Induction and repair of clustered DNA damage sites …uu.diva-portal.org/smash/get/diva2:1294853/FULLTEXT01.pdfAbramenkovs, A. 2019. Induction and repair of clustered DNA damage sites

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1548

Induction and repair of clusteredDNA damage sites after exposureto ionizing radiation

ANDRIS ABRAMENKOVS

ISSN 1651-6206ISBN 978-91-513-0591-2urnnbnseuudiva-378721

Dissertation presented at Uppsala University to be publicly examined in RudbecksalenRudbecklaboratoriet Dag Hammarskjoumllds v 20 Uppsala Monday 29 April 2019 at 1300 forthe degree of Doctor of Philosophy (Faculty of Medicine) The examination will be conductedin English Faculty examiner Professor Emeritus Peter ONeill (Department of OncologyUniversity of Oxford)

AbstractAbramenkovs A 2019 Induction and repair of clustered DNA damage sites after exposureto ionizing radiation Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1548 54 pp Uppsala Acta Universitatis UpsaliensisISBN 978-91-513-0591-2

The mechanisms that maintain genomic stability safeguard cells from constant DNA damageproduced by endogenous and external stressors Therefore this thesis aimed to specificallyaddress questions regarding the requirement and involvement of DNA repair proteins in therepair of various types of radiation-induced DNA damage

The first aim was to determine whether the phosphorylation of DNA-PKcs a major kinaseinvolved in non-homologous end joining pathway can be utilized to score the DNA double-strand break (DSB) content in cells DNA-PKcs phosphorylated (pDNA-PKcs) at T2609 wasmore sensitive to the cellular DSB content than ɣH2AX as analyzed by flow cytometry FurtherpDNA-PKcs at T2609 could discriminate between DSB repair-compromised and normal cellsconfirming that the pDNA-PKcs can be used as a DSB repair marker In paper II the DSB repairwas assessed in cells with reduced levels of DNA-PKcs The reduction in DNA-PKcs resultedin decreased cell survival and unaffected DSB repair These results clearly indicate that DNA-PKcs plays an additional role in promoting cell survival in addition to its function in DSB repair

The second part of the thesis focused on the characterization of complex DNA damage DNAdamage was investigated after exposure to α-particles originating from Ra-223 The Ra-223treatment induced a nonrandom DSB distribution consistent with damage induced by high-linear energy transfer radiation The exposure to Ra-223 significantly reduced cell survival inmonolayers and 3D cell structures The last paper unraveled the fate of heat-sensitive clusteredDNA damage site (HSCS) repair in cells HSCS repair was independent of DSB repair and theselesions did not contribute to the generation of additional DSBs during repair Prolonged heatingof DNA at relatively low temperatures induced structural changes in the DNA that contributedto the production of DNA artifacts

In conclusion these results demonstrate that DNA-PKcs can be used to monitor DSB repair incells after exposure to ionizing radiation However the functions of DNA-PKcs are not limitedto DSB repair as it can promote cell survival through other mechanisms The complexity of theDNA damage produced by high-LET radiation is a major contributor to cell death Howevernot all clusters produced in irradiated cells are converted into DSBs during repair

Keywords NHEJ DSB repair clustered DNA damage DNA repair DNA-PKcs HSCSRa-223 ionizing radiation

Andris Abramenkovs Department of Immunology Genetics and Pathology MedicalRadiation Science Rudbecklaboratoriet Uppsala University SE-751 85 Uppsala Sweden

copy Andris Abramenkovs 2019

ISSN 1651-6206ISBN 978-91-513-0591-2urnnbnseuudiva-378721 (httpurnkbseresolveurn=urnnbnseuudiva-378721)

List of Papers

This thesis is based on the following papers which are referred to in the text by Roman numerals

I Abramenkovs A Stenerloumlw B (2017) Measurement of DNA-

Dependent Protein Kinase Phosphorylation Using Flow Cytom-etry Provides a Reliable Estimate of DNA Repair Capacity Ra-diation Research 188(6) 597 ndash 604

II Gustafsson AS Abramenkovs A Stenerloumlw B (2014) Suppres-sion of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair Mutation Research 769 1 ndash 10

III Abramenkovs A Spiegelberg D Nilsson S Stenerloumlw B The α-emitter Ra-223 induces clustered DNA damage and signifi-cantly reduces cell survival Manuscript

IV Abramenkovs A Stenerloumlw B (2018) Removal of heat-sensi-tive clustered damaged DNA sites is independent of double-strand break repair PLoS ONE 13(12)e0209594

Reprints were made with permission from the respective publishers

Contents

Introduction 9 Ionizing radiation 9 DNA 10 DNA damage 11

Factors influencing DNA damage induction 12 DNA repair 13

Repair of non-DSB lesions 13 DSB signaling 14

ATM and ATR 15 Mediators of DSB repair 15

DSB repair 15 Non-homologous end joining 16 Homologous recombination 18 Alternative repair mechanisms 19

Cancer 19 Prostate cancer 20

Cancer diagnosis and treatment 21

Aim 23

Results 24 Paper I 24

Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity 24

Aim and background 24 Methods 24 Results 25 Discussion 26

Paper II 27 Suppression of DNA-dependent protein kinase sensitizes cells to radiation without affecting DSB repair 27

Paper III 30 The α-emitter Ra-223 induces clustered DNA damage and significantly reduces cell survival 30

Paper IV 33 Removal of heat-sensitive clustered damaged DNA sites is independent of double-strand break repair 33

Concluding remarks 37

Future studies 39

Acknowledgements 41

References 44

Abbreviations

53BP1 p53-Binding protein 1 8-oxoG 8-Oxoguanine ADP Adenosine diphosphate ADT Androgen deprivation therapy alt-NHEJ Alternative non-homologous end joining AP ApurinicApyrimidinic site APE ApurinicApyrimidinic endonuclease AR Androgen receptor ARv7 Androgen receptor variant 7 ATM Ataxia telangiectasia mutated ATR Ataxia telangiectasia and RAD3-related protein BER Base excision repair Bp Base pair BRCA1 Breast cancer type 1 susceptibility protein CtIP C-terminal-binding protein 1-interacting protein DDB DNA damage-binding protein DNA Deoxyribonucleic acid DNA-PKcs Deoxyribonucleic acid-dependent protein kinase catalytic

subunit DSB Double-strand break ɣH2AX phosphorylated H2A histone family member X H Histone H2AX H2A histone family member X HR Homologous recombination HSCS Heat-sensitive cluster sites KU70 X-ray repair cross-complementing 6 KU80 X-ray repair cross-complementing 5 LET Linear energy transfer MMR Mismatch repair Mre11 Meiotic recombination 11 homolog A Mut Mutator NBS1 Nijmegen breakage syndrome 1 NER Nucleotide excision repair NHEJ Non-homologous end joining PARP1 Poly [adenosine diphosphate ndash ribose] polymerase 1 PCNA Proliferating cell nuclear antigen

PIKK Phosphatidylinositol 3-kinase-related kinase Pol Polymerase RAD DNA repair protein RNA Ribonucleic acid RPA Replication protein A SSB Single-strand break ssDNA Single-stranded deoxyribonucleic acid TFIIH Transcription factor II human UV Ultraviolet XLF X-ray repair cross-complementing factor 4-like factor XPC Xeroderma pigmentosum complementation group C XRCC X-ray repair cross-complementing protein

Introduction

Ionizing radiation All organisms are constantly exposed to low levels of ionizing radiation The danger associated with exposure to ionizing radiation stems from its ability to ionize atoms and molecules that form important cellular structures Ionization events can change molecules either directly or indirectly by producing radicals that can alter surrounding molecules (1)

In biological systems the most deleterious alterations related to ionizing radiation exposure are changes in the DNA sequence However some biolog-ical effects can also be observed in cells that reside nearby exposed cells and this is referred to as the bystander effect (2)

The linear energy transfer (LET) is an important factor that determines the cell damage pattern and the consequences of that damage LET is defined as the rate of energy loss of an ionizing particle along its track Irradiation with heavy charged particles is characterized by an increase in LET at the very end of the particlersquos track (3) Consequently on a macroscopic scale the increase in LET results in a higher dose deposited at the end of the particlersquos track which differs from the dose distribution of low-LET radiation such as X-rays and ɣ-radiation (Figure 1)

Figure 1 Schematic representation of depth ndash dose distribution of photon and heavycharged particle radiation

DNA DNA was isolated for the first time in 1869 by Friedrich Miescher (4) how-ever at the time the importance of DNA was not fully recognized and only in 1944 was DNA identified as a carrier of genetic information (5) The mo-lecular composition of DNA was clarified in 1909 when it was shown that the DNA molecule was composed of 4 different nucleobases ndash adenine guanine cytosine and thymidine (6) Further studies of the DNA molecule by X-ray crystallography revealed that DNA is a double helix with inward facing bases and an outward facing sugar backbone (7) In this model the bases are stacked parallel to each other within a distance of 3 ndash 4 Aring and the interaction between bases is limited to hydrogen bonding between cytosine and guanine or adenine and thymine As the interactions between the bases are governed by noncova-lent interactions the strands can be thermally or mechanically separated (8) without causing any damage to the macromolecule

In eukaryotic cells DNA is wound around proteins to form nucleosomes (9) The proteins found in nucleosomes are called histones and each nucleo-some is an octamer consisting of a pair of H2A H2B H3 and H4 histones (10 11) and 146 base pairs (bp) of DNA (12) The nucleosomes are connected together with a DNA fragment that is associated with histone H1 (13) The nucleosomes that have undergone successive folding events create larger structures called chromosomes

Histone binding to DNA is mainly governed by noncovalent interactions such as hydrogen bonds and salt bridges (12 14) Any DNA sequence is able to bind with histones however some more flexible DNA sequences exhibit stronger interactions (15) Long amino acid side chains on histones can un-dergo posttranslational modifications such as methylation acetylation phos-phorylation ubiquitination and ADP-ribosylation (16-20) The histones and their modifications are key factors that determine DNA accessibility and thus gene transcription (21) More specifically the modifications of histone tails affect the electrostatic interactions between DNA and histones (22) which changes the DNA binding strength and determines whether a gene will be ex-pressed or silenced The modifications of histone tails are subsequently passed on to daughter cells during mitosis and are referred to as epigenetic inher-itance The flexibility of histone modifications allows an organism to develop various types of cells and tissues by altering the gene expression and molecu-lar landscape of the cell (23) In the nucleus transcriptionally repressed re-gions are referred to as heterochromatin while transcriptionally active regions are called euchromatin Regions of euchromatin and heterochromatin can eas-ily be discriminated by electron microscopy However macroscopic observa-tions of euchromatin and heterochromatin regions are not consistent with mo-lecular analysis which has identified at least five different states of chromatin organization (24)

DNA damage Every day cells are subjected to DNA damage originating from endogenous events and external stressors eg ionizing radiation and chemical exposure The majority of the damage induced by ionizing radiation is similar to damage originating from endogenous processes in terms of the chemical adducts pro-duced (25) Ionizing radiation and endogenous processes can induce a vast array of DNA lesions which include single-strand breaks (SSBs) double-strand breaks (DSBs) abasic sites base and sugar lesions protein crosslinks and interstrand crosslinks (26) Ionizing radiation induces clustered DNA damage sites at a much higher frequency than endogenous processes (27) Clustered DNA damage sites are complex lesions that have 2 or more DNA lesions within a distance of 20 - 30 nucleotides (28 29) that can be further divided into DSB and non-DSB cluster sites (Figure 2) The damage in clus-tered DNA damage sites can be found in a bistranded andor tandem configu-ration In bistranded clusters the lesions are located on the opposite strands while in the tandem clusters the lesions are distributed on the same strand The simplest bistranded cluster site is the double-strand break (DSB) which is composed of two single-strand breaks within a distance of 14 nucleotides (30) Simple DSBs or DSBs within cluster sites are the most toxic lesions as-sociated with exposure to ionizing radiation (31) Non-DSB cluster sites as

Figure 2 Clustered DNA damage sites can be separated in non-DSB cluster damage sites and DSB containing cluster sites Further the non-DSB cluster sites can be di-vided in clusters containing damage in tandem or bistranded configurations

their name implies are complex lesions that do not harbor a DSB These clus-ter sites can contain one or several SSBs apurinicapyrimidinic (AP) sites and base modifications

A specific type of non-DSB cluster site that is converted into DSBs upon heating is the heat-sensitive cluster site (HSCS) The repair mechanism for and the nature of these lesions has hitherto remained elusive however HSCSs are converted into DSBs at elevated temperatures (32) HSCS are produced after both high-LET and low-LET irradiation (33-35) The importance of the contribution of HSCSs to DSB yields was revealed when DNA extraction after irradiation was performed at 50degC and 4degC The extraction at elevated tem-peratures yields 40 ndash 50 more DSBs in the irradiated samples than extraction performed at 4degC (32 35) Further experiments revealed that HSCSs are re-paired within 1 hour after irradiation are independent of DNA-PKcs PARP1 and XRCC1 (36) and are not related with AP sites (37) As of now the con-version of HSCSs into DSBs is controversial (35 37) Therefore understand-ing the conversion of HSCSs into DSBs during the repair process in cells is crucial to correctly determine non-homologous end joining (NHEJ) repair ki-netics and the consequences of DNA repair protein deficiencies

Factors influencing DNA damage induction The quantity and complexity of DNA damage after exposure to ionizing radi-ation depend on biological chemical and physical factors The most promi-nent factors are DNA packaging and radiation quality ie LET and the oxy-gen concentration It is important to note that these factors can interact recip-rocally which is briefly discussed below

Irradiation with high-LET radiation results in more complex DNA damage clusters than exposure to low-LET radiation (Figure 3) Furthermore in con-trast to low-LET irradiation exposure to high-LET irradiation induces a nonnormal distribution of DSBs (38 39) The complexity of DSBs increases with increasing LET (40 41)

The compactization of DNA in the nucleus leads to a 5-fold decrease in the number of DSBs after irradiation and an even more pronounced effect is ob-served when the chromosomes are condensed in which a 50-fold reduction in DSB induction is observed (42) The protective effect of DNA condensation results from its reduction in the number of water molecules which in turn decreases the number of radicals produced in the vicinity of the DNA (43) this effect is observed after both high-LET and low-LET irradiation (42) Low-LET irradiation at a reduced oxygen concentration results in a decrease in DNA damage that is mediated via the reduction of oxygen-generated per-oxy radicals (44) However the reduced oxygen concentrations have no or a minimal effect on high-LET-induced DNA damage (45)

DNA repair

Repair of non-DSB lesions Due to the large number of different adducts that can be formed in DNA more than 130 different proteins have been identified that mediate its repair in hu-man cells (46) The repair of nonclustered and non-DSB clustered lesions usu-ally involves base excision repair (BER) nucleotide excision repair (NER) and mismatch repair (MMR)

Base lesions that do not distort the DNA helix are recognized by DNA gly-cosylases which cleave the N-glycosyl bond leaving an AP site The AP sites are then processed by apurinicapyrimidinic endonucleases (APEs) which in-duce an SSB Alternatively a base lesion can be processed by bifunctional DNA glycosylase which cleaves the N-glycosyl bond as well as the AP site directly producing an SSB After the formation of an SSB BER is initiated in a form of short-patch or long-patch repair (47) During short-patch repair pol-ymerase β (polβ) inserts the nucleotide and ligase IIIαXRCC1 ligates the SSB In long-patch repair a DNA fragment of 7 ndash 14 nucleotides is resynthe-sized at the AP site by polβ polδ or polε (47 48) The displaced original DNA

Figure 3 The high-LET radiation induce spatially correlated complex DSBs whilelow-LET radiation produce randomly distributed DSBs Representative images of cellsirradiated with alpha particles (high-LET) and gamma radiation (low-LET) The red dots represent DSBs (53BP1) and DNA is shown in blue (DAPI)

strand is then removed by flap endonuclease 1 (49) Finally the strand is re-joined by ligase IPCNA or ligase IIIαXRCC1 (50 51)

NER repairs base modifications that result in distorted DNA The repair is initiated by XPC-RAD32 and the DDB2DDB1 complex (52) Lesions de-tected during transcription are sensed by RNA polymerase which stalls tran-scription and repair proteins are recruited to the damage site After lesion de-tection the surrounding DNA is melted and processed by the TFIIH complex The TFIIH complex contains 10 different proteins that unwind and incise the DNA (53) Then polδ polε and polκ resynthesize the damaged DNA frag-ment (54) which is subsequently rejoined via either ligase IIIα or ligase I (55)

MMR is responsible for the removal of incorrect base pairing produced during cytosine deamination that created uracil or is the result of a misplaced nucleotide by the polymerase The repair is initiated via one of the MutS hom-ologs (56) which recruits MutLα to incise the DNA creating a SSB (57) Fur-thermore the strand is excised by exonuclease 1 and polδ synthesizes a new complementary DNA fragment (58 59) The repair is completed when ligase I rejoins the DNA strand (60)

SSBs can be repaired via several repair pathways as their formation is an intermediate step during BER NER and MMR The repair starts with recog-nition of the SSB by PARP1 After modification of the broken strand repair is then mediated by the repair proteins involved in BER (61) however com-ponents from other repair pathways can contribute

The repair of non-DSB clustered lesions depends on the configuration of the lesions their type the distance between the lesions and their complexity The lesions positioned in a tandem (62) and bistrand (63) configuration are repaired less efficiently Unlike lesions distributed in tandem the bistranded clusters have the potential to become DSBs if they are closely spaced and pro-cessed at the same time (64) As DSBs threaten the survival of cells the repair of clustered lesions follows a specific hierarchy to minimize the production of these highly toxic lesions The first lesions removed from the cluster sites are SSBs and AP sites followed by 8-oxoG (65 66) There are additional diffi-culties during repair when the lesions are closely spaced resulting in delayed processing (67)The repair and conversion of non-DSB cluster sites into DSBs have been studied in isolated systems or in plasmids transfected into cells However at this point it is not clear how cluster damage sites are repaired in different chromatin regions and how well their processing in isolated systems represents their processing in live cells

DSB signaling DSB repair is a complex process that requires lesion recognition signal prop-agation recruitment and the retention of the appropriate repair proteins at damage sites DSB sensing is poorly understood however the MRN complex

KU7080 RPA and PARP1 have been proposed to be the major DSB sensors (68)

ATM and ATR One of the major signal transducers in DSB repair is ataxia telangiectasia mu-tated (ATM) ATM is extremely sensitive to ionizing radiation and its maxi-mum activation is reached when cells are exposed to 04 Gy (69) ATM is normally a dimer that dissociates into monomers and autophosphorylates after exposure to ionizing radiation (69) The actual changes that occur in ATM after exposure are not fully understood however it has been suggested that the MRN complex is required for the complete activation of ATM (70) After its activation ATM targets and phosphorylates more than 700 molecules in-volved in cell cycle regulation chromatin remodeling and DNA damage re-pair (71) Importantly ATM activates DNA-PKcs via phosphorylation and DNA-PKcs phosphorylates ATM leading to its inactivation (72)

Ataxia telangiectasia and Rad3-related protein (ATR) autophosphorylates after it is recruited to the DNA damage site and interacts with RPA (73) ATR responds to a wide variety of lesions and is not specific to DSB repair alone The activation of ATR also occurs during replicative stress or after UV irra-diation (71 74) ATM and ATR can phosphorylate each other in response to DNA damage clearly indicating the importance of these kinases in signal transduction (72 74)

Mediators of DSB repair Prominent DSB markers are p53-binding protein 1 (53BP1) and phosphory-lated H2AX (ɣH2AX) 53BP1 is an essential mediator of DSB repair that re-localizes to DSB sites and facilitates the recruitment of chromatin remodeling proteins (75) activates checkpoint signaling (76) and participates in the choice of a DSB repair pathway (77) 53BP1 binds to H4K20me2 which is modulated by H4K16 acetylation levels near DSBs (78) Specifically the fo-cal accumulation of 53BP1 at the DSB site is dependent on H2K15ub (79)

H2AX phosphorylation at S139 (ɣH2AX) is well established marker of DSBs H2AX is phosphorylated by ATM or DNA-PKcs after the detection of a DSB (80 81) Phosphorylated H2AX molecules can span megabases away from the DSB (82) and the major role of ɣH2AX in the DSB regions is to prevent the dissociation of repair proteins from the break sites (83)

DSB repair In response to DSBs cells activate complex networks of repair and signaling pathways that are responsible for cell cycle arrest chromatin remodeling and repair The key DSB repair pathways in mammalian cells are non-homologous end joining (NHEJ) homologous recombination (HR) and alternative end

joining The first proteins to bind at the DNA termini are the KU7080 heter-odimer PARP1 and the MRN complex which is composed of Mre11 NBS1 and RAD50 (68 84) After MRN and KU7080 bind to the DNA the correct repair pathway must be selected The decision to use NHEJ or HR is depend-ent on the cell cycle stage HR is utilized during S and G2 phases while NHEJ operates in all stages of the cell cycle (85) The main proteins involved in the pathway choice are 53BP1 and BRCA1 53BP1 prevents DSB end processing and promotes NHEJ while BRCA1 promotes end processing and thus HR (86)

Non-homologous end joining The most prominent DSB repair pathway in human cells is NHEJ and the knockout of core NHEJ factors leads to severe radiosensitivity During NHEJ repair DSBs are resolved in several steps and lesions are repaired with a half-life of 75 min and 160 min (36) The repair starts with the binding of KU7080 to the ends of broken DNA followed by the recruitment of DNA-PKcs to au-tophosphorylate and activate Artemis which trims the DNA ends if necessary The DNA ends are ligated by the ligase IV XLF and XRCC4 complex (Figure 4)

KU7080 KU7080 is a heterodimer composed of KU68 and KU86 that forms a ring-like structure (87) The nucleus of human cells contains approximately 500 000 KU7080 molecules (88) KU7080 has a very high binding constant for DNA ends which has been estimated to be 24 times 109 M-1 (89) After binding to the DNA ends KU7080 rotates inwards creating a space for other repair proteins to bind (90) In addition to protecting the DNA ends from degrada-tion KU7080 has a weak AP lyase function that is important for the removal of AP sites located near DSBs (91) The deficiency of KU7080 in human cells leads to a lethal phenotype which can potentially be explained by the role of KU7080 in telomere maintenance (92)

DNA-PKcs Following KU7080 binding DNA-PKcs is recruited to the end of the DNA DNA-PKcs is a member of the phosphatidylinositol 3 kinase-like protein ki-nase (PIKK) family (93) The kinase activity of DNA-PKcs in DSB repair is stimulated by KU7080 and dsDNA (94) When activated DNA-PKcs inter-acts with all major NHEJ components and phosphorylates KU7080 (95) XLF (96) XRCC4 (97) ligase 4 (98) and Artemis (99)

An important function of DNA-PKcs is its autophosphorylation resulting in conformational changes within the protein that disrupt its interaction with KU7080 (100) The autophosphorylation of DNA-PKcs at the ABCDE clus-ter involves threonines at positions 2609 2620 2638 and 2647 and serines at positions 2612 and 2624 Defects within the ABCDE cluster result in reduced

DSB end processing and increased retention of DNA-PKcs in complex with KU7080 (101) The mutation of serines in another autophosphorylation clus-ter PRQ at positions 2023 2029 2041 2053 and 2056 results in increased end processing suggesting that the activation of the PRQ cluster counteracts the activation of the ABCDE cluster (102) DNA-PKcs in S phase is phos-phorylated at T2609 while the phosphorylation of S2056 is blocked by BRCA1 (103) indicating the potential role of T2609 in DSB end processing to initiate HR DNA-PKcs is also autophosphorylated at other residues how-ever the functions of those other autophosphorylation sites are not well un-derstood The knockout of DNA-PKcs in human cells results in compromised DSB repair (35)

In addition to its function in NHEJ DNA-PKcs plays an important role in mitosis in which DNA-PKcs phosphorylated at T2609 and S2056 localizes at centrosomes (104 105) Furthermore DNA-PKcs phosphorylated at T2609 is found in the midbody and kinetochores (105) The inhibition of DNA-PKcs during mitosis leads to atypical nuclear morphology the misalignment of chromosomes and abnormal chromosome separation (104) Additional func-tions of DNA-PKcs have been identified and include the maintenance of ge-nomic stability (106) fragmentation of the Golgi (107) and cell motility (108)

DNA processing in NHEJ Approximately 10 of DSBs induced by ionizing radiation require processing by Artemis (109) Artemis has endonuclease and exonuclease activities that are regulated by DNA-PKcs (99) At the DSB ends Artemis exhibits an en-donuclease activity that trims the DNA ends in the 3´ to 5´ direction (110) Artemis has been suggested to play a role in the pathway choice because the knockout of Artemis rescues BRCA1-deficient cells treated with a PARP1 in-hibitor (111) thus allowing the normal progression of HR Defects in Artemis lead to reduced cell survival following exposure to ionizing radiation and se-vere combined immune deficiency (112 113)

After the DNA end is bridged together by the core NHEJ proteins the pol-ymerases fill in the missing nucleotides The major polymerases involved in NHEJ are pol micro and pol λ (114 115) Interestingly cells deficient in pol micro or pol λ demonstrate no or mild radiosensitivity (116)

Ligation After the processing of DSB ends the ligase IVXRCC4XLF complex rejoins the broken DNA ends In the complex XRCC4 stabilizes and stimulates the activity of ligase IV (117) and directly interacts with XLF which orchestrates the correct positioning of the DNA ends before ligation (118) Deficiency in ligase IV XRCC4 or XLF leads to reduced DSB repair and an increased sen-sitivity to DSB-inducing agents (119-122)

Figure 4 Simplistic representation of DSB repair via NHEJ The repair process is initiated by KU7080 binding which rotates inwards on DNA strand and facilitates binding of DNA-PKcs which then autophosphorylates Afterwards Artemis process the DSB ends if necessary and Ligase IVXRCC4XLF complex rejoins the DNA ends

Homologous recombination Unlike NHEJ HR is active only when a sister chromatid is present as a tem-plate Repair via HR is more precise in terms of preserving the DNA sequence surrounding the DSB than NHEJ However DSB repair by HR is much slower than repair via NHEJ and has been estimated to take approximately 7 hours (123)

HR is initiated by MRN and CtIP which resect the DNA at DSB sites (124) The single-stranded DNA is then quickly covered by RPA which pro-tects the DNA from enzymatic degradation and annealing (125) In the next step RAD51 replaces RPA (126) and together with RAD54 initiates the in-vasion of the sister chromatid (127) After localizing the complementary DNA fragment pol η extends the DNA strand (128) and ligase I rejoins the DNA (129)

Alternative repair mechanisms

Single-strand annealing Single-strand annealing is a subpathway of HR that requires resection and RAD52 facilitated annealing of DSB ends (130) The single-strand DNA over-hang is trimmed via the ERCC1XPF complex (131) Lastly the repair is com-pleted by filling in the missing nucleotides if necessary and the DNA ends are ligated As of now the ligases and polymerases directly involved in SSA have not been identified

Alternative NHEJ When the functions of core NHEJ proteins are compromised in rodent cell lines the repair of DSBs is not fully blocked indicating the existence of an alt-NHEJ pathway Repair by alt-NHEJ involves PARP1 the XRCC1-ligase III complex and pol θ (132 133) Rejoining via alt-NHEJ relies on shorter DNA overhangs than SSA

The discrepancies in repair mechanisms in different model organisms have been poorly discussed in the scientific literature and only in recent years have few papers addressed this issue Rodent cells are able to repair DSBs to some extent in KU7080- and DNA-PKcs-deficient cells However this is not the case with human cells for several reasons First the knockout of KU7080 in human cells generates a lethal phenotype and alt-NHEJ is strongly inhibited in the presence of KU7080 (133 134) Second the knockout of DNA-PKcs in human cells results in compromised DSB repair while in rodent cells the repair is merely reduced (36) Third translocations previously attributed to alt-NHEJ in mouse cells however are mediated by NHEJ in human cells (135)

Cancer More than 141 million new cancer cases are reported every year worldwide (136) Cancer is a multifactorial disease characterized by a loss of normal cell proliferation The major factors that cause cancer are genetic factors smoking aging obesity alcohol consumption infection a lack of exercise and expo-sure to carcinogens ionizing radiation and UV radiation (136 137)

Tumor tissue is highly complex and composed of normal cells and hetero-geneous populations of malignant cells The heterogeneous populations of tu-mor cells exhibit different molecular properties and can therefore show a var-iable response to cancer treatment Malignant cells acquire one or several of the following characteristics during cancer progression abnormal prolifera-tive signaling the evasion of growth suppression immortalization escape from cell death neoangiogenetic signaling invasiveness the capacity to form metastasis atypical epigenetics the stimulation of inflammatory response ge-netic instability and the avoidance of immunogenic destruction (138)

A hyperactive DNA damage response has been observed in premalignant lesions clearly showing that the cells are experiencing elevated DNA damage and genomic instability at the early stages of tumor formation (139) In gen-eral cancer cells have higher mutation rates than normal cells (140) which eventually leads to the disruption of proteins and pathways involved in the maintenance of genomic stability Alternatively tumor cells disable the sys-tems that monitor genomic integrity that would normally trigger apoptosis or senescence in a normal cell (138)

Cancer progression and aggressiveness have been associated with deficien-cies in proteins involved in NHEJ The overexpression of KU7080 (141) DNA-PKcs (142) ligase IV (143) XLF (144) and XRCC4 (145) is linked with reduced overall survival NHEJ proteins control important functions in cells that are essential for cancer progression thus making them viable targets for cancer treatment However only a few agents targeting the major protein kinase in NHEJ DNA-PKcs have been enrolled in clinical trials as of now (146)

Prostate cancer Prostate cancer is responsible for the highest number of reported cancer cases in men with more than 109 million cases reported every year worldwide (147) The treatment of prostate cancer involves radiation therapy chemother-apy and surgery When low-risk prostate cancer is detected patients undergo watchful waiting instead of receiving aggressive treatments (148) that cause unnecessary side effects without providing any health benefits When ad-vanced prostate cancer is accompanied by metastatic lesions androgen depri-vation therapy (ADT) is prescribed ADT only slows down the progression of the disease and in the majority of cases prostate cancer eventually develops resistance The resistance associated with ADT is dependent on changes asso-ciated with androgen receptor (AR) including the upregulation of AR (149) mutations resulting in its constitutive activity (150) mutations affecting lig-and binding (151) or AR gene amplification (152) AR variant 7 (ARv7) is a truncated form of the full protein that is constitutively active and the expres-sion of ARv7 drives the proliferation of prostate cells even when the ligands that activate AR are not available (153 154) The expression of ARv7 is as-

sociated with a more aggressive disease that results in a reduced overall sur-vival (153) Approximately 90 of castration-resistant prostate cancers (CRPCs) result in bone metastases that are incurable as of now (155 156) Bone metastases are painful lesions that present as bone fractures or nerve compressions and significantly reduce quality of life (156 157)

Cancer diagnosis and treatment Cancer is usually discovered during routine examination or other tests such as blood tests positron emission tomography computed tomography magnetic resonance imaging ultrasound X-ray or endoscopic investigations Often a biopsy is acquired from the tumor site to confirm the malignant nature of the tumor After acquisition of the tumor sample the malignancy is assessed Can-cer progression is divided into 5 different stages in which stage 0 is associated with premalignancy and stage I ndash stage III characterize the invasiveness of the cancerous cells in local tissue Stage IV indicates the most advanced stage of the disease accompanied by distant metastases (158) The stage of the disease is the most important factor that determines the future prognosis The spread of cancer is a major factor contributing to mortality and morbidity (159) After diagnosis proper treatment modalities are prescribed that include surgery radiotherapy chemotherapy immunotherapy brachytherapy or a combination of treatments Tumor resection is only possible if the malfor-mation is localized and resides in a favorable position However when the disease has progressed too far or the tumor is located in an inoperable position chemotherapy or radiation therapy is utilized to attempt to cure the disease Radiation therapy is an essential tool in cancer treatment that is given in ~ 50 of cases and radiation therapy is often combined with surgery or chem-otherapy (160)

The most commonly used type of radiation in cancer treatment is high-en-ergy X-rays however in recent years particle therapy has gained popularity The major advantage of particle therapy is its ability to reduce the dose to normal tissue (161) which is critical in cases in which the tumor is localized close to radiosensitive tissue or in pediatric cancer cases in which the reduc-tion of secondary cancers and the risk of cognitive effects is of the utmost importance

When cancer has reached a more advanced stage the tumor cells spread to distant organs such as bone the liver lungs and the brain As of now meta-static cancer is not curable However several treatments focusing on alleviat-ing symptoms associated with cancer progression are employed in clinics in-cluding surgery chemotherapy hormone therapy radiation therapy and tar-geted therapy

A treatment targeting bone metastases in prostate cancer has been devel-oped that is based on the bone-seeking properties of an α-emitter Ra-223

(162) The redistribution of Ra-223 from the bloodstream to active bone re-modeling sites occurs within 2 hours after intravenous injection (163) Ra-223 treatment increases median survival and improves the quality of life of cancer patients exhibiting bone metastases (164) Unfortunately Ra-223 does not ac-cumulate in metastatic regions found in other types of tissues limiting the applicability of Ra-223 treatment

The overall aim of this thesis was to further expand our knowledge on clus-tered DNA damage repair and the consequences after exposure to ionizing radiation In the thesis the following issues were addressed more specifically

First using flow cytometry the potential use of phosphorylated DNA-PKcs as a marker of DSB repair kinetics after irradiation to provide alternative ways to estimate DSB repair kinetics was evaluated

Second the consequences of DNA-PKcs knockdown and inhibition

on cell survival and DSB repair were assessed

Third DNA damage and repair induced by Ra-223 exposure in dif-ferent prostate cancer cell lines were described

Fourth the importance of NHEJ inhibition during the repair of HSCS was investigated and HSCS were further characterized

Results

Paper I

Measurement of DNA-Dependent Protein Kinase Phosphorylation Using Flow Cytometry Provides a Reliable Estimate of DNA Repair Capacity

Aim and background Several techniques have been developed to analyze the DSB content in cells after radiation exposure including pulsed-field gel electrophoresis (PFGE) foci scoring assays live cell imaging comet assays and flow cytometry The analysis of DSB repair by biophysical assays such as PFGE provides the most reliable estimate of DSB repair kinetics in cells however this method is laborious and low-throughput Alternatively DSBs can be detected by meas-uring the phosphorylation or accumulation of DSB repair proteins at DSB sites using fluorescence microscopy or flow cytometry The drawback of the clas-sical foci scoring assay is its inability to discriminate early repair events taking place within 1 hour postirradiation similar to ɣH2AX intensity measurements with flow cytometry To address these issues more complex systems to assess foci dynamics in live cells have been developed However advanced methods require more sophisticated setups and analysis that are not available in all la-boratories

The major protein kinase modulating the repair of DSBs in cells is DNA-PKcs and upon irradiation DNA-PKcs is quickly phosphorylated at several sites One of the most prominent phosphorylation sites in DNA-PKcs is T2609 which regulates end processing activities at DSBs T2609 foci can be easily detected after irradiation using microscopy however the feasibility of the use of T2609 as a DSB marker in flow cytometric evaluation has not been investigated Therefore the aim of the study was to determine the properties of DNA-PKcs phosphorylation in irradiated cells and to evaluate the potential use of DNA-PKcs phosphorylated at T2609 as a DSB marker

Methods The DSB repair capacity of ɣ-irradiated wild-type A431 GM5758 AG07217 and HCT116 cells and ligase IV hypomorphic GM16088 cells was assessed

using PFGE foci scoring assays and flow cytometry Cell cycle phases were determined by flow cytometry using costaining with DAPI The foci were scored manually in ImageJ statistical analysis was performed using Bonfer-roni correction when necessary and p-values less than 005 indicated statisti-cal significance

Results DNA-PKcs was quickly phosphorylated at position T2609 after irradiation

reaching its maximum signal intensity within 15 ndash 30 min of irradiation (Fig-ure 5) After reaching its maximum signal intensity DNA-PKcs was rapidly dephosphorylated and a notable signal reduction was observed within one hour postirradiation in all wild-type cells The ligase IV hypomorphic cells displayed reduced dephosphorylation of DNA-PKcs Unexpectedly the dephosphorylation kinetics of DNA-PKcs were similar in all cell cycle stages

Next the levels of DNA-PKcs dephosphorylated at T2609 in the G1 phase of the cell cycle were compared to the DSB repair kinetics obtained from the PFGE assay (Figure 6) DNA-PKcs dephosphorylation at T2609 followed

Figure 5 Kinetics of DNA-PKcs phosphorylation and dephosphorylation at differentcell cycle stages in wild-type cells (A) A431 (B) AG07217 (C) HCT116 (D) GM5758and Ligase IV hypomorphic cells (E) GM16088 after irradiation with 10 Gy of gammaradiation Data from 3 ndash 8 independent experiments are shown and error bars repre-sent SEM

similar kinetics as the DSB repair kinetics observed by the PFGE assay alt-hough the repair kinetics determined with flow cytometry were slower than those determined by PFGE

Discussion The levels of DNA-PKcs phosphorylated at T2609 were able to discriminate between DSB repair-compromised and normal cells clearly showing that phosphorylated DNA-PKcs T2609 correlates with the DSB content in cells The phosphorylation of H2AX is a widely used DSB marker however H2AX phosphorylation does not reflect DSB repair within the first hour of repair Thus the use of DNA-PKcs phosphorylated at T2609 as a DSB marker at early times during repair has clear advantages over the use of ɣH2AX

Furthermore DNA-PKcs is phosphorylated at T2609 in the M phase which is unrelated to DNA damage It is worth noting that the signal intensity of T2609 phosphorylation in the M phase is low and does not interfere with the analysis Additionally DNA-PKcs phosphorylation levels in each cell cy-cle phase should be separated when DSB repair is assessed at time points longer than 4 hours because the phosphorylation of DNA-PKcs varies over the cell cycle

Figure 6 Comparison of the DNA-PKcs phosphorylation levels and DSB amount in(A) A431 (B) AG07217 (C) HCT116 (D) GM5758 and (E) GM16088 cells Datafrom 3 ndash 8 independent experiments are shown and error bars represent SEM Dottedline represents the repair in G1 cell cycle stage and is transferred from figure 5

DNA-PKcs is the key kinase regulating DSB repair in cells in the G1 and G2 phases Unexpectedly we observed similar kinetics for the dephosphory-lation of residue T2609 in the S and the G1G2 phases Previously it was observed that DNA-PKcs interacted with BRCA1 in the S phase which blocked its phosphorylation at S2056 while the phosphorylation of T2609 was not affected (103) As of now the role of DNA-PKcs phosphorylation and its involvement in DSB repair in the S phase are not well understood therefore DSB repair scoring with phosphorylated DNA-PKcs in the S phase should be used with caution

Paper II

Suppression of DNA-dependent protein kinase sensitizes cells to radiation without affecting DSB repair

Aim and background Dysfunction in normal DNA repair processes is an important factor driving cancer progression Changes in DNA repair protein expression influence can-cer aggressiveness and resistance to treatment The high expression of DNA-PKcs a major PIKK involved in NHEJ leads to reduced overall survival and poor prognosis (165 166) while the downregulation of DNA-PKcs expres-sion has been suggested to increase the risk of cancer development (167) In addition to its functions in DSB repair DNA-PKcs is an important player that orchestrates events in mitosis

The aim of this study was to determine whether the reduced survival of irradiated cells with reduced levels of DNA-PKcs is related to defective DNA repair or mediated by the alternative functions of DNA-PKcs

Methods DNA-PKcs knockdown in the A431 HCT116 and H314 cancerous cell lines and the GM5758 normal cell line was achieved using transfection of small siRNA fragments To study survival and DSB repair wild-type DNA-PKcs-expressing M059K cells and DNA-PKcs-deficient M059J cells were used The relative protein content in the cells was determined using western blot The survival of cells was assessed using a clonogenic assay The cells were synchronized with nocodazole and mitotic cells were detected using phos-phorylated H3 as a marker DSB repair was scored using a foci scoring assay and PFGE In all experiments cells were irradiated with gamma radiation

Results After transfection DNA-PKcs levels in the cells were reduced to 5 ndash 20 of the normal levels The knockdown of DNA-PKcs in combination with radia-tion exposure resulted in a significant reduction in cell survival which was comparable to the cell survival observed in cells lacking DNA-PKcs (Figure 7) To further investigate whether the reduction in DNA-PKcs affected DSB repair PFGE was performed (Figure 8) In all cell lines DNA-PKcs knock-down did not affect the DSB repair kinetics In contrast DNA-PKcs-deficient

cells showed reduced DSB repair indicating that DSB repair is differentially affected when DNA-PKcs is knocked down or not expressed The PFGE ex-periments did not indicate any DSB repair defect in mock-treated cells and in cells with reduced DNA-PKcs levels (Figure 8) Results from the PFGE ex-periments were consistent with those from the foci scoring assay in which DNA-PKcs phosphorylated at T2609 and S2056 as well as 53BP1 and the ɣH2AX foci count were not significantly different at 1 4 and 24 hours postir-radiation in mock-treated and DNA-PKcs-depleted cells

Figure 7 Knockdown of DNA-PKcs results to similar sensitivity to ionizing radiationas knockout of DNA-PKcs Clonogenic survival of mock treated and DNA-PKcs knockdown (A) A431 (B) HCT116 (C) H314 cells and (D) DNA-PKcs deficient M059J and respective wild-type cells M059K Colonies with more than 50 colonies were scored 10 ndash 15 days after irradiation Error bars represent SD from at least 3independent experiments

Then we examined how DNA-PKcs depletion affects cell cycle progression Cells with low levels of DNA-PKcs exhibited an increased amount of phos-phorylated H3 which marks cell accumulation at M phase (Figure 9 A) The

Figure 9 Suppression of DNA-PKcs leads to accumulation of cells in mitosis (A)A431 cells were irradiated with 5 Gy and after 24 hours cells with positive phosphor-ylated H3 marker were scored Error bars represent SD from 2 independent experi-ments where gt250 cells were scored each time (B) A431 cells were treated with no-codazole for 6 hours irradiated incubated for 18 hours without nocodazole and thencells positive for phosphorylated H3 were scored Error bars represent SD of twoindependent experiments where gt400 cells were scored in each experiment

Figure 8 Reduction of DNA-PKcs does not change DSB repair kinetics Comparisonof DSB repair of DNA-PKcs siRNA treated (A) A431 (B) HCT116 (C) H314 (D)GM5758 cells and DNA-PKcs deficient (E) M059J cells The DNA damage is normal-ized to the t=0 Error bars represent SD from 3 independent experiments

irradiation of synchronized cells in the G2M phase further increased the ac-cumulation of cells in M phase (Figure 9 B)

Discussion The reduction in DNA-PKcs levels in cells had no effect on DSB repair even when the cells were irradiated with high doses of ionizing radiation suggest-ing that only a few molecules of DNA-PKcs present in the nucleus are enough to repair the lesions However the depletion of DNA-PKcs had a prominent effect on cell survival which is consistent with the notion that low levels of DNA-PKcs expression in cancers correlate with the overall increased survival of patients (142) The increased accumulation of DNA-PKcs knockdown cells in mitosis indicated that DNA-PKcs is involved in processes important for cell survival independent of its function in DSB repair This was further supported by the fact that interference with normal DNA-PKcs functioning resulted in abnormal cell division and reduced the metastatic capacity of tumors (105 165) Taken together these results suggest that DNA-PKcs has an important role beyond its function in DSB repair Identification of the alternative roles of DNA-PKcs in cell survival regulation and cancer progression could poten-tially identify new targets and strategies for drug development and cancer treatment and therefore should be further explored

Paper III

The α-emitter Ra-223 induces clustered DNA damage and significantly reduces cell survival

Aim and background The most commonly diagnosed cancer in men worldwide is prostate cancer The treatment of prostate cancer involves surgery radiation therapy and an-drogen deprivation therapy (ADT) ADT is usually prescribed when the dis-ease has reached a more advanced stage and is accompanied by metastases The major target of ADT is androgen receptor (AR) which is often mutated amplified or upregulated ADT only delays the progression of prostate cancer and given enough time castration-resistant prostate cancer emerges Prostate cancers in late stages are incurable as of now and metastases originating from prostate cancer can be found in bone the lungs the liver and the brain Bone metastases are painful lesions that significantly affect a patientrsquos quality of life To combat the progression of bone metastasis Ra-223 treatment has been developed Ra-223 is an α-emitter that accumulates in bone undergoing active remodeling which also coincides with metastatic sites Ra-223 treatment has proven to be efficacious in the treatment of bone metastasis in clinics yet the

characteristics of Ra-223 binding to hydroxyapatite a mineral found in bones and the damage produced in cells have not been described Therefore the aim of this study was to investigate Ra-223 binding to hydroxyapatite DNA dam-age DNA repair and the cellular consequences of Ra-223 exposure

Methods In this study PC3 and DU145 cells which are ARv7-negative prostate cancer cells and 22RV1 cells which are ARV7 positive cells were used The ex-pression of ARv7 was validated using western blot Cell survival was deter-mined using a clonogenic assay in which colonies of more than 50 cells were considered viable The DNA damage distribution in cells was measured via PFGE or visualized by microscopy using 53BP1 and ɣH2AX as DSB markers DSB repair was determined using flow cytometry in which the levels of DNA-PKcs phosphorylated at T2609 were scored The affinity of Ra-223 was esti-mated in a hydroxyapatite-coated Petri dish using LigandTracer technology

Results Ra-223 exposure sensitized cells to the same extent independent of ARv7 ex-pression (Figure 10 A) As alpha particles have a limited range in water and tissue we investigated whether Ra-223 exposure also reduced the growth of 3D cell structures Indeed exposure to 100 kBqml significantly reduced cell survival while higher concentrations of 250 and 500 kBqml effectively in-hibited the growth of spheroids (Figure 10 B)

Unlike the DSBs induced by X-rays the alpha particles originating from the Ra-223 decay chain produced a nonrandom distribution of DSBs (Figure 11 A) The majority of the DSBs induced by Ra-223 treatment were repaired

Figure 10 Ra-223 treatment reduces cell survival in monolayers and spheroids (A)Ra-223 reduces cell survival similarly in ARv7 positive cells 22RV1 and ARv7 nega-tive cells PC3 and DU145 (B) The Ra-223 treatment decreases spheroid growth in22RV1 cells Error bars represent SEM from 3 independent experiments

within 4 hours when 6 ndash 11 of the initial amount of the damage was detected (Figure 11 B)

Binding studies revealed that Ra-223 quickly binds to the hydroxyapatite and saturates the surface within 2 hours (Figure 12 A) The estimated affinity was 192 plusmn 65 e-18 (mean plusmn SD n=9) The Ra-223-bound hydroxyapatite surface prevented spheroid expansion on the surface and reduced the cell sur-vival gt100-fold more than that observed in the unbound Ra-223 treatment (Figure 12 B and C)

Figure 11 Ra-223 induced clustered DNA damage and repair (A) Exposure to Ra-223 leads to production of non-random DSB distribution DU-145 cells were exposed to 1MBqml Ra-223 for 6 hours or irradiated with X-rays Error bars represent SEMfrom 2 independent experiments (B) The DNA-PKcs phosphorylation levels is dras-tically reduced after exposure to Ra-223 DU-145 and PC3 cells were exposed to 300kBqml Ra-223 for 1 hour allowed to repair for indicated time The signal intensity isnormalized to 15 min time point The error bars represent SEM from 3 independentexperiments

Figure 12 Ra-223 coated hydroxyapatite surface significantly reduces cell survival(A) Extravasation of 22RV1 cells are reduced on Ra-223 treated hydroxyapatite sur-face The 22RV1 spheroids were placed on hydroxyapatite coated dish and let adhereOn the next day the spheroids were treated with Ra-223 and every two days area of the extravasating cells were measured The error bars represent SEM from 3 inde-pendent experiments (B) Cell killing effect of Ra-223 is enhanced gt100-fold when Ra-223 is bound to hydroxyapatite Less than survival values represent experiments with no colonies detected and specified value is included in calculations

Discussion ARv7 expression confers resistance to ADT and promotes progression of dis-ease Here we show that treatment with Ra-223 reduces cell survival inde-pendent of ARv7 expression The damage induced by Ra-223 treatment was consistent with the damage pattern produced by high-LET irradiation indicat-ing that the major contributor to the cell-killing effect is the production of DNA damage that is difficult to repair Although the cells were able to rejoin DSBs produced by Ra-223 exposure the cell-killing effect potentially arose from misrepaired DSBs The cell-killing effect associated with Ra-223 treat-ment was observed in monolayers as well as in 3D cell structures and was further amplified when Ra-223 was bound to the hydroxyapatite surface For the first time we illustrate that Ra-223 treatment can prevent the progression of bone metastasis independent of the molecular properties of the cells and stop the seeding of new bone metastasis Ra-223 quickly associated with the bone-like structure similarly as observed in biodistribution studies (163) Here we are the first to quantify the affinity of Ra-223 for hydroxyapatite which was extremely high The advantage of high affinity compounds is the quick relocalization of compounds from blood to their target In clinics this property is particularly important because it reduces the time Ra-223 spends in the bloodstream and therefore the dose to normal tissue

Paper IV

Removal of heat-sensitive clustered damaged DNA sites is independent of double-strand break repair

Aim and background The quantification and understanding of molecular mechanisms behind DSB repair is of the utmost importance because these are the most toxic lesions that can be generated in cells Additionally to prompt DSBs a vast variety of non-DSB clusters can be produced in DNA after ionizing radiation exposure A non-DSB cluster is defined as a cluster of several lesions within 20 ndash 30 bp that do not contain DSBs A large number of in vitro experiments have con-cluded that these non-DSB cluster sites can be translated into DSBs during repair however at this stage it is not clear whether the generation of addi-tional DSBs also occurs in living cells

A specific type of non-DSB cluster damage site is heat-sensitive cluster sites (HSCSs) At elevated temperatures these lesions undergo transformation from non-DSB cluster sites to DSBs The lesions found in the clusters or dur-ing the repair of HSCSs have not been identified as of yet The release of DSBs

from HSCSs in cells has been controversial therefore the aim of this study was to clarify whether HSCSs are converted into DSBs in living cells and to further characterize the HSCS conversion process in vitro

Methods In this study we examined DSB and HSCS repair in the normal fibroblast cell line GM5758 and DNA-PKcs knockout and the corresponding wild-type cells in the HCT116 colorectal cancer cell line treated with or without the DSB repair inhibitor NVP-BEZ235 DSB repair was assessed using PFGE and 53BP1 and ɣH2AX foci scoring Additionally the presence of HSCSs in the pBR322 plasmid was measured by linear gel electrophoresis Heat-induced morphological changes were visualized in the pBR322 plasmid immobilized on mica using atomic force microscopy operating in liquid tapping mode

Figure 13 Deficiency in DSB repair does not affect the repair of HSCS Repair kinet-ics of DSB and HSCS repair in NVP-BEZ235 untreated GM5758 HCT116 andHCT116 DNA-PKcs KO cells (A C E) and NVP-BEZ235 treated cells (B D F) Therelative amount of DSBs are normalized to the DSB amount at t = 0 Error bars rep-resent SD from 3 ndash 4 independent experiments

Results

To investigate whether HSCS are converted into DSBs in human cells HSCS repair kinetics after irradiation were observed in DSB repair-com-promised cells Wild-type cells repaired the HSCSs and DSBs within 1 and 4 hours respectively (Figure 13 A and C) However a deficiency in DNA-PKcs abolished DSB repair but had no effect on HSCS repair (Figure 13 E) The wild-type cells treated with the DSB repair inhibitor exhibited proficient HSCS repair and compromised DSB repair similar to the results seen in the DNA-PKcs-deficient cells (Figure 13 B D and F) Furthermore no additional excess DSBs was observed in DSB repair inhibitor-treated or DNA-PKcs-de-ficient cells over a time period of 4 hours during which all HSCSs were re-moved The results of the 53BP1 and ɣH2AX foci scoring assay were in agree-ment with the results of PFGE experiments in which the NVP-BEZ235-treated cells displayed delayed foci formation and increased foci retention without an effect on the maximum foci count The inhibition of DSB repair by lowering of the repair temperature delayed HSCS repair without generating excess DSBs Next we examined the kinetics of HSCS conversion into DSBs At physio-logical temperatures there was a significant conversion of HSCSs into DSBs within 3 hours (Figure 14) while treatment at 50degC resulted in a gradual in-crease in additional DSBs

The analysis of DNA morphology via atomic force microscopy revealed that prolonged heating resulted in the aggregation of DNA (Figure 15) After closer inspection some DNA regions were similar to denatured DNA sug-gesting that melting of DNA sequences can occur at 50degC

Figure 14 Reduced release of HSCSs into DSBs at physiological temperaturesHCT116 (A) and GM5758 (B) cells were irradiated with 40 Gy followed by DNA ex-traction a Figure 14 Reduced release of HSCSs into DSBs at physiological tempera-tures HCT116 (A) and GM5758 (B) cells were irradiated with 40 Gy followed byDNA extraction and heating in 37degC and 50degC The error bars represent SD from twoto three independent experiments and all data is normalized to the starting level

Discussion Research focused on the translation of clustered DNA damage into DSBs after exposure to ionizing radiation in cells has been challenging In this study we show that HSCSs are not converted into DSBs in live cells This conclusion was further strengthened by the fact that no increase in the number of DSBs occurred when DNA was heated at physiological temperatures up to 3 hours a time period during which HSCSs are repaired in live cells Furthermore it has been shown that only the initial number of DSBs correlates with cell sur-vival while no association between cell survival and HSCS number has been found (168) indicating that HSCSs might not be toxic per se Surprisingly the prolonged heating of DNA at 50degC changed the structure of DNA which was a consequence of DNA melting and denaturation showing that some ar-tifacts can be produced by the prolonged exposure of DNA to heat Therefore we conclude that the heating of DNA should be avoided when DSB analysis is performed using PFGE

Figure 15 Representative images of (A) control and (B) 200 Gy gamma irradiatedpBR322 heated at 50degC

Concluding remarks

The overall goal of this thesis was to expand the knowledge of clustered DNA damage and repair More specific efforts were directed towards understanding ionizing radiation-induced DNA damage and how this damage is resolved in cells in terms of DNA-PKcs expression and modifications

In recent years particle irradiation and treatments have gained popularity in clinics It is well established that particle irradiation is able to induce DNA damage that is difficult to repair which becomes more complex with increas-ing LET An additional focus of these studies was to investigate the complex-ity and repair of DNA damage induced by Ra-223 a radionuclide used in the treatment of metastatic prostate cancer exhibiting bone metastases

The majority of the knowledge of cluster DNA damage sites comes from studying the lesions in isolation However the consequences of clustered DNA damage site repair in living cells are poorly understood The last part of this work addressed the repair of specific nonclustered DSB sites and HSCSs the conversion of HSCSs into DSBs during repair and how the HSCS content in cells affects DSB repair estimates The major findings of this thesis were as follows DNA-PKcs phosphorylation at T2609 increased with increasing radiation

dose The DNA-PKcs phosphorylation kinetics at T2609 represent early repair

events better than ɣH2AX The kinetics of DNA-PKcs dephosphorylation at T2609 should be scored

in each cell cycle phase separately if the DNA damage is determined after repair times longer than 4 hours

The kinetics of DNA-PKcs dephosphorylation were delayed in ligase IV hypomorphic cells

The reduced amount of DNA-PKcs decreased cell survival after ionizing radiation exposure without significantly affecting DSB repair

The depletion of DNA-PKcs resulted in cell accumulation in mitosis after irradiation

The inhibition of DNA-PKcs activity had a different effect than the re-duction of the DNA-PKcs levels

Ra-223 reduced cell survival independently of ARv7 expression in mon-olayers

Ra-223 induced DNA damage that was consistent with damage induced by high-LET radiation

The majority of the DSBs induced via Ra-223 exposure were repaired within 4 hours

Ra-223 strongly binds to hydroxyapatite Cells exposed to Ra-223-bound hydroxyapatite exhibited an enhanced

cell-killing effect HSCSs were not converted into DSBs in live cells and the repair of

HSCSs was independent of DSB repair The conversion of HSCSs in physiological conditions was slower than

the repair of HSCSs The prolonged heating of DNA at 50degC can alter DNA structure

The studies presented in this thesis illustrate that the phosphorylation of DNA-PKcs at T2609 can be used as a marker of DSB repair during flow cytometric evaluation It was shown that DNA-PKcs expression levels affect cell survival independent of its role in DSB repair Furthermore complex DNA damage associated with Ra-223 exposure is responsible for cell death independent of the molecular properties of the cell Lastly the heating of irradiated DNA dur-ing extraction should be avoided because it changes the structure of DNA and produces additional DSBs that do not contribute to the yield of DSBs in living cells

Future studies

The increased use of hadron therapy in cancer treatment has been shown to reduce normal tissue exposure thus minimizing the side effects associated with radiation exposure With increasing LET it is possible to overcome the challenges related to the reduced efficacy of conventional photon therapy in the hypoxic regions of the tumor The main cell-killing effects achieved in hadron therapy are due to the increased complexity of the DNA damage The ability of nonclustered DNA damage sites to convert into DSBs has been well established in isolated DNA however in cells the DNA is wrapped around histones which could potentially alter how these lesions are repaired or mod-ified The identification of the repair pathways that resolve non-DSB cluster sites and the clarification of the requirements to collapse these lesions in DSBs could potentially lead to new approaches in cancer treatment Therefore fu-ture studies to address these questions in live cells are necessary

In the first paper we identified that the phosphorylation of DNA-PKcs co-incides with the number of DSBs found in cells after exposure to low-LET However DNA-PKcs dephosphorylated at T2609 displayed similar kinetics in all phases of the cell cycle Hitherto it is not clear what functions the DNA-PKcs has in S phase and how representative is the DNA-PKcs phosphorylation at T2609 of DSB repair in the S phase Thus more extensive studies investi-gating the role of DNA-PKcs in DSB repair in S phase cells should be per-formed

The second paper illustrated the importance of DNA-PKcs in cell survival after ionizing radiation injury Surprisingly in cells with low levels of DNA-PKcs the kinetics of DSB repair were not affected even after exposure to rel-atively high doses of radiation which leads to several questions that should be addressed in future studies First does the DNA-PKcs level affect the fi-delity of DSB repair Second is the reduced survival solely the result of dys-function in mitosis Third how exactly does DNA-PKcs promote survival

In the third paper we demonstrated that cell survival is not dependent on the molecular characteristics of high-LET-irradiated cells suggesting that the complexity of DNA damage by high-LET irradiation is responsible for the cell-killing effect Interestingly it was noted that the DSB repair was not dras-tically different from the repair of low-LET-irradiated cells which is in con-trast to the general notion in the scientific literature Therefore in further stud-ies whether the DSB repair kinetics are similar following low-LET and high-LET irradiation should be addressed and a comprehensive comparison with

PFGE and flow cytometric evaluation should be made Furthermore studies should focus on the spatial distribution of the generated DSBs surrounding the core of the tracks to further elaborate the damage produced in chromatin struc-tures Additional studies should be performed to evaluate Ra-223 treatment in other types of cancers that metastasize to the bone in a clinical setting

The last paper evaluated the repair of HSCSs after irradiation Our study indicated that the process is independent of DSB repair and does not contrib-ute towards the generation of excess DSBs Because the lesions generating HSCSs and the proteins involved in the repair of these lesions are not known future studies should focus on answering these questions Understanding these processes could provide possible new targets for small inhibitors that in com-bination with radiation therapy could increase the efficacy of cancer treat-ment

Acknowledgements

This work was started in the Department of Radiology Oncology and Radia-tion Science and finalized in the Department of Immunology Genetics and Pathology with financial support from Swedish Cancer Society Swedish Ra-diation Safety Authority and Roumlnnow travel funds Many people have participated and made my road to PhD degree smooth and pleasant I would like to express my deepest thanks to colleagues and collab-orators that contributed to this thesis Many more people that are not named here deserves my sincerest gratitude My main supervisor Bo Stnerloumlw I would like to thank you for taking me into your group Without your help this thesis would not be possible Your doors have been always open for me and you have always helped me with any issue that I had regardless how busy you were I appreciate very much your patience and kindness that you have shown me over the years I have enjoyed our sci-entific discussions and you have taught me a lot about science Marika Nestor thank you for agreeing to be my co-supervisor Your positive attitude has been contagious and it is always a pleasure to discuss various sub-jects with you My co-supervisor Diana Spiegelberg it has been great sharing an office with you over the years and working on different projects Always ready with fun and discussions about science life traveling and photography and most im-portantly to lend me a helping hand and provide some advice whenever it was needed Johan Lennartsson for being my co-supervisor without you this thesis would not be possible My co-author Ann-Sofie Gustafsson it was fun sharing office working with you and having fikas together Sten Nilsson thank you for working with us on the Ra-223 project without you my project would not be feasible

Kicki I enjoyed immensely working with you and I appreciate greatly your support over the years Thank you for the care and patience that you have shown me as well as organizing awesome get together events It was truly a sad day when you decided to leave us for other ventures Vladimir whenever there was a question about the radiation protection and safety you were always there to advise Anja it was has been a delight sharing an office with you I greatly enjoyed the serious discussions we have had and the jokes and puns Sara L thanks for the afternoon fikas discussions about different topics and help whenever I needed it Maria I enjoyed greatly our discussions about science academia and life out-side work Olga for taking care of us in the labs and helping us to maintain the order Anna Orlova for sharing cells with us and good questions during my semi-nars I would like to express my gratitude to people that I met every day in the lab Piero Maryam Javad Mohamed Nakul and Anzhelika Thank you for the friendly chats during lunch time and fika To the colleagues at PPP BMS and molecular geriatrics group and especially to Bogdan Sara R and Ram Joumlrgen Johanna Hadis Sara S Amelie Sara A Joakim Silvio Maria Emma Elisabeth Ximena Gabriel and Xiaotian for the fun discussions about science life music and training Many thanks to all the people working in LigandTracer and most im-portantly to Kalle and Jos for helping with LigandTracer measurements and data analysis Hanna Jonas and Anna it was always nice to meet you in the lab or during the fika I would like to thank the students that worked on projects with me Pedram Hanna Eugenia and Martina I appreciate all your long hours spent in the lab and analyzing data Pacho Jeremy Matyas and Sara at BioVis for lending a helping hand when-ever there was trouble with the instruments or advice on image analysis was needed

Sina and Axel thank you for all the fun during the role-playing sessions and funny conversations Joatildeo for help with LigandTracer and most importantly about all the silly chats we have had and the good time watching series together To all my friends Andreas Marcus Dennis Aaron Jordi Max and Benjamin thank you for all those great years that we have had playing games watching movies and having sophisticated talks that stretched long into the nights Thanks for all the support and advice that you have given me Rasmus for all the discussions about the music and guitar stuff I have had a lot of fun playing board games watching movies and climbing with you My dear sister Anete thanks for always being there for me For all the support and care you offered me over the years I appreciate the times we have been climbing playing board games and traveling together Thanks for being the best sister Maniem vecākiem pladies par manis stimulēšanu petīt un uzzināt vairāk jau kopš bērnības Jūsu mīlestība pacietība un atbalsts visu šo gadu laikā ir bijis nebeidzams Bez jūsu palīdzības es noteikti nevarētu būt tas kas es esmu un tur kur es esmu tagad To Iris my love I appreciate and cherish your endless love and support For always encouraging me to strive for the best and making my life more com-plete

References

1 Desouky O Ding N Zhou G Targeted and non-targeted effects of ionizing ra-diation Journal of Radiation Research and Applied Sciences 20158(2)247-54

2 Nagasawa H Little JB Induction of Sister Chromatid Exchanges by Extremely Low Doses of α-Particles Cancer Research 199252(22)6394-6

3 Nelson GA Space Radiation and Human Exposures A Primer Radiation re-search 2016185(4)349-58

4 Dahm R Discovering DNA Friedrich Miescher and the early years of nucleic acid research Human Genetics 2008122(6)565-81

5 Avery OT Macleod CM McCarty M Studies on the chemical nature of the substance inducing transformation of pneumococcal types Induction of trans-formation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III The Journal of experimental medicine 194479(2)137-58

6 Levene PA Jacobs WA Uumlber die Hefe-Nucleinsaumlure Berichte der deutschen chemischen Gesellschaft 190942(2)2474-8

7 Watson JD Crick FH Molecular structure of nucleic acids a structure for deox-yribose nucleic acid Nature 1953171(4356)737-8

8 Clausen-Schaumann H Rief M Tolksdorf C Gaub HE Mechanical stability of single DNA molecules Biophysical journal 200078(4)1997-2007

9 Olins AL Olins DE Spheroid chromatin units (v bodies) Science 1974183(4122)330-2

10 Richmond TJ Finch JT Rushton B Rhodes D Klug A Structure of the nucle-osome core particle at 7 A resolution Nature 1984311(5986)532-7

11 Kornberg RD Chromatin structure a repeating unit of histones and DNA Sci-ence 1974184(4139)868-71

12 Luger K Mader AW Richmond RK Sargent DF Richmond TJ Crystal struc-ture of the nucleosome core particle at 28 A resolution Nature 1997389(6648)251-60

13 Simpson RT Structure of the chromatosome a chromatin particle containing 160 base pairs of DNA and all the histones Biochemistry 197817(25)5524-31

14 Yusufaly TI Li Y Singh G Olson WK Arginine-phosphate salt bridges be-tween histones and DNA intermolecular actuators that control nucleosome ar-chitecture The Journal of chemical physics 2014141(16)165102-

15 Segal E Fondufe-Mittendorf Y Chen L Tharingstroumlm A Field Y Moore IK et al A genomic code for nucleosome positioning Nature 2006442(7104)772-8

16 Allfrey VG Faulkner R Mirsky AE Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis Proceedings of the National Academy of Sciences of the United States of America 196451786-94

17 Mueller RD Yasuda H Hatch CL Bonner WM Bradbury EM Identification of ubiquitinated histones 2A and 2B in Physarum polycephalum Disappearance of these proteins at metaphase and reappearance at anaphase The Journal of bio-logical chemistry 1985260(8)5147-53

18 Ord MG Stocken LA Phosphate and thiol groups in histone f3 from rat liver and thymus nuclei The Biochemical journal 1967102(2)631-6

19 Levinger L Varshavsky A Selective arrangement of ubiquitinated and D1 pro-tein-containing nucleosomes within the Drosophila genome Cell 198228(2)375-85

20 Nishizuka Y Ueda K Honjo T Hayaishi O Enzymic adenosine diphosphate ribosylation of histone and poly adenosine diphosphate ribose synthesis in rat liver nuclei The Journal of biological chemistry 1968243(13)3765-7

21 Fu Y Sinha M Peterson CL Weng Z The insulator binding protein CTCF po-sitions 20 nucleosomes around its binding sites across the human genome PLoS genetics 20084(7)e1000138

22 Tse C Sera T Wolffe AP Hansen JC Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays by RNA Polymerase III Molecular and cellular biology 199818(8)4629-38

23 Britten RJ Davidson EH Gene Regulation for Higher Cells A Theory Science 1969165(3891)349-57

24 Jost KL Bertulat B Cardoso MC Heterochromatin and gene positioning inside outside any side Chromosoma 2012121(6)555-63

25 Blaisdell JO Harrison L Wallace SS Base excision repair processing of radia-tion-induced clustered DNA lesions Radiation protection dosimetry 200197(1)25-31

26 Sonntag C Free-Radical-Induced DNA Damage and Its Repair A chemical Per-spective Berlin Heidelberg Springer-Verlag 2006

27 Bennett PV Cintron NS Gros L Laval J Sutherland BM Are endogenous clus-tered DNA damages induced in human cells Free Radic Biol Med 200437(4)488-99

28 Ward JF The complexity of DNA damage relevance to biological conse-quences International journal of radiation biology 199466(5)427-32

29 Goodhead DT Initial events in the cellular effects of ionizing radiations clus-tered damage in DNA International journal of radiation biology 199465(1)7-17

30 Vispe S Satoh MS DNA repair patch-mediated double strand DNA break for-mation in human cells The Journal of biological chemistry 2000275(35)27386-92

31 Schipler A Iliakis G DNA double-strand-break complexity levels and their pos-sible contributions to the probability for error-prone processing and repair path-way choice Nucleic acids research 201341(16)7589-605

32 Rydberg B Radiation-induced heat-labile sites that convert into DNA double-strand breaks Radiation research 2000153(6)805-12

33 Benenti G Casati G Maspero G Shepelyansky DL Quantum Poincare recur-rences for a hydrogen atom in a microwave field Phys Rev Lett 200084(18)4088-91

34 Gustafsson AS Hartman T Stenerlow B Formation and repair of clustered dam-aged DNA sites in high LET irradiated cells International journal of radiation biology 201591(10)820-6

35 Stenerlow B Karlsson KH Cooper B Rydberg B Measurement of prompt DNA double-strand breaks in mammalian cells without including heat-labile sites re-sults for cells deficient in nonhomologous end joining Radiation research 2003159(4)502-10

36 Karlsson KH Radulescu I Rydberg B Stenerlow B Repair of radiation-induced heat-labile sites is independent of DNA-PKcs XRCC1 and PARP Radiation research 2008169(5)506-12

37 Singh SK Wang M Staudt C Iliakis G Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair Nucleic acids research 201139(19)8416-29

38 Hoglund E Blomquist E Carlsson J Stenerlow B DNA damage induced by radiation of different linear energy transfer initial fragmentation International journal of radiation biology 200076(4)539-47

39 Newman HC Prise KM Folkard M Michael BD DNA double-strand break distributions in X-ray and alpha-particle irradiated V79 cells evidence for non-random breakage International journal of radiation biology 199771(4)347-63

40 Gulston M Fulford J Jenner T de Lara C ONeill P Clustered DNA damage induced by gamma radiation in human fibroblasts (HF19) hamster (V79-4) cells and plasmid DNA is revealed as Fpg and Nth sensitive sites Nucleic acids re-search 200230(15)3464-72

41 Nikjoo H ONeill P Wilson WE Goodhead DT Computational approach for determining the spectrum of DNA damage induced by ionizing radiation Radi-ation research 2001156(5 Pt 2)577-83

42 Takata H Hanafusa T Mori T Shimura M Iida Y Ishikawa K et al Chromatin compaction protects genomic DNA from radiation damage PloS one 20138(10)e75622

43 Falk M Lukasova E Kozubek S Chromatin structure influences the sensitivity of DNA to gamma-radiation Biochimica et biophysica acta 20081783(12)2398-414

44 Stewart RD Yu VK Georgakilas AG Koumenis C Park JH Carlson DJ Ef-fects of radiation quality and oxygen on clustered DNA lesions and cell death Radiation research 2011176(5)587-602

45 Wenzl T Wilkens JJ Theoretical analysis of the dose dependence of the oxygen enhancement ratio and its relevance for clinical applications Radiation oncology (London England) 20116171-

46 Wood RD Mitchell M Sgouros J Lindahl T Human DNA repair genes Sci-ence 2001291(5507)1284-9

47 Frosina G Fortini P Rossi O Carrozzino F Raspaglio G Cox LS et al Two pathways for base excision repair in mammalian cells The Journal of biological chemistry 1996271(16)9573-8

48 Fortini P Pascucci B Parlanti E Sobol RW Wilson SH Dogliotti E Different DNA Polymerases Are Involved in the Short- and Long-Patch Base Excision Repair in Mammalian Cells Biochemistry 199837(11)3575-80

49 Klungland A Lindahl T Second pathway for completion of human DNA base excision-repair reconstitution with purified proteins and requirement for DNase IV (FEN1) The EMBO journal 199716(11)3341-8

50 Prasad R Singhal RK Srivastava DK Molina JT Tomkinson AE Wilson SH Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis The Journal of biological chem-istry 1996271(27)16000-7

51 Caldecott KW McKeown CK Tucker JD Ljungquist S Thompson LH An in-teraction between the mammalian DNA repair protein XRCC1 and DNA ligase III Molecular and cellular biology 199414(1)68

52 Sugasawa K Ng JMY Masutani C Iwai S van der Spek PJ Eker APM et al Xeroderma Pigmentosum Group C Protein Complex Is the Initiator of Global Genome Nucleotide Excision Repair Molecular cell 19982(2)223-32

53 Mathieu N Kaczmarek N Ruumlthemann P Luch A Naegeli H DNA Quality Con-trol by a Lesion Sensor Pocket of the Xeroderma Pigmentosum Group D Hel-icase Subunit of TFIIH Current Biology 201323(3)204-12

54 Ogi T Limsirichaikul S Overmeer RM Volker M Takenaka K Cloney R et al Three DNA Polymerases Recruited by Different Mechanisms Carry Out NER Repair Synthesis in Human Cells Molecular cell 201037(5)714-27

55 Moser J Kool H Giakzidis I Caldecott K Mullenders LH Fousteri MI Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner Molecular cell 200727(2)311-23

56 McCulloch SD Gu L Li GM Bi-directional processing of DNA loops by mis-match repair-dependent and -independent pathways in human cells The Journal of biological chemistry 2003278(6)3891-6

57 Kadyrov FA Dzantiev L Constantin N Modrich P Endonucleolytic function of MutLalpha in human mismatch repair Cell 2006126(2)297-308

58 Nielsen FC Jaumlger AC Luumltzen A Bundgaard JR Rasmussen LJ Characteriza-tion of human exonuclease 1 in complex with mismatch repair proteins subcel-lular localization and association with PCNA Oncogene 2003231457

59 Longley MJ Pierce AJ Modrich P DNA polymerase delta is required for human mismatch repair in vitro The Journal of biological chemistry 1997272(16)10917-21

60 Zhang Y Yuan F Presnell SR Tian K Gao Y Tomkinson AE et al Reconsti-tution of 5prime-Directed Human Mismatch Repair in a Purified System Cell 2005122(5)693-705

61 Caldecott KW Single-strand break repair and genetic disease Nature Reviews Genetics 20089619

62 Cunniffe SM Lomax ME ONeill P An AP site can protect against the muta-genic potential of 8-oxoG when present within a tandem clustered site in E coli DNA repair 20076(12)1839-49

63 Lomax ME Cunniffe S ONeill P 8-OxoG retards the activity of the ligase IIIXRCC1 complex during the repair of a single-strand break when present within a clustered DNA damage site DNA repair 20043(3)289-99

64 Lomax ME Cunniffe S ONeill P Efficiency of repair of an abasic site within DNA clustered damage sites by mammalian cell nuclear extracts Biochemistry 200443(34)11017-26

65 Eot-Houllier G Eon-Marchais S Gasparutto D Sage E Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins Nucleic acids research 200533(1)260-71

66 Kozmin SG Sedletska Y Reynaud-Angelin A Gasparutto D Sage E The for-mation of double-strand breaks at multiply damaged sites is driven by the kinet-ics of excisionincision at base damage in eukaryotic cells Nucleic acids re-search 200937(6)1767-77

67 Georgakilas AG Bennett PV Wilson DM 3rd Sutherland BM Processing of bistranded abasic DNA clusters in gamma-irradiated human hematopoietic cells Nucleic acids research 200432(18)5609-20

68 Yang G Liu C Chen SH Kassab MA Hoff JD Walter NG et al Super-resolu-tion imaging identifies PARP1 and the Ku complex acting as DNA double-strand break sensors Nucleic acids research 201846(7)3446-57

69 Bakkenist CJ Kastan MB DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation Nature 2003421499

70 Uziel T Lerenthal Y Moyal L Andegeko Y Mittelman L Shiloh Y Require-ment of the MRN complex for ATM activation by DNA damage The EMBO journal 200322(20)5612-21

71 Matsuoka S Ballif BA Smogorzewska A McDonald ER 3rd Hurov KE Luo J et al ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage Science 2007316(5828)1160-6

72 Zhou Y Lee JH Jiang W Crowe JL Zha S Paull TT Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM Molecu-lar cell 201765(1)91-104

73 Liu S Shiotani B Lahiri M Mareacutechal A Tse A Leung CCY et al ATR auto-phosphorylation as a molecular switch for checkpoint activation Molecular cell 201143(2)192-202

74 Stiff T Walker SA Cerosaletti K Goodarzi AA Petermann E Concannon P et al ATR‐dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling The EMBO journal 200625(24)5775-82

75 Huen MS Huang J Leung JW Sy SM Leung KM Ching YP et al Regulation of chromatin architecture by the PWWP domain-containing DNA damage-re-sponsive factor EXPAND1MUM1 Molecular cell 201037(6)854-64

76 Fernandez-Capetillo O Chen HT Celeste A Ward I Romanienko PJ Morales JC et al DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1 Nat Cell Biol 20024(12)993-7

77 Bouwman P Aly A Escandell JM Pieterse M Bartkova J van der Gulden H et al 53BP1 loss rescues BRCA1 deficiency and is associated with triple-nega-tive and BRCA-mutated breast cancers Nature Structural ampAmp Molecular Bi-ology 201017688

78 Hsiao KY Mizzen CA Histone H4 deacetylation facilitates 53BP1 DNA dam-age signaling and double-strand break repair Journal of molecular cell biology 20135(3)157-65

79 Fradet-Turcotte A Canny MD Escribano-Diacuteaz C Orthwein A Leung CCY Huang H et al 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark Nature 201349950

80 Burma S Chen BP Murphy M Kurimasa A Chen DJ ATM phosphorylates histone H2AX in response to DNA double-strand breaks The Journal of biolog-ical chemistry 2001276(45)42462-7

81 Stiff T ODriscoll M Rief N Iwabuchi K Lobrich M Jeggo PA ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ioniz-ing radiation Cancer Res 200464(7)2390-6

82 Rogakou EP Boon C Redon C Bonner WM Megabase chromatin domains in-volved in DNA double-strand breaks in vivo The Journal of cell biology 1999146(5)905-16

83 Celeste A Fernandez-Capetillo O Kruhlak MJ Pilch DR Staudt DW Lee A et al Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks Nat Cell Biol 20035(7)675-9

84 Williams RS Moncalian G Williams JS Yamada Y Limbo O Shin DS et al Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair Cell 2008135(1)97-109

85 Rothkamm K Kruumlger I Thompson LH Loumlbrich M Pathways of DNA Double-Strand Break Repair during the Mammalian Cell Cycle Molecular and cellular biology 200323(16)5706-15

86 Bunting SF Callen E Wong N Chen HT Polato F Gunn A et al 53BP1 inhib-its homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks Cell 2010141(2)243-54

87 Walker JR Corpina RA Goldberg J Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair Nature 2001412(6847)607-14

88 Hashimoto M Donald CD Yannone SM Chen DJ Roy R Kow YW A possible role of Ku in mediating sequential repair of closely opposed lesions The Journal of biological chemistry 2001276(16)12827-31

89 Blier PR Griffith AJ Craft J Hardin JA Binding of Ku protein to DNA Meas-urement of affinity for ends and demonstration of binding to nicks The Journal of biological chemistry 1993268(10)7594-601

90 Frit P Li RY Arzel D Salles B Calsou P Ku entry into DNA inhibits inward DNA transactions in vitro The Journal of biological chemistry 2000275(46)35684-91

91 Roberts SA Strande N Burkhalter MD Strom C Havener JM Hasty P et al Ku is a 5-dRPAP lyase that excises nucleotide damage near broken ends Na-ture 2010464(7292)1214-7

92 Li G Nelsen C Hendrickson EA Ku86 is essential in human somatic cells Pro-ceedings of the National Academy of Sciences of the United States of America 200299(2)832-7

93 Hunter T When is a lipid kinase not a lipid kinase When it is a protein kinase Cell 199583(1)1-4

94 Chan DW Ting NSY Lees-Miller SP Mody CH Purification and characteriza-tion of the double-stranded DNA-activated protein kinase DNA-PK from hu-man placenta Biochemistry and Cell Biology 199674(1)67-73

95 Chan DW Ye R Veillette CJ Lees-Miller SP DNA-Dependent Protein Kinase Phosphorylation Sites in Ku 7080 Heterodimer Biochemistry 199938(6)1819-28

96 Yu Y Mahaney BL Yano K-I Ye R Fang S Douglas P et al DNA-PK and ATM phosphorylation sites in XLFCernunnos are not required for repair of DNA double strand breaks DNA repair 20087(10)1680-92

97 Yu Y Wang W Ding Q Ye R Chen D Merkle D et al DNA-PK phosphory-lation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination DNA repair 20032(11)1239-52

98 Wang YG Nnakwe C Lane WS Modesti M Frank KM Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase The Journal of biological chemistry 2004279(36)37282-90

99 Goodarzi AA Yu Y Riballo E Douglas P Walker SA Ye R et al DNA‐PK autophosphorylation facilitates Artemis endonuclease activity The EMBO jour-nal 200625(16)3880-9

100 Chan DW Lees-Miller SP The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit The Journal of biological chemis-try 1996271(15)8936-41

101 Uematsu N Weterings E Yano K-i Morotomi-Yano K Jakob B Taucher-Scholz G et al Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks The Journal of cell biology 2007177(2)219-29

102 Cui X Yu Y Gupta S Cho Y-M Lees-Miller SP Meek K Autophosphorylation of DNA-Dependent Protein Kinase Regulates DNA End Processing and May Also Alter Double-Strand Break Repair Pathway Choice Molecular and cellular biology 200525(24)10842-52

103 Davis AJ Chi L So S Lee KJ Mori E Fattah K et al BRCA1 modulates the autophosphorylation status of DNA-PKcs in S phase of the cell cycle Nucleic acids research 201442(18)11487-501

104 Shang Z-F Huang B Xu Q-Z Zhang S-M Fan R Liu X-D et al Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Ca-tastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage Cancer Research 201070(9)3657-66

105 Douglas P Ye R Trinkle-Mulcahy L Neal JA De Wever V Morrice NA et al Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-de-pendent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mito-sis Bioscience reports 2014

106 Pyun BJ Seo HR Lee HJ Jin YB Kim EJ Kim NH et al Mutual regulation between DNA-PKcs and Snail1 leads to increased genomic instability and ag-gressive tumor characteristics Cell death amp disease 20134e517

107 Farber-Katz Suzette E Dippold Holly C Buschman Matthew D Peterman Mar-shall C Xing M Noakes Christopher J et al DNA Damage Triggers Golgi Dis-persal via DNA-PK and GOLPH3 Cell 2014156(3)413-27

108 Kotula E Berthault N Agrario C Lienafa MC Simon A Dingli F et al DNA-PKcs plays role in cancer metastasis through regulation of secreted proteins in-volved in migration and invasion Cell cycle 201514(12)1961-72

109 Riballo E Kuumlhne M Rief N Doherty A Smith GCM Recio Ma-J et al A Path-way of Double-Strand Break Rejoining Dependent upon ATM Artemis and Proteins Locating to γ-H2AX Foci Molecular cell 200416(5)715-24

110 Ma Y Pannicke U Lu H Niewolik D Schwarz K Lieber MR The DNA-de-pendent Protein Kinase Catalytic Subunit Phosphorylation Sites in Human Ar-temis Journal of Biological Chemistry 2005280(40)33839-46

111 Wang J Aroumougame A Lobrich M Li Y Chen D Chen J et al PTIP asso-ciates with Artemis to dictate DNA repair pathway choice Genes amp develop-ment 201428(24)2693-8

112 Moshous D Callebaut I de Chasseval R Corneo B Cavazzana-Calvo M Le Deist F et al Artemis a Novel DNA Double-Strand Break RepairV(D)J Re-combination Protein Is Mutated in Human Severe Combined Immune Defi-ciency Cell 2001105(2)177-86

113 Noordzij JG Verkaik NS van der Burg M van Veelen LR de Bruin-Versteeg S Wiegant W et al Radiosensitive SCID patients with Artemis gene mutations show a complete B-cell differentiation arrest at the prendashB-cell receptor check-point in bone marrow Blood 2003101(4)1446-52

114 Mahajan KN Nick McElhinny SA Mitchell BS Ramsden DA Association of DNA polymerase mu (pol mu) with Ku and ligase IV role for pol mu in end-joining double-strand break repair Molecular and cellular biology 200222(14)5194-202

115 Fan W Wu X DNA polymerase lambda can elongate on DNA substrates mim-icking non-homologous end joining and interact with XRCC4-ligase IV com-plex Biochem Biophys Res Commun 2004323(4)1328-33

116 Pryor JM Waters CA Aza A Asagoshi K Strom C Mieczkowski PA et al Essential role for polymerase specialization in cellular nonhomologous end join-ing Proceedings of the National Academy of Sciences 2015112(33)E4537-E45

117 Grawunder U Wilm M Wu X Kulesza P Wilson TE Mann M et al Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells Nature 1997388(6641)492-5

118 Brouwer I Sitters G Candelli A Heerema SJ Heller I de Melo AJ et al Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA Nature 2016535(7613)566-9

119 Han L Yu K Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells The Journal of experimental medi-cine 2008205(12)2745-53

120 Prakash R Zhang Y Feng W Jasin M Homologous recombination and human health the roles of BRCA1 BRCA2 and associated proteins Cold Spring Har-bor perspectives in biology 20157(4)a016600

121 Guo C Nakazawa Y Woodbine L Bjoumlrkman A Shimada M Fawcett H et al XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency Journal of Allergy and Clinical Immunology 2015136(4)1007-17

122 Zha S Alt FW Cheng HL Brush JW Li G Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells Proceedings of the National Academy of Sciences of the United States of America 2007104(11)4518-23

123 Mao Z Bozzella M Seluanov A Gorbunova V Comparison of nonhomologous end joining and homologous recombination in human cells DNA repair 20087(10)1765-71

124 Sartori AA Lukas C Coates J Mistrik M Fu S Bartek J et al Human CtIP promotes DNA end resection Nature 2007450(7169)509-14

125 Chen H Lisby M Symington LS RPA coordinates DNA end resection and pre-vents formation of DNA hairpins Molecular cell 201350(4)589-600

126 Jensen RB Carreira A Kowalczykowski SC Purified human BRCA2 stimulates RAD51-mediated recombination Nature 2010467(7316)678-83

127 Li X Heyer W-D RAD54 controls access to the invading 3-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Sac-charomyces cerevisiae Nucleic acids research 200937(2)638-46

128 McIlwraith MJ Vaisman A Liu Y Fanning E Woodgate R West SC Human DNA Polymerase η Promotes DNA Synthesis from Strand Invasion Intermedi-ates of Homologous Recombination Molecular cell 200520(5)783-92

129 Goetz JD-M Motycka TA Han M Jasin M Tomkinson AE Reduced repair of DNA double-strand breaks by homologous recombination in a DNA ligase I-deficient human cell line DNA repair 20054(6)649-54

130 Rothenberg E Grimme JM Spies M Ha T Human Rad52-mediated homology search and annealing occurs by continuous interactions between overlapping nu-cleoprotein complexes Proceedings of the National Academy of Sciences 2008105(51)20274-9

131 Motycka TA Bessho T Post SM Sung P Tomkinson AE Physical and func-tional interaction between the XPFERCC1 endonuclease and hRad52 The Jour-nal of biological chemistry 2004279(14)13634-9

132 Koole W van Schendel R Karambelas AE van Heteren JT Okihara KL Tijster-man M A Polymerase Theta-dependent repair pathway suppresses extensive ge-nomic instability at endogenous G4 DNA sites Nature communications 201453216

133 Wang M Wu W Wu W Rosidi B Zhang L Wang H et al PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways Nucleic acids research 200634(21)6170-82

134 Fattah F Lee EH Weisensel N Wang Y Lichter N Hendrickson EA Ku Reg-ulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells PLoS genetics 20106(2)e1000855

135 Ghezraoui H Piganeau M Renouf B Renaud JB Sallmyr A Ruis B et al Chro-mosomal translocations in human cells are generated by canonical nonhomolo-gous end-joining Molecular cell 201455(6)829-42

136 Torre LA Bray F Siegel RL Ferlay J Lortet-Tieulent J Jemal A Global cancer statistics 2012 CA a cancer journal for clinicians 201565(2)87-108

137 Clapp RW Jacobs MM Loechler EL Environmental and occupational causes of cancer new evidence 2005-2007 Reviews on environmental health 200823(1)1-37

138 Hanahan D Weinberg Robert A Hallmarks of Cancer The Next Generation Cell 2011144(5)646-74

139 Bartkova J Horejsi Z Koed K Kramer A Tort F Zieger K et al DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis Na-ture 2005434(7035)864-70

140 Nowell P The clonal evolution of tumor cell populations Science 1976194(4260)23-8

141 Wilson CR Davidson SE Margison GP Jackson SP Hendry JH West CM Expression of Ku70 correlates with survival in carcinoma of the cervix British journal of cancer 200083(12)1702-6

142 Abdel-Fatah TMA Arora A Moseley P Coveney C Perry C Johnson K et al ATM ATR and DNA-PKcs expressions correlate to adverse clinical outcomes in epithelial ovarian cancers BBA Clinical 2014210-7

143 Grupp K Roettger L Kluth M Hube-Magg C Simon R Lebok P et al Expres-sion of DNA ligase IV is linked to poor prognosis and characterizes a subset of prostate cancers harboring TMPRSS2ERG fusion and PTEN deletion Oncol-ogy reports 201534(3)1211-20

144 Yang S Wang XQ XLF-mediated NHEJ activity in hepatocellular carcinoma therapy resistance BMC cancer 201717(1)344-

145 Lu J Wang XZ Zhang TQ Huang XY Yao JG Wang C et al Prognostic sig-nificance of XRCC4 expression in hepatocellular carcinoma Oncotarget 20178(50)87955-70

146 Dietlein F Reinhardt HC Molecular pathways exploiting tumor-specific mo-lecular defects in DNA repair pathways for precision cancer therapy Clinical cancer research an official journal of the American Association for Cancer Re-search 201420(23)5882-7

147 Wong MC Goggins WB Wang HH Fung FD Leung C Wong SY et al Global Incidence and Mortality for Prostate Cancer Analysis of Temporal Patterns and Trends in 36 Countries European urology 201670(5)862-74

148 Coen JJ Feldman AS Smith MR Zietman AL Watchful waiting for localized prostate cancer in the PSA era what have been the triggers for intervention BJU international 2011107(10)1582-6

149 Holzbeierlein J Lal P LaTulippe E Smith A Satagopan J Zhang L et al Gene expression analysis of human prostate carcinoma during hormonal therapy iden-tifies androgen-responsive genes and mechanisms of therapy resistance The American journal of pathology 2004164(1)217-27

150 Marcelli M Tilley WD Wilson CM Griffin JE Wilson JD McPhaul MJ Def-inition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance premature termination of the recep-tor protein at amino acid residue 588 causes complete androgen resistance Mo-lecular endocrinology (Baltimore Md) 19904(8)1105-16

151 Beltran H Yelensky R Frampton GM Park K Downing SR MacDonald TY et al Targeted next-generation sequencing of advanced prostate cancer identifies potential therapeutic targets and disease heterogeneity European urology 201363(5)920-6

152 Visakorpi T Hyytinen E Koivisto P Tanner M Keinanen R Palmberg C et al In vivo amplification of the androgen receptor gene and progression of human prostate cancer Nature genetics 19959(4)401-6

153 Antonarakis ES Lu C Wang H Luber B Nakazawa M Roeser JC et al AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer The New England journal of medicine 2014371(11)1028-38

154 Antonarakis ES Lu C Luber B Wang H Chen Y Nakazawa M et al Androgen Receptor Splice Variant 7 and Efficacy of Taxane Chemotherapy in Patients With Metastatic Castration-Resistant Prostate Cancer JAMA oncology 20151(5)582-91

155 Petrylak DP Tangen CM Hussain MH Lara PN Jr Jones JA Taplin ME et al Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer The New England journal of medicine 2004351(15)1513-20

156 Tannock IF de Wit R Berry WR Horti J Pluzanska A Chi KN et al Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer The New England journal of medicine 2004351(15)1502-12

157 Fizazi K Carducci M Smith M Damiatildeo R Brown J Karsh L et al Denosumab versus zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer a randomised double-blind study Lancet (London England) 2011377(9768)813-22

158 Steele CB Li J Huang B Weir HK Prostate cancer survival in the United States by race and stage (2001-2009) Findings from the CONCORD-2 study Cancer 2017123 Suppl 245160-77

159 Seyfried TN Huysentruyt LC On the origin of cancer metastasis Critical re-views in oncogenesis 201318(1-2)43-73

160 Siegel R DeSantis C Virgo K Stein K Mariotto A Smith T et al Cancer treat-ment and survivorship statistics 2012 CA a cancer journal for clinicians 201262(4)220-41

161 Mohan R Grosshans D Proton therapy ndash Present and future Advanced Drug Delivery Reviews 201710926-44

162 Poeppel TD Handkiewicz-Junak D Andreeff M Becherer A Bockisch A Fricke E et al EANM guideline for radionuclide therapy with radium-223 of metastatic castration-resistant prostate cancer European journal of nuclear med-icine and molecular imaging 201845(5)824-45

163 Yoshida K Kaneta T Takano S Sugiura M Kawano T Hino A et al Pharma-cokinetics of single dose radium-223 dichloride (BAY 88-8223) in Japanese pa-tients with castration-resistant prostate cancer and bone metastases Annals of nuclear medicine 201630(7)453-60

164 Parker C Nilsson S Heinrich D Helle SI OSullivan JM Fossa SD et al Alpha emitter radium-223 and survival in metastatic prostate cancer The New England journal of medicine 2013369(3)213-23

165 Goodwin JF Kothari V Drake JM Zhao S Dylgjeri E Dean JL et al DNA-PKcs-Mediated Transcriptional Regulation Drives Prostate Cancer Progression and Metastasis Cancer cell 201528(1)97-113

166 He S-S Chen Y Shen X-M Wang H-Z Sun P Dong J et al DNA-dependent protein kinase catalytic subunit functions in metastasis and influences survival in advanced-stage laryngeal squamous cell carcinoma Journal of Cancer 20178(12)2410-6

167 Auckley DH Crowell RE Heaphy ER Stidley CA Lechner JF Gilliland FD et al Reduced DNA-dependent protein kinase activity is associated with lung can-cer Carcinogenesis 200122(5)723-7

168 Cheng Y Li F Mladenov E Iliakis G The yield of DNA double strand breaks determined after exclusion of those forming from heat-labile lesions predicts tu-mor cell radiosensitivity to killing Radiotherapy and oncology journal of the European Society for Therapeutic Radiology and Oncology 2015116(3)366-73

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1548

Editor The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine UppsalaUniversity is usually a summary of a number of papers A fewcopies of the complete dissertation are kept at major Swedishresearch libraries while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine (Prior to January 2005 the series was publishedunder the title ldquoComprehensive Summaries of UppsalaDissertations from the Faculty of Medicinerdquo)

Distribution publicationsuuseurnnbnseuudiva-378721