Short linear motifs (SLM) play critical roles in cell signaling and are associated with important biochemical events such as phosphorylation, glycosylation, and other post translational modifications. The primary aim of this thesis is to develop a new computational method (“ConDens”) to predict kinase substrates by assessing the evolution of phosphorylation SLM’s in a novel manner. This method could predict yeast Cdc28 kinase substrates that were not confidently detected by several other prediction methods published in literature and was demonstrated to be generalizable to other kinases. Genome-wide predictions experiments with this method also revealed potentially interesting novel substrates of Mec1, Prk1, PKA, and CKII.

Short linear motifs (SLM) play critical roles in cell signaling and are associated with important biochemical events such as phosphorylation, glycosylation, and other post translational modifications. The primary aim of this thesis is to develop a new computational method (“ConDens”) to predict kinase substrates by assessing the evolution of phosphorylation SLM’s in a novel manner. This method could predict yeast Cdc28 kinase substrates that were not confidently detected by several other prediction methods published in literature and was demonstrated to be generalizable to other kinases. Genome-wide predictions experiments with this method also revealed potentially interesting novel substrates of Mec1, Prk1, PKA, and CKII.

O Glioblastoma é um dos tumores mais agressivos do sistema nervoso central e entre as diversas neoplasias possui um dos piores prognósticos. Mesmo com as novas estratégias de tratamento, a sobrevida de pacientes portadores de glioblastoma continua sendo muito baixa, sendo a temozolomida (TMZ) o agente mais comum usado no seu tratamento. O ácido tetra-o-metil nordihidroguaiarético (M4N), é um novo agente terapêutico que funciona como um repressor transcricional global de genes dependentes do fator de transcrição Sp1, tais como Survivina e Cdk1. No presente estudo, foram investigados os níveis de expressão do gene Survivina, suas variantes gênicas por splicing alternativo e Cdk1 em amostras tumorais e linhagens celulares de GBM. Adicionalmente, foram investigados os efeitos do M4N em combinação ou não com TMZ e/ou radiação em culturas primárias e linhagens celulares de GBM. Ensaios de qRT-PCR foram realizados para determiner a expressão de mRNA das variantes gênicas de Survivina e Cdk1. A proliferação celular foi analisada pelo ensaio XTT e os niveis de apoptose e variações do ciclo celular foram determinados por citometría de fluxo. Analises de combinação de drogas utilizando diferentes estratégias de administração (simultânea e seqüencial) foram realizados baseados no método de Chou-Talalay em linhagens celulares e culturas primárias de GBM. Para os ensaios de sobrevivência clonogênica, foram utilizadas as doses de 2, 4 e 6 Gy de radiação gamma. Todas as variantes por splicing alternativo de Survivina e o gene Cdk1 foram expressos em amostras (n=16) e linhagens celulares (n=6) de GBM, exceto a variante Survivina-2B que apenas foi expressa nas linhagens celulares de GBM. O tratamento com M4N diminuiu a expressão de Cdk1, Survivina e a variante Survivina-Ex3, enquanto que houve um aumento da expressão da variante Survivina-2B. O M4N diminuiu a proliferação celular de forma isolada e sinérgicamente quando combinada com TMZ. Além disso, o M4N aumentou os efeitos da radiação, principalmente quando associado com TMZ. O M4N causou morte celular apoptótica, diminuição do índice mitótico e parada do ciclo celular principalmente na fase x G2/M. Os resultados do presente estudo sugerem a potencial aplicação clínica de M4N em combinação com TMZ e radiação no tratamento do GBM. / Glioblastoma (GBM), one of the most human malignant neoplasia, responds poorly to current treatment modalities, being temozolomide (TMZ) the most used drug in its treatment. TetraO-methyl Nordihydroguaiaretic Acid (M4N) is a global transcriptional repressor of genes dependents of Sp1 transcription factor, such as Survivin and Cdk1. In this study was evaluated the gene expression of Survivin, their spliced-variants and Cdk1 in GBM samples and cell lines. Moreover, it was investigated the effects of M4N combined or not with TMZ and/or radiation on primary cultures and cell lines of GBM. qRT-PCR assays were performed to determine the Survivin-spliced variants and Cdk1 gene mRNA expression in GBM tumor samples and cell lines. Cell proliferation was measured by XTT assay and cell cycle and apoptosis were determined by flow cytometry. Drug combination analyzes using different schedules of administration (simultaneous and sequential) were performed based in ChouTalalay method on GBM cell lines and primary cultures. For clonogenic survival, it was used the doses of 2, 4, and 6 Gy of gamma radiation. All Survivin-spliced variants and Cdk1 gene were expressed in GBM samples (n=16) and cell lines (n=6), except the Survivin-2B variant that was only expressed in GBM cell lines. M4N treatment down regulated the expression of Cdk1, Survivin and Survivin-Ex3 variant, while the Survivin-2B variant was up-regulated. M4N decreased the cell proliferation separately and synergistically with TMZ, moreover it enhanced the radiation effects, mainly when associated with TMZ. M4N also induced apoptotic cell death, decreased mitotic index and arrested the cell cycle mainly in G2/M phase. Our results suggest a potential clinical application of M4N in combination with TMZ and radiation in GB treatment.

O Glioblastoma é um dos tumores mais agressivos do sistema nervoso central e entre as diversas neoplasias possui um dos piores prognósticos. Mesmo com as novas estratégias de tratamento, a sobrevida de pacientes portadores de glioblastoma continua sendo muito baixa, sendo a temozolomida (TMZ) o agente mais comum usado no seu tratamento. O ácido tetra-o-metil nordihidroguaiarético (M4N), é um novo agente terapêutico que funciona como um repressor transcricional global de genes dependentes do fator de transcrição Sp1, tais como Survivina e Cdk1. No presente estudo, foram investigados os níveis de expressão do gene Survivina, suas variantes gênicas por splicing alternativo e Cdk1 em amostras tumorais e linhagens celulares de GBM. Adicionalmente, foram investigados os efeitos do M4N em combinação ou não com TMZ e/ou radiação em culturas primárias e linhagens celulares de GBM. Ensaios de qRT-PCR foram realizados para determiner a expressão de mRNA das variantes gênicas de Survivina e Cdk1. A proliferação celular foi analisada pelo ensaio XTT e os niveis de apoptose e variações do ciclo celular foram determinados por citometría de fluxo. Analises de combinação de drogas utilizando diferentes estratégias de administração (simultânea e seqüencial) foram realizados baseados no método de Chou-Talalay em linhagens celulares e culturas primárias de GBM. Para os ensaios de sobrevivência clonogênica, foram utilizadas as doses de 2, 4 e 6 Gy de radiação gamma. Todas as variantes por splicing alternativo de Survivina e o gene Cdk1 foram expressos em amostras (n=16) e linhagens celulares (n=6) de GBM, exceto a variante Survivina-2B que apenas foi expressa nas linhagens celulares de GBM. O tratamento com M4N diminuiu a expressão de Cdk1, Survivina e a variante Survivina-Ex3, enquanto que houve um aumento da expressão da variante Survivina-2B. O M4N diminuiu a proliferação celular de forma isolada e sinérgicamente quando combinada com TMZ. Além disso, o M4N aumentou os efeitos da radiação, principalmente quando associado com TMZ. O M4N causou morte celular apoptótica, diminuição do índice mitótico e parada do ciclo celular principalmente na fase x G2/M. Os resultados do presente estudo sugerem a potencial aplicação clínica de M4N em combinação com TMZ e radiação no tratamento do GBM. / Glioblastoma (GBM), one of the most human malignant neoplasia, responds poorly to current treatment modalities, being temozolomide (TMZ) the most used drug in its treatment. TetraO-methyl Nordihydroguaiaretic Acid (M4N) is a global transcriptional repressor of genes dependents of Sp1 transcription factor, such as Survivin and Cdk1. In this study was evaluated the gene expression of Survivin, their spliced-variants and Cdk1 in GBM samples and cell lines. Moreover, it was investigated the effects of M4N combined or not with TMZ and/or radiation on primary cultures and cell lines of GBM. qRT-PCR assays were performed to determine the Survivin-spliced variants and Cdk1 gene mRNA expression in GBM tumor samples and cell lines. Cell proliferation was measured by XTT assay and cell cycle and apoptosis were determined by flow cytometry. Drug combination analyzes using different schedules of administration (simultaneous and sequential) were performed based in ChouTalalay method on GBM cell lines and primary cultures. For clonogenic survival, it was used the doses of 2, 4, and 6 Gy of gamma radiation. All Survivin-spliced variants and Cdk1 gene were expressed in GBM samples (n=16) and cell lines (n=6), except the Survivin-2B variant that was only expressed in GBM cell lines. M4N treatment down regulated the expression of Cdk1, Survivin and Survivin-Ex3 variant, while the Survivin-2B variant was up-regulated. M4N decreased the cell proliferation separately and synergistically with TMZ, moreover it enhanced the radiation effects, mainly when associated with TMZ. M4N also induced apoptotic cell death, decreased mitotic index and arrested the cell cycle mainly in G2/M phase. Our results suggest a potential clinical application of M4N in combination with TMZ and radiation in GB treatment.

The mitotic cell cycle is fundamental to eukaryotic life. In mitosis, replicated chromosomes are segregated to form two new nuclei. This is essential to ensure the maintenance of chromosome number between parent and daughter cells. In higher eukaryotes, numerous cytological changes occur to facilitate the separation of the genetic material: the nuclear envelope breaks down, the mitotic spindle assembles, and the cell rounds-up. There is a well-conserved control network that regulates these processes to bring about the entry into mitosis, the separation of the genetic material, and the reversal of these processes during mitotic exit. To build a coherent model of these regulatory networks requires us to write the biochemical reactions in mathematical form. The work in this Thesis pertains to three fundamental switches: entry into mitosis, the metaphase-to-anaphase transition, and exit from mitosis. I present three studies from a systems-level perspective. The first investigates a novel bistable mechanism controlling mitotic entry/exit in vitro using purified proteins. Dephosphorylation of Greatwall kinase by the phosphatase PP2A-B55 creates a double negative feedback loop that gives a bistable system response with respect to cyclin-dependent kinase 1 (Cdk1) activity. The second looks at hysteresis between mitotic entry and mitotic exit in HeLa cells. Hysteresis persists when either of the regulatory loops of Cdk1 or its counter-acting phosphatase PP2A-B55 is removed, but is diminished when they are both removed. Finally, the regulation of separase in the metaphase-to-anaphase transition is analysed. Separase that is liberated from securin inhibition is isomerised by Pin1 into a conformation that can bind to cyclin B1. This binding peaks after separase has cleaved cohesin and initiated anaphase.

Tyrosine kinases regulate fundamental signal pathways in cells including cell proliferation, motility, and differentiation. The kinase activity is tightly controlled in normal cells but is usually excessive activated in cancers. Several tyrosine kinase inhibitors are used in cancer therapies nowadays. Our novel tyrosine kinase inhibitor, 1J-309, is a multiple kinase inhibitor that targets several receptors including vascular endothelial growth factor receptors (VEGFRs). We find 1J-309 dramatically reduces cell proliferation of VEGFR3+/VEGF-C+ A549 human lung cancer cells by decreasing the expression of CDK1 and cyclin B1 following growth arrest at G2/M phase. After long term drug treatment, 1J-309 causes cell death. Moreover, 1J-309 represses CDK1 expression at early stage but it does not change CDK1 RNA expression and protein stability. Additionally, 1J-309 significantly decreases the migration ability of A549 cells. 1J-309 also reduces gelatin-related invasion potency. The AKT and p38 MAPK activity are significantly repressed by 1J-309 and it dramatically drives the expression of tumor suppressor, p53, at low-dose treatment. Our results demonstrate that 1J-309 significantly attenuates cell proliferation by inducing G2/M growth arrest, reduces the invasion and migration potency, and promotes a dramatic increase of p53 in A549 cells.

Λόγω της ιδιότητάς τους να ελέγχουν τον κυτταρικό κύκλο οι CDKs έχουν προταθεί ως μοριακοί στόχοι για την ανάπτυξη αντινεοπλασματικών φαρμάκων. Σε πολλούς τύπους καρκίνου έχει παρατηρηθεί απορρύθμιση της λειτουργίας ή μεταλλάξεις στις CDKs. Μεταξύ των CDKs, η CDK1 διαδραματίζει ιδιαίτερο ρόλο μιας και είναι επαρκής για να οδηγήσει τον κυτταρικό κύκλο. Πρόσφατα η ερευνητική μας ομάδα συνέθεσε την ένωση FM-100 (2-(αιθοξυκαρβονυλο)-9-χλωρο-πυρρολο[2,3-a]καρβαζόλιο) με εκλεκτική δράση στη CDK1 (IC50 15 μΜ για το σύμπλοκο CDK1/κυκλίνη B). Στην παρούσα μελέτη έγινε αποτίμηση της βιολογικής δραστικότητας νέων πυρρολοκαρβαζολικών ενώσεων με δομή παραπλήσια του FM-100 με σκοπό την ανακάλυψη ισχυρότερων αναστολέων και ελέχθηκε η δραστικότητά τους σε καρκινικές κυτταρικές σειρές in vitro. Από τα ανάλογα που ελέγχθηκαν δύο, τα SS-261Β και SS263, εμφάνισαν αυξημένη ικανότητα αναστολής της CDK1 σε σχέση με την αρχική ένωση FM-100. Η ισχυρότερη από αυτές (SS-261Β, IC50 194nΜ) μελετήθηκε σε πέντε καρκινικές σειρές. Η SS-261Β δεν είχε καμία επίδραση στο πολλαπλασιασμό λευχαιμικών κυττάρων (Κ562), ενώ στα MCF-7 (κύτταρα καρκίνου του μαστού) η SS-261Β είχε ανασταλτική δράση στον πολλαπλασιασμό η οποία ήταν εμφανής μόνο στη μεγαλύτερη συγκέντρωση που χρησιμοποιήθηκε ο αναστολέας (10μΜ). Στη σειρά HeLa (καρκινικά κύτταρα τραχήλου μήτρας) και CaCo2 (καρκινικά κύτταρα γαστρενετρικού) η SS-261Β προκάλεσε μικρότερη αναστολή (25-30%). Μεγαλύτερη αντιμιτογόνο δράση παρουσίαση η ένωση στα PC-3 (προστατικά καρκινικά κύτταρα). Αξίζει να σημειωθεί ότι η ένωση SS-261Β είχε περιορισμένη δράση σε φυσιολογικά προστατικά επιθηλιακά κύτταρα. Παράλληλα, στα PC-3 η SS-261Β προκάλεσε μια δοσοεξαρτώμενη αύξηση της ενεργότητας της κασπάσης-3 σε συγκεντρώσεις μεγαλύτερες του 10μΜ, ενδεικτική της ικανότητάς της να προάγει την απόπτωση σε αυτά τα κύτταρα. Κάτι τέτοιο δεν παρατηρήθηκε στα HeLa. Συμπερασματικά, η ένωση SS-261Β έχει βελτιωμένες ιδιότητες σε σχέση με την FM-100, παρουσιάζει ανασταλτική δράση στο πολλαπλασιασμό καρκινικών κυττάρων, ενώ ταυτόχρονα διεγήρει την απόπτωση σε καρκινικά κύτταρα του προστάτη in vitro. / Cyclin-dependent kinases (CDKs) have been proposed as molecular targets for anti-tumour drug development due to their ability to regulate cell cycle. In many types of cancers, deregulated expression of CDKs or mutations in CDKs have been reported. Among CDKs, CDK1 is unique, as activation of this kinase is sufficient to drive cell proliferation. Recently, our research group synthesized FM-100 (ethyl 9-chloro-1H-pyrrolo[2,3-alpha]carbazole-2-carboxylate), a compound with selective inhibitory action on CDK1 (IC50 15 μΜ for the CDK1/cyclin B complex). In the present study we evaluated the biological activity of several new pyrrolocarbazole derivatives with structures similar to that of FM-100, in order to identify more potent CDK1 inhibitors. In addition, we tested the ability of the newly synthesized FM-100 analogues to inhibit cell proliferation and survival in cancer cell lines in vitro. Only two of the tested compounds, SS-261Β and SS263, exhibited a greater inhibitory action on CDK1 activity compared to the parent compound (FM-100). The most potent of the two SS-261Β had an IC50 of 194nΜ and was further studied in cell-based assays. SS-261Β had no effect on the proliferation of leukemic cells (Κ562), while SS-261B inhibited MCF-7 (breast cancer cell line) growth only at the highest concentration used (10μΜ). In HeLa (cervical cancer cell line) and CaCo2 cells (colon cancer cell line) SS-261Β had only a modest effect, reducing proliferation by 25-30%. SS-261B had a more pronounced effect on PC-3 cells (prostate cancer cell line). It should be noted that SS-261Β also exhibited anti-mitogenic effects in normal prostate epithelial cells. In a different series of experiments we observed that SS-261B dose-dependently increased caspace-3 activity in PC-3 cells when used at concentrations over 10μΜ, suggesting that it is capable of promoting apoptosis in this cell line. In summary, SS-261Β exhibits an improved profile for CDK1 inhibition compared to FM-100, exerts anti-proliferative effects in several tumour cell lines while it also inhibits survival of prostate cancer cells in vitro.

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.

Protein phosphorylation is an essential post-translational modification used in cells for regulating multiple biological processes in all organisms. Particularly, mitotic onset is regulated in all eukaryotes by an increase in cyclin-dependent kinase 1 (Cdk1) activity caused by the dephosphorylation of Cdk1 on a conserved tyrosine residue. PP2ARts1 is a phosphatase that participates in dephosphorylating the conserved tyrosine residue, tyrosine-19 (Y19). PP2ARts1 dephosphorylates phosphorylated serine and threonine residues. However, in vitro experiments suggest that in conjunction with the mammalian PP2A phosphatase activator (PTPA), PP2A gains phosphotyrosine specificity. My work indicates that Rrd1 and Rrd2 (the budding yeast homologs of PTPA) genetically interact with PP2ARts1 and the absence of these proteins cause a Swe1-dependent delay in mitosis. In parallel, utilizing a candidate approach to identify additional phosphatases specific to Cdk1-Y19, my work indicates that Ych1 and Arr2 act redundantly with Mih1 and Ptp1, and Ych1 may act downstream of PP2ARts1. In summation, my work provides the groundwork for how PP2ARts1 functions to dephosphorylate the conserved Y19 residue on Cdk1 and will lead to a better understanding of its role in regulating mitotic progression.

No description available.