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UBC Theses and Dissertations

Role of eEF2K in DNA damage response Samiei, Arash 2018

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ROLE OF EEF2K IN DNA DAMAGE RESPONSE by  Arash Samiei  BSc.  The University of British Columbia, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology & Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2018  © Arash Samiei, 2018     ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Role of eEF2K in DNA damage Response   Examining Committee: Poul Sorensen, Pathology and Laboratory Medicine Supervisor  Michel Roberge, Biochemistry Supervisory Committee Member   Supervisory Committee Member Catherine Pallen, Pathology and Laboratory Medicine Additional Examiner   Additional Supervisory Committee Members: Samuel Aparicio, Pathology and Laboratory Medicine Supervisory Committee Member Christian Steidl, Pathology and Laboratory Medicine Supervisory Committee Member submitted by Arash Samiei  in partial fulfillment of the requirements for the degree of Master of Science in Pathology and Laboratory Medicine iii  Abstract Many of the DNA damage inducing chemotherapeutic drugs preferentially kill cancer cells but they also have a negative impact on normal cells in the body. Having a better understanding of how tumors respond to the DNA damage caused by chemotherapeutic agents can improve the chemotherapy regimen and reduce the harm done on the patient. Eukaryotic elongation factor 2 kinase (eEF2K) is a regulator of mRNA translation which is over-expressed in medulloblastoma, gliomas, and some breast cancer patients with poor prognosis. Under stress conditions, such as nutrient deprivation or DNA damage, eEF2K inhibits mRNA translation elongation by phosphorylating and inhibiting the activity of eukaryotic elongation factor 2 (eEF2). It was reported that eEF2K increases cellular sensitivity to inducers of DNA damage, including hydrogen peroxide and doxorubicin. The goal of this thesis work was to define the mechanistic role of eEF2K in DNA damage response (DDR) and its role in sensitizing cells to genotoxic agents. To this aim, we used cisplatin to study the DDR in the presence and absence of eEF2K expression. We found that eEF2K enhances the overall DDR in response to cisplatin treatment and the sensitivity phenotype depends on the level of cisplatin that the cells are exposed to. When cells are treated with high levels of cisplatin, eEF2K enhances the activity of the ATM and ATR DDR pathways that lead to higher apoptosis through p53 activity. However, when treated with low levels of cisplatin, eEF2K enhances the DNA repair pathways and prevents cell death. In summary, our findings show that eEF2K boosts the DNA damage response to help repair the damaged DNA, or helps to kill the cell if the damage cannot be repaired. Overall, these results reinforce the role of eEF2K as a stress response protein.   iv  Lay Summary The DNA of the cell contains the blueprints required for the cell to function properly and survive. Many of the drugs used during chemotherapy kill cancer cells by causing DNA damage. After the DNA of the cell gets damaged, a series of safe-check mechanisms allow the cell to respond to the damage. The cell will try to repair the damage but if the damage cannot be repaired the cell will destroy itself. Very little is known about how the cell triggers these fail-safe mechanisms and how it carries the subsequent appropriate response. We discovered that the protein eukaryotic elongation factor-2 kinase, also known as eEF2K, enhances the ability of the cell to respond to DNA damage. eEF2K improves the DNA repair ability of the cell but if the damage is very high it helps the cell to destroy itself.  v  Preface The work on this dissertation was done under the supervision of Dr. Poul Sorensen. Drs. Gabriel Leprivier and Sorensen were responsible for the formation of the concept behind this project. The HEK293 cell line with stable KD of eEF2K was supplied by Dr. Daniel Radiloff. The eEF2K – and eEF2K + MEFs were provided by Dr. Gabriel Leprivier. The qRT-PCR experiment and its analysis in chapter 3.3 was carried out by Jordan Cran. I carried out all the other experiments, data analysis, and data interpretation (in collaboration with Drs. Sorensen and Leprivier) shown in this thesis. vi  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Figures ............................................................................................................................. viii Glossary ..........................................................................................................................................x Acknowledgements .................................................................................................................... xiii Dedication ................................................................................................................................... xiv Chapter 1: Introduction ................................................................................................................1 1.1 Hypothesis and aims ....................................................................................................... 1 1.2 eEF2K, eEF2, and translation elongation ....................................................................... 2 1.3 DNA damage response ................................................................................................... 6 1.3.1 ATM/ATR DDR pathway........................................................................................... 7 1.3.2 P53 response ............................................................................................................. 11 1.3.3 Cisplatin .................................................................................................................... 15 1.3.4 Repair of Cisplatin Inter-crosslinks DNA................................................................. 16 Chapter 2: Materials and methods .............................................................................................20 2.1 Cell culture .................................................................................................................... 20 2.1 Foci and micronuclei measurements ............................................................................. 21 2.2 Immunoblot analysis ..................................................................................................... 22 2.3 Quantitative RT-PCR .................................................................................................... 22 vii  2.4 siRNA Transfection ...................................................................................................... 23 2.5 MTT Assay ................................................................................................................... 23 2.6 Trypan blue cell death assay ......................................................................................... 24 2.7 Measurement of inter-crosslink repair .......................................................................... 24 2.8 Flow cytometry ............................................................................................................. 25 2.9 Statistical analysis ......................................................................................................... 26 Chapter 3: eEF2K alters the cellular response to cisplatin......................................................27 3.1 Introduction and approach ............................................................................................ 27 3.2 eEF2K enhances sensitivity to cisplatin ....................................................................... 28 3.3 eEF2K supports induction of the ATM/ATR DNA damage response ......................... 35 3.4 Role of p53 in the cellular response to cisplatin downstream of eEF2K ...................... 51 3.5 Summary ....................................................................................................................... 57 Chapter 4: eEF2K impacts DNA repair ability after cisplatin treatment ..............................64 4.1 Introduction ................................................................................................................... 64 4.2 eEF2K affects repair of DNA damage and DNA intercross links ................................ 65 4.3 eEF2K alters long term sensitivity to cisplatin ............................................................. 77 4.4 Nucleotide excision repair as a candidate for better DNA repair ability ...................... 78 4.5 Summary ....................................................................................................................... 80 Chapter 5: Conclusions and future directions ..........................................................................81 Bibliography .................................................................................................................................89 Appendices ..................................................................................................................................110  viii  List of Figures  Figure 1-1 Regulation of eEF2K activity........................................................................................ 6 Figure 1-2 The ATM/ATR DDR signaling pathway. ................................................................... 10 Figure 1-3 Upregulation of p53 with DNA damage and its downstream targets ......................... 15 Figure 1-4 Nucleotide excision repair pathway ............................................................................ 19 Figure 3-1 Cell viability of WT and eEF2K KO MEFs after 48 hours of cisplatin treatment. .... 30 Figure 3-2 Cell death of WT and eEF2K KO MEFs after 48 hours of cisplatin treatment. ......... 31 Figure 3-3 Apoptotic response of WT and eEF2K KO MEFs after 48 hours of cisplatin treatment. ...................................................................................................................................... 31 Figure 3-4 Cell viability of eEF2K-/- (MSCV)  and eEF2K-/- (rescue) MEFs after 48 hours of cisplatin treatment. ........................................................................................................................ 32 Figure 3-5 Cell viability of HEK293 cells with stable knockdown of eEF2K or with no knockdown of eEF2K after 72 hours of cisplatin treatment. ........................................................ 33 Figure 3-6 Cell viability of WT and eEF2K KO MEFs after 48 hours of camptothecin treatment........................................................................................................................................................ 34 Figure 3-7 Cell viability of WT and eEF2K KO MEFs after 48 hours of hydroxyurea treatment........................................................................................................................................................ 35 Figure 3-8 Effects of eEF2K on induction of ATM/ATR DDR signaling pathway after cisplatin treatment. ...................................................................................................................................... 40 Figure 3-9 Confirming the effects of eEF2K on induction of ATM/ATR DDR signaling pathway after cisplatin treatment in MEFs. ................................................................................................. 47 ix  Figure 3-10 Effect of eEF2K on induction of ATM/ATR DDR signaling pathway after cisplatin treatment in HEK293 cells. ........................................................................................................... 49 Figure 3-11 Effect of eEF2K on γH2AX and 53BP1 foci localization after cisplatin treatment in WT and eEF2K KO MEFs. ........................................................................................................... 50 Figure 3-12 Effect of eEF2K on tp53 mRNA levels after cisplatin treatment in WT and eEF2K KO MEFs. ..................................................................................................................................... 54 Figure 3-13 Transient p53 knockdown in MEFs. ......................................................................... 55 Figure 3-14 Effect of p53 on cell viability of WT MEFs after 48 hours of cisplatin treatment. .. 56 Figure 3-15 Effect of p53 on cell viability of eEF2K KO MEFs after 48 hours of cisplatin treatment. ...................................................................................................................................... 57 Figure 4-1 ICL DNA repair in WT and eEF2K KO MEFs. ......................................................... 69 Figure 4-2  DNA repair in WT and eEF2K KO MEFs measured through formation and resolution of γH2AX foci.............................................................................................................. 71 Figure 4-3 DNA repair in WT and eEF2K KO MEFs measured through formation and resolution of 53BP1 foci. ............................................................................................................................... 73 Figure 4-4 Formation of micronuclei in WT and eEF2K KO MEFs after cisplatin treatment. .... 75 Figure 4-5 Cell cycle progression in eEF2K WT and KO MEFs during cisplatin treatment. ...... 76 Figure 4-6 Long-term cell viability of WT and eEF2K KO MEFs pulsed with cisplatin. ........... 78 Figure 4-7 Effect of eEF2K on ERCC1 protein levels. ................................................................ 79 Figure 5-1 Model describing the role of eEF2K in DDR. ............................................................ 88 Figure Appendix-5-1 Modified alkali comet assay used to quantify ICL repair. ....................... 111  x  Glossary 2N/4N DNA  2 copies or 4 copies of the DNA 53BP1   Tumor suppressor p53-binding protein 1 ADP    Adenosine diphosphate AMP    Adenosine monophosphate AMPK   AMP activated protein kinase ATM    Ataxia Telangiectasia Mutated ATP    Adenosine triphosphate ATR    Ataxia Telangiectasia and Rad3-Related Protein BrdU   Bromodeoxyuridine CA+2   Calcium cAMP   cyclic AMP Chk1    Checkpoint kinase 1 Chk2   Checkpoint kinase 2 DDR    DNA damage response DMSO   Dimethyl sulfoxide DNA   Deoxyribonucleic acid DSB    Double stranded break eEF2    Eukaryotic elongation factor-2  eEF2K   Eukaryotic elongation factor-2 kinase eEF2K +/+    WT MEFs, cell with btwooth alleles of eEF2K eEF2K -/-  eEF2K KO MEFs  eEF2K -/- (rescue)  eEF2K KO MEF expressing eEF2K in a MSCV vector xi  eEF2K -/- (MSCV)  eEF2K KO MEF transfected with the MSCV vector   eIF   Eukaryotic initiation factor ERCC1   excision repair cross-complementation group 1 γH2AX   gamma H2A Histone Family Member X H2AX   H2A Histone Family Member X HEK293   Human embryonic kidney 293  ICL   Intercross link IF    Immunofluorescence KO    Knockout MEF    mouse embryonic fibroblast mRNA   Messenger RNA MTT    3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide nM    Nano molar P21    cyclin-dependent kinase inhibitor 1 P53    Tumor protein p53 PARP   Poly ADP ribose polymerase PI   Propidium iodide PIKK    Phosphoinositide 3-kinase related kinase  PKA    cAMP dependent protein kinase  Rad17    RAD17 Checkpoint Clamp Loader Component RNA    Ribonucleic acid Ser   Serine shRNA  Short hairpin RNA xii  siRNA   Silencing RNA Thr   Threonine tRNA    Transfer RNA UV   Ultra violet µM    Micro molar WT    Wild type xiii  Acknowledgements I would like to thank all of the past and present members of the laboratory for providing emotional and intellectual support. I particularly want to thank Dr. Gabriel Leprivier for his constant support and guidance throughout my work and studies. In addition, I would like to the Department of Pathology and Laboratory medicine and my advisor Dr. Haydn Pritchard for their support.  I would like to express gratitude my gratitude to Dr. Poul Sorensen for his mentorship and giving me the opportunity to carry out my research in his lab. I also want to thank my committee members Dr. Sam Aparicio, Dr. Michel Roberge, and Dr. Christian Steidl for their advice and guidance.  Lastly, I want to thank my family for their constant love, support and their belief in me. The work contained in this dissertation was supported by the Canadian Cancer Research Society Institute. xiv  Dedication           I dedicate this dissertation to my Mom, Dad, Sister and friends who supported me1  Chapter 1: Introduction 1.1 Hypothesis and aims All cells experience various types of stress during their lifetimes as they are exposed to the different aspects of the environment, including nutrient deprivation, genotoxic stress, hypoxia, and many others. The cell uses different genes to adapt to the stresses in the environment to prevent permanent damage to itself or the organism that it is a part of. One of these genes is the eukaryotic elongation factor 2 kinase (eEF2K) which gets activated when the cell is under certain types of stress such as nutrient deprivation and genotoxic stress1,2. Its role in helping cells to adapt to a nutrient deprived environment has been studied in great detail; however, it is not clear what role eEF2K plays during genotoxic stress2–4. We could confirm a previous study by Chu et al. (2014)2 that had demonstrated eEF2K can sensitize cells to the DNA damaging drug doxorubicin. For our purposes, we decided to use cisplatin as our drug of choice because it is an important drug used during chemotherapy of medulloblastoma patients, and eEF2K has been shown to be upregulated in medulloblastoma patients with worse prognosis1. We observed that eEF2K expression enhances cell sensitivity to high levels of cisplatin treatment. My goal was to understand how eEF2K affects sensitivity to cisplatin, such as by impacting the DNA damage response of the cell.  The hypothesis of this dissertation is that expression of eEF2K helps to sensitize cells to high levels of cisplatin by enhancing p53 expression, while at low levels of cisplatin eEF2K expression reduces sensitivity by enhancing DNA repair. The following aims will be addressed in my dissertation to support my hypothesis: 1. To determine if eEF2K influences the activation of the DNA damage response to cisplatin. 2. To determine eEF2K’s effects on the ability of the cell to repair cisplatin DNA damage. 2  3. To determine what factors are responsible for cell sensitivity to cisplatin treatment in presence or absence of eEF2K expression.  1.2 eEF2K, eEF2, and translation elongation  Protein synthesis or mRNA translation takes place in association with the ribosome, which is composed of ribosomal RNAs and proteins. Among other functions, the ribosome provides a scaffold that allows for formation of polypeptide chains as dictated by the mRNA message. There are three RNA binding sites in the ribosome, A, P, and E and they work very similar to a factory assembly line. The aminoacyl-tRNA comes into the A site, then moves in to the P site where the amino acid binds to the growing peptide chain, and lastly the tRNA moves into the E site where it is released from the ribosome. This sequence of events is divided into three different phases: initiation, elongation and termination. As the name suggests, “initiation” starts protein synthesis by assembly of the ribosome-mRNA complex and the binding of the methionine aminoacyl-tRNA to the mRNA’s AUG start codon. “Elongation” involves extending the growing peptide chain and “termination” occurs when the ribosome encounters the mRNA stop codon, which ends protein synthesis. The cell uses a variety of genes to regulate mRNA translation because it is a very energy consuming biological process5. Eukaryotic initiation factors (eIFs) and eukaryotic elongation factors (eEFs) are groups of proteins that respectively allow for efficient translation initiation or translation elongation to take place, where eIFs help with assembly of the ribosome-mRNA complex and eEFs allow the growing peptide chain to accept new amino acids. The cell can quickly turn mRNA translation off or on by activating or deactivating these factors.   We will focus on eukaryotic elongation factor 2 (eEF2) and its kinase eEF2K. The eEF2 protein allows the growing peptide chain and its tRNA to move into the P site of the ribosome by 3  hydrolyzing eEF2’s GTP 6. It plays a vital role in mRNA translation and the cell can utilize eEF2K to deactivate eEF2 through phosphorylation at the thr-56 site, which subsequently turns off mRNA translation rapidly.  eEF2K belongs to the alpha class of kinases because its sequence is not similar to other classes of kinases and has a very unique catalytic domain structure compared to other types of kinases 7,8. Its structure is composed of several different domains, the calmodulin binding domain, the catalytic domain (responsible for the kinase activity), the SEL-1 helical repeats that help to stabilize protein-protein interactions and aids in phosphorylation of eEF29,10, and the C-terminus end which has been reported to be required for phosphorylation eEF29,11. eEF2K phosphorylates eEF2 at the threonine 56 site, blocking its activity. eEF2K’s only known substrate in a cellular setting is eEF2 and similarly eEF2 does not have any other known kinases aside from eEF2K12. Although a recently published study suggests that eEF2K may have other substrates in an in vtro setting13. Therefore, from our current understanding of eEF2K we can speculate that it is a highly specialized protein involved in regulating protein synthesis. It is also worth noting that eEF2K is not an essential gene and is not required for protein synthesis since the knockout (KO) mice are able to survive and produce viable offspring 14.  eEF2K was originally discovered as a calcium/calmodulin kinase that becomes highly active in the presence of Ca+2 and cyclic AMP (cAMP) 15–17. The mechanism responsible for activation of eEF2K in presence of calcium/calmodulin kinase is not very well understood; however, Pigott et al. (2012) provide an interesting model where binding of the calmodulin/calcium complex to eEF2K enhances eEF2K’s ability to bind to ATP, which subsequently results in allosteric changes that allows the eEF2 binding domain to interact with the kinase domain. eEF2K has also been shown to be regulated independently of calcium/calmodulin 4  through its various internal phosphorylation sites. Mammalian target of rapamycin complex 1 (mTORC1) is a negative regulator of eEF2K that has been shown to inhibit eEF2K activity by direct and indirect interactions 18,19,20,21. Wang et al. (2001)19 showed that activation of mTORC1 induces p70S6 kinase activity which subsequently inhibits eEF2K by phosphorylating its ser366 site. However, they also showed that same site can get phosphorylated by an mTORC1 independent pathway, where PDK1 phosphorylation of p90RSK allows for phosphorylation of eEF2K. Another inhibitory phosphorylation on eEF2K is the ser359 site, which is a target of the p38/MAPK, IGF1, CDC2 and mTORC1 pathways 20,21. eEF2K also contains activating phosphorylation sites that allow it to be regulated independently of calcium and calmodulin.  cAMP through cAMP-dependent protein kinase (PKA) activates eEF2K by phosphorylating its ser499 site22,23. These results demonstrate eEF2K activity is not only dependent on the calcium levels of the cell and greatly expands the potential role that eEF2K could have on the cell by regulating mRNA translation elongation24. AMP activated protein kinase (AMPK) can also upregulate the activity of eEF2K by phosphorylating the ser398 site 25. The various kinases that regulate eEF2K activity are sensors of energy in the cell or play a role in cell metabolism, including AMPK, mTORC1 and PKA (Figure 1-1)26–28. Therefore, we can deduce that the cell is able to use eEF2K during times of stress to utilize its energy in the most efficient manner by turning off mRNA translation expediently at the elongation phase.  Many studies illustrate that eEF2K plays a vital role as a stress mediator protein 1,2. Leprivier et al. (2013) demonstrated that cells can use eEF2K to protect themselves during times of starvation, and cancer cells can toxic survive starvation conditions by upregulating eEF2K 1. A similar phenomenon is seen under hypoxic conditions, where eEF2K helps cells to adapt to hypoxic stress and survive in absence of efficient aerobic metabolism29.  5   The role of eEF2K under nutrient deprivation and hypoxia has been widely studied, and eEF2K expression has been shown to play a protective role under these types of stress by inhibiting mRNA translation and thereby reducing the energy that is used up by the cell1,30. eEF2K also gets activated under genotoxic stress, resulting in an upregulation of p-eEF2thr56 and slowdown of translation elongation31. However the impact of eEF2K under genotoxic stress has been studied less and includes contradictory findings 2–4. Chu et al. (2014)2 demonstrate that eEF2K plays a pro-apoptotic role when MEF cells are treated with doxorubicin; however, other studies demonstrate that eEF2K can play a pro-survival role in baby mouse kidney cells and breast cancer cells when treated with doxorubicin or ionizing radiation 3,4. Such discrepancies can be due to the use of different cell types and the assays that were used to measure cell sensitivity; for example, we demonstrate that a short-term sensitivity assay (~48-72 hour) can have very different results than a long-term cell sensitivity assay (4-7 days). Short-term assays mostly measure the activity of the pro-apoptotic pathways and less of the DNA repair ability of the cell; however, long-term sensitivity assays greatly depend on the DNA repair ability of the cell. The different types of cells can also demonstrate contradictory results, where cancer cells with faulty tumor suppresser capacities can adapt to living with some levels of DNA damage and chromatin modifications while non-cancerous cells with fully functioning tumor suppressing capabilities would be very sensitive to such effects. Therefore, great care must be taken when comparing cancer cell lines and non-cancerous cell lines.   Chu et al. (2014) demonstrated eEF2K plays a protective role for the organism by helping to rid the mice of damaged and lower quality oocytes. They also showed that eEF2K sensitizes cells to doxorubicin treatment, so this idea can be further expanded to cells with cytotoxic damage whereby eEF2K helps to get rid of the cell with highly damaged DNA to prevent further damage 6  to the organism. However, the role of eEF2K may greatly change in cancerous cells where the various DNA damage response genes and pro-apoptotic pathways have been manipulated or modified to help the cancer cells survive.   Figure 1-1 Regulation of eEF2K activity The eEF2K protein is made up of different domains that contain inhibitory and activating phosphorylation sites. The blue arrows indicate phosphorylation sites that activate eEF2K activity and red arrows indicate inhibitory phosphorylation sites. 1.3 DNA damage response  The DNA is the blueprint of the cell and its proper maintenance is essential for the function of cells in an organism. If the DNA is damaged, the cell will repair it or go through apoptosis to prevent the cell from going ‘rogue’ and causing damage to the organism. DNA damage can occur from many different sources in the environment, including normal daily human activity, cellular 7  functions, the sun’s UV radiation, smoking, medical X-ray radiation, normal cell replication, and many more 32. Therefore, our cells are equipped with a wide variety of genes and mechanisms to help the cell respond appropriately to these genotoxic stresses. Depending on the source of genotoxic stress, the DNA can be a target of different types of damage, such as double stranded breaks that arise from ionizing radiation and DNA strand crosslinks caused by UV radiation. There are also many chemicals that cause different types of DNA damage either directly or indirectly. Chemicals such as cisplatin or mitomycin c (MMC) cause crosslinks of DNA strands and chemicals such as doxorubicin cause damage by acting as intercalating agents in the DNA. However other chemicals such as camptothecin or hydroxyurea cause DNA damage indirectly by preventing proper replication or maintenance of DNA by inhibiting topoisomerase I or inhibiting nucleotide production, respectively. These drugs and ionizing radiation have been used in therapeutic treatment strategies to fight cancer for the last several decades33–35.   The high replication rate of cancer cells compared to normal cells results in their DNA going through many replication cycles which renders them much more susceptible to DNA damaging agents compared to the body’s normal cells. However, cancer cells can highjack different DNA repair or DNA damage response (DDR) pathways to resist the DNA damaging drugs, therefore it is very important to understand how the DDR pathways work and how they can lead to resistance against chemotherapeutic drugs.  1.3.1  ATM/ATR DDR pathway  The DDR signaling pathway consists of several different stages, starting with DNA damage recognition which activates the major DDR transducer kinases, ATM and ATR, that signal other downstream transducers or effectors to carry out the appropriate response (Figure 1-2)36. The 8  recognition of DNA damage is probably the least understood step in the DDR pathway. For the purposes of this thesis we will focus on recognition of DNA crosslinks and DNA double stranded breaks because they are the most relevant damages caused by cisplatin. ATM and ATR are members of the phosphoinositide 3-kinase related kinase (PIKK) family and mutations in these genes are associated with the hereditary disease Ataxia-telangiectasia, which renders afflicted people more susceptible to DNA damaging agents 37,38. ATM has been primarily associated with double stranded breaks and ATR with single stranded breaks; however, there is considerable crosstalk between the two pathways 39,40. After ATM is localized to the site of double stranded breaks by the MRN (Mre11, NBS1, Rad50) complex, it gets activated by autophosphorylation at the ser1981 site 41,42. The ATR protein is closely related to ATM; however, activation of ATR is mostly associated with single stranded DNA structures, where ATR in complex with ATRIP (ATR-interacting protein) gets localized to the RPA (replication protein A) proteins that coat  the exposed single stranded DNA 43–45. Following ATR localization to the RPA sites, it gets activated by autophosphorylation at its T1989 site46. The full activation of ATR requires the Rad9-Rad1-Hus1 (9-1-1) complex and the Rad17-Rfc2-5 complex to recruit TopBP1, which interacts with the ATR-ATRIP complex. Interaction with TopBP1 increases the activity of ATR to signal downstream effector proteins47–52. The cross talk between the ATM and ATR pathway starts at these early stages, where it has been shown that Rad17 helps to recruit the MRN complex through ATM phosphorylation and conversely the MRN complex can help activate ATR through TopBP153,54. Furthermore ATM and ATR do not solely contribute to either DSBs or SSBs; for example, ATM is able to activate the substrates CtIP and Exo1 which initiate DNA resection at the site of the double stranded breaks, resulting in exposed single stranded DNA that allows for 9  homologous recombination repair of DNA to occur in addition to getting covered by RPA proteins that help recruit ATR 55–58.  ATM and ATR can turn on a wide variety of different effectors and transducers. Their activation results in initiation of cell cycle checkpoints that allow for the repair of the damage or initiation of apoptosis through p53 stabilization 38,59,60. Two of the main downstream targets of ATM and ATR  that are involved in regulating cell cycle progression are the Chk1 and Chk2 kinases61–63. It is understood that ATM is able to directly phosphorylate and activate Chk2 (thr 68) and ATR similarly acts on Chk1 (ser 345); however, there is evidence that ATM phosphorylates Chk1 at a different site (ser 317) as well 62–65. As the gene names of Chk1 and Chk2 suggest, they play a major role in stopping cell cycle progression, and some of their downstream targets include many cell cycle regulatory genes including p53 and the Cdc25 family phosphatases 66–71. Therefore, reduced activation of the Chk1/2 proteins can result in unregulated cell cycle progression that prevents proper repair of the DNA damages or prevent apoptosis of cells with high levels of DNA damage. Unregulated cell cycle progression can potentially lead to growth of a population of cells that are not able to regulate their growth that could become cancerous in the future. Inhibition of these kinases can sensitize cells to DNA damaging agents that are used in chemotherapy because the cells cannot activate pathways that allow for DNA repair to take place72,73.  Consequently, inhibitors targeting Chk1 and Chk2 have been used in combination with DNA damaging agents for cancer treatment clinical trials with mixed results74. 10   Figure 1-2 The ATM/ATR DDR signaling pathway.  DNA damage activates the ATM and ATR kinases to initiate a signaling pathway that can initiate apoptosis, cell cycle arrest, and DNA repair. The checkpoint proteins (Chk1, Chk2, p53, and p21) can help initiate cell cycle arrest. Histone protein (H2AX) and other faciliatory proteins (Rad17) can help recruit other DDR proteins to the site of damage. The pro-apoptotic proteins (p53) will induce cell death if the DNA cannot be repaired. ATM ATR Chk2 Chk1 H2AX P53 P21 Rad17 Apoptosis Cell cycle arrest DNA repair 11  1.3.2 P53 response  Arguably one of the most important downstream proteins in the ATM/ATR DDR pathway is the transcription factor and tumor suppressor p53 (Figure 1-3). Early studies have shown that p53 null mice are highly susceptible to forming tumors both spontaneously and in response to ionizing radiation treatment, pointing to its role in protecting the organism against cancerous cells75,76. Under basal conditions the levels of the p53 protein are kept low by protein degradation in a MDM2 ubiquitin ligase regulated manner77–79. p53 is quickly upregulated by the DDR pathway through phosphorylation of its ser15 site by kinases that include ATM, ATR, Chk1, and Chk2. Phosphorylation of the ser15 sites results in protein stabilization and inhibits its degradation60,66,80. Indeed, kinases at different levels of the DDR signaling pathway stabilize p53, highlighting the importance that p53 plays in DDR. After it is activation, p53 has many downstream targets that are part of the cell cycle checkpoint, DNA repair, or apoptotic pathways (figure 1-3)81.  There is no clear understanding of how cells decide between apoptosis or cell cycle arrest and senescence; however, two models that involve p53 activity are in contention. One model proposes that high p53 protein levels induce transcription of pro-apoptotic genes with lower promoter affinity towards p53, while low levels of p53 induces transcription of pro-cell cycle arrest genes whose promoters have a higher affinity for p5382–84; however, later studies showed that promoter affinity of some genes for p53 do not fit this model85,86. The other model proposes that the decision depends strongly on the kinetic expression pattern of p53 and less so on total p53 protein levels. Therefore cells that express p53 in short pulses can induce the transcription of cell cycle arrest and DNA repair genes but not pro-apoptotic and pro-senescent genes; however, when p53 is expressed continuously over a long period of time the pro-apoptotic genes are transcribed 12  at high levels 86–89. Both models demonstrate that regulation of p53 expression is vital in deciding whether a cell can go through apoptosis or live after DNA damage and it is very likely that a combination of both models is true. These models also demonstrate how p53 expression can either increase sensitivity by inducing apoptosis or decrease sensitivity by inducing cell cycle arrest to allow for DNA repair. As Purvis et al. (2012)89 demonstrated, the p53 expression kinetics can differ between the source of gentotixc stress, and therefore the p53 induced phenotypes can be different depending on the types of DNA damage.  During DDR, p53 can halt cell cycle progression at the G1/S, S/G2, and M phase checkpoints by acting on its downstream targets90–93. Halting cell cycle progression after DNA damage detection can allow extra time for the cell to repair any potential DNA damages, while a permanent stop of cell cycle progression can lead to senescence and prevent expansion of cells with damaged DNA94,95. The most prominent of p53’s transcriptional target genes is p21, a cyclin dependent kinase (CDK) inhibitor which arrests cells at specific phases of the cell cycle96,97. p21 is required for G1/S and G2/M cell cycle arrest after genotoxic stress92,98. p53 and p21 do not activate the initial and rapid G1 cell cycle checkpoint. Instead, Chk1 and Chk2 activate CDC25A to dephosphorylate Cdk2 resulting in rapid and transient cell cycle arrest99,100. Since a phosphorylation event is a quicker than transcription and translation of p21, dephosphorylation of Cdk2 by activated CDC25A can transiently halt cell cycle progression until more stable mechanisms become active. p53 mediates a more delayed G1 cell cycle arrest response because it must transcriptionally activate its target genes, including p21, which halts cell cycle for a longer period of time or permanently 94. p53 has also been shown to play a role in S-phase progression during the DNA damage response, even though the evidence is much sparser when compared to G1, G2, and mitotic cell cycle arrest. In particular, it has been shown that p53 null cells have a 13  slower S-phase progression compared to p53 WT cells101–103. The slower S-phase progression in p53 null cells is thought to be caused by the inhibition of DNA replication of damaged DNA at the initiation and elongation phases101,103. p53 also inhibits the S-phase progression of cells with more than 4N DNA content, but at the same time it can prevent mitosis if the cell has unreplicated DNA104,105. Similarly to the G1 cell cycle checkpoint, p53 does not initiate G2 cell cycle checkpoints but keeps the cells arrested at G2 phase after the activation of the DDR92. After DNA damage induction, p53 directly represses the transcription of the G2 cell cycle progression promoter CDC25C, while promoting the transcription of the 14-3-3 gene which enhances G2 cell cycle arrest106,107. p53 also enhances G2 cell cycle arrest indirectly through its other targets, p21 and GADD4592,108,109.  The earliest studies on p53 and apoptosis demonstrated that thymocytes lacking p53 expression have reduced activation of the apoptotic pathways after exposure to ionizing radiation110,111.  p53 was shown to enhance apoptosis in response to other genotoxic agents that cause different forms of DNA damage, ranging from UV radiation to etoposide and cisplatin112–114. p53 can induce apoptosis by increasing transcriptional activity of pro apoptotic Bcl2 family genes, PAX, PUMA, and NOXA115–118. The Bcl-2 family genes are major regulators of apoptosis, where some Bcl-2 family genes act as apoptosis executioners by permeabilizing the mitochondrial membrane while other genes in that family enhance or inhibit the activity of the executioner genes119. p53 and some members of the Bcl-2 family proteins can interact directly, and this protein-protein interaction enhances mitochondrial permeabilization activity of these proteins during apoptosis120–122. p53 can also inhibit the transcriptional expression of anti-apoptotic Bcl-2 genes, leading to an enhanced pro-apoptotic response123,124. However, the pro-apoptotic activity of p53 is not limited to interaction with Bcl-2 family genes. p53 can enhance and inhibit transcriptional 14  expression of other pro-apoptotic and anti-apoptotic genes, such as PERP and Survivin respectively125–127. It also can promote survival after DNA damage by promoting the expression of its target genes that are involved in DNA repair128,129. It promotes different DNA repair pathways, including mismatch repair and nucleotide excisions repair 130,131. Some of these genes are discussed in more detail in the next section within the context of cisplatin DNA repair.  In conclusion, p53 plays a vital role in DDR and depending on the magnitude of the DNA damage, it can promote cell death or survival by enhancing apoptosis, senescence, cell cycle arrest, or DNA repair. 15   Figure 1-3 Upregulation of p53 with DNA damage and its downstream targets p53 is upregulated in response to DNA damage by the kinases ATM, ATR, Chk2, and Chk1. p53 can in turn induce apoptosis, DNA repair, and cell cycle arrest by upregulating its target genes. 1.3.3 Cisplatin As mentioned earlier cytotoxic agents that cause different types of DNA damage have been used to treat cancer patients for many decades. For the purposes of this study cis-Dichloro (ethylenediamine) platinum (II), more commonly known as cisplatin, will be discussed.  Cisplatin was originally synthesized in the 19th century by Michele Peyrone but its earliest anti-growth properties were observed in Escherichia coli by Barnett Rosenberg in 1965, which led to testing 16  its anti-tumor properties in animals and since has become one of the most widely used chemotherapeutic agents132–134. Cisplatin is included in the chemotherapeutic regiment of medulloblastoma patients, where eEF2K expression has been shown to have a negative correlation with patient outcome1. Cisplatin’s molecular structure consists of a platinum group surrounded with two chloride ions and two amine groups. It belongs to the platinum group of chemotherapeutic drugs but it is also considered an alkylating like agent because it does not carry an alkyl group but damages the DNA in a similar fashion to alkylating agents. Cisplatin’s interaction with DNA prevents proper DNA replication to take place during S-phase, resulting in inhibition of DNA replication and formation of DSBs that are the more harmful form of damaged DNA. Cisplatin interacts with DNA in three different forms, as intra-strand DNA crosslinks, inter-strand DNA crosslinks (ICLs), and DNA-protein crosslinks. It most commonly causes intra-strand crosslinks of the DNA, where the cisplatin molecule covalently binds two adjacent nucleotides, usually two guanine nucleotides, on the same DNA strand resulting in its distortion 135–137. ICLs are caused by cisplatin covalently binding to two guanine nucleotides on two opposite DNA strands 135,136,138. Lastly, it can also cause a crosslink between DNA and proteins, but this comprises the smallest group of cisplatin adducts 139. Even though each lesion occur at different frequencies, there is no clear evidence as to how much the different types of lesion are contributing to cell cytotoxicity, and it is very likely that all three contribute to cisplatin’s cytotoxic effects140. 1.3.4 Repair of Cisplatin Inter-crosslinks DNA   Nucleotide excision repair (NER) pathway is an important pathway that removes DNA lesions that are caused by different sources, including UV radiation and cisplatin (figure 1-4). The first step in NER involves the Rad23b-XPC protein complex which detects the lesion, and then recruitment of the TF2H protein complex to unwind the damaged region of DNA141–143. The DNA 17  in its open formation recruits XPA and RPA, which respectively bind to the damaged DNA strand and single stranded DNA144–146, and these factors in turn recruit exonucleases/endonucleases XPG and XPF-ERCC1 which remove the damaged region of DNA146–150. Lastly the empty region of DNA gets filled and sealed with DNA polymerase factors151,152 (figure 1-4).   The repair of crosslinks caused by cisplatin can involve different pathways153. Repair of ICLs consists of detecting the crosslink, removing and unhooking the crosslink, and lastly to repair the removed region of DNA. The NER pathway is the best understood mechanism that the cell uses to repair intra-strand crosslink adducts148,154. If the cells have entered S-phase and DNA replication is taking place, ICLs can produce DSBs indirectly through their repair process. The incision of DNA on either side of the ICL, or ‘unhooking’, during replication results in DSBs if it is left un-repaired. The NER pathway also plays a role in ICL repair, where XPF-ERCC1 and XPG nucleases excise unpaired DNA nucleotides near the ICL site155,156.  Downregulation of NER genes will reduce the ability of the cell to repair damage caused by cisplatin, and consequently upregulation of NER genes have been associated with cisplatin resistance and their downregulation linked to enhanced cisplatin sensitivity157–160 . Most of the NER genes are associated with replication independent ICL repair; however, the ERCC1-XPF complex has been shown to play a prominent role in replication dependent repair by interacting with homologous replication repair (HRR) and fanconi anemia (FA) pathways161–163. Expression of ERCC1 and XPF are required to reduce DSB levels after cisplatin treatment164. The ERCC1 gene is one of the highly-studied genes in the pathway and its upregulation is associated with cisplatin resistance in various tumors and its down regulation is linked with improved outcome in patients under cisplatin treatment157,165,166.   Other DNA repair pathways and proteins also get activated with cisplatin cytotoxicity because ICLs are complex DNA damaging structures that can give rise to single stranded DNA 18  breaks and DSBs. The FA pathway is involved in repairing ICLs caused by cisplatin while HRR and non-homologous end joining (NHEJ) repair can repair the DSBs that are indirectly formed by cisplatin167–169. One of the earliest events of the DNA repair process is the phosphorylation of H2AX at the ser139 site by ATM, this phosphorylated form of H2AX is also known as γH2AX170,171. However, ATR can also phosphorylate H2AX at the same site in the presence of single stranded DNA, so the presence of γH2AX is not restricted to DSBs172. HR, NHEJ, and FA DNA repair pathways are intensified with phosphorylation of H2AX because Brca1, 53BP1, and FANCD2 accumulate at sites of γH2AX, resulting in promotion of their respective repair pathways173–179. In summary, the full repair of DNA damages caused by cisplatin involves many different interacting pathways and proteins that repair the different intermediaries of damaged DNA. 19   Figure 1-4 Nucleotide excision repair pathway The Rad23b-XPC complex detects the DNA lesion (a), which recruits theTF2H protein complex (b) and (c). The TF2H complex unwinds the DNA (d). RPA and XPA proteins are recruited to single stranded DNA and damaged DNA strand (e). The XPF-ERCC1 and XPG protein complexes are recruited to the open DNA (e) and cut off the damaged DNA lesion (f). The DNA polymerase factors fill up and seal the empty DNA region that was cut out (g), resulting in an undamaged double stranded DNA.  20  Chapter 2: Materials and methods 2.1 Cell culture  All cells were grown in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS). The immortalized wild type (WT) and eEF2K-deficient mouse embryonic fibroblasts (MEFs) were gifts of Dr. Alexey Ryazanov (University of Medicine and Dentistry of New Jersey). The MEFs were immortalized through simian virus (SV40) large T antigen transduction. Immortalization was carried out by the Ryazanov lab. Using retroviral transduction, the MSCV plasmid expressing eEF2K and the empty MSCV vector were added into the eEF2K deficient MEFs to create the eEF2K addback MEFs referred to as eEF2K-/- (rescue) and its negative control counterpart referred to as eEF2K-/- (MSCV). Puromycin was used to select for cells that were successfully transduced and stably express eEF2K or the empty MSCV vector.  Cisplatin crystalline was bought from Sigma-Aldrich (cis-Diammineplatinum (II) dichloride- P439) and dissolved in DMSO at 50 mM stock concentration and stored at -20℃. Cisplatin stock solutions older than 2 months were not used. Camptothecin powder (Sigma-Aldrich) was dissolved in DMSO at 15 mM stock concentrations and hydroxyurea was dissolved at 658 mM stock concentrations in water and stored at -20℃. eEF2K expression was knocked down in HEK293 cells by transfection with sheEF2K lentiviral plasmid pLK0.1 (Sigma-Adlrich) with the following sequences: sheEF2K 1- CCG GCC ACT CAT ACA GTA ATC GGA ACT CGA GTT CCG ATT ACT GTA TGA GTG GTT TTT and sheEF2K 2- CCG GCG ATGA GGA AGG TTA CTT CAT CTC GAG ATG AAG TAA CCT TCC TCA TCG TTT TT. As controls, HEK293 cells were transfected with pLK0.1 lentiviral plasmid with scrambled non-targeting sequence (Sigma-Aldrich). Puromycin was used to select for the cells that were successfully transduced.  21  2.1 Foci and micronuclei measurements  Cells were plated on coverslips one day prior to the treatment. At the end of the time point the cells were fixed in 4% paraformaldehyde (PFA) in PBS for 15 minutes. The PFA was washed off and coverslips were stored in PBS (phosphate buffered saline) at 4℃ overnight. The cells were permeabilized in 0.2% PBST (PBS Triton) for 30 minutes and the cells were blocked in 5% bovine serum albumin (BSA; Thermo-Fisher) in 0.2% PBST. Cells were incubated with primary antibodies for one hour at room temperature at the desired concentration. The primary antibodies were washed off with 3 ten-minute washes with 0.2% PBST. The cells were incubated for 1 hour with the secondary antibody. The cells were washed with 3 ten-minute washes with PBST (0.2%). Cover slips were mounted on slides with mounting media containing DAPI (Vectasheild) and sealed with clear nail polish. Pictures of the fluorescent stain were taken using the Zeiss Axio Colibri fluorescent microscope. At least one hundred cells were measured for each replicate. For the quantification of the γH2AX and 53BP1 foci the ImageJ software was used to detect the maximal points of fluorescence per nucleus. The average number of foci per nucleus per biological replicate were measured. The micronuclei were visualized through the DAPI stain.  The foci measurements were carried out in technical or biological triplicates. In a technical triplicate a coverslip was divided into three equal sections and the foci of 150 cells at a minimum were counted. Subsequently the data was represented as the mean value of the three sections in a coverslip. The biological replicate represents three independently carried out experiments on three different coverslips. The foci of 150 cells per coverslip was measured and the data was represented as the mean value of the three independent coverslips.   22  2.2 Immunoblot analysis  Cells were treated with cisplatin or DMSO for the specified time, then washed with PBS. Whole cell lysates were collected using the lysis buffer (10 mM HEPES, 50 mM KCl, 5 mM MgCl2, and 0.5% NP-40). Whole cell lysates were stored at -80℃. Protein concentration was measured by using the Bradford protein assay (Bio-rad) and the lysate protein concentrations were equalized. 6X loading buffer was added to lysates to reach a final concentration of 1X. Lastly the lysates were boiled at 90℃-100℃ for 10 minutes.  Antibodies that were used are: 53BP1 (Santa Cruz), ATM (Cell Signaling), p-ATMser1981 (Human: Cell Signaling, Mouse: Millipore) , ATR (Cell Signaling), p-ATRthr1989 (Cell Signaling), Actin (Santa Cruz), cleaved Caspase 3 (Cell Signaling), PARP (Cell Signaling), Chk1 (Cell Signaling), p-Chk1ser345(Cell Signaling), Chk2 (Cell Signaling), p-Chk2thr68 (Cell Signaling), ERCC1 (Aviva Systems Biology), p21 (Santa Cruz), p53 (Cell Signaling), p-p53ser15 (Cell Signaling), H2AX (Cell Signaling), γH2AX (Cell Signaling), eEF2K (Abcam), eEF2 (Cell Signaling), and p-eEF2thr56 (Cell Signaling). Densitometry analysis of immunoblots was carried by using the ImageQuant software, where the software measures the density of each band while excluding the background. For normalization the density value of protein was divided over the density value of it respective actin band. 2.3 Quantitative RT-PCR  Wild type (WT) and eEF2K knockout (KO) MEFs were treated with 50 µM of cisplatin or DMSO. At the end point the cells were washed with PBS and total RNA was collected using a commercially available kit (Qiagen RNeasy RNA isolation kit). The cDNA was synthesized by using another commercially available kit (Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit). The following tp53 primer sequences were used to amplify the TP53 cDNA: 23  forward 5’-ACG CTT CTC CGA AGA CTG G -3, and reverse 5’- AGG GAG CTC GAG GCT GAT A -3’ and the actin cDNA was amplified by using the following primer sequences: forward 5’- GTG ACG TTG ACA TCC GTA AAG A-3’ and reverse 5’- GCC GGA CTC ATC GTA  CTC C-3’(IDT). The qRT-PCR results were analyzed comparing the cycle threshold (CT) values of tp53 to the ct values of the reference gene, actin.  2.4 siRNA Transfection  Cells were plated at ~15% confluency and transfected with 25 nM siRNA using RNAi Lipofectamine (Invitrogen). The cells were left to grow for 48 (sensitivity assays) or 72 hours (immunoblot assays) before being treated with cytotoxic drugs. Stealth RNAi negative control duplexes, medium GC Duplex; (Invitrogen) was used as a control for all of the RNAi experiments. Pooled mouse eEF2K siRNA (Santa Cruz) was used to knockdown eEF2K expression. siRNAs that were used to knockdown p53 expression include pooled mouse p53 siRNA (Dharmacon) and mouse p53 siRNA with sequence sip53-1 GUA AAC GCU UCG AGA UGU U and siP53-2 AAA UUU GUA UCC CGA GUA U (Dharmacon).  2.5  MTT Assay  Cells were plated at a confluency of 30% on a 6 well plate or 12 well plate and treated with the respective drug concentrations. At the specified time points the media containing the drug was removed. Subsequently the cells were incubated with MTT (5mg/mL) in PBS at a concentration 1 in 20 in fresh media. The cells were left in the incubator for 3-4 hours. The medium was removed and 1 mL of DMSO was added to the cells to dissolve the formazan crystals. After the crystals had fully dissolved, 80 µL of the solution was added to a 96 well plate, and absorbance was measured by a plate-reader (Molecular Devices: SpectaMax i3) at an absorbance value of 590nm and an absorbance value of 690nm was used as a control.  24  2.6 Trypan blue cell death assay  Cells were plated at a confluency of 30% on a 6 well plate or 12 well plate and treated with the respective drug concentrations. At the specified time points, all of the floating cells and adhesive cells were collected by trypsin treatment. The cells were washed with PBS and diluted at the appropriate concentration. The cells in PBS and Trypan Blue were mixed at a 1:1 concentration and 10 µL of the mixture was applied to the hemocytometer. The total number of cells and trypan blue positive dead cells were counted per 16 square grids. The percentage of dead cells was measured as the number of trypan blue positive cells over the total number of cells per 16 square grids. The average percentage of dead cells of three different grids was measured. 2.7 Measurement of inter-crosslink repair  Cells were treated with cisplatin at the specified concentration for 2 hours, followed by removal of the media and two washes of PBS. Fresh media was given to cells and they were allowed to recover from the cisplatin treatment. At the specified time the cells were treated with 5 Grays of X-ray radiation and collected immediately after with trypsinization. A modified alkaline comet assay was used to measure the repair of intercrosslinks180. About 15000 cells were suspended in 1% agarose gel and embedded on a microscope slide. The cells were lysed overnight at 4℃ in alkaline lysis solution (. The next day the lysis solution was washed off and electrophoresis was carried out in alkaline solution free of detergent for 20 minutes at 20 V. The comets were stained with PI solution. The comets were measured using a Zeiss Axio Colibri fluorescent microscope. Each condition was carried out in triplicate, and 50-100 comets were measured per replicate. The software Casplab was used to measure the comet tail moment. Tail moment is measured as: 25  DNA in the tail ∗  length of tail percent of DNA in tail ∗ length of tail= Tail moment  “DNA in the tail” represents total fluorescent signal measured in the tail of the comet. “Length of tail” is a measure of how long the comet tail is from the edge of the comet head to the tip of the comet tail. “Percent of DNA in tail” is the percentage of total fluorescent signal in the tail when compared to the combined fluorescent signal in the head and the tail.  2.8 Flow cytometry  Cell cycle profiles were measured through bivariate flow cytometry. A modified cell cycle analysis protocol was used to measure the cell cycle profile of the cells after cisplatin treatment181. The cells were plated on 4 cm plates at a confluency where they would not reach above 90% confluency throughout the treatment. They were treated with the specified concentration of cisplatin, and one hour prior to collection the cells were treated with BrdU at a final concentration of 10 µM to label the cells in S-phase. The cells were collected through trypsinization, and re-suspended in 500 µL of PBS. The cells were fixed in 70% ethanol and stored at -20℃. The ethanol was washed off the cell with PBS. BrdU-FITC conjugated antibody (BD Biosciences) was used to label the S-phase cells and PI to label the overall DNA content. Flow cytometry analysis was carried out on BD FACSCalibur machine. The FlowJo software was used to analyze the results. All doublet cells were excluded from the analysis by comparing the pulse area vs. pulse width and omitting the cell population that had higher pulse area and pulse width values than the single cell population. Sub-G1 cells and dead cells were also omitted from the analysis by excluding the population of cells that had lower pulse area values than the G1 population. 26  2.9 Statistical analysis  The mean and standard deviation (S.D.) or standard error of the mean (S.E.) were measured as the replicates of each condition allowed. GraphPad PRISM software was used to carry out unpaired Student’s t-test to assess the significance of the quantifiable differences between WT MEFs and eEF2K KO MEFs. 27  Chapter 3: eEF2K alters the cellular response to cisplatin 3.1 Introduction and approach  It has been reported that eEF2K enhances sensitivity to genotoxic stress in MEF cells treated with hydrogen peroxide and doxorubicin2. We decided to use cisplatin as the chemotherapeutic drug of choice because it is used regularly in treating medulloblastoma and eEF2K is highly expressed in medulloblastoma patients with worse outcome, as was explained in the Introduction. Since doxorubicin and cisplatin initiate DNA damage through different mechanisms of action, the sensitivity phenotype might be different as well. My first goal was to quantify how eEF2K alters sensitivity to cisplatin. eEF2K expression enhanced sensitivity to chronic or continuous cisplatin treatment which is a similar phenotype to how eEF2K affects sensitivity to doxorubicin and hydrogen peroxide treatment. The activation of ATM and ATR starts a signal transduction pathway that can lead to apoptosis. Therefore, my second goal was to examine the activation of the DDR pathway, starting with ATM, ATR and their downstream proteins. Through immunoblot analysis I determined that eEF2K can enhance the DDR pathway, starting from the initiation of the pathway with ATM and ATR to further downstream at the effector level with p53 and p21. Although there were variabilities on how effective eEF2K was in increasing the levels of activated and phosphorylated proteins in the DDR pathway, depending on the cell lines or the concentration of drugs used. p53 is a major inducer of apoptosis in response to DNA damage and its downregulation can lead to resistance to cytotoxic agents. Accordingly, my last goal was to see if p53 can act as a major inducer of apoptosis in response to cisplatin in our system. I transiently knocked down p53 prior to cisplatin treatment and confirmed its pro-apoptotic role. In conclusion, p53 enhances apoptosis in response to cisplatin treatment and eEF2K can enhance p53 activation. Therefore, eEF2K increase sensitivity to chronic or continuous cisplatin 28  treatment by helping to enhance the activity of the DDR pathway which leads to apoptosis through p53 activity.    3.2 eEF2K enhances sensitivity to cisplatin  Cells were plated at 30% confluency in 6 well plates and treated for 48 hours with a range of cisplatin concentrations (1.25 µM, 2.5 µM, 5 µM, 7.5 µM, 10 µM) and mock cells were treated with DMSO. After cisplatin induced a measurable level of cell death, cell viability was measured using MTT assays and the percentage of dead cells was measured using the trypan blue assay. The cisplatin concentrations at which cell death was observed were different between the MEF and HEK293 cell lines. This could be attributed to their baseline sensitivity to cisplatin. A 48-hour endpoint was chosen for the viability assays carried out on the MEFs because there was no major cell death at 24 hours but very high levels of cell death were observed at 72 hours in the WT and eEF2K KO MEFs. However, the end point used for HEK293 cells was 72 hours because they were more resistant to cisplatin than the MEFs. Each condition was carried out in biological triplicates. After the 48-hour treatment period the WT MEFs had a significantly lower percentage of viable cells and higher percentage of dead cells when compared to the eEF2K KO MEFs (Figures 3-1 and 3-2). Immunoblots for cleaved caspase 3 and cleaved PARP confirmed that the higher sensitivity in the WT MEFs was due to higher apoptosis (Figure 3-3). eEF2K KO cells transfected with MSCV and eEF2K expressing plasmid, respectively referred to as eEF2K-/- (MSCV) and eEF2K-/- (rescue) cells, were then used to confirm the cell sensitivity findings. The eEF2K-/- (rescue) cell line is overexpressing eEF2K, meaning it is expressing eEF2K at much higher levels than the WT MEFs. The MTT cell viability assay was repeated on these cells and confirmed that eEF2K sensitizes cells to cisplatin (Figure 3-4). To confirm these findings in another cell line, 29  HEK293 cells were transfected with two shRNA plasmids to stably knockdown eEF2K, and a non-targeting shRNA sequence was used as a negative control. HEK293 cells were treated with cisplatin for 72 hours, and again I observed that cells with eEF2K knockdown were more resistant to cisplatin treatment (figure 3-5). In conclusion, I confirmed that eEF2K expression enhances short-term cell sensitivity to cisplatin by enhancing apoptosis.   Much of the cytotoxicity caused by cisplatin occurs at S-phase because cisplatin DNA crosslinks cause extensive damage during DNA replication. In order to test if other S-phase specific cytotoxic drugs cause the same sensitivity phenotype as cisplatin I used two other drugs, camptothecin and hydroxyurea. Camptothecin is a DNA topoisomerase inhibitor which causes replication fork arrests during DNA replication, and hydroxyurea slows down DNA replication by inhibiting the production of dNTPs. The MTT assay was used again to measure the viability of the WT, and eEF2K KO MEFs that were treated with a range of drug concentration for 48 hours. The eEF2K KO cells were significantly more resistant to camptothecin (Figure 3-6). However, only the highest dosage of hydroxyurea showed a significant difference between the viability of WT and eEF2K KO MEFs (Figure 3-7), although there was a trend depicting eEF2K KO MEFs as being more resistant to hydroxyurea. Therefore, I conclude the sensitivity phenotype is not restricted to cisplatin.      30    Figure 3-1 Cell viability of WT and eEF2K KO MEFs after 48 hours of cisplatin treatment.  Immunoblot representing eEF2K expression in eEF2K +/+ MEFs and eEF2K -/- (a). MTT cell viability was performed on eEF2K WT and KO MEFs treated for 48 hours with the specified concentration of cisplatin or DMSO (b). The viability of cisplatin treated cells is presented as a percentage of DMSO treated cells. Each bar represents the mean of three biological replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate.  DMSO1.252.5 57.5 1005 01 0 0C is p la t in  (u M )Viable cells (%)e E F 2 K  + /+e E F 2 K  - / -****a) b) 31   Figure 3-2 Cell death of WT and eEF2K KO MEFs after 48 hours of cisplatin treatment.  Trypan blue assay was performed on eEF2K WT and KO MEFs treated for 48 hours with the specified concentration of cisplatin or DMSO. The percentage of trypan blue positive cells are represented as a percentage of whole population of cells that were measured. Each bar represents the mean of three technical replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate.  Figure 3-3 Apoptotic response of WT and eEF2K KO MEFs after 48 hours of cisplatin treatment.  Immunoblot analysis was performed on eEF2K WT and KO MEFs treated with 5 µM of cisplatin ranging from 0 to 24 hours. Total protein was extracted for immunoblot analysis using Cleaved PARP and caspase 3 antibodies to measure apoptotic response. Immunoblot of actin was used as a loading control. These blots are representative of three independent experiments. DMSO1.252.5 57.5 1002 04 06 08 01 0 0C is p la t in  (u M )Trypan blue positive (%)e E F 2 K  + /+e E F 2 K -/ -***32   DMSO1.252.5 57.5 1005 01 0 0C is p la t in  (u M )Viable cells (%)e E F 2 K  - / -  (M S C V )e E F 2 K  - / -  ( re s c u e )* ** * Figure 3-4 Cell viability of eEF2K-/- (MSCV)  and eEF2K-/- (rescue) MEFs after 48 hours of cisplatin treatment.  Immunoblot representing eEF2K expression in eEF2K-/- (MSCV) MEFs and eEF2K KO MEFs with addback of eEF2K expression (eEF2K -/- rescue) (a).  MTT cell viability was performed on eEF2K - and eEF2K + MEFs treated for 48 hours with the specified concentration of cisplatin or DMSO. The viability of cisplatin treated cells is presented as a percentage of DMSO treated cells (b). Each bar represents the mean of three biological replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate. a) b) 33   0152005 01 0 0C is p la t in  (u M )Viable cells (%)s h C trls h  e E F 2 K  1s h  e E F 2 K  2** ** Figure 3-5 Cell viability of HEK293 cells with stable knockdown of eEF2K or with no knockdown of eEF2K after 72 hours of cisplatin treatment. HEK293 cells with stable KD of eEF2K and a control cell line stably expressing scrambled shRNA were used (a). MTT cell viability was performed on HEK293 cells treated for 72 hours with the specified concentration of cisplatin or DMSO (b). The viability of cisplatin treated cells is presented as a percentage of DMSO treated cells. Each bar represents the mean of three biological replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate. a) b) 34   Figure 3-6 Cell viability of WT and eEF2K KO MEFs after 48 hours of camptothecin treatment.  MTT cell viability was performed on eEF2K WT and KO MEFs treated for 48 hours with the specified concentration of camptothecin. The viability of camptothecin treated cells is presented as a percentage of DMSO treated cells. Each bar represents the mean of three biological replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate.   00.1250.250.50.75 105 01 0 0C a m p th o te c in  (u M )Viable cells (%)e E F 2 K  + /+e E F 2 K  - / -****35   Figure 3-7 Cell viability of WT and eEF2K KO MEFs after 48 hours of hydroxyurea treatment.  MTT cell viability was performed on eEF2K WT and KO MEFs treated for 48 hours with the specified concentration of hydroxyurea. The viability of camptothecin treated cells is presented as a percentage of DMSO treated cells. Each bar represents the mean of three biological replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate. 3.3 eEF2K supports induction of the ATM/ATR DNA damage response  The enhanced sensitivity of the eEF2K expressing cells to cisplatin could be due to a downstream effect of the ATM and ATR DDR pathways. Activated ATM, ATR, and their downstream targets, Chk1 and Chk2, stabilize the p53 protein by phosphorylating it on the ser15 site60–67,80. Stabilized p53 transcriptionally upregulates different genes, many of which include pro-apoptotic proteins such as PAX, PUMA, and NOXA115–118. Therefore, in response to DNA damage an enhanced ATM/ATR activity results in upregulation of p53 and its pro-apoptotic targets, which can result in higher cell death.   WT and eEF2K KO MEFs were treated with cisplatin at a concentration of 5 µM and 50 µM or DMSO for up to 6 hours and whole cell lysates were collected at 2-hour intervals. 50 µM 050758510015005 01 0 0H y d ro x y u re a  (u M )Viable cells (%)e E F 2 K  + /+e E F 2 K  - / -*36  is a high concentration of cisplatin and it was chosen to ensure there is substantial activation DDR pathway. Time points earlier than the cell death assays were chosen because it allows me to look at the initial activation of the DNA damage response, before cell cycle arrest, DNA repair, and cell death pathways were activated. Immunoblot analysis targeting proteins in the ATM/ATR signaling pathway were used to assess pathway activation (Figure 3-8). The WT MEFs show higher expression of the total levels of ATM and ATR proteins, as well as of their activated phosphorylated forms. The eEF2K KO MEFs show reduced expression of ATM and ATR substrates that have been phosphorylated, including p-Rad17, p-Chk1, p-p53, p21 and γH2AX, as well as reduced expression of total protein levels of Chk1 and p53. I observed a difference in DDR signaling starting from the top transducer kinases of the pathway (ATM and ATR) to the downstream effector proteins that carry out the appropriate responses, where KO cells exhibit low activation of proteins in the ATM/ATR DDR pathway as compared to WT cells. Unfortunately, I was not able to assess activation of Chk2 because antibodies targeting mouse Chk2 and p-Chk2thr68 did not produce measurable immunoblots.  To confirm that these findings are not caused by adaptation of the eEF2K KO MEFs, we used a non-targeting siRNA and siRNA targeting eEF2K to knockdown its expression in WT MEFs. After KD, the MEFs were treated with 5 µM of cisplatin for up to 6 hours. The knockdown of eEF2K was not very strong but subdued differences were still observed in the expression levels of the activated forms of the proteins involved in the ATM/ATR pathway (Figure 3-9c). Cells with eEF2K KD show lower expression of many of the activated proteins in the ATM/ATR pathway, as was observed with lower expression of p-Chk1, p53, p-p53, and p21. I specifically carried out a densitometry analysis of p53 and p-p53 immunoblots in Figure (3-9c) and confirm that p53 and p-p53 levels are higher in siCtrl cells at 6 hours after cisplatin treatment (Figure 3-9d).However, I 37  cannot conclude if there are differences in expression levels of γH2AX, p-ATR, and p-ATM between the two cell lines. In addition, there were no differences in the total protein levels of ATM, ATR, and Chk1 (Figure 3-9c). To see if adding back eEF2K expression to the eEF2K KO MEFs can reactivate these pathways, we treated eEF2K-/- (MSCV)  and eEF2K-/- (rescue) MEFs with 5 µM, 10 µM, and 50 µM of cisplatin or DMSO for 6 hours. When treated with 5 µM of cisplatin the eEF2K-/- (rescue) cells expressed higher levels of the activated proteins in the ATM/ATR pathway as compared to the eEF2K-/- (MSCV)  cells but minor differences were observed at 10 µM and no differences were observed between the two cell lines at 50 µM. Also, there was no difference in the total protein expression levels of ATM, ATR, and Chk1 (Figure 3-9a), indicating that eEF2K does not increase translation of these proteins but promotes their activation. I specifically carried out a densitometry analysis of p53 and p-p53 immunoblots in Figure (3-9a) and confirm that p53 and p-p53 levels are higher in eEF2K-/- (rescue) cells with the strongest difference being at 5 µM.  To confirm that our findings were not restricted to MEF cells, HEK293 cells were treated with 10 µM, 20 µM, and 50 µM of cisplatin or DMSO for up to 6 hours. Despite the weak knockdown of eEF2K there was a stronger downregulation of the p-eEF2 protein, indicating that the knockdown is downregulating eEF2K activity. HEK293 cells with eEF2K KD had lower activation of most of the proteins in the ATM/ATR pathway compared to Scr cells, including p-Chk1, γH2AX, p53, p-p53, p-Rad17 and in addition lower Chk2 activation; however, I do not observe any differences in the expression of p-ATR and p-ATM between the Scr cells and eEF2K KD cells unlike what was observed in the MEF immunoblots (Figure 3-10a). There was no difference in the total protein levels of ATM, ATR, and Chk1 but the total Chk2 levels were lower in the eEF2K KD cells as compared to Scr cells. The HEK293 cells were treated with 50 µM of 38  cisplatin for up to 6 hours and whole cell lysates were collected at 2-hour intervals, 50 µM of cisplatin was chosen because that was the concertation that showed the biggest difference in signaling. Immunoblot analysis using antibodies targeting p53, p-p53ser15, and p21 confirmed that in response to cisplatin, eEF2K expression enhances p53 levels and enhances the levels of p53 target protein p21 in non-MEF cell lines as well (Figure 3-10b).  Localization of DDR proteins to the site of DNA damage is one of the methods used to measure activity of DDR. Two of these proteins that are regularly used to measure DNA damage activity are γH2AX and 53BP1175. γH2AX foci localization is one of the earliest DDR processes that can be visualized, while 53BP1 localization occurs later and it is very important in NHEJ repair175,182. Immunofluorescence (IF) microscopy was next used to look at the localization of γH2AX and 53BP1 foci, since the eEF2K expressing cells have a higher level of γH2AX expression as observed in the immunoblots. The eEF2K WT and KO MEFs were treated for 2 hours with 50 µM of cisplatin to allow for γH2AX and 53BP1 foci localization. After measuring the foci that were present in the DMSO treated cells, all cells with more than ten γH2AX foci and five 53BP1 foci were considered positive for foci localization in response to cisplatin. There were more eEF2K WT MEFs positive for γH2AX and 53BP1 foci localization compared to eEF2K KO MEFs, confirming the immunoblot finding that WT MEFs have enhanced DDR activity (Figure 3-11).  39  ATM p-ATM(ser1981) ATR p-ATR(ser428) Chk1 p-Chk1(ser345) eEF2K P53 γH2AX p-Rad17(ser645) Rad17 eEF2K +/+    0      2       4       6 eEF2K -/- 50 µM Cisplatin (hrs):   0       2       4       6 p-P53(ser15) Actin P21 a) 40   Figure 3-8 Effects of eEF2K on induction of ATM/ATR DDR signaling pathway after cisplatin treatment. eEF2K WT and KO MEFs were treated with (a) 50 µM or (b) 5 µM of cisplatin for up to 6 hours. The control cells were treated with DMSO for 6 hours. Whole cell lysates were collected at 2-hour intervals. Immunoblot analysis was done on the whole cell lysates using antibodies targeting proteins related to the ATM/ATR signaling pathway. Immunoblot of actin was used as a loading control. These blots are representative of two independent experiments.   ATM p-ATM(ser1981) ATR p-ATR(ser428) Chk1 p-Chk1(ser345) γH2AX P53 p-P53(ser15) P21 eEF2K Actin eEF2K +/+  0        2      4      6 eEF2K -/- 5 µM Cisplatin (hrs):   0      2      4       6 b) 41     a) 42  DMSO 5 105001234p 5 3  d e n s ito m e tr yC is p la t in  (µ M )Arbitrary Units (A.U) p53/actine E F 2 K  - / -  (M S C V )e E F 2 K  - / -  ( re s c u e )DMSO 5 10500 .00 .20 .40 .6p -p 5 3  d e n s ito m e tryC is p la t in  (µ M )Arbitrary Units (A.U) p-p53/actine E F 2 K  - / -  (M S C V )e E F 2 K  - / -  ( re s c u e ) b) 43  DMSO2 Hours4 Hours6 Hours0 .00 .51 .01 .5p -A T R  d e n s ito m e tryC is p la t in  (µ M )Arbitrary Units (A.U) p-ATR/actine E F 2 K -/-  (M S C V )e E F 2 K -/-  ( re s c u e ) DMSO2 Hours4 Hours6 Hours0 .00 .10 .20 .30 .40 .5p -A T M  d e n s ito m e tr yC is p la t in  (µ M )Arbitrary Units (A.U) p-ATM/actine E F 2 K -/-  (M S C V )e E F 2 K -/-  ( re s c u e ) 44   Ctrl siRNA    0      2      4       6 sieEF2K  Cisplatin (hrs): 0      2      4       6 ATM p-ATM(ser1981) ATR p-ATR(ser428) Chk1 p-Chk1(ser345) γH2AX P53 p-P53(ser15) P21 eEF2K Actin c) 45  DMSO2 Hours4 Hours6 Hours012345p 5 3  d e n s ito m e tr yH o u rsArbitrary Units (A.U) p53/actins iC tr lS ie E F 2 K DMSO2 Hours4 Hours6 Hours012345p -p 5 3  d e n s ito m e tryH o u rsArbitrary Units (A.U) p-p53/actins iC tr lS ie E F 2 K d) 46  DMSO2 Hours4 Hours6 Hours0 .0 00 .0 50 .1 00 .1 5p -A T R  d e n s ito m e tryH o u rsArbitrary Units (A.U) p-ATR/actins iC tr lS ie E F 2 K DMSO2 Hours4 Hours6 Hours0 .00 .10 .20 .30 .4p -A T M  d e n s ito m e tr yH o u rsArbitrary Units (A.U) p-ATM/actins iC tr lS ie E F 2 K   47  Figure 3-9 Confirming the effects of eEF2K on induction of ATM/ATR DDR signaling pathway after cisplatin treatment in MEFs. siRNA targeting eEF2K was used to KD eEF2K expression in WT MEFs and non-targeting siRNA was used as control.  After KD of eEF2K expression was established the cells were treated with 5 µM of cisplatin for up to 6 hours. Whole cell lysates were collected at 2-hour intervals (a). Densitometry analysis of p53 and p-p53 immunoblots shown in 3-9a (b). eEF2K-/- (MSCV)  and eEF2K-/- (rescue) cells were treated with 5 µM, 10 µM, and 50 µM cisplatin. After 6 hours of treatment whole cell lysates were collected (c). Densitometry analysis of p53, p-p53, p-ATR, and p-ATM immunoblots shown in 3-9c (d). The untreated cells were treated with DMSO for 6 hours. Immunoblot analysis was done on the whole cell lysates using antibodies targeting proteins related to the ATM/ATR signaling pathway. Immunoblot of actin was used as a loading control. These blots are representative of two independent experiment. 48   eEF2K γH2AX P53 Chk2 eEF2 P-eEF2(thr56) ATM p-ATM(ser1981) ATR p-ATR(ser428) Chk1 p-Chk1(ser345) p-P53(ser15) P21 Actin p-Chk2(thr68) p-Rad17(ser645) Rad17 ShScramble    0         10       20       50 SheEF2K 1 Cisplatin (µM): SheEF2K 2    0       10       20       50     0        10      20       50 a) 49   Figure 3-10 Effect of eEF2K on induction of ATM/ATR DDR signaling pathway after cisplatin treatment in HEK293 cells. HEK293 cells with stable KD of eEF2K and a control cell line stably expressing scrambled shRNA were treated with 10 µM, 20 µM, and 50 µM cisplatin. After 6 hours of treatment whole cell lysates were collected. Immunoblot analysis was done on the whole cell lysates using antibodies targeting proteins related to the ATM/ATR signaling pathway (a). To confirm p53 activation in the HEK293 cell line, they were treated with 50 µM of cisplatin for up to 6 hours. Immunoblot analysis was done on the whole cell lysates using antibodies targeting p53, p-p53ser15, and P21 (b). The control cells were treated with DMSO for 6 hours. Immunoblot of actin was used as a loading control. These blots are representative of two independent experiments.  50 µM Cisplatin (hrs): Actin eEF2K P21 P53 ShScramble SheEF2K 1 0        2        4       6 SheEF2K 2 0       2        4       6 0      2        4        6 p-P53(ser15) b) 50     eEF2K WT and KO MEFs were plated on coverslips and treated with 50 µM of cisplatin or DMSO for 2 hours.  The cells were fixed in PFA (4%) and IF microscopy was done using antibodies targeting γH2AX (a, b) and 53BP1 (c, d). Cells with more than 10 γH2AX foci and 5 53BP1 foci were considered positive. Figures a and b are representative images of γH2AX foci (a) and 53BP1 foci (b) that were quantified under each treatment. Each bar represents the mean of three technical replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate. DMSOCisplat in01 02 03 04 05 0 % cellsH2AX foci positivee E F 2 K  + /+e E F 2 K  - / -*DMSOCisplat in01 02 03 04 05 05 3 b p 1  fo c i c e lls % cells 53BP1 foci positivee E F 2 K  + /+e E F 2 K  - / -**eEF2K +/+ eEF2K -/- Cisplatin DMSO eEF2K +/+ eEF2K -/- Cisplatin DMSO b) a) d) c) Figure 3-11 Effect of eEF2K on γH2AX and 53BP1 foci localization after cisplatin treatment in WT and eEF2K KO MEFs. 51   3.4 Role of p53 in the cellular response to cisplatin downstream of eEF2K   eEF2K expressing cells have higher expression levels of p-p53ser15 and its downstream protein p21 after cisplatin treatment compared to their eEF2K deficient counterparts. The higher sensitivity of eEF2K expressing cells compared to eEF2K deficient cells can be due to higher apoptosis caused by the higher p53 levels. The difference in p53 levels is also observed at the mRNA level, as measured by qRT-PCR analysis, where higher p53 transcript levels are present in WT MEFs under basal conditions and under cisplatin treatment. (Figure 3-12). Since p53 regulates its own gene transcription we cannot confirm if higher levels of p53 in eEF2K proficient cells originates from mRNA differences or from differences in p53 protein stability due to ser-15 phosphorylation. There are reduced levels of ATM, ATR, Chk1 (both total and phosphorylated) and γH2AX in the WT compared to the eEF2K KO MEFs. We see similar reduced levels, yet not as large differences, in MEFs with transient knockdown of eEF2K, eEF2K-/- (MSCV)  MEFs, and HEK293 eEF2K KD cells compared to their control counterparts. Indicating that differences in p53 phosphorylation are partly due to reduced activation of ATM/ATR; however, other biochemical pathways can also be playing a role in regulating levels of p53 such as the pathways that feed into MDM2 regulation and p53 acetylation183. The reduced activation of p53 in cells lacking eEF2K expression is a potential explanation for the higher resistance of these cells, since higher p53 activation is associated with increased apoptosis. To investigate this, the next step is to knockdown p53 in the WT MEFs and see if this enhances resistance to cisplatin.  p53 can promote apoptosis during DDR by acting on many of its targets that carry out programmed cell death in response to DNA damage115–118. In the previous section the higher p53 and p-p53 expression in eEF2K expressing cells was demonstrated, and in this section I will 52  provide evidence that p53 expression enhances resistance to continuous or chronic cisplatin treatment.  Cells were plated in 12 well plates at 15% confluency. Two siRNA sequences targeting p53 were used to knock down p53 expression in WT MEFs and nontargeting siRNAs were used as control (Figure 3-13a). When p53 expression was knocked down in eEF2K KO MEFs and WT MEFs, the eEF2K KO MEFs express lower levels of the p53 protein and demonstrate lower p53 upregulation after cisplatin treatment when compared to the WT MEFs (Figure 3-13b). MTT assays were used to measure cell viability in cells with and without p53 knockdown. p53 expression was knocked down and the cells were treated with DMSO or cisplatin at concentrations of 1.25 µM, 2.5 µM, and 5 µM for 48 hours. WT MEFs with p53 KD showed higher viability and resistance to cisplatin than cells treated with nontargeting. siRNAs (Figure 3-14). This suggests that p53 plays a pro-apoptotic role after cells are treated with cisplatin for 48 hours. In future studies, the proapoptotic role of p53 in our model can be investigated by looking at the cleaved caspase 3 and cleaved PARP levels of the MEFs after cisplatin treatment. It is worth mentioning that p53 could play a pro-survival role at lower concentrations of cisplatin or pulse treatments of cisplatin, since it can also promote cell cycle arrest and activate DNA repair pathways82–84.  p53 was also transiently knocked down in eEF2K KO MEFs, siRNA sequences previously used in WT MEFs, to measure their sensitivity after 48 hours of cisplatin treatment. Unlike WT MEFs, the difference in sensitivity to cisplatin of eEF2K KO cells with and without p53 knockdown was not as significant, and only one of the siRNAs at one concentration resulted in a P value that was considered significant (Figure 3-15). Considering that under basal conditions eEF2K KO MEFs have lower p53 expression than WT MEFs, further lowering p53 expression using siRNAs, might not affect the sensitivity phenotype (Figure 3-13b). A potential cause for this 53  difference is that p53 expression levels in the eEF2K KO MEFs could be below the threshold that causes apoptosis, and thus it will not lead to a phenotypic difference when its expression is further reduced. However, we only measured sensitivity after 48 hours of treatment, and it is possible that after longer time points, reduced activation of cell cycle checkpoint and DNA repair pathways influenced by p53 could play a bigger role in cell survival.    These results indicate that eEF2K may be increasing sensitivity to cisplatin through p53. However, they fall short of demonstrating a direct causation between eEF2K and p53, but instead suggest that eEF2K can have downstream effects which directly or indirectly lead to p53 activation and cell death. To confirm our findings, high p53 expression needs to be established in eEF2K KO MEFs prior to cisplatin treatment. If high p53 levels in eEF2K KO MEFs increase cell death, then eEF2K utilizes p53 to promote cell death. 54   DMSO 2 4 60 .0 0 00 .0 0 50 .0 1 00 .0 1 50 .0 2 0T im e  (h o u rs )Arbitrary Units (A.U)e E F 2 K  + /+e E F 2 K  - / -R e la t iv e  p 5 3  m R N A  le v e ls* Figure 3-12 Effect of eEF2K on tp53 mRNA levels after cisplatin treatment in WT and eEF2K KO MEFs. eEF2K WT and KO MEFs were treated with 50 µM of cisplatin for up to 6 hours. The untreated cells were treated with DMSO for 6 hours. Whole cell lysates were collected at 2 hour intervals for qRT-PCR analysis. Primers against tp53 (a) and actin (b) transcript were used to carry out the analysis. Actin is used as the control housekeeping gene DMSO 2 4 60 .00 .20 .40 .6T im e  (h o u rs )Arbitrary Units (A.U)R e la t iv e  A c t in  m R N A  le v e lsb) a) 55  and p53 transcript levels are normalized to the actin levels. Each bar represents the mean of 2 technical replicates. P-value of ≤ 0.05 is represented by (*). The error bars denote S.D of each triplicate.     Figure 3-13 Transient p53 knockdown in MEFs. Two siRNA targeting p53 were used to knockdown p53 expression. Immunoblot analysis of whole cell lysate using p53 targeting antibody was used to confirm the down regulation of the p53 protein and non-targeting siRNA was used as control (a). Pooled siRNA targeting p53 expression was used to downregulate the expression of p53 in WT and eEF2K KO MEFs. Immunoblot analysis on whole cell lysates was done to compare the p53 expression levels in the WT and eEF2K KO MEFs (b). Antibody targeting actin was used as a loading control. These blots are representative of three independent experiments. Si-p53                  -        1            2 p53   Actin b) a) 56  DMSO1.252.5 505 01 0 01 5 0C is p la t in  (µ M )Viable cells (%)e E F 2 K  + /+  s iC tr le E F 2 K  + /+  s i-p 5 3  1e E F 2 K  + /+  s i-p 5 3  2**** Figure 3-14 Effect of p53 on cell viability of WT MEFs after 48 hours of cisplatin treatment.  Two siRNA targeting p53 was used to knockdown p53 expression in WT MEFs and non-targeting siRNA was used as control.  After knockdown of p53 expression was established the cells were treated with the specified concentration of cisplatin or DMSO for 48 hours. Phase-contrast images of MEFs treated for 48 hours 2.5 µM and 5 µM of cisplatin (a). MTT cell viability was performed on MEFs with and without knockdown of p53 treated for 48 hours with the specified concentration of cisplatin or DMSO (b). The viability of cisplatin treated cells is presented as a percentage DMSO 2.5µM 5µM Ctrl siRNA Si-p53 b) a) 57  of DMSO treated cells. Each bar represents the mean of three biological replicates. P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate.  DMSO1.252.5 505 01 0 01 5 0C is p la t in  (µ M )Viable cells (%)e E F 2 K  - /-  s iC tr le E F 2 K  - / -  s i-p 5 3  1e E F 2 K  - / -  s i-p 5 3  2* Figure 3-15 Effect of p53 on cell viability of eEF2K KO MEFs after 48 hours of cisplatin treatment.  Two siRNA targeting p53 was used to knockdown p53 expression in eEF2K KO MEFs and non-targeting siRNA was used as control.  After knockdown of p53 expression was established the cells were treated with the specified concentration of cisplatin or DMSO for 48 hours. MTT cell viability was performed on MEFs with and without knockdown of p53 treated for 48 hours with the specified concentration of cisplatin or DMSO. The viability of cisplatin treated cells is presented as a percentage of DMSO treated cells. P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate.  3.5 Summary  I conclude that eEF2K expression helps sensitize cells to consistent cisplatin treatment as measured by the cell viability assays. Under basal conditions, expression of eEF2K did not have a noticeable impact on cellular proliferation rates, meaning that in the MEF and HEK293 cell line models the eEF2K deficient cell lines proliferated as the same rate as its respective eEF2K 58  proficient cell line. Even though the sensitivity phenotype seen in the eEF2K WT and KO MEFs was confirmed in the eEF2K-/- (MSCV)  and eEF2K-/- (rescue) MEFs and the HEK293 cells by the MTT assay, the cleaved PARP and caspase 3 assays need to be carried out in these cell lines to fully confirm that eEF2K enhances sensitivity and promotes apoptosis in response to cisplatin treatment. The sensitivity to cisplatin could also be attributed to slower apoptotic process in the eEF2K deficient cells since the cell sensitivity assays depict 48 hours. In future experiments by showing more time points we will be able to have better understanding of the cell death kinetics. The camptothecin and hydroxyurea experiments were only carried out in the WT and eEF2K KO MEFs, therefore my findings might be caused independently of eEF2K. To address the role of eEF2K in response to these drugs, the sensitivity experiments need to be repeated in the eEF2K-/- (MSCV) and eEF2K-/- (rescue) MEFs or the HEK293 cell line. Cellular senescence could be contributing to the differences that we observed in cell sensitivity, therefore in future studies senescent cells after cisplatin treatment will be labeled using β-galactosidase to measure how eEF2K can affect cell senescence.   eEF2K is also playing a role in enhancing the ATM and ATR pathways, but my findings suggest that it does not directly regulate their total protein expression levels. The differences between ATM and ATR protein levels is only observed between WT and eEF2K KO MEFs and it is not observed in the other cell models, I do not fully understand why we see these inconsistent results but it might be due to adaptive mechanisms, mice of origin, or other unknown mechanisms. Therefore, the difference observed in the WT and eEF2K KO MEFs is probably independent of eEF2K and caused by other differences between two cell lines. The lower protein expression levels of ATM and ATR seen in eEF2K KO MEFs compared to WT MEFs could have been acquired as an adaptive mechanism in the eEF2K KO MEFs to downregulate the pro-apoptotic pathways 59  promoted by ATM and ATR, but this is only speculation and needs to be further tested. This follows findings of a previous study that demonstrated oocytes from eEF2K KO mice have a higher percentage of low quality cells and demonstrate a lower apoptotic response2. Therefore, in an in vitro setting the eEF2K KO MEFs with lower quality and intact ATM and ATR expression would go through apoptosis at a higher rate while the cells with lower levels of ATM and ATR would be less likely to go through apoptosis and have a higher chance of surviving through each passage. This results in an indirect selection of cells that have lower ATM and ATR protein levels. Alternatively, this difference could also be due to the different mice expressing different levels of these proteins eEF2K is helping to boost the DDR pathway instead of activating it because at high cisplatin concentrations (10 µM and 50 µM) I see minor differences in the expression of the activated proteins in the ATM/ATR pathway of eEF2K knockdown models and the rescue model (figure 3-9).  The weaker differences depicted in Figure (3-9c) between siCtrl and siEF2K treated cells can also be attributed to an incomplete knockdown of eEF2K in addition to high cisplatin concentration (50 µM) as was observed in Figure (3-9a). It should be noted that minor changes in the expression of these proteins could put the cells over the threshold to induce apoptosis and make the difference in the cell either promoting death or survival, as was observed in previous studies concerning p5382–89.   The expression of phosphorylated proteins in the ATM/ATR DDR pathway in the eEF2K-/- (MSCV) and eEF2K-/- (rescue) MEFs matches the WT/KO cell results at 5 µM but at 50 µM we do not see a difference between the phosphorylated protein levels of eEF2K-/- (MSCV) and eEF2K-/- (rescue) MEFs. This discrepancy in findings between the WT/KO MEFS and eEF2K-/- (MSCV) /eEF2K-/- (rescue) could be attributed to the differences in levels of the ATM and ATR proteins and the level of genotoxic stress the cell is experiencing. The role of eEF2K becomes 60  minimized at very toxic doses of cisplatin (e.g. 50 µM) (Figure 3-9a), but if there are also different levels of ATM, ATR, and Chk1 being expressed a difference in signaling will be observed (Figure 3-8a). At less toxic doses of cisplatin (e.g. 5 µM), the cell does not initiate a strong DDR signal and can eEF2K play a bigger role in helping to boost the DDR pathway. Therefore, at lower dosages of cisplatin, the role of eEF2K as an enhancer of DDR pathway becomes evident. This is reflected in the MEF cisplatin sensitivity assays, where differences in sensitivity was observed between concentration of 1.25 µM to 10 µM (Figures 3-1 to 3-4), but at 50 µM all cells were dead after 24 hours (data not available).  Even though the strongest difference between ATM/ATR signaling activity is seen in the WT and eEF2K KO MEFs, weaker differences are still observed in the other cell line models. Where we see differences p-Chk1, p-Chk2, p-Rad17, p53, and p-p53 to varying levels (Figures 3-8 to 3-10). This points to eEF2K playing the role of a modulator in the DDR pathway, which helps to increase the overall signaling output as opposed to activating the pathway. However, it would be very interesting to look at how the rescue MEF model and HEK293 cell line models would be affected if ATM and ATR levels were knocked down, and whether the signaling pathway immunoblots would match our findings in the WT/KO MEFs.  Figure (3-11) is a visualization of two DDR proteins localizing to the site of damage, and we concluded that WT MEFs compared to eEF2K KO MEFs are able to localize γH2AX and 53BP1 with higher efficiency. This follows what was observed in the immunoblots of WT and eEF2K KO MEFs (Figure 3-8), where WT MEFs show an upregulation of the active proteins in the DDR pathway. However, Figure (3-11) is a representation of one independent experiment and to be fully confident in our conclusion it needs to repeated at least two more times.  Phosphorylation of ATM and ATR shows inconsistencies between the different experiments, despite consistent differences between the phosphorylation of DDR proteins 61  downstream of ATM and ATR. In figure (3-10a) I see no upregulation p-ATR with cisplatin treatment in HEK293 cells with or without eEF2K KD but its direct downstream proteins Rad17 and Chk1 are more phosphorylated with cisplatin treatment. In addition, HEK293 cells with eEF2K KD express lower levels of p-Rad17 and p-Chk1 compared to the Scr control. ATM is the only known kinase that phosphorylates Chk2 at the thr 68 site, and ATR is the only known kinase that phosphorylates Chk1 at the ser 345 site and Rad 17 at the ser 646 site61,63,184. Since phospho-sites of proteins that are specifically phosphorylated by ATM or ATR are consistently upregulated in eEF2K proficient cells I can be confident that there is an upregulation of the ATM/ATR pathway in eEF2K proficient cells. In Figure (3-10a) we also see inconsistent p21 expression, where there is no upregulation with cisplatin treatment in HEK293 cells with or without eEF2K KD. This can be indicative of no p21 upregulation in response to cisplatin (Figure 3-10a) but that is unlikely because the other DDR proteins are upregulated in response to cisplatin. However, in Figure (3-10b) I see an upregulation of p21 with cisplatin treatment over time. In an ideal situation, more quantitative methods should be used to measure protein and phosphorylated protein levels with the use of mass spectrometry. In future studies to fully understand the role of p21, one needs to examine the activity of p21’s target proteins which include the CDKs and PCNA. Another discrepancy was observed in the relative levels of p53 under basal conditions between WT and eEF2K KO MEFs, where p53 levels in figure (3-13b) are much higher in the WT MEFs compared to the eEF2K KO MEFs in figures (3-8a and b). These differences can be due to differences in the cells at the time of the experiment, since all of these experiments were carried out at different times and with cells at different passage numbers. This shows that eEF2K has minimal effects on p53 levels under basal conditions. The more consistent difference is observed when the MEFs are treated with 5 µM of cisplatin and HEK293 cells with a range of cisplatin concentrations, where 62  eEF2K expressing cells compared to their eEF2K deficient counterparts show higher upregulation of p53 which correlates with an upregulation of p-p53 and its active upstream kinases p-Chk1 and p-Chk2.  In order to validate that eEF2K helps to enhance DDR pathway a cleaner cell model is required. The use of WT and KO MEFs can result in differences due adaptation and differences in mice and knockdown models can produce incomplete knockdowns or mixed cell populations that can alter the results. Therefore, in future studies CRISPR can be utilized to completely knockout eEF2K out of cell lines that have minimal genome instability and minimal alterations to the DDR pathway. The assays carried out in this chapter will be repeated on these new cell lines to confirm my findings.  It is should also be mentioned that our cell lines have been immortalized through SV40 large T antigen transduction, which inhibits the activity of some tumor suppressors including p53. However, I was able to demonstrate in my findings that p53 is not fully deactivated and it still is promoting cell death in response to cisplatin, as I show with p21 upregulation in response to cisplatin and the transient knockdown of p53 in the WT MEFs increasing resistance to cisplatin. In order to fully understand the interaction of p53 and eEF2K, non-immortalized primary cell lines need to be used, however many technical difficulties are associated with them as well.  Two possible conclusions can be drawn from the results of this chapter; a) the KO cells have lower levels of DNA damage compared to WT cells, as they can repair the cisplatin damage much faster due to their higher repair abilities; or b) the eEF2K KO cells may have the same amount of DNA damage than WT cells but are not able to recruit DDR factors, such as 53BP1 or γH2AX, as efficiently. The second conclusion is more likely because Figure (3-11) demonstrates 63  WT MEFs are more efficient than eEF2K KO MEFs at recruiting 53BP1 and γH2AX. The higher repair ability of the WT MEFs is confirmed in chapter 4.  In this chapter, I show that eEF2K expression sensitizes cells and increases apoptosis in response to continuous cisplatin treatment over a 48-hour period. eEF2K WT MEFs are also more sensitive than eEF2K KO MEFs to other drugs, including camptothecin and hydroxyurea. eEF2K proficient also helps to enhance the ATM/ATR DDR signaling, particularly by increasing p-Chk2, p-Chk1, p53, p-p53, and p-Rad17. The increased signaling of the DDR pathway in the eEF2K WT MEFs was visualized by higher γH2AX and 53BP1 foci localization after cisplatin treatment in the WT MEFs compared to the eEF2K KO MEFs. The lower p53 upregulation can be responsible for the resistance of the eEF2K KO MEFs to continuous cisplatin treatment, especially since eEF2K KO MEFs also have lower p53 mRNA levels. In order to confirm the role of p53 in enhancing sensitivity to cisplatin, I observed that knock down of p53 expression makes cells more resistant to cisplatin treatment when compared to cells that have no knockdown of p53 expression. Interestingly, knocking down of p53 expression in eEF2K KO MEFs did not increase resistance to cisplatin treatment. In summary the enhanced sensitivity of eEF2K expressing cells can be in part due to the enhanced activity of the ATM/ATR DDR pathway which phosphorylates and stabilizes the p53 protein, leading to apoptosis and cell death. It should be emphasized that this does not indicate that eEF2K is the only activator of DDR or that its expression is required to activate the entire pathway. My data show that eEF2K helps to enhance the activity of the pathway, and higher DDR activity can signal the cell to activate apoptosis.    64  Chapter 4: eEF2K impacts DNA repair ability after cisplatin treatment 4.1 Introduction  If there is a high level of damage and the cell cannot repair the damage DNA damage response pathway can initiate apoptosis. It would be more beneficial for the organism to get rid of damaged cells to prevent “rogue” cells from taking root in the host organism. However, if the magnitude of DNA damage is low, DNA repair pathways can repair the DNA and apoptosis does not get initiated. Cell sensitivity to low levels of DNA damage can be enhanced by inhibition of the DNA repair pathways and allowing damage to build up over time. If the damage reaches high levels it can result in cell death through necrosis or apoptosis, while cells that have fully functional DNA repair pathways can repair the damage before it become too detrimental.  Different assays can be used to measure repair of cisplatin-caused DNA crosslinks and double stranded breaks that form indirectly due to the crosslinks. The repair of inter-crosslinked DNA strands can be measured through a modified alkaline comet assay (Figure Appendix 5-1). Preparation of a comet assay involves lysing the cell in an agarose gel and using electrophoresis to separate the short and long strands of DNA. The shorter strands of DNA which correspond to damaged DNA run further away from the nucleus while the longer strands of DNA migrate more slowly through the agarose gel and stay close to the nucleus. This can be visualized under the microscope by staining the DNA with a fluorescent dye. However, the process is reversed when the DNA strands are inter-crosslinked, since the cross linked DNA strands are longer than the undamaged DNA and travel more slowly through the agarose gel. In the modified alkali comet assay an extra step is added prior to the collection of the cells, where they are treated with a dosage of X-ray radiation. The X-ray radiation can break apart the DNA but it has no effects on the cisplatin crosslinks. Therefore, DNA free of ICLs will consist of smaller DNA strands that have 65  been broken apart by IR and can travel further along the agarose gel, while DNA with ICLs will travel slower due to the long strands of DNA connected by the intercross linking agents.    γH2AX and 53BP1 proteins become localized at DNA DSB sites. Immunofluorescence microscopy can be used to visualize γH2AX and 53BP1 localization and is used as a general measure of DNA repair activity, albeit it is an indirect measurement of DNA repair since it does not measure the damage to the DNA directly. However, γH2AX foci localization is not limited to DNA DSB sites and it can localize to other sites of damage as well. The DNA staining dye DAPI is also used to visualize nuclear micronuclei abnormalities that can form because of DNA damage.  In the following chapter I demonstrate how eEF2K affects DNA repair. First, I used a modified comet assay and IF microscopy to show that eEF2K KO MEFs are less efficient at repairing ICLs and DNA damage. Next immunoblot analysis was used to show that eEF2K enhances expression of the NER protein ERCC1, potentially explaining the enhanced DNA repair abilities. Lastly the long-term cell sensitivity assay demonstrates that WT MEFs can better withstand pulsed treatment of cisplatin. In conclusion, eEF2K enhances DNA repair abilities by potentially upregulating ERCC1, which results in higher resistance to pulse treatment of cisplatin. 4.2 eEF2K affects repair of DNA damage and DNA intercross links  For the following DNA repair assays 5 µM of cisplatin was chosen to carry out the experiments because very little cell death was observed after a 2-hour pulse treatment in both the WT and eEF2K KO MEFs at 48 hours and after 24 hours of continuous 5 µM cisplatin treatment relatively low cell death was observed. WT and eEF2K KO MEFs were treated with a pulse of 5 µM cisplatin for 2 hours, and then the cells were allowed to recover in fresh media until they were collected at the specified time points.  66   To measure ICL repair the cells were given 2 hours or 24 hours to recover from the cisplatin treatment and DMSO was used as mock treatment. Prior to collecting the cells for the comet assay, all cells were treated with 5 grays of X-ray radiation to induce DSBs. We could confirm that the assay worked because the negative control cells had very large tail moments, confirming that IR was able to break apart the DNA (Figure 4-1a). The cells that were only allowed 2 hours of recovery had very small tail moments, demonstrating that the cisplatin was crosslinking the DNA (Figure 4-1b). At the 24-hour time point, the eEF2K KO MEFs had significantly smaller tail moments than the WT MEFs, meaning that the WT MEFs can repair ICLs faster than eEF2K KO MEFs (Figure 4-1b). The direct repair of ICLs is enhanced in WT MEFs, which will reduce the more detrimental forms of DNA damage that can form at later stages of the cell cycle.   The general DNA repair abilities of the eEF2K KO and WT MEFs was measured by quantifying the formation and resolution of γH2AX and 53BP1 foci after the cells were pulsed with cisplatin for 2 hours. There was no significant difference in the number of γH2AX foci between the eEF2K KO and WT MEFs treated with DMSO, and up to three hours after the cisplatin was removed (Figure 4-2). γH2AX foci in the WT MEFs increased 6 hours after treatment, and by 16 hours after treatment the foci count decreases (Figure 4-2). However, foci count in the eEF2K KO MEFs rise at 16 hours and decrease by 24 hours (Figure 4-2). The foci levels peak and decrease faster in the WT MEFs, indicating that they can repair the damage faster than the eEF2K KO MEFs. γH2AX foci form during the excision step of ICL repair, therefore the faster increase and decrease of γH2AX foci levels in WT MEFs indicates higher repair efficiency.  The 53BP1 focus counts show a different kinetic pattern than γH2AX. The number of 53BP1 foci in the WT MEFs peak at 16 hours and gradually decrease from that point (Figure 4-3). 53BP1 foci count in the eEF2K KO MEFs is not regular, as it peaks at 3 hours, decreases by 6 67  hours and increases again at 16 hours (Figure 4-3). The gradual increase and decrease of 53BP1 in WT MEFs shows a regulated DNA repair pathway, but the seemingly random pattern of foci levels in the eEF2K KO MEFs could depict an inability to properly respond to DNA damage. Since 53BP1 does not directly repair cisplatin damage, the irregular pattern of 53BP1 foci formation and resolution could be due to misregulation of previous DNA repair steps. The difference between γH2AX and 53BP1 foci kinetics could be explained by 53BP1 proteins only being able to localize to sites of DNA DSBs, but γH2AX proteins can localize at other forms damaged DNA in addition to DNA DSB sites. At the 16-hour time point the increase of γH2AX foci and the static level of 53BP1 foci in the eEF2K KO MEFs can be attributed to an increase in DNA damages that are not DNA DSBs. However very little is known about the relationship between 53BP1 and ICL repair, so it is difficult to relate the misregulation of 53BP1 localization and slower response of γH2AX foci localization with our current knowledge of DDR.   If DNA damage is not repaired, it can result in more damaging chromosomal abnormalities during cell metaphase due to abnormal chromatid separation. Abnormal chromatid separation results in formation of micronuclei, which are tiny segments of chromosomes that end up in daughter cells due to either breaking off or being mis-segregated. We used DAPI staining and IF microscopy to measure micronuclei, which can be visualized as tiny circles surrounding the cell nucleus (Figure 4-4). Micronuclei were only present after 16 and 24 hours of cisplatin pulse treatment in eEF2K WT and KO MEFs. The eEF2K KO MEFs carry more micronuclei at the 16 and 24-hour time points, pointing to inefficient DNA repair resulting in problems during mitosis and chromatid segregation that lead to chromosomal breakage and aneuploidy.   To determine the impact of eEF2K-mediated DNA damage mitigation on mitosis and cell cycle, the cell cycle profile of the eEF2K KO and WT MEFs after cisplatin treatment was 68  measured. We were expecting to observe deregulated cell cycle checkpoints after cisplatin treatment in eEF2K KO MEFs compared to WT MEFs since the immunoblots indicated downregulation of activated cell cycle checkpoint proteins in the eEF2K KO MEFs. An inactive G1 or G2 checkpoint can prevent proper repair of the DNA and allow potential cancerous cells to survive. However, under our conditions we did not see major differences in G1 and G2 cell cycle checkpoints but there was a difference in S-phase. The MEFs were treated with 5 µM of cisplatin for up to 24 hours and collected at 8-hour intervals. The cells were also labeled with BrdU to distinguish cells in S-phase. There was no difference between the cell cycle profiles under basal conditions, and after cisplatin treatment both cell lines showed a delayed S-phase progression. However, the eEF2K KO MEFs remained in S-phase longer than the WT MEFs (Figure 4-5). Considering the prior data pointing to inefficient DNA repair in the eEF2K KO MEFs, the KO cells may be getting delayed in S-phase because they are not able to repair the ICLs caused by cisplatin. In order to test this idea, the cell cycle analysis needs to be repeated with cisplatin pulse treatment instead of continuous treatment because a pulse treatment would allow the cell to repair its damage and it will not overwhelm the DNA repair machinery. If DNA repair can function efficiently, cisplatin damage will be repaired and the cell cycle will continue to progress. However, if DNA cannot be repaired then cells could stay arrested at cell cycle checkpoints permanently.  69   Figure 4-1 ICL DNA repair in WT and eEF2K KO MEFs. Representative IF pictures of PI labeled MEFs used to quantify the comet assay results. The DMSO treated MEFs have distinctive comet shape, while the cells collected 2 hours after pulse treatment have a circular shape (a). The MEFs were pulsed for 2 hours with 5 µM of cisplatin or the equivalent volume of DMSO. Then the cells were allowed to recover in fresh media for 2 hours and 24 hours. The cells were exposed to 5 gray of X-ray radiation to induce DSBs.  Level of ICLs was quantified by alkaline comet assay. Mean tail moment was quantified as the mean of 100 cells per condition and the data are the average of 3 biological replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate (b).  DMSO 2 2405 01 0 01 5 02 0 0T im e  (h o u rs )Tail momente E F 2 k  + /+e E F 2 K  - / -*eEF2K +/+ eEF2K -/- DMSO 2 Hours 24 Hours a) b) 70   eEF2K +/+ eEF2K -/- DMSO 0 Hours3 Hours6 Hours16 Hours24 Hours48 Hours a) 71   Figure 4-2  DNA repair in WT and eEF2K KO MEFs measured through formation and resolution of γH2AX foci. The MEFs were plated on coverslips and pulsed for 2 hours with 5 µM of cisplatin or the equivalent of DMSO. Then the cells were allowed to recover in fresh media for up to 48 hours after pulse treatment and they were fixed in PFA (4%) at the specified time points. The foci were visualized by IF microscopy using antibodies targeting γH2AX. Panel (a) shows representative images of γH2AX foci that were quantified under each treatment where the blue colour is the DAPI DNA stain and the bright green dots are the γH2AX foci. The ImgJ software was used to count the number of foci per cell (b). Each bar represents the mean of three biological replicates and at least 100 cells were measured per replicate. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate. DMSO 0 3 6 16244801 02 03 0T im e  (h o u rs )Number of foci/celle E F 2 K  + /+e E F 2 K  - / -*b) 72   73   Figure 4-3 DNA repair in WT and eEF2K KO MEFs measured through formation and resolution of 53BP1 foci.  The MEFs were plated on coverslips and pulsed for 2 hours with 5 µM of cisplatin or the equivalent of DMSO. Then the cells were allowed to recover in fresh media for up to 48 hours after pulse treatment and they were fixed in PFA (4%) at the specified time points. The foci were visualized by IF microscopy using antibodies targeting 53BP1. Panel (a) shows representative images of 53BP1 foci that were quantified under each treatment, where the blue colour is the DAPI DNA stain and the bright green dots are the 53BP1 foci. The ImgJ software was used to count the number of foci per cell (b). Each bar represents the mean of three biological replicates and at least 100 cells were measured per replicate. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate. DMSO 0 3 6 162448051 01 5T im e  (h o u rs )foci/celle E F 2 K  + /+e E F 2 K  - / - *b) 74    eEF2K +/+ eEF2K -/- 24 hours DMSO a) 75   Figure 4-4 Formation of micronuclei in WT and eEF2K KO MEFs after cisplatin treatment.  The MEFs were plated on coverslips and pulsed for 2 hours with 5 µM of cisplatin or the equivalent of DMSO. Then the cells were allowed to recover in fresh media for up to 48 hours after pulse treatment and they were fixed in PFA (4%) at the specified time points. The micronuclei and nuclei were visualized by the DAPI DNA stain. The larger circles are the cell’s nuclei while the smaller dots around the nuclei are the micronuclei. The orange arrows point to cells that were micronuclei negative and the red arrows point to cells that were micronuclei positive (a). Any cell that had more than 2 micronuclei was considered micronuclei positive. Cells with abnormal looking nuclei were not counted because they might be going through apoptosis. Each bar represents the mean of three biological replicates and at least 100 cells were measured per replicate. A P-value of ≤ 0.05 is represented by (*) and A p-value of ≤ 0.1 is represented by (**). The error bars denote S.D. of each triplicate (b).  0162402 04 06 08 0T im e  p o s t c is p la tin  (h o u rs )Micronuclei positive cells (%)e E F 2 K  + /+e E F 2 K  - / -***b) 76    eEF2K +/+ Untreated 8 hours 16 hours 24 hours eEF2K -/- Figure 4-5 Cell cycle progression in eEF2K WT and KO MEFs during cisplatin treatment. Cell cycle distribution of asynchronously growing MEFs under basal conditions and under 5 µM cisplatin treatment. Figure (a) depicts the bivariate cell cycle profile. The x-axis represents the DNA stained with PI and the overall DNA content separating the cells in G1 and G2/M phase. The higher population on the y-axis represents the BrdU positive cells that are in S-phase. The Graphpad software was used to depict the data of figure (a) in chart format (b). a) b) 77   4.3 eEF2K alters long term sensitivity to cisplatin   Since the eEF2K WT cells have better DNA repair capabilities, they would also carry less DNA damage to the next stages of their cell cycle or pass on to their daughter cells. Consequently, if the cells are treated with a low concentration of cisplatin for a limited amount of time without initiating an apoptotic pathway, then the cells that have more efficient DNA repair should have higher resistance to cisplatin. We carried out this experiment on the WT and eEF2K KO MEFs, where the cells were treated with 2.5 µM and 5 µM of cisplatin for 2 hours, and then we washed off the cisplatin containing media and replaced it with fresh media. Cells were then allowed to recover for 5 days. The cells were not exposed to very high levels of cisplatin and no major apoptosis was observed after 2 days in order to reduce input from pro-apoptotic pathways and allow DNA repair pathways to be the prominently active pathway. At the 5-day end point MTT assays were used to measure cell viability. WT MEFs showed significantly higher cell viability than the eEF2K KO MEFs (Figure 4-6). This stands in contrast to when the cells were treated continuously with cisplatin, demonstrating that high or low magnitude of genotoxic drug used in an experiment can result opposite sensitivity results. This agrees with the finding that WT MEFs showed more efficient DNA repair because it would allow them to recover from their DNA damage and survive. 78   Figure 4-6 Long-term cell viability of WT and eEF2K KO MEFs pulsed with cisplatin. eEF2K WT and KO MEFs treated pulsed for 2 hours with 5 µM of cisplatin with or DMSO. MTT cell viability was performed on the MEFs after were allowed to grow for 5 more days in fresh media. The viability of cisplatin treated cells is presented as a percentage of DMSO treated cells. Each bar represents the mean of three biological replicates. A P-value of ≤ 0.05 is represented by (*). The error bars denote S.D. of each triplicate.  4.4 Nucleotide excision repair as a candidate for better DNA repair ability   Nucleotide excision repair pathway is one of the main pathways involved in repair of cisplatin caused DNA damage. ERCC1-XPF complex acts as an endonuclease that is active in this pathway and its up regulation has been associated with resistance to cisplatin treatment. ERCC1 as previously mentioned also interacts with different ICL DNA repair pathways, and a decrease in its expression would inhibit repair abilities150,154,155,158,163,164. As mentioned in the Introduction chapter, ERCC1 is one of the most studied genes in the ICL repair pathway and it has been demonstrated to act as one of the most prominent genes in DNA repair pathway, therefore its lower expression level could explain the differences I see in cisplatin DNA repair. Immunoblots for DMSO2.5 505 01 0 01 5 0C is p la t in  (u M )Viable cells (%)e E F 2 K  + /+e E F 2 K  - / -* *79  ERCC1 expression show higher levels of ERCC1 in the WT MEFs when compared to eEF2K KO MEFs, under both basal and cisplatin treatment conditions (Figure 4-7a). Similarly, increased levels of ERCC1 protein were observed in cells treated with ctrl siRNAs as opposed to sieEF2K, and in control HEK293 cells compared to those with eEF2K knockdown (Figure 4-7 b and c). Therefore, one of the potential causes for the reduction of DNA repair in the eEF2K KO MEFs could be the lower expression of ERCC1. We need to emphasize that lower ERCC1 protein expression is only a potential candidate for the DNA repair abilities in the eEF2K KO MEFs. Further experiments need to be carried to confirm the role of ERCC1 in our model and if other genes are also contributing to reduced DNA repair abilities.    Figure 4-7 Effect of eEF2K on ERCC1 protein levels. 5 µM Cisplatin (hrs): Actin eEF2K +/+    0          2          4       6 eEF2K -/-   0      2         4         6 ERCC1      0       2        4       6 5 µM Cisplatin (hrs):      0      2       4       6     0       2       4        6 shCtrl sheEF2K 1 sheEF2K 2 Actin ERCC1 5 µM Cisplatin (hrs): Actin siCtrl      0       2        4       6 sieEF2K   0      2       4       6 ERCC1 a) b) c) 80  eEF2K WT and KO MEFs (a) and WT MEFs treated with siRNA targeting eEF2K or non-targeting siRNA (b) were treated with 5 µM of cisplatin for up to 6 hours. HEK293 cells with stable KD of eEF2K and a control cell line stably expressing scrambled shRNA were treated with 5 µM of cisplatin for up to 6 hours (c). The untreated cells were treated with DMSO for 6 hours. Whole cell lysates were collected at 2-hour intervals. Immunoblot analysis was done on the whole cell lysates using antibodies targeting the ERCC1 protein. Antibody targeting actin was used as a loading control. These blots are representative of three independent experiments. 4.5 Summary  In this chapter, I show that eEF2K WT MEFs have higher DNA repair abilities than eEF2K KO MEFs. I showed more efficient repair of ICLs caused by cisplatin in WT MEFs compared to eEF2K KO MEFs as measured by the comet assay. Similarly, the γH2AX and 53BP1 IF foci experiments and the micronuclei measurements point to better DNA repair in WT MEFs compared to eEF2K KO MEFs. The more efficient DNA repair in the WT MEFs is reflected in the long-term sensitivity studies, where the WT MEFs are less sensitive than the eEF2K KO MEFs, while the cell cycle studies point to slower S-phase progression in the eEF2K KO MEFs after cisplatin treatment, potentially caused by less efficient DNA repair. Lastly, I show that the difference in DNA repair could be explained by the higher expression of the DNA repair protein ERCC1 in cells with higher eEF2K expression when compared to their KO or KD counterparts. 81  Chapter 5: Conclusions and future directions  eEF2K’s role in adaptation to cellular stress by nutrient deprivation has been well documented1,13. However, the role of eEF2K in genotoxic stress has not been studied in depth, and its exact function is not very clear2,3,31. A previous study had demonstrated that eEF2K sensitizes MEF cells to doxorubicin treatment2. I expanded on these findings and showed that eEF2K expression enhances sensitivity to cisplatin, camptothecin, and potentially hydroxyurea. Enhanced sensitivity can be either due to higher apoptosis or reduced DNA repair ability, since at high levels of DNA damage the pro-apoptotic pathways will kill the cell to prevent more damage to the organism or the cell dies from a highly damaged and unusable DNA. I provide evidence that eEF2K enhances general DDR resulting in both higher apoptosis and higher DNA repair, where eEF2K sensitizes cells that are under higher levels of cisplatin stress due to continuous/chronic treatment but increases the resistance of cells that are treated under lower levels of cisplatin stress due to pulse/acute treatment (Chapters 3.2 and 4.3). Without eEF2K expression there is less apoptosis and DNA repair, meaning under high DNA damage levels the cells would go through apoptosis more slowly, but under low DNA damage levels they will repair their DNA less efficiently. Therefore, under genotoxic stress DNA damage will build up in cells lacking eEF2K expression, which will either result in their cell death in a certain population of the cells while other cells will adapt to the DNA damages and survive. The population of cells that survive such genotoxic conditions could lose the regulatory pathways that prevent unregulated cell proliferation and become cancerous because they may accumulate mutations and increase genomic instability (Figure 5-1b).   The eEF2K expressing cells show higher activation of ATR/ATM DDR signaling pathway compared to their eEF2K deficient counterparts as I see higher levels of p-ATMser1981, p-ATRthr1989, 82  p-Chk1ser345, p-Chk2thr68, and p-p53ser15. The levels of p-p53ser15 are perhaps the most relevant to the enhanced sensitivity phenotype seen with eEF2K expression, because p53 promotes the expression of many pro-apoptotic downstream targets115–118. The sensitivity of the WT MEFs to cisplatin decreased when we knocked down p53 expression, confirming that in the WT MEFs, p53 promotes cell death in response to cisplatin. In conclusion, I provide evidence to show that eEF2K enhances sensitivity to high levels of cisplatin by activating p53 potentially through the ATM/ATR DDR signaling pathway. However, further studies are necessary to determine if eEF2K could be enhancing other signaling pathways that promote cell death through p53 or independent of p53.  When I treated cells only with a 2-hour pulse of cisplatin we saw the opposite sensitivity phenotype, whereby the WT MEFs were more resistant to cisplatin than the eEF2K KO MEFs. Unlike the continuous cell sensitivity results the WT and KO MEFs that were pulsed with cisplatin did not show major cell death up to 48-72 hours after the treatment. In addition, the WT MEFs were observed to have higher p53 induction compared to the eEF2K KO MEFs, yet they were more sensitive to low levels of cisplatin treatment. Therefore, the activation of the ATM/ATR pathway that was previously observed probably cannot explain the sensitivity difference in cells that were pulsed with cisplatin.  However, the DNA repair arm of the DDR pathway could be responsible for the enhanced resistance seen in the WT MEFs under those conditions, and we provide evidence that eEF2K enhances DNA repair ability in response to cisplatin. The modified alkali comet assay shows that eEF2K KO MEFs specifically have lower ICL repair capabilities. The levels of γH2AX and 53BP1 foci and micronuclei point to higher general DNA repair capabilities in the eEF2K WT MEFs compared to eEF2K KO MEFs. The cell cycle profile after cisplatin treatment also points better DNA repair in the WT MEFs compared to the eEF2K KO MEFs, because the slower S-phase progression of the eEF2K KO MEFs could be indicative of 83  slower DNA repair. If a cell repairs the ICL DNA damage slower during S-phase then it will also experience slower DNA replication because the replication machinery will be slowed down by the damaged regions of DNA. Since we provide evidence that eEF2K KO MEFs have less efficient DNA repair then it could explain the slower S-phase progression in eEF2K KO MEFs.  There are many genes involved in repair of DNA damage caused by cisplatin. ERCC1 interacts with different DNA repair pathways that repair DNA crosslinks, and its downregulation greatly sensitizes cells to ICL inducing agents, including cisplatin and MMC153,157,165,166. We showed that eEF2K proficient cells express higher levels of ERCC1 protein compared to their KO or knockdown counterparts, so ERCC1 could at least partially be responsible for the enhanced DNA repair activity in WT MEFs. In order to understand if ERCC1 is responsible for the observed differences in DNA repair and sensitivity, DNA repair assays and MTT cell viability assays need to be repeated with a knockdown of the ERCC1 gene. Even though we show that WT MEFs have higher DNA repair capabilities than eEF2K KO MEFs, we have not shown this in other cell line models. The difference in DNA repair capacity might not be dependent on eEF2K activity but dependent on compensatory mechanisms that have resulted in DNA repair to be downregulated in the eEF2K KO MEFs. However, eEF2K is very likely to enhance DNA repair because we confirmed that eEF2K increases expression of the ERCC1 DNA repair protein in the HEK293 cells and transient knockdown of eEF2K in the WT MEFs.  The two different sensitivity phenotypes observed depend on the magnitude of cisplatin the cells are exposed to, where cells under continuous treatment experience higher magnitude of damage than pulse treatment. Which leads us to ask why does eEF2K sensitize cells to cisplatin treatment by inducing apoptosis through p53, while at the same time it protects cells by enhancing DNA repair abilities. One possible explanation is that eEF2K’s role is not confined to protecting 84  a single cell but rather to protect the entire organism. When cells are treated with high levels of cisplatin they may not be able to repair all the damaged DNA, which could lead to formation of pre-cancerous cells. Therefore, eEF2K may prevent accumulation of these damaged cells, that could be detrimental to the entire organism, by using p53 to induce apoptosis 82–84, 86–89. However, when the cells are exposed to a lower magnitude of cisplatin, high levels of p53 do not get activated to induce apoptosis and the DNA repair machinery of the cell is able repair the damage 82–84, 86–89. If any damaged DNA is not repaired it could deregulate the cells signaling machinery, leading to unregulated cell proliferation  and potential formation of  cancerous cell populations; for example, unrepaired ICLs can induce double stranded breaks or chromosome abnormalities that can become detrimental to the cell and the entire organism164,185,186. eEF2K helps to enhance DNA repair ability to fully repair the DNA and prevent any damaged DNA from being missed. In conclusion, there is a balance between DNA repair and apoptosis induced by cisplatin treatment that is dependent on the total level of cisplatin the cell is exposed to, and eEF2K appears to enhance both pathways through ERCC1 and p53, respectively. If the degree of DNA damage is too high, the cell should ideally commit to apoptosis to prevent damage to the organism.   In a clinical setting eEF2K activity can be very important because if p53 cannot induce apoptosis due to a mutation or lower protein expression, cells with high eEF2K expression will have enhanced DNA repair capabilities that can lead to resistance to chemotherapeutic drugs. eEF2K activity and expression levels could be used to modify the dosage of patients; for example, patients with high eEF2K levels and low p53 activity need to be exposed to higher concentrations of chemotherapeutic drugs to induce cell death, and cells with high eEF2K levels and high p53 activity could be treated with a lower dosage of those drugs. 85   The main question that has yet to be answered is how does eEF2K interact with the DDR machinery. The best known role of eEF2K is to inhibit mRNA translation by deactivating eEF2, and its activity increases after a stress is put on the cell31. Therefore, it is reasonable to hypothesize that eEF2K enhances DDR through deactivation of eEF2 and inhibition of mRNA translation at the elongation phase. The only proteins that consistently had higher levels within eEF2K proficient cells were ERCC1 and p53. ERCC1 is a DNA repair protein and currently it is not known to play a role in initiating DNA damage response. However, the differences in total levels of p53 could be caused by differences in mRNA translation, but higher stabilization by phosphorylation at the ser15 site cannot be ruled out. Therefore, eEF2K could be promoting translation of one gene or groups of genes that promote DDR activation. In a recent in vitro study it was shown that eEF2K could have other substrates that it can  phosphorylate, therefore eEF2K’s role in DNA damage response could be independent of eEF2 and dependent on phosphorylation of another potential target13.  There is contradictory data on whether eEF2K selectively inhibits translation of certain mRNAs or if it inhibits overall mRNA translation, and this inconsistency could be due to the type of stress the cells were exposed to (i.e. nutrient deprivation or genotoxic stress)1,2. It has been shown that under UV radiation there is a general reduction of mRNA translation and reprogramming of the translation machinery, where certain mRNAs are preferentially translated over others, resulting in upregulation of some DDR proteins187. Two  of these proteins were also upregulated in our studies, ERCC1 which we found to be upregulated in all eEF2K proficient cells, and ATM which we found to be upregulated only in the WT MEFs187. There is evidence that eEF2K by inhibiting eEF2 activity and translation elongation, could be promoting translation of specific mRNAs. A similar reprogramming of translation machinery could be occurring under 86  cisplatin treatment, whereby translation of  DDR mRNAs is enhanced and the translation of other genes is inhibited188–190.   We demonstrated that eEF2K KO MEFs have lower ICL DNA repair capabilities, but cisplatin causes intra-crosslinks of DNA strands as well. The repair of intra-crosslinks caused by cisplatin can be studied by using antibodies targeted against cisplatin induced DNA adducts191. We also demonstrated through foci formation and dissolution that eEF2K KO MEFs cannot repair DNA damage as efficiently as WT MEFs, but we were not able to single out specific pathways that might underlie these observations. In future studies, specific DNA repair pathways can be studied, particularly the fanconi anemia DNA repair pathway because it plays a major role in the repair of ICLs163,192. In addition, it would be interesting to look at activity of DNA homologous repair recombination because this pathway is involved in the later stages of ICL repair161.   The role of eEF2K in DDR also needs to be tested in vivo. Such in vivo experiments would include establishing xenografts of cancer cells with and without stable knockdown of eEF2K. The mice would then be treated with cisplatin when the tumors have reached an appropriate size. Radiation and other chemotherapeutic agents could also be used to simulate a chemotherapeutic regimen used in the clinic. To better simulate the application of these findings to the clinic, this study can also be expanded to the use of eEF2K inhibitors in place of stable knockdown; however, it should be noted that there currently is no eEF2K inhibitor that is being used in the clinic.   Together, these experiments provide new insights into how the cell uses eEF2K under genotoxic stress conditions to enhance DDR pathways and to promote the appropriate response, whether it is apoptosis, cell cycle changes, checkpoint activation, or DNA repair.  87                               eEF2K DNA damage (cisplatin) DDR Apoptosis P53 DNA repair Cell survival eEF2K Low level of DNA damage (cisplatin) DDR Apoptosis P53 DNA repair Cell survival eEF2K High level of DNA damage (cisplatin) DDR Apoptosis P53 DNA repair Cell survival a) 88     Figure 5-1 Model describing the role of eEF2K in DDR.  (a) eEF2K can enhance DDR, resulting in higher DNA repair and p53 activation. The cell sensitivity phenotype will depend on the magnitude of cisplatin the cell is exposed to. If the magnitude of cisplatin is high, the cell cannot fully repair the high levels of DNA damage it experiences. This results in eEF2K to upregulate DDR and p53 resulting apoptosis to prevent cells with damaged DNA to live inside the organism. If the magnitude of cisplatin is high enough to initiate the apoptotic pathway then eEF2K will help upregulate the DNA repair pathways to help fully repair the DNA. (b) In cells with reduced or non-existent expression of eEF2K, genotoxic stress will be very damaging. The cell is less efficient at repairing DNA or initiating apoptosis which will result can result in cells that have adapted to live with a damaged DNA to live inside the organism. The regulatory mechanism of these could become deregulated and the cells can become cancerous.    eEF2K DNA damage (cisplatin) DDR Apoptosis P53 DNA repair Cell survival Build up of DNA damage Cell death Adaption to DNA damage Cell survival b) 89  Bibliography 1. Leprivier, G. et al. The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation. Cell 153, 1064–79 (2013). 2. 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(c) the cell is either able to repair the damage (left image) or the ICLs are not removed (right image). The cells are treated with IR prior to collection. (d) DNA that was not exposed to IR remains intact (far left image). The IR causes DSBs which break apart the DNA (middle image). If ICLs are present the smaller DNA strands stay attached together with the cisplatin ICL (far right image). (e) The alkali comet assay is carried out, and the samples are lysed in agarose gels and by electrophoresis the large and small DNA strands are separated. Intact DNA is composed of large strands of DNA that moves slowly through agarose and stay close together (far left image). DNA exposed to IR is mostly composed of small DNA strands that run faster through the agarose gel, forming a large tail like shape (middle image). DNA that is connected with ICLs is mostly composed of larger strands that also move slowly through the agarose gel, mostly staying close together at the head of the comet shape.    Figure Appendix-5-1 Modified alkali comet assay used to quantify ICL repair.  

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