Comparison of DNA Repair Kinetics after Radio- and Particle-Therapy in P53 Relevant B-cell
Lymphoma cell models. Abstract The induction of DNA-double-strand-breaks (DSB) is a hallmark of radiation induced cytotoxicity. Upon induction of DSBs, the cellular DNA repair machinery detects these lesions and activates signaling cascades which induce the repair of DSBs. A key component in DNA repair is the histone protein H2AX, which becomes rapidly phosphorylated on a serine four residues from the carboxyl terminus forming ?H2AX at nascent DSB sites. After exposure of cells to ionizing radiation (IR) or heavy ion particles, large number of ?H2AX molecules form so called radiation induced foci (RIF) where DNA repair proteins accumulate to promote the repair of DSB.
The microscopic visualization of RIF using immunohistochemistry is therefore considered a surrogate for measuring the radio biological effects (RBE) of radiation therapy. Sequential quantification of RIF after the exposure to IR can also serve as a surrogate for measuring DNA repair kinetics which is proportional to the complexity of DNA damage, and the intrinsic ability of the cell to efficiently repair RIF. Human and mouse temperature sensitive p53 B-cell lymphoma cell model was used to measure and compare the induction of IR induced DSB between photon, proton and carbon beams. The uniqueness of this approach relies on the fact that the effects of p53 mutant (p53-Val135) and p53 wild type in myc-driven lymphomas can be measured in parallel and with the same genetic background. Moreover, the Heidelberg-ion-beam therapy center (HIT) is one of few locations in the world where radiobiological effects of photon, proton and especially carbon beams can be measured simultaneously. Frequency of RIF with high-throughput microscopy-based immunohistochemistry was measured at 1h, 24h and 72 hours post exposure to IR on ?H2AX. In the first step the cytotoxic effects of photon, proton and carbon beams have been compared after isoeffective doses using in house developed macros and the Java-based image processing program ImageJ. Secondly, residual RIF frequencies were quantified 24 and 72 hours post IR to compare DNA repair kinetics among radiation qualities, p53 mutant, and p53 wild type conditions.
The results indicate a higher degree of complex damage of particle compared to photon beams. In addition, an increase in cytotoxicity can be measured based on the function of p53 wild type. In summary, the quantification of RIF has been used as molecular surrogate to measure the RBEs of photon, proton and carbon beams as a function of DNA repair kinetics and functionality of p53 providing molecular and radiobiological insights for radio- and particle-therapy for b-cell lymphomas. Our results purpose that effect of loss-of-function mutations p53 in B-cell lymphoma cells after IR with monitoring ?-H2AX foci. Furthermore, a variance between the quality of three radiation particles (photon, proton, carbon) was studied with dose-response and time-course experiments using gamma H2AX foci immunofluorescence method. Finally, this study aims to show the role of p53 and the effect of radiation on xxxxxxxxx , which could be a new treatment method for patients with lymphoma in the future. 1. Introduction 1.1.Quantification of DNA damage repair kinetics Previous studies show that DNA damage caused cancer. Besides that, DNA damage occurs every second evoked by many factors. As protection, DNA damage determination and repair mechanisms are in cells. It is called repair pathway (DDR/R) and it provides an activation of DNA damage checkpoints. These damages result in mutations creating single strand breaks (SSBs) and double strand breaks (DSBs), which are depending on source of factor. Various chemicals are used in the treatment produces DNA lesions. Besides that, there are some factors such as radiation which causes physically damage. It is shown that as a result of irradiation, cancer can initiate in a human body depending on the doses. Lesions cause clustered DNA damage which is also called multiple damaged sites. These sites also contain DSB regions (1).
DNA damage contain different types of DNA lesions such as non-DSB lesions, DSB lesion, and oxidative clustered DNA lesions (OCDLs). There are many studies which show that DNA lesions occur in both strands of DNA while one strand has tandem lesions. These lesions produce clusters of DNA damage which are difficult to repair. At the same time, in case of repairing of OCDLs, some of them can turn to de novo DSB over repair. Although it wasn’t shown which type of clusters of lesions can converted dangerous type of DSB, in case of these clusters present in one strand SSB can caused a delay of lesion repair process on the other strand which depends critical parameters such as nucleotide distance of lesions and 3’ or 5’ direction. In early studies shown that under high doses of radiation using pUC18 plasmid, lesion clusters can be more than three based and that these bases separate from each other can turn into DSB by a glycosylase (2). Another step of repair is the conversion of MDS into DSB in clustered damage region (3).
If it is not repaired, in case of activation with radiation, it causes chromosomal breaks (4). Studies show that there are two main problems for investigation of DNA damage: On the one hand there is measurement of DSB and OCDL levels, and types in cellular level and on the other hand there is reliable quantification (5). To detect these damages, ?H2AX methodology is getting more popular year by year. (6). Although the determination of non-DSB lesions with fluorescence microscopy, some of DNA lesions cannot be monitored such as 1-20 bp DNA lesions (7). In addition to that, gel electrophoresis with damage probes, and immunofluorescence microscopic approaches using targeted antibodies could be used to visualize the DNA lesions. (5) 1.2. Using a biomarker ?H2AX for determination of DNA double strand breaks So far it is shown that for the determination of DSB the ?H2AX method is the most reliable and quantitative technique. H2AX has a phosphorylation site at serine 139 and belongs to the H2A histone family. Following the DSB in DNA, this site starts being phosphorylated and this phosphorylation is called as “?-phosphorylation” (8).
The advantage using ?H2AX is that it has ?-phosphorylation level in megabase-long sites of chromatin. This ?-phosphorylation occurs after producing DSBs. Besides that, phosphorylation does not occur only in these sites, it extends in both sides of damage (9). Owing to ?-phosphorylated H2AX nucleosomes, DSB can be able to be monitored by microscopy (see Figure 1). Additionally, in case of using confocal microscopy H2AX foci roughly damage can be seen in cell cycle phases. Figure 1. Determination of fluorescence microscopy images There are three channels for fluorescent images detection (Blue: DAPI, Red: DSB foci and Green: non-DSB lesions), for the geometrical areas; foci area-without foci area. Pclc investigate that Intensity of a given fluorescent channel based on foci area. (Figure 1.A)DNA damage were calculated based on non-DSB lesions mean intensity. In case of Pclc values higher than 1 means colocalization and that results with the determination of DNA lesions (c). The calculation was made intensity of DAPI channel (blue) area divided by the mean intensity of the same channel over the rest of cell nucleus (after excluding any nucleoli areas). (5) Even if H2AX amount and percentages of H2A differ based on cell types, the phosphorylated average of DSB is similar. This way, ?H2AX foci can be measured in cell lines. Comparing with other techniques such as ELISA, flow cytometry and immunoprecipitation, which also use unique antibodies against the ?H2AX epitope, this method has a high impact for determination of DSBs. (10) Besides that, after cells are subjected to IR, ?H2AX foci occurred in the euchromatin (11). Generally, this method plays in sensitivity, reliability detection of DSBs. The sensitivity is feature is the detection of DSBs more than other damages (8). However, it has been reported that ?H2AX can be formed at other types of lesions. E.g. in high frequencies in S-phase of cell replication (12) or in some other cell types undergoing for chromatin remodeling. (13).
Nevertheless, the reason for high sensitivity is monitoring of ?-phosphorylation in the whole nucleus. There are evidences which provide increasing numbers of ?H2AX foci related the chromosomal instability (14). Additionally, Banath et al. show determination of DSBs monitoring ?H2AX foci. They measured that different DNA damaging therapeutic agents by measuring the fraction of cells (15). IR treatment which is against to cancer, has a high effect in cells and causes damage which cannot be repaired (16). Against to cancer cells (especially lymphoma cancer) surgeries, chemotherapie’s or RT’s are important treatments. Depending on dose and type of RT effects damage of DNA level. As it was already mentioned DNA lesions is most difficult damage type. We provide that dose and type dependent effect of RT in this thesis. In addition, there is evidence that after this sort of therapy, secondary cancer development may occur. But using latest technologies in radiation with chemotherapeutic agent, level of success is increased. Examples of these agents might be platinum based on microtubule inhibitors, DNA alkylating agents, topoisomerase poisons, antimetabolites, antitumor antibiotics, proteasome inhibitors etc. (17). Early studies show the importance of ROS at the region of DNA lesion (18). low levels of ROS results with Genomic instability (GI) via NHEJ and DNA repair (19). As shown RT is one of major cure in cancer which has the effect to cluster DNA damage (20). Among these, the use of proton beams as an alternative to traditional high energy electrons has, at least in theory, improved accuracy of targeting and reduction in surrounding tissue toxicity (21). Dose and quality of radiation depends on the DNA damage’s complexity. Increasing the dose and Linear Energy Transfer (LET) indicates that accumulation of clustered lesions in cancer cells (22). In addition, depending on level of LET, cells can decide whether it needs DNA repair kinetics proteins or not (23).
Advantages of LET IR is that there are extensive amounts of clustered damage leading to increased relative biological effectiveness (RBE) versus both Photon based and even Proton-based modalities (24). In addition to that, Carbon ion based RT applied first time in the Heavy Ion Medical Accelerator (HIMAC) in Chiba, Japan in 1994 (25). After Japan, Germany has the Heidelberg Ion Therapy Center (HIT) in 2009. Our treatment using Carbon-ion based IR were performed in HIT. Cells control their genomic integrity by monitoring the DNA repair mechanism in case of DNA damage. One of the damage is the DSBs and cell cycle progress is arrested after that. This mechanism run until the deficiency in the genome is fixed. If it consists of aberrant DNA repair mechanism, this can cause cancer development (26). During the DNA repair mechanism, the histone protein H2AX has a significant role. It is transformed to ?H2AX at nascent DSB site by phosphorylation through carboxyl terminus of H2AX on serine four residues. This conversion enables to use of ?H2AX as a biomarker for detection of DSBs (27). H2AX can also be phosphorylated by serine residue, however, its function is unclear. Although DSB caused cancer, it is used as a treatment of cancer. In cancer cells, DSBs are present for the activation of cell death pathways by therapeutic agents (28). DSBs can be i) produced by these agents directly or ii) can occur in case of repairing (29). Monitoring ?H2AX focus formations allow us to detect DSBs. This can be used as a treatment and will be investigated in this thesis. detection of DSBs and this can be used as a treatment. In this thesis we investigated ?H2AX focus formation depending on DSBs (30). H1, H2A, H2B, H3, and H4 histone protein families take a role in chromatin organization by packaging eukaryotic DNA. H2AX is one of the H2A family members, and two of H2A are found in each nucleosome which is basic subunit of chromatin (31). There is a switch the ratio of existing H2AX on the diverse type of cells.
While normal fibroblasts contain nearly 10% of H2AX, lymphocytes and SF68 glioma cells implicate minimum 2% and maximum 20% of H2AX respectively (32). Association between replication and synthesis of H2AX, depends on amount of H2AX for using determination of DSBs or G1/G0 phase cells on replication (33). DNA repair mechanism and H2AX molecules are associated. DSBs trigger the necessary and activation of PI3K-like kinases such as ataxia telangiectasia mutated (ATM), ATR (ataxia telangiectasia and Rad3-related), and DNA-PK (DNA-dependent protein kinase) for H2AX-phosphorylation (?H2AX). The researches indicate that the ?H2AX foci size and brightness can rise depending on the exposure time of ionizing radiation (IR) on mitotic chromosomes of fixed Indian muntjac cells. Whereas the resemble ?H2AX foci sizes are observed on other mammalian cells (5). As the ?H2AX is formed upon DSBs, repair of DSB gives rise to the disappearance of ?H2AX. The rejoining of DSBs leads to a decrease in the amount of ?H2AX foci, and this mechanism programmed such two ways as ?H2AX dephosphorylation or its removal from chromatin (34). In addition to DSB dependent H2AX phosphorylation, the ?H2AX formation can be induced by ATR kinase because of the DNA single-stranded breaks (SSBs) created via ultraviolet C irradiation (35). Figure 2. Results of DSBs related to ?H2AX signaling pathways. a. Mutation of DNA-damage repair proteins present producing DSBs, besides that represen-ting of telomere shortening, viral infection and is subjected to UV radiation can called as an indirect causes of DSB damage.(36) b. ?H2AX has related to three kinases which are ataxia telangiectasia mutated (AtM), ataxia telangiectasia rad3-related (Atr) and, DNA dependent protein kinase (DNA-PK). These kinases are responsible from DNA damage repair. After the determination of DSB, they response phosphorylation of H2AX . Nibrin protein (NBS1) and mediator protein 1 (MDc1) alerts AtM and it stimulates the H2AX phosphorylation.(37) PP2A and PP4c phosphatases have role bind to and dephosphorylate to ?H2AX (38). c. Binding of NBS1 and MDc1 to ?H2AX which lets the accumulation of repair proteins at the DSB sites. rNF8 protein which has roles in the activity of immune system functions, DNA repair, and ubiquitination of ?H2AX (39). Additionally, ?H2AX causes accumulation of McPH1 proteins at DSB sites and this protein has interactions with 53BP1, BrcA1, cHK1, NBS1, MDc1, Atr and AtM which takes the cell to cell cycle arrest. (40). Via McPH1 protein; 53BP1, BrcA1, cHK1 and NBS1 were accumulated. It shows the role of ?H2AX in DNA repair. (41)
Nevertheless, ?H2AX has interaction with the human histone acetyltransferase tiP60 complex which interacts with UBc13 protein. Both of the proteins regulate ?H2AX acetylation which is important for ubiquitination (Ub). It can be later released from the chromatin. Furthermore, it both plays role in interaction of two sister chromatids in homologous recombination and during apoptosis. (42) As mentioned before there are many reasons for producing DSBs. As an example of direct causes, irradiation and drugs can be given. As it can be seen ROS molecules also cased DSBs. In addition, indirect pathways such as TOP isomerases can be given as an example which is used in replication. H2AFX, which is human H2AX gene, is located on 11q23. This region faces often mutation or deletion in a vast variety of human cancer types such as acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) (43). The mutation in H2AX is related to the contribution of cancer development and progression, also it affects resistance to treatment of different cancers (44). Besides, the recent studies display that H2AX has the tumor suppressor gene capacity because H2ZX homozygote and heterozygote knockout mice are not liable to cancer development. However, these mice show an ability to cancer development in a p53-null background (45). DSB is induced not only by cancer development but also by cancer treatment, and this leads to the use of ?H2AX as a potential biomarker for diagnosis, and target for treatment (46). Modifying the genome and creating DSBs exhibit ?H2AX formation. Thus, ?H2AX can be used for interpretation of treatment progress and cancer growth (47). Physicians can detect DSBs in different types of patient samples by quick and cheap assays focusing on the ?H2AX formation. Therefore, they can decide the level of influence of the therapeutic agent to damage. Furthermore, there exist also many environmental factors causing stress on both animal and human, and this stress induces ?H2AX formation. Due to that, these analyses can be utilized to determine the effects of different factors on stress. As a conclusion, ?H2AX can be used for both diagnosis or treatment of the diseases, and evaluation of the impact of diverse factors to the DNA damage. 1.3.
Role of p53 protein in DNA repair mechanism One of the most known tumor suppressors is p53 which has very low concentration because of short half-life time. Reason for this short half-life is E3 ubiquitin ligase MDM2. MDM2 is one of the genes regulated by transcription which p53 regulates. Increased p53 levels result also increased of MDM2 transcription rate, followed by this, it results the degradation of the p53 protein, forming an autoregulatory circuit in cells. There are various stress situations that can cause this such as telomere erosion, hypoxia, the mutational activation. Stress can be recognized by protein set which signal MDM2 or p53. As a results, depending on cell type and modifications, there is p53 transcription program. However, this also can cause many ways of situation such as apoptosis, cell cycle arrest, metabolic alterations or extrinsic signaling to and from the immune system. The extraordinary diversity with p53 protein responds to a wide variety of stresses, either repairing the resultant defects or killing the defective cells, which gives rise to the tumor suppressor phenotype of the TP53 gene. In addition, the wild type p53 protein responds to a wide variety of cancer treatments including chemotherapy and irradiation, often killing and repairing the normal cells (homeostasis) with wild-type p53. As such, it also provides the differential toxicity observed with therapies between normal cells and cancer cells, leading to cell death upon treatment. A mutant p53 gene and protein fails at both prevention and therapy. (48) The induction of p53 in response to DNA damage is coordinated by the ataxia–telangiectasia mutated (ATM) and ataxia–telangiectasia and Rad3-related (ATR) protein kinases, which mediate the rapid destruction of MDM2 and MDM4.
ATM and ATR are members of the phosphatidylinositol-3 kinase-like kinase family. They coordinate a complex signaling network in response to various forms of DNA damage. ATM plays a crucial part in the immediate response to double-strand breaks by coordinating the activation, execution of checkpoint pathways, and repair pathways. ATR plays role in replication stress, and DNA crosslinking. These pathways share downstream targets in the repair and checkpoint pathways such as the transducer kinases CHK1, CHK2, and components of the p53 pathway. ATM and ATR induce one damage signal various post-transcriptional modification which is an appropriate and proportionate manner according to the nature of the damage and intensity of the stress. One of these modifications has relationship with tumor suppression and the roles of these modifications’ determinate by studies generation of p53 knocking mice which has substitutions at major sites of posttranslational modification in p53. There are two important phosphorylation sites which called Serine 15, threonine 18. S20 and T18 have role both inducing the interaction of p53 with the transcriptional machinery and can inhibit interaction of p53 and MDM2. Besides that, phosphorylation of p53 might has role of p53 induction and this important for determination of developing tumor cells. (49) 1.4. P53 and radiation In case of radiation, p53 is main regulator in DNA repair mechanism and doing so by promoting protein translation and inhibition of protein degradation. After damage in nucleus, there will be accumulation of p53.
As a result of signaling pathway activation, apoptosis can be induced which also takes to cell to replication arrest. Therefore, p53 plays a crucial role in controlling cellular fate after irradiation. For example, activation of p53 results in dramatically increased pre-mitotic apoptosis in tissues that have a rapid turnover rate such as the hematopoietic system and the gastrointestinal epithelium. To the contrary, in tissues with a slower turnover rate, such as the myocardium, accumulation of p53 following radiation does not cause a significant increase in pre-mitotic apoptosis. Instead of that, it induces genes that control cell-cycle checkpoints such as the cyclin-dependent kinase inhibitor p21. (50) In this thesis, searching for role of p53 lymphoma cancer cells were used.
B-cell lymphomas which are belongs to Hodgkin’s lymphomas and most non-Hodgkin lymphomas, and they are “blood cancers” in the lymph nodes and they occurred frequently in older adults. Following this, our main aim is investigation of DNA repair kinetics using ?H2AX detection which shows DSBs after irradiation in wild type and mutant p53 lymphoma cell lines.