ABSTRACT Individuals that carry deleterious mutations in the breast and ovarian cancer susceptibility gene 1 (BRCA1) have much more elevated risk to develop breast and/or ovarian cancer than the individuals from the general population. The BRCA1 gene product has been implicated in several aspects of the DNA damage response, but its biochemical function in these processes has remained elusive. In order to probe BRCA1 functions we conducted a yeast two-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central region of BRCA1. In this dissertation, we report the identification of the actin-binding protein Filamin A (FLNA) as a BRCA1 partner and demonstrate that FLNA is required for the efficient regulation of DNA repair process at its early stages. Cells lacking FLNA display a diminished ionizing radiation (IR)-induced BRCA1 focus formation and a slow kinetics of Rad51 focus formation. In addition, our data demonstrate that FLNA is required to stabilize the interaction between DNA-PK holoenzyme components such as DNA-PKcs and Ku86 in a BRCA1-independent manner. Our data are consistent with a model in which the absence of FLNA compromises homologous recombination and non-homologous end joining. Our findings have implications for our understanding of the response to irradiation-induced DNA damage.

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.