In somatic cells, checkpoint pathways trigger cell cycle arrest in response to unreplicated or damaged DNA by inhibiting the activity of cyclin-dependent kinases (Cdks). In the Xenopus laevis embryo, checkpoints are not operational until the midblastula transition (MBT). Studies in cell-free egg extracts indicate that a threshold concentration of nuclei, which approximates the MBT concentration, is required to elicit a checkpoint. The checkpoint response to unreplicated DNA in the extract prevents transition into mitosis by inhibiting Cdk1/cyclin B, causing an increase in the minimum amount of cyclin B necessary to enter mitosis, termed the cyclin threshold. Once the threshold of cyclin is maintained or exceeded, the system will proceed into mitosis after a lag time. We have investigated the relationship between nuclear concentration and cell cycle regulation in the extract. By precisely regulating the concentration of cyclin B and nuclear content in extract samples, we have found 1) the concentration of nuclei affects cyclin B thresholds and lag time of entry into mitosis, 2) elevated cyclin thresholds caused by DNA replication blocks are further increased by increasing the concentration of nuclei, and 3) double-stranded DNA breaks in the extract system do not affect cyclin thresholds or lag time of entry into mitosis within the range of nuclear concentrations that can be efficiently replicated. This data provides evidence of the importance of the nucleocytoplasmic ratio in normal cell cycle progression and its importance for checkpoint acquisition during early Xenopus laevis development. / Master of Science

Das Prostatakarzinom (PCa) ist die am häufigsten diagnostizierte Krebsneuerkrankung bei Männern in Deutschland sowie die dritthäufigste Ursache krebsbedingter Sterbefälle. Die Therapiemöglichkeiten für das PCa sind sehr beschränkt und mit erheblichen Nebenwirkungen verbunden. Daher wurden in dieser Arbeit neue Therapieansätze verfolgt: zum einen wurde das Phytoöstrogen Tectorigenin (TG) und zum anderen der Histon-Deacetylase-Inhibitor (HDI) Valproinsäure (VPA) auf die jeweilige Wirksamkeit gegen das PCa untersucht. Das Phytoöstrogen Tectorigenin wird aus dem Gesamtextrakt von Belamcanda chinensis-Rhizomen isoliert. Dieser Gesamtextrakt wurde zunächst in PCa-Zellen in vitro getestet. Sowohl in den humanen PCa-Zellen LNCaP als auch in den murinen PCa-Zellen 2E, die aus dem Prostatatumor einer Maus des TRAMP-(transgenic adenocarcinoma of mouse prostate) Mausmodells neu etabliert wurden, konnte eine Inhibition der Zellproliferation sowie eine Veränderung der Expression ausgewählter PCa-relevanter Gene detektiert werden. Anschließend wurde eine Microarray-Analyse durchgeführt, bei der einige differentiell exprimierte Gene nach der Behandlung der humanen LNCaP-Zellen mit Tectorigenin identifiziert werden konnten. Neben einer Reihe von Androgen-induzierten Genen traten u.a. der IGF-IR (insulin-like growth factor I receptor) und PSA (prostate specific antigen) auf. In vivo-Studien in TRAMP-Mäusen konnten zudem eine langsamere Progression des PCa nach Behandlung mit Tectorigenin zeigen. In den in vivo-Studien an subkutanen LNCaP-Tumoren im Nacktmaus-Modell konnte sowohl ein vermindertes Tumorgewicht als auch eine verminderte Tumorgröße nach der Behandlung der Mäuse mit Tectorigenin beobachtet werden. Im zweiten Teil dieser Arbeit wurde die Wirkung des HDI VPA auf PCa-Zellen untersucht. Funktionell konnte eine erhöhte Acetylierung des Histons 3 an Lysin 9 sowie eine Verringerung der Zellproliferation, -migration und -invasion nach der Behandlung der primären PCa-Zellen 2E mit VPA gezeigt werden. Molekular konnten einige differentielle Genexpressionen nach der Behandlung der 2E-Zellen mit VPA detektiert werden. Es konnte z.B. eine Abnahme der Androgenrezeptor-Expression, eine Zunahme der Expression des proapoptotischen Proteins Bim sowie eine Verringerung der Expression von Foxo1 und Gsk3β gezeigt werden. Weiterhin wurde eine Deregulation von Zellzyklus- und Angiogenese-relevanten Genen wie z.B. Cdkn1a, Cdk4, Vegfa und Hif1α beobachtet. Des Weiteren wurden Kandidatengene aus einer früheren Microarray-Analyse untersucht. Für alle sieben untersuchten Kandidatengene wurde die differentielle Genexpression nach der Behandlung der 2E-Zellen mit VPA sowohl konzentrations- als auch zeitabhängig mittels quantitativer real time PCR bestätigt. Für die verstärkt exprimierten Gene konnte ein Zusammenhang mit einer erhöhten Acetylierung des Promotorbereichs der jeweiligen Gene nach VPA-Behandlung dargestellt werden. Das Kandidatengen Cyclin D2 wurde für weiterführende Untersuchungen ausgewählt. Als einziges Mitglied der D-Typ-Cycline lag Cyclin D2 nach VPA-Behandlung verstärkt exprimiert vor. Diese Re-Expression konnte auch durch die Behandlung mit HDIs verschiedener Wirkstoffklassen hervorgerufen werden. Die Untersuchung der PCa-Zelllinien PC-3, DU145 und LNCaP nach VPA-Behandlung zeigte ebenfalls, wie bei den primären PCa-Zellen 2E, eine Re-Expression von Cyclin D2 sowie eine Inhibition der Proliferation. Nicht-maligne Zelllinien, die bereits eine hohe Basalexpression von Cyclin D2 aufweisen, veränderten durch VPA die Cyclin D2-Expression nicht und auch der Effekt auf die Zellproliferation war nur moderat vorhanden. Die Verringerung der Cyclin D2-Expression mittels siRNA in den murinen Fibroblastenzellen NIH/3T3 führte zu einem Anstieg der Zellproliferation. Dass Cyclin D2 im PCa einen Tumorsuppresor darstellt, zeigt auch die immunhistochemische Analyse von humanen PCa-Gewebeschnitten, bei der keine Expression von Cyclin D2 im PCa-Gewebe nachgewiesen werden konnte. In gesundem Prostatagewebe hingegen wurde Cyclin D2 in den proliferierenden Zellen exprimiert. Im Gegensatz dazu wurde Cyclin D1 im PCa-Gewebe stärker exprimiert als im gesunden Prostatagewebe. Schließlich wurden in vivo-Studien an TRAMP-Mäusen mit VPA-Gabe über das Trinkwasser durchgeführt. Es wurden drei verschiedene Versuchsgruppen untersucht. In der Überlebensstudie, welche VPA präventiv ab einem Alter von sechs Wochen erhielt, konnte ein höheres Überlebensalter, ein geringerer Tumoranteil sowie ein geringeres Tumorgewicht verzeichnet werden. Nach präventiver Gabe von VPA ab einem Alter von sechs Wochen bis zu einem Alter von 30 Wochen konnte kein Unterschied zwischen der VPA- und der Kontrollgruppe detektiert werden. In der Überlebensstudie nach therapeutischer Gabe von VPA ab einem Alter von 16 Wochen konnte ein höheres Überlebensalter und ein geringerer Tumoranteil, jedoch kein Unterschied im Tumorgewicht gezeigt werden.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2007. / Typescript. Abstract available online via ProQuest Digital Dissertations. Includes bibliographical references (leaves 207-223).

INTRODUÇÃO: O melanoma acral lentiginoso (MAL) tem freqüência expressiva entre os casos de melanoma observados no nosso meio e difere dos outros tipos clinicopatológicos de melanoma cutâneos por não ter a exposição solar como fator predisponente. Poucos trabalhos da literatura enfocam as alterações dos genes supressores de tumores e a expressão de suas proteínas nas lesões de MAL. Por esses motivos propusemo-nos a realizar um estudo retrospectivo visando uma melhor compreensão das proteínas envolvidas na via p16 INK4a /ciclina D1/CDK4/pRb do ciclo celular, no MAL em casuística brasileira. MÉTODOS: Através de técnica de imunoistoquímica foi demonstrada a expressão das proteínas p16, ciclina D1, CDK4 e pRb em 32 lesões de MAL. Comparou-se a freqüência de expressão desses marcadores nos tumores delgados (espessura de até 1,0mm) e espessos (mais de 1,0 mm de espessura), assim como de acordo com a presença ou ausência de ulceração. RESULTADOS: Houve expressão de p16 em 78% das lesões, de ciclina D1 em 61,5%, CDK4 em 84% e pRb em 85% dos MAL analisados. O padrão de imunomarcação das células neoplásicas foi nuclear para todos os anticorpos. Expressão nuclear associada à citoplasmática foi observada em 76% dos tumores positivos para p16 e 81% dos casos positivos para CDK4. Não houve diferenças significativas entre as freqüências de expressão de cada um dos marcadores quando comparados de acordo com sua espessura (delgados e espessos) e presença de ulceração. CONCLUSÕES: A expressão de ciclina D1 e CDK4, nos melanomas acrais lentiginosos, pode ser considerada aberrante e reflexo de provável alteração na via supressora de tumores p16/ciclinaD1/CDK4/pRb. A expressão tecidual de p16 e pRb, demonstrada pela técnica imunoistoquímica, pode não refletir as prováveis alterações da via supressora de tumores estudada. Esses marcadores, isoladamente, não devem ser considerados como fatores prognósticos no melanoma acral lentiginoso / INTRODUCTION: Acral lentiginous melanoma (ALM) has an expressive frequency between melanoma cases in our country, and it differs from the others clinical pathological types of cutaneous melanoma, because it does not have sun exposure as a predisposing factor. There are few publications in the literature addressing the expression of tumor suppressor pathway proteins in ALM. For these reasons we proposed to carry out a retrospective study aiming at a better understanding of p16 INK4a /cyclin D1/CDK4/pRb pathway involvement in ALM lesions. METHODS: Expression of p16, cyclin D1, CDK4 and pRB in 32 ALM lesions was displayed by immunohistochemistry. We compared the frequency of the expression of these markers in thin (thickness up to 1.0 mm) and thick lesions (thickness above 1.0 mm), as well as in accordance with the presence or absence of ulceration. RESULTS: 76% of the tumors were positive to p16 and 81% of the cases were positive to CDK4. There was no significant difference between the frequency of the expression of each marker when comparing in accordance with the thickness (thin and thick lesions) and the presence of ulceration. CONCLUSIONS: The expression of cyclin D1 and CDK4 in acral lentiginous melanoma may be considered aberrant and as reflex of a probable alteration in the p16/cyclin D1/CDK4/pRb tumor suppressor pathway. p16 and pRb tumor expression displayed by immunohistochemistry may not reflect probable alterations of the tumor suppressor pathway studied. The expression of these proteins, per se, may not be considered as prognostic factor in acral lentiginous melanoma

INTRODUÇÃO: O melanoma acral lentiginoso (MAL) tem freqüência expressiva entre os casos de melanoma observados no nosso meio e difere dos outros tipos clinicopatológicos de melanoma cutâneos por não ter a exposição solar como fator predisponente. Poucos trabalhos da literatura enfocam as alterações dos genes supressores de tumores e a expressão de suas proteínas nas lesões de MAL. Por esses motivos propusemo-nos a realizar um estudo retrospectivo visando uma melhor compreensão das proteínas envolvidas na via p16 INK4a /ciclina D1/CDK4/pRb do ciclo celular, no MAL em casuística brasileira. MÉTODOS: Através de técnica de imunoistoquímica foi demonstrada a expressão das proteínas p16, ciclina D1, CDK4 e pRb em 32 lesões de MAL. Comparou-se a freqüência de expressão desses marcadores nos tumores delgados (espessura de até 1,0mm) e espessos (mais de 1,0 mm de espessura), assim como de acordo com a presença ou ausência de ulceração. RESULTADOS: Houve expressão de p16 em 78% das lesões, de ciclina D1 em 61,5%, CDK4 em 84% e pRb em 85% dos MAL analisados. O padrão de imunomarcação das células neoplásicas foi nuclear para todos os anticorpos. Expressão nuclear associada à citoplasmática foi observada em 76% dos tumores positivos para p16 e 81% dos casos positivos para CDK4. Não houve diferenças significativas entre as freqüências de expressão de cada um dos marcadores quando comparados de acordo com sua espessura (delgados e espessos) e presença de ulceração. CONCLUSÕES: A expressão de ciclina D1 e CDK4, nos melanomas acrais lentiginosos, pode ser considerada aberrante e reflexo de provável alteração na via supressora de tumores p16/ciclinaD1/CDK4/pRb. A expressão tecidual de p16 e pRb, demonstrada pela técnica imunoistoquímica, pode não refletir as prováveis alterações da via supressora de tumores estudada. Esses marcadores, isoladamente, não devem ser considerados como fatores prognósticos no melanoma acral lentiginoso / INTRODUCTION: Acral lentiginous melanoma (ALM) has an expressive frequency between melanoma cases in our country, and it differs from the others clinical pathological types of cutaneous melanoma, because it does not have sun exposure as a predisposing factor. There are few publications in the literature addressing the expression of tumor suppressor pathway proteins in ALM. For these reasons we proposed to carry out a retrospective study aiming at a better understanding of p16 INK4a /cyclin D1/CDK4/pRb pathway involvement in ALM lesions. METHODS: Expression of p16, cyclin D1, CDK4 and pRB in 32 ALM lesions was displayed by immunohistochemistry. We compared the frequency of the expression of these markers in thin (thickness up to 1.0 mm) and thick lesions (thickness above 1.0 mm), as well as in accordance with the presence or absence of ulceration. RESULTS: 76% of the tumors were positive to p16 and 81% of the cases were positive to CDK4. There was no significant difference between the frequency of the expression of each marker when comparing in accordance with the thickness (thin and thick lesions) and the presence of ulceration. CONCLUSIONS: The expression of cyclin D1 and CDK4 in acral lentiginous melanoma may be considered aberrant and as reflex of a probable alteration in the p16/cyclin D1/CDK4/pRb tumor suppressor pathway. p16 and pRb tumor expression displayed by immunohistochemistry may not reflect probable alterations of the tumor suppressor pathway studied. The expression of these proteins, per se, may not be considered as prognostic factor in acral lentiginous melanoma

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

Previous studies suggest that the IGF-I stimulates the elastin gene transcription through the unique responsive sequence on the elastin promoter, which is a putative retinoblastoma control element (RCE). This site interacts with (Sp)-family transcription factors whose delivery is mediated by the retinoblastoma protein (Rb). Since Rb (phosphorylated on serine 780) has been implicated in the initiation of the cell cycle, we speculated that a different phosphorylation of Rb might determine Rb involvement in elastogenesis. Obtained results demonstrated that, IGF-I-induced elastogenic signaling pathway in human dermal fibroblasts includes activation of cyclinE/cdk2 causing a site specific phosphorylation of Rb on threonine 821. This permits the sequestration of Sp1 by Rb before it could bind the elastin promoter, thereby allowing the elastin gene transcription. We also found that blocking of H-Ras in Costello syndrome fibroblasts (characterized by heightened proliferation and impaired elastogenesis), selectively down-regulated Rb phosphorylation on serine 780 and normalized impaired elastogenesis.

Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. The goal of my dissertation research is to identify the MEK kinase responsible for activation of the MAPK cascade during mitosis and to elucidate the biochemical mechanisms that regulate its activity. In the described herein work I purified and characterized the MEK kinase activity present in M-phase arrested Xenopus egg extracts. I demonstrate that B-Raf is the critical MEK kinase required for activation of the MAPK pathway at mitosis. Consistent with this, I show that B-Raf is activated in an M-phase dependent manner. Further, I provide data linking Cdk1/cyclin B to mitotic activation of B-Raf. Cdk1/cyclin B associates with and phosphorylates B-Raf in M-phase arrested extracts and directly targets Xenopus B-Raf in vitro at a conserved Ser-144 residue. Phosphorylation at Ser-144 is critical for M-phase dependent activation of B-Raf and for B-Raf's ability to trigger activation of the MAPK cascade at mitosis. Finally, I demonstrate that mitotic B-Raf undergoes feedback phosphorylation by MAPK at its conserved C-terminal SPKTP motif. Mutation of both phosphorylation sites within the SPKTP sequence to alanines increases activity of mitotic B-Raf. Further, inhibition or over-activation of MAPK during mitosis enhances or diminishes B-Raf activity, respectively. These results indicate that MAPK-mediated feedback phosphorylation negatively regulates B-Raf activity. Additionally, I show that active mitotic B-Raf exists in large multi-protein complex(s). By utilizing a proteomics approach I identify a set of proteins, which potentially associate with B-Raf at M-phase. Future studies are necessary to elucidate the involvement of these proteins in regulating B-Raf mitotic functions. In summary, my dissertation studies demonstrate that B-Raf activates MAPK signaling at mitosis and undergoes an M-phase dependent regulation. I propose that B-Raf has important functions at mitosis that contributes to its overall role in promoting cell proliferation.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 144-170). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Cette thèse a pour objectif l’identification de nouveaux inhibiteurs de kinase et plus particulièrement de kinases dépendantes de cyclines (CDKs). Des inhibiteurs de CDKs sont en essais cliniques depuis une dizaine d’année. Un faisceau d’informations récentes montre que cette nouvelle classe pharmacologique pourrait prochainement occuper une place prépondérante dans la thérapie antitumorale. L’introduction de cette thèse décrit les principaux inhibiteurs de CDKs en se focalisant sur ceux dont le développement clinique est en cours et sur les structures les plus récemment divulguées (2009 à 2013). Trois molécules avancées en études cliniques s’avèrent intéressantes et s’approchent de la mise sur le marché : la Roscovitine (phase clinique IIb), le Dinaciclib (phase clinique III) et le Palbociclib (phase clinique III). D’un point de vue expérimental, cette thèse se décompose en deux parties principales. La première modulation a consisté à rechercher des nouveaux groupements qui pourraient sur des squelettes déjà connus comme les purines apporter un avantage en ce qui concerne l’activité des produits. Les structures cristallines des complexes inhibiteur-enzymes et notamment celles de Roscovitine-CDK2 et CR8-CDK2 ont guidé la conception des nouvelles molécules. C’est ainsi que sur les structures biaryliques déjà connues, un groupement phénol a été greffé sur l’un des cycles conduisant ainsi à de nouveaux inhibiteurs de kinases. Ces molécules sont plus puissantes que les produits non hydroxylés. L’augmentation de l’activité est particulièrement sensible au niveau de la kinase CDK2 qui est impliquée notamment dans régulation du cycle cellulaire. La seconde partie du travail porte sur la recherche de structures isostères des purines. Ainsi, le squelette thieno[3,2-d]pyrimidines a été développé de novo. Deux types d’intermédiaires produits ont été préparés afin de permettre la diversification des structures. En premier temps la voie de synthèse via l’intermédiaire thiométhyle a été conduite, néanmois cette voie présente certaines limites. Le deuxième intermédiaire trihalogéné a permi d’optimiser la préparation des molécules thieno[3,2-d]pyrimidines. Les évaluations des produits préparés ne sont pas totalement terminées. Ces molécules sont moins puissantes que les purines sur les CDKs mais agissent au niveau d’autres kinases. / In the introduction, the main functions of cyclin dependent kinases are detailed. Whenever it was possible the link with pathologies where these kinases are overexpressed is presented. This is followed by the description of the inhibitors which are actually undergoing clinical testing. Most of these clinical studies are targeting cancer and leukemia. Impressive clinical results have been disclosed for Dinaciclib, Palbociclib and Roscovitine. The synthesis of two series of compounds is then envisioned. The first series of products are purine derivatives bearing a hydroxybiarylmethyl group on the 6 position of the purine scaffold.  Two approaches were compared in the synthesis of the hydroxylbiarylmethylamino group. In both approaches the key step was the orthoformylation of phenols using magnesium chloride as catalyst. The prepared compounds were evaluated against kinases and a tumor cell line. They were found more potent than homologous products without hydroxyls. The second families of products are thieno[3,2-d]pyrimidines. A new general route to these products based on the preparation of 7-bromo-2,4-dichloro-thieno[3,2-d]pyrimidine which can allow the synthesis of a large diversity of trisubstituted derivatives.