Cells respond to the induction of DNA damage with activation of the DNA damage response (DDR), a complex signalling network which orchestrates cell cycle arrest and DNA repair in order to maintain genomic stability and cell viability. Activation of these signalling pathways enables cancer cells to survive DNA damaging chemo- or radiotherapy and contributes to the development of therapy resistance. Therefore, components of the DDR have become attractive targets for chemo- or radiosensitisation. Furthermore, cancer cells frequently exhibit defects in certain DDR components and may, as a consequence, become highly dependent on remaining DDR pathways to survive DNA damage. Two apical mediators of the DDR are the serine/threonine protein kinases ATM and ATR. ATM is frequently mutated in non-small cell lung cancer (NSCLC), and defects in ATM may render the tumour cells dependent on ATR signalling for survival. In this study, we characterised the functional consequences of ATM mutations in NSCLC cell lines and established an immunohistochemistry-based assay to identify patients with loss of ATM expression. As a single agent, pharmacological ATR inhibition (ATRi) was selectively cytotoxic for cells deficient in both ATM and p53. Furthermore, ATRi in combination with either ATM or PARP inhibition selectively killed tumour cells with mutant p53. We show that following ATR inhibition, ATM and p53 perform critical cell cycle checkpoint functions, independently of each other. Our results suggest that while retained function in any of these pathways is sufficient to maintain cell viability, functional loss of ATM, ATR and p53 results in premature mitotic entry, chromosome fragmentation and mitotic catastrophe. We conclude that in NSCLC the functional status of both ATM and p53 determines the cellular response to ATR inhibition, and propose that a combination of ATR inhibition with ATM or PARP inhibition may have broad utility for the treatment of p53-mutated NSCLC.

by Brian C. Freeman. / Typescript. / Thesis (M.S.)--Florida State University, 1991. / Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:24:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-21Bitstream added on 2014-06-13T19:51:38Z : No. of bitstreams: 1 cezario_gag_me_botfm.pdf: 488938 bytes, checksum: 0f674e5f7e12d7ce0a1eabedb5c6a4bb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecules and influence on immunity to control the host-parasite interaction. Our objective was to evaluate the mRNA expression of TLR-2 and 4, expression and production of IL-12, IFN-γ, TNF-α, IL-17, IL-10 and TGF-β and NO production during infection with Leishmania chagasi and correlate TLR2 and 4 expressions with cytokines production and NO. Infection resulted in increased TLR2, TLR4, IL-17, TNF-α and TGF-β expression at the beginning of infection, with a decrease at the final phase in according the parasitic load; IFN- γ and IL-12 decreased at the peak of parasitemia and increased at the final phase; IL-10 increased during the whole period under analysis. With respect to cytokines and NO production, TGF-β, TNF-α and IL-17 showed high rates at the initial phase, and IFN-γ and IL-12 showed high rates at the final phase; IL-10 and NO showed increasing production during the infection period evaluated. There was a positive correlation of TLR2 and 4 with TNF-α, IL-17, NO, IL-10 and TGF-β at the beginning of infection, and with TNF-α, IL-17 and TGF-β at the end. Our data suggest that L. chagasi was in contact with host’s cells via TLR2 and 4, which resulted in cytokine modulation. This interaction could be considered as pathogenic mechanism in visceral leishmaniasis

Masters of Science / The three-dimensional structures of proteins are important in understanding their function and interaction with ligands and other proteins. In this work, the structures of two proteins, ImuA’ from mycobacterium tuberculosis and GH9 C1 cellulase from a metagenomic library, were analysed using structural biological and modelling techniques. The gene encoding ImuA’ was amplified by two-step PCR, cloned, and expressed in E. coli. The recombinant ImuA’ produced was found to be largely insoluble. The insoluble protein was successfully solubilized in 8M urea but refolding the protein to its native structure was unsuccessful. By homology modelling, a 3D model of ImuA’ was obtained from a partly homologous protein RecA. In comparison to RecA, ImuA’ appears to lack some loop amino acids critical for DNA binding. Hence ImuA’ is postulated to not bind DNA. The second protein, GH9 C1 cellulase, was produced in E. coli. The protein was purified by chromatographic techniques and crystallized in a precipitant to protein ratio of 1:2 by hanging and sitting drop crystallization methods. The reservoir solution was made up of 15-30% (w/v) PEG 3350, 200 mM salt and 100 mM Tris-HCL pH 7.5-8.5. The protein crystals only diffracted x-rays to 4 å resolution which could not be used to obtain a crystal structure of the protein. The diffraction data, however, showed the crystal to be monoclinic with space group P2. Homology modelling revealed GH9 C1 cellulase to be a two domain protein with a smaller N-terminal Ig-like domain and a larger catalytic domain.The catalytic domain retains two ca2+ binding sites, which potentially stabilize the active site conformation and increase thermostability of the protein. Overall GH9 C1 cellulase is structurally similar to other GH9 cellulases, suggesting that its catalytic mechanism may be conserved.

No description available.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22736号 / 医博第4654号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 武藤 学, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...

Chromosomal translocation is a characteristic feature of human lymphoid malignancies and a driver of the initiation and progression of the disease. They arise from the mis-repair of physiological DNA double-strand breaks (DSBs) generated during the assembly and subsequent modifications of the antigen receptor gene loci, namely V(D)J recombination and class switch recombination (CSR). Mammalian cells have three DSB repair pathways –classical non-homologous end-joining (cNHEJ), alternative end-joining (A-EJ), and homologous recombination. DNA end-resection that generates a single-strand 3’ overhang is a critical regulator for the repair pathway choice. Specifically, localized end-resection prevents cNHEJ and exposes flanking microhomology (MH) to promote error-prone A-EJ. In addition to DNA repair, DNA end-resection generates extended single-strand DNA, which activates the ATR-mediated cell cycle checkpoint and indirectly contributes to genomic integrity. The central goal of my thesis research is to investigate the physiological role of DNA end-resection initiation in lymphocyte development and lymphomagenesis. DNA end-resection in mammalian cells is mostly initiated by the endonuclease activity of MRE11-RAD50-NBS1 (MRN) complex aided by CtIP. In addition, MRN protein also recruits EXO1 and DNA2 nucleases in combination with Top3 helicase complex for more extensive resection. The CtIP protein is essential for the endonuclease activity of the MRN complex that initiates DNA end-resection. CtIP is essential for embryonic development. Here I utilized B cell-specific conditional deletion models and loss-of-function mutations to investigate the role and regulation of CtIP and CtIP-mediated DNA end-resection in lymphocyte development and tumorigenesis. The level and extent of CtIP-mediated resection are tightly regulated. For the first aim, we applied the ATAC-Seq and EndSeq methods to test whether chromatin accessibility determines the level of DNA end-resection. Specially, we found that chromatin-bound DNA damage response factors – H2AX and 53BP1- reduced the accessibility of the DNA around the DSBs and antagonized end-resection. Our data also suggest that during DNA damage response, the nucleosome-free or accessible regions are more prone to secondary DNA breakages. Mechanistically, the preferential vulnerability is correlated with the availability of chromatin-bound DNA damage response factor 53BP1, which protects the nucleosome covered region at the price of the nucleosome-free regions. The work provides one explanation for tissue and cell type-specific translocations in transcriptionally active regions and super-enhancers. For the second and third aims, I investigated the role of CtIP and CtIP-mediated end-resection in lymphocyte development and lymphomagenesis in vivo using the conditional deletional CtIP allele and a phosphorylation-deficient CtIP-T855A mutant. T855 phosphorylation promotes end-resection but is not essential for cellular viability. I identified a sequence-context-dependent role of CtIP and end-resection in A-EJ mediated repair. We found that the reduced level of end-resection did not alter the frequency of the A-EJ mediated joining during B cell CSR, nor the levels of micro-homology at the junction, a defining feature of A-EJ mediated repair. These findings, for the first time, showed that DNA end-resection is not essential for A-EJ-mediated chromosomal DSBs repair nor for the generation of MH at the junction in vivo. This unexpected observation also highlights a tissue- and cell type-specific regulation of A-EJ and the importance of sequence context for A-EJ. Moreover, we found that ATM kinase suppresses A-EJ mediated translocation and reported the very first cell cycle-dependent analyses of CSR junctions. In cNHEJ-deficient B cells (e.g., Xrcc4- or DNA-PKcs- deficient), the A-EJ pathway is responsible for both the residual CSR events and the generation of the oncogenic IgH-Myc chromosomal translocations. In the last chapter, I determined how CtIP contributes to oncogenesis using the CtIP-T855A phospho-deficient mouse model. The result showed that CtIP T855 phosphorylation is critical for the neonatal development of Xrcc4-/-p53-/- mice and IgH-Myc translocation driven lymphomagenesis in DNA-PKcs-/-Tp53-/- mice. Mechanistically, phospho-deficient CtIP compromises the extent of end-resection without affecting the initiation. Reduced end-resection in CtIP-T855A mice and cells attenuated G2/M checkpoints and reduced the tolerance to the oncogene-induced replication stress, thereby limit lymphomagenesis. Collectively, our data provided the first in vivo characterization for the role of CtIP and its related end-resection pathway in lymphocyte development and lymphomagenesis. The results highlight the importance of end-resection for checkpoint maintenance (§ 4) and the context-dependent regulation of A-EJ and DNA repair pathway choice in vivo (§ 3), explaining why A-EJ is more robust at the repetitive switch regions. Finally, the application of HTGTS, EndSeq, and ATAC-Seq technologies in lymphocyte-specific gene rearrangements markedly improved the analysis depth and sensitivity while reducing the cost of repair-junction sequencing, allowing the quantitative detection of subtle changes and additional mechanistic insights. Specifically, we showed that end-resection could be regulated at the level of chromatin accessibility, which is determined by both baseline chromatin occupancy and DNA damage-induced changes (§ 2). These findings provide one explanation for the tissue and cell type-specificity of translocation targeting. These techniques can be used to analyze the impact of other DNA repair factors during lymphocyte development and lymphomagenesis and in translocation in general.

This work aimed to better understand the early molecular events occasioned in human thyrocytes following exposure to genotoxic agents than can lead to thyroid mutagenesis and cancer. Similar in vitro data in human physiological models remain scarce.The reported incidence of papillary thyroid carcinoma (PTC), has been increasing worldwide but the pathogenesis of sporadic PTC remains unravelled. H2O2 has long been postulated by the host laboratory and by other groups as a potential thyroid mutagen since it is capable to cause DNA damage and it affects γ radiation-induced DNA damage in thyrocytes. Thyrotropin (TSH) has recently been advocated as a potential risk factor for PTC: it has been associated with advanced stage disease and with progression of microcarcinoma during active surveillance. TSH signalling was found to be essential for BRAF-induced thyroid carcinogenesis in mouse models. Radiation (γ and β) is the only clearly established environmental risk factor for PTC; the cancer relative risk is rising linearly beyond a threshold of 100mGy thyroid absorbed dose. We studied in human thyrocytes in primary cultures the early molecular events following 131I exposure (β radiation) in comparison to γ radiation and H2O2 exposure, and their modulation by TSH. We assessed cell survival, DNA damage, DNA repair, gene expression, cell proliferation and apoptosis. Although we globally observed that the thyrocyte responses following exposure to β, γ radiation or H2O2 showed similarities, TSH, unlike other proliferative agents tested, specifically increased DNA damage both in non-exposed and in 131I-exposed thyrocytes. TSH did not influence γ radiation- or H2O2-induced damage. The effect of TSH on DNA damage in non-exposed thyrocytes decreased after incubation with an antioxidant agent and in 131I-exposed thyrocytes was partially alleviated after inhibition of iodide uptake. Therefore, in our experimental conditions, TSH seemed to predispose thyroid cells to greater DNA damage after exposure to 131I via Gαq-mediated increase in ROS/H2O2 levels, independent of its action on iodide uptake or proliferation status. DNA repair timing was similar between β, γ radiation and H2O2. Both β and γ radiation resulted in an extended thyroid cell cycle arrest but no apoptosis. In contrast, H2O2 did not appear to affect cell cycle at the same extent.This works includes the first, to our best knowledge, RNA sequencing to obtain the gene expression profile of human thyrocytes following 131I exposure. Overall transcriptional responses of thyrocytes exposed to 131I, γ radiation and H2O2 overlapped. At this stage, we were not able to attribute a distinct molecular signature to each agent, pointing that they probably employ similar cellular toxicity mechanisms. Regulated genes were involved in inflammatory response, cytokine signaling, DNA repair, apoptosis and cell cycle. Again, TSH yielded a more prominent transcriptomic response, both in number and in expression level of regulated transcripts, exclusively following 131I exposure. Better understanding of the early stages of thyroid carcinogenesis could lead to a more precise, personalized management of thyroid cancer patients and even in novel therapeutic strategies. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished