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.

P53 est mutée dans plus de la moitié des cancers humains et son inactivation est souvent associée à une résistance à la thérapie anti-cancer. Notre équipe a montré que dans le cas de tumeurs où p53 est inactive, la surexpression de la phosphatase Wip1 permettait la restauration de l’efficacité de la chimiothérapie, et une protection des tissus sains face aux effets secondaires du traitement. Afin d’améliorer cette stratégie, deux objectifs principaux ont été étudiés : trouver une protéine qui peut interagir avec la voie de signalisation de Wip1 et potentialiser son action dans cette stratégie thérapeutique, et trouver des molécules chimiques pouvant activer Wip1. Nous avons réalisé un criblage d’interférence à ARN de l’intégralité du kinome humain afin d’identifier plusieurs kinases, dont l’inhibition potentialise l’action anti-tumorale de Wip1. Cela a ainsi mis en évidence l’action de Wee1 et Hipk2, dont des inhibiteurs existent, sur la voie de signalisation de Wip1. Inhiber Wee1 avec une faible dose de MK-1775, un inhibiteur spécifique de cette kinase, a permis de réduire la concentration efficace de cisplatine, en entraînant une apoptose caspase-3-dépendante. De plus, combiner MK-1775 avec la surexpression de Wip1 n’a pas d’impact sur l’effet protecteur de cette phosphatase envers les tissus sains. Nous avons ensuite montré que le Vorinostat, un inhibiteur des histone-déacétylases, permettait une augmentation de la transcription de Wip1 dans des cellules de cancer du sein mutées pour p53. Ce travail de thèse a permis de mettre en évidence un moyen de potentialiser la stratégie de traitement des tumeurs p53-négatives basée sur la phosphatase Wip1. / P53 is mutated in more than half of human cancers and when inactivated is often associated with a resistance to anti-cancer therapy. Our team has hown that in the case of p53-negative tumors, overexpression of Wip1 phosphatase sensitizes tumor cells to chemotherapy, while protecting normal tissues from the side effects of the treatment. To improve this strategy, two main objectives were studied. Firstly, to find a protein which can interact with Wip1 pathway and potentiate its action in this therapeutic strategy. Secondly, to find a molecule which can activate Wip1. We realized a siRNA screening of the whole human kinome to identify several kinases, whose inhibition could potentiate anti-tumoral action of Wip1. We have shown that Wee1 and Hipk2, which both have available inhibitors, have an action on Wip1 pathway. Inhibiting Wee1 with a low dose of MK-1775, a specific inhibitor of this kinase, allowed us to decrease effective cisplatin concentration, inducing a caspase-3-dependent apoptosis. Moreover, the combination of MK-1775 with Wip1 overexpression does not impair the protective effect that this phosphatase provides towards normal tissues. We then showed that the Vorinostat, an HDAC inhibitor, induces an increase in Wip1 transcription in breast cancer cells with inactive p53. This work uncovered a way to potentiate the Wip1-based therapeutic strategy of p53-negative tumors.

In this study we found that tumour cells can be radiosensitised by targeting the DNA damage response kinases, ATM and ATR. Furthermore, we highlight that Wee1 inhibitors, which are already under the clinical trials need to be further investigated in combination with radiation in the context of tumour hypoxia. In addition, we observed that induction of autophagy using STF-62247 can lead to radiosensitisation of VHL deficient RCC cells. Our studies with the rapamycin analogue temsirolimus, already in the clinic for the treatment of various cancers, can be a potential candidate as a radiosensitiser for RCC cells. Overall, these finding led us to investigate further whether autophagy inducing compounds, which are either in clinic or in clinical trials, can effect the response to radiation. From a panel of candidate drugs which are known to induce autophagy we identified an aminopeptidase inhibitor, CHR-2797. CHR-2797 induces autophagy in the oesophageal cancer cell lines FLO-1 and OE21. Although, our results with CHR-2797 demonstrate it as a potential radiosensitiser, the mechanism of its radiosensitisation needs to be established. Our results from CHR-2797-induced radiosensitisation, further led us to investigate if other aminopeptidase inhibitors have a role in radiosensitisation. Therefore, we selectively screened candidate aminopeptidase inhibitors and identified some promising effects on radiosensitivity.

No description available.

The effect of FdUrd, zeocin, caffeine and their combination on the cell cycle of green algae Chlamydomonas reinhardtii and Scenedesmus quadricauda and response of these model organisms to DNA damage were studied. Both, FdUrd and zeocin, caused DNA damage that led to cell cycle arrest in these algae. In contrast, caffeine partially abolished G2 phase block imposed by zeocin. Protein levels of three crucial cell cycle regulators - CdkA, CdkB and Wee1 kinases were measured to identify mechanisms controlling reaction to DNA damage.

Cdk1 is an important cell cycle regulator that, in association with different cyclin regulatory subunits, is responsible for signaling important cell cycle events in all eukaryotic cells. In budding yeast, inhibition of Cdk1 by selective deletion of cyclin subunits has been shown to prevent anaphase onset, suggesting that Cdk1 activity is critically important for triggering anaphase onset. In many eukaryotes, Cdk1 has been shown to phosphorylate subunits of the anaphase promoting complex (APC), an E3 ubiquitin ligase which directly signals anaphase onset by triggering the degradation of the anaphase inhibitor securin. It is currently unclear, however, whether the APC is the sole essential substrate of Cdk1 in anaphase onset or if Cdk1 triggers anaphase onset by phosphorylating additional proteins. Eukaryotic Cdk1 is regulated by the Wee1 family of tyrosine kinases and the Cdc25 family of phosphatases which directly oppose Wee1 activity. Wee1 phosphorylation of Cdk1 on a single tyrosine residue inhibits Cdk1 and has been shown to prevent or delay mitotic entry. In this work we sought to further elucidate the mechanism through which Cdk1 regulates anaphase onset. We showed that, in addition to regulating mitotic entry, the budding yeast Wee1 kinase and Cdc25 phosphatase (Swe1 and Mih1 respectively in S. cerevisiae) regulate anaphase onset by modulating Cdk1 activity. Activation of Swe1 delays anaphase onset and cells lacking SWE1 enter anaphase prematurely, demonstrating that Swe1 regulates anaphase onset in unperturbed cell cycles. Deletion of the CDC55 regulatory subunit of PP2A has been shown to bypass cell cycle delays due to Swe1 activation. We showed that this is due, in part, to PP2ACdc55 dephosphorylation of Cdk1 sites on the APC. We have also shown that Cdk1 directly phosphorylates separase, the protease that dissolves sister chromatid linkages upon release from inhibitory securin/separase complexes upon APC-mediated securin degradation. Similar to phosphoregulation of the APC, we showed that Cdk1 phosphorylation of separase is opposed by PP2ACdc55. Phosphoregulation of separase appears to be important for regulation of the separase substrate Slk19 which cooperates with the conserved kinesin-5 Cin8 and microtubule bundling protein Ase1 to regulate spindle elongation at the spindle midzone.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24130号 / 医博第4870号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中島 貴子, 教授 万代 昌紀, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

During the early development of Xenopus laevis embryos, cells divide without checkpoints. At the midblastula transition (MBT), the cell cycle is remodeled as the division time lengthens and checkpoints are acquired. Initiation of the MBT depends upon the degradation of maternally supplied cyclin E, which is the regulatory partner of the cyclin dependent kinase, Cdk2. To study the program that drives cyclin E degradation and cell cycle remodeling at the MBT, embryos were treated with two cell cycle inhibitors, GST-D34Xic1 and XChk1. Injection of embryos with GST-D34Xic1, a stoichiometric inhibitor of cyclin E/Cdk2, delays degradation of cyclin E and onset of the MBT. GST-D34Xic1 lowers Wee1 level, a kinase that maintains Cdks in an inactivate state. Eventual degradation of cyclin E is preceded by degradation of GST-D34Xic1. The mathematical modelers, Andrea Ciliberto and John Tyson, incorporated the data into a kinetic model and set of ordinary differential equations. The model accurately described the experimental data and made additional predictions, which were tested experimentally. Additionally, embryos were injected with mRNA encoding XChk1, a kinase that activates Wee1 and inhibits Cdc25, the phosphatase opposing Wee1. Like GST-D34Xic1, XChk1 inhibits cyclin E/Cdk2 and delays the degradation of cyclin E. In contrast to GST-D34Xic1, XChk1 elevates the level of Wee1 at a time when sibling controls begin the MBT, despite cell cycle arrest. Since XChk1 inhibits both Cdk1 and Cdk2, and GST-D34Xic1 inhibits only Cdk2, we propose Cdk1 destabilizes Wee1, whereas Cdk2 elevates Wee1 level. Prior to the MBT, when cyclin E/Cdk2 is active, Wee1 is maintained. After cyclin E/Cdk2 is destroyed at the MBT, Wee1 is degraded. / Master of Science

Die Zellreplikation ein kontrollierter Prozess aus sequentieller und zeitlich koordinierter Aktivierung und Abbau von Zyklinen, die einen schnellen Übergang zwischen den Zyklusphasen ermöglichen. Dabei ist der Erfolg bei der Ermittlung der wichtigsten Komponenten und Aufgliederung der Schaltmechanismen im Wesentlichen auf die gleichzeitige Anwendung von Modellsystemen wie Hefe, Frosch und Fliege zurückzuführen. Das heutige Verständnis des Zellzyklus muss erweitert werden, um zu überprüfen ob die Erkenntnisse auch auf in-vivo Modelle von Säugetieren wie der Maus zutreffen. Es existieren solche Modelle, die sich auf spezifische Kontrollpunkte oder Übergänge konzentrieren, allerdings noch kein integriertes Modell, in dem der Zellzyklus durch eine Verletzung im Säugetier induziert wird. Das Modellsystem der Leberregeneration bei Nagern wurde gewählt, da es sich durch das am höchsten verbreitete Phänomen der Synchronisation der Zellproliferation auszeichnet. Mit dem Fokus auf die Frage, wie die Zellen durch pro-inflammatorische Signale nach Verletzungen ins Priming in der G1/S Phase eintreten, gingen wir in einen durch Zytokine und Wachstumsfaktoren induzierten Säugetier-Zellzyklus über. Weiterhin wurden mitotische Ereignisse modelliert, die zum Alles-oder-Nichts G2/M Übergang und dem mitotischen Ausgang führen. Wir konzentrieren uns auf die vielversprechende Funktion von Cdh1 in der Zellzykluskontrolle, welches bekanntlich eine Schlüsselrolle in der G1 Phase spielt. Weiterhin haben wir dessen Rolle bei der Verzögerung der G2 Phase untersucht. Wir vermuten eine zentrale Rolle von Cdh1 im Zellzyklus durch die Kontrolle der Dynamik der Zykline. Das Modell ist ein Versuch, die Kernmechanismen der Zellzykluskontrolle bei Säugetieren zu verstehen. Besseres Verständnis der Mechanismen in der Säugetierzelle würde das Studium der Zellphysiologie im Hinblick auf Störungen der humanen Zellzyklusmaschinerie, welche zu Krankheiten wie Krebs führen. / Cell replication is a controlled process with sequential and timely activation and degradation of cyclins leading to swift transitions between the phases of the cell cycle. The essential achievement in identifying the key components and in dissecting the mechanisms of the cell cycle circuitry has been attributed to the simultaneous use of model systems like yeast, frogs, and flies. Present understanding of the cell cycle needs to be extended to investigate whether those findings also apply to mammalian in-vivo models like mice. We chose liver regeneration in mammals as the model system because it is the most synchronised cell proliferation phenomenon, where 95\% of the cells simultaneously enter cell cycle. The G1-S phase transition was modelled, focusing on how injury induced pro-inflammatory signals \textit{prime} the cells in G1 phase and consequently both cytokine and growth factor induced pathways lead to further cell cycle progression. The model was further extended to mitotic events leading to the all-or-none G2-M transition and mitotic exit. I focussed on the emerging role of Cdh1 in the mammalian cell cycle. Cdh1 known for its role in G1 phase was further investigated for its role G2 delay. Cdh1 was suggested to be at the core of the cell cycle machinery controlling cyclin dynamics. This model is an attempt in understanding core machinery of the mammalian cell cycle. Better understanding of the cell cycle control system in mammalian cells would enable understanding perturbations of the human cell cycle machinery which lead to diseases like cancers.

The childhood cancer neuroblastoma mostly affects children under the age of 2 and comprises 6% of all childhood cancers. Neuroblastoma has very diverse phenotypes caused by both inter- and intra-tumour heterogeneities. The phenotypes are classified as being either low- or high-risk. This project focuses on high-risk NB cell lines with various chemotherapy sensitivity. Titration studies with chemotherapy agents cisplatin or doxorubicin showed a proneness of p53 mutated cell lines to arrest in either the S- and/or the G2/M-phase, depending on the drug and the drug dosage, indicating on a dose-dependent cell cycle response. To potentially inhibit the cells from arresting a treatment assay with 3 cell cycle key-components, pATM, Chk1 and Wee1 inhibitors was done. An initial immunocytochemistry staining of the expression levels of pATM and Wee1 showed that pATM was upregulated for 5 out 7 tested cell lines, namely SK-N-SH, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C, upon chemotherapy treatment with doxorubicin. Wee1 was however only upregulated for 3 out 7 cell lines; Kelly, SK-N-DZ and BE(2)-C. The upregulation of pATM and Wee1 showed a potential confirmation of their involvement in CT induced cell cycle arrest. Upon inhibition of pATM, Chk1 and Wee1 diverse effects were observed for each cell line (SK-N-SH, SK-N-AS, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C). Wee1 showed the most promising results were the cell viability decreased for all 5 p53 mutated cell lines and the confluency over time decreased for 4 out 5 p53 mutated cell lines. The p53 wild type cell line SK-N-SH was less sensitive towards Chk1 and Wee1 inhibition indicating that cell lines with functional p53 might not be as dependent on the Chk1 and Wee1 pathways compared to cell lines with non-functional p53. Thus, targeting the cell cycle arrest might be a promising therapeutic target for high-risk neuroblastoma. / Barndomscancern neuroblastom utgör 6% av all barncancer. Majoriteten av de drabbade är under 2 år. Neuroblastom har en stor mångfald av fenotypiska utryck som orsakas av dess inter- och intra-tumör heterogenitet. Fenotyperna klassificeras antigen som låg- eller högrisk. Här har 7 högrisks-neutoblastom cellinjer med varierande grad av känslighet mot kemoterapi analyserats. Titreringsstudier med kemoterapierna cisplatin och doxorubicin påvisade en benägenhet för de p53 muterade cellinjerna att arrestera i S- och/eller i G2/M-fasen, beroende på behandlingen samt behandlingsdosen, vilket indikerar på en dos-beroende cellcykel respons. En behandlingsanalys med de 3 nyckelkomponenterna fosforylerat ATM, Chk1 samt Wee1 gjordes för att potentiellt inhibera cellerna från att arrestera. Efter en initial immunocytokemi infärgning av pATM samt Wee1 visade 5 av 7 cellinjer (SK-N-SH, SK-N-FI, Kelly, SK-N-DZ samt BE(2)-C) en uppreglering av pATM-uttryck till följd av doxorubicin behandling. Däremot var Wee1 endast uppreglerat för 3 av 7 cell linjer (Kelly, SK-N-DZ samt BE(2)-C). Uppregleringen av pATM och Wee1 påvisar ett potentiellt samband mellan kemoterapi-inducerad cellcykelarrest och ökat utryck av pATM och Wee1. Vid inhibering av pATM, Chk1 samt Wee1 gav Wee1 de mest lovande resultaten där cellviabiliteten minskade för samtliga 5 p53-muterade cellinjer och där konfluensen över tid minskade för 4 av 5 p53-muterade cellinjer. SK-N-SH med funktionerande p53 var mindre känslig gentemot Chk1 och Wee1 inhibering, vilket indikerar att cellinjer med funktionerande p53 inte är lika beroende av reaktionsvägarna för Chk1 och Wee1 jämfört med cellinjer som har icke-funktionerande p53. Därmed kan riktad behandling mot cellcykelarrest vara en lovande behandling för högrisks-neuroblastom.