Chromatin structure and radiation-induced DNA damage

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The International Journal of Biochemistry & Cell Biology 49 (2014) 84–97

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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel

Review

Chromatin structure and radiation-induced DNA damage: From structural biology to radiobiology Christophe Lavelle a,b,c,d,∗ , Nicolas Foray d,e a

Genome Structure and Instability, National Museum of Natural History, Paris, France CNRS UMR7196, Paris, France INSERM U1154, Paris, France d Nuclear Architecture and Dynamics, CNRS GDR 3536, Paris, France e INSERM, UMR1052, Radiobiology Group, Cancer Research Centre of Lyon, Lyon, France b c

a r t i c l e

i n f o

Article history: Received 13 September 2013 Received in revised form 13 January 2014 Accepted 18 January 2014 Available online 29 January 2014 Keywords: Chromatin Nucleosome DNA damage Radiation Radiobiology

a b s t r a c t Genomic DNA in eukaryotic cells is basically divided into chromosomes, each consisting of a single huge nucleosomal ﬁber. It is now clear that chromatin structure and dynamics play a critical role in all processes involved in DNA metabolism, e.g. replication, transcription, repair and recombination. Radiation is a useful tool to study the biological effects of chromatin alterations. Conversely, radiotherapy and radiodiagnosis raise questions about the inﬂuence of chromatin integrity on clinical features and secondary effects. This review focuses on the link between DNA damage and chromatin structure at different scales, showing how a comprehensive multiscale vision is required to understand better the effect of radiations on DNA. Clinical aspects related to high- and low-dose of radiation and chromosomal instability will be discussed. At the same time, we will show that the analysis of the radiation-induced DNA damage distribution provides good insight on chromatin structure. Hence, we argue that chromatin “structuralists” and radiobiological “clinicians” would each beneﬁt from more collaboration with the other. We hope that this focused review will help in this regard. © 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural aspects of radio-induced DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Mechanisms of DNA attack and protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Ionizing radiation as a probe for chromatin conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Chromatin conformation inﬂuence on DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Damage at the DNA scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Damage at the nucleosome scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Damage at the chromatin ﬁber scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Damage at the chromosome scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical aspects of radio-induced DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Radiosensitivity tissue reactions after radiotherapy: chromatin and high-dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. From clinical tissue reactions to the notion of radiosensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. DNA repair and chromatin techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85 85 85 85 87 87 87 88 88 88 89 89 89 89 90

Abbreviations: AR, adaptive response; BD, base damage; bp, base pair; CT, computed tomography [or] chromosome territory; DDR, DNA damage response; DSB, double strand break; FISH, ﬂuorescence in situ hybridization; HDC, highly damaged cells; HR, homologous recombination; HRS, hyper-radiosensitivity; IR, ionizing radiation; IRR, induced radio-resistance; LET, linear energy transfer; LINE, long repetitious interspersed sequence; LNT, linear no threshold; LORD, low and repeated doses; NCP, nucleosome core particle; NHEJ, non-homologous end joining; NLT, non-linear threshold; PFGE, pulsed-ﬁeld gel electrophoresis; SF, surviving fraction; SB, strand break; SINE, short repetitious interspersed sequence; SSB, single strand break. ∗ Corresponding author. E-mail address: [email protected] (C. Lavelle). 1357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2014.01.012

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3.2.3. DNA breaks repair deﬁciencies and chromatin organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Impact of the cell cycle on chromatin organization and DNA damage response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Chromatin and radiation-induced cancers after exposure to low-dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. The hypersensitivity to low-doses (HRS) effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. The low and repeated doses (LORD) effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. The adaptive response (AR) phenomenon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Our genome is constantly attacked by cellular metabolic products and environmental agents (e.g. chemical agents or ionizing radiation (IR)), triggering a DNA damage response (DDR) as a series of coordinated events that allow DNA damage detection, signalling, repair and, ultimately, survival, death or transformation. This response occurs in the context of chromatin, a dynamic entity comprising DNA associated with histone octamers and other non-histone components. Far from being a “repetitive unit” of chromatin, nucleosomes show various characteristics (sliding propensity, conformational dynamics) which are conferred by histone variants and their post-translational modiﬁcations along with the physical properties of the DNA (Lavelle, 2009). The local structure of chromatin and its dynamical features inﬂuence the outcome of environmental aggression. The presence of a ligand (mainly histones) reduces the probability of strand break (SB) production by a local masking of the attack sites. The way nucleosomes can change conformation and interact with neighbouring nucleosomes inﬂuences the higher structure and dynamics of chromatin. The distribution of damage along the DNA molecule depends on these features. Indeed, the pattern of discrete energy deposition along the track of the ionizing radiation combined with local chromatin structure deﬁnes the spatial distribution of the lesions induced in DNA (Fig. 1). Clustered damage sites are usually a signature of IR, and are known to produce complex damage–including doublestrand breaks (DSB)–which are more difﬁcult to repair than other lesions (Eccles et al., 2011; Schipler & Iliakis, 2013). More precisely, low linear energy transfer (LET) radiation (e.g. X-rays, beta or gamma emissions) induces lower concentrations of ionization events, hence less complex DNA damage – including DSB – than high LET (e.g. alpha emissions and heavy ions) (Nikjoo et al., 1998). Now, IR can be considered as a ‘two-edged sword’ in that it may lead to the formation of mutations and genetic instability in normal tissue while favouring cell killing in tumour cells after radiotherapy (Eccles et al., 2011). Therefore, a precise knowledge of both track structure and chromatin organization are required not only for the understanding of the mechanisms of radiation effect in cells but also in practical aspects of radiotherapy. There exist already many good reviews on chromatin and DNA repair protein choreography (Altmeyer & Lukas, 2013; Bao, 2011; Goodarzi & Jeggo, 2013; Gospodinov & Herceg, 2013; Lisby & Rothstein, 2009; Loizou et al., 2006; Lukas & Bartek, 2004; Papamichos-Chronakis & Peterson, 2013; Price & D’Andrea, 2013; Soria et al., 2012) with some speciﬁcally focusing on DSB and other radiation-induced damage (Hunt et al., 2013; Lomax et al., 2013). Larry Thompson recently presented a quite comprehensive review addressing the biochemistry of repair pathways after ionizing radiation (Thompson, 2012); we wish here to build on this with a complementary perspective, addressing the relationship between chromatin research and radiobiological research from a physical point of view, mainly focusing on chromatin 3D organization.

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An interesting aspect of IR is that it can serve both as a curing or investigating device (Fig. 1). Indeed, experimentally induced DNA damage is a useful tool to study chromatin structure at its various levels of organization (Rydberg, 2001). On the other hand, radiotherapeutic protocols usually based on empirical knowledge on DNA repair would beneﬁt from a more thorough utilization of the knowledge of the spatio-temporal organization of repair processes in the context of higher-order chromatin structure (Jezkova et al., 2013). Both aspects are discussed in this review. Section 2 is dedicated to structural (fundamental) issues, discussing (1) how a good knowledge of DNA organization at various scales can indeed help to understand better the effect of IR on DNA and (2), the other way round, how the data collected from radiobiological studies might feed models of chromatin structure models and yield a better knowledge of DNA organization at all scales. Section 3 is dedicated to clinical issues; a general approach consists in increasing the severity of DNA damage in tumors and facilitating their repair in normal tissues. However, individual factors and other technical parameters such as dosage and the repetition of doses during radiotherapy or radiodiagnosis must be taken into account to better evaluate risk and anti-tumor efﬁciency. The linkage between structural and clinical issues is clear in vivo, even if it is somewhat neglected in the scientiﬁc and medical literature, where papers dealing with most aspects of radiation/radiotherapy are published mostly in specialized journals -even when they provide valuable information on genome structure and properties, that would interest a broader audience. We argue in this review that the radiobiology and chromatin ﬁelds should be intrinsically coupled. As we will see from Sections 2 and 3 below, both domains have different aims but rest upon common ground. Since the outcome of radio-induced damage (including potential chromosome instabilities) is inﬂuenced by local chromatin conformation, chromatin “structuralists” and radiotherapists would each beneﬁt from tighter collaborations with the other. We hope that this focused review will help in this regard, and anticipate that within ten years Sections 2 and 3 will truly merge, and that the fuzzy “because of chromatin structure” argument used anytime/anywhere will be replaced with a precise description of what actually happens in the nucleus upon radioinduced DNA damage and repair. 2. Structural aspects of radio-induced DNA damage 2.1. General considerations 2.1.1. Mechanisms of DNA attack and protection IR induces strand breaks (SB) along with sugar and base modiﬁcations in DNA (Fig. 2). SB occur mainly via the reaction of OH radicals produced by water radiolysis with the H atoms of the sugar moiety (Balasubramanian et al., 1998). Interestingly, the SB occurrence is not the same at all nucleotide sites: for example, reduction is obtained in regions where the local structure gives a narrow minor groove, leading to a decrease in the accessibility of these particular attack sites to the radicals (Isabelle et al.,

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Fig. 1. Chromatin and ionizing radiation. DNA breaks upon ionizing radiation (IR) are expected to depend on the chromatin local structure at energy deposition spots (blue circles), in respectively (a) no chromatin (inter chromosomal territory (CT) space), (b) open chromatin (or “euchromatin”) and (c) dense chromatin (or “heterochromatin”) environments. Hence, the analysis of DNA fragments collected after in vitro irradiation reﬂects the various levels of chromatin organization. In return, the ﬁne knowledge of chromatin structure and dynamical properties is required to understand radiation effects in vivo.

Base Damage

Single-strand breaks

Double-strand breaks

Energy Microdeposition required

>1 eV/nm3

Incidence per Gy per human cell

~ 10000

~ 1000

~ 40

50% repaired in:

5-10 min

10-20 min

> 50 min

>10 eV/nm3

>100 eV/nm3

End-Joining

Repaired by :

Recombination Excision-Resynthesis Fig. 2. Biochemical and biological features of the major radiation-induced DNA damage.

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Fig. 3. Multiscale organization of the genome. From naked DNA to chromosomes, the chromatin shows various stage of compaction that vary during the cell cycle (e.g; during transcription or, even more dramatically, after replication). (A) Crystal structure of a nucleosome; B: electron microscopy image of a chromatin ﬁber extracted from CHO cell; C and C’: FISH image of CHO chromosomes during mitosis (C) and interphasis (C’). DNA is stained with DAPI (blue); interstitial telomeric sequences (ITS, known as radiosensitive regions in CHO cells) are stained in red (Revaud et al., 2009). (For interpretation of the color information in this ﬁgure legend, the reader is referred to the web version of the article.)

1995; Sy et al., 1997). Also the presence of a ligand reduces the probability of SB production in a sequence-dependent manner, either by a ligand-induced sequence-dependent modiﬁcation of DNA structure (leading to changes of accessibility of attack sites) or the sequence-dependent binding of the ligand to electronegative spots (leading to local masking of the attack sites, local scavenging and local chemical repair of primary lesions) (Chiu & Oleinick, 1998; Spotheim-Maurizot et al., 1995; Warters et al., 1999). Noteworthy, proteins that do not bind to DNA protect against radiation-induced strand breakage owing to their ability to scavenge bulk OH radicals, but more efﬁcient protection is afforded by DNA binding proteins.

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et al., 1994; Svoboda & Harms-Ringdahl, 2005; Takata et al., 2013; Warters & Lyons, 1992; Xue et al., 1994). Histones and non-histone proteins contribute to the radioprotection of DNA, core histones being the major radioprotectors (Franchet-Beuzit et al., 1993; Xue et al., 1994). The dynamical feature of chromatin, e.g; upon transcription or replication, has also to be considered. Regarding both situations, it was shown for instance that actively transcribed genes are surrounded by large-scale domains of radiosensitive chromatin (Bunch et al., 1995), and that replicating DNA with open chromatin structure is more sensitive to DSB induction by IR (Sak et al., 2000). If one can logically expect that the accessibility of damaging agents decreases with chromatin compaction rate, one has to keep in mind that the same is true for the access of repair enzymes. Indeed, several studies have shown that both the sensitivity to DSB induction and efﬁciency of DSB repair differ for distinct chromatin domains (Falk et al., 2010; Slijepcevic & Natarajan, 1994), which in turn inﬂuence the tendency to form chromosomal aberrations (Falk et al., 2007; Falk et al., 2008; Kruhlak et al., 2006; Magnander et al., 2010; Nikiforova et al., 2000). This double-edge effect could also explain why some studies reported a rather counterintuitive hypersensitivity of condensed chromatin regions (Biade et al., 2001; Chapman et al., 2001; Stobbe et al., 2002). On the other hand, limited movement of DNA in chromatin dense regions could limit chromosomal rearrangements and favor faithful repair; some related observations have been made in bacteria, where it was shown that a packed chromatin organization enhances radiation tolerance (Lieber et al., 2009). In eukaryotes, a complex ballet of chromatin factors, including numerous nucleosome remodelers, promotes the formation of open, relaxed chromatin structures at DSBs allowing the DNA-repair machinery to access the surrounding region; readers interested in the underlying mechanisms should reference to the many recent and comprehensive reviews on the subject (see for instance (Price & D’Andrea, 2013; Smeenk & van Attikum, 2013; Thompson, 2012)). We will now give a brief overview of the consequence of ionizing radiation observed at various (and arbitrary) scales: naked DNA, nucleosome core particle (NCP), nucleosomal arrays and chromosome territories. 2.2. Damage at the DNA scale

2.1.2. Ionizing radiation as a probe for chromatin conformation Chromatin can be seen as a multiscale architecture, with compaction stages that range from naked DNA to nucleosomes, chromatin ﬁbers (or nucleosomal arrays) and mitotic chromosomes (and/or chromosome territories during interphase) (Fig. 3). The analysis of the distribution of DNA damage after irradiation (mostly high LET) provides valuable information to ﬁt in chromatin ﬁber models. By depositing energy on water molecules, IR can generate radicals that are then able to diffuse a few nm. In the nucleus, the radicals can attack bases, leading to the formation of base adducts, or sugars, resulting in SB. Similar strand breaks can also arise as a consequence of direct deposition of energy on the sugar moieties (Chatterjee & Holley, 1991). Hence, DNA lesions induced by ionizing radiation in cells are clustered and not randomly distributed (Fig. 1). For low LET radiation, this clustering occurs mainly on the small scales of DNA molecules and nucleosomes. For high LET radiation, clustering also occurs on larger scales and reﬂects higher order chromatin 3D organization (Folle et al., 1998; Radulescu et al., 2004; Rydberg, 2001). 2.1.3. Chromatin conformation inﬂuence on DNA damage Many studies show that the induction of DSB by IR is inﬂuenced by the chromatin structure. Hence, the number of DSB is enhanced by chromatin relaxation and chromatin-proteins depletion (by high salt or proteolysis treatments) before irradiation (Elia & Bradley, 1992; Murakami et al., 1995; Nygren et al., 1995; Oleinick

As a ﬂexible polyelectrolyte, the DNA double helix has elastic and electrostatic properties which govern its intrinsic dynamics, as well as its capacity to form alternative structures (Potaman & Sinden, 2005) and the way it interacts with proteins (Carrivain et al., 2012). Since ionizing radiation induces strand breaks mainly via the reaction of OH radicals, break occurrence depends on the local sequence whose structure may change the accessibility of particular sites to attack by radicals. Several studies have addressed the intrinsic radiosensitivity of naked DNA with regard to sequence and/or applied geometrical and topological constraints. There is for instance some evidence that AATT sequences are less attacked than average DNA (Franchet-Beuzit et al., 1993) or that DSB induced in a plasmid preferentially affects GC-rich motifs (Humtsoe et al., 2003). However, by bombarding an 80 base pair (bp) fragment with fast neutrons, Spotheim-Maurizot and colleagues have shown that the breakage probability at a given nucleotide site is not determined by the chemical nature of the nucleotide per se, but mainly by the local sequence-modulated intrinsic structure (Sy et al., 1997). Since the intrinsic structure is potentially affected by mechanical and topological constraints on DNA, one would expect supercoiling to inﬂuence radiosensitivity; this is indeed the case, and it was shown that the more relaxed (or less densely supercoiled) a plasmid is, the greater is its radiosensitivity (Swenberg & Speicher, 1995). This enhanced radiation sensitivity of underwound

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DNA is consistent with the transient denaturation of the double helix which increases the susceptibility of individual strands to free radical attack (Miller et al., 1991); on the other hand, others studies failed to conﬁrm such an effect (Culard et al., 1994; Milligan et al., 1992). 2.3. Damage at the nucleosome scale DNA-protein complexes offer radioprotection of DNA through local scavenging of OH radicals and reduced accessibility of the attack sites (Spotheim-Maurizot et al., 1995). This is the case in nucleosomes, where radiolytic footprinting of NCP showed that histones tend to mask the attack sites (Franchet-Beuzit et al., 1993). Namely, the pattern of SB induced by the in vitro irradiation of chicken NCP displays periodically alternating sensitive and protected zones (a “wave-like” form) with a 10.39 ± 0.16 bp periodicity, consistent with the number of base pairs per helical turn of DNA duplex in a nucleosome formerly measured by nuclease digestion (Prunell, 1983) or chemical footprinting (Hayes et al., 1990). We can indeed expect that the histones will protect the DNA facing it, the outer face of the wrapped DNA being more likely to be damaged than the inner face. Interestingly, further analysis of these data by modeling showed that bending of the DNA around the nucleosome also contributes in itself (although to a less extent) to radioprotection (Begusova et al., 2000). For this, the expected SB of the “NCP-DNA” (that correspond to the palindromic sequence used by Luger et al. to crystallize NCP (Luger et al., 1997)), the “naked NCP-DNA” (same path of DNA, but with the histones removed from the structure) and the “linear NCP-DNA” were compared by calculating the accessibility of a given atom as the area of the surface generated by the center of a sphere of 1.2 A˚ radius (OH radical size) rolling on the van der Waals surface of this atom and simulating the diffusion motion of the radicals by Monte-Carlo. These in silico results showed that the accessibility of attack sites in the NCP-DNA is mainly modulated through masking by histones, and only slightly through bending. 2.4. Damage at the chromatin ﬁber scale Nucleosomes are more or less regularly spaced along the genome forming a beads-on-a-string ﬁlament that was ﬁrst visualized by electron microscopy about 40 years ago (Olins & Olins, 1974; Oudet et al., 1975; Woodcock et al., 1976). Under appropriate conditions, the beads-on-a-string ﬁlament (or “10 nm nucleoﬁlament” (Finch & Klug, 1976)) fold into a compact ﬁbrous structure of roughly 25–30 nm in diameter (Finch & Klug, 1976; Olins & Olins, 1979; Pooley et al., 1974; Ris & Kubai, 1970; Worcel & Benyajati, 1977). This so-called “30 nm chromatin ﬁber” is usually considered as the next discrete packaging level of DNA after the nucleosome formation. However, after about 40 years of intense experimental (biochemical, biophysical) and modeling (from the ﬁrst hand-made models to most recent all-atoms computations) efforts, chromatin structure and the hypothetical “30 nm ﬁber” remains a controversial issue (Grigoryev & Woodcock, 2012; Joti et al., 2012; Mozziconacci & Lavelle, 2008; Tremethick, 2007; van Holde & Zlatanova, 1995; van Holde & Zlatanova, 2007). Amazingly, ﬁrst in vivo evidence for a regular chromatin superstructure came from radiation-damage experiment. Indeed, the precise distribution of breaks along the DNA molecule after traversal by a single particle is expected to depend on the position of the nucleosomes relative to each other, giving a size distribution of fragments that should exhibit a pattern characteristic of the chromatin structure. An advantage of this method is that the cells are alive and unﬁxed at the time the structure is monitored during irradiation. Such experiments were carried by Chatterjee and colleagues on human ﬁbroblasts and Chinese hamster ovary (CHO)

cells, irradiated with X-rays or accelerated ions (Rydberg et al., 1998). Characteristic size distributions of short DNA fragments were obtained, with a prominent peak at 78 bases (corresponding to one turn of DNA around the nucleosome) and broader peaks at about 185, 290, 370 and 450 bases (respectively 78, 175, 270, 350 and 430 for CHO cells which repeat length has been formerly reported to be about 10 bp shorter than for human ﬁbroblast (Compton et al., 1976; Revaud et al., 2009), which may account for the shorter fragments founded here). Comparison of the experimental data with simulation support a zig-zag model of the chromatin ﬁber (Bernhardt et al., 2003; Rydberg et al., 1998), rather than the simple solenoidal model historically favored (Finch & Klug, 1976). Interestingly, the observation that the structure of the chromatin ﬁber appears to be the same for cells in G0 and cells in mitosis suggests that the compaction of chromatin that occurs in mitosis reﬂects changes mainly at higher levels of chromatin organization (Rydberg et al., 1998). Such studies show that radiation-induced damage analysis can provide information on chromatin structure in the living cell; on the other hand, papers aimed at predicting the impact of radiation on DNA need to consider precise models of DNA organization within the chromatin ﬁber (Bernhardt et al., 2003; Friedland et al., 1998; Valota et al., 2003). This is also true for higher levels of DNA organization in the nucleus (Friedland et al., 2003; Friedland et al., 1999; Friedland and Kundrat, 2013; Friedland et al., 2008; Rydberg, 2001), which is the subject of the next section. 2.5. Damage at the chromosome scale As evidenced by ﬂuorescence in situ hybridization (FISH) with speciﬁc chromosome probes, the nucleus is a highly ordered and compartmentalized structure with deﬁned chromosome territories (Cremer & Cremer, 2001). The same way radiation studies coupled with chromatin ﬁber modeling help to distinguish between various potential models (see former section), a similar approach at the nuclear scale can help to test various models of chromosome organization, with a better understanding of the formation of chromosomal aberrations as an important potential outcome (Ballarini et al., 2002; Ballarini et al., 1999; Cremer et al., 1996; Edwards et al., 1994; Folle, 2008; Folle et al., 1998; Holley et al., 2002; Kreth et al., 1998; Kreth et al., 2007; Ottolenghi et al., 2001; Ottolenghi et al., 1999; Sachs et al., 1997a,b). Indeed, the signiﬁcance of chromatin structure and nuclear architecture in the localization of breakpoints, and the subsequent potential chromosomal aberrations, is still not well understood. Again, chromatin models can help to understand radiation outcomes better, just as radiation data (combined with simulation) can help to understand nuclear architecture better; two pioneering studies illustrating both objectives are given below. In a “radiobiological” perspective, Folle and colleagues compared the breakpoint patterns produced by nucleases (restriction endonucleases and DNase I) and ionizing radiations (neutrons and gamma-rays) in CHO cells during G1 and S-phase (Folle et al., 1998; Martinez-Lopez et al., 1998). In the Chinese hamster genome, SINEs (short repetitive interspersed sequences, the major one being the Alu sequence family) and LINEs (long repetititive interspersed sequences) are partitioned in G-light (which represent the active housekeeping subgenome) and G-dark (which pertain to the mostly inactive tissue-speciﬁc subgenome) bands. This partitioning allows speciﬁc targeting of DSB induced by AluI or BamHI to the housekeeping and tissue-speciﬁc subgenomes respectively. A co-localization of breakpoints was found with a preferential occurrence (>70%) in G-light bands independent of the cell cycle stage in which aberration production took place. Similar results were found with DNaseI and radiation, suggesting that active chromatin from G-light bands is more prone to damage. In other words,

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chromosome domains with active genes could be in a more open conformation leaving DNA loops more accessible to radical attack (Folle et al., 1998; Lobrich et al., 1996). However, one should keep in mind that, as discussed before, the repair process (hence rearrangement proﬁles) are also expected to depend on chromatin conformation and dynamics, so it is usually difﬁcult, without a clear mechanistic framework, to attribute this correlation between banding patterns and breakage occurrence to either a true damage protecting effect in dense regions or a repair bias that would favor rearrangements in more open chromatin regions. This could indeed rather reﬂect the relative roles of slow and fast repair components of induced DSBs leading to chromosome aberrations (Mosesso et al., 2010). It should also be noted that, while nonrandom distributions of DSBs are usually observed with high-LET radiation, several studies using low-LET radiation showed no differences in DSB yields or distributions in different parts of the genome (Prise et al., 2001; Rothkamm & Lobrich, 1999). In a “structural” perspective, Kreth and colleagues modeled the extent of radiation induced chromosome damage under certain geometrical constraints and compared it to experimental data in order to make the distinction between various competing models: namely territorial and non-territorial models (Kreth et al., 1998). For this, localized irradiation with a focused UV-laser beam ( = 257 nm) was conducted and simulated, considering that if the chromosome arrangement in interphase was a non-territorial one, more chromosomes would be damaged by the localized irradiation than in case of a territorial organization. The difference was obvious and discredited non-territorial models. Since then, remarkable efforts have been made to get true multiscale models ranging from DNA double-helix at atomic resolution to chromatin ﬁber loops, heterochromatic/euchromatic regions and chromosome territories, and to assess radiation outcomes by overlapping radiation track structures with these models (Friedland and Kundrat, 2013). At the same time, the ability to follow DSB repair with imaging approaches has revolutionized our understanding of the repair process and its regulation in space and time (Lisby & Rothstein, 2009); the emerging picture is consistent with dynamical radio-induced foci exploring the nuclear space during repair (Aten et al., 2004; Chiolo et al., 2011; Dion & Gasser, 2013; Jakob et al., 2011; Krawczyk et al., 2012) (see (Chiolo et al., 2013) for a recent review). The local and global chromatin changes that contribute to these dynamics have still to be characterized.

3. Clinical aspects of radio-induced DNA damage 3.1. General considerations As speciﬁed above, like any other DNA breaking agents, IR disturb chromatin and trigger DNA damage recognition, DNA repair, cell cycle arrest and cellular death or transformation throughout a complex cascade of phosphorylation of signaling proteins (Foray et al., 2003; Lomax et al., 2013). Radiation dose, expressed in Grays (Gy; 1 Gy = 1 J/kg)) is one of the major parameters that logically conditions radiation-induced effects. However, while the radiation-induced production of DNA damage is linear, the radiobiological effects are generally non-linear, and one of the recurrent difﬁculties in radiobiology is indeed to determine the actual nature of the dose-dependence effect. In human quiescent diploid cells, a dose of 1 Gy X-rays induces about 10000 base damage (BD), 1000 DNA single-strand breaks (SSB) and 40 DSB (Frankenberg-Schwager, 1989) (Fig. 2). Among all the radiation-induced DNA damage, SSB and DSB affect chromatin integrity most dramatically, although BD and acetylation may play a signiﬁcant role (Szumiel & Foray, 2011). Research about DNA break repair and chromatin integrity

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has considerably increased in the last two decades thanks to technological advances and because of societal outcomes: - immunoﬂuorescence techniques and discovery of early biomarkers of DNA break repair and signaling are revolutionizing radiobiology, by allowing the detection of individual DNA breaks in each individual cell nucleus. This technique permits investigation of the effects of low-dose (<0.2 Gy) on the scale of those applied in radiodiagnosis such as mammography or Computed Tomography (CT) scan (Rothkamm & Lobrich, 2003). The plethora of data accumulated with immunoﬂuorescence has also signiﬁcantly contributed to building molecular models of the radiation response (Bodgi et al., 2013); - medical exposure to radiation has increased by a factor of 2 in the last 10 years, raising the question of the occurrence of radiosensitive tissue reactions after radiotherapy and the incidence of radiation-induced cancers potentially caused by radiodiagnosis and by non-tumoral irradiated tissues during radiotherapy. Estimation of the yields of DNA breaks induced by radiation and the functionality of DNA break repair is therefore at the basis of most of predictive approaches that may concern millions individuals per year (UNSCEAR, 2011). 3.2. Radiosensitivity tissue reactions after radiotherapy: chromatin and high-dose 3.2.1. From clinical tissue reactions to the notion of radiosensitivity About 1-15% cancer patients treated by radiotherapy can show some early or late signiﬁcant tissue reactions of different grades (severity), which may limit the application of their treatment and increase morbidity. These reactions are for instance erythema and dermatitis for breast cancer, proctitis for prostate cancers, ﬁbrosis in oesophageal cancer and lymphomas (Dorr & Hendry, 2001; ICRP, 2007). Some rare cases of fatal reactions have been also reported and are mainly due to mutations of genes essential for DSB repair and signalling like ATM or LIG4 (Attard-Montalto et al., 1996; Pietrucha et al., 2010). For other radiosensitive tissue reactions, the spectrum of their occurrence, severity and macroscopic features is so large that their deﬁnition of reactions is still not consensual: in practice, recognition that a patient effectively suffers from a signiﬁcant tissue reaction still relies solely on subjective clinical judgement. Discounting dosimetry errors, these tissue reactions are the clinical signs of one common biological feature: individual radiosensitivity. Here, the radiosensitivity term is associated with the notion of radiation-induced cellular death and not with that of cancer proneness (Foray et al., 2012). In 1956, Puck and Markus proposed the deﬁnitive loss of clonogenic capacity as a deﬁnition of radiobiological death since it is a common point of the major radiation-induced cellular death encountered after irradiation: senescence, mitotic death and apoptosis (Puck & Marcus, 1956). Such deﬁnition generated a standard assay based on the measurement of the number of colonies of irradiated cells. In 1981, Fertil and Malaise pointed out a correlation between the radiocurability of tumors and the surviving fraction at 2 Gy (SF2) (2 Gy generally corresponds to a daily radiotherapy session) (Fertil & Malaise, 1981). Some years after, some research groups extended this correlation to normal tissues and established the ﬁrst lists of genetic syndromes associated with radiosensitivity. Interestingly, a majority of these syndromes are caused by DSB repair deﬁciencies and were shown to be associated to impairment of radiation-induced chromatin re-condensation (Deschavanne & Fertil, 1996). Besides, the shape of cell survival curves was shown to be strongly affected by chromatin decondensers. This is notably the case of sodium chloride whose concentration progressively

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depletes histones, rendering DNA more accessible to radiation and cells more and more radiosensitive. At the beginning of the 70s, chromatin relaxation, whether due to physical, chemical or genetic reasons, was already known for its radiosensitization power (Iliakis et al., 1988) (see discussions in Section 2). 3.2.2. DNA repair and chromatin techniques Chromatin disorganization after irradiation is inextricably linked to SSB and DSB repair deﬁciency. Although radiationinduced SSB are more frequent than DSB, DSB repair half-time is about 50 min in human radioresistant individuals while SSB are repaired more rapidly (SSB repair half-time is about 10–20 min) (Foray et al., 2005). Hence, some hours post-irradiation are required for a full chromatin re-condensation even in apparently healthy patients. Besides, one will see below that dose repetition can produce supra-additive effects with severe clinical consequences likely because a second dose applied when chromatin condensation has not recovered, renders more severe newly induced DNA damage (see below). The ﬁrst techniques for assessing DNA breaks were also used for estimating chromatin condensation/decondensation, as well (Fig. 4). This is notably the case of the halo assay that enables the visualization of DNA loops undergoing supercoiling changes (Roti Roti & Wright, 1987). The halo assay consists of incubating cell nuclei with a ﬂuorescent DNA intercalating agent like propidium iodide and in measuring the increase of nuclear size caused by the radiation-induced induction of SSB. Chromatin relaxes with increasing supercoils up to the level above which chromatin winds in the opposite sense so rewinds. The assessment of the radiationinduced increases of halo diameter made possible the detection of chromatin condensation impairment in a number of genetic syndromes (like AT) and permitted correlation of DSB repair and chromatin response to radiation. Progressively, the halo assay was replaced by the comet assay that combines halo assay and gel electrophoresis (Olive, 2009). Hence, DNA fragment migrate via an electrical ﬁeld and form the tail of a comet-like-shaped ﬂuorescence signal while the yield of unrepaired DNA breaks increases the size of the head of the comet. Unfortunately, a real confusion appeared in literature between comet assays performed in alkaline solution that concerns SSB and DSB (denatured as SSB), whereas neutral comet, more difﬁcult to develop, reﬂects DSB response only (Olive, 2009). Since DSB repair deﬁciencies were shown to be more highly correlated with radiosensitivity than SSB repair impairment, a number of comet data obtained in alkaline conditions (mainly reﬂecting SSB repair) were in discrepancy with the clonogenic survival data. In the 90’s, pulsed-ﬁeld gel electrophoresis (PFGE) became more popular than comet. Such technique enables the migration of 15-megabase sized DNA fragments, which provided an interesting precision for studying DNA scaffolds. However, using either alkaline or neutral conditions, PFGE had the disadvantage of requiring very high doses of radiation (more than 10 Gy, which does not correspond to any single medical exposure), in order to get a sufﬁcient number of small DNA fragments (Cedervall et al., 1995; Iliakis, 1991). Some variant of PFGE techniques with speciﬁc non-ionic lysis solution preserving DNA organization also helped to reveal chromatin impairment in human tumor cells but, again, only at considerably higher doses (100 Gy at least) (Woudstra et al., 1996). Hence, the combination of techniques assessing DSB and salts (like sodium butyrate) and non-ionic detergents that retain the higher-order nuclear matrix organization, including chromatin loops, seemed to show that DSB present in points of attachment to the nuclear matrix may be more severe than other DSB occurring in loops. However, the relevance of such conclusions was mitigated by the values of the radiation doses (Johnston & Bryant, 1994; Nackerdien et al., 1989; Olive & Banath, 1995). Such conclusions point out a major and recurrent problem of chromatin and radiation research: reconcile the necessity for a rigorous quantiﬁcation

of radiosensitivity by distinguishing the radiation response at the level of the patient for medical conditions on the one hand, and the use of non-clinically relevant radiation doses (because of being too high) and cellular models that monitor events in chromatin, on the other. In parallel, the accumulation of data at the end of the 90’s highlighted that both chromatin impairment and DSB repair deﬁciency contribute independently to the intensity of radiation response: both DSB repair deﬁciency and chromatin decondensation cause hyper-radiosensitivity while DSB repair deﬁciency or chromatin decondensation causes moderate radiosensitivity separately (Chavaudra et al., 2004). Finally, there is to date evidence that histone deacetylase inhibitors can be used as radiosensitisers in radiotherapy (Groselj et al., 2013). 3.2.3. DNA breaks repair deﬁciencies and chromatin organization A considerable technological advance came from Löbrich and Bonner’s labs, showing that the phosphorylated forms of the variant H2AX histone relocalize as nuclear foci and may serve as a sensor of DSB recognized by the non-homologous end-joining pathway (Rogakou et al., 1999; Rothkamm & Lobrich, 2003). For the ﬁrst time in radiation research, a technique has the considerable advantage to detect radiation-induced DSB at clinically-relevant dose (i.e. some Grays). Our group proposed a classiﬁcation of human radiosensitivity on the basis of unrepaired DSB remaining 24 h after irradiation assessed with PFGE or H2AX immunoﬂuorescence. By contrast, the variety of H2AX foci patterns, notably in non-irradiated cells, revealed inter-individual differences in the chromatin condensation that were not necessarily linked with radiosensitivity but spontaneous genomic instability (Joubert et al., 2008). In fact, the aging syndromes, that generally show a signiﬁcant yield of spontaneous SSB and sometimes DSB, elicit a spontaneous chromatin disorganization that does not facilitate DSB repair but does not necessarily prevent it completely: in the survey of human radiosensitivity published by Deschavanne and Fertil (Deschavanne & Fertil, 1996), the aging syndromes (Werner, Cockayne, Xeroderma Pigmentosum, . . .) are associated with a moderate but not a hyper-radiosensitivity, with the notable exception of progeria that seems to combine both chromatin disorganization and DSB repair defect (Varela et al., 2008). Despite its usefulness to assess individual radiosensitivity, the clonogenic survival assays cannot be applied as a routine in radiotherapy departments. Unfortunately, molecular endpoints predictive of radiosensitivity remain to be deﬁned clearly. With regard to genomics (gene expression and mutations assays), a quantitative correlation between individual radiosensitivity, microarrays and polymorphisms respectively, still remains to be established (Barnett et al., 2012). Conversely, the study of radiobiological features of some human genetic syndromes associated with radiosensitivity (e.g. ataxia telangiectasia, Nijmegen’s syndrome, xeroderma pigmentosum, etc. . .) has consolidated the conclusion that predictive assays based on the functionality of the DNA repair pathways permit quantiﬁcation and predict the severity of acute reactions to radiotherapy. However, again, there is to date a gap between molecular studies involving non-human cellular models and clinical reality. Indeed, although many works on genetically modiﬁed yeasts and rodents have undoubtedly contributed to increase our knowledge of stress response, they have led to an underestimation of the continuum of responses to radiation: a number of works have caricatured the response to radiation as an all-or-none phenomenon with very radio-resistant and hyperradiosensitive cases. The most hyper-radiosensitive genetically modiﬁed yeast or rodent models show mutations of proteins like Ku70, Ku80, Rad51, Rad52, etc. . . and have suggested the dual model of DSB repair “non-homologous end-joining (NHEJ) or else homologous recombination (HR)”. However, there is no known

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Fig. 4. Schematic representation of the major techniques related to both chromatin relaxation and DSB repair.

radiosensitive human syndromes associated with impairment of HR, likely because this repair pathway is not active in quiescent cells that represent the majority of normal tissue potentially irradiated during radiotherapy (Massart et al., 2009). Furthermore, the proteins that are essential for NHEJ (Ku70, Ku80, DNA-PKcs) are so important for cell viability that there is no known radiosensitive human syndromes associated with mutations in these proteins. By contrast, there are no viable animal models that mimic genetic syndromes of clinical interest such as BRCA1, BRCA2, ATM heterozygous mutations that confer high risk of breast or ovarian cancer. Hence, the obvious necessity to quantify and to describe the spectrum of radiation responses disappeared behind a number

of “monogenic” studies that represent, still to date, the great majority of papers in radiobiology. A global and multigenic approach is therefore needed to better reﬂect the large spectrum of radiosensitivity reactions observed in clinic (Massart et al., 2009). When the list of the most radiosensitive syndromes is established as a function of survival fraction at 2 Gy (SF2) (Table 1), some interesting conclusions can be made: the great majority of radiosensitive syndromes are associated with moderate radiosensitivity and not hyper-radiosensitivity (that leads to death after radiotherapy session). All these genetic syndromes that a radiation oncologist can encounter in his career are caused by mutations of genes involved in DSB repair and signalling but not necessarily the most

Table 1 major genetic syndromes associated with intrinsic radiosensitivity reﬂected by the surviving fraction at 2 Gy (SF2). Genetic syndromes

Mutated gene

SF2 (%)

Role of the protein

Chromatin impairment?

Ataxia telangiectasia (“classical” homozygous mutation)

ATM

1–5

Known

Ligase 4 syndrome Nijmegen’s syndrome Progeria (Hutchinson-Gilford syndrome) Ataxia telangiectasia («variant» homozygous mutations)

LIG4 NBS1 Lamin A ATM

2–6 5–9 8–19 10–15

Usher’s syndrome Cockayne’s syndrome

USH genes CS genes

15–20 15–30

Xeroderma Pigmentosum

XP genes

15–30

ATLD syndrome Huntington Chorea Gardner’s syndrome Turcot’s syndrome Fanconi anemia BRCA2

15–40 18–30 20–30 20–30 20–40

BRCA1 Artemis syndrome Cernunnos syndrome Omenn’s syndrome

MRE11 IT15 APC hMSH2 FANC genes BRCA2 BRCA1 Artemis XLF/Cernunnos RAG1, RAG2

Rothmund-Thomson’s syndrome Werner’s syndrome Bloom’s syndrome

RecQ4 WRN BLM

30–50 30–50 30–50

Signalling, DSB recognition and repair DSB repair DSB repair Nuclear membrane Signalling, DSB recognition and repair ? Helicases, nucleases BD repair Helicases, nucleases BD repair Endonuclease ? Mismatch repair? Mismatch repair? Base damage repair Scaffold protein Scaffold protein NHEJ NHEJ NHEJ? Immunoglobulin production Helicase Helicase Helicase

20–40 20–40 20–40 30–50

Known Known Known (aging) Known Unknown Known (aging) Known (aging) Unknown Known (aging) Unknown Unknown Known Known Unknown Unknown Unknown

Known (aging) Known (aging) Known (aging)

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essential genes acting upstream of NHEJ (Deschavanne & Fertil, 1996). As a result, only little is known about the chromatin scaffold of the syndromes of real clinical interest whose total incidence may reach 5–15% individuals. The most common radiotherapy-induced reactions are not necessarily associated with massive defect in DSB repair but with mild deﬁciencies ranging about 1–10% of unrepaired DSB after 2 Gy gamma-rays (Foray et al., 2012). These remarks suggest therefore that efforts of chromatin and repair research should focus on syndromes of clinical interest despite their lack of spectacular radiosensitivity. Lastly, a complete radiotherapy treatment is generally made of successive sessions of radiotherapy (e.g. 2 Gy every open day with about 40 Gy for breast cancer and 70 Gy for prostate cancer treatment). The questions raised by the dynamics of the chromatin organization during the repetition of doses should also be the subject of interesting research.

3.2.4. Impact of the cell cycle on chromatin organization and DNA damage response The previous sections concern mostly chromatin organization in quiescent cells and, therefore, the radiation response of normal tissues and non-proliferating tumors. Cell cycle impacts on both the relative contribution of the DNA damage repair pathways and the level of chromatin condensation: (1) HR and alternative NHEJ pathways become preponderant in G2/M cells (Iliakis, 2009); (2) the average number of DSB induced per Gy per human diploid cell is 80 in S cells (most decondensed chromatin) and 20 in G2/M (most condensed chromatin) whereas it is about 40 in G0/G1 cells (see above; (Frankenberg-Schwager, 1989)). Hence, since DNA break repair pathways are activated by the natural SSB in replication forks, S phase is long known to be the most radioresistant cell cycle phase while it is associated to the highest radiation-induced DSB yield. This conclusion strongly suggests that: (1) the number of induced DSB is less predictive of the radiation response than and the number of unrepaired DSB (Joubert et al., 2008); (2) unlike chemotherapy, and the false Tribondeau and Bergonié’s law, radiotherapy preferentially targets quiescent rather than proliferating cells (Vogin & Foray, 2013). Besides, it is noteworthy that severe tissue reactions potentially caused by radiotherapy concern most likely quiescent tissues at lower dose: for example, while intestine cells grow faster that skin ﬁbroblasts, dermatitis are observed after radiotherapy of breast cancer before a total dose of 40 Gy while radiation-induced rectites are observed after prostate cancer irradiation that may reach 70 Gy, at least. Because cell cycle impacts on both DSB repair and chromatin condensation, it is frequently the source of technical artefacts as illustrated by the controversy with the elution technique in the 1980s: the lysis solution used in the experimental protocol was differentially efﬁcient according to the cell cycle distribution (Okayasu & Iliakis, 1989). Similarly, clonogenic cell survival or DSB repair rate may be biased by different cell cycle distribution (Chavaudra et al., 2004). Independently of technical artefacts, it is more and more documented that cell cycle dependent chromatin decondensation will inﬂuence DNA repair. The alternative NHEJ pathways together with cohesin complexes, nucleases, helicases and histone deacetylases appear to be the major actors of the speciﬁc response of irradiated proliferating cells: their identiﬁcation will contribute in the development of new chemotherapy drugs (Moscariello & Iliakis, 2013; Sjogren & Strom, 2010). This is notably the case of histone deacetylase (HDAC) inhibitors. Since HDACs are highly expressed in cancer cells, HDAC inhibitors, which promote chromatin expansion and transcription, appear as interesting agents to favor radiosensitization and limit cancer progression if applied in combination with radiotherapy (Groselj et al., 2013).

3.3. Chromatin and radiation-induced cancers after exposure to low-dose The term “individual radiosensitivity” historically reﬂects the risk of post-radiotherapy tissue reaction and must be distinguished from the notion of susceptibility to radiation-induced cancer. However, to date, there is a real confusion between these two notions (Foray et al., 2012). After exposure to low-dose of radiation (i.e. those, below 0.1 Gy, applied in radiodiagnosis like mammography and CT scan), the occurrence of tissue reactions diminishes drastically and the radiation-induced genomic instability and cell transformation remains the major clinical concern. The question of the biological effects of low-dose of X-rays exposure, whatever its source, are debated abundantly because their understanding is a societal and economical issue. As an example, with regard to the risk of radiation-induced cancer, two different models are opposed: the linear no threshold (LNT) model that describes a risk that is only nil when dose is nil. This precautionary principle is supported by the International Commission for radiological Protection (ICRP, 2007). Conversely, the non-linear threshold (NLT) model describes a cancer risk that is nil below a threshold dose value and increases with dose at higher values. Radiobiological investigations are needed to elucidate molecular and cellular mechanisms speciﬁc to low-dose in order to estimate the potential risk that would be eventually associated with these irradiations. Few studies of chromatin organization have addressed the range of low-dose response to irradiation (Belyaev et al., 1996). Three major radiobiological effects speciﬁc to low-dose have emerged to date: the hypersensitivity to low-doses (or hyperradiosensitivity, HRS) effect, the low and repeated doses (LORD) effect, and the adaptive response (AR) phenomenon. The chromatin organization changes during the particular irradiation conditions where such effects are observed are still to be characterized. 3.3.1. The hypersensitivity to low-doses (HRS) effect The number of radiation-induced DNA damage, directly linked to physical energy deposition, is systematically proportional to the dose. Conversely, repair and signaling activities and clonogenic survival are generally not linearly linked to the dose. The phenomenon of hypersensitivity to low dose (HRS) followed by induced radioresistance (IRR) is a representative example of a non-linearly dosedependent event (Fig. 5). In fact, HRS is generally observed between 1 mGy and 50 cGy with a maximum peak at a cell-line-dependent threshold of about 10–30 cGy. At this peak, clonogenic survival may reach similar values as for doses of 1–3 Gy (i.e. at dose about 100 times higher!). Although a number of molecular models have been proposed, mechanisms of HRS are however unclear. It has been suggested that HRS may depend upon changes in chromatin conformation, failure of the ATM-dependent G2/M checkpoint, or DNA repair defects. More recently, we have shown that HRS may be explained by the lack of recognition of DSB by the repair and signaling pathway. In fact, all the irradiation conditions where HRS is observed correspond to exposure duration of less than 30 s during which the DSB recognition does not seem to occur (Thomas et al., 2013). Interestingly, during our investigations, cells with an unexpectedly high number of DNA breaks have been observed between 1 mGy to 30 cGy. These so called “highly damaged cells” (HDC) might correspond to the multi-aberrant (rogue) cells observed in cytogenetics. Investigations are therefore needed to explain how and in what conditions HDC phenomenon is linked to HRS and what are the molecular and cellular mechanisms behind the occurrence of these speciﬁc low-dose events. Two types of data suggest that chromatin organization inﬂuences HRS effect: (1) inhibitors of the poly(ADP-ribose) Polymerase (PARP) that is required for chromatin integrity, would facilitate the occurrence of HRS (Chalmers

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Fig. 5. Hypersensitivity to low-dose. Different illustrations of the hypersensitivity to low-dose (HRS) phenomenon as observed with clonogenic assays (A; courtesy from (Xue et al., 2009)), micronuclei (B; courtesy from (Slonina et al., 2007)) or unrepaired DSB (C).

et al., 2004); (2) while chromatin changes together with variation in DNA-proteins interactions can be measured through the viscosity of cell lysates, it was shown that low-dose X-rays affect the viscosity of human cells (Belyaev & Harms-Ringdahl, 1996). 3.3.2. The low and repeated doses (LORD) effect Preliminary results from our lab indicate that X-ray DSB induced by two identical high doses (e.g; 1 Gy) separated by a time interval ranging from minutes to hours) are repaired more slowly than after 2 Gy in human primary ﬁbroblasts (Fig. 6). Similar observations have been performed on doses up to 2 mGy (Colin et al., 2011). Between the two doses, during some minutes, there is no signiﬁcant repair of DSB, but initial SSB may be repaired and secondary SSB due to BD excision can appear and increase the chromatin decondensation. Such a chromatin decondensation may make slower the DSB repair process and lead to the accumulation of unrepaired

DSB. Such accumulation of unrepaired DSB may be at the origin of the production of multi-aberrant (rogue) cells. In 2011, to assess in vitro mammographic radiation-induced DNA damage in mammary epithelial cells from 30 patients with low (LR) or high (HR) family risk of breast cancer, radiation-induced DSB were quantiﬁed by using ␥H2AX immunoﬂuorescence in different conditions of mammography irradiation: the dose repetition (2 + 2 mGy) provided more induced and more unrepaired DSB than 4 mGy at one time, and was exacerbated in HR patients. This study highlights the existence of a LOw and Repeated Dose (LORD) effect. These ﬁndings may lead us to re-evaluate the number of views performed in screening using a single view (oblique) in women whose mammographic beneﬁt has not properly been proved, such as HR patients (Colin et al., 2011). Some other manifestations of the deleterious effect of repeated doses have been published. The so-called “W” effect is one of the most representative examples. From a linear

Fig. 6. Repeated dose effect. Two radiation doses separated by an interval of time t may trigger synergistic effects, notably with regard to the dynamics of the chromatin recondensation linked to DSB repaired. For t lower than 24 h and according to the genetic status considered, the repeated dose effect may lead to increase the severity of DSB.

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electron accelerator providing pulsed irradiation separated by time intervals of a second to a few minutes, the V. Favaudon’s group observed a tetraphasic, W-shaped time-dependent dose-response curve strongly dependent on the cell line and on the PARP status, suggesting again the inﬂuence of chromatin organization (Ponette et al., 1996). Questions about synergy between these particular effects with HRS phenomenon (described in the section above) are of clinical interest. Since it is long known that time between splitdose conditions the chromatin recondensation and the DSB repair (see for instance (Wierowski et al., 1984)), a better understanding of the dynamics of chromatin condensation/decondensation in these particular but clinically relevant conditions would be therefore a very useful research axis. 3.3.3. The adaptive response (AR) phenomenon The term radioadaptive response is generally used for describing increased radioresistance and/or reduced carcinogenicity after a priming dose followed by a challenging high-dose. However, behind the same term, very different protocols of irradiation are applied. The most frequently used AR protocol is a challenging exposure to high-dose of more than 1 Gy due to a conditioning exposure to low-dose (0.001–200 mGy) generally delivered at dose-rate higher than 100 mGy min−1 and with an interval period (4–6 h) between the priming dose and the challenging dose (Calabrese, 2009). Very few research groups have focused on human cells. Furthermore, some genetic statuses, some cell cycle distributions appear to be more prone to AR revealing that it is not necessarily the most radioresistant cells that show AR, but to the contrary, a minimal radiosensitivity is required to observe its reduction by AR. However, care must be taken with regard to the potential biases linked to the different cellular models. Hence, dose and time interval between doses condition both DSB repair and chromatin condensation/decondensation that appear therefore two essential causes and consequences of AR phenomenon. Interestingly, it is noteworthy that, like for HRS phenomenon, the method of anomalous viscosity time dependence contributed to establish quantitative relationship between AR and chromatin response (Belyaev et al., 1996). Again, the development of research in this ﬁeld is required, inasmuch as AR phenomenon is at the crossroads of the debates about low-dose effects and the LNT/NLT models controversy (Feinendegen, 2005). 4. Conclusions In 1908, Claudius Regaud, a pioneer in radiotherapy and founder of the Curie Institute in Paris (France), stated: “C’est la chromatine nucléaire qui est la partie la plus sensible des cellules” (“it’s nuclear chromatin that is the most sensible part of the cell”) (Vogin & Foray, 2013). More than a century after, we are still struggling to understand the precise link between chromatin architecture and dynamics and the effect of radiation. While everybody agrees that chromatin inﬂuences both the amount and location of breakages produced by IR as well as the efﬁciency of the repair process, the mechanisms are still misunderstood. New advances in the knowledge of chromatin organization and dynamics may be of high clinical interest: - with regard to the improvement of anticancer treatments modality: as clustered damage sites produced by IR are difﬁcult to repair, this reduced repairability is exploitable in radiotherapy throughout innovative strategies: one interesting approach is to enhance radiosensitivity of cancer cells by targeting the repair pathways of radiation-induced clustered damage and complex DSB through inhibition of speciﬁc proteins that are not required in the repair pathways for endogenous damage (Lomax et al.,

2013). The association with epigenetic factor targeting also offers promising perspectives (Banuelos et al., 2007; Cerna et al., 2006; Groselj et al., 2013; Kim et al., 2006; Kim et al., 2013; Ren et al., 2013); - with regard to basic questions raised by new radiotherapy techniques: the development of radiotherapy modalities like Cyberknife (Dieterich & Gibbs, 2011), hypo- or hyper-fractioned dose modalities raise to date the question of repeated doses whose effects may be directly related to the chromatin dynamics response to IR. Such question is also raised by radiodiagnosis such as mammography. Hence, the development of the so basic and fundamental research about chromatin may have a number of applications in clinics: we argue this should be taken more in consideration for future projects. Acknowledgements We thank Jean-Marc Victor and David Levens for critical reading of the manuscript and helpful comments. C.L., formerly a pure chromatinist, would also like to thank Chantal Desmaze and Laure Sabatier for introducing him some times ago to the fascinating world of radiobiology. References Altmeyer M, Lukas J. To spread or not to spread–chromatin modiﬁcations in response to DNA damage. Curr Opin Genet Dev 2013;23:156–65. Aten JA, Stap J, Krawczyk PM, van Oven CH, Hoebe RA, Essers J, Kanaar R. Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains. Science 2004;303:92–5. Attard-Montalto SP, Saha V, Kingston J, Plowman N, Taylor M, Arlett C, Bridges B, Eden O. Increased radiosensitivity in a child with T-cell non-Hodgkin’s lymphoma. Med Pediat Oncol 1996;27:564–70. Balasubramanian B, Pogozelski WK, Tullius TD. DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc Natl Acad Sci U S A 1998;95:9738–43. Ballarini F, Biaggi M, Ottolenghi A. Nuclear architecture and radiation induced chromosome aberrations: models and simulations. Radiat Prot Dosimetry 2002;99:175–82. Ballarini F, Merzagora M, Monforti F, Durante M, Gialanella G, Grossi GF, Pugliese M, Ottolenghi A. Chromosome aberrations induced by light ions: Monte Carlo simulations based on a mechanistic model. Int J Radiat Biol 1999;75:35–46. Banuelos CA, Banath JP, MacPhail SH, Zhao J, Reitsema T, Olive PL. Radiosensitization by the histone deacetylase inhibitor PCI-24781. Clin Cancer Res Off J Am Assoc Cancer Res 2007;13:6816–26. Bao Y. Chromatin response to DNA double-strand break damage. Epigenomics 2011;3:307–21. Barnett GC, Coles CE, Elliott RM, Baynes C, Luccarini C, Conroy D, Wilkinson JS, Tyrer J, Misra V, Platte R, Gulliford SL, Sydes MR, Hall E, Bentzen SM, Dearnaley DP, Burnet NG, Pharoah PD, Dunning AM, West CM. Independent validation of genes and polymorphisms reported to be associated with radiation toxicity: a prospective analysis study. Lancet Oncol 2012;13:65–77. Begusova M, Sy D, Charlier M, Spotheim-Maurizot M. Radiolysis of nucleosome core DNA: a modelling approach. Int J Radiat Biol 2000;76:1063–73. Belyaev I, Harms-Ringdahl M. Effects of gamma rays in the 0.5-50-cGy range on the conformation of chromatin in mammalian cells. Radiat Res 1996;145:687–93. Belyaev I, Spivak IM, Kolman A, Harms-Ringdahl M. Relationship between radiation induced adaptive response in human ﬁbroblasts and changes in chromatin conformation. Mutat Res 1996;358:223–30. Bernhardt P, Friedland W, Jacob P, Paretzke HG. Modeling of ultrasoft X-ray induced DNA damage using structured higher order DNA targets. Int J Mass Spectrom 2003:579–97. Biade S, Stobbe CC, Boyd JT, Chapman JD. Chemical agents that promote chromatin compaction radiosensitize tumour cells. Int J Radiat Biol 2001;77:1033–42. Bodgi L, Granzotto A, Devic C, Vogin G, Lesne A, Bottollier-Depois JF, Victor JM, Maalouf M, Fares G, Foray N. A single formula to describe radiationinduced protein relocalization: Towards a mathematical deﬁnition of individual radiosensitivity. J Theor Biol 2013;333:135–45. Bunch RT, Gewirtz DA, Povirk LF. Ionizing radiation-induced DNA strand breakage and rejoining in speciﬁc genomic regions as determined by an alkaline unwinding/Southern blotting method. Int J Radiat Biol 1995;68:553–62. Calabrese E. Hormesis, non-linearity, and risk communication. Human Exp Toxicol 2009;28:5–6. Carrivain P, Cournac A, Lavelle C, Lesne A, Mozziconacci J, Paillusson F, Signon L, Victor JM, Barbi M. Electrostatics of DNA compaction in viruses, bacteria

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Sequence-driven telomeric chromatin structure