Benzene is an ubiquitous pollutant and industrial solvent that has been identified as a human leukemogen. Early exposure to environmental carcinogens such as benzene has been postulated to play a role in the etiology of childhood leukemia, however the association remains controversial. Genotoxic agents such as benzene can cause an increase in the frequency of DNA double-strand breaks, which may remain unrepaired or result in the initiation of DNA recombinational repair mechanisms. The first objective was to investigate the induction of DNA double-strand breaks following in utero treatment to 200 mg/kg and 400 mg/kg benzene i.p. using the phosphorylated histone γ-H2A.X as a marker. Using immunoblotting, treatment with benzene did not increase the formation of γ-H2A.X in bone marrow cells of adult C57Bl/6N male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells following in utero exposure to 200 mg/kg or 400 mg/kg benzene throughout gestational days 7 to 15. Secondly, the study investigated the induction of micronuclei following in utero exposure to benzene. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells. In utero exposure to 400 mg/kg benzene throughout gestational days 7 to 15 also caused a statistically significant increase in the percentage of micronucleated cells in maternal bone marrow and post-natal bone marrow cells. Fetal liver cells also demonstrated a statistically significant increase in the percentage of micronucleated cells following 200 mg/kg and 400 mg/kg benzene. The third objective was to investigate the initiation of DNA recombination following in utero exposure to benzene using the pKZ1 mutagenesis mouse model as a surrogate marker for non-homologous end joining activity. Adult pKZ1 mouse tissue yielded no recombination events; however, post-natal bone marrow cells did contain detectable recombination frequencies. iii In utero benzene exposure did cause an increasing trend in recombination events, and upon analysis of only the samples containing detectable levels of recombination, in utero exposure to 400 mg/kg of benzene caused a statistically significant increase in recombination frequency within this group. These results demonstrate that benzene does not increase the formation of γ-H2A.X after acute and in utero exposure, however, the induction of micronuclei following acute and in utero benzene exposure confirmed that benzene is a genotoxic agent causing chromosomal breaks. In utero benzene exposure increased the frequency of DNA recombination in bone marrow from post-natal day 9 pups exhibiting detectable levels of recombination. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-08-22 11:07:49.162

No description available.

Radiation therapy targets cancer cell death by overwhelming cells with harmful DNA damage. Understanding how cells repair radiation damage and in particular how they become resistant to radiation therapy is important for effective cancer treatment. Our lab made the novel discovery that Bcl10, a cytoplasmic protein important for NF-B activation, localizes to endogenous H2AX foci in the nucleus of breast cancer cells. We determined that following radiation treatment Bcl10 is recruited to ionizing radiation-induced foci in a dose-dependent matter and that it is important for the repair of radiation-induced DNA damage. We also observed that breast cancer cells are extremely sensitive to Bcl10 knockdown, causing cellular senescence, while normal breast epithelial cells are insensitive. Our findings identify Bcl10 as potent anti-cancer target. / Experimental Oncology

No description available.

Nucleotide synthesis is a conserved and highly regulated response to DNA damage, required for the efficient repair of DNA double strand breaks (DSB) by homologous recombination (HR). This is essential to prevent loss of heterozygosity (LOH) and maintain genome stability. The aim of this study was to identify new genes important for HR through roles in damage-induced nucleotide synthesis. A screen was performed to identify S. pombe gene deletion strains whose DSB sensitivity was suppressed by deleting the ribonucleotide reductase (RNR) inhibitor spd1⁺ to promote nucleotide synthesis. The screen identified a number of genes including ddb1⁺, cdt2⁺, rad3⁺ and csn1⁺ which have known roles in nucleotide synthesis. Distinct roles were identified for the DNA damage checkpoint in suppressing LOH. rad3⁺, rad26⁺, rad17⁺ and the rad9⁺, rad1⁺ and hus1⁺ genes encoding the 9-1-1 complex were required for DNA damage-induced nucleotide synthesis through Cdt2 induction to promote Spd1 degradation. The HR repair defect of rad3⁺ and rad26⁺ deletion strains was partially suppressed by spd1⁺ deletion. However, the HR repair defect of rad17⁺, rad9⁺, rad1⁺ and hus1⁺ deletion strains was not suppressed. An additional role was confirmed for Rad17 and the 9-1-1 complex in preventing LOH by promoting DSB resection. A role was identified for the Gcn5 histone acetyl transferase (HAT) protein module, consisting of Gcn5, Ngg1, Ada2 and Sgf29, in suppressing DSB sensitivity by promoting nucleotide synthesis. This was independent of Cdt2 or RNR protein levels. The Gcn5 HAT module was also found to regulate DSB repair pathway choice consistent with previous observations. Deletion of gcn5⁺, ngg1⁺ or ada2⁺ decreased HR and increased non-homologous end joining. Surprisingly, deletion of spd1⁺ in a gcn5∆, ngg1∆ or ada2∆ background also promoted HR. This predicts a role for nucleotide pools in regulating DSB repair pathway choice. Eleven other candidates showed repeatable suppression of DSB sensitivity following spd1⁺ deletion. However many of these candidates did not show reduced nucleotide levels. This suggests deleting spd1⁺ may also suppress DSB sensitivity by a different mechanism.

Plants have developed intricate mechanisms to control growth in response to a variety of environmental cues, to compensate its immobility and to survive in both normal and adverse conditions. The TCP proteins are a family of plant-specific, basic helix-loop-helix (bHLH) transcription factors that involve in different aspects in plant growth and developmental control. The Arabidopsis TCP20 has been shown to involve in coordinating cell growth and proliferation, and in growth arrest in response to DNA double-stranded breaks (DSB). In this thesis, the main interest is to examine the function of Arabidopsis TCP6, which shares the highest homology with TCP20, and like TCP20, contains a putative ATM phosphorylation motif that suggests potential involvement in the ATM/ATR-mediated DSB responses. Expressional analysis including transcript measurement and reporter gene tagging demonstrated that TCP6 is expressed in flowers, in particular in the first mitotic event of pollen and ovule/embryo sac development, indicating that TCP6 potentially involves in regulating the mitotic cell cycle during gametophyte development. Yet no gametophytic or fertility-affecting mutant phenotype was observed in the tcp6 single and tcp6/tcp20 double mutants, which may be due to high functional redundancy. The tcp6/tcp20 double mutant seedlings exhibited significantly higher growth performances in true leaf growth compared to wild type when treated with gamma radiation, implying that both functional TCP6 and TCP20 are involved in response to gamma radiation-generated DSBs. The work of this thesis provides the first expressional and functional characterizations of TCP6, with the results suggesting that TCP6 and other class I TCPs play a role in regulating growth under both normal and stress conditions.

Au cours de la réplication, la réparation des cassures double brin (CDB) par recombinaison homologue (RH), basée sur la synthèse d’ADN à partir de la chromatide sœur, permet le maintien de la stabilité du génome. La religature d’extrémités (EJ) éloignées de CDB peut quant à elle générer des réarrangements menaçant son intégrité. Nous avons étudié le mécanisme de réparation par EJ en fonction de la distance séparant deux cassures double brin. En utilisant des substrats intra-chromosomiques permettant la mesure de l’efficacité et de la fidélité du EJ après ligature d’extrémités éloignées ou proximales, nous avons mis en évidence l’implication du complexe de cohésion dans l’inhibition du EJ d’extrémités distales. Le complexe de cohésion joue donc un rôle central dans l’interface réplication/réparation ; la cohésion des chromatides sœurs favorise la réparation par RH et permet l’inhibition spécifique du EJ d’extrémités éloignées, probablement en limitant la mobilité de la chromatine endommagée et la formation d’une synapse propice au rapprochement des extrémités. La religature d’extrémités éloignées est également nécessaire aux mécanismes de diversification des gènes des immunoglobulines tels que la recombinaison V(D)J et la commutation de classe. L’étude de souris Rad21+/- a également démontré une implication du complexe de cohésion dans ces mécanismes essentiels à la diversité de l’information génétique. Le complexe de cohésion étant impliqué dans ces mécanismes et dans l’inhibition des réarrangements complexes tels que les translocations et insertions il est un acteur essentiel de la diversité et de la stabilité génomique. / DNA double-strand breaks (DSBs) repair is essential for genome stability/diversity, but can also generate genome rearrangements. Although non-homologous end-joining (NHEJ) is required for genome stability maintenance, the joining of distant double strand ends (DSE) should inexorably lead to genetic rearrangements. We analyzed the efficiency and accurency of close or distal EJ repair. Our data show that global end-joining is more efficient on close ends (34bp) compared to distal ends (3200bp) and that C-NHEJ is favored on close ends, resulting in more accurate outcome, compared to distal ends where more mutagenic A-EJ events takes place. In addition, the joining of distal ends favors the insertion/capture of DNA sequences. These data show only few kb distances between two DSEs are sufficient to jeopardize DSB repair efficiency and accuracy, leading to complex scars at the re-sealed junctions, and cell response is sufficiently sensitive to differently process such distal ends. We next addressed the question of the mechanisms preventing the joining of distant DSE. We show that depletion of the cohesin complex proteins specifically stimulates the end-joining of I-SceI-induced DSBs distant of 3200bp, while the joining of close DSEs (34bp) remained unaffected. Consistently, exome sequencing and cytogenetic analysis revealed that RAD21 ablation generates large chromosome rearrangements and a strong induction of replication stress-induced chromosome fusions. These data reveal a role for the cohesin complex in the protection against profound genome rearrangements arising through ligation of distant DSEs.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

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.