu80) in A549 and

NCI-H446 cells irradiated with low-dose-rate 125I seeds and high-dose-rate

g-ray with and without wortmannin (WM) treatment. An increase of

pKu80 in A549 cells was observed 3 h after exposure to 125I seed and

g-ray radiation. Exposure to 2 Gy 125I seed and 2 and 4 Gy g-ray

irradiation induced a slight increase of pKu80 in NCI-H446 cells.

Exposure to 4 Gy 125I seed irradiation decreased the pKu80 expression

in NCI-H446. Treatment of WM with 20 mM reduced the pKu80

expression in A549 and NCI-H446 nonirradiated and irradiated with 125I

seeds and g-ray.

In summary, our results demonstrated that NCI-H446cells with a low proliferation rate become more sensitiveto LDR 125I seed irradiation than HDR irradiation withdose. Furthermore, the radiosensitization effect of WM onNCI-H446 cells exposed to LDR 125I seed is greater thanthat exposed to HDR g-irradiation. However, thisphenomenon is not occurred to A549 cells with a highproliferation rate. The constitutive level of Ku70/Ku80proteins and the postirradiation change of phosphorylatedKu80 protein determining the efficacy of DNA DSBs repairwere the important factor affecting the sensitivity of tumorcells to continuous LDR 125I seed irradiation. Theseproteins together with cell proliferation rate are goodindicators of cell-killing efficiency with respect to LDR125I seed irradiation.

Acknowledgments

We acknowledge the support of the Key TechnologiesResearch and Development Program (No. 045211026) ofthe Science and Technology Commission of ShanghaiMunicipality and National Natural Science Foundation ofChina (Nos. 31070758, 30970870).

Conflicts of interest notification: Any actual of potentialconflicts of interest do not exist.

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