**DNA Radiosensitization: The Search for Repair Refractive Lesions Including Double Strand Breaks and Interstrand Crosslinks**

Tsvetan G. Gantchev1,2, Marie-Eve Dextraze1 and Darel J. Hunting1

*1Department of Nuclear Medicine & Radiobiology, Faculté de medicine, Université de Sherbrooke, Sherbrooke, Québec, 2Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, 1Canada 2Bulgaria* 

### **1. Introduction**

More than half of all cancer patients receive radiotherapy (RT) as part of their treatment regimens. The cytotoxicity of ionizing radiation is mainly mediated by the ensuing DNA damage. Double-stranded DNA breaks (DSB) are generally accepted to be the most important lesions for the induction of cell death by ionizing radiation because they are much more difficult to repair than single strand breaks, although their radiation yield is very low, at two orders of magnitude less than that of single strand breaks (SSB) (Hempel & Mildenberger, 1987). The role of other DNA lesions such as base damage and interstrand crosslinks in cell killing has not been yet fully elucidated. Gamma-radiation inflicts DNA damage *via* two separated processes: i) direct interaction with DNA and; ii) indirect damage produced by secondary radicals ( OH, H and e¯aq) generated after water radiolysis (Michaels & Hunt, 1978). Among the H2O derived radicals, hydroxyl radicals ( OH) are the species primarily responsible for strand break formation and DNA base damage (> 35%). Hydrated electrons, e¯aq, which are generated at a yield comparable to the OH (G-values of ~ 2.8x10-7 mol.J-1), participate in only ~ 8% of the total damage and the exact nature of DNA damage formed *in vivo* by e¯aq is obscure (Nabben *et al*., 1982). In vertebrate cells, the majority of RT-induced DSB are repaired by non-homologous end joining (NHEJ) with some contribution from homologous recombination repair (HRR) during the late S and G2 phases of the cell cycle (Jackson, 2002; Takata *et al.*, 1998). The repair of other DNA damages, such as interstrand crosslinks (ICL) is more complicated and less understood (Wang, 2007; Moldovan & D'Andrea, 2009). ICLs can be recognized by the nucleotide excision repair (NER) system and it is accepted that ICL repair involves nucleolytic cleavage at or near the site of ICL to produce a suitable substrate that can subsequently be repaired by homologous recombination (HR) (D'Andrea & Grompe, 2003; Moldovan & D'Andrea, 2009a, Liu *et al.*, 2010).

An approach to improve the effectiveness of RT is either to enhance the formation of lethal DNA lesions, or to use inhibitors of DNA repair pathways (or both) and thus to render tumor

DNA Radiosensitization: The Search for Repair Refractive

the formation of a guanine cation radical, G+

U and

radical species (

available.

**strand breaks** 

the polymer after gamma-irradiation.

Lesions Including Double Strand Breaks and Interstrand Crosslinks 419

deoxyribose to form a strand break or accepts an electron from a neighboring base (interbase chain e¯ transfer (ET), generating an oxidized base), which eventually terminates with

reactions may result in DNA crosslinks (Fig. 1), but this was only recently shown experimentally (Zeng & Wang, 2007). New findings show that electron capture and migration along BrdU-substituted DNA might be contra-thermodynamically selective and, thus far more complicated (Yoshioka *et al.*, 1999; Aflatooni *et al.*, 1998). In cells, radiosensitization by BrdU (typically at ~20-30% thymine replacement) gives about 2-3 fold radiotoxicity enhancement and in most cell types parallels similar increase in the DSB yield, and/or the decreased rate of their repair (Sawada & Okada, 1972). Further characteristics of BrdU-mediated DSB and other DNA damages *in vivo* (*e.g.* DNA crosslinks) are scarcely

U abstracts an H-atom from 2'-

U-radical addition

(Nese *et al.*, 1992).

Br). It is generally assumed that

**2. DNA-structure/conformation dependent BrdU-sensitized formation of** 

A crucial difference between the radiosensitization of single and double stranded DNA by BrdU was not known until our work with purified *semi*-complementary (mismatched) DNA showing the single stranded specificity of BrdU-induced DNA damage (Cecchini *et al.*, 2004). In parallel experiments, using BrdU-substituted (or not) single-stranded (ssDNA), double-stranded (dsDNA) and mismatched (wobble) semi-complementary (scDNA) DNA we have found that BrdU efficiently sensitizes single stranded BrdU-substituted (brominated) oligonucleotides, but not when these are hybridized to completely complementary oligonucleotides to form normal dsDNA duplexes. We estimate that BrdU radiosensitization efficiency in dsDNA drops up to 20-fold compared to that in ssDNA. Comparative measurements of radiolytic loss of the bromine atom in ssDNA *vs*. dsDNA likewise indicate that this process is greatly suppressed in dsDNA (Cecchini *et al.*, 2004, 2005a). In mismatched, scDNA duplexes, strand brakes are formed in loci encompassing nucleotides surrounding BrdU. However, high efficiency single strand break formation takes place on the brominated strand, or on the opposite, non-brominated strand but have not been detected on both, suggesting that they are mutually exclusive events (Hunting *et al.*, unpublished; Cecchini *et al.*, 2005b). Experiments performed with scDNA bearing variable number of mismatches (from one to five) and containing a single BrdU substitution gave qualitatively similar results. The radiation dose-response for strand break formation was linear for both the brominated and the opposite, non-brominated strand within the single-stranded regions of a standard model scDNA containing a bulge formed by up to five mismatched bases (Dextraze *et al.*, 2007). Interestingly, UVB-irradiation of BrdU-substituted DNA also demonstrated DNA-structure specificity, but in this situation BrdU greatly enhances breakage of only the brominated strand in dsDNA, or either the brominated or the non-brominated strand in the case of scDNA (Cecchini *et al.*, 2005b; Chen *et al.*, 2000). The different effects initiated by radiolysis and photolysis, especially with BrdU-dsDNA, underline the role of DNA structure-conformation properties in solution as a prerequisite for the initial electron-capture by BrdU and/or the propagation of an excess-electron along

The importance of DNA structure during sensitization by BrdU is further demonstrated by comparison of the damages induced in A- and B-form DNA. Using brominated 25-mer

cells more sensitive to ionizing radiation. A novel strategy to inhibit radiation-induced double strand break repair was recently promoted by using short modified DNA molecules that mimic double strand breaks (Dbaits) and artificially activate the DNA-PK pathway (Quanz *et al.*, 2009). Likewise, using siRNA screening of genes involved in DNA damage repair, Higgins *et al*. (2010) identified POLQ (DNA polymerase θ) as a potential tumor-specific target whose knockdown led to tumor cell–specific radiosensitization. However, with the exception of oxygen-enhancing or mimetic agents and platinum derivatives such as cis-platinum, which amplify DNA damage, the only direct DNA radiosensitizing agents known to date are halogenated uracils, such as 5-bromodeoxyuridine (BrdU). BrdU administration to cultured cells, animals and humans leads to replacement of isosteric thymine by 5-bromouracil during replication and excision repair of DNA. The basic pathway of BrdU radiolysis and DNA strand break formation in solution was described many years ago (Zimbrick *et al.*, 1969a,b). It involves dissociative e¯aq attachment to BrdU, followed by the formation of a 5-uracil-yl σradical ( U) and a bromine ion (Br¯, Fig. 1). The reaction is very efficient in air-free solutions, with k(e¯aq+ BrU) = 2.6x1010 M-1s-1 and G(U) = 2.4x10-7 mol.J-1, but interactions with OH may intervene and can lead to somewhat different products. BrdU is also an efficient UV-light absorbing phtotosensitizer; the homolytic Br−C bond cleavage, however, results in two

Fig. 1. Primary reaction mechanism of 5-BrdU sensitization and subsequent damages in wobble scDNA. The dynamic structure of scDNA facilitates electron (hydrogen atom) transfer (ET) between the two strands. The ET direction depends on the BrdU local sequence context (e.g. purine *vs*. pyrimidine environment). In the scheme the 5-uracil-yl radical (3) and its products are denoted by X. Modified bases (reduced or oxidized; red and blue), uracil and/or 2'-deoxyribose may further interact between and/or with undamaged bases and oxygen to form various products, including AP-sites, intra- and inter-strand crosslinks (ICL) and strand breaks (SB).

cells more sensitive to ionizing radiation. A novel strategy to inhibit radiation-induced double strand break repair was recently promoted by using short modified DNA molecules that mimic double strand breaks (Dbaits) and artificially activate the DNA-PK pathway (Quanz *et al.*, 2009). Likewise, using siRNA screening of genes involved in DNA damage repair, Higgins *et al*. (2010) identified POLQ (DNA polymerase θ) as a potential tumor-specific target whose knockdown led to tumor cell–specific radiosensitization. However, with the exception of oxygen-enhancing or mimetic agents and platinum derivatives such as cis-platinum, which amplify DNA damage, the only direct DNA radiosensitizing agents known to date are halogenated uracils, such as 5-bromodeoxyuridine (BrdU). BrdU administration to cultured cells, animals and humans leads to replacement of isosteric thymine by 5-bromouracil during replication and excision repair of DNA. The basic pathway of BrdU radiolysis and DNA strand break formation in solution was described many years ago (Zimbrick *et al.*, 1969a,b). It involves dissociative e¯aq attachment to BrdU, followed by the formation of a 5-uracil-yl σ-

U) and a bromine ion (Br¯, Fig. 1). The reaction is very efficient in air-free solutions,

**-**

**- Br . .**

OH may

with k(e¯aq+ BrU) = 2.6x1010 M-1s-1 and G(U) = 2.4x10-7 mol.J-1, but interactions with

**X** H/e

mismatch

**N N O**

**dR**

**1 2 3**

**O \_ Br**

**UV/ -Br.**

3'

**Br**

**eaq**

5'

intra

3'

ICL

scDNA

**N N O**

**dR**

**O**

5'

(ICL) and strand breaks (SB).

**X X**

Fig. 1. Primary reaction mechanism of 5-BrdU sensitization and subsequent damages in wobble scDNA. The dynamic structure of scDNA facilitates electron (hydrogen atom) transfer (ET) between the two strands. The ET direction depends on the BrdU local sequence context (e.g. purine *vs*. pyrimidine environment). In the scheme the 5-uracil-yl radical (3) and its products are denoted by X. Modified bases (reduced or oxidized; red and blue), uracil and/or 2'-deoxyribose may further interact between and/or with undamaged bases and oxygen to form various products, including AP-sites, intra- and inter-strand crosslinks

5'

3'

SB

AP

**N N O**

**dR**

**O**

**addition**

**H-abstr**

ICL

SB

ICL

AP

intervene and can lead to somewhat different products. BrdU is also an efficient UV-light absorbing phtotosensitizer; the homolytic Br−C bond cleavage, however, results in two

radical (

radical species ( U and Br). It is generally assumed that U abstracts an H-atom from 2' deoxyribose to form a strand break or accepts an electron from a neighboring base (interbase chain e¯ transfer (ET), generating an oxidized base), which eventually terminates with the formation of a guanine cation radical, G+ (Nese *et al.*, 1992). U-radical addition reactions may result in DNA crosslinks (Fig. 1), but this was only recently shown experimentally (Zeng & Wang, 2007). New findings show that electron capture and migration along BrdU-substituted DNA might be contra-thermodynamically selective and, thus far more complicated (Yoshioka *et al.*, 1999; Aflatooni *et al.*, 1998). In cells, radiosensitization by BrdU (typically at ~20-30% thymine replacement) gives about 2-3 fold radiotoxicity enhancement and in most cell types parallels similar increase in the DSB yield, and/or the decreased rate of their repair (Sawada & Okada, 1972). Further characteristics of BrdU-mediated DSB and other DNA damages *in vivo* (*e.g.* DNA crosslinks) are scarcely available.
