Exposure to low dose ionising radiation: Molecular and clinical consequences

Cancer Letters 349 (2014) 98–106

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Exposure to low dose ionising radiation: Molecular and clinical consequences Lynn M. Martin a, Brian Marples b, Thomas H. Lynch a, Donal Hollywood a, Laure Marignol a,⇑ a Urologic and Radiation Oncology, Applied Radiation Therapy Trinity and Prostate Molecular Oncology Research Group, Discipline of Radiation Therapy and Institute of Molecular Medicine, Trinity College Dublin, Ireland b Department of Radiation Oncology, William Beaumont Hospital, 3811 W. Thirteen Mile Road, Royal Oak, MI 48073, USA

a r t i c l e Keywords: Ionising radiation Low dose Hypersensitivity Cell cycle arrest DNA damage

i n f o

a b s t r a c t This review article provides a comprehensive overview of the experimental data detailing the incidence, mechanism and significance of low dose hyper-radiosensitivity (HRS). Important discoveries gained from past and present studies are mapped and highlighted to illustrate the pathway to our current understanding of HRS and the impact of HRS on the cellular response to radiation in mammalian cells. Particular attention is paid to the balance of evidence suggesting a role for DNA repair processes in the response, evidence suggesting a role for the cell cycle checkpoint processes, and evidence investigating the clinical implications/relevance of the effect. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Radiotherapy (RT) is currently utilised in the treatment of approximately half of all oncology patients during the course of their illness. The therapeutic intent of radiation oncology is to deliver a sufficiently lethal dose to the target volume to achieve local tumour control while minimising the harmful effects to normal tissues, in order to avoid treatment-related acute side effects and late morbidity. The radiation doses prescribed in current practice are based on the clinically determined radiation tolerance of the surrounding normal tissues, and a trade-off between normal tissue toxicity and tumour control is often required. A number of factors can influence normal tissue tolerance including dose, fractionation, the volume irradiated as well as individual variation in radiation sensitivity [1]. RT protocols have evolved to limit the proportion of highly radiosensitive adverse reactions to about 0.5–5% of cases [2]. Despite great clinical progress in the field there remain a small proportion of individuals who develop severe normal tissue reactions, the underlying molecular basis for which are currently imperfectly understood. Accumulating evidence indicates that in certain tumours, RT needs to be delivered in higher than ‘conventional’ fractionated doses in order to achieve improved tumour control probability

DOI of original article: http://dx.doi.org/10.1016/j.canlet.2013.05.021

⇑ Corresponding author. Address: Radiation and Urologic Oncology, Prostate Molecular Oncology Research Group, Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine, Trinity College Dublin, Dublin 8, Ireland. Tel.: +353 1 8963255; fax: +353 1 8963246. E-mail address: [email protected] (L. Marignol). 0304-3835/$ - see front matter Ó 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.12.015

(TCP). To achieve this, an increasing number of RT techniques including 3-dimensional conformal radiotherapy (3D-CRT), intensity modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT), use multiple beams of radiation to conform the dose to the three-dimensional shape of the tumour, allowing an increased dose to be delivered to the target volume, while minimising the dose to the surrounding normal tissue. However, concern has been raised regarding the carcinogenic potential of exposing a large volume of normal tissue to such low doses of ionising radiation (IR) [3–7]. The rate of radiation-induced clonal inactivation is dose and genotype dependent [8]. Low dose hyper-radiosensitivity (HRS) is characterised by an increased sensitivity to radiation doses less than 0.3 Gy, which is followed by a more radioresistant response per unit dose between 0.3 and 0.6 Gy termed increased radioresistance (IRR). The HRS/IRR response can be defined or confirmed mathematically using the induced repair model (Fig. 1), but a modified model was recently proposed [9]. Since its identification more than two decades ago [10], it has been demonstrated in vitro in approximately 75% of the 50 mammalian normal and malignant cell lines tested to date (Table 1) [11]. Malignant cells lines tested included glioma [12–18], colorectal [19–24], prostate [23,25–29], bladder [22,30], cervix [23], lung [15,23,31,32], breast [15,31], melanoma [15,21,22], head and neck [33], oral [16], neuroblastoma [34], sarcoma [13,28] and Chinese hamster ovarian [35] cancer cells. The majority of normal cell lines examined were fibroblasts (Chinese hamster [20,26,36–38], rat [17], human [11,15,17,28,31,39–41], but human keratinocytes [28] and lung epithelial cells [28,42] were also tested. HRS has also been demonstrated in vivo in skin [43], in lung and kidney tissue,

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mechanism as HRS/IRR, and in fact, IDRE is thought to be a dose rate-dependent manifestation of HRS/IRR [50]. In both instances, irradiated cells experience radioprotective transitions in cell killing from hypersensitive states to radioresistant states at discrete dose rate (for IDRE) and dose (for HRS/IRR) thresholds. Leonard et al. have demonstrated that IDRE only occurs in cell lines that express HRS [50]. 3. HRS and increased tumour cell kill

Fig. 1. Typical cell survival curve with evidence of hyper-radiosensitivity (HRS). Broken line shows low-dose extrapolation from linear quadratic (LQ) model applied to high-dose survival data. Solid line shows induced repair fit. Image adapted from [69].

metastatic tumour nodules [44], and normal human epidermis [45]. The mechanisms underlying the cell-type specific expression of HRS are still being investigated, but appear related to defective DNA repair systems and cell cycle regulation. HRS may represent an exploitable mechanism for increased tumour cell kill, and is thought to be one of the mechanisms that may increase normal tissue reactions and protect against carcinogenesis following low dose IR [46]. This review discusses the experimental data detailing the incidence, mechanism and significance of HRS in radiotherapy. 2. HRS/IRR at high doses and low dose rates HRS appears to be a widespread phenomenon in the low dose radioresponse of mammalian cells. It has been observed in response to acute dose rate negative pi-mesons [38], high linear energy transfer (LET) radiation given at a low dose rate [47], low dose neutrons [48], protons [49] and carbon ions [41]. IRR however, is only evident after low and intermediate LET radiation exposures. Moreover, it has been demonstrated that the excess in cell killing observed at very low dose rates termed the ‘‘inverse dose rate effect’’ (IDRE), appears to be derived from the same radioprotective

The clinical relevance of HRS in tumour control is a matter of debate. While HRS may increase tumour cell kill and improve the therapeutic ratio of radiotherapy, the ominous presence of tumour hypoxia in solid tumours may prevent its induction [51]. Radiotherapy delivery techniques, such as IMRT and VMAT, involve the complex arrangement of a number of external radiation beams to shape the dose distribution to the treatment volume. The therapeutic effectiveness of IMRT in the treatment of prostate cancer and head and neck tumours for instance has been well documented [52,53]. IMRT delivers dose using fields at fixed gantry angles, either dynamically or statically (step and shoot) using many beam apertures (segments) that are shaped with multileaf collimators. The number of beams used in IMRT plans can range from 5 to 15 with the daily dose maintained at 2 Gy. Similarly VMAT delivers daily doses through variable gantry rotation [54]. The standard daily dose (fraction) of radiation is thus delivered by a number of external radiation beams building up to a total dose of 2 Gy. The dose contributed by each of these beams is part of the daily fraction and represents a partial fraction (PF). The PF may be within the dose range for induction of HRS. Lin and Wu demonstrated that delivery of PF of a RT treatment such that the smaller fractions (<0.5 Gy) are delivered before larger fractions (>0.5 Gy), can induce a small increase in cell kill in vitro [29]. Further evidence of this phenomenon has however not been since reported and in vivo data is currently lacking to support potential clinical relevance. The hypothesis that reducing the dose per fraction to doses within the HRS region may improve tumour control has been tested. In vivo studies utilising such ultrafractionated protocols (3 fractions of 0.4 Gy per day, interval 4 h, 7 days per week) in xenografts derived from HRS + HGL21- and T98G glioblastoma cells however failed to show improved tumour control when compared

Table 1 Prevalence of HRS by tumour origin/cell type. Cell origin Malignant Glioma cells Colorectal Prostate Bladder Cervix Lung Breast Melanoma Head and neck squamous Oral squamous Neuroblastoma Human sarcoma Chinese hamster ovarian Normal cells

HRS + cell lines

HRS

T98G, CAL58, A7, HGL21, U123, BMG1, U87-MG, DBTRG, MO59K, MO59J/Fus1, G5, G111, G142, G152 HT29, RKO DU145, PC3, LnCaP RT112

U373, MO59J, CL35 (subclone of G5)

[12–18]

HCT116, SW48

[19–24] [23,25–29] [22,30] [23] [15,23,31,32] [15,31] [15,21,22] [33]

A549 MCF7 MeWo, Be11, M4Be, A375P, SKMel2 SCC-61, SQ20B

cell lines

Siha H460 U1

PECA-4451, PECA-4197 HS633T (soft tissue sarcoma) CHOAA8

Chinese hamster V79, V79379A, fibroblasts Rat fibroblasts MR4 (oncogene-transformed) Human fibroblasts MSU-1, GS3, GM0639 cells (ATM+/+, termed GMcells), MRC5, HeLax skin human fibroblast hybrid cells (CGL1, CGL3), AT22IJE-TJ EBS7YZ5 (ATM complemented) Human Keratinocyte Lung epithelial L132

HX142 ATBr1(osteosarcoma) CHO

References

[16] [34] [13,28] [35] [20,26,36–38]

3.7

[17]

2800T, AT5BIVA cells (ATM / , termed AT cells) AT22IJE-TJ EBS7 (AT)

[11,15,17,28,31,39– 41]

HaCAT, HPV-G

[28] [28,42]

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to conventional irradiation [55]. Nonetheless, the potential contribution of HRS to enhance radiation-induced tumour cell kill should be further examined. In vitro experiments showed that low dose fractionated radiotherapy (LDFRT) can improve the efficacy of chemotherapeutic agents such as paclitaxel [33], taxotere [56] and cisplatin [57]. At a low nontoxic concentration (1 lM), cisplatin indeed appears to abolish the increased radioresistant portion of the survival response [58]. The feasibility of the combination of LDFRT with chemotherapy has been confirmed in patients with optimally debulked stage III/IV carcinoma of the endometrium [59], relapses or metastases of epithelial tumours [60], stage IIA/B-IIIA breast cancer [61] and glioblastoma multiforme [62]. The benefit appears consistent with hypersensitive growth arrest response and cell killing [59] and phase II studies are underway. 4. HRS and normal tissue reactions Much of what is known regarding the effects of HRS on normal tissues stems from studies utilising human skin as a model for normal tissues. The importance of studying the effect of low radiation doses in human skin is clear; irradiation of skin is inevitable in RT

treatments, it is readily observable and the propensity of skin to develop early and late reactions at radiation doses >1 Gy/fraction following RT has been well documented. While skin does not always represent the critical limiting factor for RT protocols, it serves as a valid model which will inform the radioresponse of other acute-reacting normal tissues. Using a variety of endpoints (kidney function, clonogenic survival, basal cell density, proliferation, erythema, micronucleus assay, growth arrest, cH2AX foci), HRS has been demonstrated in lung epithelial cells and murine kidney, salivary glands [11] as well as in various components of human skin including the epidermis, keratinocytes, and fibroblasts [59,63–65,44,66–68,45,69] (Table 2). This HRS response was observed after both single and repeated low dose fractions, indicating that HRS reactions are likely to occur following RT protocols incorporating low doses of radiation (e.g. IMRT). Modelling has shown that HRS in normal tissues will tend to increase the normal tissue complication probability, particularly in tissues with a pronounced volume effect and may thus affect treatment planning for IMRT [70]. However, the potential implications of this response in terms of normal tissue reactions are unclear. A low-dose hypersensitivity loss in basal skin keratinocytes was reported throughout

Table 2 Studies investigating the incidence of HRS in irradiated normal tissues using various endpoints. Model

No. of subjects

Murine skin

Fractionation

Murine kidney

Human skin

Functional endpoint

Results

0.1–0.2 Gy/#, 8 h interval Kidney function: 5ICr– EDTA clearance, Urine output, hematocrit 16– 50 weeks post IR 117 pts total: 65 pts 52 pts

Human epidermis of prostate ca patients

Authors [17,18]

Increased X-ray effectiveness at low [19] doses and deviation from LQ

Skin doses between 0.4 Treated palliatively with 5, 10, 12 and 20 daily and 5.2 Gy evaluated for treatment fractions (varying thicknesses of bolus, various body sites) undergoing prostatic irradiation erythema for localised carcinoma of the prostate (no bolus, 30– 32 fractions)

[63] LQ model significantly underpredicted peak erythema values at doses less than 1.5 Gy per fraction

0.45 Gy/fraction v 1.1 Gy/fraction

Basal cell density Ki67, p53, p21

Greater loss of basal cells post 0.45 Gy

[64]

Greater reduction in BCD after 0.48 Gy skin doses

[65] Study 1 of 2

Human skin

24 (23 male, 1 female) 48 biopsies

3-Field technique, 6–12 MV photons bolus used to correct skin dose to 0.48 Gy, 1.22 Gy and 0.74 Gy

Basal cell density

Human skin

8 pts with metatstatic tumour nodules

1  daily dose 0.5 Gy and >1 Gy UF (0.5 Gy  12 daily doses)

BCD and proliferation (Ki- Greater BCD reduction in high dose/ [44] 67, Cyclin A) # arm Study 2 of 2

Normal cells of Skin fibroblasts and Survival curve 0.05–4 Gy keratinocytes cervical ca derived from 40 pts patients

The fraction of binucleated Low incidence of HRS in fibroblasts [66,67] cells with micronuclei (2/40) and keratinocytes (4/40)

Keratinocytes and fibroblasts of cervical ca patients

Skin fibroblasts from 0.25 Gy TDS, 4 h interval, 0.75 Gy  1, 0.5 Gy TDS, 4 h Micronucleus assay interval, 1.5 Gy  1 21 pts and keratinocytes derived from 23 pts

Human epidermis

64 Skin biopsies from 5 PCa pts

Inverse fractionation effect observed [68] in pts expressing HRS after multiple low doses

Samples taken 30 min after the first dose fraction at H2AX foci, apoptotic cells cH2AX foci pattern in biopsies taken [45] locations of the skin corresponding to approximately 30 min after a single fraction 0.1, 0.2, 0.45 and 1.1 Gy revealed HRS below 0.3 Gy No decrease in foci observed at 2hr Additional biopsies, corresponding to 0.45 and 1.1 Gy, post IR were taken 2 h after the first fraction and after 1 week of txt

40 pts, 32 of which Keratinocytes received RT and fibroblasts of cervical ca patients

0.25 Gy TDS, 4 h interval, 0.75 Gy  1, 0.5 Gy TDS, 4 h Micronucleus assay Maximum grade of acute interval, 1.5 Gy  1 and late tissue reactions

No significant relationship found between MN induction, either in fibroblasts or keratinocytes, and acute and late effects

[69]

Keratinocytes of 89 pts prostate ca 25 pts patients

7 weeks, EBRT

HRS induced increase in p21 decrease in mitosis persistent foci

[59]

1 and 6.5 weeks after txt Growth arrest (p21) mitosis (p-H3) cell death (cH2AX)

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Fig. 2. Evidence for HRS in vitro by cell origin. To date, 53 cell lines from 16 different cell types (14 mammalian, 2 hamster) have been investigated for evidence of HRS. The most recent data indicate that 75% of cell lines (40/53) tested to date express the response.

7 weeks of radiotherapy in skin punch biopsies from prostate cancer patients undergoing radiotherapy with a curative intent [64]. In skin, HRS however appears to be relatively rare, occurring in only 6 of 40 cervical cancer patients treated with IR [71]. Furthermore it did not correlate with acute or late normal tissue complications in these patients [69]. The influence of HRS as regards normal tissue complications in other models remains to be seen. Normal tissue complications are associated with an inflammatory response [72] that could be reduced with the use of LDFRT. LDFRT was indeed shown to modulate inflammatory processes and as a result is used to treat inflammatory and degenerative diseases [73]. Low radiation doses lowered the amount of secreted IL1beta in a NF-kappaB dependent manner in activated macrophages [74]. The dose dependency of the anti-inflammatory properties of radiation appears to follow a relationship similar to that observed in HRS [75]. Further research is required to evaluate the potential significance in normal tissue complications in radiation therapy. 5. Low dose radiation and carcinogenesis Clinical exposure to low doses of IR was originally considered harmless. Estimations of the stochastic effects were based on linear extrapolation from high-dose survival data as is currently recommended by standards organizations, such as the International Commission on Radiological Protection [76]. The data, on which these estimates are based, are derived from the A-bomb database, a record of the health and mortality of the survivors of the Hiroshima and Nagasaki atomic bombs – which is considered the gold standard when assessing the health risks of radiation over the dose range of 0.1–2.5 Gy. Data from this database suggest that the underlying dose–response relationship of IR and its stochastic effects is linear without any thresholds. However, a number of recently identified phenomena specific to the low dose region of the dose response curve have brought into question the validity of the linear no threshold model to predict cancer risk in the low dose region [77]. Whereas the adaptive response suggests that this procedure would overestimate the risks of low dose exposures, the bystander effect, and concept of sensitive subpopulations suggest that such approximations may grossly underestimate the risk of low doses of radiation. The nature of the dose-response relationship in this low dose region is thus a topic of lively debate. Low-dose radiation exposure has been linked to increased cancer risk [78]. The excess relative risk (ERR), which is a measure of the size of the increase in cancer risk in the study population due to the radiation at given doses, has been used to examine the relationship between radiation doses and the risk of cancer induction. Results from the Life Span Study in the survivors of the atomic

bombings at Hiroshima and Nagasaki estimated ERR for solid cancers at 0.47/Gy [79]. However, the task of relating HRS to cancer risk is difficult due to the lack of reliable biomarkers. The European project DoReMi (Low Dose Research towards Multidisciplinary Integration) aimed to identify the most appropriate biomarkers of exposure for use in population studies. Their report highlighted that the majority of techniques reviewed do not have sufficient sensitivity to detect doses <0.5 Gy, although good candidates (comet assay, Y-H2AX immunostaining) exist. H2AX function is indeed essential for mammalian DNA repair and genomic stability [80]. Mutation and/or transformation in vitro studies comparing high and low radiation doses are required to examine the mechanisms of radiation-induced carcinogenesis at low doses and elucidate the potential role of HRS (see Fig. 2). 6. The mechanism of HRS It is generally believed that defects in a number of components of the DNA damage response to IR may be responsible for the HRS response, and while the underlying mechanism remains to be elucidated, the consensus is that the relative increase in cell kill observed is derived from a protective mechanism that has evolved to prevent genomic instability by removing those cells at risk of mutation. At higher doses then, where the entire population is at risk, the biological focus is thought to switch from that concerning potentially lethal damage to a means to preserve the entire population [46]. The studies in the field fall into one of three categories: (1) those suggesting a role for DNA repair (2) those suggesting a role for the cell cycle, and (3) those investigating the clinical implications/relevance of the effect (Fig. 3). 6.1. Evidence supporting the involvement of DNA repair processes Extensive evidence suggests a role for DNA repair in the HRS/ IRR response. Support for the involvement of DNA repair processes stems primarily from work demonstrating the dependence of HRS on LET, a role for non homologous end-joining (NHEJ) repair pathway in IRR, and evidence of persistent RAD51 foci, an essential component of the HR double strand break (DSB) repair pathway, following exposure to low radiation doses (Fig. 4). 6.2. DNA double strand breaks and HRS A particular type of DNA damage has yet to be definitively affiliated with HRS. While, it has been demonstrated that HRS is not the result of a failure to recognise DNA DSBs [17], a number of observations implicate DNA DSB repair pathways in the HRS/IRR response. These include an inducible NHEJ response associated

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Fig. 3. Time line of studies informing the current understanding of the mechanism of low dose radiation hypersensitivity.

with IRR, and persistent RAD51 foci evident at late time points after low dose IR. In addition, a hypersensitive response for H2AX has been observed in skin biopsies [45] suggesting a role for DSBs specifically in HRS in epidermal cells at least. 6.3. HRS is associated with persistent RAD51 foci Evidence that persistent RAD51 foci are common at late time points after low dose IR exposure implicates a role for DNA repair

in HRS (Fig. 4A). DNA damage-induced Rad51 nuclear foci are thought to reflect repair of DNA double-strand breaks by homologous recombination, a repair pathway mainly activated in G2 phase cells following irradiation [81]. Short et al. [82] reported that RAD51 foci co-localised with BRCA2 foci are common following low doses of radiation in glioma cells hypersensitive to low radiation doses. Co-localisation of RAD51/BRCA2 foci is thought to be indicative of HR repair. Consistent with this observation, Thomas et al. demonstrated a higher frequency of unrepaired DNA DSB

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cycle (reviewed in [89]). A specific role for two components of this pathway; PARP (Poly-ADP ribose polymerase) [90] and DNA-PK (DNA-dependent Protein Kinase) has been demonstrated [14,34] in IRR, following observations that the IRR response is eliminated in PARP and DNA-PK deficient cell lines. Vaganay-Juery et al., reported decreased DNA-PK activity in association with HRS (Fig. 4B). Indeed, a number of other studies also suggest that the transition to IRR is the result of an inducible repair mechanism; a response similar to IRR is triggered by non-lethal doses of DNAdamaging agents [37]. Moreover, the IRR response has been demonstrated to require de novo protein synthesis [91]. 6.5. DNA MMR and HRS

Fig. 4. DSB DNA repair and G2/M cell cycle arrest are implicated in HRS Exposure to low dose radiation induces DNA damage. While a particular type of DNA damage has yet to be definitively affiliated with HRS a number of observations implicate DNA double strand break (DSB repair) pathways in the HRS/IRR response. (A) In G2 phase cells, activation of homologous recombination is associated with persistent BRCA2/RAD51 foci, residual unrepaired DSB, activation of apoptosis and expression of HRS. (B) In G1/S phase cells, radiation-induced DNA damage is likely linked to the Non-Homologous-End-Joining (NHEJ) DSB DNA repair pathway. HRS+ cells were associated with repressed PARP-1 and DNA-PK activity and residual unrepaired DSBs. HRS+ cells have repeatedly been reported to evade the early G2 cell cycle arrest following irradiation. (C) In G2 phase cells, ATM-dependent phosphorylation of Chk2 leads to activation of Cdc25c, prevention of Cdc2-cyclinB activation and early G2 cell cycle arrest. A role for the mismatch repair pathway (MMR) has been implicated in the evasion of this arrest by HRS+ cells, and in the induction of persistent RAD51 foci. (D) In G1/S phase cells, recognition of radiation-induced damage leads to ATR-dependent activation of Chk1, activation of Cdc25c, prevention of Cdc2-cyclinB activation and late G2 cell cycle arrest. The late G2 checkpoint has not to date been associated with HRS. (E) Expression of HRS is associated with increased induction of apoptosis following evasion of G2 cell cycle arrest and incomplete repair of DNA damage.

processed by the NHEJ and by the RAD51-dependent HR pathways in hypersensitive compared with non-hypersensitive cells derived from the same tumour [83]. However, it may also represent sites of inefficient or dysregulated HR, for example sites at which there has been failure to locate a homologous partner for exchange, inappropriate RAD51 binding or self-self interaction as a result of high RAD51 protein levels [84,82]. While not the most widely demonstrated response associated with hypersensitive cells, a model in which the RAD51 recombination pathway may have an important influence on the survival following low doses of radiation is consistent with observations that HRS is most pronounced in G2 phase cells [85,86]. Moreover, it may provide an explanation for the prevalence of HRS in radioresistant cancer cells [11] given that RAD51 is over-expressed in many tumours [87]. 6.4. HRS is associated with reduced NHEJ response The dependence of IRR on a functional NHEJ response was one of the first reported attributes of the HRS/IRR phenomena [88]. NHEJ is the primary DNA double strand break repair pathway in mammalian cells active during G0, G1 and early S phases of the cell

DNA mismatch repair (MMR) was most recently implicated in HRS following observations that an absent HRS response was associated with MMR-deficiency in a panel of 3 cell lines [92,93]. DNA MMR is a highly conserved DNA repair pathway that recognises and repairs base-pairing errors that arise during DNA replication or recombination [94,95]. This pathway has been largely studied in the DNA damage response to chemotherapeutic agents, but the activation of the MMR system after IR remains controversial [96,97]. Accumulating data suggest that MMR proteins may be involved in the DNA damage response to IR, via recognition of IR induced DNA damage including oxidative damage, clustered base damage, and DSBs, either directly or indirectly as a component of the BRCA1 Associated Genome Surveillance (BASC) complex (reviewed in [98]). Recognition of these lesions may then promote differential cell cycle and apoptotic responses. The MMR repair pathway may indeed be required for the ATM-mediated activation of chk2 and subsequent induction of the early G2 cell cycle arrest following irradiation [99] (Fig. 4C). We have recently identified a role of MSH2 in expression of HRS in endometrial carcinoma cells [100]: HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint persistent MRE11, cH2AX, RAD51 foci at 2 h after irradiation. A role for MMR-proficiency was also observed in the preferential response of cancer cells to prolonged low dose rate irradiation [101]. 6.6. Evidence supporting the involvement of the cell cycle The dependence of IRR on functional DNA repair and the importance of cell cycle phase for the efficiency and propensity for particular types of DNA repair [102] prompted investigation into the differential low dose radiosensitivity of cells in various phases of the cell cycle. Early studies demonstrated that G1 phase cells were less sensitive to low dose IR than were the entire population [12], later studies investigated the HRS effect in each cell cycle phase and demonstrated a more pronounced HRS response in G2 phase enriched cells, with little [103] or no evidence of HRS in other cell cycle phases [86]. Mechanistic studies have also revealed a role for cell cycle checkpoints in HRS. Cell cycle checkpoints are surveillance mechanisms that block cell cycle transitions, that function to block cell cycle progression to allow repair of DNA damage prior to cell entry and transit through mitosis [104]. This arrest is only released when repair is completed. Where repair is not possible, damaged cells are removed by programmed cell death (apoptosis) [105]. Two distinct G2/M phase cell cycle checkpoints are known to be activated following exposure to IR [81], namely the early and late G2/M phase checkpoints. The first and so-called early G2/M checkpoint can be initiated in G2 phase cells following activation of ATM and subsequent phosphorylation of Chk2 (Fig. 4C). It reflects the response to DNA damage in cells that are already in G2 at the time of irradiation, and reflects the failure of these cells to progress to mitosis. The second G2/M checkpoint is activated at late time points after exposure

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and reflects the delay of cells in other phases of the cell cycle (G1, S) at the time of irradiation. It is associated with ATR-dependent phosphorylation of Chk1 and Cdc25c (Fig. 4D). Both pathways converge on Cdc25c, a positive regulator of cell cycle progression, which is inhibited by Chk1-mediated or Chk2-mediated phosphorylation. Activation of cdc25c prevents the dephosphorylation of cdc2-cyclinB and progression of cells into mitosis [106]. HRS has been repeatedly correlated with evasion of the early G2 checkpoint [41,85,86,92,107–109], with the transition from HRS to IRR reflective of the activation of the early G2 checkpoint, and thus inducible repair mechanisms [41,85,92,109]. However, a maintained growth arrest has also been associated with HRS in keratinocytes [59,64], and in the A549 cell line, the mechanism of HRS/ IRR in was recently proposed to result from early G2/M checkpoint arrest and enhanced DNA DSBs repair through Chk2 activation [110]. In these cells, dose-dependent repression of Cdc25c phosphatase expression was associated with early G2 checkpoint arrest and may represent an alternative mechanism to HRS [111]. These data contradict the most recent working hypothesis for the mechanism of HRS indicating that HRS is the apoptotic response of cells that evade the early G2/M checkpoint [85], and raises the question of whether the mechanism of HRS may be cell type dependent. It will therefore be important for future studies to resolve the involvement of cell cycle responses and reconcile this with DNA repair processes. 7. Conclusion The HRS/IRR phenomenon has been extensively demonstrated in the past decade. The balance of evidence suggests it may have varied implications for radiotherapy practices and public health. While in vivo studies continue to provide insight into the potential clinical implications of the effect in terms of exploitation of the response for a therapeutic benefit and implications for normal tissue reactions, no changes in current practices can be made until the mechanism is fully understood. Indeed, if the HRS response is mostly related to growth arrest and cell survival as is suggested by the most recent data in epidermal cells, the risk of mutagenesis is potentially greater than expected in patients lacking HRS. This may allow time for alternative more error-prone repair mechanisms to be triggered or may trigger a senescent state. On the other hand, if cell death governs the HRS response to low doses, this risk is eliminated. Thus, the most pressing and fundamental issue to resolve is how the DNA damage response affects life or death versus genomic stability to low doses of radiation. Conflict of Interest The authors declare no conflict of interest. Acknowledgments We would like to acknowledge the following organisations for support: Cancer Research Ireland, Higher Education Authority Program for Research in Third Level Institutions and Trinity College Dublin. In addition, we acknowledge Science Foundation Ireland for their support of the Flow cytometry facility in the Institute of Molecular Medicine, Trinity College Dublin. References [1] M. McKay, L. Peters, Genetic determinants of radiation response. Report on a symposium held at Peter MacCallum Cancer Institute, Melbourne, Australia, 8 August 1996, Int. J. Radiat. Biol. 71 (1997) 225–229. [2] A. Norman, A.R. Kagan, S.L. Chan, The importance of genetics for the optimization of radiation therapy. A hypothesis, Am. J. Clin. Oncol. 11 (1988) 84–88.

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