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International Journal of Radiation Biology, 2013; Early Online: 1–9© 2013 Informa UK, Ltd.ISSN 0955-3002 print / ISSN 1362-3095 onlineDOI: 10.3109/09553002.2013.835503
Correspondence: Prof. Carine Michiels, University of Namur, URBC, NARILIS, 61 rue de Bruxelles, Namur, 5000 Belgium. Tel: 32 8172 4131. Fax: 32 8172 4135. E-mail: [email protected]
(Received 20 March 2013; revised 18 July 2013; accepted 30 July 2013)
Low dose hypersensitivity following in vitro cell irradiation with charged particles: Is the mechanism the same as with X-ray radiation?
Anne-Catherine Heuskin1, Carine Michiels2 & Stephane Lucas1
1Namur Research Institute for Life Sciences (NARILIS), Research Center for the Physics of Matter and Radiation (PMR), University of Namur, 2Namur Research Institute for Life Sciences (NARILIS), Unité de Recherche de Biologie Cellulaire (URBC), University of Namur, Namur, Belgium
Introduction
In the field of radiobiology, several low dose effects have been highlighted over the last decade. Low dose hypersen-sitivity (HRS), induced radioresistance (IRR) at higher doses, bystander effect, or adaptive responses can be mentioned. Among these, the HRS effect, followed by the IRR phenom-enon as the dose increases, could be of great interest for the fine tuning of new modalities for cancer treatment. The HRS effect has been thoroughly demonstrated for a variety of biological systems, such as bacteria, yeast, algae or in in vitro
rodent and human cell lines (both cancer and normal), and in studies with in vivo animal normal-tissue model.
Mathematical modelling of low dose hypersensitivity
A great amount of data about HRS after irradiation with sparsely ionizing radiation, like X- or g-rays is available in the literature (Vaganay-Juery et al. 2000, Rothkamm and Löbrich 2003, Wykes et al. 2006, Thomas et al. 2008). Usually, clonogenic assays following in vitro cell irradiation result in a linear quadratic behavior of the survival fraction (SF):
SF e D D a b 2 (1)
where a and b are the radiosensitivity parameters for a given cell line, and a given type of radiation. For most of the cell lines tested, a deviation from the linear quadratic model is observed in the low dose region (below ∼ 0.5 Gy). Cell killing is enhanced per unit dose of radiation but this effect is lost at higher doses (∼ above 0.5 Gy), and the survival curve follows the linear quadratic behavior again.
A series of theoretical approaches have been proposed to model this phenomenon.
The first model proposed is the Induced Repair (IndRep) model developed by Marples and Joiner (1993). In this approach, all the cells are hypothesized to be initially sensi-tive and then become resistant at higher doses, the a param-eter being dependent of the dose D in this case:
S e
D
D D D
r r sD Dc
=
with
( ) 2
a b
a a a a( ) ( ) /= e (2)
The value of a starts at as in the low dose region and gradu-ally changes to ar. The dose Dc is the transition dose between the low dose hypersensitivity effect and the linear quadratic behavior. The Dc parameter may be also defined as the dose
REVIEW
AbstractPurpose: Among the low dose effects that have been discovered during the past decade, the low dose hypersensitivity (HRS) is of prime importance. This phenomenon, compared to irradiation at higher doses used in conventional radiotherapy, enhances cell killing per unit dose at low doses and is followed by an induced radioresistance (IRR) effect. On survival fraction curves, a devia-tion from the linear quadratic model can be observed. HRS has mainly been studied after irradiation with sparsely ionizing radiation. Little work has been done to check its actual existence after irradiation with medium and high linear energy transfer (LET) particles. This article reviews recent studies involving HRS following irradiation of rodent and human cells with protons, alpha particles and carbon ions and assesses the applicability of a photon HRS model to charged particles.Conclusion: We propose that the HRS threshold dose and the radiosensitive parameter as may be LET and deoxyribonucleic acid (DNA) damage-clustering dependent. Combining the use of high-LET particles at low doses and chemotherapy strategies increasing the proportion of HRS-sensitive cells could become a good candidate treatment for radioresistant cancers.
Keywords: Charged particles, low dose hyper-radiosensitivity, ATM activation, cell radiobiology
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2 A.-C. Heuskin et al.
where the conversion of as to ar is 63% complete. The extent of HRS is sometimes characterized by the quantity g:
g s
r
aa
1. (3)
g is the amount by which a, at very low doses, is larger than at high doses. A positive value suggests enhanced cell killing and HRS.
Wouters et al. (1996) proposed the accumulated damage induced radioresistance model, describing the survival frac-tion by the following equation:
S f e f es D D D a a b( )12
r (4)
where
faD e
n
n aD
n
c
( )
!0
1
∑ (5)
A fraction f of the total cell population is considered as sensi-tive at low doses and is characterized by the as parameter. The other part of the cell population follows the usual linear quadratic model characterized by the ar and b radiosensitiv-ity parameters. The two subpopulation proportions change with the dose, as can be seen in equation (5). This model states that there is an average number a of events per gray and that damage distribution follows the Poisson statis-tics. To overcome HRS and trigger IRR, a cell must sustain a critical number c of damage. The sensitive population f diminishes as a increases: A lot of events per gray are more susceptible to induce IRR; on the contrary, f increases as c increases because more events are required to overcome HRS. Although this model is useful to obtain the number of critical events necessary to trigger IRR, the dose at which this phenomenon occurs is not provided.
The HRS phenomenon is not limited to survival curve shapes. For instance, it is also observed in mutation yields, i.e., there is an enhanced production of mutations per unit dose at very low doses compared to higher doses (Hooker et al. 2004). In 2005, Scott used their NEOTRANS3 model, which enabled to fit mutation yield and neoplastic trans-formation data at very low doses (Scott 2005). The expected biological effects frequency Y(D) depends on the dose D and is given by:
Y D Y Y k D D
Y D f Y Y kD D D D
Y
( ) (1 ) D
( ) 1 { (1 ) }0 0 PAM
0 0 0 PAM off
<≤ <= ( )
(( ) ( ) .0 0 offD Y 1 Y kD D D ≥ (6)
Y(D) is used either for mutation or neoplastic transformation and Y0 is the spontaneous yield present before irradiation. The production of mutations or neoplastic transformations is modulated through a competition of three different pro-cesses: A p53-dependent apoptosis pathway, repair with low or high fidelity and a p53-independent protective apoptosis mediated (PAM) process. The PAM process is only activated between DPAM and Doff, whereas p53-dependent apoptosis is functional for all doses. The fraction of lesions removed
by the PAM process is represented by f0. Below DPAM, it is suggested that lesions are repaired with low fidelity. For all other dose ranges, it is hypothesized that high fidelity repair occurs. It follows that the slope parameter k is different in the first zone D DPAM. In addition, k depends on the incidence of genomic damage induction, repair, misrepair, p53-depen-dent apoptosis and the studied endpoint.
In brief, in the first dose zone, the PAM process is absent and the fidelity of repair is low. Consequently, the biologi-cal effects of radiation are enhanced. This can be compared to HRS. In the second dose zone, the PAM and high fidelity repair processes are active, resulting in a decreased effect of radiation, analogous to IRR. In the third dose zone, the PAM process is inactive while fidelity of repair is high. From this zone, yields match the ones predicted by the linear- no-threshold (LNT) model (National Research Council [NRC] 2006). Although this model is of great interest, it is not directly related to survival fraction analysis and therefore will not be further discussed in the remaining of this work.
Although there is an extensive amount of data in literature about HRS following exposure to sparsely ionizing radiations, little work has been done to check the actual existence of the HRS phenomenon after irradiation with charged particles, such as light ions or carbon ions. This paper proposes to review the existing information linking low dose hypersensi-tivity and charged particles. We first examine the status of the rodent V79 cell line, two human cell lines and normal human fibroblasts following irradiation with charged particles. The HRS mechanism demonstrated for sparsely ionizing radia-tion will be then discussed. Evidence from literature shows that it could also be applied to charged particles. From these data, a dependence of the as and Dc parameters on the linear energy transfer is proposed.
Can HRS be observed after exposure to medium and high LET particles?
Chinese hamster V 79 cellsThe majority of studies linking HRS and particle-irradiations have been conducted on the V79 cell line. This cell line was developed from the lung tissue of a young male Chinese hamster and is p53 mutant. The cells have a high plating efficiency (80%), and a generation time of 12–14 h, mak-ing them convenient to perform quick clonogenic assays (around 3–4 days compared to ∼ 10 days for human cells). One of the first studies performed with charged-particles involved negative pi-mesons (Marples et al. 1994). V79 cells were irradiated as monolayers with plateau or peak pions (10–20 keV/mm and 35 keV/mm, respectively), and with 250 kV X-rays, as the reference radiation considered having a low linear energy transfer (LET). Cell survival was assessed after 84 h growth. Data were fitted using the Induced Repair model (Equation 2). No HRS was shown for peak pions: the Dc value was inferior to 0.2 Gy, which is the lowest dose used in this experiment (g 2.31 3.68 and Dc 0.09 0.11 Gy). Indeed, the interaction of peak pions with matter is mainly constituted of ‘star’ events: A process by which atom nuclei present in the surrounding medium absorb low energy pions and then fragment in a ‘star’ fashion (Raju 1980). The nuclear
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In vitro hypersensitivity after high-LET irradiation 3
fragments produced are mostly protons, alpha particles and heavier particles of high LET, leading to more severe and clustered damage than plateau pions, which simply lose their energy by slowing down. Due to the higher LET of fragments and clustered lesions, it is possible that IRR is triggered at the lowest dose used in this experiment, prevent-ing the observation of HRS for peak pions. Although survival curves obtained with peak pions did not exhibit HRS, the survival curve obtained for plateau pions was very similar to the X-ray curve, exhibiting enhanced cell killing below 0.3 Gy (g 8.31 8.95). However, the error is of the magnitude of g and more data points out that lower doses would have been useful to assess the radiosensitivity more accurately. The threshold dose Dc value for plateau pions was less than for X-rays (0.27 0.19 Gy compared to 0.38 0.07 Gy for X-rays), suggesting that the response varies with the LET.
Schettino et al. (2001) performed low dose V79 cell irra-diations with a charged particle microbeam. Briefly, 9 103 cells were seeded on 3 mm thick Mylar-base dishes, were allowed to attach for 4 h and were immediately exposed to protons (3.2 or 1 MeV at the cell entrance). The authors do not state the LET of the used particles. For comparison purpose, we estimated the LET at the cell entrance at 11 and 24 keV/mm, respectively using the SRIM (Stopping and Range of Ions in Solids) program (Ziegler et al. 1985). Cell posi-tions were recorded during the irradiation procedure and revisited after a 3-day incubation period. The use of a micro-beam set-up allows us to know the exact number of particles delivered to the cell nucleus. In this way, any bystander effect, (i.e., the phenomenon by which non-hit cells located in the close vicinity of irradiated cells suffer from the effect of irradiation), which could have arisen at such low doses is eliminated. For both LET values, the survival fraction shows a deviation from the linear quadratic model, an enhanced cell killing effect being observed between 10 and 50 particles per cell nucleus. This HRS phenomenon was more pronounced after 11 keV/mm proton irradiation. In this experiment, g was 3.6 1.2 for 11 keV/mm protons and only 0.6 0.3 for 24 keV/mm protons, showing that HRS was minimal in the lat-ter. The Dc values were similar: 0.35 0.11 Gy for 11 keV/mm protons and 0.4 0.3 Gy for 24 keV/mm protons, although the threshold dose determination is not obvious in the latter. Indeed, no clear regimen change can be clearly seen on the survival curve and this is reflected on the Dc error, which has a magnitude equal to the parameter value. This is consistent with the data obtained with pi-mesons: HRS diminishes as LET increases. Analyzing the two sets of proton data using the accumulated damaged induced radioresistance model (see Equations 4 and 5) gave evidence for HRS being LET depen-dent. As expected, the average number of damage events per proton (a) increased with the LET (0.262 0.04 proton1 for 11 keV/mm protons and 0.44 0.15 proton1 for 24 keV/mm protons). However, when this quantity is converted to Gy1, the dependence is reverted: 11.98 for 11 keV/mm protons and 8.56 for 24 keV/mm protons.
A partial explanation for this is the nature of the conversion from proton1 to Gy1: The LET value is taken into account and more 11 keV/mm protons are required to achieve a dose of 1 Gy, resulting in a higher value for a. However, this also
indicates that 11 keV/mm protons inflict more damage per gray than 24 keV/mm protons, which is contrary to all DNA damage simulation studies. The number of critical damage (c) needed to induce IRR decreased with LET (6 for 11 keV/mm protons and 4 for 24 keV/mm protons). The LET dependence of c suggests that the clustering of deoxyribonucleic acid (DNA) damage likely plays an important role in the triggering of IRR (Nikjoo et al. 1999). Interestingly, the group of Cheru-bini et al. (2011) also irradiated confluent monolayers of V79 cells with proton broad beams of similar energies (28.5 and 7.7 keV/mm). The cells were irradiated in a confluent state in the dose range of 0.1–5 Gy. Although radiosensitivity param-eters at high doses and energies were very similar to those published in the microbeam study, no effect was observed at low doses, even with 7.7 keV/mm protons. However, it may be difficult to compare data obtained with microbeam and broad beam irradiation, due to dosimetry issues to convert a particle number into a macroscopic dose, and due to the fact that a microbeam shoots every particle exactly at the same location in the cell nucleus, which is very different from a homogeneous nucleus irradiation. One of the possibilities to explain this apparent discrepancy is to hypothesize that, in the broad beam experiment, IRR is triggered below 0.1 Gy, the lowest dose used in the study. Alternatively, in the broad beam study, cells were irradiated in a confluent state. Con-tact inhibition may alter the transition from G2 to mitosis, a process involved in HRS.
Although the HRS status in V79 cells is unclear after irradiation with protons, alpha particles and carbon ions seem to be good candidates to induce this phenomenon. Böhrnsen et al. (2002a, 2002b) irradiated V79 cells with broad beams of low-energy (153 keV/mm) and high-energy carbon ions (27.5 keV/mm). Although the survival curve for low-energy carbon ions was purely linear, a significant hypersensitivity was observed around a 0.1 Gy dose of high-energy carbon ions (ar 0.12 0.06 Gy1, as 3.73 2.28 Gy1 and Dc 0.085 0.034 Gy). From 0.5 Gy the transi-tion to the linear quadratic behavior was observed. This dis-crepancy between high energy 27.5 keV/mm carbon ions and low energy carbon ions could be explained by differences in microscopic energy deposition events. Indeed, low energy particles such as 153 keV/mm carbon ions tend to have a nar-rower track, so that the energy deposition is concentrated in localized regions. As for peak pions and 24 keV/mm protons, this may lead to more clustered DNA damage. Hence, no HRS is observed. On the other hand, for high energy carbon ions, the track is considerably larger (around 120 mm), leading to a less concentrated ionization density which is comparable to photons. In this case, HRS occurs. The group of Tsoulou et al. (2001) also performed clonogenic assays on the V79 cell line following irradiation with broad beams of a-particles. They used three different LET: 58.9, 79.3 and 101.7 KeV/mm. Cells were seeded 18 h before irradiation on Mylar-based specially designed Petri dishes. Doses ranged from 0.1–3 Gy at a dose-rate of 1 Gy/min. Colonies were scored after a 7-day growth and the Induced Repair model was used to fit the data. HRS was detected for all radiation energies. Interestingly, as, which is associated with the depth of the deviation from the LQ model in the very low-dose region, appeared to increase
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2004, Marples and Collis 2008). Most results were reported after X-ray or g-ray irradiation. DNA double-strand breaks are generally accepted to be the most biologically significant lesions induced by ionizing radiation; consequently a lot of studies focus on molecular complexes involved in the repair of DSB. The molecular basis of HRS was tentatively studied by the Vaganay-Juéry group (Vaganay-Juery et al. 2000). The DNA-dependent protein kinase (DNA-PK) complex activity (Ku DNA-end binding activity and kinase activity of the whole complex) was studied in 10 human cancer cell lines (6 HRS and 4 HRS cell lines). Strikingly, a decrease in the DNA-PK activity following a 0.2 Gy irradiation was only found in the six cell lines exhibiting HRS, whereas the DNA-PK activity increased in the four cell lines which did not exhibit HRS. This transient decrease in the DNA-PK activity within the first hours following low-dose irradiation could contribute to increase the amount of unrepaired damage, and lead to enhanced cell killing. Moreover, another group (Rothkamm and Löbrich 2003) investigated the kinetics of g-H2AX foci disappearance in confluent MCR-5 cells after irradiation with doses of 20, 5, and 1.2 mGy of X-rays, and observed a decreasing capacity for DSB repair with a decreasing irradia-tion dose. This observation may link the presence of residual DSB to the phenomenon of HRS, despite that the dose range used is somewhat different from the cGy range where HRS is usually observed. Although DSB repair seems to be com-promised at low doses, HRS does not seem to be linked with a failure to detect DNA double-strand breaks (Wykes et al. 2006). Xu et al. (2002) recently demonstrated the existence of two molecularly distinct G2/M checkpoints, which are acti-vated by ionizing radiation. The G2 accumulation, also known as the ‘Sinclair’ checkpoint (typically assessed by propidium iodide staining) begins to be measurable only several hours after irradiation. It is ataxia telangiectasia mutated (ATM)-independent, dose dependent, and represents the arrest of cells which were in earlier phases of the cell cycle at the time of irradiation. In contrast, the second checkpoint is the ‘early’ G2/M checkpoint; it occurs very early after irradiation, is very transient, has been shown to be ATM-dependent and repre-sents the failure of cells which were in G2 phase at the time of irradiation to progress into mitosis. To assess this checkpoint properly, a distinction has to be made between cells in the G2 phase of the cell cycle and cells undergoing mitosis. The most common way would be to stain cells for phosphorylated his-tone H3 and assess the DNA content with propidium iodide. The mitotic ratio is chosen as the endpoint (i.e., the ratio of irradiated/unirradiated cells stained positive for phospho-rylated histone H3). No dependence on dose was evidenced in the 1–10 Gy range for this early G2 checkpoint, but below 0.4 Gy (which is similar to the Dc dose for sparsely ionizing radiation), a decrease in the arrest became apparent. Failure to arrest would lead to chromatin condensation and conver-sion of unrepaired DSB into chromosomal breaks during the G2 to M transition, and cells would then undergo mitosis with damage to DNA, which could cause lethality. The existence of a checkpoint threshold was also confirmed in the study of Deckbar et al. (2007). G2-irradiated human fibroblasts were monitored for chromosome breakage 4–6 h after exposure to X-rays. Strikingly, almost all the cells released from the G2
with LET and the most pronounced HRS effect was observed for 101.7 keV/mm a-particles (as 1.8 0.7 Gy1, 2.1 0.6 Gy1 and 5.3 0.7 Gy1 for increasing LET values). This might be related to the greater complexity of damage produced by higher LET particles, e.g., double-strand breaks (DSB) very close to each other or associated with other types of dam-age, such as single strand breaks or base damage (Nikjoo et al. 2001). These clustered damaged sites are more difficult to repair and lead to a greater cell killing efficiency. On the contrary, the transition dose Dc decreased with increasing LET and IRR was triggered at lower doses (Dc 0.7 0.6 Gy, 0.55 0.26 Gy and 0.34 0.05 Gy for increasing LET values). However, the transition dose for 58.9 keV/mm a particles is hardly determined, especially because the data could be more appropriately fitted with a simple linear quadratic (LQ) model.
Human cellsLittle work has been done to evidence HRS in human cells after exposure to medium and high-LET radiation. Two human cell lines already known for HRS following X-ray irra-diation have been studied so far. Unpublished data from the group of Cherubini et al. (2010) showed that T98G human glioblastoma cells exhibit moderate enhanced cell killing below 0.7 Gy following irradiation with a broad beam of 28.5 keV/mm protons (ar 0.97 0.01 Gy1, as 1.1 0.4 Gy1, Dc 0.7 0.45 Gy). The obtained g ratio is equal to 0.1 and the ranges determined for ar and as overlap, which suggests that no HRS is taking place. This is consistent with the data obtained with protons of similar energies from Schettino et al. and Cherubini et al. as discussed in the previous sec-tion on V79 cells. However, in the work of Wera et al. (2013), A549 lung cancer cells were irradiated with a broad beam of 10 keV/mm protons. Interestingly, HRS was evidenced below 0.2 Gy (ar 0.824 0.03 Gy1, as 11.6 5.24 Gy1 and Dc 0.052 0.015 Gy). Moreover, this marked response was also highlighted in a g-H2AX assay (the phosphorylated form on serine 139 of the X variant of histone H2A). Cells were labeled with a g-H2AX antibody 24 h after irradiation, ana-lyzed by confocal microscopy and considered as positive if their intensity was significantly higher than control samples. The relation between the dose and the percentage of posi-tive cells appeared to be linear in a 0–2 Gy range. However at very low doses, this percentage was higher than predicted by linear extrapolation, suggesting a greater radiation effect.
Finally, it has also been shown by Xue et al. (2009) that normal cells also exhibit HRS after high-LET radiation. GM0639 normal human fibroblasts were irradiated with a broad beam of 70 keV/mm carbon ions and a region of greater sensitivity was identified at low doses (ar 1.57 0.08 Gy1, as 3.31 0.92 Gy1 and Dc 0.17 0.08 Gy).
Are HRS mechanisms the same after low LET radiation than after medium and high LET radiation?
A great amount of work has been performed to identify the mechanisms of low dose hypersensitivity and increased radioresistance (see reviews: Joiner et al. 2001, Marples
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In vitro hypersensitivity after high-LET irradiation 5
checkpoint exhibited three or four premature chromosome condensation (PCC) breaks. Pulsed field gel electrophoresis studies reported a 1:3–6 relationship between the DSB yield and PCC breaks, suggesting a checkpoint sensitivity level of 10–20 DSB. Consistent with these findings, they observed that cells were released from the G2 checkpoint harboring ∼ 20 g-H2AX foci. Although being rapidly activated, the G2/M checkpoint is not maintained until complete repair. Follow-ing the discovery of these two G2 checkpoints, Marples et al. (2003) proposed and investigated the hypothesis that HRS would be a manifestation, at low doses, of the absence of arrest of cells irradiated in the G2 phase of the cell cycle. Cell survival assays following X-ray irradiations were performed for asynchronous populations, as well as for G1, S and G2 enriched populations of V79 cells using flow cytometry cell sorting. Interestingly, the G1 and S enriched populations did not exhibit HRS, whereas G2 phase cells showed a response that was markedly more prevalent than the one observed for the asynchronous population. Since the exact cell cycle phase composition was known and using a three compo-nent Induced Repair model (G1 and S cell response being only described by ar and b; the G2 cell response described by the whole set of parameters), they were able to fit not only the asynchronous curve, but also the survival curves of the enriched populations. From this data, they concluded that the HRS phenomenon is completely dominated by the complex response of G2 cells. The mitotic ratio was also inves-tigated for HRS positive and HRS negative cells. For HRS cell lines, no arrest was observed at low doses, whereas HRS cell lines displayed an arrest event at the lowest dose point. A study from Krueger et al. (2010) confirmed that HRS is mainly displayed by G2 phase cells. A double thymidine block was performed to synchronize cells in the G2 phase, and this resulted in an exaggerated HRS response in clono-genic cell survival data. The mitotic ratio was also shown to be constant in the low-dose region, a decrease occurring only at doses above 0.6 Gy. In the HRS region, the number of g-H2AX foci was also assessed over time. As expected, the number of foci remained stable for 60 min after treatment, indicating a lack of DSB repair.
Several studies pointed out that a lack of ATM-activation might be the underlying mechanism of HRS, because the dose-activation profile for both ATM and the ‘early’ G2 checkpoint matches the transition in cell survival from low-dose HRS to IRR (Krueger et al. 2007a). However, the same ATM activation pattern was observed in cell lines that do not exhibit HRS. It was suggested that the few HRS negative cell lines (about 20% of the 40 tested to date) undergo a disso-ciation between ATM activity and the ‘early’ G2 checkpoint, producing an aberrant ‘early’ G2 checkpoint response that evades dose-dependent ATM regulatory control.
Enns et al. (2004) gave some insight about the death type by which HRS positive cell lines would eliminate damaged G2 cells. Three cell lines, two HRS positive (A549 [wt p53] and T98G [p53 mutant]) and one HRS negative (MCF7 [wt p53]), were tested for apoptosis in the low dose range follow-ing irradiation with 137Cs g-rays. A first marker of apoptosis, cleaved caspase-3, was assessed 24 h after irradiation. A549 and T98G showed a marked activation of caspase-3 at the
doses that induce HRS, whereas no significant caspase-3 activation was seen for the MCF7 cell line. A second marker of apoptosis, the translocation of phosphatidylserine to the exterior leaflet of the plasma membrane, can be detected by binding of fluorescein-tagged Annexin V. As it is considered as an early-to-intermediate event in the apoptotic process, the assay was performed from 2–24 h post irradiation. Con-firming the data obtained for caspase-3 assay, A549 and T98G cell lines showed a maximal binding at 4 and 6 h post irradiation, respectively, whereas no binding was observed for MCF7 cells.
Next, the potential role of the tumor suppressor p53 was investigated. Cells were irradiated in the presence of pifithrin, a p53-activity inhibitor and hence of p53-induced apoptosis and then assessed for Annexin V binding and clonogenic survival. Not only was the Annexin V binding eliminated, but the HRS response was completely ablated. This data indicate that p53-dependent apoptosis is the main cell death type in the low dose region for HRS positive cell lines. Moreover, the data from Krueger et al. (2007b) closely relate apoptosis and G2 phase cells irradiated in a low dose range. However, it should be mentioned that G0 lymphocytes exhibit HRS evidenced by an increase in chromosomal aberrations (Nasonova et al. 2006). The irradiation of G0 lymphocytes should only produce chromosome-type aberrations, since a double-strand break produced in G0–G1 would be replicated in the S-phase and would be present on both sister chroma-tids. Chromatid-type aberrations can only be produced dur-ing S–G2, involve one sister chromatid, and are believed to be mainly induced by endogenous reactive oxygen species (ROS). In this experiment, the dose dependence of the total aberration yield had a linear shape except at very low doses where a peak was identified around 5–7 cGy. At low doses, the total aberration yield was interestingly dominated by chromatid-type aberrations. Oxidatively damaged sites of the DNA generated in the G1 phase could be converted into DNA breaks and subsequently, into chromatid-type aberrations later in the cell cycle (i.e., late S–early G2). It was suggested that the high yield of chromatid-type aberration registered at very low doses was a consequence of an endogenously generated ROS elevation. Indeed, close correlation between radiosensitivity and ROS formation following exposure to radiation was evidenced in (Ogawa et al. 2003). However, this interpretation is questionable as the amount of radiation-induced ROS at such low doses may be low compared to the endogenous level.
Xue et al. (2009) checked whether the photon-induced HRS mechanism could be applied to high-LET particles: ATM/ and ATM/ fibroblasts were irradiated with 70 keV/mm carbon ions. ATM/ fibroblasts exhibited HRS in the low-dose region for both survival and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutation assays (Dc 0.17 and 0.2 Gy, respectively) whereas ATM/ fibro-blasts did not, although they were more radiosensitive. Then, the ATM activity was modulated by two agents: chloroquine, known to induce ATM activation, and KU55933, a specific inhibitor of ATM kinase. Activation of ATM with chloroquine eliminated the low-dose HRS for ATM/ cells, whereas in -hibition of ATM activity by KU55933 prevented the initiation
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affected by the HRS process. The use of this accumulated dam-age model is not straightforward. Indeed, this is not a unique solution and a variety of approaches are possible. For instance: (i) The parameters a and c can be set as free (as in Schettino et al. 2001); (ii) c is fixed at 1; (iii) a is fixed as the average num-ber of double strand breaks per gray for the used radiation; and (iv) c is fixed at 20. Approaches (ii) and (iii) were already used in Wouters et al. (1996): Setting the parameter c to 1 means that one lesion is sufficient to trigger IRR, whereas parameter a may be fixed as the average number of DSB/Gy if already known or modelled. Although approaches (i) to (iii) have a physical meaning, the resulting fits for high-LET data were not satisfactory: Errors on parameters were huge and (iii) leads to discontinuities in the fits because of the high number of DSB produced by high-LET radiation. Besides, no LET dependency was evidenced. The rationale for approach (iv) lies in the exis-tence of a double-strand break G2/M checkpoint threshold, as described in Deckbar et al. (2007). In the light of this study, we state that IRR is triggered for all radiation protocols, when DNA harbors ≈ 20 DSB (thus setting c to 20). This fitting method gave the best results. Table I shows our fitting results for proton, pion and alpha data when using approach (iv) and the results of the IndRep model extracted from (Marples et al. 1994, Schettino et al. 2001, Tsoulou et al. 2001). In some of these works, the parameter as is not readily accessible and g is given instead. Equation 3 was used to derive as and error propagation was performed to assess the parameter accuracy.
For radiations where HRS is barely detectable, errors on parameters are sometimes important (i.e., 58.9 keV/mm alpha particles, peak pions, 24 keV/mm protons). The LET dependency of the different parameters is best represented in Figures 1 and 2.
In Figure 1, parameters as and Dc, which are extracted from the IndRep model, are plotted as a function of the LET. For a given radiation type, as seems to increase with increasing LET. However, due to error bars, only alpha particle data are reliable enough to confirm this conclusion. On the contrary, the threshold dose Dc seems to decrease with increasing LET. But once again, due to statistical errors, this conclusion might be invalid. The transition dose Dc between HRS and IRR is supposed to be reached after a defined amount of DNA damage is induced in the cell nucleus (10–20 DSB according to studies using X-rays). However, this does not take cluster-ing and track structure effects into account: The clustering
of the IRR response. This data suggest that the ATM status and the ATM activity modification are likely to affect the occur-rence of HRS/IRR, following irradiation with sparsely ioniz-ing radiation as well as with high-LET charged particles.
The involvement of ATM was confirmed by Western blot-ting: compared with higher doses, the ATM phosphorylation level was much weaker at doses less than 0.2 Gy (same dose as threshold dose for the HPRT assay). Phospho-histone H3 staining was also performed for 0.2, 0.5 and 1 Gy of carbon ions. Although the arrest was visible for 0.5 Gy and higher, the mitotic ratio was similar to control samples for a 0.2 Gy irradiation. Furthermore, this group also pointed out that ATM activation modulation could affect this ‘early’ G2 checkpoint at low doses, chloroquine significantly reducing the mitotic ratio, and KU55933 keeping the mitotic ratio at control level. This data suggest that evading the ‘early’ G2 checkpoint would correspond to insufficient ATM activation at low doses. Furthermore, g-H2AX staining was performed at 0.2, 0.5 and 1 Gy of carbon ions. The disappearance of foci was much slower after a 0.2 Gy exposure, indicating a lower efficiency of DSB repair in the low dose region.
The mechanisms mentioned above were thoroughly dem-onstrated after irradiation with sparsely ionizing radiation, and most of them are now confirmed for heavy ion irradia-tions (Xue et al. 2009), although DNA-PK involvement and apoptosis were not investigated. Therefore, it is very likely that HRS mechanisms are identical for sparsely ionizing radiation and charged particles, although HRS may be not observed if LET is too high.
Is HRS LET-dependent?
From the clonogenic assay studies presented above, it seems likely that HRS parameters are LET-dependent. The IndRep model was invariably used to fit the clonogenic data for V79 cells in the various studies reviewed above (except the micro-beam proton data, Schettino et al. 2001). Here, we reanalyzed the alpha, proton and pion data for V79 cells in the light of the accumulated damage induced radioresistance model (Equa-tions 4 and 5) and compared the results of our fits with the results already published, using of the IndRep model, in order to seek LET dependence. For all survival curves, the param-eters ar and b were kept identical to the values already pub-lished, because the high dose data were supposed not to be
Table I. Fitting results for V79 cells exposed to protons, pions and alpha particles of various LET using IndRep and accumulated damage models.
Particle LET (keV/mm)
IndRepAccumulated damage model
(c 20)
as (Gy1) Dc (Gy) as (Gy1) a (Gy1)
Alpha particlesa 58.9 1.8 0.7 0.7 0.6 1.38 0.55 15.34 32.779.3 2.1 0.6 0.55 0.26 1.47 0.33 16.87 6.28
101.7 5.3 0.7 0.34 0.05 3.22 0.78 38.8 5.3Pionsb 10–20 0.74 1.81d 0.27 0.19 0.34 0.12 60.67 11.25
35 0.89 1.58d 0.09 0.11 0.43 1.42 76.91 35.84Protonsc 11 0.92 0.49d 0.35 0.11 0.75 0.13 57.3 6.36
24 1.12 1.2d 0.4 0.3 1.07 0.19 45.99 13.45
Errors indicate the standard error of the mean (SEM).aIndRep results from Tsoulou et al. (2001).bIndRep results from Marples et al. (1994).cIndRep results from Schettino et al. (2001).dWith Equation 3 and error propagation.
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In vitro hypersensitivity after high-LET irradiation 7
et al. 2001, Semenenko and Stewart 2006). The exception is the proton data: here, a decreases as LET increases, as it was already the case in the original study when values were con-verted into Gy1 (Schettino et al. 2001).
Overall and at present, it is not possible to confirm or disprove a LET dependence for HRS related parameters, although some tendencies are observed. More data are needed to support our conclusions; it is especially neces-sary that an extensive number of cell lines are tested, for the majority of the available data were obtained in V79 cells. In the case of broad beam experiments, it should be kept in mind that a possible bystander effect can not be excluded, given the very low doses used. Indeed, if LET is too high, a smaller number of particles is needed to achieve a given dose and some cells might not be irradiated. In some cases, it is possible that the bystander effect is mistaken for HRS.
Perspectives for cancer treatment
Clinical trials on HRS following charged particle irradiation have not been undertaken yet. However attempts are cur-rently being made to take advantage of the HRS phenom-enon in X-ray radiotherapy. The pulsed reduced dose rate (PRDR) radiotherapy (Ma et al. 2011) consists in delivering 10 X-ray pulses of 0.2 Gy with 3-min intervals daily, resulting in an effective dose rate of 0.067 Gy/min. The idea behind PRDR is to take advantage of both tumor cells HRS below their
of lesions becomes more severe for identical doses as the LET increases, in a way that the cell can no longer ignore the damage and triggers the G2/M checkpoint, even when doses get weaker and weaker. Therefore, the Dc might decrease with increasing LET. This conclusion was reached previously by Edin et al. (2012). In their work, breast cancer cells were exposed to low LET radiations: a 60Co source (≈ 1.8 keV/mm) or 220 kV X-rays (≈ 3.6 keV/mm). Interestingly, the Dc value was smaller for X-irradiated cells than for g-irradiated cells. The higher ionization density of secondary electrons origi-nating from X-rays was thought to be responsible for this diminution, because it could result in a higher level of DNA damage locally. It is also possible that no HRS is observed when LET is too high [for instance ≈ 30 keV/mm protons and low energy carbon ions (Marples et al. 1994, Schettino et al. 2001, Böhrnsen et al. 2002a, Cherubini et al. 2010, 2011)]: In such a situation, only one particle track would induce suf-ficient clustering, due to low energy and narrower track, and would immediately trigger IRR.
In Figure 2, the HRS parameters as and a, for the accumu-lated damage model with c 20, are plotted as a function of the LET. Similarly to the IndRep model, as seems to increase with increasing LET, although the peak pion fitting is highly inaccurate. Parameter a increases with increasing LET: Indeed high LET radiations are supposed to create more events per gray as supported by Monte Carlo studies (Nikjoo
Figure 1. LET dependence for parameters of the IndRep model (a) as (Gy1) (b) Dc (Gy). Values for pions (triangles), protons (squares) and alpha particles (stars) were extracted from Marples et al. (1994), Schettino et al. (2001) and Tsoulou et al. (2001), respectively.
Figure 2. LET dependence for parameters of the accumulated damage model (a) as. Gy1 (b) a (Gy1): pions (triangles), protons (squares) and alpha particles (stars).
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8 A.-C. Heuskin et al.
The high quality of charged particle dose distribution, the so-called Bragg peak, combined to the potential HRS previously described benefits could be of great interest for the treatment of radioresistant or recurrent cancers. More data are thus needed to fully exploit the biology of HRS and charged particle radiations and check whether the clinical benefits demonstrated for X-rays are also present.
Conclusion
Low dose hypersensitivity has been thoroughly studied during the past decade, mainly following X-ray irradiation. However, several groups have also shown the existence of HRS after irra-diation with pi-mesons, protons, alpha particles, as well as car-bon ions on Chinese hamster V79 cell line, on human cancer cell lines and on normal fibroblasts. It is therefore very prob-able that HRS mechanisms are identical for sparsely ionizing radiation and for charged particles. HRS would be a manifesta-tion of the absence of cell cycle arrest of cells irradiated in the G2 phase, which would be due to insufficient ATM activation. The data available in literature for rodent and human cell lines and our own experience may indicate that, for a given type of particle, the threshold dose Dc and the radiosensitive param-eter as are LET-dependent. The lesions to DNA being more complex with increasing LET, the full activation of ATM would be reached at a lower dose than for X-rays. Therefore the tran-sition dose Dc between low dose hypersensitivity and induced radioresistance would decrease with LET. The initial portion of the survival curve slope at low doses as would increase with the severity of the lesions, and thus with increasing LET up to the maximum relative biological effectiveness (RBE). How-ever, due to statistical errors, it is not possible to confirm this conclusion at present. Further work on different cell lines and radiation types is required to refine this hypothesis.
Finally, the combination of the ability of charged particles to induce HRS at low doses and the chemical strategies to increase the proportion of cells in G2 phase inside a tumor might represent a more effective way to address radioresis-tant cancers.
Acknowledgements
The authors are grateful to Dr Pavel Moskovkin for his assis-tance in data analysis and programming.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
A.C. Heuskin is a PhD fellow funded by the Belgian Fund for Scientific Research (F.R.S. – FNRS).
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