Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical

Nuclear Medicine and Biology 39 (2012) 1142–1151

Contents lists available at SciVerse ScienceDirect

Nuclear Medicine and Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n u c m e d b i o

Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical☆ Bart Cornelissen, Sonali Darbar, Veerle Kersemans, Danny Allen, Nadia Falzone, Jody Barbeau, Sean Smart, Katherine A. Vallis ⁎ Department of Oncology, CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, OX3 7LJ Oxford, UK

a r t i c l e

i n f o

Article history: Received 13 April 2012 Received in revised form 11 May 2012 Accepted 2 June 2012 Keywords: Radioimmunoconjugate γH2AX Auger electrons DNA damage Ionizing radiation

a b s t r a c t 111 In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, 111In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether 111 In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6 MBq/μg) can amplify treatment-related DNA damage for therapeutic gain. Methods: MDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with 111InDTPA-anti-γH2AX-Tat (3 MBq, 6 MBq/μg) or a control radioimmunoconjugate, 111In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. 111In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies. Results: The number of γH2AX foci was greater after exposure of cells to IR (10 Gy) plus 111In-DTPAanti-γH2AX-Tat compared to IR alone (20.6±2.5 versus 10.4±2.3 foci/cell; Pb.001).111In-DTPA-anti-γH2AXTat resulted in a reduced surviving fraction in cells co-treated with IR (4 Gy) versus IR alone (5.2%±0.9% versus 47.8%±2.8%; Pb.001). Similarly, bleomycin (25–200 μg/mL) plus 111In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10 Gy) plus 111InDTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either 111In-DTPA-anti-γH2AX-Tat or IR alone (−0.002±0.004 versus 0.036±0.011 and 0.031±0.014 mm 3/ day, respectively, Pb.001). Conclusion: 111In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Approximately 50% of all cancer patients receive radiotherapy either alone or in combination with other treatments such as surgery or chemotherapy [1]. Outcomes after radiotherapy have improved recently as a result of greater spatial precision of radiation delivery to tumor, allowing dose-escalation in the tumor and avoidance of normal tissue. However, some tumors remain poorly responsive to radiation therapy. Recent efforts have been directed towards radiosensitization using, amongst others, antimetabolites, histone deacetylation inhibitors, hypoxia-selective toxins or vascular normalisation [2–4]. As the formation of DNA double-strand breaks (DNA dsbs) is ☆ Financial support: This research was supported through the CR-UK/EPSRC/MRC/ NIHR Oxford Cancer Imaging Centre, the Oxford Experimental Cancer Medicine Centre, the NIHR Oxford Biomedical Research Centre and through a grant to KAV from Cancer Research-UK (C14521/A6245). ⁎ Corresponding author. CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Radiobiology Research Institute, Churchill Hospital, OX3 7LJ Oxford, UK. Tel.: +44 0 1865 255850; fax: +44 0 1865 857533. E-mail address: [email protected] (K.A. Vallis). 0969-8051/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nucmedbio.2012.06.001

one of the most deleterious consequences of exposure to ionizing radiation (IR) and to some chemotherapeutic agents [5], several radiosensitization approaches are designed to inhibit DNA dsb repair signalling or to enhance radiation-induced DNA damage by increasing the formation of reactive oxygen species [1]. Here, we propose a novel and fundamentally different strategy to increase the effectiveness of external beam radiotherapy (IR) and other anti-cancer therapies that cause DNA dsb damage. By targeting the DNA damage response (DDR) protein, γH2AX, with anti-γH2AX antibodies conjugated to the therapeutic radioisotope ( 111In), Auger electrons are emitted at sites of DNA dsb. Since Auger electrons are low-energy, short-range and densely-ionizing radiation, pre-existing DNA dsb damage can be transformed into complex, irreparable DNA damage [6]. The phosphorylation of histone H2AX on serine S-139 by the PI3Klike kinases, ATM, ATR or DNA-PKcs, to form phospho-H2AX (γH2AX) is an early step in the repair of DNA dsbs [7]. γH2AX accumulates and forms foci at sites of DNA dsb where it plays a role in the recruitment of DNA repair proteins, rapid re-joining of DNA ends and the promotion of error-free repair [8]. γH2AX foci can be detected in vitro using an immunofluorescence assay which has been used widely

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151

in the study of DNA damage and its repair following IR and other genotoxic agents [9]. Marked induction of γH2AX follows exposure of cancer cells to genotoxic treatment such as IR. In general, γH2AX foci are slower to resolve in tumor versus normal tissues [10]. These differences in the magnitude and temporal dynamics of γH2AX expression in malignant compared to normal tissues have stimulated interest in γH2AX as a therapeutic target [11]. We previously synthesized 111In-labelled anti-γH2AX antibodybased radioimmunoconjugates (RICs) with the goal of in vivo imaging of DNA dsbs. We have shown that these RICs track to sites of γH2AX foci in vitro and in vivo [12]. Anti-γH2AX-based conjugates produced fluorescent and single photon emission computed tomography (SPECT) signals that could be monitored non-invasively. The problem of delivery of an antibody-based RIC to an intranuclear target was met through incorporation of the cell-penetrating peptide, Tat, into RIC design. Tat is derived from the transactivator of transcription protein of the HIV-1 virus and mediates transport of various cargos (ranging from peptides to nanoparticles) across the cell membrane. The Tatpeptide possesses a nuclear localization sequence, through which it binds to importins for transport into the nucleus [13]. These previous findings raised the question of whether a Tat-modified,γH2AXseeking antibody, labelled at higher specific activity with the Augerelectron-emitting isotope, 111In, could be used to deliver a significant amount of therapeutic radioisotope directly to sites of pre-existing DNA strand breaks. The use of an Auger electron-emitting isotope is particularly advantageous in this setting. Auger electrons are low energy particles that are released from atoms that decay by electron capture. They have a very short track length (in the nm to μm range) and are densely ionizing and so cause intense biological damage within a small volume surrounding the decay site [14,15]. Thus an Auger electron-emitting radionuclide that accumulates at sites of DNA dsbs, through association with γH2AX, would likely increase the damage at that site, converting simple dsbs into more complex damage. This would have the effect of reducing the probability of successful repair of the DNA dsb and of increasing the likelihood of cell kill. In this report it is shown that the radioimmunoconjugate, 111InDTPA-anti-γH2AX-Tat, when radiolabelled to specific activity of 6 MBq/μg, causes additional DNA damage in cells exposed to a genotoxic insult. This amplification of DNA damage translates into significantly increased cancer cell kill both in vitro and in vivo. 2. Materials and methods 2.1. Cell culture MDA-MB-468 breast cancer cells were obtained from the CR-UK cell services laboratories. MDA-MB-231 cells, stably transfected with HER2 (231-H2N), were a gift of Dr Robert Kerbel (Sunnybrook Health Sciences Centre, Toronto, ON) [16]. Cells were cultured in 5% CO2 in DMEM (Sigma-Aldrich, Dorset, UK) supplemented with 10% fetal calf serum (Invitrogen, Paisley, UK) and penicillin/streptomycin, 100 U/ mL (Invitrogen, Paisley, UK). All cell lines were tested and authenticated by the provider. The cumulative culture length of all cells was less than 6 months after retrieval from liquid nitrogen storage. 2.2. Synthesis of radioimmunoconjugates 111 In-DTPA-anti-γH2AX-Tat and 111 In-DTPA-mouseIgG-Tat ( 111In-DTPA-mIgG-Tat) were synthesized as described previously [12]. Anti-γH2AX antibodies were purchased from Merck (product no. DR1017). Antibodies were raised against a synthetic phosphopeptide corresponding to amino acids surrounding the Ser 139 phosphorylation site of human H2AX and has been shown to cross-react with mouse γH2AX. Tat-peptide(GRKKRRQRRRPPQGYG) incorporation was achieved using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/

1143

N-hydroxysuccinimide (EDC/NHS; Pierce, Rockford, IL, USA) activation. Unconjugated Tat was removed using sephadex G50 gel filtration columns. Anti-γH2AX-Tat conjugate was conjugated to the activated metal ion chelator isothiocyanatobenzyl-DTPA (p-SCN-Bn-DTPA) (Macrocyclics, Dallas, TX). Excess unconjugated p-SCN-Bn-DTPA was removed using G50 SEC. BnDTPA-anti-γH2AX-Tat conjugates were radiolabelled with 111In by addition of 111In chloride. Addition of increasing amounts of 111In to BnDTPA-anti-γH2AX-Tat resulted in final product with specific activity ranging from 0 to 6 MBq/μg. Radiolabeling yield (RLY) was determined by instant thin layer chromatography (ITLC) and was≥95%. Chemical and radiochemical purity as determined by size exclusion radio-HPLC of final product was≥95%. 2.3. Retention assay Internalization, nuclear localization and retention of 111In-DTPAanti-γH2AX-Tat in MDA-MB-468 cells were determined as previously described [12]. Briefly, cells were exposed for 1 h to 111In-DTPAanti-γH2AX-Tat (0.5 μg; in 2 mL of medium), labelled at various specific activities (0–6 MBq/μg). Then, cells were irradiated (10 Gy) or mock irradiated (0 Gy), washed, and supplied with fresh growth medium. At 4 h after irradiation, the amount of 111In retained in cells was determined, and expressed as a fraction of the 111In present in cells immediately before irradiation. 2.4. γH2AX assay MDA-MB-468 cells (2×10 5) were seeded on cover slips in 6-well plates, allowed to adhere overnight and then incubated with 111InDTPA-anti-γH2AX-Tat (0.5 μg; 6 MBq/μg in 2 mL of medium) or 111InDTPA-mIgG-Tat (0.5 μg; 6 MBq/μg in 2 mL of medium) for 1 h at 37 °C to allow internalization and nuclear accumulation of RICs. Cells were mock-treated (0 Gy) or X-irradiated (4 or 10 Gy) using a 137Cs irradiator (IBL-637; Cisbio International; dose rate 1.0 Gy/min) and then incubated for 1 h to allow formation of γH2AX foci, or incubated for 24 h to allow repair of DSBs and dissolution of γH2AX foci. Cells were stained for γH2AX foci and mounted with Vectashield® with DAPI (Vector Laboratories, Peterborough, UK), as previously described [12]. 2D Images were acquired using a fluorescence microscope (Carl Zeiss, Welwyn Garden City, UK), and the number of foci per cell was counted. In parallel experiments, cells were exposed to bleomycin (20 μg/mL) for 16 h. 111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgGTat (0.5 μg; 0, 1 or 6 MBq/μg) was added and after 2 h cells were immunostained for γH2AX as described above. The spatial distribution ofγH2AX foci was analyzed using a Matlab tool developed in house, incorporating Ripley's K function [17]. For analysis of the grouping of γH2AX foci Z-stacks (i.e. 3D images) were acquired using a confocal microscope equipped with a piezo-electric element controlled stage (Zeiss 510, Zeiss). On the resulting images, nuclei were delineated in silico based on DAPI signal. γH2AX foci were localized based on immunostaining intensity. The size of the groups of foci was measured as the average ratio of the sphere containing the group. To quantify clustering of γH2AX foci, i.e. the grouping of γH2AX foci in space, an analysis method based on Ripley's K function was used [18]. The Ripley K function is defined as the density of objects in space based on the distance between the objects. When clustering of γH2AX foci occurs, the density at small distances is much greater that that at larger distances. We quantified the size and significance of this difference, compared to randomly distributed γH2AX foci, as a normalized p-value. A p-value of 100% indicates a random distribution of foci within a nucleus, whereas smaller pvalues denote clustering. Corrections were made to account for edgeeffects (i.e. clusters around foci located near the perimeter of the nucleus are limited by the nuclear envelope) and for the absolute number of foci per cell.

1144

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151

2.5. Comet assays Cell suspensions (5×10 5 cells in 500 μL medium) were incubated with 111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgG-Tat (0.5 μg; 6 MBq/μg) for1 h at 37 °C to allow internalization and nuclear accumulation of RICs. Cells were exposed to γ-radiation (0, 4 or 10 Gy), incubated for 1 h at 37 °C to allow for γH2AX formation and then harvested for neutral comet assays using the Trevigen COMETS kit (Trevigen, Helgerman, CT, USA). Briefly, cells were mixed with low melting point agarose which was spread on microscope slides and allowed to set. Cells were lysed with buffer provided in the COMETS kit. In some cases, base damage and AP sites were evaluated by adding glycosylases to cells as described by Collins et al. [19]. The enzymes used were Fpg (New England Biolabs, Ipswich, MA, US; 1:10,000 in 10 mM bis-tris-propane.HCl, 10 mM MgCl2, 1 mM DTT, pH 7.0), Endo III (New England Biolabs, Ipswich, MA, US; 1:10,000 in 20 nM Tris.HCl, 1 mM DTT, 1 mM EDTA, pH 8.0) and Endo IV (New England Biolabs, Ipswich, MA, US; 1:10,000 in 50 nM Tris.HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, pH 7.9). Slides were rinsed in TBE buffer (10.8 % [w/v] tris base, 5.5% [w/v] boric acid, 0.93% [w/v] EDTA). Electrophoresis was performed in TBE buffer for 30 min at 1 V/cm. Slides were washed in water and dehydrated with ethanol, air dried overnight and then treated with Sybr green (Stratagene, La Jolla, CA, USA) to stain DNA. Comets were imaged by confocal microscopy on a Leica confocal system (Leica Microsystems, Heidelberg, Germany), and analyzed using software developed inhouse [20]. The Olive tail moment (OTM) was calculated as the average of at least 100 comets per data point. 2.6. Clonogenic assays Cell suspensions (2×10 5 cells in 200 μL of medium) were incubated with 111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgG-Tat (0.25 μg/mL, specific activities 0–6 MBq) for 1 h at 37 °C to allow internalization and nuclear accumulation of RICs. Cells were exposed to γ-radiation (0 or 10 Gy) and incubated for 24 h at 37 °C. An aliquot of cells was plated in DMEM with 10% FBS (20% for MDA-MB-468 cells) and incubated at 37 °C and 5% CO2. Colonies were counted after 1–2 weeks and the surviving fraction (SF) calculated. In parallel experiments, cells were incubated with bleomycin (0–200 μg/mL) for 4 h, exposed to 111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgG-Tat for 24 h and then harvested for clonogenic assays. 2.7. Monte Carlo simulation The distribution of radiation deposited dose around a single decaying 111In atom, or dose point kernel, was calculated. The transport of Auger electrons (0.345–25.58 keV) and internal conversion electrons (144.6–245 keV) was simulated by a Monte Carlo code, PENELOPE (PENetration and Energy Loss of Positrons and Electrons) [21]. Electron energy spectra were obtained from Eckerman et al. [22]. Electron transport was simulated using a mixed (class II) scheme, where hard collisions were simulated individually from the differential cross sections, while the global effect of the soft interactions between consecutive hard collisions was simulated as a single artificial event using multiple-scattering approximations. The energy threshold for transported particles was set at 100 eV, energies smaller than this cut-off were deposited locally. A concentric spherical geometry was used, with the smallest sphere around the decaying nucleus having a radius of 10 nm. The average dose deposition in each sphere was calculated for 2.6×10 6 decays. 2.8. Tumor growth inhibition All animal procedures were carried out in accordance with the UK animals (Scientific Procedures) Act 1986 and with local ethical

committee approval. 231-H2N xenografts were established in female BALB/c nu/nu mice (Harlan, UK) by subcutaneous (s.c.) injection in the right flank of 1.5×10 6 cells in DMEM:matrigel 1:1 (Matrigel, BD, Oxford, UK). Tumor volume was calculated as V= 0.5(a 2 ×b), where a and b are the short and long axis, respectively. When the tumor volume reached approximately 100 mm 3, animals were randomized into groups of seven. 111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgG-Tat (10 μg/mouse, 6 MBq/μg) was injected intravenously (i.v.) 1 h before animals were anesthetized using a ketamine/xylazine mixture. A dose of 10 Gy was delivered to the tumor using 300 kV X-rays (Gulmay 320 kV irradiator; 2 Gy/min). The radiation set-up allowed irradiation of the right hind quarter, including the tumor and right leg, only. Control mice were anesthetized and sham-irradiated. Tumor size was monitored using caliper measurements for up to 60 days. Body weight was measured weekly. Tumor growth rate was determined by nonlinear curve fitting of an exponential growth model. Kaplan–Meier survival curves were generated taking either tumor volume doubling time (V=2 V0) or time at which animals were euthanized as the endpoint. Animals were euthanized when the tumor diameter reached 12.5 mm.

2.9. Statistical analysis All statistical analysis and curve fitting were performed using the Graphpad Prism software package (Graphpad Software, San Diego, CA, USA). Tabular results were compared using 1-way ANOVA with post-hoc Tukey test. Fitted parameters from curve fits were compared using the F-test. Kaplan–Meier survival curves were compared using the log-rank test with Bonferroni correction for multiple comparisons. A P value of b0.05 was considered significant. In figures, statistical significance was denoted: *Pb0.05, **Pb0.01: ***Pb0.001.

3. Results 3.1. 111In-DTPA-anti-γH2AX-Tat increases IR- and bleomycin-associated induction of γH2AX foci To investigate the ability of 111In-DTPA-anti-γH2AX-Tat to augment radiation- and chemotherapy-induced DNA dsb, the number of γH2AX foci was counted following combined exposure of MDA-MB-468 cells to genotoxic treatment plus RIC. The induction of γH2AX foci in MDA-MB-468 cells was significantly greater after exposure to a combination of IR (10 Gy) plus 111InDTPA-anti-γH2AX-Tat (6 MBq/μg) compared to IR alone (20.6±2.5 versus 10.4±2.3 foci/cell, respectively; Pb.001) (Fig. 1A). This phenomenon was also observed when a lower dose (4 Gy) of IR was combined with 111In-DTPA-anti-γH2AX-Tat versus IR alone (18.9±1.9 versus 7.9±1.7 foci/cell, respectively; Pb.001). The combination of IR (4 or 10 Gy) plus 111In-DTPA-mIgG-Tat (6 MBq/ μg) had little effect on the number of γH2AX foci compared to IR alone (after 4 Gy, 7.3±1.1 versus 7.5±1.7 foci/cell, PN.05; after 10 Gy, 11.8±2.2 versus 10.4±2.3 foci/cell, PN.05) (Fig. 1A). 111InDTPA-anti-γH2AX-Tat alone did not increase the number of foci/cell compared to control, untreated cells (PN.05). In MDA-MB-468 cells, at both 1 h and 24 h after exposure to IR plus 111In-DTPA-anti-γH2AX-Tat (6 MBq/μg), the number of γH2AX foci/cell was significantly higher compared to IR-only samples (16.9±1.7 and 9.9±1.4 versus 7.2±1.1 and 4.0±0.8 foci/cells, respectively [Pb0.001 for both comparisons]) ( Fig. 1B). In contrast the number of foci/cell was not significantly induced at 1 h or 24 h when 111In-chloride ( 111InCl3) was combined with IR compared to IR only. Neither 111InCl3 nor 111In-DTPAanti-γH2AX-Tat caused a significant increase in the number of γH2AX foci in non-irradiated cells at 1 h or 24 h (PN.05).

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151

ns

25

***

*** Control 111 In-DTPA-mIgG-Tat

20

111

15

In-DTPA-anti-γH2AX-Tat

10 5 0

0

4

10

IR (Gy)

Control

***

20

111 111

ns 15

In In-DTPA-anti-γH2AX-Tat

*

60

ns

ns

ns

ns

5 0

IR 1h

***

***

no IR

IR

24 h

Fig. 1. (A) MDA-MB-468 cells were exposed to 111In-DTPA-anti-γH2AX-Tat or 111In-DTPAmIgG-Tat (0.25μg/mL; 6MBq/μg) for 1h to allow internalization and nuclear accumulation of RICs and then irradiated (0, 4 or 10Gy). After 1h, cells were immunostained for γH2AX and the number of γH2AX foci/cell was counted in N100 cells. Results are expressed as the mean number of foci/cell in three independent experiments±standard error (SE). (B) MDA-MB-468 cells were exposed to 111In-DTPA-anti-γH2AX-Tat (0.25μg/mL; 6MBq/μg) or an equivalent amount of 111InCl3 and irradiated (0 or 4Gy) 1h later. After incubation for 1 or 24h, cells were processed as in (A).

3.2. 111In-DTPA-anti-γH2AX-Tat enhances IR-induced DNA double strand breaks Neutral comet assays were performed as a first step towards characterization of the DNA damage caused when 111In-DTPAanti-γH2AX-Tat is combined with IR. The Olive Tail Moment (OTM) is a measure of the number of DNA dsb. There was a significant increase in the OTM in MDA-MB-468 cells treated with a combination of IR plus 111 In-DTPA-anti-γH2AX-Tat (6 MBq/μg) compared to IR alone (at 10 Gy, 40.2±14.1 versus 16.0±3.4; at 4 Gy, 24.8±11.4 versus 15.3± 3.5 [Pb0.001, for both comparisons]) (Fig. 2A). This effect was not observed when 111In-DTPA-anti-γH2AX-Tat was added to nonirradiated cells or when 111In-DTPA-mIgG-Tat (6 MBq/μg) was combined with IR (Fig. 2A). The addition of Fpg, Endo III and Endo IV to transform oxidized purines, pyrimidines and AP-sites into DNA dsb respectively, showed that 111In-DTPA-anti-γH2AX-Tat, when combined with IR (4 Gy), results in increased formation of oxidized purines compared to IR alone but does not influence the formation of oxidized pyrimidines or AP-sites (Fig. 2B). When 111In-DTPA-anti-γH2AX-Tat was combined with IR, the OTM in comet assays with added Fpg, Endo III or Endo IV was 21.3±2.8, 14.9±1.10 and 17.4±0.69, respectively, versus 14.7±0.74 when no glycosylase was added (Pb0.01, PN.05, PN.05, respectively). Exposure of cells to 111In-DTPA-anti-γH2AX-Tat only also induced oxidized purines, but to a lesser extent than when 111 In-DTPA-anti-γH2AX-Tat was combined with IR. There was no significant formation of oxidized purines, oxidized pyrimidines or AP-sites in cells exposed to IR only (P N.05). 111In-DTPA-mIgG-Tat had no effect on the formation of oxidized bases or AP-sites either alone or in combination with IR (Fig. 2C). 111

In-DTPA-anti-γH2AX-Tat results in clustered γH2AX foci

To determine whether the spatial distribution of γH2AX foci is influenced by the addition of 111In-DTPA-anti-γH2AX-Tat to a

Control Olive tail moment

10

no IR

3.3.

A

111

ns

40

111

In-DTPA-mIgG-Tat In-DTPA-anti-γH2AX-Tat

ns 20

0

0

B

4

10

IR (Gy)

No enzyme Fpg Endo III Endo IV

30

Olive tail moment

Number of γH2AX foci/cell

B

genotoxic treatment, the position of foci within individual nuclei of 231-H2N cells was mapped in three dimensions using Z-stacks. In cells treated with a combination of IR or bleomycin plus 111In-DTPAanti-γH2AX-Tat, foci tended to occur in groups. Grouping was quantified by calculating Ripley's K-value, which showed a significant difference between cells treated with IR alone versus IR plus 111InDTPA-anti-γH2AX-Tat, Pb.001 (Fig. 3A, 3B). Groups of foci spanned approximately 1 μm in diameter. To exclude the possibility that clustering of foci was a result simply of the greater number of γH2AX foci in cells exposed to both IR and 111In-DTPA-anti-γH2AX-Tat, the relationship between the significance level of the Ripley K-value and the number of foci/cell was investigated in silico, but no statistical

20

ns

**

ns

***

10

0

no IR

IR

no IR 111

Control

IR

In-DTPA-anti-γH2AX-Tat

C No enzyme 30

Fpg Endo III

Olive tail moment

Number of γH2AX foci/cell

A

1145

Endo IV 20

ns

ns

no IR

IR

ns

ns

no IR

IR

10

0

Control

111

In-DTPA-mIgG-Tat

Fig. 2. (A) MDA-MB-468 cells were exposed to 111In-DTPA-anti-γH2AX-Tat or 111InDTPA-mIgG-Tat (0.25 μg/mL; 6 MBq/μg) for 1 h to allow internalization and nuclear accumulation of RICs and then irradiated (0, 4 or 10 Gy). Cells were incubated for 1 h and neutral comet assays were then performed. Results are expressed as the mean OTM for N100 cells±SE. (B) MDA-MB-468 cells were exposed to 111In-DTPA-anti-γH2AXTat (0.25 μg/mL; 6 MBq/μg) and irradiated (4 Gy) as in (A). Comet assays were performed with the addition of an endonuclease digestion step, using Fpg, Endo III, and Endo IV, to determine the extent of clustered breaks. (C) Comet assays were performed following exposure of cells to 111In-DTPA-mIgG-Tat.

1146

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151 111In-DTPA-anti-

111In-DTPA-

Control

A

γH2AX-Tat

mIgG-Tat

Control

B Control 111

21 ± 2

16 ± 3

22 ± 3

111

In-DTPA-mIgG-Tat In-DTPA-anti-γH2AX-Tat

IR (4 Gy)

*** 138 ± 10

143 ± 9

355 ± 14

105 ± 3

120 ± 9

196 ± 11

p-value on Ripley's K-value

Bleomycin

ns 100

50

0

IR (4 Gy)

Bleomycin

10 5

DSB likely 10 0

7.10-3 Dose (Gy)

Chance of a DSB occurring @ distance

Control

C

*** ns

10 -5

DSB unlikely

10 -10

10 -15 10 -8

10 -7

10 -6

5.10-5

10 -5 Radius (m)

10 -4

10 -3

10 -2

Fig. 3. (A) 231-H2N cells were incubated either in the presence of 111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgG-Tat (0.25 μg/mL; 6 MBq/μg) for 1 h to allow internalization and nuclear accumulation of RICs and then irradiated (4 Gy), or in the presence of bleomycin (20 μg/mL) for 16 h followed by addition of111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgGTat for 2 h. γH2AX immunostaining was performed. Representative images are shown. The mean number of foci/cell±SE is shown in each panel. (B) Using confocal microscopy, the number and spatial distribution of γH2AX foci were determined. Ripley's K value was determined for 10 cells per condition, and the statistical significance of the K-value was determined using Monte Carlo simulations. Results are shown as the average relative significance levels of Ripley K values±SE. (C) The radial distribution of radiation deposited dose around a single decaying 111In atom, as calculated by Monte Carlo simulation.

dependence between these 2 variables was found (Spearman's rank correlation coefficient, PN.05). 3.4. Micro-dosimetry: dose point kernel of

111

In

The radial distribution of dose around a decaying 111In atom, as calculated by Monte Carlo simulation, is depicted in Fig. 3C. 3.5. 111In-DTPA-anti-γH2AX-Tat enhances the cytotoxicity of IR and bleomycin The combination of IR (4 Gy) plus 111In-DTPA-anti-γH2AX-Tat (6 MBq/μg) resulted in a lower SF compared to either treatment alone in MDA-MB-468 cells (5.2%±0.6% versus 60.5%±4.2% and 47.8%± 1.1%, respectively; Pb0.001)(Fig. 4A). In contrast, the combination of IR plus 111In-DTPA-mIgG-Tat was not more cytotoxic than IR alone (PN.05). In 231-H2N cells, 111In-DTPA-anti-γH2AX-Tat plus IR also reduced clonogenic survival more significantly than either IR or 111InDTPA-anti-γH2AX-Tat alone (16.6%±0.4% versus 91.6%±7.2% and

82.7%±11.3%, Pb.01) (Fig. 4B). In both cell lines, enhancement of radiation-induced cytotoxicity by 111In-DTPA-anti-γH2AX-Tat was dependent on the specific activity of the radioimmunoconjugate (Fig. 4C, 4D). In MDA-MB-468 and 231-H2N cells the combination of bleomycin (25–200 μg/mL) plus 111In-DTPA-anti-γH2AX-Tat resulted in lower SF compared to bleomycin alone (F test, Pb.0001 and P= .0082, respectively) (Fig.4E, 4F). This effect was not observed when IR was combined with 111In-DTPA-mIgG-Tat (results not shown). 3.6. 111In-DTPA-anti-γH2AX-Tat enhances the antitumor effect of IR in vivo The anti-tumor effect of 111In-DTPA-anti-γH2AX-Tat plus IR was investigated in 231-H2N xenograft-bearing mice. The tumor growth rate decreased significantly in mice treated with a single dose of IR (10 Gy) plus 111In-DTPA-anti-γH2AX-Tat (10 μg, 6 MBq/μg) (average growth rate −0.002±0.004 mm 3/day) compared to untreated controls (0.040±0.018 mm 3/day; Pb.001) or either agent alone (IR alone, 0.036±0.011 mm 3/day, Pb.001; 111In-DTPA-anti-γH2AX-Tat alone,

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151

Control

111

111

In-DTPA-mIgG-Tat

111

In-DTPA-anti-γH2AX-Tat (6 MBq/μg)

In-DTPA-mIgG-Tat In-DTPA-anti-γH2AX-Tat (6 MBq/μg)

B

**

100

Surviving fraction (%)

Surviving fraction (%)

Control 111

A

* 50

no IR

50

IR (4 Gy)

no IR no IR 4 Gy IR

100

50

IR (4 Gy) no IR 10 Gy IR

D Surviving fraction (%)

C Surviving fraction (%)

100

0

0

0

100

50

0 0.5

1.0

2.0

3.0

4.0

6.0

Specific activity 111In-DTPA-anti-γH2AX-Tat (MBq/μg)

E

PBS 0.0

bleomycin + 111In-DTPA-anti-γH2AX-Tat 100

50

0

1.0

2.0

3.0

4.0

6.0

bleomycin

F

bleomycin

0.5

Specific activity 111In-DTPA-anti-γH2AX-Tat (MBq/μg)

Surviving fraction (%)

PBS 0.0

Surviving fraction (%)

1147

bleomycin + 111In-DTPA-anti-γH2AX-Tat 100

50

0 0

20

50

100

200

0

Bleomycin (μg/mL)

20

50

100

200

Bleomycin (μg/mL)

Fig. 4. (A) MDA-MB-468 or (B) 231-H2N cells were exposed to 111In-DTPA-anti-γH2AX-Tat or 111In-DTPA-mIgG-Tat (0.25 μg/mL; 6 MBq/μg) for 1 h and then irradiated (0, 4or 10 Gy). After 24 h, clonogenic assays were performed. (C) MDA-MB-468 or (D) 231-H2N cells were exposed to PBS or 111In-DTPA-anti-γH2AX-Tat at increasing specific activities (0–6 MBq/ μg) followed by IR after 1 h. After 24 h clonogenic survival was determined. (E) MDA-MB-468 or (F) 231-H2N cells were exposed to increasing concentrations of bleomycin (0– 200 μg/mL). Four hours later, cells were exposed to 111In-DTPA-anti-γH2AX-Tat and clonogenic survival was determined. Results are shown as the relative number of colonies determined in three independent experiments±standard deviation (SD).

0.031±0.014 mm 3/day, Pb.001) (Fig. 5). In contrast the growth rate of tumors exposed to cold (non-radiolabelled) DTPA-anti-γH2AX-Tat plus IR was not significantly different to that of tumors exposed to IR only (0.026±0.007 versus 0.036±0.011 mm 3/day; PN.05). The tumor growth rate was not significantly altered by treatment with 111InDTPA-mIgG-Tat compared to controls (0.045±0.023 versus 0.040± 0.018 mm 3/day; PN.05). The combination of IR plus 111In-DTPA-mIgGTat had a significantly less potent effect on tumor growth rate than IR plus 111In-DTPA-anti-γH2AX-Tat (0.017±0.004 versus −0.002± 0.004 mm 3/day; Pb.05). Log-rank analysis of Kaplan–Meier curves based on tumor-doubling time (V=2 V0) showed the same trend. Median V=2 V0 time was significantly longer for the combination of IR plus 111In-DTPA-anti-γH2AX-Tat (N 53 days), compared to untreated controls (13.2 days; P=.039), IR alone (5.4 days), 111InDTPA-anti-γH2AX-Tat alone (15.7 days), 111In-DTPA-mIgG-Tat alone (9.0 days), the combination of IR plus 111 In-DTPA-mIgG-Tat (31.8 days) or the combination of IR plus unlabelled DTPA-

anti-γH2AX-Tat (30.7 days). None of the treatment regimens resulted in a significant effect on body weight, compared to nontreated animals (data not shown). 3.7. Retention assay The relative amount of 111In retained in irradiated MDA-MB-468 cells, was dependent on the amount of 111 In conjugated to anti-γH2AX-Tat. An increase in specific activity led to more prolonged retention in the irradiated cells (correlation coefficient R=0.94, P= .02). No such effect was observed in unirradiated cells, which expressed only baseline levels of γH2AX (R=0.87, PN.05). 4. Discussion Auger electron-emitting radionuclides incorporated into radiopharmaceuticals that target cancer cell surface receptors, proteins or

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151

0.08

*** ***

0.06 0.04

** *

0.02 0.00

t ro l 2A TP XIR Ta A+ 1 t 11 m Ig In G -D -T TP at IR A + 1 -an IR 11 tiIR γH + In co 2 -D ld TP AXD Ta ATP t m AI g an G -T tiat γH 2A XTa t

-0.02

11 1

In

-D

B

-D

In

11 1

TP Aa

nt

i-γ H

C

on

A

Growth rate constant (mm3 day-1)

1148

Volume (mm3) normalized

700

Control IR

600

111

In-DTPA-anti-γH2AX-Tat IR + cold DTPA-anti-γH2AX-Tat

500 400

IR + 111In-DTPA-mIgG-Tat 111 In-DTPA-mIgG-Tat IR + 111In-DTPA-anti-γH2AX-Tat

300 200 100 0 0

20

40

60

Time (days) Fig. 5. Mice bearing 231-H2N xenografts were exposed to IR (10 Gy) directed at the tumor or sham irradiated, and then received PBS, DTPA-anti-γH2AX-Tat, 111In-DTPAanti-γH2AX-Tat or 111In-DTPA-mIgG-Tat i.v. Tumor size was measured for 53 days. Results are expressed as the mean tumor growth rate for 7 mice/group±SE (A) or the average volume in mm3 (B).

other antigens have been investigated extensively as molecular radiotherapy agents [23]. Because of the short range of Auger electrons, it is recognized that the cytotoxicity of Auger electronemitting radiopharmaceuticals is strongly influenced by their intracellular distribution, especially the extent of intranuclear accumulation [14,24]. Therefore, strategies have been developed to enhance nuclear accumulation after tumor cell binding. Such strategies include the incorporation of cell-penetrating peptides, nuclear localizing sequences (NLS), or both into radiopharmaceutical design, as used by Reilly et al. [25–27]. Others have used radiolabelled oligonucleotides that deliver radiation to specific gene sequences or peptide ligands that bind a cell surface receptor which itself translocates to the nucleus [14]. In the current study a wholly different and novel approach was taken: here, the target was an exclusively DNAassociated intra-nuclear protein,γH2AX, which is only expressed in cells in which DNA dsbs exist. The phosphorylated histone isoform, γH2AX, is a DNA damage signaling protein that is formed in cells following DNA dsb damage after exposure to genotoxic agents, including ionizing radiation and many anti-tumor chemotherapy agents. Furthermore, γH2AX baseline expression levels are increased in many types of cancers and their precursors compared to normal cells [28–31]. γH2AX is expressed in foci, with many copies of the protein accumulating at sites of DNA dsb [8]. It was calculated previously that up to 1.2×10 6 copies of γH2AX could be present in an irradiated tumor cell [12]. The abundance of γH2AX and its location in the immediate proximity to DNA render it an attractive target for Auger-electron radioimmunotherapy, given

the short range yet densely ionizing nature of Auger electrons. Using the Auger electron-emitting RIC, 111In-DTPA-anti-γH2AX-Tat, we investigated whether it is possible to target γH2AX for therapeutic gain. We reasoned that an Auger electron-emitting radionuclide that accumulates sufficiently at DNA dsbs through association with γH2AX, could amplify the damage at those sites and in so doing convert non-lethal damage to irreparable and potentially lethal damage. By augmenting DNA dsb damage, the γH2AX-targeting agent would lead to further phosphorylation of H2AX and, therefore, formation of its own target resulting in subsequent rounds of increasing amounts of damage, eventually leading to cell death. A proposed mechanism for the action of 111In-DTPA-anti-γH2AXTat, as we have used it in the proof-of-principle studies in this manuscript, is shown in Fig. 6. First, the 111In-DTPA-anti-γH2AX-Tat is loaded into cells, so that it is in place at the time that γH2AX starts to be formed. Cells are then irradiated to cause DNA dsb which leads to phosphorylation of H2AX to form γH2AX, to which 111In-DTPAanti-γH2AX-Tat binds. This brings 111In close to DNA which is wrapped around a histone hetero-octamer. As 111In decays, it is likely that Auger electrons will cause additional, and possibly more complex (clustered) DNA damage. Auger-electron-induced dsbs will then, in turn, cause the formation of more γH2AX, and so binding of 111InDTPA-anti-γH2AX-Tat. This runaway effect would be expected to eventually lead to cell death. The first step in this proposed chain of events is the internalization of an anti-γH2AX antibody into cells. The challenge of producing a RIC capable of traversing the cytoplasmic membrane and nuclear

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151

1149

Secondly, the decay of 111In inside the cell nucleus, close to DNA causes additional DNA damage. Exposure of irradiated cells to 111InDTPA-anti-γH2AX-Tat was associated with a significant increase in the number of γH2AX foci and DNA fragmentation (using the neutral comet assay), compared to IR alone (Fig. 1 and 2). Importantly, exposure of unirradiated cells to 111In-DTPA-anti-γH2AX-Tat did not result in an increase of γH2AX foci after 24 h (Fig 1B), suggesting that toxicity to normal tissues with low γH2AX expression would be

envelope was met by conjugation of a cell-penetrating peptide, Tat, to an anti-γH2AX antibody. We have previously demonstrated that 111 In-DTPA-anti-γH2AX-Tat internalizes in tumor cells and tumor xenografts, where it co-localizes with γH2AX foci [12]. If the cell does not express γH2AX, then 111In-DTPA-anti-γH2AX-Tat is not retained and is soon transported out of the cell. The rapid and active exocytosis of 111In-DTPA-anti-γH2AX-Tat is likely mediated by interaction of the Tat-moiety with phospho-inositol-(4,5)-bisphosphonate [32].

A iii. Irradiation

iv. IR-induced γH2AX foci

TAT1h

0.5 h TAT-

TAT-

TAT-

ii. 111In-anti-γH2AX-Tat internalized

i. Cells incubated with 111In-anti-γH2AX-Tat

v. In nucleus: 111In-anti-γH2AX-Tat

co-localizes with γH2AX foci

TAT-

TAT-

TAT-

viii. More γH2AX foci form around original focus

Repeated rounds of internalization/retention of 111In-anti-γH2AX-Tat and γH2AX formation

TAT-

vii. 111In decay

vi. 111In-anti-γH2AX-Tat binds to γH2AX foci

B

% Retention at 4h

100

50

0

1

2

3

4

0 Gy

6

1

2

3

4

6

10 Gy

SA (MBq/μg) IR dose

Fig. 6. (A) A schematic overview of events during 111In-DTPA-anti-γH2AX-Tat amplification of DNA damage. (i–ii) 111In-anti-γH2AX-Tat is internalized into cells, mediated by Tatpeptides. (iii) Cells are irradiated or exposed to bleomycin causing DNA dsbs. (iv) γH2AX foci are induced by IR or bleomycin. (v–vi) 111In-DTPA-anti-γH2AX-Tat binds to γH2AX foci via the IgG moiety. (vii) 111In decays and Auger-electrons are emitted where 111In-DTPA-anti-γH2AX-Tat is concentrated at γH2AX foci, resulting in more DNA dsbs locally. (viii) More γH2AX foci are formed in the vicinity of the original one. (B) MDA-MB-468 cells were exposed to 111In-DTPA-anti-γH2AX-Tat (0.5 μg/mL, 6 MBq/ μg) for 1 h, and then mockirradiated (0 Gy) or irradiated (10 Gy). Cells were washed thrice with PBS and supplied with fresh medium. After 4 h, the fraction of 111In remaining in the cells was determined, as described in Materials and methods.

1150

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151

limited. Furthermore, it was shown that the DNA damage resulting from 111In Auger electron emissions was complex and clustered, with mostly oxidized purines (Fig. 2B and 2C). This observed preponderance of purine base lesions is consistent with previous reports in which Auger electron-emitting isotopes were found to be associated with an increased yield of this type of damage [33]. We predicted that the DNA damage caused by Auger electrons would lead to formation of more γH2AX. Interestingly, the γH2AX foci that formed after exposure of irradiated or bleomycin-treated cells to 111In-DTPA-anti-γH2AX-Tat appeared to be grouped in clusters (Fig. 3). This is consistent with the hypothesis that the absorbed radiation dose from Auger electron is deposited in very restricted volume around the decay site [15]. Furthermore, the size (radius) of the groups of foci (0.5 μm) corresponds to the range/dose threshold around a decaying 111In atom within which DNA dsb formation is calculated to be most likely (7 10 −3 Gy; Fig 3C). We showed previously that 111In-DTPA-anti-γH2AX-Tat is retained up to 16 times longer in cells in which γH2AX has been induced by irradiating the cells (10 Gy) [12]. 111In-DTPAanti-γH2AX-Tat radiolabelled to high specific activity shows similar behaviour, i.e. it is retained in cells expressing γH2AX, but now with the added effect that it leads to more γH2AX foci, thereby enhancing its own retention. The data in Figure 6 corroborate this: The higher the specific activity, the greater the retention of 111InDTPA-anti-γH2AX-Tat. This protracted retention increases the probability that 111In will decay close to DNA. Whether this occurs while 111In-DTPA-anti-γH2AX-Tat is bound to γH2AX or simply through the mass effect of more 111In atoms being retained in the nucleus, the increased Auger electron emission will lead to increased DNA damage and chance of cell kill. This model of the mechanism of action of 111In-DTPA-anti-γH2AXTat is supported by the observation that when labelled at high specific activity (6 MBq/μg) it significantly reduced the clonogenic survival of irradiated and bleomycin-treated cells (Fig.4). A specific activity-dependent decrease in clonogenic survival suggests that this is 111In-dependent, and that it is the Auger electrons that are responsible for the cell killing effect of 111In-DTPA-anti-γH2AX-Tat. Furthermore, a specific activity-dependent increase in cellular retention of 111In-DTPA-anti-γH2AX-Tat was observed (Fig. 6B) suggesting that by inducing additional DNA dsb and γH2AX foci, 111 In-DTPA-anti-γH2AX-Tat modulates its own nuclear accumulation and retention, further enhancing its own lethal effects. These data also suggest that using 111In-DTPA-anti-γH2AX-Tat to image DNA damage using specific activities up to 1 MBq/μg, as previously reported, does not cause additional DNA damage or cause cytotoxicity [12]. Several investigators have used Monte Carlo simulation to calculate the radiation absorbed dose from Auger electron-emitting isotopes within a cell [34,35]. However, the focal accumulation of 111 In at sites of DNA damage implies that the nuclear radiation absorbed dose as calculated using these methods, which assume homogeneous distribution of radioisotopes in cell compartments, may not faithfully reflect the biological effects caused by agents such as 111 In-DTPA-anti-γH2AX-Tat. Indeed, the local dose around a decaying 111 In atom reaches levels of 5000Gy, but quickly decreases with increasing distance (Fig.3C). Therefore, knowledge of the precise location of 111In atoms on the nanometre scale is necessary to provide truly meaningful dose calculations. Because this information cannot be obtained using current state-of-the-art techniques, no micro-dosimetric calculations are presented here, and no correlation was made between radiation absorbed dose and DNA damage or cell survival. In this proof-of-principle study, RICs were added to cells 1 h before irradiation to allow internalization and nuclear accumulation so that newly-formed γH2AX foci would be immediately bound by RICs. Variation of the length of time between exposure to 111In-DTPAanti-γH2AX-Tat and irradiation (15 min to 4 h) had no significant

influence on survival (data not shown). This suggests there is a wide therapeutic time-window and that the time between administration of 111In-DTPA-anti-γH2AX-Tat and genotoxic insult is not a major determinant of cytotoxicity. In in vivo studies of mice bearing 231-H2N xenografts, administration of 111In-DTPA-anti-γH2AX-Tat (6 MBq/μg) was found to significantly slow tumor growth when combined with IR but not when delivered as a single agent (Fig. 5). Interestingly, cold (nonradiolabelled) DTPA-anti-γH2AX-Tat also reduced tumor growth rate when combined with 10 Gy IR, albeit to a much lesser extent than 111 In-DTPA-anti-γH2AX-Tat. The gross body weight of 111In-DTPAanti-γH2AX-Tat-treated animals was measured for 53 days and there was no difference compared to controls. This suggests that the expression level of γH2AX in dividing normal tissues, such as skin and gut mucosa, is not high enough to present a target for 111In-DTPA-anti-γH2AX-Tat therapy [36]. There are several potential clinical applications for an Augeremitting radiopharmaceutical such as 111In-DTPA-anti-γH2AX-Tat which targets DNA dsb. The results presented here suggest that the concentration of genotoxic anti-cancer drugs, such as bleomycin, or the dose of external beam radiotherapy, could be reduced when used in combination with 111In-DTPA-anti-γH2AX-Tat (Fig. 4E and 4F). This would reduce the toxic side-effects of these treatments, while inflicting DNA damage in tumor tissue. The combination of 111InDTPA-anti-γH2AX-Tat and IR or genotoxic drugs would be expected to cause more extensive DNA damage, and so a greater likelihood of tumor control, compared to monotherapy. Cancer patients could be treated with radiotherapy or chemotherapy to induce γH2AX in tumor, followed by 111 In-DTPA-anti-γH2AX-Tat administration. 111 In-DTPA-anti-γH2AX-Tat at lower specific activity (0.5–1 MBq/ μg) could be used for imaging to select those patients who would benefit from Auger electron therapy in a theranostic approach. 5. Conclusion The use of short range electron-emitting isotopes to target an intranuclear protein and to convert sites of existent but sublethal DNA damage into irreparable damage represents a new approach to the radiotherapy of cancer. 111In-DTPA-anti-γH2AX-Tat binds to γH2AX and so is retained at sites of DNA damage. Through the emission of Auger electrons, decay of 111In at these sites resulted in an increase of DNA dsb, an increase in the amount of complex DNA damage, a reduction in clonogenic survival in vitro, and a marked reduction in tumor growth rate in vivo. References [1] Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer 2011;11:239–53. [2] Camphausen K, Tofilon PJ. Inhibition of histone deacetylation: a strategy for tumor radiosensitization. J Clin Oncol 2007;25:4051–6. [3] Overgaard J. Hypoxic radiosensitization: adored and ignored. J Clin Oncol 2007;25: 4066–74. [4] Shewach DS, Lawrence TS. Antimetabolite radiosensitizers. J Clin Oncol 2007;25: 4043–50. [5] Helleday T, Petermann E, Lundin C, Hodgson B, Sharma RA. DNA repair pathways as targets for cancer therapy. Nat Rev Cancer 2008;8:193–204. [6] Kassis AI, Adelstein SJ. Radiobiologic principles in radionuclide therapy. J Nucl Med 2005;46(Suppl. 1):4S–12S. [7] Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 1998;273:5858–68. [8] Nakamura AJ, Rao VA, Pommier Y, Bonner WM. The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks. Cell Cycle 2010;9:389–97. [9] Nakamura A, Sedelnikova OA, Redon C, Pilch DR, Sinogeeva NI, Shroff R, et al. Techniques for gamma-H2AX detection. Methods Enzymol 2006;409:236–50. [10] Sedelnikova OA, Bonner WM. GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence. Cell Cycle 2006;5:2909–13. [11] Kao J, Milano MT, Javaheri A, Garofalo MC, Chmura SJ, Weichselbaum RR, et al. gamma-H2AX as a therapeutic target for improving the efficacy of radiation therapy. Curr Cancer Drug Targets 2006;6:197–205.

B. Cornelissen et al. / Nuclear Medicine and Biology 39 (2012) 1142–1151 [12] Cornelissen B, Kersemans V, Darbar S, Thompson J, Shah K, Sleeth K, et al. Imaging DNA damage in vivo using γH2AX-targeted immunoconjugates. Cancer Res 2011;71:4539–49. [13] Costantini DL, Hu M, Reilly RM. Peptide motifs for insertion of radiolabeled biomolecules into cells and routing to the nucleus for cancer imaging or radiotherapeutic applications. Cancer Biother Radiopharm 2008;23:3–24. [14] Cornelissen B, Vallis KA. Targeting the nucleus: an overview of Auger-electron radionuclide therapy. Curr Drug Discov Technol 2010;7:263–79. [15] Kassis AI. The amazing world of auger electrons. Int J Radiat Biol 2004;80: 789–803. [16] du Manoir JM, Francia G, Man S, Mossoba M, Medin JA, Viloria-Petit A, et al. Strategies for delaying or treating in vivo acquired resistance to trastuzumab in human breast cancer xenografts. Clin Cancer Res 2006;12:904–16. [17] Ripley BD. Modelling spatial patterns. J R Stat Soc Series B Stat Methodol 1977;39: 172–212. [18] Ripley BD. Modelling spatial patterns. J R Stat Soc Series B Stat Methodol 1977; 39:21. [19] Collins AR, Duthie SJ, Dobson VL. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 1993;14: 1733–5. [20] Vojnovic B, Barber PR, Johnston P, Gregory HC, Marples B, Joiner, MC, et al. A high sensitivity, high throughput, automated single-cell gel electrophoresis (‘Comet’) DNA damage assay Available online at: http://users.ox.ac.uk/~atdgroup/ technicalnotes/Comet%20analysis.pdf. [21] Salvat F, Fernandez-Varea JM, Sempau J. PENELOPE-2008: A Code System for Monte Carlo Simulation of Electron and Photon Transport. Barcelona, Spain: PENELOPE-2008; 2008. p. 336. [22] Eckerman KF, Endo A. MIRD: radionuclide data and decay schemes. Reston, VA: Society of Nuclear Medicine; 2008. [23] Howell RW. Auger processes in the 21st century. Int J Radiat Biol 2008;84:959–75. [24] Falzone N, Cornelissen B, Vallis K. Auger emitting radiopharmaceuticals for cancer therapy. In: García Gómez-Tejedor G, Fuss MC, editors. Radiation damage in biomolecular systems. Netherlands: Springer, Dordrecht; 2012. p. 246–52. [25] Cornelissen B, McLarty K, Kersemans V, Scollard DA, Reilly RM. Properties of [(111)In]-labeled HIV-1 tat peptide radioimmunoconjugates in tumor-bearing

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34]

[35]

[36]

1151

mice following intravenous or intratumoral injection. Nucl Med Biol 2008;35: 101–10. Kersemans V, Cornelissen B, Minden MD, Brandwein J, Reilly RM. Drug-resistant AML cells and primary AML specimens are killed by 111In-anti-CD33 monoclonal antibodies modified with nuclear localizing peptide sequences. J Nucl Med 2008; 49:1546–54. Chen P, Wang J, Hope K, Jin L, Dick J, Cameron R, et al. Nuclear localizing sequences promote nuclear translocation and enhance the radiotoxicity of the anti-CD33 monoclonal antibody HuM195 labeled with 111In in human myeloid leukemia cells. J Nucl Med 2006;47:827–36. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907–13. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864–70. Oka K, Tanaka T, Enoki T, Yoshimura K, Ohshima M, Kubo, M, et al. DNA damage signaling is activated during cancer progression in human colorectal carcinoma. Cancer Biol Ther 9. Wasco MJ, Pu RT, Yu L, Su L, Ma L. Expression of gamma-H2AX in melanocytic lesions. Hum Pathol 2008;39:1614–20. Rayne F, Debaisieux S, Yezid H, Lin YL, Mettling C, Konate K. Phosphatidylinositol(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells. EMBO J 2010;29:1348–62. Datta K, Jaruga P, Dizdaroglu M, Neumann RD, Winters TA. Molecular analysis of base damage clustering associated with a site-specific radiation-induced DNA double-strand break. Radiat Res 2006;166:767–81. Goddu SM, Howell RW, Rao DV. Cellular dosimetry: absorbed fractions for monoenergetic electron and alpha particle sources and S-values for radionuclides uniformly distributed in different cell compartments. J Nucl Med 1994;35:303–16. Cai Z, Pignol JP, Chan C, Reilly RM. Cellular dosimetry of 111In using monte carlo Nparticle computer code: comparison with analytic methods and correlation with in vitro cytotoxicity. J Nucl Med 2010;51:462–70. Bonner WM, Redon CE, Dickey JS, Nakamura AJ, Sedelnikova OA, Solier S, et al. GammaH2AX and cancer. Nat Rev Cancer 2008;8:957–67.