<p>Approximately 50% of all patients with cancer receive radiation therapy at some point during the course of their illness. Despite advances in radiation delivery and treatment planning, normal tissue toxicity often limits the ability of radiation to eradicate tumors. The tumor microenvironment consists of tumor cells and stromal cells such as endothelial cells that contribute to tumor initiation, progression and response to therapy. Although endothelial cells can contribute to normal tissue injury following radiation, the contribution of stromal cells to tumor response to radiation therapy remains controversial. To investigate the contribution of endothelial cells to the radiation response of primary tumors, we have developed the technology to contemporaneously mutate different genes in the tumor cells and stromal cells of a genetically engineered mouse model of soft tissue sarcoma. Using this dual recombinase technology, we deleted the DNA damage response gene <italic>Atm</italic> in sarcoma and heart endothelial cells. Although deletion of <italic>Atm</italic> increased cell death of proliferating tumor endothelial cells, <italic>Atm</italic> deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. In addition, the ATM inhibitor NVP-BEZ235 selectively radiosensitized primary sarcomas, demonstrating a therapeutic window for inhibiting ATM during radiation therapy. Sensitizing tumor endothelial cells to radiation by deleting <italic>Atm</italic> prolonged tumor growth delay following a non-curative dose of radiation, but failed to increase local control. In contrast, deletion of <italic>Atm</italic> in tumor parenchymal cells increased the probability of tumor eradication. These results demonstrate that tumor parenchymal cells rather than endothelial cells are the critical targets that regulate tumor eradicaiton by radiation therapy.</p> / Dissertation

It has been reported that locus coeruleus (LC) degeneration precedes the degeneration of other neurons in the brain in some neurodegenerative diseases like Alzheimer’s disease (AD) and Parkinson’s disease (PD). However, the precise mechanisms of neurodegeneration remain to be elucidated. N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) has been widely used as a noradrenergic neurotoxin in the development of AD and PD animal models for specific LC degeneration. However, the precise mechanism of action of DSP4 remains unclear. An increased systemic DNA damage caused by neurotoxin or oxidative stress has been found to be related to the pathogenic development of neurodegeneration. The process of neurodegeneration is not well understood, so current therapeutic approaches are limited to disease management and symptoms relief, such as using antidepressants for depression symptoms, which often accompany neurodegenerative disorders. To date, few studies have explained why different groups of antidepressants have similar clinical effects on relieving depression. Our data demonstrate that DSP4 induces the DNA damage response (DDR) and results in down-regulation of dopamine β-hydroxylase (DBH) and the norepinephrine transporter (NET), which are 2 noradrenergic phenotypes. DSP4 results in cell cycle arrest in S and G2/M phases, which is reversible. The comet assays verify that DSP4 induces single-strand DNA breaks (SSBs). Furthermore, the neurotoxins camptothecin (CPT) and DSP4 were used to induce the DDR in SH-SY5Y cells, fibroblast cells, and primary cultured neurons. Data show that both CPT and DSP4 induce the DDR in SH-SY5Y cells and primary cultured LC neurons. Compared to fibroblast cells, SH-SY5Y cells and LC neurons are more sensitive to the accumulation of DNA damage when treated with CPT or DSP4. Persistent phosphorylated H2AX (γH2AX) and p53 (p-p53ser15) levels indicate a deficient repair in noradrenergic SH-SY5Y cells and LC neurons. In addition, the current study demonstrates that some antidepressants reduce the DDR induced by DSP4 or CPT in SH-SY5Y cells. Flow cytometry data show that selective antidepressants protect cells from being arrested in S-phase. Together, these effects suggest that blocking DNA damage is one important pharmacologic characteristic of antidepressants, which may explain why different antidepressants could alleviate depression symptoms in neurodegenerative patients.

Liver cancer is the fourth leading cause of cancer-associated deaths globally, and among primary liver cancers, hepatocellular carcinoma (HCC) encompasses 75-85% of all cases. HCC is a highly lethal disease due to limited treatment options – only a small subset of patients qualify for surgical resection or transplantation; the remaining patients often display resistance to radiation therapy or chemotherapy. Overexpression of the oncogene astrocyte elevated gene-1 (AEG-1) is associated with poorer survival and increased tumor recurrence in HCC, and numerous studies show its role in initiation of hepatocarcinogenesis. A prior study also demonstrated AEG-1 expression inhibits senescence by diminishing the ATM/Chk1/Chk2/p53/p21 DNA damage response (DDR) pathway. The aim of this study is to understand if AEG-1 expression promotes radioresistance in HCC. A CRISPR/Cas9 plasmid system was used to delete AEG-1 in the QGY-7703, HuH7 and DihXY cell lines, which model HCC. The cell lines were then treated with ionizing radiation (IR). We find that knockout of AEG-1 in these cell lines induces sensitivity to IR at 2.5 Gy. In response to radiation, AEG-1 wildtype cells more profoundly upregulate ATR, Chk1, and Chk2 signaling; and also more rapidly induce γH2AX, ATM, and BRCA1 signaling, which sense dsDNA breaks to initiate homologous recombination repair. We conclude that AEG-1 expression protects HCC cells from IR through two mechanisms: 1) rapidly initiating the DNA damage response; and 2) increasing replication fork stabilization. These findings indicate AEG-1 can be a therapeutic target in combination with radiation treatment to improve outcomes for HCC patients who demonstrate radioresistance.

The contents of nuclei are highly organized. Nuclear organization is facilitated by a dense protein network, called the nuclear lamina, which underlies the nuclear envelope. The nuclear lamina is composed of filamentous lamins and more than eighty lamin-associated proteins (LAPs). Among the first LAPs identified are LEM Domain (LEM-D) proteins, named after LAP2, emerin and MAN1. LEM-D proteins have many shared and unique functions that include providing structural support to the nucleus, regulating signal transduction pathways and gene expression, facilitating proper progression through the cell cycle and maintaining chromatin attachments at the nuclear periphery. Despite requirements for these processes in all cell types, loss of globally expressed LEM-D proteins causes tissue-restricted defects. Identification of the primary function in tissues susceptible to LEM-D protein loss is a persistent challenge in the nuclear lamina field. Research described here uses Drosophila as a model to understand LEM-D protein function. Loss of the Drosophila emerin homologue Otefin (Ote) causes a complex phenotype in the ovary wherein both somatic and germline cells are compromised. Using tissue-restricted expression experiments, it was determined that Ote function is only required in germline stem cells (GSCs) to maintain all cells in the ovary. Developmental, molecular and genetic analyses revealed that the primary defect in ote mutant ovaries is an early block in germline differentiation, followed by GSC death. Genetic rescue experiments revealed that both of these GSC defects are due to the activation of the DNA Damage Response (DDR) proteins ATR and Chk2. Interestingly, activation of ATR and Chk2 occurs independent of detectable canonical DDR triggers, including DNA damage. Immunohistochemical analyses suggest that Ote might be regulating chromatin condensation and/or heterochromatin organization in GSCs. Through studies of Ote, a rescue method was discovered that involves culturing animals at elevated temperatures. This novel rescue strategy, termed hyperthermia, acts independent of ATR or Chk2 inhibition. Interestingly, elevated temperatures leads to chromatin decondensation in Drosophila, suggesting that hyperthermia may rescue oogenesis by alleviating chromatin defects observed in ote mutant germ cells. Together, results from experiments discussed herein dissect a complex ovary phenotype to reveal the critical requirement for a nuclear lamina LEM-D protein. Investigations into Ote function have revealed several aspects of GSC biology. The ATR/Chk2 checkpoint activated in the absence of Ote uncovered a previously unidentified cause of female GSC death. Further, findings that ATR and Chk2 are activated in the absence of canonical triggers suggest that GSCs possess a system to monitor defects or changes in the nucleus that do not involve DNA damage. Therefore, studies of Ote function and ote mutant phenotypes have uncovered valuable insights into LEM-D protein function and revealed the existence of novel conditions required for GSC maintenance.

Replication Protein A (RPA), the major eukaryotic single-strand DNA (ssDNA) binding protein, is essential for replication, repair, recombination, and checkpoint activation. Defects in RPA-associated cellular activities lead to genomic instability, a major factor in the pathogenesis of cancer. The ssDNA-binding activity of RPA is primarily mediated by two domains in the RPA1 subunit. I characterized mutant forms of RPA to elucidate the contribution of specific residues in the high affinity DNA binding domains to the cellular function of RPA. These studies enhance the understanding of the properties of RPA that contribute to DNA repair and cellular checkpoints. Mutation of a conserved leucine residue to proline in the high-affinity DNA binding site of RPA (residue L221 in human RPA) has been shown to have a high rate of chromosomal rearrangements in yeast and mice. I characterized the equivalent mutation in human RPA. My studies show that the mutation causes a defect in ssDNA binding and a nonfunctional protein. Combined with the mice studies, the data suggest that haploinsufficiency of RPA causes an increase in DNA damage and in the incidence of cancer. The ssDNA-interactions of the high affinity binding domains in RPA1 are mediated by several residues including four highly conserved aromatic residues. Mutation of these residues had no effect on DNA replication but caused defects in DNA repair pathways. I conclude that DNA intermediates in different DNA metabolic pathways require different RPA binding functions and that the aromatic residues are indispensable for binding in DNA repair. These studies illustrate that different DNA metabolic pathways have distinct requirements for RPA function. A decrease in binding to ssDNA of any length has specific consequences in vivo. These data also demonstrate that a single mutation in RPA in a residue that does not even contact ssDNA can result in a non-functional RPA complex. I conclude that even a modest decrease in RPA protein levels is not compatible with long term cell survival. Taken together, these studies highlight the importance of proper regulation of RPA protein levels and its ssDNA binding affinity to proper maintenance of the integrity of the genome.

<p>The eukaryotic genome integrity is safeguarded by the DNA damage response, which is composed of a network of signal transduction pathways that upon genotoxic stresses, arrest cell cycle progression, motivate repair processes, or induce apoptosis or senescence when cells incur irreparable DNA damage. During this process, DNA damage-induced post-translational modifications, most notably protein phosphorylation, of a variety of DNA damage-responsive proteins has been shown to mediate the initiation, transduction and reception of the DNA damage signals, resulting in alterations of their stability, activities or subcellular localizations, ultimately leading to activation of various downstream effector pathways. </p><p>While a lot has been elucidated on the downstream events of the DNA damage response, little is known about how DNA damage is detected. Two still ongoing studies of this dissertation attempt to address this question. Our preliminary work on ATM indicates that serine 2546 is critical for its kinase activity. Substitution of this residue with phosphomimetic aspartate, but not nonphosphorylable alanine, abrogates the kinase activity of ATM and fails to rescue the checkpoint-deficient phenotype exhibited by the ATM-deficient cells, suggesting that removal of an inhibitory phospho group at S2546 might be required for the activation of ATM. In another study, we identified a novel DNA-damage responsive threonine residue (T622) in Rad17, which undergoes ATM/ATR-dependent phosphorylation in vitro and in vivo. Ectopic expression of a phosphodeficient mutant (T622A) of Rad17, but not its wild-type control, shows a pronounced defect in sustaining Chk1 phosphorylation and the corresponding G2/M checkpoint upon DNA damage, suggesting that phosphorylation at T622 might complement that on the two previously reported phosphorylation sites, S635 and S645, to mediate G2/M checkpoint activation while the latter is primarily responsible for intra-S phase checkpoint. </p><p>Although a large amount of knowledge has been accumulated about the initiation and activation process of the DNA damage response, how cells recover, the equally important flip side of the response, has remained poorly understood. We have found that in cells recovering from replication stress, RPA32 phosphorylation at ATM/ATR-responsive sites T21 and S33, which reportedly suppresses DNA replication and recruiting other checkpoint and repair proteins to the DNA lesions, is reversed by the serine/threonine protein phosphatase 2A (PP2A). Cells with a RPA32 persistent-phosphorylation mimic (T21D/S33D) exhibit normal checkpoint activation and re-enter the cell cycle normally after recovery, but display a pronounced defect in the repair of DNA breaks. These data indicate that PP2A-mediated RPA32 dephosphorylation may be a required event during the repair process in the DNA damage response. </p><p>In summary, these studies in this dissertation highlight the importance of reversible phosphorylation and dephosphorylation in the modulation of the DNA damage response. What's more, they also extend our knowledge and deepen our understanding of this process by revealing that dephosphorylation may positively regulate the activation of cell cycle checkpoints, which is seemingly dominated by protein phosphorylation upon DNA damage, that phosphorylation of certain checkpoint proteins at different sites may result in distinct consequences, and that dephosphorylation of some activated checkpoint/repair proteins may function as an important mechanism for cells to recover from the DNA damage response.</p> / Dissertation

No description available.

Checkpoint kinase 2 (CHK2) is an effector kinase of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'α subunit of protein phosphatase 2A (PP2A), a major protein serine/threonine phosphatase of the cell, as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'α binds to the SQ/TQ cluster domain of CHK2, which is a target of ATM phosphorylation. CHK2 is able to bind to many B' subunits as well as the PP2A C subunit, indicating that it can bind to the active PP2A enzyme. The induction of DNA double-strand breaks by ionizing radiation (IR) as well as treatment with doxorubicin causes dissociation of the B'α and CHK2 proteins, however, it does not have an effect on the binding of B'α to CHK1. IR-induced dissociation is an early event and occurs in a dose-dependent manner. CHK2 and B'α can re-associate hours after DNA damage and this is not dependent upon the repair of the DNA. Dissociation is dependent on ATM activity and correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after IR. CHK2 is able to phosphorylate B'α in vitro; however, in vivo, irradiation has no effect on PP2A activity or localization. Alternatively, PP2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity and protein stability. These data reveal a novel mechanism for PP2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP2A/CHK2 complex in later time points after damage, which may help to attenuate the signal. This mechanism of CHK2 negative regulation by PP2A joins a growing list of negative regulations of DNA damage response proteins by protein serine/threonine phosphatases.

<p>The interaction of human tumor viruses with host growth suppressive pathways is a fine balance between controlled latent infection and virus-induced oncogenesis. This dissertation elucidates how Epstein-Barr virus interacts with the host growth suppressive DNA damage response signaling pathways (DDR) in order to transform infected human B lymphocytes. </p><p> Here I report that the activation of the ATM/Chk2 branch of the DDR in hyper-proliferating infected B cells results in G1/S cell cycle arrest and limits viral-mediated transformation. Similar growth arrest was found in mitogen-driven proliferating of B cells that sets the DDR as a default growth suppressive mechanism in human B cells. Hence, the viral protein EBNA3C functions to attenuate the host DDR and to promote immortalization of a small portion of infected B cells. Additionally, the pharmacological inhibition of the DDR in vitro increases viral immortalization of memory B cells that facilitates the isolation of broadly neutralizing antibodies to various infectious agents. Overall, this work defines early EBV-infected hyper-proliferating B cells as a new stage in viral infection that determines subsequent viral-mediated tumorigenesis.</p> / Dissertation

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.