The goal of my research was to obtain a quantitative understanding of the mechanisms of DNA double-strand break (DSB) repair, and the activation of the tumor suppressor p53 in response to DSBs in human cells. In Chapter 2, we investigated how the kinetics of repair, and the balance between the alternate DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), change with cell cycle progression. We developed fluorescent reporters to quantify DSBs, HR and cell cycle phase in individual, living cells. We show that the rates of DSB repair depend on the cell cycle stage at the time of damage. We find that NHEJ is the dominant repair mechanism in G1 and in G2 cells even in the presence of a functional HR pathway. S and G2 cells use both NHEJ and HR, and higher use of HR strongly correlates with slower repair. Further, we demonstrate that the balance between NHEJ and HR changes gradually with cell cycle progression, with a maximal use of HR at the peak of active replication in mid-S. Our results establish that the presence of a sister chromatid does not affect the use of HR in human cells. Chapter 3 examines the sensitivity of the p53 pathway to DNA DSBs. We combined our fluorescent reporter for DSBs with a fluorescent reporter for p53, to quantify the level of damage and p53 activation in single cells. We find that the probability of inducing a p53 pulse increases linearly with the amount of damage. However, cancer cells do not have a distinct threshold of DSBs above which they uniformly induce p53 accumulation. We demonstrate that the decision to activate p53 is potentially controlled by cell-specific factors. Finally, we establish that the rates of DSB repair do not affect the decision to activate p53 or the dynamical properties of the p53 pulse. Collectively, this work emphasizes the importance of collecting quantitative dynamic information in single cells in order to gain a comprehensive understanding of how different DNA damage response pathways function in a coordinated manner to maintain genomic integrity.

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2007. / Title from second page of PDF document. Includes bibliographical references (p. 91-102).

The clearance of viruses is largely dependent upon the activation of T cells to generate a robust immune response. However, host responses are suppressed during chronic viral infections. In this thesis, we explored the role of Top2α in DNA topology in individuals with chronic HBV, HCV, and HIV infections. We found that Top2α protein expression and activity were low in T cells derived from chronically virus-infected individuals compared to healthy subjects. Using CD4+ T cells treated with Top2α inhibitor or poisoner as a model, we demonstrated that Top2α inhibition disrupts the DNA topology, suppresses DNA repair kinase (ATM), and telomere protein (TRF2) expression, and induces T cell dysfunction. These findings reveal that Top2α inhibition is a mechanism by which viruses evade the host responses and establish persistent infection, and thus, restoring Top2α levels could be a way of boosting immune responses during chronic viral infections.

In recent years, a great effort has been deployed towards a better understanding of the molecular changes in cells and in the bone marrow (BM) environment that contribute to the development and progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). Among others, the aberrant hematopoietic stem cells in MDS often display increase in DNA double strand breaks, genomic instability with common loss or rearrangement of chromosomes and an ineffective response to DNA damage, a phenomenon that has been linked to the onset of cellular senescence. Additionally, the BM microenvironment can become more pro-inflammatory. In our effort to better understand the contribution of the BM microenvironment on MDS progression, we analyzed the expression profiles of cytokines in the BM microenvironment in all stages of MDS/AML and found several proinflammatory cytokines that increase with disease progression. Also, by repeated sampling of patients over the course of 5-azacytidine therapy, we were able to assess the changes in the proinflammatory cytokine milieu with the progression of the disease. Additionally, we aimed to identify the candidate markers for the improvement of MDS prognosis. We focused on naturally occurring germline polymorphism of NAD(P)H dehydrogenase (quinone 1) gene (NQO1*2)...

CD4 T-cell depletion is a hallmark of HIV/AIDS, but the underlying mechanism is still unclear. We have recently shown that ataxia-telangiectasia-mutated (ATM) deficiency in CD4 T cells accelerates DNA damage, telomere erosion, and cell apoptosis in HIV-infected individuals on antiretroviral therapy (ART). Whether these alterations in ART-treated HIV subjects occur in vitro in HIV-infected CD4 T cells remains unknown. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the telomeric DNA damage response (DDR) and cellular apoptosis in highly permissive SupT1 cells, followed by the validation of our observations in primary CD4 T cells with active or drug-suppressed HIV infection. Specifically, we established an in vitro HIV T-cell culture system with viral replication and raltegravir (RAL; an integrase inhibitor) suppression, mimicking active and ART-controlled HIV infection in vivo. We demonstrated that HIV-induced, telomeric DDR plays a pivotal role in triggering telomere erosion, premature T-cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This in vitro model provides a new tool to investigate HIV pathogenesis, and our results shed new light on the molecular mechanisms of telomeric DDR and CD4 T-cell homeostasis during HIV infection. IMPORTANCE The hallmark of HIV infection is a gradual depletion of CD4 T cells, with a progressive decline of host immunity. How CD4 T cells are depleted in individuals with active and virus-suppressed HIV infection remains unclear. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the chromosome end (telomere) DNA damage response (DDR) and cellular apoptosis in a T-cell line (highly permissive SupT1 cells), as well as in primary CD4 T cells with active or drug-suppressed HIV infection. We demonstrated that HIV-induced telomeric DDR plays a critical role in inducing telomere loss, premature cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This study sheds new light on the molecular mechanisms of telomeric DDR and its role in CD4 T-cell homeostasis during HIV infection.

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DNA damage in spermatozoa is a crucial contributor to spontaneous abortion, severe genetic disease in the offspring and infertility. The chromatin of spermatozoa is highly compacted, transcriptionally and translationally silent, hence lacking DNA damage response (DDR). DDR foci follow within seconds after a DNA double strand break (DSB) and correlate to an abortive topoisomerase-IIb activity during spermiogenesis. When comparing the DSB frequencies at the two most fragile genomic loci (fragile sites FRA3B, FRA16D) in human and murine spermatozoa with lymphocytes, significantly increased DSB levels were detected in spermatozoa in both species. This corroborates that spermatozoa are more prone to DSBs than somatic cells. When comparing the DSB frequencies at FRA3B/FRA16D in spermatozoa of smokers with non-smokers, two-fold increases were found, probably caused by cigarette smoke components triggering abortive topoisomerase-IIβ activity. The phosphorylated DDR proteins H2AX and ATM were identified in human spermatozoa and murine spermatids using multicolour immunostaining with laser-scanning confocal microscopy (LSCM) and Western blots. Based on significantly increased DDR foci in spermatozoa of smoking men, but lacking DDR foci in response to in vitro challenge with H2O2, an abortive topoisomerase-IIb activity is the likely cause of DDR foci in spermatozoa. As DDR foci are susceptible to cigarette smoke, they can potentially be used as a novel biomarker. When comparing paternal spermatozoa, and lymphocytes as well as maternal and cord lymphocytes from 39 families for DSBs (via high-throughput LSCM pH2AX detection) and DNA fragmentation (Comet assay), significant increases were found in newborns of mothers exposed to environmental tobacco smoke and smoking fathers. When challenging lymphocytes and spermatozoa to different genotoxicants, significantly increased DNA damage in newborns compared to adults was found. This confirms an exceptional vulnerability in newborns, believed to cause increased susceptibly to disease in later life, including cancer.

Introduction: Au Canada, le cancer de la prostate est le cancer le plus fréquemment diagnostiqué chez les hommes et le plus mortel après les cancers du poumon et du côlon. Il y a place à optimiser le traitement du cancer de la prostate de manière à mettre en œuvre une médecine personnalisée qui s’adapte aux caractéristiques de la maladie de chaque patient de façon individuelle. Dans ce mémoire, nous avons évalué la réponse aux dommages de l’ADN (RDA) comme biomarqueur potentiel du cancer de la prostate. Les lésions potentiellement oncogènes de l'ADN déclenche une cascade de signalisation favorisant la réparation de l'ADN et l’activation des points de contrôle du cycle cellulaire pour préserver l’intégrité du génome. La RDA est un mécanisme central de suppression tumorale chez l’homme. La RDA joue un rôle important dans l’arrêt de la prolifération des cellules dont les génomes sont compromis, et donc, prévient la progression du cancer en agissant comme une barrière. Cette réponse cellulaire détermine également comment les cellules normales et cancéreuses réagissent aux agents utilisés pour endommager l'ADN lors du traitement du cancer comme la radiothérapie ou la chimiothérapie, en plus la présence d,un certain niveau de RDA dans les cellules du cancer de la prostate peuvent également influer sur l'issue de ces traitements. L’activation des signaux de la RDA peut agir comme un frein au cancer dans plusieurs lésions pré-néoplasiques de l'homme, y compris le cancer de la prostate. Il a été démontré que la RDA est augmentée dans les cellules de néoplasie intra- épithéliale (PIN) comparativement aux cellules prostatiques normales. Toutefois, le devient de la RDA entre le PIN et l’adénocarcinome est encore mal documenté et aucune corrélation n'a été réalisée avec les données cliniques des patients. Notre hypothèse est que les niveaux d’activation de la RDA seront variables selon les différents grades et agressivité du cancer de la prostate. Ces niveaux pourront être corrélés et possiblement prédire les réponses cliniques aux traitements des patients et aider à définir une stratégie plus efficace et de nouveaux biomarqueurs pour prédire les résultats du traitement et personnaliser les traitements en conséquence. Nos objectifs sont de caractériser l'activation de la RDA dans le carcinome de la prostate et corréler ses données avec les résultats cliniques. Méthodes : Nous avons utilisé des micro-étalages de tissus (tissue microarrays- TMAs) de 300 patients ayant subi une prostatectomie radicale pour un cancer de la prostate et déterminé le niveau d’expression de protéines de RDA dans le compartiment stromal et épithélial des tissus normaux et cancéreux. Les niveaux d’expression de 53BP1, p-H2AX, p65 et p-CHK2 ont été quantifiés par immunofluorescence (IF) et par un logiciel automatisé. Ces marqueurs de RDA ont d’abord été validés sur des TMAs-cellule constitués de cellules de fibroblastes normales ou irradiées (pour induire une activation du RDA). Les données ont été quantifiées à l'aide de couches binaires couramment utilisées pour classer les pixels d'une image pour que l’analyse se fasse de manière indépendante permettant la détection de plusieurs régions morphologiques tels que le noyau, l'épithélium et le stroma. Des opérations arithmétiques ont ensuite été réalisées pour obtenir des valeurs correspondant à l'activation de la RDA qui ont ensuite été corrélées à la récidive biochimique et l'apparition de métastases osseuses. Résultats : De faibles niveaux d'expression de la protéine p65 dans le compartiment nucléaire épithélial du tissu normal de la prostate sont associés à un faible risque de récidive biochimique. Par ailleurs, nous avons aussi observé que de faibles niveaux d'expression de la protéine 53BP1 dans le compartiment nucléaire épithéliale du tissu prostatique normal et cancéreux ont été associés à une plus faible incidence de métastases osseuses. Conclusion: Ces résultats confirment que p65 a une valeur pronostique chez les patients présentant un adénocarcinome de la prostate. Ces résultats suggèrent également que le marqueur 53BP1 peut aussi avoir une valeur pronostique chez les patients avec le cancer de la prostate. La validation d'autres marqueurs de RDA pourront également être corrélés aux résultats cliniques. De plus, avec un suivi des patients plus long, il se peut que ces résultats se traduisent par une corrélation avec la survie. Les niveaux d'activité de la RDA pourront éventuellement être utilisés en clinique dans le cadre du profil du patient comme le sont actuellement l’antigène prostatique spécifique (APS) ou le Gleason afin de personnaliser le traitement. / Background: Prostate cancer is the most frequently diagnosed cancer in Canadian men and is the third deadliest after lung and colon cancers. Currently, prostate cancer treatments are based on results obtained of digital rectal exam, Gleason scores from biopsy specimens and serum PSA (Prostatic Specific Antigen) levels. The identification of specific biomarkers for diagnosis and prognosis, as well as new therapeutic targets, is quickly paving the way for personalized medicine. Ideally, in the future, patient care will include molecular signature of a patient's disease to guide for a more efficient treatment. In this thesis, we evaluated the DNA damage response (DDR) as a potential biomarker in prostate cancer. DNA lesions in mammalian cells trigger the DDR signalling cascade that orchestrates DNA repair and activate cell cycle checkpoints to preserve genome integrity. Loss of genome stability is usually associated with cancer development, and activated DDR signalling in cells with genomic instability act as a cancer barrier in several pre-neoplastic human lesions, including prostate cancer. Thus, the DDR is an important cancer suppression mechanism. The DDR is also activated in response to anti- cancer agents including radiation therapy (RT) and DNA-damaging chemotherapies. Pre- existing DDR levels in prostate cancer cells may influence the outcome of these cancer treatments. DDR signalling has been detected during human prostate cancer progression from low levels in normal prostate cells to high levels in high-grade prostatic intraepithelial neoplasia (HG-PIN). However, DDR signalling variations detected from HG-PIN to adenocarcinoma remain unclear, and no correlations were performed with patient clinical outcome data. Our hypothesis is that the levels of persistent DDR signalling activity will be variable with different grades and aggressiveness of prostate cancer. The levels of this activity could be correlated with the clinical responses to treatments and could even predict this process. We believe that having new biomarkers will help personalizing cancer treatment and certainly increase treatments’ efficiency. Our objectives are to characterize the occurrence of DDR activation in prostate carcinoma and to correlate it with patients’ survival and responsiveness to treatment. Methods: We used tissue microarrays (TMAs) from human radical prostatectomy specimens of 300 men with prostate cancer and estimated the level of DDR protein expression in the stromal and epithelial compartments of normal and aggressive cancer tissues. The expression level of the DDR markers p53 binding protein-1 (53BP1), phosphorylated H2AX (p-H2AX), p65 (p65 subunit of Nuclear Factor (NF-κB) and phosphorylated checkpoint kinase-2 (p-CHK2) was quantified using immunofluorescence (IF) coupled to high-content automated imaging. The quantification of our DDR markers was first validated on an experimental TMA (TMA-cell) including normal and irradiated (to induce DDR signalling) cultured human fibroblasts. The data was quantified using binary layers commonly used to classify pixels in an image so areas could be analysed independently allowing the segregation of specific compartments including nuclei, epithelia and stroma. Arithmetic operations were performed to render values corresponding to DDR activation that were then correlated with clinical outcomes such as biochemical recurrence and occurrence of bone metastasis. Results: We found that low levels of p65 protein expression in the nuclear epithelial compartments of normal prostate tissue were associated with a reduced probability of biochemical failure (which corresponds to a rise in the serum level of PSA in prostate cancer patients following treatment, surgery in this cohort of patients). Moreover, we also observed that low levels of 53BP1 protein expression in the nuclear epithelial compartments of normal and cancerous prostate tissue were associated with a lower incidence of bone metastasis. Conclusion: These results confirm that p65 has prognostic value in patients with prostate adenocarcinoma. Based on our results, we suggest that 53BP1 marker may have a prognostic value as well. The validation of other markers and particularly DDR markers may correlate with patients’ outcome. With longer follow-up, it may translate into correlation with survival. Levels of DDR activity in cancer tissue could be used in daily clinic as part of the patient’s diagnostic profile as much as his prostatic specific antigen (PSA) or Gleason score in order to predict response and personalize the treatment in order to guide the patients towards the most appropriate treatment amongst all those available for their prostate cancer.