Dissertations / Theses on the topic 'G1 Phase Cell Cycle Checkpoints'

Progression through the cell cycle is tightly controlled, and the decision whether or not to enter a new cell cycle can be influenced by both internal and external cues. For budding yeast one such external cue is pheromone treatment, which can induce G1 arrest. Two distinct mechanisms are known to be involved in this arrest, one dependent on the arrest protein Far1 and one independent of Far1, but the exact mechanisms have remained enigmatic. The studies presented here further elucidate both of these mechanisms. We looked at two distinct aspects of the Far1-independent arrest mechanism. First, we studied the role of the G1/S regulatory system in G1 arrest. We found that deletion of the G1/S transcriptional repressors Whi5 and Stb1 compromises Far1-independent arrest, but only partially, and that this partial arrest failure correlates to partial de-repression of G1/S transcripts. Deletion of the CKI Sic1, however, is more strongly required for arrest in the absence of Far1, though not when Far1 is present. Together, this demonstrates that functionally overlapping regulatory circuits controlling the G1/S transition collectively provide robustness to the G1 arrest response. We also sought to reexamine the phenomenon of pheromone-induced loss of G1/S cyclin proteins, which we suspected could be another Far1-independent arrest mechanism. We confirmed that pheromone treatment has an effect on G1 cyclin protein levels independent of transcriptional control. Our findings suggest that this phenomenon is dependent on SCFGrr1but is at least partly independent of Cdc28 activity, the CDK phosphorylation sites in Cln2, and Far1. We were not, however, able to obtain evidence that pheromone increases the degradation rate of Cln1/2, which raises the possibility that pheromone reduces their synthesis rate instead. Finally, we also studied the function of Far1 during pheromone-induced G1 arrest. Although it has been assumed that Far1 acts as a G1/S cyclin specific CDK inhibitor, there has been no conclusive evidence that this is the case. Our data, however, suggests that at least part of Far1’s function may actually be to interfere with Cln-CDK/substrate interactions since we saw a significant decrease of co-pulldown of Cln2 and substrates after treatment with pheromone. All together, the results presented here demonstrate that there are numerous independent mechanisms in place to help robustly arrest cells in G1.

Progression through the cell cycle is tightly controlled, and the decision whether or not to enter a new cell cycle can be influenced by both internal and external cues. For budding yeast one such external cue is pheromone treatment, which can induce G1 arrest. Two distinct mechanisms are known to be involved in this arrest, one dependent on the arrest protein Far1 and one independent of Far1, but the exact mechanisms have remained enigmatic. The studies presented here further elucidate both of these mechanisms. We looked at two distinct aspects of the Far1-independent arrest mechanism. First, we studied the role of the G1/S regulatory system in G1 arrest. We found that deletion of the G1/S transcriptional repressors Whi5 and Stb1 compromises Far1-independent arrest, but only partially, and that this partial arrest failure correlates to partial de-repression of G1/S transcripts. Deletion of the CKI Sic1, however, is more strongly required for arrest in the absence of Far1, though not when Far1 is present. Together, this demonstrates that functionally overlapping regulatory circuits controlling the G1/S transition collectively provide robustness to the G1 arrest response. We also sought to reexamine the phenomenon of pheromone-induced loss of G1/S cyclin proteins, which we suspected could be another Far1-independent arrest mechanism. We confirmed that pheromone treatment has an effect on G1 cyclin protein levels independent of transcriptional control. Our findings suggest that this phenomenon is dependent on SCFGrr1but is at least partly independent of Cdc28 activity, the CDK phosphorylation sites in Cln2, and Far1. We were not, however, able to obtain evidence that pheromone increases the degradation rate of Cln1/2, which raises the possibility that pheromone reduces their synthesis rate instead. Finally, we also studied the function of Far1 during pheromone-induced G1 arrest. Although it has been assumed that Far1 acts as a G1/S cyclin specific CDK inhibitor, there has been no conclusive evidence that this is the case. Our data, however, suggests that at least part of Far1’s function may actually be to interfere with Cln-CDK/substrate interactions since we saw a significant decrease of co-pulldown of Cln2 and substrates after treatment with pheromone. All together, the results presented here demonstrate that there are numerous independent mechanisms in place to help robustly arrest cells in G1.

Cellular checkpoints are typically considered to both facilitate the ordered execution of the cell cycle and to act as a barrier to oncogene driven cell cycles and the transmission of unresolved genetic lesions from one phase to the next. Furthermore, these mechanisms are also believed to underpin the responses of cells, both in normal and cancerous tissues, to those therapies that either directly or indirectly generate DNA damage. In recent studies however, it has become clear these checkpoints permit the passage of significant genomic aberrations into subsequent cell cycle phases and even descendant cells, and that heterogeneous responses are apparent amongst genetically identical cells. The consequences of this checkpoint ‘negligence’ remain relatively uncharacterised despite the importance of checkpoints in current models for how genomic instability is avoided in the face of ubiquitous DNA damage. Unresolved DNA damage is presumably inherited by subsequent cell cycle phases and descendant cells yet characterisation of the consequences of this has been relatively limited to date. I therefore utilised microscopy-based lineage tracing of cells expressing genetically encoded fluorescent sensors, particularly the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes (Sakaue-Sawano et al., 2008), with semi-automated image analysis to characterise the response of single cells and their descendants to DNA lesions across multiple cell cycle generations. This approach, complemented by generational tracing by flow cytometry, permitted me to characterise the timing of cell fate determination in treated and descendant cells, the non-genetic heterogeneity in checkpoint responses and overall lineage behaviour, correlations between cells (similarly to Sandler et al., 2015) and cell cycle timing dependencies in the response to DNA damaging agents. With these single cell analytical approaches I show that the consequences of DNA damage on descendant cell fate is dramatic, suggesting checkpoint mechanisms may have consequences and even cooperate across phases and generations. U2OS cell lineages traced for three generations following the induction of DNA damage in the form of strand breaks showed greatly induced cell death in the daughters and granddaughters of DNA damaged cells, termed delayed death. Furthermore, lineage behaviour was characterised as highly heterogeneous in when and whether cell death occurred. Complementary flow cytometric approaches validated the findings in U2OS cells and suggested HeLa cells may show similar behaviour. These findings indicate that checkpoint models need to incorporate multigenerational behaviour in order to better describe the response of cells to DNA damage. Understanding the processes governing cell fate determination in descendant cells will impact upon our understanding of the development of genomic instability during carcinogenesis and how DNA-damaging chemotherapeutics drive cells to ‘quit’ the cell cycle.

Les mécanismes développementaux qui spécifient le nombre et le phénotype laminaire des neurones du cortex cérébral jouent un rôle essentiel dans l’établissement de la cytoarchitecture corticale. Le nombre de neurones dans chaque couche d'une aire donnée est déterminé par le taux de production neuronale, qui dépend étroitement de l'équilibre entre les divisions prolifératives et différenciatives. Des observations clés suggèrent que la durée de la phase G1 (TG1) ferait partie intégrante d'un mécanisme cellulaire régulant le mode de division des précurseurs du cortex. Nous avons testé cette hypothèse par l'accélération expérimentale de la progression dans la phase G1 de précurseurs corticaux de souris in vivo, via la surexpression des cyclines E1 et D1. A E15, la réduction de TG1 promeut la rentrée dans le cycle cellulaire aux dépens de la différenciation neuronale, résultant en une modification de la cytoarchitecture du cortex adulte. Des données de modélisation confirment que les effets induits par la réduction de TG1 sont médiés par des changements du mode de division. Les effets de la surexpression des cyclines E1 et D2 à E13 sont plus modérés qu'à E15, indiquant des différences intrinsèques entre les précurseurs corticaux précoces et tardifs. La mesure des phases du cycle cellulaire des populations de précurseurs corticaux à l’aide de différentes techniques révèle un niveau important d’hétérogénéité et souligne la nécessité de prendre en compte la diversité des précurseurs co‐existant dans les zones germinales du télencéphale
In the cerebral cortex, area‐specific differences in neuron number and phenotype are distinguishing features both within and across species. The developmental mechanisms that specify the number of neurons and their laminar fate are instrumental in specifying cortical cytoarchitecture. Neuron number in layers and areas correlate with changes in the rate of neuron production, largely determined by the balance between proliferative and differentiative divisions in cortical precursors. Key observations suggest a concerted regulation between the duration of the G1 phase (TG1) and mode of division and have led to the hypothesis that TG1 could be an integral part of a cellular mechanism regulating the mode of division of cortical precursors. To test this hypothesis we experimentally accelerated TG1 in mouse cortical precursors in vivo, via the forced expression of cyclinE1 and cyclinD1. At E15, TG1 reduction promoted cell‐cycle re‐entry at the expense of differentiation and led to cytoarchitectural modifications. Modeling confirms that the TG1‐induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. Forced expression of G1 cyclins was also applied to early cortical precursors. The effects of cyclinD1 and cyclinE1 up‐regulation at E13 were milder than those observed at E15, pointing to intrinsic differences between early and late cortical precursors. The used of various techniques to measure cell‐cycle kinetics in distinct precursor populations underlined the necessity of taking the full diversity of neural precursors co‐existing in the GZ of the telencephalon into account when performing cellcycle kinetics analysis

X&barbelow;IAP a&barbelow;ssociated f&barbelow;actor 1&barbelow; (XAF1) was initially isolated as novel 34 kDa protein which bound XIAP in a two-hybrid screening. The XAF1A protein consists of 301 a.a. and contains seven potential zinc finger domains. Two alternatively splice variants of XAF1 were later isolated. One isoform (XAF1B) was formed by the removal of a 57 bp exon, which leads to an in-frame deletion of the third zinc finger and the creation of a shorter 32.5 kDa protein. The other splice variant (XAF1C) contains a 154 bp exon insertion, which truncates the sixth and seventh zinc fingers to produce an 18.7 kDa protein. XAF1A and XAF1B, but not XAF1C, bound XIAP in in vitro pull down assays. Northern blot analysis showed at least four distinct sizes of xaf1 mRNA ranging between 3.9 and 7.0 kb, which may indicate other XAF1 isoforms yet to be discovered. Though the possible role of these zinc fingers on the XAF1/XIAP interaction has yet to be determined, recent experiments indicate that XAF1A can block the ability of XIAP to inhibit caspase-3 in vitro. Furthermore, overexpression of XAF1A in HEL299 cells triggered a G1 cell cycle arrest. This G1 arrest coincides with an increase in p21, but not p53. The ability of XAF1 to block XIAP function and induce cell cycle arrest suggests a role for XAF1 in the control of both apoptosis and cell growth. The coding regions of XAF1A, B and C are encoded on a total of 9 exons within a span of 20 kb. The single copy xaf1 gene has been mapped, using FISH analysis, distal to the TP53 gene on 17p13.2. Southern blot analysis of YACS within this region further localizes the xaf1 gene on YAC 746 C 10, which contains the markers D17S1831, D17S796, and D17S1881. These markers are located approximately 3 cM telomeric to the TP53 gene. Since the xaf1 gene is located in a region commonly deleted in numerous types of cancers, this may suggest a tumour suppressor role for XAF1 in cancer. To test this theory, a 60 cell line panel from the NCl was analyzed for xaf1 RNA expression by Taqman and heterozygosity status of markers proximal to xaf1. Taqman analysis indicated that the majority of cell lines expressed little or no xaf1 RNA while xiap levels were relatively high. A PCR study of markers near xaf1 showed significant loss of heterozygosity (LOH) in this region. The loss of xaf1 expression and significant LOH near the xaf1 gene indicate that the down-regulation of XAF1 may be important in the development of the transformed phenotype.

Interferons (IFNs) are a class of structurally related proteins first discovered to be produced by virus-infected cells. By now, several other inducing agents have been described. IFNs exert multiple effects on cells exemplified by the establishment of an antiviral state, inhibition of cell proliferation and alteration of different immune reactions. In the present thesis the inhibition of cellular growth concentrated on effects in the early cell cycle have been studied. The human glioma cell line 251 MG was found to be blocked in the S phase of the cell cycle upon addition of IFN both to exponentially growing and growth-factor depleted, synchronized cells. Thymidine kinase and DNA-polymerase activities were reduced in parallel with the S phase effect. 2-5 oligo Anucleotides transfected into glioma cells lead to inhibition of cell growth, exponentially growing cells being blocked in the S phase as during IFN treatment. In contrast, synchronized, restimulated cells were blocked in the cellcycle phase where they resided at the time of transfection. As 2-5 oligo A synthetase activity was induced in the middle of the Gl phase, these results might indicate that the kinetics of expression of oligonucleotides after IFN additiondetermines the type of cell cycle block obtained in differenttumor cells. IFN inhibited preferentially proteins originating from newly synthesized mRNA in Sw 3T3 cells, c-mvc did not seem to be included among these proteins. In both cell systems c-myc expression was unaltered after IFN treatment. In clone T1 selected from the the Sw 3T3 cell line , c-mvc expression was uncoupled to growth and seemed to be growth factor independent. The change in c-myc expression in clone T1 compared to SW 3T3 cells did not render the cells sensitive to IFN. Hence, c-myc regulation does not seem to be the mechanism by which IFN regulates cell growth in this system. The proliferation marker KI-67 antigen was shown not to be causatively involved in growth inhibition of IFN. The reduced levels of the antigen was proposed to be a secondary effect caused by the G0/G1 arrest.

Diss. (sammanfattning) Umeå : Umeå universitet, 1991, härtill 6 uppsatser

digitalisering@umu

Cyclin B1 is a cell cycle protein typically associated with the regulation of cellular division (mitosis). Previous studies in this laboratory involving preimplantation mouse embryos found that cyclin B1, or a cyclin B 1-related protein, were present at both G1 and G2/M phase of the cell cycle. Not only was cyclin Bi detected during G1 phase in this study, it was found to be present in higher concentrations at G1 phase through the first three cell cycles. These findings were unexpected, because most of the literature suggests that cyclin B1 is normally degraded during G1 phase. Using immunoprecipitation and immunoblot techniques, a more detailed study of cyclin B1 expression was inititated. Using two different primary antibodies direct against cyclin B1, a 48.97 kDa protein band, which is believed to be cyclin B1, was detected at both G1 and G2/M phases in 1-cell mouse embryos. Using another antibody directed against Cdk1, the kinase that forms a complex with cyclin B1 in order to direct the G2/M transition, a 37 kDa protein band was also detected at both G1 and G2/M phases in 1-cell mouse embryos. In order to determine whether cyclin B1 was present as a complex with Cdk1, immunoblotting with the anti-Cdk1 antibody. Again, a 37kDa protein band was detected at both G1 and G2/M phases. Finally, in order to determine whether the cyclin B1/Cdk1 complex exists in its active form, histone kinase assays were performed using anti-cyclin B1 immunoprecipitates. Kinase activity was detected in immunoprecipitates collected from G2/M phase 1-cell embryos, but no kinase activity was detected from immunoprecipitates collected from G1 phase 1-cell embryos. These data indicate that cyclin B1 and Cdk1 are present and exist as a complex in both G1 and G2/M phases of 1-cell mouse embryos, although the complex only appears to be active at the G2/M phase.
Department of Biology

Das humane Zytomegalievirus, ist ein ubiquitäres Pathogen, welches akute und persistierende Infektionen verursacht. Bei immunsupprimierten Patienten kann das Virus zu schweren Erkrankungen, wie Hepatitis, Pneumonie und bei kongenitaler Infektion außerdem zu Schädigungen des ZNS führen. HCMV blockiert die Zellproliferation durch einen Arrest am G1/S-Übergang des Zellzyklus, andererseits wird aber gleichzeitig die Expression S-Phase spezifischer Gene aktiviert. Teilweise lässt sich dies durch eine Virus vermittelte spezifische Inhibition der zellulären DNA-Repliaktion sowie durch eine massive Deregulation Zyklin-assozzierter Kinasen erklären. Zellkulturexperimente deuten darauf hin, dass die Zellzyklusalterationen wichtige Voraussetzungen für eine erfolgreiche Virusreplikation darstellen. Es ist hingegen nicht bekannt, welche Relevanz sie für die Virusvermehrung in vivo und das pathologische Erscheinungsbild im erkrankten Organismus besitzen. Diese Frage kann nur in einem Tiermodell sinnvoll angegangen werden. Aufgrund der Wirtsspezifität der Zytomegalieviren, ist man dabei auf die Verwendung der jeweiligen artspezifischen CMV angewiesen. Murines CMV (MCMV) und Ratten-CMV (RCMV) sind dabei die bislang bestuntersuchtesten Systeme. Das Anliegen dieser Arbeit war es zu prüfen, inwieweit die für HCMV beschriebenen Zellzyklusregulationen in MCMV und RCMV auf Zellkulturbasis konserviert sind. Es konnte gezeigt werden, dass sowohl RCMV als auch MCMV einen antiproliferativen Effekt auf infizierte Zellen besitzen und ebenso wie HCMV zu einem Zellzyklusarrest führen. Nager-Zytomegalieviren können Zellen auch in der G2-Phase arretieren und in dieser Zellzyklusphase auch effizient replizieren können. Die Infektion mit Nager-CMV führt außerdem auf breiter Basis zur Veränderung Zyklin-assoziierter Kinaseaktivitäten. Allen Zytomegalieviren ist die Hemmung der zellulären DNA-Synthese am G1/S-Übergang durch die Inhibition des replication licensing, dem Beginn der DNA-Synthese gemein. Durch diese vergleichende Studie wird einerseits deutlich, dass wesentliche funktionelle Schritte der Zellzyklusregulation zwischen den Zytomegalieviren konserviert sind, aber andererseits die zu Grunde liegenden molekularen Mechanismen zum Teil deutlich variieren.
Human Cytomegalovirus (HCMV) is an ubiquitous, species-specific beta-herpesvirus that, like other herpesviruses, can establish lifelong latency following primary infection. HCMV infection becomes virulent only in immunocompromised patients such as premature infants, transplant recipients and AIDS patients where the virus causes severe disease like hepatitis, pneumonitis and retinitis. Congenital infection produces birth defects, most commonly hearing loss. To develop rational-based strategies for prevention and treatment of HCMV infection, it is crucial to understand the interactions between the virus and its host cell that support the establishment and progression of the virus replicative cycle. In general, herpesviruses are known to replicate most efficiently in the absence of cellular DNA synthesis. What is more, they have evolved mechanisms to avoid the cell´s DNA replication phase by blocking cell cycle progression outside S phase. HCMV has been shown to specifically inhibit the onset of cellular DNA synthesis resulting in cells arrested with a G1 DNA content. Towards a better understanding of CMV-mediated cell cycle alterations in vivo, we tested murine and rat CMV (MCMV/RCMV), being common animal models for CMV infection, for their influence on the host cell cycle. It was found that both MCMV and RCMV exhibit a strong anti-proliferative capacity on immortalised and primary embryonic fibroblasts after lytic infection. This results from specific cell cycle blocks in G1 and G2 as demonstrated by flow cytometry analysis. The G1 arrest is at least in part caused by a specific inhibition of cellular DNA synthesis and involves both the formation and activation of the cells’ DNA replication machinery. Interestingly, and in contrast to HCMV, the replicative cycle of rodent CMVs started from G2 as efficiently as from G1. Whilst the cell cycle arrest is accompanied by a broad induction of cyclin-cdk2 and cyclin-cdk1 activity, cyclin D1-cdk4/6 activity is selectively suppressed in MCMV and RCMV infected cells. Thus, given that both rodent and human CMVs are anti-proliferative and arrest cell cycle progression we found a surprising divergence of some of the underlying mechanisms. Therefore, any question put forward to a rodent CMV model involving cell cycle regulation has to be well defined in order to extrapolate meaningful information for the human system.

In this work we have assessed the possible contribution of the human chromosome-21 gene DYRK1A in the developmental cortical alterations associated with Down Syndrome using the mBACTgDyrk1a mouse, which carries 3 copies of Dyrk1a, and a trisomic model of the syndrome, the Ts65Dn mouse. We show that trisomy of Dyrk1a changes the cell cycle parameters of dorsal telencephalic radial glial (RG) progenitors and the division mode of these progenitors leading to a deficit in glutamatergic neurons that persist until the adulthood. We demonstrate that Dyrk1a is the triplicated gene that causes the deficit in early-born cortical glutamatergic neurons in Ts65Dn mice. Moreover, we provide evidences indicating that DYRK1A-mediated degradation of Cyclin D1 is the underlying mechanism of the cell cycle defects in both, mBACTgDyrk1a and Ts65Dn dorsal RG progenitors. Finally, we show that early neurogenesis is enhanced in the medial ganglionic eminence of mBACTgDyrk1a embryos resulting in an altered proportion of particular cortical GABAergic neuron types. These results indicate that the overexpression of DYRK1A contributes significantly to the formation of the cortical circuitry in Down syndrome.
En aquest treball s'ha avaluat la possible contribució del gen DYRK1A, localitzat en el cromosoma humà 21, en les alteracions del desenvolupament de l’escorça cerebral associades a la Síndrome de down (SD) mitjançant l’estudi de dos models murins: el ratolí mBACTgDyrk1a, el qual conté 3 còpies de Dyrk1a, i el ratolí Ts65Dn, un dels models trisòmics de la SD més ben caracteritzats. Els nostres resultats mostren que la trisomia de Dyrk1A altera alguns paràmetres del cicle cel•lular i el tipus de divisió dels progenitors neurals del telencèfal dorsal, donant lloc a un dèficit de neurones glutamatèrgiques que persisteix fins l’edat adulta. Hem demostrat que Dyrk1a és el gen triplicat responsable del dèficit inicial en la generació de neurones glutamatèrgiques corticals observat en el ratolí Ts65Dn. A més a més, hem proporcionat evidències de que la degradació de Ciclina D1 induïda per DYRK1A és el mecanisme molecular subjacent a les alteracions de cicle cel•lular observades en els progenitors neuronals dels embrions mBACTgDyrk1a i Ts65Dn. Per altra banda, hem demostrat que la neurogènesis inicial està incrementada en l’eminència ganglionar medial dels embrions mBACTgDyrk1a, fet que altera la proporció de subtipus específics d’interneurones GABAèrgiques en l’escorça cerebral adulta. En conclusió, els nostres resultats indiquen que la sobreexpressió de DYRK1A contribueix significativament a la formació dels circuits cortical en la SD.

STAUFEN1 (STAU1) est une protéine de liaison à l’acide ribonucléique (ARN) double brin jouant un rôle important dans le contrôle post-transcriptionnel de nombreux ARN messager (ARNm). Sa déplétion diminue la prolifération des cellules non cancéreuses en altérant les transitions G1/S et G2/M. En revanche, Ceci n’a aucun impact sur la prolifération des cellules tumorales. La déplétion de STAU1 module le niveau d’expression des transcrits et/ou des protéines impliquées dans la régulation des points de contrôle des transitions de phase. Notamment, STAU1 module le niveau d’expression de la protéine CDK4 ainsi que l’abondance de l’ARNm E2F1, deux régulateurs indispensables de la transition G1/S. Le transcrit de ces deux gènes possède un site de liaison à STAU1 ou STAU1 binding site (SBS) dans la région codante ou coding sequence (CDS) et dans la région non codante en 3’ (3’UTR), respectivement. Cependant, l’importance de la liaison de STAU1 à ces transcrits n’a pas encore été étudiée. Étonnamment, la sensibilité des cellules non tumorales et tumorales à l’expression de STAU1 est inversée lors de la surexpression de STAU1. En effet, sa surexpression altère l’entrée en mitose des cellules cancéreuses et diminue leur prolifération, alors qu’elle n’a aucun effet sur la prolifération des cellules non tumorales. Lors de la mitose, STAU1 s’associe au fuseau mitotique (FM), ce qui lui permet de localiser des ARNm et de contrôler leur séquestration et/ou leur traduction locale. Cependant, le mécanisme qui permet à STAU1 de lier le FM est encore inconnu. Pour ce mémoire, nous avons donc poursuivi deux objectifs. Le premier but est de comprendre la régulation post-transcriptionnelle médiée par STAU1 des transcrits essentiels à la transition G1/S chez les cellules non tumorales. Notre hypothèse est que STAU1 par sa liaison directe à ses transcrits cibles via le SBS module leur expression. Pour ce faire, des cellules de type sauvage ou déplétées en STAU1 étaient transfectées par des plasmides exprimant les transcrits de CDK4 et d’E2F1 contenant un SBS endogène ou muté de telle sorte qu’il ne reconnait plus STAU1. L’expression des protéines CDK4 et E2F1 est dosée par un essai luciférase ou un immunobuvardage de type western ou western blot (WB). Nous avons observé que STAU1 régule négativement et positivement l’expression endogène de CDK4 et d’E2F1, respectivement, ce qui contribue au passage de la transition G1/S, donc à la prolifération cellulaire non tumorale. Les essais luciférases ont confirmé le rôle de STAU1 dans la régulation positive d’E2F1 lorsque liée au SBS dans le 3’UTR du transcrit E2F1. Malheureusement, les plasmides utilisés pour l’expression de CDK4 se sont avérés non fonctionnels, ce qui nous a forcés à mettre de côté cette expérience. Le deuxième but est d’étudier les déterminants qui régulent la localisation de STAU1 au FM chez les cellules tumorales. Pour ce faire, la localisation de STAU1 ou des mutants au FM est détectée par WB à partir de préparations des FM purifiés. Nos données montrent que le déterminant est composé de plusieurs acides aminés (aa) situés entre le 26ème et 37ème aa du côté N-terminal de la protéine STAU1. En somme, nos résultats montrent les différents rôles de STAU1 dans les cellules tumorales vs cellules non tumorales. De ce fait, STAU1 pourrait être une cible thérapeutique spécifique potentielle dans le traitement du cancer.
STAUFEN1 (STAU1) is a double stranded RNA binding protein that plays an important role in the post-transcriptional control of many mRNAs. Its depletion decreases the proliferation of non-cancer cells by altering G1/S and G2/M transitions. In contrast, this has no impact on the proliferation of tumor cells. The decrease of STAU1 expression modulates the level of transcripts/proteins of several genes involved in phase transition checkpoints, including CDK4 and E2F1, two essential regulators in G1/S transition. In addition, CDK4 and E2F1 transcripts have a STAU1 binding site (SBS) in the coding sequence (CDS) and the non-coding region in 3’ (3’UTR), respectively. However, the molecular consequence of STAU1 association with the SBS is not yet studied. Surprisingly, the sensibility of non-cancer and cancer cells to STAU1 expression is reversed following STAU1 overexpression. Indeed, its overexpression alters the entry into mitosis of cancer cells and decreases their proliferation, while it has no effect on non-cancer cells. During mitosis, STAU1 associates with the mitotic spindle, which allows it to localize mRNAs and other non-coding RNAs. STAU1 likely controls their sequestration and/or local translation during mitosis. However, the molecular determinant involved in STAU1-spindle association is still not known. Therefore, for this master thesis, we had two objectives. The first goal is to understand the post-transcriptional regulation mediated by STAU1 on transcripts that are essential for G1/S transition in non-tumor cells. Our hypothesis is that STAU1, by its direct binding to the SBS of its target transcripts, modulates their expression. To do this, plasmids coding for CDK4 and E2F1 containing a wild-type or mutated SBS that does not recognized STAU1 were transfected in wild-type and STAU1-depleted cells. Expression of CDK4 and E2F1 was detected by dual luciferase assay and western blot (WB). Our results first indicate that STAU1 negatively and positively regulates the endogenous expression of CDK4 and E2F1, respectively, which contributes to the passage of G1/S transition, and therefore to the proliferation non-tumor cells. Then, the luciferase assays confirm the role of STAU1 in E2F1 expression, depending on STAU1 binding to E2F1 SBS in its 3’UTR. Unfortunately, the plasmids used for CDK4 expression turned out to be non-functional. The second goal is to identify the molecular determinants responsible for the localization of STAU1 to the mitotic spindle in tumor cells. To this end, the localization of STAU1 or of several mutants was measured by WB using purified spindle preparations. Our data show that the determinant is composed of several amino acids (aa) located between the 26th and 37th aa at the N-terminal end of STAU1. In summary, our results show the different roles of STAU1 in tumor and non-tumor cells. Therefore, STAU1 could be a potential specific therapeutic target in cancer treatments.

碩士
長庚大學
生物醫學研究所
99
Klebsiella pneumoniae（KP）, a gram-negative bacterium, normal intestinal microflora, usually causes nosocomial infection. In Taiwan. KP infection causes severe liver abscess, especially in patients with diabetes mellitus. Many researches indicate that cells execute apoptosis and necrosis during bacterial infection, and furthermore influence the cell cycle. But the molecular mechanism is still not clear in KP. Previous study in our laboratory found that chromatin condensation, DNA fragmentation and accumulation sub-G1 phase in KP infected HepG2 cells. My results demonstrated that cell viability decreased during KP infection, flowcytometry data showed increased PS exposure, ATP analysis showed that ATP concentration was dramatically decreased after 4 hours KP infection. Apoptosis：The AIF and Endo G were released from mitochondria and translocated to nucleus post-infection. Cyt C also released from mitochondria. Bad, Apaf-1 and m-Calpain were increasd post KP infection. Caspase-9 and Caspase-7 activation, PARP inactivation and α-fodrin cleavage, DFF40 increase and DFF45 decrease were observed during KP infection. Cell cycle molecules including Cyclin D1, Cyclin D2, p-Rb and E2F were decreased during KP infection. Moreover, p-p53 was increased post KP infection. Our data suggest that the KP infection causes HepG2 cells to undergo early apoptosis and late necrosis, and the decrease in the molecules involved in the G1 checkpoint contributes the cell cycle arrest in G1 phase.

碩士
中國醫藥大學
醫學研究所
92
Recently, Herbal medicines are increasingly becoming a popular project. Gynostemma pentaphyllum MAKINO is noted for its functions of treating hepatitis and cardiovascular diseases in Asia. Gypenosides are the major components that are extracted from Gynostemma pentaphyllum MAKINO. However, the molecular mechanism underlying the gypenosides-induced cell cycle arrest and apoptotic process is unclear. In this study, we have evaluated the chemopreventive role of gypenosides in human lung cancer (A549) cells in vitro by studying the regulation of proliferation, cell cycle and apoptosis. Gypenosides inhibited cell proliferation, induced G0/G1 arrest and apoptosis in A549 cells. Investigation on the levels of CDKIs (p21 and p27) by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) and Western Blotting showed that p21 and p27 were increased with the increasing doses of gypenosides in A549 cells. The levels of p21 and p27 increased after A549 cells were cotreated with various concentrations of gypenosides. The increase of the levels of p21 and p27 may be the major factor for gypenosides to cause G0/G1 arrest in the examined cells. Flow cytometric assay and gel electrophoresis of DNA fragmentation also confirmed that gypenosides induced apoptosis in A549 cells. Our data demonstrated that gypenosides-induced apoptotic cell death was accompanied by up-regulation of Bax, NF-κB, caspase-3 and caspase-9, while it had no effect on the levels of Bcl-2 and p53. Taken together, gypenosides therefore appears to exert its anticarcinogenic properties by inhibiting proliferation, inducing G0/G1 phase arrest and apoptosis underwent activation of NF-κB, Bax and caspase-3 in human lung A549 cancer cell line.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3511. Adviser: Paul J. Hergenrother. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

碩士
國立中興大學
生命科學系所
94
Retinoic acid (RA) and its derivatives, retinoids, are common used in cancer research with a long history. RA enables to turn-off cancer cell proliferation, induces cell cycle arrest and leads to differentiation or apoptosis in many kinds of cancer, such as leukemia, hepatoma, breast cancer, and prostate cancer. The effects of RA in cancer biology are varied and still need further investigation. Cyclin-dependent kinase 5 (Cdk5), a member of Cdk family, binds to its specific activators, p35 or p39, and gets itself activated. The researches of Cdk5 have been focused on neuronal and muscle tissues because of the activators’ distribution. Although the structure of Cdk5 is similar to Cdk1 (cdc2), it has not been considered to involve in the regulation of cell cycle. Our previous study shows that the presences of Cdk5 and p35 in prostate cancer cell lines (LNCaP, PC3 and DU145) play important roles in digoxin-induced apoptosis. Here, I first identified that treatment with 1 μM ATRA in DU145 cells could increase protein expressions of Cdk5, p35, Egr1, p21 and p27. I also observed that ATRA could affect the morphological change, decreases of growth and viability, and cell cycle G1 phase increase in DU145 cells. Cotreatment with 1 μM ATRA and 1 μM roscovitine, a specific inhibitor of Cdk5, could reversely decrease the expressions of p21 and p27, the G1 phase ratio and rescue the drop of viability caused by ATRA. Taken together, my present results suggest that Cdk5 probably involves in ATRA-induced cell cycle G1 phase increase in DU145 cells through the regulation of p21 and p27.

Aim: Cisplatin is the first platinum drug that shows promising anti-tumor effect clinically. Oxaliplatin, a third-generation platinum drug that incorporates a diaminocyclohexane (DACH) structural entity, can overcome cisplatin resistance. R,R-5, a novel platinum compound that integrates the DACH entity with a demethylcantharidin (DMC) component that is derived from a traditional Chinese medicine (TCM) , can also overcome cisplatin resistance. The principal objectives of this study was to investigate in detail, the effect of these compounds at the antephase and G2 checkpoints of the cell cycle, and to establish the relationship (if any) between different structural entities with checkpoint activation. The ultimate aim of the study was to ascertain the potential for the development of novel checkpoint abrogators as anti-tumor agents.
Background: A common procedure in current cancer chemotherapy is to induce genomic stress in cancer cells, leading to irreparable DNA damage and eventually cell death. However, there are several DNA repair mechanisms in cancer cells to maintain genomic stability, which require cell cycle checkpoints to stop cell proliferation for DNA damage repair, thereby avoiding errors in cellular events like DNA replication, transcription and mitosis. Among these cell cycle checkpoints, antephase and G2 checkpoints are two gate checkpoints for mitosis. Abrogation of G2 checkpoint has been reported to give rise to synergistic cytotoxic effect with DNA damaging agents, representing a means of circumventing drug resistance in chemotherapy.
Conclusions: Acute stress to cisplatin can activate the MMR/c-Abl/MEKK1/p38MAPK pathway, leading to the activation of antephase checkpoint, and stop cells from entering mitosis immediately. DACH-containing platinum compound oxaliplatin fails to activate this antephase checkpoint. However, both cisplatin and oxaliplatin can activate the G2 checkpoint, which can be abrogated by DMC. In contrast, RR-5 can bypass both the antephase and G2 checkpoints. In summary, novel TCM-platinum compound R,R-5 can bypass mitotic DNA damage checkpoints in cancer cells and thus has the potential for further development as an anti-cancer drug.
Methods: Microarray analysis was used to detect gene transcription profiles after drug treatments. The activation of mitotic checkpoints was inspected by counting mitotic cells and utilizing flow cytometry. Using Western blotting, the activation of certain key players in the antephase and G2 checkpoint was revealed. MTT assays were performed to show the outcome of checkpoint activation.
Results: In HCT116 cells, 35 genes that facilitate G2/M transition were found to be up-regulated after R,R-5 treatment compared with oxaliplatin in the microarray analysis, implying the bypass of mitotic checkpoints by R,R-5 rather than oxaliplatin. Acute stress (2 hour) of cisplatin activated the antephase checkpoint, resulting in a rapid decrease in mitotic index and phosphorylation of histone H1, which avoided mitotic catastrophe and promoted cell survival in HeLa cells. Further experiments demonstrated that this antephase checkpoint could be abrogated by c-Abl and p38MAPK inhibitors, or siRNAs against c-Abl or MEKK1, suggesting that this checkpoint may be controlled by an MMR/c-Abl/MEKK1/p38MAPK pathway. In contrast, oxaliplatin and R,R-5 did not activate this antephase checkpoint. Moreover, after 24 hour oxaliplatin treatment in HeLa cells, the mitotic index and CDK1 activity were decreased, which could be restored by concomitant treatment with ATM/ATR inhibitor and DMC. This indicated the activation of G2 checkpoint by oxaliplatin and implied that DMC can abrogate oxaliplatin-activated G2 checkpoint by restoring CDK1 activity. Cisplatin could also activate G2 checkpoint, whereas R,R-5 apparently bypassed this G2 checkpoint.
Guan, Huaji.
Adviser: Vincent Hon Leung Lee.
Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 212-249).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Yes
Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.

Les protéines sont les produits finaux de la machinerie génétique. Elles jouent des rôles essentiels dans la définition de la structure, de l'intégrité et de la dynamique de la cellule afin de promouvoir les diverses transformations chimiques requises dans le métabolisme et dans la transmission des signaux biochimique. Nous savons que la doctrine centrale de la biologie moléculaire: un gène = un ARN messager = une protéine, est une simplification grossière du système biologique. En effet, plusieurs ARN messagers peuvent provenir d’un seul gène grâce à l’épissage alternatif. De plus, une protéine peut adopter plusieurs fonctions au courant de sa vie selon son état de modification post-traductionelle, sa conformation et son interaction avec d’autres protéines. La formation de complexes protéiques peut, en elle-même, être déterminée par l’état de modifications des protéines influencées par le contexte génétique, les compartiments subcellulaires, les conditions environmentales ou être intrinsèque à la croissance et la division cellulaire. Les complexes protéiques impliqués dans la régulation du cycle cellulaire sont particulièrement difficiles à disséquer car ils ne se forment qu’au cours de phases spécifiques du cycle cellulaire, ils sont fortement régulés par les modifications post-traductionnelles et peuvent se produire dans tous les compartiments subcellulaires. À ce jour, aucune méthode générale n’a été développée pour permettre une dissection fine de ces complexes macromoléculaires. L'objectif de cette thèse est d'établir et de démontrer une nouvelle stratégie pour disséquer les complexes protéines formés lors du cycle cellulaire de la levure Saccharomyces cerevisiae (S. cerevisiae). Dans cette thèse, je décris le développement et l'optimisation d'une stratégie simple de sélection basée sur un essai de complémentation de fragments protéiques en utilisant la cytosine déaminase de la levure comme sonde (PCA OyCD). En outre, je décris une série d'études de validation du PCA OyCD afin de l’utiliser pour disséquer les mécanismes d'activation des facteurs de transcription et des interactions protéine-protéines (IPPs) entre les régulateurs du cycle cellulaire. Une caractéristique clé du PCA OyCD est qu'il peut être utilisé pour détecter à la fois la formation et la dissociation des IPPs et émettre un signal détectable (la croissance des cellules) pour les deux types de sélections. J'ai appliqué le PCA OyCD pour disséquer les interactions entre SBF et MBF, deux facteurs de transcription clés régulant la transition de la phase G1 à la phase S. SBF et MBF sont deux facteurs de transcription hétérodimériques composés de deux sous-unités : une protéine qui peut lier directement l’ADN (Swi4 ou Mbp1, respectivement) et une protéine commune contenant un domain d’activation de la transcription appelée Swi6. J'ai appliqué le PCA OyCD afin de générer un mutant de Swi6 qui restreint ses activités transcriptionnelles à SBF, abolissant l’activité MBF. Nous avons isolé des souches portant des mutations dans le domaine C-terminal de Swi6, préalablement identifié comme responsable dans la formation de l’interaction avec Swi4 et Mbp1, et également important pour les activités de SBF et MBF. Nos résultats appuient un modèle où Swi6 subit un changement conformationnel lors de la liaison à Swi4 ou Mbp1. De plus, ce mutant de Swi6 a été utilisé pour disséquer le mécanisme de régulation de l’entrée de la cellule dans un nouveau cycle de division cellulaire appelé « START ». Nous avons constaté que le répresseur de SBF et MBF nommé Whi5 se lie directement au domaine C-terminal de Swi6. Finalement, j'ai appliqué le PCA OyCD afin de disséquer les complexes protéiques de la kinase cycline-dépendante de la levure nommé Cdk1. Cdk1 est la kinase essentielle qui régule la progression du cycle cellulaire et peut phosphoryler un grand nombre de substrats différents en s'associant à l'une des neuf protéines cycline régulatrice (Cln1-3, Clb1-6). Je décris une stratégie à haut débit, voir à une échelle génomique, visant à identifier les partenaires d'interaction de Cdk1 et d’y associer la cycline appropriée(s) requise(s) à l’observation d’une interaction en utilisant le PCA OyCD et des souches délétées pour chacune des cyclines. Mes résultats nous permettent d’identifier la phase(s) du cycle cellulaire où Cdk1 peut phosphoryler un substrat particulier et la fonction potentielle ou connue de Cdk1 pendant cette phase. Par exemple, nous avons identifié que l’interaction entre Cdk1 et la γ-tubuline (Tub4) est dépendante de Clb3. Ce résultat est conforme au rôle de Tub4 dans la nucléation et la croissance des faisceaux mitotiques émanant des centromères. Cette stratégie peut également être appliquée à l’étude d'autres IPPs qui sont contrôlées par des sous-unités régulatrices.
Proteins are the end-products of gene interpretative machinery. Proteins serve essential roles in defining the structure, integrity and dynamics of the cell and mediate most chemical transformations needed for everything from metabolic catalysis to signal transduction. We know that the central dogma of molecular biology, one gene = one mRNA = one protein is a gross simplification and that a protein may do different things depending on the form in which its mRNA was spliced, how and where it is post-translationally modified, what conformational state it may be in or finally, which other proteins it may interact with. Formation of protein complexes may, themselves, be governed by the states in which proteins are expressed in specific cells, cellular compartments or under specific conditions or dynamic phases such has growth or division. Protein complexes involved in mitotic cell cycle regulation are particularly challenging to dissect since they could only form during specific phases of the cell cycle, are highly regulated by post-translational modifications and can be found in any subcellular compartments. To date, no general methods have been developed to allow fine dissection of these protein complexes. The goal of this thesis was to establish and demonstrate a novel strategy for dissecting protein complexes regulating the budding yeast Saccharomyces cerevisiae (S. cerevisiae) mitotic cell cycle. In this thesis, I describe my development and optimization of a simple survival-selection Protein-fragment Complementation Assay using the prodrug-converting enzyme, yeast cytosine deaminase as reporter (OyCD PCA). I further describe, in a series of proof of principle studies, applications of the OyCD PCA to dissect the mechanism of transcriptional activation by key mitotic transcription factors and to dissect protein-protein interactions (PPIs) among regulators of the mitotic cell cycle. A key feature of the OyCD PCA is that it can be used to detect both formation and disruption of PPIs by virtue of having positive readouts for both assays. I applied the OyCD PCA in a strategy to dissect interactions between the key transcription factors of the G1/S phase: SBF and MBF. These two heterodimeric transcription factors are composed of, respectively, two distinct DNA-binding subunits named Swi4 and Mbp1 and a common transcription activation subunit called Swi6. I took advantage of the dual selection by OyCD PCA to engineer a specific mutant of Swi6 in order to demonstrate the rewiring of a transcriptional network. We isolated Swi6 with mutations found in its C-terminal domain previously identified for binding Swi4 and Mbp1 and important for SBF and MBF activities. Our results support a model where Swi6 undergoes a conformational change upon binding to Swi4 or Mbp1. In addition, this Swi6 mutant was used to dissect the regulatory mechanism that governs the entry of S. cerevisiae to a new round of cell division also known as START. We found that the SBF and MBF repressor Whi5 directly binds to the C-terminal domain of Swi6. Finally, I applied the OyCD PCA to dissect the yeast cyclin dependent kinase Cdk1-protein complexes. Cdk1 is the essential kinase that regulates cell cycle progression and can phosphorylate a large number of different substrates by teaming up with one of nine cyclin regulatory proteins (Cln1-3, Clb1-6). I describe a strategy to identify interaction partners of Cdk1 that can easily be scaled up for a genome-wide screen and associate the complexes with the appropriate cyclin(s) required for mediating the interaction using the OyCD PCA and deletion of the cyclin genes. My results allow us to postulate which phase(s) of the mitotic cell cycle Cdk1 may phosphorylate proteins and what function potential or known substrates of Cdk1 may take on during that phase(s). For example, we identified the interaction between Cdk1 and the γ-tubulin (Tub4) to be dependent upon Clb3, consistent with its role in mediating nucleation and growth of mitotic microtubule bundles on the spindle pole body. This strategy can also be applied to study other PPIs that are contingent upon accessory subunits.