Repair of oxidatively-induced clustered DNA lesions, after exposure of human cancer cells to ionizing radiation

Abstract

In the present thesis, the induction and repair of complex DNA lesions, double strand breaks (DSBs) of high complexity accompanied by oxidatively-induced base lesions in their close vicinity were studied after exposure to ionizing radiation (IR) of varying quality i.e. linear energy transfer (LET). These lesions are considered the hallmark of ionizing radiation as well as of the high levels of oxidative stress. Complex DNA damage may be consisted by DSBs, single strand breaks (SSBs) and base lesions within a very short DNA fragment up to 10-15 base pairs. Clustered DNA damage challenges the cell DNA repair machinery and therefore the eventual misrepair could lead to mutations, genomic instability or even carcinogenesis. The in situ immunofluorescence-based detection of this kind of damage is generally accepted as a very challenging goal and can be performed currently only at the chromosomal level and within DNA regions much larger than the above mentioned above. The main experimental method followed in this thesis, it is the in situ indirect immunofluorescence, using specific antibodies against several human DNA repair proteins i.e. for DSBs (γ-Η2ΑΧ), for oxidative base lesions (OGG1) and for abasic DNA sites (hAPE1). Moreover complementary meta-analysis of experimental results was performed, achieving the systematical review of underlying mechanisms. The scientific interest for DNA damage as well as for the consequential cellular response can be attributed to the dual action of ionizing radiation. On one hand, coincidental exposure to ionizing radiation could disrupt DNA integrity, causing mutations, sometimes beneficial, promoting evolution or detrimental causing genomic instability and sometimes cancer. On the other hand, one of the most important therapies against cancer, radiotherapy utilizes ionizing radiation to target the DNA of tumor cells and thus to induce programmed cell death (apoptosis). In the framework of the present thesis, several human cancer cell lines originating from breast cancer (MCF7), hepatocellular carcinoma (HepG2), lung carcinoma (A549), glioblastoma (MO59K and their isogenic MO59J) but also normal immortalized keratinocytes (FEP18-11-T1), as well as lymphocytes from healthy donors were studied. Different types of ionizing radiation and LET, electromagnetic (Low LET: γ- and X- rays) and charged particles (high-LET: a- particles and Argon ions) were used. Increasing complexity of the induced damage was detected with the increase of LET of the incident radiation. Regarding the repair of this damage, a significant delay in the process of complex lesions was also observed. Chemical inhibition of the DNA repair protein DNA-PK was induced in cell cultures, using two different and highly specific inhibitors, namely the NU7026 and the NU7441. NU7441 was found to induce stronger inhibition in double strand break repair process than NU7026. DNA-PK was also tested by using a pair of cell lines (MO59K and MO59J), MO59J are DNA-PKcs deficient. DNA repair was delayed but finally was achieved. In order to detect complex DNA lesions that also include oxidative base lesions in the vicinity of a DSB, we defined the proper protocol which allows their visualization as colocalized fluorescence signal coming from fluorescent antibodies that target the DSB repair proteins (γ-Η2ΑΧ or 53BP1) and the base lesion repair proteins (OGG1 or APE1).In order to evaluate the colocalization levels based on “fluorescent” images, as a result of the present work, we introduced a criterion-Parameter called Pclc (colocalization parameter). Our criterion conserves the focus topology for the DSBs, which are considered the ‘centers’ of repair, and regarding the oxidative base lesions, it uses their mean luminosity. Pclc compares the surface (or spatial in the case of 3D images) luminosity of the signal coming from the labeled antibody that targets the base repair enzymes, considered over the DSB foci area with the same value considered over the rest of the cell nucleus area. Using our suggested parameter Pclc, normalization of any fluctuation among images has been achieved, making possible the comparison among images resulting by different experiments. The main advantage of this consideration is that it allows unbiased the decision whether or not the occurring colocalization is random or of biological importance. Significant Pclc increases were detected with increasing LET and this trend was verified with some minor variations for all radiations used. In addition, specific correlations were made with repair time and focus size synonymous with damage complexity. Approaching the DNA repair of complex DNA lesions on organism level (systems biology), data analysis coming from universal data bases was performed, utilizing bioinformatics. For this purpose several open source tools were utilized, and multilevel systematic proofing of the results was performed. Proper gene nomenclatures were case –specifically used. Gene ontology terms hold the key role in the gene screening that was performed, making the results reliable. This analysis has been based exclusively on experimentally evaluated data. Specific correlations were done between DNA damage response (DDR) and repair genes with the immune system and inflammatory response especially in the case of radiation-induced systemic effects which consisted a significant part of our bioinformatical work. The idea of radiation non-targeted effects holds currently a prominent position in radiation biology and our main hypothesis has been that complex DNA lesions and their repair can be regarded as the first ‘danger’ signal due to the difficulty the cell encounters in processing and amending these genome problems. Bioinformatics analysis revealed numerous (human and plant) genes that we believe that are also participating in DNA repair machinery, following the exposure to IR and especially to the repair of clustered DNA lesions. Several other genes where identified and are proposed as potential biomarkers of exposure to endogenous (replication, oxidative) and to exogenous stress (ionizing radiation), being also applicable to biological dosimetry. Regarding cancer therapy, the role of DNA repair genes was explored and several genes were suggested as potential targets for chemical/pharmaceutical inhibition in order to enhance radiotherapy in the frame of Synthetic Lethality, moreover the idea of Multi-pathway Lethality was also proposed. Summarizing, the results of this study are centered on the biophysical mechanisms induction and repair of complex DNA lesions. They led to the development of protocols for the detection of these lesions on the cellular level, by using in situ immunofluorescence. The scientific importance of these findings relies primarily on two aspects: 1. the technical improvements and advancement of the field of in situ detection of complex damage as well as 2. the biophysical/biological importance of the findings on the processing of complex DNA damage consisting of a DSB and neighboring oxidative lesions challenging the repair machinery of the cell. More importantly, the biophysical experimental and complementary bioinformatics work has been published in the following papers: [1]Z. Nikitaki, V. Nikolov, I. V. Mavragani, I. Plante, D. Emfietzoglou, G. Iliakis, and Alexandros G. Georgakilas, "Non-DSB clustered DNA lesions. Does theory colocalize with the experiment?," Radiation Physics and Chemistry, 2016. 128: p. 26-35.AbstractIonizing radiation results in various kinds of DNA lesions such as double strand breaks (DSBs) and other non-DSB base lesions. These lesions may be formed in close proximity (ie, within a few nanometers) resulting in clustered types of DNA lesions. These damage clusters are considered the signature of ionizing radiation, notably charged particles of high linear energy transfer (LET). Accumulating theoretical and experimental evidence suggest the induction of these clustered lesions appears under various irradiation conditions but also as a result of high levels of oxidative stress. The biological significance of these clustered DNA lesions pertains to the inability of cells to process them efficiently as isolated lesions. The results in the case of unsuccessful or erroneous repair can vary from mutations up to chromosomal instability. In this mini review, we discuss of several Monte Carlo simulations codes and experimental evidence regarding the induction and repair of radiation-induced non-DSB complex DNA lesions. We also critically discuss the most widely used methodologies (ie, gel electrophoresis and fluorescence microscopy [in situ colocalization assays]). Based on the comparison of different approaches, we present examples and suggestions for the improved detection of these lesions in situ. Based on the current status of knowledge, we conclude that there is a great need for improvement of the detection techniques at the cellular or tissue level, which will provide valuable information for understanding the mechanisms used by the cell to process clustered DNA lesions.[2]Z. Nikitaki, V. Nikolov, I. V. Mavragani, E. Mladenov, A. Mangelis, D. A. Laskaratou, Georgios I. Fragkoulis, Christine E. Hellweg, Olga A. Martin, Dimitris Emfietzoglou, Vasiliki I. Hatzi, Georgia I. Terzoudi, George Iliakis and Alexandros G. Georgakilas, "Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)," Free Radic Res, pp. 1-45, Sep 5 2016.AbstractDetrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of double strand breaks (DSBs), single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase of the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing linear energy transfer (LET) i.e., γ-, X-rays 0.3-1 keV/μm, α-particles 116 keV/μm and 36Ar ions 270 keV/μm. Using ɣ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5-16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1) i.e. damage foci as probes for oxidized purines or abasic sites respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also made correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage. [3]Z. Nikitaki, I. V. Mavragani, D. A. Laskaratou, V. Gika, V. P. Moskvin, K. Theofilatos Konstantinos Vougas, Robert D. Stewart and Alexandros G. Georgakilas., "Systemic mechanisms and effects of ionizing radiation: A new 'old' paradigm of how the bystanders and distant can become the players," Semin Cancer Biol, vol. 37-38, pp. 77-95, Jun 2016.AbstractExposure of cells to any form of ionizing radiation (IR) is expected to induce a variety of DNA lesions, including double strand breaks (DSBs), single strand breaks (SSBs) and oxidation, as well as loss of bases, i.e. abasic sites. The damaging potential of IR is primarily related to the generation of electrons, which through their interaction with water produce free radicals. In their turn, free radicals attack DNA, proteins and lipids. Damage is induced also through direct deposition of energy. These types of IR interactions with biological materials are collectively called ‘targeted effects’, since they refer only to the irradiated cells. Earlier and sometimes ‘anecdotal’ findings were pointing to the possibility of IR actions unrelated to the irradiated cells or area, i.e. a type of systemic response with unknown mechanistic basis. Over the last years, significant experimental evidence has accumulated, showing a variety of radiation effects for ‘out-of-field’ areas (non-targeted effects-NTE). The NTE involve the release of chemical and biological mediators from the ‘in-field’ area and thus the communication of the radiation insult via the so called ‘danger’ signals. The NTE can be separated in two major groups: bystander and distant (systemic). In this review, we have collected a detailed list of proteins implicated in either bystander or systemic effects, including the clinically relevant abscopal phenomenon, using improved text-mining and bioinformatics tools from the literature. We have identified which of these genes belong to the DNA damage response and repair pathway (DDR/R) and made protein-protein interaction (PPi) networks. Our analysis supports that the apoptosis, TLR-like or NOD-like receptor signaling pathways are the main pathways participating in NTE. Based on this analysis, we formulate a biophysical hypothesis for the regulation of NTE, based on DNA damage and apoptosis gradients between the irradiation point and various distances corresponding to bystander (5 mm) or distant effects (5 cm). Last but not least, in order to provide a more realistic support for our model, we calculate the expected DSB and non-DSB clusters along the central axis of a representative 200.6 MeV pencil beam calculated using Monte Carlo DNA damage simulation software (MCDS) based on the actual beam energy-to-depth curves used in therapy.  [4]A. G. Georgakilas, A. Pavlopoulou, M. Louka, Z. Nikitaki, C. E. Vorgias, P. G. Bagos, Ioannis Michalopoulos,"Emerging molecular networks common in ionizing radiation, immune and inflammatory responses by employing bioinformatics approaches," Cancer Lett, vol. 368, pp. 164-72, Nov 28 2015.AbstractEfficient radiation therapy is characterized by enhanced tumor cell killing involving the activation of the immune system (tumor immunogenicity) but at the same time minimizing chronic inflammation and radiation adverse effects in healthy tissue. The aim of this study was to identify gene products involved in immune and inflammatory responses upon exposure to ionizing radiation by using various bioinformatic tools. Ionizing radiation is known to elicit different effects at the level of cells and organism i.e. DNA Damage Response (DDR), DNA repair, apoptosis and, most importantly, systemic effects through the instigation of inflammatory ‘danger’ signals and innate immune response activation. Genes implicated both in radiation and immune/inflammatory responses were collected manually from the scientific literature with a combination of relevant keywords. The experimentally validated and literature-based results were inspected, and genes involved in radiation, immune and inflammatory response were pooled. This kind of analysis was performed for the first time, for both healthy and tumor tissues. In this way, a set of 24 genes common in all three different phenomena was identified. These genes were found to form a highly connected network. Useful conclusions are drawn regarding the potential application of these genes as markers of response to radiation for both healthy and tumor tissues through the modulation of immune and/or inflammatory mechanisms.[5]N. Hekim, Z. Cetin, Z. Nikitaki, A. Cort, and E. I. Saygili, "Radiation triggering immune response and inflammation," Cancer Lett, vol. 368, pp. 156-63, Nov 28 2015.AbstractRadiation therapy (RT) is a well-established but still under optimization branch of Cancer Therapy (CT). RT uses electromagnetic waves or charged particles in order to kill malignant cells, by accumulating the energy onto these cells. The issue at stake for RT, as well as for any other Cancer Therapy technique, is always to kill only cancer cells, without affecting the surrounding healthy ones. This perspective of CT is usually described under the terms “specificity” and “selectivity”. Specificity and selectivity are the ideal goal, but the ideal is never entirely achieved. Thus, in addition to killing healthy cells, changes and effects are observed in the immune system after irradiation. In this review, we mainly focus on the effects of ionizing radiation on the immune system and its components like bone marrow. Additionally, we are interested in the effects and benefits of low-dose ionizing radiation on the hematopoiesis and immune response. Low dose radiation has been shown to induce biological responses like inflammatory responses, innate immune system activation and DNA repair (adaptive response). This review reveals the fact that there are many unanswered questions regarding the role of radiation as either an immuneactivating (low dose) or immunosuppressive (high dose) agent.[6]Z. Nikitaki, I. Michalopoulos, and A. G. Georgakilas, "Molecular inhibitors of DNA repair: searching for the ultimate tumor killing weapon," Future Med Chem, vol. 7, pp. 1543-58, Aug 2015.AbstractDNA repair (DR) inhibitors are small molecules that interact with DR proteins in order to disrupt their function and induce a ‘strike’ to the high fidelity of the mammalian DNA repair systems. Many anticancer therapies aim to harm the DNA of the usually highly proliferative cancer cell, causing it to undergo apoptosis. In response to this, cancer cells attempt to fix the induced lesion and reconstitute its genomic integrity, in turn reducing the efficacy of treatment. To overcome this, DR inhibitors suppress DNA repair proteins’ function, increasing the potency and tumor killing effect of chemotherapy or radiotherapy. In this review, we discuss clinically applied novel inhibitors under translational investigation and we apply bioinformatic tools in order to identify repair proteins implicated in more than two phenomenically distinct DNA repair pathways (e.g., base excision repair and nonhomologous end joining), that is, the concept of ‘synthetic lethality’. Our study can aid towards the optimization of this therapeutic strategy and, therefore, maximizing treatment effectiveness like in the case of radiation therapy.[7]Z. Nikitaki, C. E. Hellweg, A. G. Georgakilas, and J. L. Ravanat, "Stress-induced DNA damage biomarkers: applications and limitations," Front Chem, vol. 3, p. 35, 2015.Abstract A variety of environmental stresses like chemicals, UV and ionizing radiation and organism’s endogenous processes such as replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damage plays a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g., X- or α-,γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e., single, complex DNA lesions etc. that can be used as DNA damage biomarkers. We critically compare DNA damage detection methods and their limitations. In addition, we suggest the use of DNA repair gene products as biomarkes for identification of different types of stresses i.e., radiation, oxidative, or replication stress, based on bioinformatic approaches and meta-analysis of literature data.[8]V. I. Hatzi, D. A. Laskaratou, I. V. Mavragani, Z. Nikitaki, A. Mangelis, M. I. Panayiotidis, et al., "Non-targeted radiation effects in vivo: a critical glance of the future in radiobiology," Cancer Lett, vol. 356, pp. 34-42, Jan 1 2015.AbstractRadiation-induced bystander effects (RIBE), demonstrate the induction of biological non-targeted effects in cells which have not directly hit by radiation or by free radicals produced by ionization events. Although RIBE have been demonstrated using a variety of biological endpoints the mechanism(s) of this phenomenon still remain unclear. The controversial results of the in vitro RIBE and the evidence of nontargeted effects in various in vivo systems are discussed. The experimental evidence on RIBE, indicate that a more analytical and mechanistic in depth approach is needed to secure an answer to one of the most intriguing questions in radiobiology.Στη διατριβή αυτή μελετήθηκαν οι σύνθετες οξειδωτικές βλάβες DNA, δίκλωνες θραύσεις αυξημένης πολυπλοκότητας που συνοδεύονται και από βλάβες βάσεων στην εγγύς τους περιοχή. Οι βλάβες αυτές θεωρούνται η υπογραφή των ιοντιζουσών ακτινοβολιών αλλά και του ισχυρού οξειδωτικού στρες στο κύτταρο. Αποτελούνται από διάφορες βλάβες, όπως δίκλωνες θραύσεις, μονόκλωνες και αλλοιώσεις βάσεων πολύ κοντά η μία στην άλλη εντός 10-15 ζευγών βάσεων DNA. Λόγω της μεγάλης δυσκολίας επιδιόρθωσής τους μπορούν να οδηγήσουν πολύ εύ