Benzene is a ubiquitous environmental pollutant and a known human leukemogen. Early-life exposure to environmental carcinogens, including benzene, may lead to genomic instability in the fetus, ultimately leading to an increased risk for the development of childhood cancers including leukemia. It is possible that exposure to benzene results in DNA damage that may either be left unrepaired or be repaired erroneously causing genotoxicity. The first objective of this study was to determine if exposure of fetal hematopoietic cells to p-benzoquinone, a known toxic metabolite of benzene, increased DNA recombination in the pKZ1 model of mutagenesis. A significant increase in recombination was observed following exposure to 25 μM and 50 μM p-benzoquinone for 2, 4, 8, and 24 hours. A significant increase in recombination was also observed following exposure to 25 μM p-benzoquinone for 30 min, 45 min, and 1 hour, but not 15 min as compared to vehicle alone. Secondly, this study determined if exposure of fetal hematopoietic cells to p-benzoquinone resulted in DNA damage using γ-H2A.X as a marker for DNA double strand breaks and 8-hydroxy-2’-deoxyguanosine as a marker of oxidative DNA damage. A significant increase in γ-H2A.X foci formation was observed following exposure to 25 μM p-benzoquinone for 30 min, 45 min and one hour. Exposure of fetal hematopoietic cells to 25 μM p-benzoquinone did not significantly increase oxidative DNA damage at any of the examined time points. The third objective of this study was to determine whether or not reactive oxygen species were involved in the observed increase in DNA damage and recombination. Exposure to 25 μM p-benzoquinone for 15 min and 30 min, but not 45 min or one hour, led to an increase in reactive oxygen species production as measured by 5-(and-6)-chloromethyl-2-7-dichlorodihydrofluorescein diacetate fluorescence. Additionally, pretreatment with 400 U/mL PEG-catalase, an antioxidative enzyme, attenuated the increases in both DNA recombination and DNA double strand breaks as compared to treatment with p-benzoquinone alone. These studies indicate that p-benzoquinone is able to induce DNA damage and recombination in fetal hematopoieitic cells and that reactive oxygen species and oxidative stress may be important in the mechanism of toxicity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2010-07-23 15:44:05.381

Dephosphorylation of the Cdc2 kinase by the Cdc25 tyrosine phosphatase is the universally conserved trigger for mitotic entry. Cdc25 is also the point of convergence for checkpoint signaling pathways which monitor the genome for damaged DNA and incomplete replication. In addition, Cdc25 is inhibited by a MAP kinase cascade in the event of osmotic, oxidative and/or heat stress. These pathways inhibit cell cycle progression by phosphorylating Cdc25 resulting in its association with 14-3-3 and nuclear export. Although Cdc25 can be observed leaving the nucleus following inhibitory signals it is controversial whether phosphorylation, 14-3-3 binding or export itself is required for checkpoint proficiency. In fission yeast, Cdc25 is phosphorylated in vitro on 12 serine and threonine residues by the effector kinase of the DNA replication checkpoint, Cds1. Nine of these residues reside in the N-terminal regulatory region, while three are found in the extreme C-terminus of the protein. We show here that phosphorylation the nine N-terminal residues, nor any of the 12 in vitro sites, are required for enforcement of the DNA replication checkpoint. In lieu of Cdc25 phosphorylation the phosphatase is rapidly degraded and mitotic entry prevented by the action of the Mik1 kinase, targeting Cdc2. Thus, multiple mechanisms exist for preventing mitotic entry when S-phase progression is inhibited. The three C-terminal in vitro phosphorylation sites have not previously been examined in fission yeast. However, homology exists between the S. pombe protein and the Cdc25 orthologues in humans, Xenopus and Drosophila in this region. We report here that in S. pombe these sites are required to prevent mitotic entry following replication arrest in the absence of Mik1, and in the maintenance, but not establishment, of arrest following DNA damage. Our previous work showed that Cdc25 nuclear import requires the Sal3 importin-β but at the time we were unable to show a direct interaction between these two proteins. The final chapter of this thesis proves physical interaction by co-immunoprecipitation. Cdc25 mutants lacking all twelve putative Cds1 sites show nuclear localization during mitosis in a sal3- background, effectively reversing the cell cycle regulated pattern of accumulation of the phosphatase. / Thesis (Ph.D, Biology) -- Queen's University, 2011-06-20 12:16:15.71

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This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.

This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.

Loss of function of the WRN protein causes the genetic disorder Werner Syndrome that is characterized by increased cancer and premature aging. WRN belongs to the RecQ helicase family that plays key roles in preventing genome instability. In response to treatment with genotoxins, WRN is subject to post-translational modification. The relationship of post-translational modification of WRN with its function in DNA metabolism is unknown. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in DNA replication. Consistent with this notion, WS cells are sensitive to DNA replication inhibitors and DNA damaging agents that tend to block replication fork progression. The cells exhibit an extended S phase, as well as defects in normal bi-directional progression of replication forks diverging from the majority of replication origins. To elucidate the relationship between post-translational modifications of WRN with its function in DNA metabolism, here the acetylation of WRN was studied. In our studies, we provide evidence that WRN acetylation is a dynamic process that strongly correlates to blockage of replication by persistent DNA damage. We also determined the effect of WRN acetylation on its specificity and enzymatic functions. In addition, our studies reveal how agents that block replication regulate the nature of WRN interactions with RPA, a factor known to bind to single-stranded DNA generated at blocked replication forks. Our results demonstrated that WRN and RPA form a stable direct association under normal physiological conditions and treatments that block replication fork progression increase their association, further supporting the idea that WRN is involved in DNA replication through its action at blocked or stalled replication forks. Thus, these studies point to both 1) an important role for acetylation of WRN and 2) its interaction with RPA in the putative function of WRN in response to blocked replication. Overall, our results impact knowledge regarding the relationship between DNA damage, genome instability and the development and progression of aging and cancer.

Ruthenium complexes show promise as light activated photodynamic therapy (PDT) prodrugs. Strained octahedral complexes were synthesized that produce a cytotoxic species upon light activation. pUC19 DNA damage in vitro experiments were carried out to determine the type of damage observed. In vivo cell experiments were carried out on the non-small lung cancer A549 cell line to determine the phototherapeutic window of the synthesized complexes. One mechanism of drug resistance via elevated levels of glutathione was addressed through in vitro binding studies carried out with UV-Vis spectroscopy and in vivo glutathione titrations in the A549 cell line. Several complexes were shown to be potential PDT agents with light-activated activities greater than cisplatin and 10-100 fold lower dark toxicities.

The cell cycle is a highly complex process that is under the control of several pathways.  Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.

Gene regulatory networks are dynamic and continuously remodelled in response to internal and external stimuli. To understand how these networks alter cellular phenotype in response towards specific challenges, my first project sought to develop a methodology to explore how the strength of genetic interactions changes according to environmental context. Defined as sensitivity-based epistasis, the results obtained using this methodology were compared to those generated under the conventional fitness-based approach. By integrating this information with gene expression profiles and physical interaction datasets, we demonstrate that sensitivity-based epistasis specifically highlights genetic interactions with a dynamic component. Having investigated how an external stimulus regulates network dynamics, we next sought to understand of how genome positioning impacts transcription kinetics. This feat was accomplished by cloning two gene-reporter constructs, representing contrasting promoter architectures, across 128 loci along chromosome III in S.Cerevisiae. By comparing expression and noise measurements for promoters with “covered” and “open” chromatin structures against a stochastic model for eukaryotic gene expression, we demonstrate that while promoter structure regulates burst frequency (the rate of promoter activation), positional effects in turn appear to primarily modulate burst size (the number of mRNA produced per gene activation event). By integrating these datasets with information describing global chromatin structure, we suggest that the acetylation state of chromatin regulates burst size across the genome. Interestingly, this hypothesis is further supported by nicotinamide-mediated inhibition of Sir2 which would appear to modulate burst size globally across the genome.