Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer (2024)

Ionizing radiation effects in the cell

Ionizing radiation is a type of high-energy radiation that is able to releaseelectrons from atoms and molecules generating ions which can break covalent bonds.Ionizing radiation directly affects DNA structure by inducing DNA breaks,particularly, DSBs. Secondary effects are the generation of reactive oxygen species(ROS) that oxidize proteins and lipids, and also induce several damages to DNA, likegeneration of abasic sites and single strand breaks (SSB). Collectively, all thesechanges induce cell death and mitotic failure.

Ionizing radiation can be divided into X-rays, gamma rays, alpha and beta particlesand neutrons. Quiescent and slowly dividing cells are less radiosensitive, like thoseconstituting the nervous system, while cells with high proliferation rates are moreradiosensitive, like bone marrow, skin, and epithelial cells of the gastro-intestinaltract, among others. The radiation dose is measured in units gray (Gy), a measure ofthe amount of radiation absorbed by 1 kg of tissue (Dunne-Daly, 1999).

Radiotherapy

Radiotherapy is a treatment aimed at shrinking the tumor mass or at eliminatingresidual tumor cells by exposing the tumor to ionizing radiation. Radiotherapyregimes mostly use X- and gamma radiation (Masuda andKamiya, 2012). Radiation affects tumor and healthy irradiated cellsindistinctly. Radiotherapy is used as the standard treatment for breast cancer aftermastectomy; but this therapy may be also used prophylactically or palliatively toreduce the risk of tumor recurrence or to relieve symptoms caused by tumor growth andassociated metastases, respectively (Delaneyet al., 2005). Radiation therapy can be delivered byexternal-beam radiation or internal radiation. External-beam radiation therapy iscreated electronically by a linear accelerator which produces photon beams known asX-rays, with electric potentials in the range of 4 to 20 mega Volts. Patients receiveradiation doses in daily sessions for several weeks, and the radiation dose may beadministered in three different schemes: accelerated fractionation,hyperfractionation and hypofractionation. Accelerated fractionation means a radiationscheme in which the total dose of radiation is divided into small doses, and thetreatments are given more than once per day. The total dose of radiation isadministered in a shorter period of time (fewer days) compared to standard radiationtherapy (weeks). A reduction in the treatment time may reduce the repopulation oftumor cells, resulting in a better locoregional control. In hyperfractionedtreatment, the total radiation dose is divided into smaller doses, and it isadministered more than once a day; but in the same period as standard radiotherapy(days or weeks). Dose reduction may reduce the toxicity risk, although the total doseis increased. Hypofractionated radiation treatment is given once a day or less often.The total dose is divided into larger doses and is administered over a shorter periodthan standard radiotherapy. This scheme reduces patient visits and cost, and fewerside effects are noticed when compared to conventional radiation therapy.

The internal radiation therapy, also called brachytherapy, is released fromgamma-radiation sources such as radioactive isotopes like ⁶⁰Co and¹³⁷Cs, which are placed within the patient's body. This type ofradiation can deliver high doses of focalized radiation with an electric potential inthe range of 0.6 to 1 megaVolt and causes less damage to normal tissues (Patel and Arthur, 2006).

DNA repair after ionizing radiation

Ionizing radiation causes DSBs directly, but in addition base damages due to indirecteffects are also induced. This radiation causes formation of ROS (reactive oxygenspecies) which are indirectly involved in DNA damage. These ROS generates apurinic /apyrimidinic (abasic) sites in the DNA, SSBs, sugar moiety modifications, anddeaminated adducted bases (Redon etal., 2010; Aparicio etal., 2014). When DNA is damaged, the repair machinery of thecell is activated and stops the cell cycle at specific control checkpoints to repairDNA damage and prevent continuation of the cycle. It is known that the intrinsicradiosensitivity of tumor cells is strongly influenced by the cells DSB repaircapability (Mladenov et al.,2013). If tumor cells are able to efficiently repair the radiation damage,resistance to radiation develops, enabling cells to survive and replicate. If thedamage remains unrepaired, these mechanisms induce programmed cell death or apoptosisto prevent accumulation of mutations in daughter cells (Deckbar et al., 2011; Guo et al., 2011).

As mentioned, ionizing radiation inevitably reaches normal tissue, inducing bystandereffects in tumor-adjacent normal cells that may contribute to chromosomal aberrationsand to increase the risk for new malignancies. High doses of radiation may producetoxicity and reduce the patient's prognosis (Brownet al., 2015). Individual radiation treatment based onDSB repair capability could predict toxicity to surrounding tissues, therebyimproving treatment safety. DSB repair capability depends not just on gene integrity,but also on gene expression. In addition to germinal mutations affecting genes likeBRCA 1 and 2 or other related genes, genetic and epigeneticmechanisms may reduce or abrogate the expression of genes involved in DSB repair(Bosviel, et al., 2012).The DNA repair capability could be relevant to decide on the appropriate treatmentfor cancer patients, and functional tests may provide valuable information for theseclinical decisions.

DSB repair pathways

DSB repair is achieved in three ways: non-hom*ologous end joining (NHEJ), conservativehom*ologous recombination (HR) and single-strand alignment, also callednon-conservative hom*ologous recombination (SSA) (Langerak and Russell, 2011). HR is considered an error-free mechanismbecause it uses an undamaged DNA guide strand to repair the DSB, and the original DNAis reconstituted without loss of genetic information, but this mechanism proceedsslowly and is only exerted at the S/G2 phases of the cell cycle. NHEJ and SSA areconsidered error-prone and mutagenic mechanisms because the processing of DNA endsmay incur in loss or modification of genetic information at the repaired DSB ends.NHEJ is the most common mechanism of DSB repair in eukaryotic cells. In thismechanism, the DNA strands at the DSB are cut or modified, and the ends are ligatedtogether regardless of hom*ology, generating deletions or insertions. Although thisprocess is error-prone, this mechanism can fix the DNA damage quickly, because it isnot restricted to a single cell cycle phase, thus preventing increased geneticinstability (Do et al.,2014). These mechanisms are detailed below and in the Figure 1. The main proteins involved in the early steps of DSBdetection, chromatin remodeling and DNA repair are listed in Table 1.

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Figure 1

DSB repair pathways. In NHEJ, the KU70/KU80 heterodimer binds to the DSB,protects it from degradation by exonucleases, and acts as a repressor of HR.The KU70/80 heterodimer recruits and activates the DNA-PKcs and KU70 interactswith XRCC4. Then, the DNA ligase IV interacts with the KU heterodimer to ligatethe DNA ends. If required for ligation, PNKP binds to phosphorylated XRCC4 toprocess the DNA ends. In the HR pathway the MRN complex is recruited at the DSBends and CtIP binds to the MRN complex activating an exonuclease activity whichcreates single strand segments at the borders of the DSB that are extended bythe EXO1 3′- 5′ exonuclease. Then, hSSB1 binds to free ends and RPA (anheterometic complex formed by RPA70, RPA32 and RPA14) protects againstdegradation. RPA is replaced by RAD51-BRCA2. RAD51 nucleoprotein searches forand invades the hom*ologues sequences, from sister chromatid, to form a Hollidayjunction. The sister chromatids are joined by cohesin proteins to facilitatethe interconnection of the DSB to the hom*ologous recombination. Subsequently,RAD51 is removed leaving a free 3′-OH and DNA is synthesized by the DNApolymerase δ using the hom*ologous chromatid as a template. Resolvase enzymessolve the Holliday junction and the DNA ends are joined by DNA ligase I. TheSSA pathway is not conservative and depends on the presence of repeatedsequences flanking the DSB. In this mechanism, the MRN complex joined to CtIPcleaves the 5′-end of one strand of DNA to expose microhom*ology sequences.hom*ologous sequences are aligned, while nonaligned regions are removed by theERCC1/XPF nucleases. Then, DNA ends are joined by DNA ligase III.

Radiosensitivity in Breast Cancer Patients

Radiosensitivity is the susceptibility of the cells or tissues to ionizing radiation.Some patients may be more sensitive to radiation. Sensitivity results from the toxiceffects of radiotherapy resulting in lesions of the patient's normal tissues. Theseeffects may be acute or late, depending on the time of their manifestation. Acuteeffects occur during the treatment or shortly after and they are usually reversibleand occur in rapidly proliferating tissues, like skin, gastrointestinal tract andhematopoietic tissues. Late effects manifest six months or later after the treatment.Late effects can be permanent, mainly affecting slowly proliferating tissues such askidneys, heart, and the nervous system, and may involve systemic deregulations of theendocrine system (Barnett et al.,2009). Radiation promotes DSB as mentioned above, and this damage isdetrimental for genome integrity (Chistiakovet al., 2008; Rübeet al., 2008; Henríquez-Hernández et al., 2011).

Mechanisms of hypersensitivity to ionizing radiation are still unclear, but isestimated that 70% of hypersensitivity cases are due to genetic variants (Turesson et al., 1996). Asmentioned above, mutations in the ATM gene are associated withextreme hypersensitivity to ionizing radiation (Masuda and Kamiya, 2012), and polymorphisms in genes likeXRCC3 and RAD51 increase the risk ofradiosensitivity (Vral et al.,2011). These genes are also implicated in breast cancer. Mayer et al. (2011) analyzedgene expression in peripheral blood lymphocytes of breast and cervical cancerpatients. They identified 153 genes altered by ionizing radiation. These genes areinvolved in cell cycle control and apoptosis in response to radiation. Of these, 67genes were useful to discriminate between normal reacting patients and subjects withsevere radiosensitivity. However, the analyses were performed on lymphocytes, and theauthors comment that an analysis of expression in different tissues would be requiredto define a more precise gene signature (Mayeret al., 2011).

The 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) base damage is produced byionizing radiation and is repaired by nucleotide excision followed by removal of thisabnormal deoxynucleoside out of the cell (Evanset al., 2010). 8-oxo-dG has been used as a urinarymarker of oxidative stress and has been associated with lung cancer (Il'yasova et al., 2012) andgastrointestinal diseases (Ock etal., 2012). It has also been proposed as a marker forradiosensitivity (Erhola et al.,1997, Roszkowski and Olinski, 2012).Haghdoost et al. (2001)studied 8-oxo-dG urinary levels in breast cancer patients before and after adjuvantradiotherapy (4 to 6 Gy). Radiosensitive patients showed skin redness in the radiatedareas and significantly increased urinary levels of 8-oxo-dG, and these authorsproposed the use of this deoxynucleoside as a urinary biomarker for radiosensitivity.This biomarker facilitates the study of individual radiosensitivity, since theabnormal metabolite maybe measured by ELISA (Haghdoost et al., 2001). In a study by Skiöld et al. (2013),radiation-induced oxidative stress response was analyzed by the 8-oxo-dG biomarker inserum from ex-vivo irradiated leukocytes samples obtained frombreast cancer patients that developed severe acute skin reactions (RTOG [RadiotherapyOncology Group Criteria] grade 3-4) during radiotherapy and from patients with breastcancer showing no early skin reactions after radiotherapy (RTOG grade 0). The authorsdemonstrated that patients with RTGO grade 0 showed increased extracellular serumlevels of 8-oxo-dG, in contrast with the significantly low serum levels observed inpatients with RTOG grades 3 and 4, indicating that 8-oxo-dG is a useful biomarker toanalyze cellular responses to ionizing radiation (Skiöld et al., 2013). Nonetheless, 8-oxo-dG can alsoresult from cell exposure to oxidative stress by ROS, as may occur when tissues areexposed to environmental pollutants (Hecht,1999). For these reasons this biomarker is not specific for ionizingradiation but, as in the case of the studies by Skiöld et al. (2013), it is helpful as a comparativeex vivo test of irradiated cells to define the biological effectsof ionizing radiation. Extracellular levels of 8-oxo-dG are appropriate indicators ofthe cells capability to repair the DNA damage caused by ROS.

Certain phenotypes of breast cancer have been associated with locoregional recurrence(LRR). Brollo et al. (2013)suggested that HER2+ tumors are more susceptible to ionizing radiation, while Voduc et al. (2010) observedthat LRR seemed higher in patients with triple negative marker breast cancer,although the number of LRR events was small. At present, there are no molecularmethods to discriminate between patients with high and low LRR (Britten et al., 2013). In addition, there is notenough information regarding the possible adverse effects of radiotherapy that mayinduce genomic and epigenetic modifications and changes in gene-expression profilesin breast cancer.

Henríquez-Hernández et al.(2011) analyzed isolated peripheral blood lymphocytes (PBLs) from patientswith advanced breast cancer treated ex vivo with high radiotherapydoses to study ionizing radiation resistance. They showed that lymphocytes frompatients with low DNA damage and high apoptosis rates had low risks of radiationadverse events.

Studies analyzing the type of repair that occurs when cells are exposed to radiationand the correlation with abnormal expression of certain genes involved in DSB repairhave also been conducted. In vitro studies of Bca11 (familial breastcancer cell line) and Bca10 (sporadic breast cancer cell line) cell lines showed highNHEJ repair activity and direct HR non-conservative repair in the Bca11 cell line.The Bca10 cell line also showed an increase in non-conservative repair of direct HR,but to a lesser degree than Bca11. Consequently, repair mechanisms in these celllines may cause deletions in the DNA sequence and cell cycle deregulation (Keimling et al., 2008). Theseauthors performed a study in PBLs from patients with sporadic breast cancer, healthywomen with familial risk of breast cancer, and healthy controls, and theydemonstrated increased NHEJ and SSA in both, cancer patients and subjects athereditary risk, vs. the healthy controls. This study suggested thatthese two groups are prone to extended non-conservative DSB repairing mechanisms.Based on these results, Keimling etal. (2012) implemented a test to analyze DSB repair invitro.

Techniques for DSB Repair Analysis

Some tests have been devised to assess DNA damage in response to diverse substances,microorganisms, or environmental conditions. Some of these tests are describedbelow.

Comet assay

The alkaline comet assay involves measurement of DNA damage in SSB and DSB. Thismethod is fast and cheap. It provides important information about the risk ofdiseases related to oxidative stress (Alapetiteet al., 1999; Dusinskaand Collins, 2008). In this assay, cells are embedded in a thin layer ofa*garose on a thin glass slide, cells are lysed in a solution containing detergent andNaCl, releasing the DNA from the proteins bound to it, but leaving DNA fragmentsstill attached to the nuclear membrane. Then, the plate is incubated in an alkalinesolution, an electrophoresis is run and DNA is stained with ethidium bromide. DNAfragments travel to the anode forming a comet-like image when viewed by fluorescencemicroscopy (Fikrová et al.,2011, Baumgartner et al.,2012). The image of the comet head denotes the DNA content and the tail thefrequency of DNA breaks (Figure 2B). Softwareprograms designed to analyze the comet image allow measurement of DNA content andtail length. The length of the comet tail correlates with the level of DNAdamage.

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Figure 2

General assays for detecting DNA damage (A)Immunohistochemistry with antibodies directed against γ-H2AX: peripheral bloodmononuclear cells are isolated, nuclei are stained with DAPI and withantibodies directed at γ-stained H2AX and visualized under fluorescentmicroscopy. (B) Comet assay: the comet assay is also performed onmononuclear cells. The cells are embedded in agarose on a thin glass slide,cells are lysed and incubated in an alkaline solution. Subsequently, DNAfragments are separated by electrophoresis and stained with ethidium bromide.The comet-like image is viewed under a fluorescence microscope. The length ofthe comet tail indicates the frequency of DNA breaks

Hair et al. (2010) used amodified comet assay method in which slides with cells embedded in agarose wereincubated with three different treatments: 1) alkaline electrophoresis to detect SSBinduced radiation and alkaline-labile sites; 2) electrophoresis of cells treated withformamidopyrimidine [Fapy] -DNA glycosylase (Fpg); this releases the damaged purines,leaving apurinic sites (AP sites) that are subsequently cleaved with the cellular APlyase, producing single strand fragments which can be visualized in the comet assay,and 3) electrophoresis after treatment of the cells with bacterial endonucleaseEndoIII, which cleaves the damage strands at sites presentingoxidized pyrimidines, thus increasing the sensitivity of the comet assay by leavinggaps in mutated bases (Hair et al.,2010).

Some disadvantages of the comet assay are the variability between different protocolsand between laboratories, which makes it difficult to define ionizing radiationtoxicities, so this issue will require adoption of standardized and comparableprotocols (Forchhammer et al.,2010; Henríquez-Hernández etal., 2012; Azqueta etal., 2014). Sirota etal. (2014) studied inter-laboratory variation of comet assayfactors, like slide brands, duration of alkali treatment and electrophoresisconditions, and they found that laboratory differences were associated withelectrophoresis conditions, especially the temperature during alkalineelectrophoresis, which affects the rate of conversion of alkali labile sites tosingle stranded breaks (Sirota etal., 2014). Additionally, it has been suggested thatimplementation of a standard software will be required for comet assay interpretation(Fikrová et al.,2011).

γ-H2AX

The histone H2AX variant of the histone H2A is present in subsets of nucleosomes (2to 25% of the total H2A) and has been implicated in DSB repair. When H2AX isphosphorylated at the serine residue 139 by phosphoinositide-3-kinase-related proteinkinases (PIKKs), the phosphate group adopts a γ position in the protein, constitutingthe gamma H2AX (γ-H2AX) configuration (Rogakouet al., 1998; Rothkammand Horn, 2009). This phosphoprotein acts in early events of DNA repair bydecondensing the chromatin near the DSB (Kruhlaket al., 2006). Additionally, γ H2AX joins to the DSBends forming a “γH2AX focus” which is extended for several Mb at the sides of theDSB. A method used for the analysis of DNA damage is the measurement of γ-H2AX usingantibodies against

In the γ-H2AX assays, peripheral blood is collected and mononuclear cells areseparated and fixed on a glass surface. Then, an immunohistochemistry withanti-γ-H2AX antibody is performed and the results are analyzed by fluorescencemicroscopy in which fluorescent foci are measured (Figure 2A). This test may be also analyzed by flow cytometry or by westernblot (Kinner et al., 2008;Dickey et al., 2009; Podhorecka et al., 2010).

γ-H2AX foci measurements in patients before and after radiotherapies using low andhigh doses of ionizing radiation have shown a linear relationship between DNA damageand exposure to radiation. The initial number of γ-H2AX foci is consistent with DSBsin the cells. After a while, the γ-H2AX foci disappear due to the DNA repair (Rübe et al., 2008; Horn et al., 2011). This methodis sensitive for measuring DNA repair in patients undergoing radiotherapy, but it isalso applied in other fields, such as DNA damage analysis due to occupationalexposure or contact with environmental pollutants, cigarette smoke, drugs, etc‥ It isimportant to note that these co-exposures may affect the results in radiotherapypatients and, hence, should be considered on an individual basis. Furthermore,phosphorylation of H2AX is observed in the absence of DSB in the replication process,in mitosis and during DNA fragmentation in apoptosis. Therefore, the test must beable to distinguish between apoptotic and non-apoptotic cells (Dickey et al., 2009).

Comet assay and γ-H2AX methods described above help to assess DNA damage and repair,but do not allow discrimination of the type of damage, like SSB or DSB. It is alsoimportant to analyze whether the damage is repaired and what kind of repair mechanismis operating to assess whether cells are sensitive or resistant to ionizingradiation.

Engineered proteins to detect spontaneous DSB

Shee et al. (2013) developeda new synthetic technology to quantify DSBs in bacterial and mammalian cells. Thismethod use the green fluorescent-protein (GFP) fused to the GAM protein (GAM-GFP), aviral protein from bacteriophage Mu, which shares sequence hom*ology with theeukaryotic proteins KU80 and KU70 involved in NHEJ (Aparicio et al., 2014). Unlike the KU protein, the GAMprotein is not involved in DNA repair reactions. GAM binds to DNA and inhibits avariety of exonucleases involved in DNA repair (Abraham and Symonds, 1990; fa*gagnaet al., 2003; Sheeet al., 2013). This advance allows the study andquantification of DNA breaks. In this method, the I-SceIendonuclease is used to make site specific DSBs and cells are transfected with a MuGAM-GFP fusion expression vector. The GAM-GFP protein joins the DSBs formed by theI-SceI treatment, generating fluorescence at the damaged siteswhich can be analyzed by fluorescence microscopy. Since the GAM-GFP protein competeswith KU proteins, this results in low levels of DNA damage, thus limiting thistechnology to the study of DSB repair by HR (Sheeet al., 2013).

Identification of repair mechanisms by specific DNA substrates

As mentioned above, Keimling et al.(2012) developed an in vitro method in which PBLs aretransfected with marker plasmids for enabling discrimination of the mechanismsinvolved in DSB repair: HR, NHEJ, and SSA (Figure3A). In this procedure, PBLs are transduced in three different experimentswith separate plasmids, each containing the EGFP reporter gene followedby differentsequences amenable to undergo one of the different mechanisms of DNA repair definedabove. Cells in the three groups are co-transduced with a plasmid codifying forI-SceI as the inductor of DSB repair events. Fluorescencedetection after 24 h by flow cytometry in any of the three transduced cells of thepanel measures the events of each individual operating mechanism, allowing moredetailed information about DSB repair in individual patients (Figure 3B). This test is amenable for high-throughput sampleprocessing and analysis (Boehden etal., 2002; Keimling etal., 2012).

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Figure 3

Specific assays for detecting DNA damage (A) The EJ-EGFPplasmids contains a mutated version of the EGFP gene (green light bar) createdby inserting a restriction site for the meganuclease I-SceIflanked by a 5 bp microhom*ology sites (black arrows); this plasmid was designedto be repaired by NHEJ. The Δ-EGFP/3’EGFP and Δ-EGFP/5’EGFP plasmids contain anarray of an EGFP mutated gene containing an I-SceI site (greenlight bar) followed by a spacer (purple bar) and EGFP gene versions truncatedat their flanking 3’ and 5’ ends, respectively (dark green bars) which allowthe reconstitution of the wild-type version of the marker geneby SSA and HR, respectively. (B) Analysis of DSB repair: The assayis performed in three cultures of peripheral blood lymphocytes (PBLs),transduced separately with each of the plasmid versions designed fordiscrimination of SSA, NHEJ and HR. The cultures are co-transduced with anadditional plasmid expressing the I-SceI enzyme. Aftergenerating DBS in the target plasmids by the expressed restriction enzyme, DNArepair in PBLs repair by each of the different DNA repair pathway may bemonitored by restoration of the wild-type version of EGFP 24 h aftertransduction by measuring EGFP florescence by flow cytometry.

Associate Editor: Carlos F. M. Menck

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