Hypoxia and Cellular Localization Influence the Radiosensitizing Effect of Gold Nanoparticles (AuNPs) in Breast Cancer Cells Author(s): Lei Cui, Kenneth Tse, Payam Zahedi, Shane M. Harding, Gaetano Zafarana, David A. Jaffray, Robert G. Bristow, and Christine Allen Source: Radiation Research, 182(5):475-488. 2014. Published By: Radiation Research Society DOI: http://dx.doi.org/10.1667/RR13642.1 URL: http://www.bioone.org/doi/full/10.1667/RR13642.1

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RADIATION RESEARCH

182, 475–488 (2014)

0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13642.1

Hypoxia and Cellular Localization Influence the Radiosensitizing Effect of Gold Nanoparticles (AuNPs) in Breast Cancer Cells Lei Cui,a Kenneth Tse,b,c Payam Zahedi,a Shane M. Harding,b,c Gaetano Zafarana,e David A. Jaffray,b,d,e,1 Robert G. Bristowb,c,e and Christine Allena,e,1 a Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada; b Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, Canada; c Ontario Cancer Institute/Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada; d TECHNA Institute and Department of Radiation Physics, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada; and e STTARR Innovation Centre, Radiation Medicine Program, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada

are the need to limit dose to spare surrounding healthy tissue (3) and radiation resistance of hypoxic cells in solid tumors (4). Recent physical targeting advancements such as image guidance and intensity modulation have allowed higher radiation dose delivery to tumors while avoiding healthy tissues resulting in improvements in the therapeutic ratio (5). Radiosensitization is another promising strategy designed to increase the biological effect of radiation therapy in a tumor through concurrent treatment with chemical sensitizing agents (i.e., radiosensitizers) such as nitroimidazoles (6), cisplatin (7), iodinated DNA targeting agents (6) and gold nanoparticles (AuNPs) (8). As originally proposed by Boag, the effect of ionizing radiation on biological systems can be divided into three phases: physical, chemical and biological (1, 9). The physical phase is the period in which biological molecules are ionized or excited by radiation to generate free radicals. In the chemical phase, these highly reactive free radicals react with other molecules to ‘‘restore electronic charge equilibrium’’ (1). The biological phase refers to the stage in which the effects of radiation on cells lead to events such as irreparable DNA damage, permanent cell cycle arrest and finally, cell death (1). AuNPs were initially recognized as a potent radiosensitizer due to their significantly larger X-ray cross section (i.e., probability of physical interaction with radiation) in comparison to soft tissues (10). The radiosensitizing effects of AuNPs have been demonstrated both in vitro (11–21) and in vivo (8, 10, 22, 23). However, the experimentally determined radiation dose enhancement factor (DEF) values for AuNPs in biological systems have been significantly higher than those predicted by consideration of physical interactions alone (i.e., calculations based on mass attenuation or Monte Carlo methods) (24). As more studies have been undertaken, it has become evident that as radiosensitizers, AuNPs are also involved in the chemical phase of radiation exposure (25). Additionally, biological pathways through which AuNPs sensitize radiation have been

Cui, L., Tse, K., Zahedi, P., Harding, S. M., Zafarana, G., Jaffray, D. A., Bristow, R. G. and Allen, C. Hypoxia and Cellular Localization Influence the Radiosensitizing Effect of Gold Nanoparticles (AuNPs) in Breast Cancer Cells. Radiat. Res. 182, 475–488 (2014).

Hypoxia exists in all solid tumors and leads to clinical radioresistance and adverse prognosis. We hypothesized that hypoxia and cellular localization of gold nanoparticles (AuNPs) could be modifiers of AuNP-mediated radiosensitization. The possible mechanistic effect of AuNPs on cell cycle distribution and DNA double-strand break (DSB) repair postirradiation were also studied. Clonogenic survival data revealed that internalized and extracellular AuNPs at 0.5 mg/ mL resulted in dose enhancement factors of 1.39 6 0.07 and 1.09 6 0.01, respectively. Radiosensitization by AuNPs was greatest in cells under oxia, followed by chronic and then acute hypoxia. The presence of AuNPs inhibited postirradiation DNA DSB repair, but did not lead to cell cycle synchronization. The relative radiosensitivity of chronic hypoxic cells is attributed to defective DSB repair (homologous recombination) due to decreased (RAD51)-associated protein expression. Our results support the need for further study of AuNPs for clinical development in cancer therapy since their efficacy is not limited in chronic hypoxic cells. Ó 2014 by Radiation Research Society

INTRODUCTION

Radiation therapy is a critical component in the disease management of over half of all cancer patients (1, 2). Two major challenges that limit the efficacy of radiation therapy 1 Addresses for correspondence: Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada, M5S 3M2; e-mail: [email protected] or [email protected]. ca and Princess Margaret Hospital, 5th Floor, Room 631, 610 University Ave., Toronto, Ontario, Canada, M5G 2M9; e-mail: david. [email protected].

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FIG. 1. AuNPs are involved as radiosensitizers in the physical, chemical and biological phases of the effects of radiation on cells. [Timescale adapted from Joiner and van der Kogel, 2009 (1).]

discovered (15). These additional mechanisms may explain the disparity between the experimental measurements and theoretical predictions of DEF. Low-energy photoelectrons and Auger electrons produced during the physical interaction between AuNPs and radiation have a short effective range that is on the nanoscale (26–28). Indeed, in their study, Kong et al. reported that the radiosensitization effect of intracellular AuNPs was more significant than that of AuNPs associated with the cell membrane (11). To date, the contribution of extracellular AuNPs has not been quantitatively assessed. During the chemical phase, a recent study showed that O2– produced by irradiation could bind to the reactive surface of AuNPs, forming AuNP-O2– intermediates. These intermediates can then act as a catalyst to further increase reactive oxygen species (ROS) generation (25), leading to greater cell kill (19). Since oxygen acts as both a substrate and intermediate in ROS generation, lack of intratumoral oxygen could theoretically diminish the radiosensitization effect of AuNPs. Due to the radioresistance of cells under hypoxia it is important to understand the radiosensitization effect of AuNPs under both oxic and hypoxic conditions. Furthermore, biological mechanisms have also been shown to be involved in the radiosensitization effects of AuNPs (24). For instance, Roa et al. demonstrated that cell cycle synchronization caused by AuNPs was the mechanism underlying their radiosensitization, although this phenomenon has not been observed widely (15). To this point, the effects of hypoxia, DNA repair and cell cycle on AuNPassociated radiosensitization have not simultaneously been reported within a specific tumor cell model. As shown in Fig. 1 the aim of the current study was to evaluate aspects of the three phases of radiation effects on

cells with AuNPs as a radiosensitizer. Because of the reported ease with which small-sized nanoparticles penetrate the tumor interstitium, relative to larger particles (29), AuNPs with an average diameter of 2.7 nm were employed. Their radiosensitizing effect was assessed in the human breast cancer cell line MDA-MB-231, which has been used extensively in previous studies investigating AuNPs as a radiosensitizer (16, 20). Specifically, the influence of the cellular localization of AuNPs on their radiosensitizing effect was determined. Given that cell uptake of nanoparticles relies greatly on energy dependent endocytosis (30), which can be impeded under hypoxia (31), uptake of AuNPs was evaluated under both oxic and hypoxic conditions. In addition, the radiosensitization effect of AuNPs was compared under these conditions. To the best of our knowledge, this is the first report of the radiosensitization effects of AuNPs under oxia, acute and chronic hypoxia, as well as the biological effects of these conditions on the radiosensitization by AuNPs. Lastly, the effects of these AuNPs on cell cycle distribution and radiationinduced DNA double-strand breaks (DSB) were evaluated. METHODS All chemicals were purchased from Sigma-Aldrich (Oakville, Canada) and used as received unless otherwise noted. Preparation and Characterization of AuNPs Materials and methods used for the preparation and characterization of the AuNPs can be found in our previously published article (32). In brief, AuNPs with an average diameter of 2.7 nm were prepared by reducing Au3þ using NaBH4 as the reducing agent and tiopronin as the surfactant. The purity of the AuNPs was verified using 1H NMR and the morphology and size were assessed by transmission electron

HYPOXIA INFLUENCES THE RADIOSENSITIZATION BY AuNPs

microscope (TEM) analysis. The stability of AuNPs in water and cell culture media were evaluated using inductively coupled plasma atomic emission spectroscopy (ICP-AES) and TEM analysis (32).

477

cell surviving fraction (SF) was expressed as the plating efficiency achieved with treatment in comparison to that for nontreated cells (1). Cytotoxicity and Radiosensitizing Effects of AuNPs under Oxia

Cell Culture and Hypoxia MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection (ATCCt, Rockville, MD). Cells were cultured in Dulbecco’s modified Eagle medium/Ham’s F12 1:1 Mix media. The cell culture media was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were grown as monolayers at 378C in 5% CO2 and 90% relative humidity. To achieve acute or chronic hypoxia, cells were plated under oxia and incubated for 24 h prior to transfer to a hypoxia chamber with 0.2% O2 for 4 or 72 h incubation, respectively (33).

To evaluate the cytotoxicity of the AuNPs, cells were treated with various concentrations of AuNPs (0–2.0 mg/mL) suspended in cell culture media. After 4, 8 or 24 h of incubation, cells were seeded for clonogenic assessment. Surviving fraction of each treatment group was reported. To characterize the influence of the concentration of AuNPs on their radiosensitizing effect, cells were treated with various concentrations of AuNPs (0–1.0 mg/mL). After 4 h of incubation, cells were exposed to radiation (4 Gy) and seeded for clonogenic assays. The results were plotted as SF ratio using the following equation: SFratio ¼

Quantitative Assessment of the Cellular Accumulation of AuNPs

SFIRþAuNPs =SFAuNPs SFIR

ð1Þ

6

For quantitative analysis of cellular accumulation, cells (1 3 10 cells/well, 6-well plates) were treated with two different concentrations of AuNPs (0.25 and 0.5 mg/mL, which is equivalent to 1.61 and 3.21 lM, respectively) for 1, 4, 8, 16, 24 or 48 h. At each time point cell media was removed, cells were washed three times with phosphate buffered saline (PBS) and then harvested with 0.25% trypsin. Cells were counted using a hemocytometer, centrifuged to a pellet, digested with HNO3 at 908C for 60 min and diluted with distilled deionized (dd) H2O. The amount of gold (Au) was measured by ICP-AES and normalized to the number of cells in each sample. The results were reported as the amount of Au (pg) per cell. For concentration dependent uptake, cells (under oxia, acute hypoxia and chronic hypoxia) were treated with different concentrations of AuNPs (0.01–1 mg/mL) for 4 h, and the amount of Au was evaluated by ICPAES analysis. Qualitative Assessment of the Cellular Accumulation of AuNPs Transmission electron microscopy (TEM) was employed for visualization of the cellular accumulation and intracellular distribution of the AuNPs as outlined elsewhere (34). In short, cells were plated into 6-well plates at a density of 1 3 106 cells/plate. After 24 h of incubation, cells were treated with 0.5 mg/mL AuNPs in cell media. At different time points (20 min, 1 or 4 h) cells were washed twice with PBS, fixed and sectioned. Each section was placed onto a copper grid and imaged by TEM. Alternatively, cells were transferred to the hypoxia chamber and incubated for 4 (acute hypoxia) or 72 h (chronic hypoxia), treated with a 0.5 mg/mL AuNP solution for 4 h, washed, fixed and sectioned for TEM analysis.

where SFIRþAuNPs represents the SF of cells treated with AuNPs prior to irradiation, SFAuNPs is the SF of cells treated with AuNPs alone, and SFIR represents the SF of cells treated with radiation alone. SFAuNPs was included in the equation to exclude the toxic effect of AuNPs. The impact of incubation time on the radiosensitizing effect of the AuNPs was investigated. After plating and incubation, the cell culture media was replaced with fresh media containing 0 or 0.25 mg/mL of AuNPs. Cells were exposed to radiation (0, 2, 4, 6 Gy) post 20 min, 1, 4, 8, 16 or 24 h of incubation with the AuNPs. Survival curves were produced for each time point with or without pretreatment with AuNPs. The data were fitted with a linear-quadratic model: SF ¼ exp (–aD – bD2), as previously reported (17). The DEF of the AuNPs was calculated as the ratio of the radiation doses, which resulted in 0.1 SF with or without AuNPs (17). For studies assessing the dependence of the radiosensitizing effect of AuNPs on their localization with respect to cells (i.e., intracellular or extracellular; shown in Fig. 6A), four treatment groups were used: 1. control (no AuNPs); 2. AuNPs (0.5 mg/mL) added to cells immediately prior to irradiation; 3. cells pretreated with 0.5 mg/mL AuNPs for 4 h, washed twice with PBS, cell media replaced with fresh media without AuNPs followed by irradiation; and 4. cells pretreated with 0.5 mg/mL AuNPs for 4 h then exposed to radiation with the AuNPs remaining in the cell media. For all treatment groups, cells were kept on ice prior to addition or removal of AuNPs to avoid energy-dependent endo- or exocytosis. Cells were then irradiated at 0, 2, 4 and 6 Gy for colony formation and calculation of clonogenic survival. Final survival curve data was fitted to the linear-quadratic model of cell kill. Radiosensitizing Effects of AuNPs under Acute and Chronic Hypoxia

Radiation Source and Dose Calculations for Cell Irradiation Studies The energies used were obtained from a Gulmay D3225 orthovoltage unit at dose rates of 3.47 Gy/min at 225 kVp, or 2.97 Gy/min at 225 kVp for experiments investigating radiosensitization under hypoxia. Dose calculations for cell irradiation were performed as previously described (34). Clonogenic Survival Assays Cell survival was evaluated by clonogenic assay as described previously (35). In brief, cells were plated in 6-well plates at a density of 1 3 106 (unless otherwise noted) and incubated for 24 h prior to treatment. After treatment (e.g., addition of AuNPs and/or radiation), cells were washed twice with PBS and trypsinized. For each treatment, cells were counted and seeded into cell culture dishes at different cell densities to produce an appropriate number of colonies. After 14 days, the colonies were fixed and stained with 1% methylene blue in 50% ethanol. The number of colonies (at least 50 cells) was counted. The

Hypoxia in solid tumors can exist as acute or ‘‘cycling’’ hypoxia due to altering and dynamic flow in blood vessels (in which cells undergo cycles of hypoxia and reoxygenation over minutes to hours) and chronic or diffusion-limited hypoxia as a function of proliferation and increasing distance from blood vasculature (lasting hours to days) (36, 37). To evaluate the effect of different types of hypoxic conditions on the radiosensitizing effect of the AuNPs, five different experimental conditions were used (38): 1. incubation and irradiation under oxia (oxia!oxia); 2. incubation under chronic hypoxia and irradiation under hypoxia (chronic hypoxia!hypoxia); 3. incubation under chronic hypoxia followed by reoxygenation and irradiation under oxia (chronic hypoxia!oxia); 4. incubation under acute hypoxia and irradiation under hypoxia (acute hypoxia!hypoxia); and 5. incubation under acute hypoxia followed by reoxygenation and irradiation under oxia (acute hypoxia!oxia). Cells were seeded in 6-well plates at a density of 0.3 3 106 or 1 3 106 cells/well, and after 24 h of incubation under oxia, the plates containing 1 3 106 cells were transferred to the hypoxia chamber. After 72 h, the plates originally containing 0.3 3 106 cells were

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transferred into the same hypoxia chamber. For both groups, cell culture media was replaced with fresh media (pre-degassed in the hypoxia chamber) containing 0 or 0.5 mg/mL AuNPs, and the cells were further incubated in the chamber for an additional 4 h. In parallel, another group of cells was incubated with AuNPs under oxia. For the hypoxia!hypoxia group, hypoxia was maintained during irradiation by enclosing plates within a container degassed in the hypoxia chamber that was completely sealed. For the hypoxia!oxia group, cells were exposed to air for reoxygenation 10–15 min prior to irradiation, as described in previous publications (39, 40). After 5 Gy irradiation, cell survival was evaluated by clonogenic assay. The radiosensitizing effects were calculated as SF ratios, as described above in Eq. (1). The influence of oxygen on radiation was calculated in two different ways. In the first approach the SF of the hypoxia!hypoxia treatment group was compared to that of the oxia!oxia group. In the second approach the SF of the hypoxia!hypoxia treatment group was compared to that of the hypoxia!oxia group. In the current study, oxygen enhancement ratios (OER) of Cell Kill 1 and 2 (i.e., OER-K1 and OER-K2) were used to describe the effects of oxygen and hypoxia on radiation-induced cell kill as follows: OER  K1 ¼

SFhypoxia!hypoxia SFoxia!oxia

ð2Þ

OER  K2 ¼

SFhypoxia!hypoxia SFhypoxia!oxia

ð3Þ

where SFhypoxia!hypoxia, SFoxia!oxia and SFhypoxia!oxia refer to the SF of cells after irradiation for the hypoxia!hypoxia, oxia!oxia and hypoxia! oxia groups, respectively. Western Blot Analysis Cells were seeded in 15 cm tissue culture dishes at densities of 2 3 106 or 6 3 106 cells/dish. After incubation under oxia, the dish with 6 3 106 cells was transferred to the hypoxia chamber. After 72 h, the dish originally containing 2 3 106 cells was transferred into the same hypoxia chamber. For the latter group, the cell culture media was replaced with pre-degassed fresh media. After an additional 4 h of incubation under hypoxia, the cells were washed with degassed PBS and collected using a cell scraper. The cells were lysed using a lysis buffer (50 mM Tris-HCl pH 7.5/150 mM NaCl/1 mM EDTA/1% NP40/1 unit/mL benzonase]) (Novagen, Billerica, MA) containing protease (1 mM benzamidine hydrochloride hydrate/1 lg/mL antipain/ 5 lg/mL aprotinin/1 lg/mL leupeptin/1 mM phenylmethanesulfonyl fluoride) and phosphatase (10 mM sodium fluoride/2 mM imidazole/ 1.15 mM sodium molybdate/4 mM sodium tartrate/2 mM sodium pyrophosphate/2 mM b-glycerophosphate/2 mM sodium orthovanadate) inhibitors (41). For Western blots, an 8% running gel was cast (8% 37.5:1 acrylamide:bisacrylamide/200 mM pH 7.0 Tris-acetate buffer/0.75 mg mL-1 ammonium persulfate/0.125% TEMED), and overlayed with 6% stacking gel. Fifteen microliters of sample in the sample buffer solution (6% glycerol/0.83% b-mercaptoethanol/1.71% SDS/0.058 M Tris-HCl pH 6.8/0.002% Bromophenol Blue) was loaded into each well. Gels were run at 100 V in Tris-acetate running buffer (50 mM tricine/50 mM Tris/0.1% SDS/5 mM sodium bisulfite). The proteins were then transferred onto a PVDF membrane in transfer buffer (0.145% Tris/0.72% glycine/25% methanol) at 25 V and 100 mA in an ice packed bucket for 1 h. The membrane was blocked in Odysseye Blocking Buffer for 1 h, and incubated in primary antibodies [Rad51 (H-92) sc-8349, rabbit polyclonal IgG; Ku70 (A-9) sc-5309, mouse monoclonal IgG2a] (Santa Cruz Biotechnology, Santa Cruz, CA) 1,000 times diluted in Tris-buffered saline (TBS)/0.1% Tweent 20 overnight at 48C. After washing, the membrane was incubated in secondary antibodies (donkey anti-rabbit IRDyet 800 CW 926–32213, Donkey anti-mouse IRDyet 680 926–32222, LICORt Biosciences, Lincoln, NE), which were diluted 20,000-fold in

Odyssey Blocking Buffer containing 0.1% Tween 20. After washing with 0.1% Tween 20 in TBS, blots were imaged using the Odyssey scanner, and densitometry was conducted using an Odysseyt Infrared Imaging System (LI-COR Biosciences). The total amount of proteins in each sample was normalized to 1 by the amount of Ku70, and the amount of Rad51 was normalized to the total amount of proteins. Cell Cycle Analysis Cells were plated in 6-well plates at a density of 1 3 106 cells/well. After 24 h of incubation, the cell culture media was replaced with fresh media containing no or 0.5 mg/mL AuNPs. BrdU was added to the wells 1 h prior to fixation at a final concentration of 50 lM and after 1, 4, 8, 16, 24 or 48 h of incubation, cells were harvested using trypsin. Cells were then centrifuged to a pellet and washed twice with PBS. The pellet was resuspended in 100 lL of PBS and fixed in 5 mL of 75% ethanol. Fixed cells from early time points were kept at 208C for storage. After all the samples were collected, the cells were centrifuged to a pellet, the supernatant was removed and the pellet was loosened by vortexing. The samples were then incubated in 1 mL of 2 N HCl with 0.5% Triton X-100 for 30 min to denature the DNA. The cells were centrifuged again, and resuspended in 1 mL of 0.1 M Na2B4O7 to neutralize the acid. For each sample, an aliquot of 106 cells was put into a new tube, centrifuged and resuspended in 50 lL of 0.5% Tween 20 (v/v) plus 1.0% BSA (Rockland Immunochemicals Inc., Gilbertsville, PA) (w/v) in PBS. Twenty microliters of anti-BrdU FITC (BD Biosciences, Mississauga, Ontario, Canada) was added to each sample and then all the samples were incubated at room temperature for 30 min in the dark. The cells were centrifuged, resuspended and incubated in 1 mL of 5 lg/mL of PI (Life Technologies Inc., Burlington, CA) and 10 lM of RNAse A in PBS for 15–30 min. Cell cycle analysis was performed on a FACSCalibure flow cytometer (Canto II FCF, BRV), and the data were analyzed using FlowJo software. Immunofluorescence Assay The immunofluorescence assay used was previously described (41). Cells were seeded onto 18 3 18 mm glass cover slips in 6-well plates. After incubation, the cell culture media was replaced with fresh media containing no or 0.50 mg/mL AuNPs and incubated for 4 h. Cells were then exposed to radiation (0, 2 or 4 Gy), and further incubated at 378C for 30 min or 24 h before fixation. To exclude cells in the S phase, which contain endogenous DSB, 5-ethynyl-2 0 deoxyuridine (EdU) (Invitrogene, Burlington, CA) was added to the cells at a final concentration of 10 lM 1 h prior to fixation (41). Cells were washed with PBS and fixed with 4% paraformaldehyde and 0.2% Triton X-100 in pH 8.2 PBS at room temperature for 20 min, and were then washed with PBS and permeabilized with 0.5% Nonidet P40 (NP 40) in PBS. After another rinse with PBS, cells with incorporated EdU were labeled using the Click-iTt EdU Alexa Fluort 647 kit (Invitrogen) following the manufacturer’s protocol with slight modification (41). Cover slips were inverted onto parafilm containing 50 lL of reaction solution and incubated for 30 min. The staining was followed by 3 washes with PBS. Cells were then blocked with 1% normal donkey serum (Jackson Immunoresearch Laboratories Inc., West Grove, PA) and 2% BSA (Rockland Immunochemicals) in PBS for 1 h at room temperature and incubated on parafilm with primary antibodies in 3% BSA/PBS at 48C overnight. The primary antibody used in this study was c-H2AX (mouse monoclonal, JBW301 05–636 1:800, Millipore, Billerica, MA). After primary antibody incubation the coverslips were washed three times for 5 min each with 0.175% Tween 20 and 0.5% BSA in PBS, then incubated in secondary antibodies, donkey anti-mouse Alexa 488 (Invitrogen), on parafilm for 45 min at room temperature with light shielding. The coverslips were washed again three times for 5 min each with 0.175% Tween 20 and 0.5% BSA in PBS, and

HYPOXIA INFLUENCES THE RADIOSENSITIZATION BY AuNPs

479

incubated in 0.1 lg/mL 4 0 ,6-diamidino-2-phenylindole (DAPI, Invitrogen) for 10 min at room temperature. After rinse with PBS, the cover slips were mounted onto glass slides with Vectashieldt anti-fade (Vector Laboratories, Burlingame, CA). Cells were imaged as previously described using a 603 oil immersion objective (41). For each treatment group, at least 50 nuclei were analyzed using Image-Prot Plus software (Media Cybernetics Inc., Rockville, MD). For foci counting, cells in their S phase (EdU positive stain) were excluded, and manually adjusted thresholds were maintained for treatment groups with or without AuNPs. For samples fixed 30 min post 4 Gy irradiation, the ‘‘top hat’’ filter was applied to increase the resolution of foci. Images for publication were prepared using ImageJ software (NIH, Bethesda, MD). Results are reported as number of foci per nucleus (Fig. 9B) and SEM values represent experiment-to-experiment variability. Statistical Analysis Statistical analyses were performed using the Statistical Package for the Social Sciences V16.0 (SPSS Inc.). A two-sample t test was used to measure statistical significance between pairs of results. For statistical analyses among three or more groups, one-way analysis of variance (ANOVA) was used and subsequent multiple comparisons with Bonferroni correction were performed when statistical significance was detected by the ANOVA F test. A P value ,0.05 was considered to be significant (P , 0.05).

RESULTS

Cytotoxicity of the AuNPs

Cytotoxicity of AuNPs has been reported by several groups (20, 42, 43), therefore, an initial study was conducted to identify an optimal concentration of AuNPs that killed no more than 60% of cells to observe an additional effect of ionizing radiation and determine clonogenic cell radiosensitization based on combined agent studies. As shown in Fig. 2, a concomitant increase in the cytotoxicity of the AuNPs was observed with both an increase in the concentration of particles and incubation time. Since a concentration of 0.5 mg/mL AuNPs showed significantly lower cytotoxicity relative to higher concentrations (1.0 and 2.0 mg/mL) after a 4 h incubation period, this concentration was selected for studies examining the influence of localization of AuNPs on their radiosensitizing effect. A lower concentration of 0.25 mg/mL AuNPs was selected to investigate time-dependent radiosensitization, as it led to no more than 60% clonogenic kill on its own. Cellular Accumulation of the AuNPs

The cellular uptake of AuNPs was both concentrationand time-dependent (Fig. 3A). Statistically significant differences (P , 0.05) in the cellular levels of AuNPs were observed at all time points after incubation with the two different concentrations of particles (0.25 and 0.5 mg/ mL). The intracellular level of Au after 20 min of incubation was below the detectable limit of the ICP method employed for analysis (Fig. 3C). The cellular level of Au was found to increase in the first 8 h of incubation then decreased in the

FIG. 2. Surviving fraction after 4, 8 or 24 h of treatment with different concentrations of AuNPs. *Significant difference between groups. Data represents mean 6 SEM (n ¼ 3).

ensuing 40 h. After a 4 h incubation period, the amount of Au in cells was approximately 5% of the total amount of Au used for treatment (i.e., 95% of Au remained in the cell culture media) for both concentrations evaluated. Results from the concentration dependent uptake studies (Fig. 3B) revealed that for cells under oxia, the level of AuNP cell uptake increased for concentrations up to 0.5 mg/ mL, then began to plateau at higher concentrations (i.e., 0.75 and 1.0 mg/mL). For cells under hypoxia, uptake of AuNPs was much lower compared to cells under oxia. Incubation of cells with 0.5 mg/mL AuNPs under oxia resulted in intracellular levels of Au that were over threefold higher than levels achieved under chronic and acute hypoxia. Cells under both chronic and acute hypoxia showed similar uptake patterns over a broad concentration range of AuNPs (Fig. 3B). The cellular localization of the AuNPs was visualized by TEM analysis (Fig. 3C). From these images it can be seen that after 20 min of incubation no cellular uptake of AuNPs is evident. Upon entering the cells, after 1 h of incubation, the AuNPs are mostly visible in the perinuclear region and are sequestered in large clusters in vacuoles such as endosomes and/or lysosomes. No localization of the AuNPs in organelles such as the nucleus or mitochondria is evident in the images. Similar observations were made after TEM analysis of cells incubated with AuNPs for 4 h under both oxia and hypoxia. The Influence of Time, Concentration and Cellular Localization on the Radiosensitizing Effect of AuNPs =SFAuNPs Figure 4 shows the SF ratio ðSFratio ¼ SFIRþAuNPs Þ as SFIR a function of the concentration of AuNPs. The SF ratio reached a minimum at a concentration of 0.5 mg/mL

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FIG. 3. Panel A: Cellular uptake of the AuNPs after incubation over 48 h. *Statistically significant differences between the two concentrations (P , 0.05). Panel B: The cellular level of Au after a 4 h incubation period with seven different concentrations of AuNPs under oxia, chronic hypoxia and acute hypoxia. *Statistically significant differences between oxia and hypoxia (P , 0.05). #Statistically significant differences between 0.5 mg/mL and other concentrations under oxia (P , 0.05). Data represents mean 6 SEM (n ¼ 3). Panel C: TEM images of cells after incubation with AuNPs under oxia 20 min (I and II); 1 h (III and IV); 4 h (V and VI); 4 h under chronic hypoxia (VII and VIII); and 4 h under acute hypoxia (IX and X). II, IV, VI, VIII and X represent high magnification images of selected views in I, III, V, VII and IX. The scale bar represents 2 lm in images I, III, V, VII and IX, and 500 nm in images II, IV, VI, VIII and X.

AuNPs. The radiation dose-response curves for cells after different incubation times with AuNPs are shown in Fig. 5. The fit parameters (a and b), goodness of fit (R2) for the radiation dose-response curves and the values for DEF at 0.1 SF are summarized in Table 1. The data show that an incubation time of 1 h or longer results in similar values for DEF. The impact of cellular localization of AuNPs on their radiosensitizing effect is shown in Fig. 6B. The fit parameters (a and b) and goodness of fit (R2) for the radiation dose-response curves, as well as the values for DEF at 0.1 SF are summarized Table 2. AuNP Radiosensitization under Acute and Chronic Hypoxia

FIG. 4. The radiosensitizing effect of AuNPs after a 4 h incubation period prior to irradiation (4 Gy). The SF ratio is described by the following equation: (SFIRþAuNPs / SFAuNPs)/ SFIR. *Statistically significant differences in the SF ratio at 0.5 mg/mL AuNPs and other concentrations.

The surviving fraction of cells treated with AuNPs and radiation under oxic and hypoxic conditions is plotted in Fig. 7A. Chronic hypoxia alone caused significant cell death (SF ¼ 0.47 6 0.04), while acute hypoxia was less damaging (SF ¼ 0.93 6 0.04). To exclude the effects of hypoxia, cell survival was replotted in Fig. 7B, with normalization for the toxicity associated with each hypoxic condition. The

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HYPOXIA INFLUENCES THE RADIOSENSITIZATION BY AuNPs

FIG. 5. Radiation dose-response curves for cells incubated with AuNPs for different periods of time (i.e., 20 min, 1, 4, 8, 16 or 24 h) and irradiated at 0, 2, 4 and 6 Gy. Data points represent mean 6 SEM (n ¼ 3).

TABLE 1 Fitted Parameters Obtained Using the LQ model, and DEF Calculated at 0.1 SF for Experimental Data Shown in Fig. 5 Radiation þ AuNPs

Radiation a

Incubation time 20 min 1h 4h 8h 16 h 24 h

0.59 0.66 0.73 0.50 0.47 0.33

6 6 6 6 6 6

b 0.06 0.07 0.08 0.10 0.06 0.09

0.049 0.033 0.022 0.047 0.056 0.073

6 6 6 6 6 6

R 0.010 0.012 0.014 0.018 0.010 0.017

2

0.984 0.978 0.971 0.939 0.981 0.942

a 0.62 0.99 1.03 0.90 0.69 0.58

6 6 6 6 6 6

0.06 0.08 0.08 0.12 0.07 0.06

b

R2

0.055 6 0.011 1.35*1016 4.07*1014 0.023 6 0.022 0.068 6 0.013 0.082 6 0.010

0.982 0.915 0.869 0.945 0.980 0.984

DEF at 0.1 SF 1.04 1.31 1.26 1.44 1.33 1.31

6 6 6 6 6 6

0.02 0.05 0.01 0.02 0.08 0.06

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radiosensitizing effects of AuNPs were calculated as SF =SFAuNPs ratios ðSFratio ¼ SFIRþAuNPs Þ as shown in Table 3. SFIR Reduced Expression of Rad51 in Cells under Chronic Hypoxia

Chronic hypoxia for up to 72 h has previously been shown by our laboratories to lead to decreased translation of expression of RAD51, a master protein involved in the homologous recombination DSB repair pathway. This led to a decreased OER when compared to acute hypoxic treatments (37). We therefore evaluated RAD51 expression relative to Ku70 (a protein used in the nonhomologous recombination pathway that is not affected by chronic hypoxia) as a protein expression control, (37). Consistent with our previous studies, we observed reduced RAD51 expression in chronic hypoxia-treated cells compared to oxic or acute hypoxia-treated cells (Fig. 7C). For chronic hypoxic cells the amount of Rad51 decreased to 43%, while for cells exposed to acute hypoxia the value was 95%. The Effect of AuNPs on Cell Cycle Distribution and Postirradiation DNA Double-Strand Breaks

FIG. 6. Panel A: Treatment groups to assess the dependence of the radiosensitizing effect of AuNPs on their localization with respect to cells. Panel B: Radiation dose-response curves for cells with no AuNPs or intracellular and/or extracellular AuNPs. Data points represent mean 6 SEM (n ¼ 3).

Figure 8 summarizes the cell cycle distribution of cells treated with AuNPs for up to 48 h. Statistical analysis revealed that at each time point the percentage of cells in the G2/M phase was similar for the control and the AuNPs treated groups. Figure 9A shows the representative images from the immunofluorescence assay. Cells in their S phase containing endogenous DSB (EdU positive) were excluded from foci counting. Treatment of cells with AuNPs alone for 24 h did not cause DSB. With the presence of the AuNPs, there was no increase in the number of foci (c-H2AX) 30 min postirradiation. The residual breaks (24 h postirradiation) were increased from 10.41 6 0.66 to 13.98 6 0.37 when cells were irradiated with 2 Gy, and from 23.17 6 1.04 to 34.71 6 2.01 for 4 Gy irradiation (Fig. 9B). DISCUSSION

As shown in the Introduction section and Fig. 1, the effect of radiation on biological systems can be divided into TABLE 2 Fitted Parameters Obtained Using the LQ Model, and DEF Calculated at 0.1 SF for Experimental Data Shown in Fig. 6 AuNPs Treatment group

Extracellular

Intracellular

1 2 3 4

– þþþ – þþ

– – þ þ

a 0.69 0.74 1.00 1.02

6 6 6 6

0.02 0.04 0.13 0.17

b

R2

DEF at 0.1 SF

0.012 6 0.004 0.019 6 0.007 1.50e – 16 1.57e – 16

0.997 0.992 0.940 0.900

1 1.09 6 0.01* 1.39 6 0.07* 1.41 6 0.08

Note. Treatment groups: 1. No AuNPs; 2. AuNPs added immediately prior to irradiation; 3. Removal of AuNPs in cell culture media after 4 h of pretreatment; 4. AuNPs remained in cell culture media after 4 h of pretreatment. þ Relative amount of AuNPs. – Absence of AuNPs. * Statistically significant difference between the DEF values of treatment groups 2 and 3 (P , 0.05).

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HYPOXIA INFLUENCES THE RADIOSENSITIZATION BY AuNPs

TABLE 3 Surviving Fraction Ratio at 5 Gy Treatment groups Oxia!oxia Chronic hypoxia!hypoxia Chronic hypoxia!oxia Acute hypoxia!hypoxia Acute hypoxia!oxia

FIG. 7. Panel A: Survival of cells after irradiation and treatment with AuNPs under oxia or hypoxia as measured by clonogenic assay. ‘‘ þ’’ indicates cells receiving AuNPs or radiation (IR) treatment; ‘‘–’’ indicates absence of the treatment. Blue squares ‘‘ þ’’ indicate hypoxia!hypoxia groups; red squares ‘‘ þ’’ indicate hypoxia!oxia groups. SF is reported as plating efficiency compared to the control group under oxia. Data represents mean 6 SEM (n ¼ 3). Panel B: Survival of cells with toxicity of hypoxia normalized. Data represents mean 6 SEM (n ¼ 3). Panel C: Protein expression levels of Ku70 and Rad51 in cells under oxia, chronic hypoxia and acute hypoxia. Numbers in parentheses indicate the relative amount of Rad51 in cells after normalization with the corresponding Ku70 level.

Surviving fraction ratio 0.11 0.22 0.13 0.61 0.12

6 6 6 6 6

0.09 0.08 0.02 0.07 0.02

physical, chemical and biological phases. AuNPs have become a radiosensitizer of significant, widespread interest and likely impact these three phases. The physical interaction between AuNPs and radiation is known to generate large numbers of electrons and photons (10). The photoelectric effect and subsequent Auger cascade occur at kVp radiation energies producing numerous photoelectrons and Auger electrons. These electrons then interact with the biological components of cells and transfer their energies to the latter by producing radicals and ions, leading to increased cell damage and ultimately radiosensitization (24). Using plasmid DNA as a model, previous studies have shown that most electrons released from AuNPs irradiated by kVp X rays are associated with low energy that result in ‘‘localized energy deposition’’ at the nanoscale (26, 28). In vitro studies have further demonstrated that AuNPs in the cytoplasm of cells are more effective radiosensitizers than those attached to the cell membrane, highlighting the role of short-range electrons under kVp X rays. These observations emphasize the importance of targeting AuNPs to cellular components for achieving maximal radiosensitization (11, 13). The pioneering in vivo study by Hainfeld et al. showed the significant radiosensitizing effect of AuNPs at 2 min post intravenous administration of the formulation in mice using a 250 kVp X-ray source (8). Although no information on the intratumoral distribution of the AuNPs was provided, it is likely that the AuNPs were localized primarily in the extracellular matrix of tumors at the time of irradiation. Thus, these results indicate that extracellular AuNPs can significantly enhance the effect of radiation. Pignol et al. have suggested that AuNPs outside cells may elicit a ‘‘crossfire’’ effect in which longer range electrons released from AuNPs travel a distance of several cell diameters to interact with cell nuclei, resulting in radiosensitization (44). Further investigation is needed to quantitatively verify the effect of long-range electrons. Also notably, in several in vitro studies, radiation has been applied to cells without prior removal of AuNPs from the cell culture media (12, 15–18, 20). Although cellular uptake of AuNPs was evaluated in some of these studies, the contribution of the AuNPs outside the cells to radiosensitization was not quantitatively analyzed. Given that the amount of AuNPs remaining in the cell culture media could be significantly higher than that inside cells, it is important to delineate and

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FIG. 8. Cell cycle distribution in cells exposed to AuNPs (0.5 mg/mL) for 1, 4, 8, 16, 24 or 48 h.

understand the effect of AuNPs in cell culture media versus that associated with AuNPs in cells. In our study, cellular uptake of AuNPs was evaluated prior to quantitatively analyzing the impact of the cellular localization of AuNPs on radiosensitization. The kinetics of cellular uptake of AuNPs is a combined effect of endocytosis, exocytosis and cell proliferation (32). Internalization of the AuNPs under oxia was found to be dependent on time and concentration (Fig. 3A). This is in agreement with our previous study and findings by others (32, 45). As shown in Fig. 3B, concentration-dependent cell uptake under oxia showed that uptake increased almost linearly with AuNP concentrations up to 0.5 mg/mL followed by a plateau. A similar trend was observed by Chithrani et al. and was attributed to saturation of receptormediated endocytosis, since uptake of these AuNPs was shown to be mediated by nonspecific adsorption of serum proteins onto the particles (45). Interestingly, in our study at the same concentration of AuNPs, the level of uptake was much lower for hypoxic cells compared to oxic cells. This phenomenon can be attributed to decelerated endocytosis, which has been shown to result under hypoxia due to impeded fusion of early endosomes and the prolonged halflife of receptor tyrosine kinases (31, 46). Studies examining the radiosensitizing effect of AuNPs (Fig. 4) revealed that radiosensitization increased with AuNP concentrations up to 0.5 mg/mL. The lower radiosensitizing effect seen at higher concentrations (0.75 and 1.0 mg/mL) is due to saturated uptake, as well as the higher toxicity of the AuNPs at these concentrations. As shown in Table 1, similar values of DEF were observed for incubation times of 1 h or longer, but the DEF was much

lower for 20 min of incubation. Given that no effective uptake was detected 20 min post incubation, it can be concluded that intracellular AuNPs play the most significant role in radiosensitization. A similar observation was made by Zhang et al., in which a higher DEF was observed for intracellular AuNPs compared to those attached to the cell membrane (13). The key role of AuNPs inside cells was further verified, as shown in Fig. 6. Although the AuNPs in the cell culture media comprised about 95% of the total Au content, radiation enhancement was not significant (DEF ¼ 1.09 6 0.01), while AuNPs inside cells led to a significantly higher DEF of 1.39 6 0.07. These findings suggest that efficient delivery of AuNPs into target cells is crucial for full exploitation of their radiosensitization effects. For future in vivo and clinical applications, parameters such as route of administration of AuNPs and timing sequence of AuNPs and radiation therapy should be carefully considered to achieve the maximum level of cell uptake in the target tissue at the time of irradiation. Aside from enhanced physical interaction with radiation, AuNPs are also involved in the chemical phase of radiation through the generation of elevated ROS levels (19, 47). The increase in ROS generation is partly due to the secondary electrons emitted from AuNPs, which subsequently interact with molecules in close proximity to produce ROS (47). Cheng et al. have demonstrated the concept of ‘‘chemical enhancement’’ achieved by the reactive surface of AuNPs (25). In this study, the enhanced hydroxylation of coumarin carboxylic acid under irradiation was attributed to the ‘‘increased conversion of intermediates to the products’’. It was proposed that superoxide produced by radiation exposure activates the slightly electronegative surface atoms

HYPOXIA INFLUENCES THE RADIOSENSITIZATION BY AuNPs

FIG. 9. Panel A: Representative images from the immunofluorescence assay. Panel B: Number of c-H2AX foci 30 min or 24 h postirradiation (0, 2, 4 Gy). *Statistically significant difference between the treatment groups. Data represents mean 6 SEM (n ¼ 3).

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TABLE 4 Effect of Oxygen on Radiation-Induced Cell Kill OER-K1 OER-K2

Chronic hypoxia

Acute hypoxia

1.47 6 0.31a,b 3.38 6 0.24

4.36 6 0.70 4.35 6 1.34

a Statistically significant difference between OER-K1 and OER-K2 for the chronic hypoxia group. b Statistically significant difference between the values of OER-K1 for the chronic and acute hypoxia group.

of the nanoparticles by forming AuNPs-O2–. These reactive molecules then catalyze the reactions between radical intermediates and other molecules to produce more ROS (25). Due to the important role of oxygen in ROS generation as both substrate and intermediate, lack of oxygen will lower the level of ROS generation and therefore the radiosensitization effect of AuNPs. In the current study, the radiosensitizing effect of AuNPs was investigated under hypoxia to confirm its oxygen dependence. The role of oxygen in enhancing the effects of radiation by permanently ‘‘fixing’’ DNA damage has been well established (48), and hypoxia has been long recognized as the main reason for radioresistance in cancer cells (49). In 2002, Zolzer et al. showed that chronic hypoxia can increase the radiosensitivity of cells and reduce the effect of oxygen (38). In their study two different OER values were calculated by using oxic and reoxygenated hypoxic cells as references to distinguish the effect of oxygen on cell physiology and radiochemistry. A pure radiochemical oxygen effect was obtained when the hypoxia!hypoxia group was compared to the hypoxia!oxia group, and the combination effects of oxygen on cell physiology and radiochemistry were observed when oxic!oxia group was used as the reference. As the chronic hypoxic cells were observed to be less radioresistant, the value of combined OER was lower than the pure radiochemical OER (38). In our current study, for chronic hypoxic cells, the combined oxygen effect (OER-K1) was lower than the pure radiochemical effect (OER-K2) (1.47 6 0.31 vs. 3.38 6 0.24), while for acute hypoxic cells OER-K1 and OER-K2 had similar values (4.36 6 0.70 vs. 4.35 6 1.34) (Table 4). These observations confirmed that chronic hypoxia reduces radioresistance of cells. As expected, the values of OER-K2, which represent the pure radiochemical OER, were statistically similar for chronic and acute hypoxic cells (3.38 6 0.24 vs. 4.35 6 1.35). The reduced radioresistance of cells under chronic hypoxia has been related to the decreased capacity for homologous recombination due to decreased translation of homologous recombination-related proteins such as RAD51 (37). To confirm this, the level of RAD51 was evaluated for cells under oxia and hypoxia. Ku70, a protein involved in nonhomologous end joining (NHEJ), was used as a positive control, as levels of this protein should not be affected by hypoxia (37). The fact that the level of Rad51 was

decreased to 43% in chronic hypoxic cells and was relatively unchanged in acute hypoxic cells supports the observation that reduced radioresistance of cells under chronic hypoxia is most likely due to a functional homologous recombination deficiency, since the latter has been observed in similarly treated cells of varying histologic background (37). Table 3 shows that the radiosensitizing effects of AuNPs were lowest for the hypoxia!hypoxia group under acute hypoxia (SF ratio ¼ 0.61 6 0.07), while the value was greater for the hypoxia!hypoxia group under chronic hypoxia (SF ratio ¼ 0.22 6 0.08). The greater radiosensitizing effect of AuNPs for cells under chronic hypoxia was the consequence of homologous recombination deficiency. When cells were exposed to radiation after reoxygenation (hypoxia!oxia), the radiosensitizing effects were similar for both chronic and acute hypoxic groups (0.13 6 0.02 vs. 0.12 6 0.02). Unlike the hypoxia!hypoxia group, the effect of homologous recombination deficiency was not detected and this is likely due to overkill by the presence of AuNPs, which leads to more than a sevenfold increase in cell death when irradiated under oxia. There is growing evidence that biological interactions between AuNPs and cells also contribute to their radiosensitizing effects (24). Roa et al. observed accumulation of cells in G2/M phase after 2 h of exposure to AuNPs, suggesting that cell cycle synchronization is an important biological pathway for radiosensitization (15). In the current study it was shown that cells treated with 2.7 nm AuNPs for up to 48 h did not lead to cell cycle synchronization. Similarly, Butterworth et al. observed no change in cell cycle distribution when cells were treated with 1.9 nm AuNPs for 24 or 48 h (16). Another possible biological mechanism of radiosensitization is inhibition of DNA repair (6). Previous studies have reported different outcomes for the effect of AuNPs on DNA damage postirradiation. Chithrani et al. showed 50 nm AuNPs lead to an increased number of residual foci after 4 Gy irradiation in HeLa cells, however, initial DNA DSBs were not evaluated (17). Jain et al. observed that in MDA-MB-231 cells, there was no increase in number of either initial (1 h postirradiation) or residual foci (24 h postirradiation) using 1.9 nm AuNPs at 1 Gy (20). In our study, no increase in the number of initial foci was observed in the presence of AuNPs, however, the number of residual foci increased significantly. These results indicate that the 2.7 nm AuNPs inhibit DNA repair processes postirradiation. Further studies are needed to elucidate the molecular pathways involved in the inhibited DNA repair that is observed the presence of the AuNPs. It is likely that the physico-chemical properties of AuNPs (i.e., size, surface chemistry), cell line, incubation conditions and radiation dose influence the biological interactions between AuNPs and cellular components and therefore, may explain the different results observed by various groups. Overall, the current study highlights the importance of cell uptake of AuNPs as a means to fully exploit their

HYPOXIA INFLUENCES THE RADIOSENSITIZATION BY AuNPs

radiosensitization effects. In addition, oxygen was shown to play a critical role in determining the extent of radiosensitization by AuNPs. Although cells under acute hypoxia showed the greatest degree of radioresistance, they were still radiosensitized by AuNPs, and therefore should not limit their use as a novel agent in vivo. Furthermore, although the use of AuNPs inhibited postirradiation DNA repair, it did not lead to cell cycle synchronization. Findings from these studies should help guide the design of AuNPs as radiosensitizers and aid in the selection of parameters for further in vitro and in vivo evaluation of their radiosensitization effects.

12.

13. 14. 15. 16.

ACKNOWLEDGMENTS This research was funded by an operating grant from CIHR to D.A. Jaffray, R.G. Bristow and C. Allen and a Terry Fox New Frontiers Program grant. L. Cui has been funded by the MDS Nordion Graduate Scholarship in Radiopharmaceutical Sciences, Hoffmann-La Roche/ Rosemarie Hager Graduate Fellowship and an Ontario Graduate Scholarship. Kenneth Tse has been funded by an Ontario Graduate Scholarship, the Princess Margaret Hospital Foundation and the Terry Fox Foundation Strategic Training Initiative for Excellence in Radiation Research for the 21st Century, CIHR. S. Harding has been funded by an Ontario Graduate Scholarship. L. Cui thanks summer students, Kaiyin Zhu and Kaitlynn Almeida for assistance with cell studies. R.G. Bristow is a Canadian Cancer Society research scientist. Received: December 17, 2013; accepted: August 4, 2014; published online: October 31, 2014

17. 18. 19. 20.

21.

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