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1
Oehler, Verena. "Role of Chk1 and Chk2 in mitotic checkpoint control in vertebrate cells." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/148/.
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Two conserved protein kinases, Chk1 and Chk2, are activated in response to genotoxic stress and mediate multiple cell cycle checkpoint mechanisms that ensure genomic integrity. The establishment of mitotic checkpoint delay in response to DNA damage or incompletely replicated DNA is conventionally thought to be accomplished through inhibition of the cyclin-dependent kinase, Cdc2. Both Chk1 and Chk2 have the potential to operate in this pathway. Therefore, the initial aim of this study was to investigate the relative requirement of Chk1 and Chk2 for mitotic delay mechanisms triggered by DNA damage and DNA replication arrest in avian and human cells. These studies demonstrated that Chk1 is the principal regulator of the G2/M checkpoint with a direct role in the establishment and maintenance of the mitotic delay in response to DNA damage. Chk1 was also found to be required for the S/M checkpoint in response to DNA replication arrest; however, detailed analysis indicated that its role is to maintain rather than initiate this checkpoint, as cells lacking functional Chk1 can initially delay mitosis for many hours before they enter a premature mitosis with unreplicated DNA. In avian cells, mitotic phosphorylation of cyclinB2 was found to be mediated by cyclin dependent kinases and suppressed by checkpoint signalling. However, accumulation of potentially active phospho-cyclinB2/Cdc2 complexes was observed during the initial mitotic delay in the absence of functional Chk1, suggesting that other factors apart from the conventionally known mechanisms can restrain mitotic Cdc2 activity. In addition, avian cells were able to delay mitosis effectively during replication arrest in the presence of the ATM/ATR inhibitor caffeine, further emphasizing the possibility of mitotic delay mechanisms that operate independently of ATM/ATR and Chk1. Furthermore, this study revealed that endogenous Cdc6 accumulates in a Chk1-dependent manner during replication arrest. To test whether Cdc6 might function upstream or downstream of Chk1 in the replication checkpoint pathway, Cdc6 was ectopically expressed in both checkpoint-proficient and checkpoint-deficient Chk1-depleted cells. The results from these intervention experiments give preliminary evidence that places Cdc6 downstream of Chk1 in the S/M checkpoint response. The ability of cells to delay the onset of mitosis while DNA replication is stalled independently of ATM/ATR/Chk1 is consistent with the general idea of an inherent relationship between the process of DNA replication and mitosis. The replication machinery might be able to signal either normal DNA replication in progress or the presence of stalled replication structures and thereby intrinsically link the successful completion of DNA synthesis to progression into mitosis.
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Höglund, Andreas. "Regulation of DNA damage responses by the Myc oncogene : implications for future anti-cancer therapies." Doctoral thesis, Umeå universitet, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-44284.
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Myc is a transcription factor frequently found deregulated in human cancer. Cells with deregulated expression of Myc carry a selective advantage against its neighbours due to the fact that Myc-mediated transcription governs crucial cellular events such as proliferation and growth. In addition, Myc has been implicated in several other aspects of tumour biology like cellular immortality, the formation of new blood vessels and the colonization of distant tissues through the process of metastasis. Therapy aimed at disrupting essential pathways regulated by Myc is important because of the many different types of cancers that depend on continued signalling along these pathways. This thesis describes new treatment opportunities for cancers with a high Myc signature. In Paper Ι, we describe a new role for the DNA methyltransferase inhibitor Decitabine in the treatment of Myc transformed tumours cells. We show that the therapeutic potential of Decitabine in the treatment of Burkitt Lymphoma relies not only on its ability to cause reactivation of silenced genes such as pro-apoptotic PUMA, but also on the DNA damage that this drug induces. In vivo, Decitabine delays disease progression of transplanted lymphoma cells. In Paper ΙΙ, we identify the DNA damage checkpoint kinase Chk1 as a therapeutic target in Myc overexpressing cancers. We show that targeting Chk1 with shRNA or with a novel small molecule inhibitor cause a delay in disease progression of transplanted lymphoma cells in vivo. In Paper ΙΙΙ, the Chk1-related kinase Chk2 is evaluated as a therapeutic target in Myc overexpressing cancers. Myc overexpressing cells are not dependent on Chk2 but we show that Chk2 abrogation using shRNA causes polyploidization and protection against DNA damage. However, Chk2-targeted therapy elicits a synergistic lethal response in combination with inhibition of the DNA repair associated protein PARP. In conclusion, this thesis shows the potential of targeting the DNA damage machinery and the functional hubs important for maintenance of genomic stability in tumours with a deregulated expression of Myc.
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Qin, Dongyan. "Specificity and structural modeling of FHA domain of CHK2 and a general characterization of FHA domain of caenorhabditis elegans CHK2." The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1061304007.
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Freeman, Alyson. "CHK2 is Negatively Regulated by Protein Phosphatase 2A." Scholar Commons, 2010. http://scholarcommons.usf.edu/etd/3443.
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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.
5
Ghelli, Luserna di Rorà Andrea <1987>. "The Inhibition of Chk1/Chk2 and Wee-1 Kinases as a Promising Therapy for the Treatment of Adult Acute Lymphoblastic Leukemia." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7529/1/Ghelli_Luserna_di_Rora%27_Andrea_Tesi.pdf.
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Due to inadequate treatments, the survival rate of adult patients with acute lymphoblastic leukemia (ALL) is still very poor. Thus there is a need to improve the efficacy of conventional therapy. In this study we evaluated the effectiveness of checkpoint kinase inhibitors (Chk-i) in single agent and in combination with different compounds conventionally used for the treatment of B-/T-ALL. We showed that Chk1 and Chk2 kinases are highly expressed and hyper-activated in tumor samples in comparison to normal tissue. On these bases we speculate that the inhibition of these kinases could mine the genetic stability and enhance cell death in ALL cells. We firstly evaluate the efficacy in single agent of the Chk1/Chk2 (PF-0477736 and LY2606368) and of the Wee1 (MK-1775) inhibitors on different cell lines and on primary cells isolated from adult B-ALL patients. We demonstrated that the inhibition of Chk1/Chk2 kinases reduces of the cell viability, activates the apoptosis and modify the expression of different elements of the G2/M checkpoint. To assess the chemo-sensitizer activity of different checkpoint kinase inhibitors, several combination studies were performed. To this purpose, LY2606368 and MK-1775 were combined with different tyrosine kinase inhibitors (imatinb, dasatinib and bosutinib) and with the purine nucleoside analogue, clofarabine. The efficacy of the combinations was not only evaluated in term of reduction of the cell viability but also in term of induction of apoptosis and induction of DNA damages. The results found were then confirmed on primary cells of B-ALL patients. Finally different class of checkpoint kinase inhibitors were combined together in order to evaluate their interaction. In our opinion the preclinical data presented in this study are the basis for a future evaluation of this class of compound in clinical trials in the treatment of adult ALL patients.
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Ghelli, Luserna di Rorà Andrea <1987>. "The Inhibition of Chk1/Chk2 and Wee-1 Kinases as a Promising Therapy for the Treatment of Adult Acute Lymphoblastic Leukemia." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2016. http://amsdottorato.unibo.it/7529/.
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Due to inadequate treatments, the survival rate of adult patients with acute lymphoblastic leukemia (ALL) is still very poor. Thus there is a need to improve the efficacy of conventional therapy. In this study we evaluated the effectiveness of checkpoint kinase inhibitors (Chk-i) in single agent and in combination with different compounds conventionally used for the treatment of B-/T-ALL. We showed that Chk1 and Chk2 kinases are highly expressed and hyper-activated in tumor samples in comparison to normal tissue. On these bases we speculate that the inhibition of these kinases could mine the genetic stability and enhance cell death in ALL cells. We firstly evaluate the efficacy in single agent of the Chk1/Chk2 (PF-0477736 and LY2606368) and of the Wee1 (MK-1775) inhibitors on different cell lines and on primary cells isolated from adult B-ALL patients. We demonstrated that the inhibition of Chk1/Chk2 kinases reduces of the cell viability, activates the apoptosis and modify the expression of different elements of the G2/M checkpoint. To assess the chemo-sensitizer activity of different checkpoint kinase inhibitors, several combination studies were performed. To this purpose, LY2606368 and MK-1775 were combined with different tyrosine kinase inhibitors (imatinb, dasatinib and bosutinib) and with the purine nucleoside analogue, clofarabine. The efficacy of the combinations was not only evaluated in term of reduction of the cell viability but also in term of induction of apoptosis and induction of DNA damages. The results found were then confirmed on primary cells of B-ALL patients. Finally different class of checkpoint kinase inhibitors were combined together in order to evaluate their interaction. In our opinion the preclinical data presented in this study are the basis for a future evaluation of this class of compound in clinical trials in the treatment of adult ALL patients.
7
Zannini, Laura. "Analysis of the functional domains of the cell cycle checkpoint kinase Chk2." Thesis, Open University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441156.
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LeBron, Cynthia. "Regulation of MDMX nuclear import and degradation by Chk2 and 14-3-3." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0001992.
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Wroble, Brian Noel. "The Role of Chk2 and Wee1 Protein Kinases during the Early Embryonic Development of Xenopus laevis." Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/29723.
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In somatic cells, when DNA is damaged or incompletely replicated, checkpoint pathways arrest the cell cycle prior to M or S phases by inhibiting cyclin-dependent kinases (Cdks). In Xenopus laevis, embryonic cellular divisions (2-12) consist of rapid cleavage cycles in which gap phases, checkpoint engagement, and apoptosis are absent. Upon the completion of the 12th cellular division, the midblastula transition (MBT) begins and the cell cycle lengthens, acquiring gap phases. In addition, cell cycle checkpoint pathways and an apoptotic program become functional. The studies described here were performed to better understand the roles of two protein kinases, Chk2/Cds1 and Wee1, during checkpoint signaling in the developing embryo.
The DNA damage checkpoint is mediated by the Chk2/Cds1 kinase. Conflicting evidence implicates Chk2 as an inhibitor or promoter of apoptosis. To better understand the developmental function of Chk2 and its role in apoptosis, we expressed wild-type (wt) and dominant-negative (DN) Chk2 in Xenopus embryos. Wt-Chk2 created a pre-MBT checkpoint by promoting degradation of Cdc25A and phosphorylation of Cdks. Embryos expressing DN-Chk2 developed normally until gastrulation and then underwent apoptosis. Conversely, low doses of wt-Chk2 blocked radiation-induced apoptosis. These data indicate that Chk2 inhibits apoptosis in the early embryo. Therefore, Chk2 operates as a switch between cell cycle arrest and apoptosis in response to genomic assaults.
In Xenopus laevis, Wee1 kinase phosphorylates and inhibits Cdks. To determine the role of Wee1 in cell cycle checkpoint signaling and remodeling at the MBT, exogenous Wee1 was expressed in one-cell stage embryos. Modest overexpression of Wee1 created a pre-MBT cell cycle checkpoint, similar to Chk2, characterized by cell cycle delay and phosphorylation of Cdks. Furthermore, overexpression of Wee1 disrupted remodeling events that normally occur at the MBT, including degradation of Cdc25A, cyclin E, and Wee1. Interestingly, overexpression of Wee1 also resulted in post-MBT apoptosis. Taken together, these data suggest the importance of Wee1 as not only a Cdk inhibitory kinase, but also potentially as a promoter of apoptosis during early development of Xenopus laevis. The studies described here provide evidence that Chk2 and Wee1 have both similar and distinct roles in the developing embryo.Ph. D.
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Lopergolo, A. "CHK2 PHOSPHORYLATION OF SURVIVIN-DEX3 CONTRIBUTES TO A DNA DAMAGE-SENSING CHECKPOINT IN CANCER." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/171952.
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Survivin is a pivotal cancer gene with multiple roles in cell viability and mitosis, but the function(s) of its alternatively spliced isoforms has remained elusive. Here, we show that a survivin spliced variant that lacks exon 3 and contains a unique -COOH terminus sequence,
survivin-DEx3, is differentially expressed in cancer, compared to normal tissues, and correlates with aggressive disease and markers of unfavorable prognosis, by genome-wide bioinformatics analysis. Unlike other survivin variants, survivin-DEx3 localizes exclusively to nuclei in tumor cells, and is phosphorylated by the DNA damage checkpoint kinase, Chk2, on residues located in its unique -COOH terminus. Ala mutagenesis of the Chk2 phosphorylation sites prolongs survivin-DEx3 stability in tumor cells, inhibits the expression of Ser139 phosphorylated H2AX in response to double strand DNA breaks, and impairs colony formation in soft agar after DNA damage. Active Chk2 was detected at the earliest stages of the colorectal adenoma-to-carcinoma transition, persisted in advanced tumors, and correlated with increased survivin expression, in vivo. These data suggest that Chk2 phosphorylation of survivin-DEx3 contributes to a DNA damage-sensing checkpoint in tumor cells, which may affect sensitivity to genotoxic therapies.
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Rice, Ian S. "Honors Thesis: BRCA1 Interactions with BACH1, BARD1, and CHK2: Recent Evidence and Potential Developments in the Diagnosis, Treatment, and Prevention of Human Breast Cancer." Miami University Honors Theses / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=muhonors1114185039.
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Bindermann, Robert [Verfasser]. "Mutationsanalyse der Zellzyklus-Kontrollpunktkinase Chk2 in humanen Tumorzelllinien : Bedeutung für Tumorgenese, -Biologie und Therapieresistenz / Robert Bindermann." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2013. http://d-nb.info/1031098070/34.
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Cardoso, Suziene Caroline Silva. "Análise da expressão imunoistoquímica da Checkpoint Kinase - 2 no Carcinoma epidermóide da cavidade oral." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17143/tde-08012019-155708/.
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O carcinoma epidermóide da cavidade oral (CEO) é o tumor maligno mais frequente da região da cabeça e pescoço. O Chk2 (Checkpoint kinase 2) é considerado um supressor tumoral que atua na resposta celular ao dano do DNA. Entretanto, a relação do Chk2 entre CEO ainda não está compreendida. O objetivo deste estudo foi avaliar a imunoexpressão do Chk2 nos CEOs e associar sua expressão com parâmetros clínico-patológicos de importância prognóstica, incluindo a sobrevida global, sobrevida livre de doença e livre de metástase. A expressão de Chk2 foi analisada em 104 amostras de pacientes com CEO por meio da técnica de imunoistoquímica e foi positiva em 97,11% dos nossos casos com CEO, com isso, estratificamos apenas os casos de marcação positiva, e dividimo-los em alta expressão (> 66%) e baixa expressão (<66%), e excluímos os casos negativos de nossa análise, pois o número de casos com expressão negativa para Chk2 seria inclusivo para realizarmos as análises estatísticas. A positividade de Chk2 na maioria dos nossos casos sugere que o Chk2 possa estar envolvido na patogênese desses tumores, porém em nosso trabalho, a expressão de Chk2 não se associou com os parâmetros prognósticos. Não houve diferença entre a sobrevida global, sobrevivência livre de metástases e sobrevida livre de doença de acordo com a marcação de Chk2. Em conclusão, em nossos achados, o Chk2 não pode ser considerado como um biomarcador prognóstico do carcinoma de células epidermóides da cavidade oral.Squamous cell carcinoma of the oral cavity (CEO) is the most frequent malignant tumor of the head and neck region. Chk2 (Checkpoint kinase 2) is considered a tumor suppressor that acts on the cellular response to DNA damage. However, the Chk2 relationship between CEO is not yet understood. The objective of this study was to evaluate the Chk2 immunoexpression in the CEOs and to associate their expression with clinical-pathological parameters of prognostic importance, including global survival, disease-free survival, and metastasis-free. The expression of Chk2 was analyzed in 104 samples of patients with CEO using the immunohistochemistry technique and was positive in 97.11% of our cases with CEO, with that, we stratified only the cases of positive marking, and we divided them into high expression (> 66%) and low expression (<66%), and we excluded the negative cases from our analysis, since the number of cases with negative expression for Chk2 would be inclusive for the statistical analysis. The positivity of Chk2 in most of our cases suggests that Chk2 may be involved in the pathogenesis of these tumors, but in our study, the expression of Chk2 was not associated with the prognostic parameters. There was no difference between overall survival, metastasis-free survival, and disease-free survival according to the Chk2 labeling. In conclusion, in our findings, Chk2 cannot be considered as a prognostic biomarker of oral squamous cell carcinoma.
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Aljohani, Saad Ali. "The human DNA damage sensor RAD9B affects CHK2-T68 phosphorylation, p21 stability and cell survival in G1." Thesis, Bangor University, 2016. https://research.bangor.ac.uk/portal/en/theses/the-human-dna-damage-sensor-rad9b-affects-chk2t68-phosphorylation-p21-stability-and-cell-survival-in-g1(1880e1f7-bd30-4dd1-9517-f3ab6ade0d4a).html.
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The RAD9 protein assembles with RAD1 and HUS1 in the 9-1-1 DNA damage sensor complex (RAD9-HUS1-RAD1) which plays a crucial role in the activation of the DNA damage checkpoints by recruiting repair enzymes to DNA lesions and by initiating a cell cycle arrest. Why human cells express two RAD9 proteins, RAD9A and RAD9B, which share significant similarity is still very enigmatic. Moreover, both genes encode several splice variants with unknown function. While RAD9A is intensively studied, very little information is available about RAD9B. To get a deeper insight into the biology of RAD9B, two stable HEK293 cell lines were constructed which either over-express full-length human RAD9B-002 (417aa) or its N-terminally truncated splice variant RAD9B-001 (345aa) from an inducible promotor. These studies were complemented by the expression analysis of all five RAD9B splice variants at mRNA level in human tissues. This study revealed for the first time specific activities of the two RAD9B variants. While both proteins target the CHK2-p21 signalling module, which regulates cell cycle progression from G1 into S phase and the response to DNA damage in G1/S, they do affect CHK2 kinase and the cell cycle regulator p21WAF1/CIP1 in distinct ways. Elevated protein levels of full-length RAD9B initiate cell death in G1, cause the moderate hyperposphorylation of CHK2 and delay the degradation of p21 in the presence of oxidative stress. On the contrary, high levels of the N-terminally truncated variant RAD9B-001 only transiently block G1-S transition, cause a strong hyper-phosphosphorylation of CHK2 and delay p21 degradation in the response to UV irradiation. While the shorter variant may block p21 degradation in a PCNA-dependent manner in S phase, the full-length protein may act through the anaphase promoting complex (APC) when cells exit mitosis. The work provides also evidence that the hyper-phosphorylation of CHK2 is not dependent on the normal upstream kinase ATM, but on the mitotic regulator MPS1/TTK kinase. Dissecting this novel regulatory network formed by the two splice variants of the human RAD9B gene may help to understand why RAD9B expression levels change in several human cancers including testicular cancer, myelogenous leukaemia, ovarian epithelial carcinoma and cervical carcinoma (HeLa) cells.
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Martínez, Marchal Ana. "Regulation of the oocyte pool in mammals." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667797.
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Durant la oogènesi dels mamífers, les oogònies proliferen forman els anomenats cists. Les oogònies entren en meiosis progressant en la profase I i els cists es trenquen al mateix temps que es produeix una mort massiva perinatal dels oòcits. En la profase I, s’indueixen trencaments de doble cadena (DSBs) per tot el genoma, que son reparats per recombinació homòloga per a promoure la sinapsi dels cromosomes homòlegs. Existeixen diferents mecanismes que s’activen en resposta a errors en aquests processos i que aturen el cicle cel·lular i produeixen l’apoptosi de les cèl·lules danyades. La resposta al dany al DNA (DDR) es activada en presència d’oòcits i d’espermatòcits amb errors de recombinació en l’anomenat checkpoint de recombinació. Per l’altre banda, errors en la sinapsi activen el checkpoint de sinapsi. El nostre objectiu era caracteritzar les funcions de la DDR i del checkpoint de sinapsi durant l’oogènesi en mamífers. Contràriament al que succeeix en espermatòcits, els oòcits presenten un alt número de DSBs no reparats a l’estadi de paquitè en el moment en que es produeix la mort oocitària massiva i el trencament del cists. Per tal d’esbrinar si el checkpoint de recombinació participa en la regulació del número d’oòcits en mamífers, hem analitzat el número de DSBs, el número d’oòcits en femelles perinatals i adultes, el trencament dels cists, la formació de fol·licles i la vida reproductiva de femelles de ratolí control i mutants per a la quinasa efectora de la via de la DDR, la proteïna CHK2. Les nostres dades han revelat la implicació de CHK2 en la regulació del número d’oòcits, però només en ovaris fetals, obrint la possibilitat de l’existència d’una via alternativa regulant el número d’oòcits després del naixement. Els nostres estudis utilitzant ovaris cultivats in vitro en presència d’inhibidors, suggereixen que CHK1 podria compensar l’absència de CHK2 in vivo. Per tant, la via de la DDR controlaria el número d’oòcits en mamífers. A més, hem trobat un augment del número d’oòcits en adultes velles mutants per CHK2 suggerint que la DDR controla la llargada de la vida reproductiva en mamífers. Finalment, hem estudiat el possible paper de TRIP13 en el checkpoint de sinapsi. La proteïna TRIP13 es necessària per a la recombinació, però també per a la sinapsi dels cromosomes sexuals i per a la formació de la vesícula sexual, suggerint un possible rol al checkpoint de sinapsi. Hem analitzat el número d’oòcits en ovaris Spo11-/- Trip13mod/mod i Dmc1-/- Chk2-/- Trip13mod/mod per a esbrinar si TRIP13 es necessària per a activar el checkpoint de sinapsi en femelles. Les nostres dades han revelat un rescat en el número d’oòcits en el triple mutant, però no en el doble. Aquest resultats obren la possibilitat de que TRIP13 participi en el checkpoint de sinapsis, però com a alternativa, proposem que aquesta participació podria ser compatible amb una possible regulació per part de TRIP13 de la elecció de la via de reparació dels DSBs.During mammalian oogenesis, oogonia proliferate forming the so-called cysts. The oogonia enter meiosis progressing through prophase I and the cysts break down concomitantly to massive perinatal oocyte death. During meiotic prophase I, double strand breaks (DSBs) are induced throughout the genome and repaired by homologous recombination to promote the synapsis of the homologous chromosomes. In response to errors in these processes, different response pathways are activated triggering cell cycle arrest or even apoptosis. The DNA damage response (DDR) is activated in response of meiocytes with recombination failure in the recombination checkpoint; while errors in synapsis trigger the synapsis checkpoint. We aimed to characterize the roles of the DDR and synapsis checkpoint in mammalian oogenesis. Contrary to what occurs in spermatocytes, oocytes present high numbers of unrepaired DSBs at pachynema, at the time of the massive oocyte death and cyst breakdown. In order to know if the recombination checkpoint participates in the regulation of the oocyte number in mammals, we analyzed the presence of DSBs, the oocyte number in both perinatal and adult females, the cyst breakdown, the formation of follicles and the reproductive lifespan using control and mutant mice for the effector kinase of the DNA damage response pathway, CHK2. Our data revealed the involvement of CHK2 in the regulation of the oocyte number but only in fetal ovaries prior to birth, raising the question of a possible alternative regulator acting just after birth. Our studies using in vitro ovarian cultures using inhibitors, suggest that CHK1 may compensate the loss of CHK2 perinatally in vivo. Thus, revealing that the DDR pathway controls the oocyte number in mammals. Furthermore, we found an increased number of oocytes in elder Chk2 mutant females suggesting that the DDR controls the reproductive lifespan extension in mammals. Finally, we studied the possible involvement of TRIP13 in the synapsis checkpoint. The protein TRIP13 is required for recombination, but it is also needed for the synapsis of sex chromosomes and the sex body formation. Thus, suggesting a possible role in the synapsis checkpoint. We analyzed the oocyte number in females from Spo11-/- Trip13mod/mod and Dmc1-/- Chk2-/- Trip13mod/mod ovaries in order to infer if TRIP13 is required to implement the synapsis checkpoint in females. Our data revealed a rescue in the number of oocytes in the triple mutant, but not in the double mutant. These results leave open the possibility of a participation of TRIP13 in the synapsis checkpoint, but as an alternative, they could be compatible with a possible role of TRIP13 regulating the DSB repair pathway choice.
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Behnfeldt, Julia. "Chk2 phosphorylation of the BLM helicase promotes its interactions with topoisomerase IIa and the resolution of chromosome breakage." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429263006.
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Masrouha, Nisrine. "Functional analysis of the Drosophila chk2 gene, loki : analysis of novel genetic interactors of Bic-D in Drosophila melanogaster." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80329.
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Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the cell cycle proceeds. The Chk2 family of kinases plays a central role in mediating responses to DNA damage or DNA replication blocks in various organisms. My functional analysis of the Drosophila serine/threonine kinase Loki/Chk2 shows that fly chk2 monitors double-strand breaks caused by irradiation during S and G2 phases and induces cell cycle arrest in embryonic cells around cellularization.loki is also required for the normal number of germ line cells to form in the embryo, and for normal modification of Vasa, a crucial factor in germ cell formation. However, during normal oogenesis loki expression is suppressed by orb. Another group described the involvement of Drosophila loki/chk2 in the meiotic pachytene checkpoint. Using our loki·null mutant, I obtained the opposite result: loki/chk2 does not have an essential function in this process.
The second part of my thesis deals with the question of how cells are instructed about their identity in a developing organism. (Abstract shortened by UMI.)
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Pansani, Fabianna. "Expressão imunohistoquímica do Chk2 e associação com características clínico-patológicas e desfecho em pacientes com câncer de cólon metastático." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17154/tde-15062015-090143/.
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INTRODUÇAO: O câncer de cólon é a terceira neoplasia mais prevalente no país, com aumento progressivo da incidência associada ao envelhecimento populacional. Os avanços nos tratamentos local e sistêmico do câncer de cólon metastático tem aumentado significativamente o tempo de sobrevida global. Entretanto, ainda não existem biomarcadores consolidados na literatura, capazes de predizer resposta a estes tratamentos ou o prognóstico. No processo da carcinogênese, uma das importantes vias que se encontra alterada é a via de reparo do DNA. A Chk2 é uma proteína quinase com atividade no reparo celular atuando de forma supressora no processo da carcinogênese, sendo que alterações em sua expressão e/ou função têm sido associadas à progressão tumoral em outras neoplasias como no câncer de mama, pulmão, vulva, bexiga, cólon, ovário, osteossarcoma e linfomas. OBJETIVO: Avaliar a expressão imunohistoquímica do Chk2 no câncer de cólon metastático e correlacionar sua expressão com características clínico-patológicas e sobrevida. PACIENTES E MÉTODOS: Foram incluídos 58 pacientes com diagnóstico confirmado de câncer de cólon metastático, tratados em primeira linha com quimioterapia baseada em fluorouracila e oxaliplatina. O tempo mínimo de seguimento foram de 2 anos pós-diagnóstico. Para análise da expressão do Chk2 foram utilizadas as técnicas de tissue microarray e imunohistoquímica. Estes resultados foram correlacionados com características clínicas, patológicas e de sobrevida. Para análise estatística, foi utilizado o programa SPSS17 e o valor de p<0,05 foi considerado estatisticamente significativo. RESULTADOS: A expressão de Chk2 foi positiva em 69% dos pacientes. Houve associação entre a expressão de Chk2 e o status linfonodal (p = 0,012) e entre a sobrevivência (p=0,034). A expressão negativa de Chk2 aumentaram as chances de envolvimento linfonodal (OR:10,2, p=0,03). O tempo de sobrevida global de pacientes Chk2 negativo foi maior (72 versus 59 meses, p=0,155), o mesmo foi observado com o tempo sobrevida livre de progressão (19 versus 13 meses, p=0,293). As curvas de sobrevida foram diferentes de acordo com a expressão do Chk2 em pacientes com ou sem envolvimento linfonodal, sendo menor nos pacientes com Chk2 positivo, p=0,028. Houveram mais óbitos em pacientes com Chk2 positivo. Análise multivariada identificou o performance status segundo a escala de ECOG (p=0,001 ); metástase sincrônica (p=0,037); diferenciação das células tumorais (p=0,029) e expressão de Chk2 (p=0,020) como fatores independentes para sobrevida global. CONCLUSÃO: A expressão positiva do Chk2 no adenocarcinoma de cólon metastático foi indicativa de maior agressividade e disseminação tumoral, impactando de forma negativa na sobrevida e desfecho dos pacientes.INTRODUCTION: The DNA damage checkpoint pathway has been of interest to the field of cancer biology, since checkpoint defects result in the accumulation of altered genetic information, a central feature of carcinogenesis. Little is known about the role of Chk2 in colorectal cancer tumorigenesis. OBJECTIVE: The purpose of this study was to evaluate Chk2 expression in metastatic colon cancer and correlate this with clinicopathological features and patient survival. PATIENTS AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer diagnosis, treated with capecitabine and oxaliplatin chemotherapy as standard doses. Patients included had, at least, 2 years post diagnosis of clinical following. The tissue microarray immunohistochemistry was the technic to evaluate Chk2 expression. Statistics analysis used SPSS 17. A p-value <0,050 was considered to be statistically significant. Immunohistochemical expression of Chk2 and its relationship with clinical and pathological characteristics and survival data was reported. RESULTS: The expression of Chk2 was positive in 69%. There was association between expression of Chk2 and Iymph node status (p=0.012) and between survival (p=0.034). The negative expression of Chk2 enhanced the chances of linfonodal involvement (OR:10,2, p=0.03). The global survival time of Chk2 negative patients was higher (72 versus 59 months, p= 0.155); the same was observed with progression-free survival time (19 versus 13 months, p=0.293). The survival curves were different according to Chk2 expression in patients with or without Iymph node involvement, being lower in patients with Chk2 positive, p=0.028. There were more deaths in patients with Chk2 positive. Multivariate regression analysis identified performance status ECOG (p=0.001), synchronous metastasis (p=0.037), tumor cell differentiation (p=0.029) and expression of Chk2 (p=0.020) as independent factors to overall survival. CONCLUSION: This study demonstrated that the Chk2 positive expression in colon cancer indicates increased tumor spread and tumoral aggressiveness, impacting negatively on survival and outcome of patients.
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Rainey, Michael David. "Genetic analysis of the role of Chk2 in the DNA-integrity checkpoints in the somatic DT40 B-lymphoma cell line." Thesis, University of Glasgow, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402009.
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Oliva, Trastoy Manel. "Rôle de la protéine phosphatase 2C Wip1 dans la régulation du suppresseur de tumeurs Chk2 en réponse au stress génotoxique." Paris 11, 2006. http://www.theses.fr/2006PA112053.
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Les cellules de mammifère possèdent des mécanismes de défense garantissant la correcte transmission de l'information génétique. L'ensemble des réponses cellulaires mises en œuvre lors de l'endommagement de l'ADN est appelé " checkpoint de l'ADN ". Notre étude porte sur la protéine kinase suppresseur de tumeurs Chk2. Les mutations du gène CHEK2 ont été corrélées au syndrome tumoral de Li-Fraumeni chez l'Homme. Pré-activée par phosphorylation de façon ATM-dépendante sur le résidu thréonine 68 (Thr68) lors d'un stress génotoxique, puis finalement activée par autophosphorylation sur la boucle T, Chk2 phosphoryle à son tour ses substrats qui sont impliqués dans l'arrêt du cycle cellulaire, la réparation des dommages de l'ADN et l'apoptose. Les mécanismes d'activation de Chk2 sont globalement bien connus, mais on ne sait toujours pas comment se déroule sa désactivation lorsque les lésions de l'ADN ont été réparées. Le laboratoire a montré en 2002, chez la levure, que les protéines phosphatases 2C de levure Ptc2/3 sont nécessaires à l'inactivation du checkpoint induit par une cassure double brin. Nous montrons ici que cette voie de signalisation est conservée dans les cellules humaines : après une irradiation, la protéine Wip1, ou PP2Cδ, codée par l'oncogène PPM1D, se lie à Chk2 et la déphosphoryle, principalement sur la phospho-Thr68. Wip1 est donc capable de s'opposer à l'activation de Chk2 par ATM. De plus, en utilisant deux lignées tumorales humaines HCT15, l'une n'exprimant pas d'activité Chk2, et l'autre exprimant la protéine HA-Chk2 à des niveaux endogènes, nous avons montré que la surexpression de Wip1 supprime la contribution de Chk2 dans l'arrêt du cycle en phase G2Mammalian cells possess several mechanisms which guarantee the proper transmission of genetic material. The whole of these cellular answers induced by DNA damage is called “DNA checkpoint”. Our study relates to the tumour suppressor kinase protein Chk2. Mutations in the CHEK2 gene have been reported in families with Li-Fraumeni syndrome. Pre-activated by phosphorylation on threonine 68 (Thr68) by ATM at the time of a genotoxic stress, Chk2 is finally activated by autophosphorylation on its T-loop. Chk2 then phosphorylates its substrates which are involved in stopping cell cycle progression, and promoting DNA repair and apoptosis. Whereas Chk2 activating mechanisms are overall well known, little is known about how the return to the physiological state is held when DNA has been repaired. Our laboratory showed in 2002, in the yeast, that type 2C protein phosphatases Ptc2/3 are required for the inactivation of the checkpoint induced by a double strand break. Our current study puts in obviousness that this mechanism is preserved in human cells. In response to DNA damage, Wip1 protein, also called PP2Cδ and coded by the PPM1D oncogene, binds and dephosphorylates Chk2, mainly on the phospho-Thr68. Wip1 is thus able to antagonise ATM activation of Chk2 by ATM after an ionizing radiation. Moreover, by using two HCT15 tumoral human cell lines, one possessing no Chk2 activity, and the other expressing at endogenous levels the HA-Chk2 protein, we have shown that Wip1 overexpression annihilates the Chk2 contribution to the stop in G2 phase. Thus, our results indicate that Wip1 phosphatase is able to negatively regulate the Chk2 activity upon a genotoxic stress
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Lossaint, Gérald. "Mécanismes orchestrant la sortie du cycle cellulaire opérant en G2." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20040/document.
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La dérégulation du système de surveillance qui bloque la prolifération lorsque l'intégrité du génome est compromise fait partie intégrante de la cancérogenèse. Nous cherchons à décortiquer les mécanismes qui, en phase G2, orchestrent l'arrêt du cycle cellulaire, irréversible, en présence des lésions de l'ADN (sénescence) ou réversible (quiescence), en absence de signaux mitogéniques ou confluence. L'objectif du premier volet fut d'élucider les rôles respectifs de l'inhibiteur de CDK (CKI) p21Waf1 et des kinases Chk1 et Chk2 dans l'arrêt en G2 dû au stress génotoxique menant à la sénescence. Nous avons montré que dans les cellules humaines normales cet arrêt nécessite l'action de p21 et Chk1 tandis que Chk2 n'est pas requise. Au contraire, dans plusieurs lignées cancéreuses, malgré la présence de p53, ce rôle de p21 est compromis à cause d'une activation inefficace de la kinase ATM. Par conséquent, en dépit d'une forte activation de Chk1 bloquant la mitose, ces cellules ne parviennent pas à initier la sénescence (Lossaint et al., soumis). L'objectif du deuxième volet fut de mettre en évidence le programme déclenchant la quiescence lors de confluence ou en absence de sérum. Les travaux antérieurs de l'équipe ont montré que cette décision pouvait être prise avant la mitose même si l'arrêt du cycle n'a lieu qu'en phase G1 suivante. En étudiant les fibroblastes synchronisés nous avons trouvé que la quiescence est précédée par l'inhibition pré-mitotique de la phosphorylation de pRb due à une diminution de cycline D1 et une stabilisation du CKI p27Kip1 (Chassot et al., 2008). De plus, nos résultats récents montrent que la présence de sérum entre le point R et la mitose est requise pour initier la réplication de l'ADN au cycle suivant. Les travaux futurs devraient élucider comment différentes voies de signalisation, via la voie cycline D-pRb, affectent divers composants de l'appareil de réplication de l'ADN pour inhiber la progression du cycle de façon réversible ou irréversibleCancer is a multi-step process resulting from abrogation of several barriers to uncontrolled proliferation. They include inhibitory pathways with appropriate checkpoints that lead to reversible (quiescence) or irreversible (senescence, apoptosis) block of cell proliferation. We are especially interested in pathways orchestrating cell cycle exit that operate in the G2 phase. The first objective of this thesis was to decipher mechanisms that prevent mitosis in response to DNA damage. We found that Cdk inhibitor p21Waf1 plays a crucial role in blocking mitotic onset in normal cells; acting in tandem with checkpoint kinase Chk1, p21 inactivates mitotic Cdks and inhibits pRb phosphorylation, thereby irreversibly blocking mitotic entry. In contrast, in p53-proficient transformed cells, the induction of p21 in G2 is impaired, most likely because of deficient ATM activation. While, in some cases, Chk1 hyper-activation prevents mitosis, the absence of p21 compromises the senescence program from G2. Finally, we showed that Chk2 is dispensable for G2 arrest in both non-transformed and transformed cells (Lossaint et al., submitted). Our second objective was to elucidate the pathways that induce quiescence (G0). This reversible cell cycle exit occurs in G1, requires pRb family members and p27Kip1-dependent Cdk inactivation. Based on observations obtained in our team and the data in the literature, we hypothesized that reversible cell cycle exit program might be launched before mitosis. By using an in vitro wounding model, we showed that confluence-driven quiescence is preceded by pre-mitotic CDK inhibition by p27, cyclin D1 downregulation and reduced pre-mitotic pRb phosphorylation (Chassot et al., 2008). Moreover, our results obtained in synchronized fibroblasts that were serum-starved after release from G1/S block suggest that cyclin D1 might stimulate proliferation by keeping pocket proteins phosphorylated during G2/M progression (Lossaint et al., in preparation)
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Bouzid, Hana. "Réponse cellulaire induite par les dommages de l'ADN créés par les ecteinascidines, une classe unique de médicaments anticancéreux." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066502.
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Les ectéinascidines (la trabectédine, la lurbinectédine) sont de nouveaux dérivés de produits naturels marins qui se lient de façon covalente à l'ADN, actifs contre les cancers chimio-résistants. L'objectif de ma thèse est 1) d'identifier les principales voies de transduction activées en réponse à l'apparition des lésions de l'ADN induites par les ETs 2) d'établir si l'abrogation pharmacologique de la réponse cellulaire induite par l'endommagement de l'ADN (ATM, ATR, Chk1, Chk2) peut moduler l'activité thérapeutique des ETs. Dans un premier temps, nous avons montré que la voie ATR/Chk1 activée principalement en réponse à l'apparition d'un stress réplicatif et la voie ATM/Chk2 qui initie la réponse cellulaire suite à la formation de lésions double-brins, sont activées en réponse aux adduits créés par les ETs. Dans un second temps, nous avons montré que les combinaisons des ETs avec les inhibiteurs Chk1/Chk2 ou les inhibiteurs ATR ou ATM seuls s'accompagnent d'une modeste potentialisation. Inversement, la combinaison simultanée des ETs avec les inhibiteurs ATR et ATM entraine une forte synergie dans les modèles du cancer de l'utérus et de l'ovaire sensibles ou résistants au cisplatine. Enfin, nous avons montré que cette potentialisation passe par l'inhibition du recrutement des protéines impliquées dans l'initiation et la réalisation des mécanismes de réparation par recombinaison homologue. Ces résultats suggèrent qu'en inhibant simultanément les vois initiés par ATR et ATM, l'activité thérapeutique des ETs pourrait être potentialisée en cliniqueEcteinascidins (Trabectedin, Lurbinectedin) are novel marine derived natural products, DNA minor groove binders and active against chemo-resistant cancers. The purpose of my thesis was to 1) characterize the DNA damage response (DDR) to both trabectedin and lurbinectedin 2) to establish whether the pharmacological abrogation of cell response induced by DNA damage (ATM, ATR, Chk1, Chk2) can modulate the therapeutic activity of ETs. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, the pharmacological inhibition of either Chk1/2, ATR or ATM kinases is not accompanied by a significant improvement of either trabectedin or lurbinectedin cytotoxic activity. However, the simultaneous inhibition of both ATM and ATR strongly potentiates the activity of both ETs. Importantly, these results are not restricted to HeLa cells but can also be extended to cisplatin-sensitive or -resistant ovarian carcinoma cell lines. Finally, we showed that the concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the initiation and realization of homologous recombination repair mechanisms. Together, our data identify ATR and ATM as central coordinators of the DDR to trabectedin and lurbinectedin and provide a mechanistic rational for combinations of these compounds with both ATR and ATM inhibitors
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Munoz, Marcia Medicine UNSW. "The EDD protein is a critical mediator in the DNA damage response." Awarded by:University of New South Wales. Medicine, 2006. http://handle.unsw.edu.au/1959.4/25977.
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An intact cellular response to DNA damage is important for the maintenance of genomic stability and tumour prevention. EDD, the human orthologue of Drosophila melanogaster ???hyperplastic discs???, is over-expressed or mutated in a number of common human cancers. EDD is a progestin regulated gene that encodes an E3 ubiquitin ligase involved in cell communication and cell adhesion, and although it has also been implicated in the DNA damage response through its association with DNA damage proteins, a definitive role has yet to be demonstrated. The work presented herein shows that EDD is necessary for an adequate cellular response to double-strand DNA breaks. Cells depleted of EDD exhibit reduced survival, radio-resistant DNA synthesis and failure to maintain G2/M arrest following DNA damage induced by phleomycin exposure. Furthermore, EDD-depleted cells display impaired activating phosphorylation and kinase activity of the checkpoint kinase CHK2 after DNA damage. These effects appear to be largely modulated through a phospho-dependent interaction involving the CHK2 FHA domain and a region of EDD spanning a number of putative FHA-binding threonines. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasise the potential importance of EDD in cancer.
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Lemaire, Matthieu. "Contrôle du cycle cellulaire : les phosphatases CDC25 et la protéine SvCds1/SvCHK2." Toulouse 3, 2006. http://www.theses.fr/2006TOU30167.
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Les phosphatases CDC25 sont des acteurs majeurs de l'activation des complexes CDK-Cycline dans les cellules eucaryotes, et constituent une cible essentielle des points de contrôle du cycle cellulaire. (i) Nous avons examiné le rôle des CDC25 humaines au cours du cycle cellulaire et lors de l'activation des points de contrôle, en remplaçant le gène cdc25 unique de la levure S. Pombe par les séquences d'ADN codant différentes phosphatases CDC25 humaines. (ii) Ensuite nous avons caractérisé la phosphorylation in vitro de CDC25B par les kinases p38 et MK-2, deux acteurs de la réponse cellulaire aux radiations ultraviolettes. (iii) CDC25 est phosphorylée et inhibée par la kinase Cds1/CHK2 lors de l'activation des points de contrôle, et nous avons montré l'existence d'un variant d'épissage de cette kinase, conservé de la levure à l'homme. (iv) Enfin nous présentons des travaux suggérant la participation de Cdc25 à des complexes de haut poids moléculaire dans la levure S. PombeCDC25 phosphatases are key players in the activation of CDK-Cyclin complexes in eukaryotic cells, and as such, are major targets of the cell cycle checkpoint pathways. (i) We have examined and compared the role of human CDC25 phosphatases during the cell cycle and in response to checkpoint activation, by generating fission yeast strains expressing the human CDC25 isoforms in place of endogenous Cdc25. (ii) We have studied the in vitro phosphorylation of CDC25B by MK-2 and p38, two kinases implicated in the UV-induced DNA damage response. (iii) CDC25 is phosphorylated and inhibited by the Cds1/CHK2 kinase during checkpoint activation, and we have discovered the existence of an evolutionarily conserved splice variant of this kinase. (iv) We also present some data suggesting that Cdc25 is associated with high density complexes in the fission yeast S. Pombe
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Taste, Corinne. "Fonctions et régulation de la protéine suppresseur de tumeurs BRCA1 dans la réponse cellulaire aux poisons du fuseau mitotique." Toulouse 3, 2007. http://www.theses.fr/2007TOU30065.
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Le gène BRCA1 est un suppresseur de tumeurs prédisposant au cancer du sein. Des données récentes ont suggéré l'implication de BRCA1 dans la réponse aux agents anti-mitotiques. Durant ma thèse, j'ai étudié les fonctions et la régulation de BRCA1 au cours de cette réponse cellulaire. Dans une première partie, j'ai mis en évidence le rôle de BRCA1 dans la régulation du checkpoint mitotique activé après dommages aux microtubules. Puis, j'ai analysé sa régulation dans ces conditions et ai montré que la protéine kinase Chk2 phosphoryle BRCA1 sur la sérine 988 après dommages aux microtubules et ainsi module le rôle de BRCA1 dans la nucléation des microtubules. Enfin, mes travaux suggèrent que la kinase mitotique Plk-1 pourrait également phosphoryler BRCA1 après traitement au paclitaxel. Ces travaux montrent que BRCA1 est impliquée dans la réponse cellulaire aux poisons du fuseau mitotique et suggèrent que Chk2 et Plk-1 régulent les fonctions de la protéine impliquées dans cette réponseInherited mutations of the tumor suppressor gene BRCA1 confer an increase risk for breast and ovarian cancer. BRCA1 has been recently implicated in the response to anti-mitotic agents. In the present report, we studied BRCA1 functions ans regulation in this response. In a first part, we demonstrated that BRCA1 is involved in the regulation of the mitotic checkpoint activated by microtubules damage. Then, we have analyzed its regulation in this conditions and we have showed that Chk2 phsophorylates BRCA1 on serine 988 after mitotic spindle disruption and thus, regulates microtubule nucleation activity of BRCA1. Further data also suggest that BRCA1 is a potential target of the mitotic kinase Plk-1 after paclitaxel treatment. Our findings show that BRCA1 is involved in the cellular response to agent that disrupt tje mitotic spindle and suggest that Chk2 and Plk1 regulate functions of BRCA1 in this response
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Mamouni, Kenza. "Rôle de la GTPase RhoB dans la réponse aux dommages à l'ADN induits par la camptothécine." Toulouse 3, 2013. http://thesesups.ups-tlse.fr/2031/.
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RhoB est une GTPase impliquée dans diverses fonctions intracellulaires comme l'organisation du cytosquelette. En plus de ses rôles bien établis, RhoB a récemment émergé comme un gène de réponse précoce aux dommages à l'ADN. RhoB est surexprimée et activée en réponse à divers génotoxiques bien que les mécanismes d'induction et la relevance fonctionnelle de cette induction restent mal compris. RhoB possède également des propriétés de suppresseur de tumeurs. Son expression diminue lors de la progression tumorale et la perte de RhoB favorise la prolifération cellulaire et l'invasion. Pour étudier le rôle de RhoB dans la réponse aux dommages à l'ADN et son implication potentielle dans la progression tumorale, nous avons utilisé la camptothécine (CPT), un inhibiteur sélectif de la topoisomérase I qui produit des cassures double-brin (DSBs) de l'ADN. Nous montrons que, dans les cellules traitées par la CPT, les DSBs induisent l'expression de RhoB par un mécanisme qui dépend de Chk2 et de son substrat HuR qui se lie à l'ARNm de RhoB et prévient sa dégradation. Des cellules déficientes en RhoB présentent un défaut de déphosphorylation de gamma-H2AX après le retrait de la CPT, suggérant un défaut de réparation des DSBs. Ces cellules présentent également une diminution de l'activité de PP2A, une phosphatase pour gamma-H2AX et d'autres protéines de la signalisation et de la réparation des dommages à l'ADN. Nous proposons que les DSBs activent une voie Chk2-HuR-RhoB qui favorise la déphosphorylation de gamma-H2AX par PP2A. Enfin, nous montrons que les cellules déficientes en RhoB accumulent du gamma-H2AX endogène et des anomalies chromosomiques, suggérant que la perte de RhoB augmente l'instabilité génomique induite par les DSBs et la progression tumoraleRhoB is a GTPase implicated in various intracellular functions such as cytoskeletal organization. Besides its well-established roles, RhoB recently emerged as an early DNA damage-inducible gene. RhoB is overexpressed and activated in response to various genotoxics although the mechanism of induction and functional relevance remain unclear. RhoB also possesses tumor suppressor properties. Its expression decreases during tumor progression and loss of RhoB promotes cell proliferation and invasion. To study the role of RhoB in the DNA damage response and its potential implication in tumor progression, we used camptothecin (CPT), a selective inhibitor of topoisomerase I that produces DNA double-strand breaks (DSBs). We show that, in CPT-treated cells, DSBs induce RhoB expression by a mechanism that depends on Chk2 and its substrate HuR that binds to and protects RhoB mRNA against degradation. RhoB deficient cells fail to dephosphorylate gamma-H2AX following CPT removal suggesting defective DSB repair. These cells also show decreased activity of PP2A, a phosphatase for gamma-H2AX and other DNA damage signaling and repair proteins. We propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of gamma-H2AX. Finally, we show that RhoB deficient cells accumulate endogenous gamma-H2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression
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Lima, Joel Fernandes. "Caracterização funcional de componentes da resposta ao dano DNA em \'Aspergillus nidulans\': os genes chkA, chkB e ddbA." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-06062008-162010/.
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A constante exposição dos diferentes organismos a agentes que danificam a estrutura da molécula do DNA fez com que a célula desenvolvesse mecanismos de reparo que se mostraram conservados durante a evolução. Em células de mamíferos, as vias de reparo ao dano ao DNA e a regulação dos pontos de checagem do ciclo celular atuam de forma coordenada no reparo do dano a fim de evitar uma progressão do ciclo celular antes do reparo e uma possível perpetuação do dano. Além disso, alguns dos componentes dessas vias metabólicas também atuam em outros processos, como replicação, transcrição, recombinação meiótica e silenciamento gênico. NER é um importante mecanismo no processo que reconhece e remove dímeros de ciclobutano e 6-4 pirimidina-pirimidona da estrutura do DNA. Em mamíferos foram identificados sete grupos de complementação para células deficientes de XP (XPA-G). Um desses grupos é XPE, conhecido por possuir forte afinidade ao dano ao DNA causado por luz UV, sendo formado por duas subunidades, DDB1 e DDB2. Uma busca no banco de dados de Aspergillus nidulans utilizando uma seqüência DDB1 de Homo sapiens, revelou uma única seqüência com similaridade relevante denominada como DdbA que não possui nenhuma similaridade com a proteína DDB2. Em Aspergillus nidulans, a proteína DdbA também está envolvida no reparo do dano ao DNA causado por luz UV e 4-NQO, no entanto, vimos aqui, que DdbA está interagindo com as proteínas UvsBATR, H2AX e CshBCSB no reparo ao dano ao DNA causado por MMS, Bleomicina, 4-NQO e luz UV. Além disso, uma análise na expressão do gene ddbA mostrou que ele é induzido por estas drogas, no estresse oxidativo e nos processos de desenvolvimento assexual e sexual de A. nidulans. Nós também vimos que a localização celular de DdbA não foi afetada durante a resposta ao dano ao DNA causado por luz UV e 4-NQO indicando que a proteína DdbA está presente no núcleo independentemente do dano. Em S. pombe, as proteínas serina-treonina quinases CHK1 e CHK2 foram identificadas como essenciais para o bloqueio do ciclo celular na fase S em resposta ao dano ao DNA ou em resposta ao estresse replicacional. Essas quinases são fosforiladas pelas quinases ATM e ATR e tem sido extensivamente caracterizadas em A. nidulans. Neste fungo, as proteínas ChkACHK1 e ChkBCHK2 estão envolvidas na reposta ao dano ao DNA e estão interagindo de forma epistática e sinergística com as proteínas quinases AtmAATM e UvsBATR. Nossos resultados também sugerem que as proteínas ChkA e ChkB podem estar envolvidas em meiose, e atuam em vias complementares durante o bloqueio da fase S do ciclo celular. Além disso, as proteínas AtmA, ChkA, ChkB e UvsB são redundantemente complementares na manutenção do crescimento polar das hifas em Aspergillus nidulans.The constant exposure of different organisms to agents that damage the DNA structure, has provided the cells with repair mechanisms that are conserved during evolution. In mammal cells, the DNA damage repair pathways and the cell cycle checkpoint regulation act together to prevent cell cycle progression before the repair is performed avoiding mutation fixaxion. However these responses are complex and demand overlapping functions and the intersection of many metabolic pathways. NER is an important mechanism in the process that recognize and remove cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidone photoproduct from the DNA structure. In mammals seven complementation groups for XP deficient cells were identified. One of these groups is XPE, known for having strong affinity to the DNA damage caused by UV light and is formed by two subunits DDB1 and DDB2. A search on the Aspergilus nidulans database using a Homo sapiens DDB1 sequence, revealed a single ORF with relevant similarity. The A. nidulans homologue was deleted and named DdbA. ddbA does not have significant similarity to DDB2 protein. In A. nidulans the protein DdbA is involved on the DNA damage repair caused by UV light and 4NQO. Additionaly ddbA is genetically interacting with uvsBATR, histone H2AX and cshBCSB the damage repair caused by MMS , BLEO, 4NQO and UV light. Also, an analysis of the gene ddbA expression indicated that it is induced by MMS, BLEO, 4-NQO, oxidative stressing agents and by the assexual and sexual development processes of A. nidulans. We also verified that the sub-cellular localization of DdbA was not affected by the presence of UV light or 4-NQO indicating that the protein DdbA is constitutively present in the nucleus. In S. pombe, the serine treonine kinases CHK1 and CHK2 proteins were identified as essential to the Sphase blockage in response to the DNA damage or replicational stress. These kinases are phosphorilated by ATR and ATM kinases, respectively and have been extensively characterized in A. nidulans. In this fungus, the proteins ChkACHK1 and ChkBCHK2 are involved on the DNA damage response and are genetically interacting in an epistatic and/or synergistic manner with the AtmAATM and UvsBATR kinases. Our results also sugest that the proteins ChkA and ChkB may also be involved in meiosis and act in a complementary way during the S-phase block. Furthermore the AtmA, ChkA, ChkB e UvsB proteins are complementary redundant for the maintenance of the polar growth in A. nidulans.
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Garner, Sarah. "The molecular biology of Chp2." Thesis, University of Southampton, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398831.
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Schadel, Vanessa. "Identifikation neuer Komponenten der Chk1 vermittelten Signaltransduktion." [S.l. : s.n.], 2009. http://nbn-resolving.de/urn:nbn:de:bsz:16-opus-88315.
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Martinho, Rui Goncalo V. R. C. "Analysis of Rad3 and Chk1 checkpoint protein kinases." Thesis, University of Sussex, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297946.
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Conchon, Elizabeth. "Synthèse de nouveaux carbazoles inhibiteurs de la Chk1." Clermont-Ferrand 2, 2006. https://theses.hal.science/tel-00717338/document.
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La granulatimide et l'isogranulatimide, molécules naturelles isolées d'une ascidie Didemmum granulatum sont connues pour être des inhibiteurs de la Chk1, enzyme régulatrice du point de contrôle en G2 du cycle cellulaire. La première partie de ce travail est consacrée à la présentation du point de contrôle en G2 du cycle cellulaire et plus particulièrement à la checkpoint 1 kinase Chk1. Dans une seconde partie, la synthèse de nouveaux carbazoles analogues de la granulatimide et l'isogranulatimide sera décrite. Parmi ces analogues l'hétérocycle imidique sera tout d'abord remplacé par un lactame, une pyrazolinone, une pyridazine et l'hétérocycle imidazole sera quand à lui remplacé par des carbocycles à 5 ou 6 chaînons fonctionnalisés. Une troisième partie présente les résultats des tests d'inhibition de la Chk1 et les activités antiprolifératives sur trois lignées cancéreuses effectuées sur les analogues synthétisés. Tous les tests biologiques ont été réalisés au sein des laboratoires Servier
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Řeháková, Jana. "Udržitelný rozvoj cestovního ruchu v CHKO Železné hory." Master's thesis, Vysoká škola ekonomická v Praze, 2017. http://www.nusl.cz/ntk/nusl-358834.
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The thesis deals with the issue of sustainable tourism development in protected landscape area of the Iron Mountains. The main objective is to evaluate the current status and potential of the region on issues of sustainable tourism development, sustainable forms of tourism and evaluate conservation in the PLA Iron Mountain. The theoretical part is devoted to sustainable development, sustainable development of tourism and the area of the Iron Mountains. The practical part is to map the nature and landscape protection in the PLA Iron Mountain with regard to small-scale protected areas and analyze friendly forms of tourism. Followed by evaluation of the awareness of sustainable development of the territory by questionnaire from the perspective of local public administration in tourism, residents PLA and business owners. Last theoretical part is the SWOT analysis and recommendations for the future.
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Vaculíková, Klára. "Znečištění vnášené do CHKO Moravský kras povrchovými toky." Master's thesis, Vysoké učení technické v Brně. Fakulta stavební, 2020. http://www.nusl.cz/ntk/nusl-409737.
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The Diploma Thesis deals with the water pollution entering the Protected Landscape Area Moravian Karst via surface streams. In order to quantify the extent of the pollution surface streams entering the Moravian Karst were selected. A survey of a region of interest was done. Actual volume flow rate, measurements of physicochemical parameters, water samplings, laboratory analyses of samples were done monthly from April to November 2019. Conductivity, pH, temperature, oxidation-reduction potential, concentration of dissolved oxygen, and oxygen saturation were measured in situ. Afterwards, COD, BOD, phosphate, total phosphorus, ammonia, nitrites, nitrates and Kjeldahl nitrogen were determined in the laboratory. Organic nitrogen and total nitrogen were also calculated. The results were evaluated with a respect of Government Decree No. 401/2015 Coll. Material flows were calculated for selected parameters and used as other criterion that evaluate the water pollution of monitoring surface streams. Areas out of the PLA needing an enhanced protection of watercourses were proposed based on quantified pollution. Cooperation with two primary schools in the Moravian Karst supported the research. Pupils and their teachers were trained, and they measured phosphate in surface streams from April to November 2019. The website related to phosphorus in surface water were created and results of phosphate measurement were uploaded there.
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Ameline, Baptiste. "Evaluation du transfert d'optogènes pour le traitement par thérapie génique d'un modèle canin de dystrophies rétiniennes héréditaires." Thesis, Nantes, 2016. http://www.theses.fr/2016NANT1001/document.
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La cécité ou la très grande malvoyance peut résulter de différentes pathologies comme les dystrophies rétiniennes héréditaires (DRH) caractérisées par la perte des photorécepteurs. Une des approches pour traiter les DRH est la thérapie génique spécifique, c’est à dire le remplacement du gène défectueux par un gène sain. Des études chez des modèles animaux de DRH ont démontré l’efficacité de la thérapie géniques spécifique, et conduit au lancement d’essais cliniques.Malgré des résultats encourageants, la thérapie génique spécifique n’est pas toujours applicable, en particulier quand la dégénérescence est trop avancée ou si le gène muté est inconnu. Pour traiter tous les cas de DRH, un nouvel axe de thérapie génique est envisagé : le transfert d’optogène.Cette stratégie consiste à réactiver la rétine devenue aveugle par l’expression de protéines photosensibles dans la rétine. Notre objectif est d'évaluer l'efficacité du transfert d'optogène chez un modèle canin naturellement déficient pour le gène Rpe65, provoquant une forme sévère de DRH proche de celles retrouvées chez l’homme. La stratégie thérapeutique retenue est : L'injection intravitréenne, après vitrectomie, d'un vecteur recombinant dérivé du virus adéno-associé de sérotype2 (rAAV2/2), portant le transgène optogénétique sous contrôle d'un promoteur fort et spécifique des tissus neuronaux : hSyn. Le but de ce projet est de transduire efficacement les cellules ganglionnaires rétiniennes d'un modèle canin déficient pour le gène Rpe65 et d'évaluer la photosensibilité des cellules transduitesInherited retinal diseases (IRD) affect about 2 million people worldwide, leading to severe visual impairment.Specific gene addition therapy is one of the most promising strategies to treat these patients. Howevermany of them are not eligible for specific gene therapy,such as.1) Patients with unknown deficient genes.2) Patients beyond the therapeutic window.3) Patients whose the deficient gene is too large forAAV encapsidation.4) Patients undergoing a dominant form of IRD.Therefore, the aim of this project is to develop analternative strategy, independent of the mutation and the retinal degeneration kinetic: the optogene transfer. In context of IRD, it will consist to convert survivingretinal ganglion cells into sensitive light cells followingthe transfer of ChR2 or Opn4 optogene. Several rodentmodels of IRD have been successfully treated usingthese optogenes. Nevertheless, this approach hasnever been evaluated in large animal models. The objective of our study will be to define the feasibility ofoptogene transfer to restore vision in blind patients by evaluating the safety and the efficacy of AAV-mediated gene transfer of ChR2, eNpHR or Opn4, after vitrectomy, in ganglion cells of a canine model of IRD, the Rpe65-deficient dog
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Platel, Marie. "Régulation du programme spatio-temporel de la réplication de l'ADN lors du développement précoce du Xénope." Thesis, Université Paris-Saclay (ComUE), 2015. http://www.theses.fr/2015SACLS043.
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Chez les eucaryotes supérieurs, la réplication de l'ADN est initiée à partir de plusieurs milliers d'origines. Mais la régulation spatio-temporelle de leur activation reste mal caractérisée. Une voie de contrôle (checkpoint) de la phase S est activée lorsque les fourches de réplication sont bloquées, inhibant ainsi l'activation d'origines tardives. L'objectif de ma thèse consistait à étudier deux facteurs essentiels dans le programme spatio-temporel de la réplication, dans le système du xénope : la protéine « checkpoint » Chk1, qui est un facteur inhibiteur de l'activation des origines, et les désoxyribonucléotides (dNTPs), précurseurs de la synthèse de l'ADN. Chez le xénope, après douze divisions embryonnaires, a lieu la transition mid-blastuléenne (MBT). A cette étape, une augmentation du ratio nucléo-cytosolique va entrainer la titration des facteurs de réplication, ce qui active le point de contrôle et ralentit la phase S. Il est possible de mimer in vitro les phases S rapides des embryons pendant le développement précoce en augmentant la concentration en noyaux dans l'extrait d'œufs.Nous avons pu voir par l'inhibition, la déplétion ou la surexpression de Chk1 que cette protéine régulait l'activation des origines lors d'un stress, mais également dans une phase S non perturbée, grâce à la technique du peignage moléculaire. Ce résultat montre que le niveau de Chk1 doit être finement régulé pour permettre une réplication correcte dans une phase S non perturbée, chez les eucaryotes supérieurs. Nous avons ensuite cherché à savoir si la concentration en dNTPs pouvait être limitante pendant le développement et comment elle modulait le programme de réplication. Nous avons comparé l'effet de l'ajout de dNTPs sur la réplication en mimant plusieurs stades précoces du développement pré-MBT. La variation de la concentration en dNTPs agit sur la réplication en augmentant à la fois l'activation des origines et, en fonction de la concentration en noyaux, aussi la vitesse des fourches. Cet effet est indépendant du checkpoint de la réplication dans ce système et d'autres études sont nécessaires pour comprendre les mécanismes moléculairesDNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal regulation program which is not well characterized. The S phase checkpoint is activated when replication forks are blocked which inhibits the firing of late origins. The aim of my thesis consisted to study two essentials factors in spatio-temporal replication program in Xenopus system: the checkpoint protein Chk1, inhibitor of origin activation, and the deoxyribonucleotides (dNTPs), DNA synthesis precursors. In Xenopus, the mid-blastula transition (MBT) occurs after twelve embryonic divisions. An increase of the nucleo-cytosolic ratio induces a titration of replication factors, that activates the checkpoint and slows down the S phase. It is possible to mimic in vitro the rapid S phases of early Xenopus development stages by increasing the nuclei concentration. By DNA combing combined with Chk1 inhibition, depletion and overexpression experiments, we show that Chk1 controls origins activation in perturbed but also unperturbed S phase. My results show that Chk1 levels needs to be tightly regulated in order to properly control the replication program during normal S phase in higher eukaryotes. In order to determine whether the concentration of dNTPs could be another limiting replication factor, we compared the effect of dNTPs addition on replication by mimicking in vitro several early stages of pre-MBT development. Addition of dNTPs affects DNA replication, by increasing origin activation and, dependent on nuclei concentration, also the fork speed. This effect is independent of the S phase checkpoint and further studies are needed in order to understand the molecular mechanisms behind
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Svobodová, Eliška. "Udržitelný rozvoj cestovního ruchu ve vybrané destinaci - CHKO Kokořínsko." Master's thesis, Vysoká škola ekonomická v Praze, 2012. http://www.nusl.cz/ntk/nusl-197277.
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This thesis is concerned with the sustainable tourism development in the Protected Landscape Area Kokořínsko. The aim is to evaluate current state of sustainable tourism in this area and outline the perspectives of its further development. The theoretical part is dedicated to the definition of the concept of sustainable development and its application to the tourism in protected areas. The beggining of the practical part describes the Protected Landscape Area Kokořínsko. The practical part is also concerned with the organization of tourism and an analysis of offer of environmentally friendly forms of tourism. The thesis is completed with an overall evaluation based on the SWOT analysis, chosen indicators of sustainable tourism development and a questionnaire survey among tourists, residents, entrepreneurs in the tourism industry and municipalities in the region of the Protected Landscape Area Kokořínsko.
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Petzová, Terezie. "Možnosti rozvoje udržitelného cestovního ruchu na území CHKO Brdy." Master's thesis, Vysoká škola ekonomická v Praze, 2017. http://www.nusl.cz/ntk/nusl-359466.
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The diploma thesis deals with the development of sustainable tourism in protected areas. The aim of the thesis is to evaluate the possibilities of sustainable tourism development in the protected area. The theoretical part of the thesis deals with the issue in general terms. The problem is practically solved on the example of Brdy Protected Landscape Area (PLA). An identification for localization and implementation conditions for the development of sustainable tourism has been realized within the analysis of the examined areas potential. This identification was conducted as an empirical investigation with the representatives of the interested municipalities, local action groups and other organizations. The acquired knowledge has been reviewed with the PLA representatives opinions. Summarizing all gained knowledge, the PLA Brdy has been assessed as area that fulfils the prerequisites for the sustainable tourism development. Due to the character of the area, the development must be implemented only in the environment friendly forms.
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Gysin, Romana. "Struktura a vývoj autochtonních bukových porostů v CHKO Broumovsko." Master's thesis, Česká zemědělská univerzita v Praze, 2016. http://www.nusl.cz/ntk/nusl-260480.
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The thesis describes the horizontal and vertical structure of two permanent research plots with the developmental stages of the tree layer of native beech stands in NPR Broumov in the area CHKO Broumov nature reserve in the Sudetenland. The horizontal structure of the tree layer is random and in the developmental stages of natural regeneration. The average number of individuals natural recovery is around 15 100 individuals per hectare.
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Chouinard, Guillaume. "Localisation et fonction de CHK2 en mitose." Thèse, 2012. http://hdl.handle.net/1866/10024.
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Les centrosomes dont le rôle principal est d’organiser le cytosquelette de microtubules et le fuseau mitotique servent aussi de sites d’interaction pour plusieurs protéines régulatrices du cycle cellulaire et de la réponse aux dommages à l’ADN. Une de ces protéines est la kinase CHK2 et plusieurs publications montrent une sous-population de CHK2 localisée aux centrosomes dans les cellules en interphase et en mitose. Toutefois, la localisation de CHK2 aux centrosomes demeure controversée, car des doutes subsistent en ce qui concerne la spécificité des anticorps utilisés en immunocytochimie. En utilisant des lignées cellulaires du cancer du côlon, les cellules HCT116 sauvages et HCT116 CHK2-/- ainsi que différentes lignées d’ostéosarcome humain dans lesquelles l’expression de CHK2 a été inhibée par ARN interférence, nous montrons que les anticorps anti-CHK2 qui donnent un signal centrosomal sont non spécifiques et reconnaissent un antigène inconnu sur les centrosomes. Cependant, par des expériences d’immunofluorescence réalisées avec des cellules U2OS qui expriment les protéines de fusion GFP-CHK2 ou FLAG-CHK2, nous révélons une localisation centrosomale de CHK2 dans les cellules en mitose, mais pas en interphase. Ce résultat a été confirmé par vidéomicroscopie dans les cellules vivantes exprimant GFP-CHK2. Pour déterminer le ou les rôles potentiels de CHK2 en mitose nous avons réalisé des expériences pour explorer le rôle de CHK2 dans la progression de la mitose, la nucléation des microtubules aux centrosomes et la progression de la mitose en présence de problèmes d’attachement des chromosomes où de lésions génotoxiques. Nos données suggèrent que CHK2 n’est pas impliquée dans la régulation de la mitose dans les cellules U2OS.Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. Here, we clarify that CHK2 only localized at centrosomes during mitosis. Using wild-type and CHK2-/- HCT116 human colon cancer cells, or human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs, we show that several CHK2 antibodies are non-specific for immunofluorescence and cross-react with an unknown centrosomal protein(s). To analyse further CHK2 localization, we established cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with centrosomes in mitotic cells, from early mitotic stages until cytokinesis. In contrast to previous data obtained by A. Stolz and colleagues with the human colon carcinoma HCT116 cell line, our experiments exploring the possible functions for CHK2 during mitosis did not support a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis in human osteosarcoma U2OS cells.
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Hsu, Pei-Ching, and 徐珮菁. "CHK2-mediated Regulation of PARP1 in Oxidative DNA Damage Response." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/346y29.
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博士國防醫學院
生命科學研究所
106
Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor, which upon activation, recruits downstream proteins by poly(ADP-ribosyl)ation (PARylation). However, it remains largely unclear how PARP1 activity is regulated. Interestingly, the data obtained through this study revealed that PARP1 was co-immunoprecipitated with checkpoint kinase 2 (CHK2), and the interaction was increased after oxidative DNA damage. Moreover, CHK2 depletion resulted in a reduction in overall PARylation. To further explore the functional relationship between PARP1 and CHK2, this study employed H2O2 to induce an oxidative DNA damage response in cells. Here, we showed that CHK2 and PARP1 interact in vitro and in vivo through the CHK2 SCD domain and the PARP1 BRCT domain. Furthermore, CHK2 stimulates the PARylation activity of PARP1 through CHK2-dependent phosphorylation. Consequently, the impaired repair associated with PARP1 depletion could be rescued by re-expression of wild-type PARP1 and the phospho-mimic but not the phospho-deficient mutant. Mechanistically, we showed that CHK2-dependent phosphorylation of PARP1 not only regulates its cellular localization but also promotes its catalytic activity and its interaction with XRCC1. These findings indicate that CHK2 exerts a multifaceted impact on PARP1 in response to oxidative stress to facilitate DNA repair and to maintain cell survival.
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Wu, Meng-Shan, and 吳夢珊. "The Pathological Significance of Chk2 in Non-Small Cell Lung Cancer." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/huf9rn.
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碩士國立中興大學
生命科學院碩士在職專班
98
Lung cancer is the leading cause of cancer death in many developed country including Taiwan. Many doctors and scientists have focused their time towards the prevention and treatment of lung cancer. However, the effect is minimal. Chk2 kinase is an important cell cycle regulator in the DNA damage response pathway. In response to exogenous or endogenous DNA damage agents, both ATR-Chk1（Ataxia telangiectasia mutated and Rad3-related-checkpoint kinase 1）and ATM-Chk2（Ataxia telangiectasia mutated-checkpoint kinase 2 ）are activated.Chk2 is mainly phosphorylated and activated by ATM kinase. In this study, we detected the expression of Chk2 mRNA by RT-PCR（13.33％）, the expression of Chk2 protein in lung cancer cell lines and patients’ samples by Western blotting（68％）, and that of Chk2 in lung cancer pathology tissue（84.75％）and tissue-array（65％）by Immunohistochemistry. Our results showed that Chk2 protein is highly expressed in lung cancer tissue and cell line.
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Ting-YuChen and 陳亭羽. "Chk2 regulates the cell cycle progression by controlling primary cilia acetylation." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/hf6p9b.
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Pamidi, Ashwin. "Functional and genetic interactions of Mus81 with the Chk2-p53 tumor suppressor pathway." 2007. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=742438&T=F.
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Wang, Hui-Chun, and 王惠君. "ATM and Chk2 regulate BRCA1 in DNA end-joining repair of double strand breaks." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/34098351681590542452.
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博士國防醫學院
生命科學研究所
94
Homologous recombination (HR) and non-homologous end-joining (NHEJ) are the two mechanisms responsible for repairing DNA double strand breaks (DSBs) and act in either a collaborative or competitive manner in mammalian cells. DSB repaired by NHEJ, may be more complicated than the simple joining of the ends of DSB, since, if nucleotides were lost, it would result in error-prone repair. This has led to the proposal that a subpathway of precise NHEJ exists which can repair DSBs with higher fidelity; this is supported by recent findings that the expression of the HR gene, BRCA1, is causally linked to in vitro and in vivo precise NHEJ activity. To further delineate this mechanism, the present study explored the connection between NHEJ and the cell-cycle checkpoint proteins, ATM and Chk2, known to be involved in activating BRCA1, and tested the hypothesis that ATM and Chk2 promote precise end-joining by BRCA1. Support for this hypothesis came from the observations that (a) knock-down of ATM and Chk2 expression affected end-joining activity; (b) in BRCA1-defective cells, precise end-joining activity was not restored by a BRCA1 mutant lacking the site phosphorylated by Chk2, but was restored by wild-type BRCA1 or a mutant mimicking phosphorylation by Chk2, (c) Chk2 mutants lacking kinase activity or with a mutation at a site phosphorylated by ATM had a dominant negative effect on precise end-joining in BRCA1-expressing cells. These results suggest that the other two HR-regulatory proteins, ATM and Chk2, act jointly to regulate the activity of BRCA1 in controlling the fidelity of DNA end-joining by precise NHEJ.
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Chen, Chiao-Yu, and 陳巧育. "Analysis of the status of TTK/hMps1、CHK2 and Ramp/L2DTL in breast cancer." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/78022853878870666773.
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碩士國立臺灣大學
生物化學暨分子生物學研究所
93
Worldwide, breast cancer is one of the most common cancer among women, and is the second leading cause of cancer death in women as well. In Taiwan, breast cancer ranks fourth among the caures of overall female cancer-related deaths and the incidence of age in this disease tends to early onset. It is important to understand tumorgenesis and systemic recurrence in breast cancer. Our laboratory has studied the breast carcinogenesis for few years. Lymph node metastasis is a bio-marker for poor prognosis in breast cancer. However, approximately 6.4% of lymph node negative patients that acquire systemic recurrence. We are particularly interested in setting up new supplemental bio-markers for systemic recurrence. According to the previous study, p53 mutation has shown a strong correlation with recurrence (p=0.00193). p53 is a supplemental bio-marker useful for clinical diagnosis as well as lymph node metastasis. Nevertheless, there are a few wild-type p53 patients that acquired recurrence. Therefore, we examine the status of other molecular components such as CHK2 and TTK/hMps1 in the p53 signaling pathway. Inaddition, we have also analyzed the status of Ramp/L2DTL, a nuclear protein Ramp/L2DTL has shown the correlation with early metastasis of hepatoma (personal communication with Dr. H-J Hsu, NTUH). By sequence analysis, we identified two TTK/hMps1 polymorphism, Ｉ５２７Ｉand Ｐ７８９Ｐ. We determined the expression level of TTK/hMps1 by semi-quantitative RT-PCR. According to the statistical analysis, high expression level of TTK/hMps1 correlatd strongly both with systemic recurrence (p=0.0022) and p53 mutation (p=0.0001). TTK/hMps1 can be a supplemental bio-marker useful for clinical diagnosis. We also determined the expression level of Ramp/L2DTL by semi-quantitative RT-PCR. According to the resules of statistical analysis, high expression level of Ramp/L2DTL showns correlation with systemic recurrence (p=0.0247). We identified one CHK2 polymorphism (E84E) by sequencing analysis. According to the study by other groups, CHK2 with 11 major splice variants were detected in their tumor series. We have found some splice variants in breast cancer specimens. How the splice variants of CHK2 influence breast carcinogenesis remain to be addressed.
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CHANG, HUEI-CIH, and 張惠慈. "Etoposide induces cellular senescence by activating DNA-PK/Chk2/autophagy signaling in adrenocortical tumor." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/mckken.
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碩士國立臺南大學
生物科技學系碩士班
107
The anti-tumor drug etoposide (ETO) is wildly used in treating several cancers, including adrenocortical tumor (ACT). ETO treatment induces DNA damage response and increases the malignancy of recurring tumor. Therefore, it is important to understand the effect and the underlying molecule mechanism of ETO treatment in ACT cells. ETO treatment inhibited ACT cell growth by arresting the cell at G0/G1 phase rather than induced cell death. In addition, several markers of cellular senescence, including the enlarged nuclei, activated acidic β-galactosidase activity, up-regulation of p53 and p21, or down-regulation of Lamin B1, were observed suggesting that ETO treatment induced cellular senescence. Centrosome is composed of two centrioles and the surrounding pericentriolar material (PCM), and its amplification induces cellular senescence. Upon ETO treatment, centrosome amplification was observed. However, each amplified PCM contained only one centriole, suggesting that ETO-induced centrosome amplification is caused by centriole splitting. The activation of DNA damage response was observed, and ETO-induced centrosome amplification was inhibited by the inhibition of DNA-PK, but not ATM and ATR. We further confirmed that DNA-PK/Chk2 signaling reduced ETO-induced centrosome amplification followed by inhibiting cellular senescence. Furthermore, DNA-PK/Chk2 signaling trigged autophagy that contributed centrosome amplification and senescence upon ETO treatment. In addition, primary cilia was observed upon ETO treatment. To further confirm the role of primary cilia upon ETO treatment, primary cilia were disrupted by shRNA against intraflagellar transport protein IFT88, that DNA-PK/Chk2/autophagy signaling reduced ETO-induced primary cilia followed by inhibiting cellular senescence in cilia deficient cells. In summary, we have elucidated that ETO inhibits ACT via inducing centrosome amplification , primary cilia and cellular senescence, and these events were triggered by DNA-PK/Chk2/autophagy signaling.
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Wu, Hsin-Hui, and 吳信輝. "Structural and Functional Study of FHA Domain of MDC1 and CHK2 in DNA Damage Response." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/53624064955081379577.
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博士國立清華大學
生物資訊與結構生物研究所
103
This work focuses on the structural basis of the FHA domain (forkhead-associated domain) in the interaction between MDC 1 (mediator of DNA damage checkpoint 1) and CHK2 (checkpoint kinase 2) upon DNA damage. The MDC1 protein functions as a key mediator that interacts with multiple proteins involved in DNA damage response (DDR) pathway. It binds to not only sensor proteins like ATM and MRE11, but also effector proteins such as CHK2. The complicated phospho-signaling network controls the sensing, initiating, and mediating steps that lead to downstream repair pathways, cell-cycle checkpoints, and apoptosis. Previously, the CHK2 binding site for MDC1-FHA was shown to be pThr68 (the same site recognized by the FHA domain of CHK2 for dimerization and activation of CHK2). To elucidate the molecular mechanism of MDC1-CHK2 interaction, we solved crystal structures of mouse MDC1-FHA and its complex with a human CHK2 peptide containing pThr68. Surprisingly, MDC1-FHA exists as an intrinsic dimer in solution and in crystals. Structural and binding analyses support the pThr+3 ligand specificity of FHA domains, and provide structural insight into MDC1-CHK2 interaction. In order to test whether the dimerization of MDC1-FHA directs MDC1 function in vivo, we selected different MDC1-FHA mutants with disrupted dimerization while maintaining the pThr-binding ability. The full-length MDC1 protein containing such mutations not only failed to dimerize in vivo as suggested by split-GFP system, but also failed to rescue cellular radio-sensitivity caused by MDC1 knockdown. In addition, our result shows that the dimeric feature affects the MDC1 protein turnover rate on DNA lesion sites by which the accurate DNA damage signal can be executed. It implies that the dimeric feature may play a role of super-scaffold to interact with other proteins after DNA damage. In addition, dimerization-dependent trans autophophorylation is a common mechanism to active kinase. Activated ATM kinase phosphorylates CHK2 on Thr68 to trigger CHK2 activation in DNA damage. The pThr68 interacts with its FHA domain, leading to dimerization and T-loop autophosphorylation. However, the molecular basis of this activation process remains unclear. Here we use site-specifically pThr68 CHK2 for biophysical characterization by AUC and provide structural explanation by SAXS analyses. The results show that pThr68 plays a critical role to stabilize CHK2 dimerization. The SAXS results show that pThr68-mediated dimerization is possible to bring two kinases to correct orientation for efficient activation loop trans autophosphorylation.
48
Shao, Wei-Syun, and 邵韋勳. "Activation of Chk2 contributes to baicalein-induced G2/M cell cycle arrest in human ovarian cancer cells." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/93393769472037450885.
Full textAbstract:
碩士淡江大學
化學學系碩士班
99
Baicalein has been reported to exhibit anti-tumor activities. In this study, we examined the anti-proliferating effects of baicalein on ovarian cancer cells. We found that baicalein inhibited the cell growth of ovarian cancer cells. Besides, our results demonstrate that baicalein caused reactive oxygen species (ROS) generation, H2AX phosphorylation and Chk2 activation in ovarian cancer cells. Furthermore, we also observed that G2/M regulatory molecules such as Cdc25C, Cdk1, cyclin B1 were down-regulation by baicalein. Taken together, these data suggest baicalein may generate ROS to cause DNA damage and then results in G2/M arrest in ovarian cancer cells.
49
Nikitin, Pavel A. "DNA Damage Response Suppresses Epstein-Barr Virus-Driven Proliferation of Primary Human B Cells." Diss., 2012. http://hdl.handle.net/10161/6183.
Full textAbstract:
The interaction of human tumor viruses with host growth suppressive pathways is a fine balance between controlled latent infection and virus-induced oncogenesis. This dissertation elucidates how Epstein-Barr virus interacts with the host growth suppressive DNA damage response signaling pathways (DDR) in order to transform infected human B lymphocytes.
Here I report that the activation of the ATM/Chk2 branch of the DDR in hyper-proliferating infected B cells results in G1/S cell cycle arrest and limits viral-mediated transformation. Similar growth arrest was found in mitogen-driven proliferating of B cells that sets the DDR as a default growth suppressive mechanism in human B cells. Hence, the viral protein EBNA3C functions to attenuate the host DDR and to promote immortalization of a small portion of infected B cells. Additionally, the pharmacological inhibition of the DDR in vitro increases viral immortalization of memory B cells that facilitates the isolation of broadly neutralizing antibodies to various infectious agents. Overall, this work defines early EBV-infected hyper-proliferating B cells as a new stage in viral infection that determines subsequent viral-mediated tumorigenesis.
Dissertation
50
ČANDA, Jan. "Obojživelníci CHKO Blaník." Master's thesis, 2007. http://www.nusl.cz/ntk/nusl-46689.
Full textAbstract:
The principal aim of this diploma thesis was the mapping of amphibian species inhabiting the Protected Landscape Area Blaník. The field research was carried out during the years 2005 to 2006. The occurrence of following 8 species and 1 synklepton was confirmed: Smooth Newt (Triturus vulgaris), Warty Newt (Triturus cristatus), Alpine Newt (Triturus alpestris), Fired Bellied Toad (Bombina bombina), Common Spadefood (Pelobates fuscus), Common Toad (Bufo bufo), European Tree Frog (Hyla arborea), Grass Frog (Rana temporaria) and Water Frogs (Rana synklepton esculenta). The determination was performed on the basis of morphologic characters and voice expressions. During the field research suitable reproductive habitats of particular species were studied with respect to numbers of specimens found there. Based on presented results, habitats valuable for protection mere marked. The records of particular species were written into maps of the species occurance.