Carcinoma de Células Renais (CCR) representa uma família de tumores distintos com evolução clínica imprevisível. Uma variedade de moléculas tem sido avaliada como marcadores prognósticos para CCR. Ciclina D1, uma proteína reguladora do ciclo celular, encontra-se superexpressa em vários tumores primários. Nosso objetivo é avaliar sua expressão como marcador prognóstico em CCR. Antes disso, traçamos um perfil clínico e histopatológico da amostra e verificamos sua relação com os fatores prognósticos considerados clássicos pela literatura. 109 espécimes de pacientes diagnosticados com CCR foram obtidos entre 2005 e 2010 no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto USP e submetidos à análise imunoistoquímica juntamente com 07 amostras de tecido renal normal. A maior parte das características epidemiológicas e clínicas de nossa amostra foi similar àquelas descritas na literatura mundial. Houve predomínio do gênero masculino, da raça branca, com idade próxima a 60 anos, frequência de pacientes assintomáticos em torno de 36% e grande prevalência do CCR de células claras (71,55%). A mortalidade específica da doença foi de 13,76%, sendo o CCR de células claras o tipo mais frequente entre os óbitos e casos metastáticos. Os casos que exibiram má evolução clínica, definida pela ocorrência de metástase e/ou óbito por CCR (22,01%), estiveram associados à presença de sintomas ao diagnóstico, maior tamanho tumoral, grupo de estágio alto (III ou IV), grau nuclear de Fuhrman alto (3 ou 4), presença de necrose e de diferenciação sarcomatóide no tumor, além de outros fatores histológicos desfavoráveis (p < 0,01). Isso indica que as variáveis utilizadas na avaliação de prognóstico em países desenvolvidos podem ser aplicadas aos nossos pacientes. Não houve expressão imunoistoquímica de Ciclina D1 nos casos de tecido renal normal. Observou-se heterogeneidade de marcação nuclear intratumoral no total de casos e menor expressão proteica entre os CCR papilífero e cromófobo. Pacientes com tumores com Ciclina D1baixa (até 30% de células positivas) apresentaram má evolução clínica (p = 0,03), maior tamanho tumoral (p = 0,01), presença de sintomas ao diagnóstico (p = 0,04), grau nuclear alto (p = 0,001), presença de necrose (p = 0,004) e de diferenciação sarcomatóide (p = 0,04) no tumor, além de menor sobrevida sem metástase e/ou óbito por CCR (p = 0,03). Após análise multivariada, a expressão de Ciclina D1 não apresentou valor prognóstico independente para má evolução clínica, embora tenha aumentado levemente a acurácia prognóstica do modelo adotado. Em todas as análises realizadas para o CCR de células claras isoladamente, observamos significância estatística semelhante à do total de casos (CCR). Nosso estudo demonstrou que: a proteína Ciclina D1 encontra-se superexpressa em CCR; os tipos de CCR parecem exibir diferentes padrões de marcação imunoistoquímica da Ciclina D1; alta marcação da proteína (acima de 30% de células positivas) esteve associada à boa evolução clínica e à maioria dos fatores prognósticos favoráveis bem estabelecidos na literatura. Novas investigações são necessárias para descobrir que mecanismos levam a seu acúmulo nas células neoplásicas e quais outros eventos podem estar contribuindo para a progressão da doença. / Renal Cell Carcinoma (RCC) is a family of distinct tumors with unpredictable clinical outcome. A variety of molecules have been evaluated as prognostic markers for RCC. Cyclin D1, a cell cycle regulatory protein, is overexpressed in several primary tumors. Our purpose is to evaluate its expression as a prognostic marker in RCC. Before that, we drew a clinical and histopathological profile of the sample and verified its relationship with prognostic factors regarded as classics in literature. 109 specimens from patients diagnosed with RCC were obtained between 2005 and 2010 at Hospital das Clínicas - Ribeirão Preto School of Medicine USP and submitted to immunohistochemical analysis, along with 07 normal kidney tissue samples. Most epidemiological and clinical characteristics of our sample were similar to those described in the literature. There was a predominance of male, Caucasian, aged about 60 years, the frequency of asymptomatic patients around 36%, and high prevalence of clear cell RCC (71.55%). The disease-specific mortality was 13.76%, being the clear cell RCC the most frequent type among deaths and metastatic cases. Cases that exhibited poor clinical outcome, defined by the occurrence of metastasis and/or death by RCC (22.01%), were related to the presence of symptoms at diagnosis, larger tumor size, high stage group (III or IV), high Fuhrman nuclear grade (3 or 4), presence of necrosis and sarcomatoid differentiation in the tumor and other unfavorable histological factors (p < 0.01). This indicates that the variables used in the assessment of prognosis in developed countries can be applied to our patients. There was no immunohistochemical expression of Cyclin D1 in cases of normal kidney tissue. There was intratumoral heterogeneity in nuclear staining in all cases and lower protein expression among papillary and chromophobe RCC. Patients with Cyclin D1low tumors (up to 30% positive cells) showed poor clinical outcome (p = 0.03), larger tumor size (p = 0.01), presence of symptoms at diagnosis (p = 0.04), high nuclear grade (p = 0.001), presence of necrosis (p = 0.004) and sarcomatoid differentiation (p = 0.04) in the tumor and lower survival without metastasis and/or death by RCC (p = 0.03). After multivariate analysis, the expression of Cyclin D1 showed no independent prognostic value for poor clinical outcome, although it has slightly increased the prognostic accuracy of the model adopted. In all analyzes performed for clear cell RCC alone, we observed statistical significance similar to that of the total cases (RCC). Our study showed that: Cyclin D1 protein is overexpressed in RCC; RCC types seem to exhibit different patterns of immunohistochemical staining for Cyclin D1; high protein expression (over 30% positive cells) was related to good clinical outcome and to most favorable prognostic factors well established in the literature. Further investigations are necessary to reveal which mechanisms lead to its accumulation in neoplastic cells and what other events might be contributing to the progression of the disease.

Neoplasias malignas das glândulas salivares são lesões raras e o mecanismo pelo qual esses tumores progridem ainda não está completamente esclarecido na literatura. A galectina-3 é uma proteína multifuncional expressa em uma grande quantidade de tecidos normais, mas que também tem sido associada à progressão tumoral de neoplasias malignas da tireóide, próstata e neoplasias gástricas. Estudos prévios demonstraram que a galectina-3 está também expressa em algumas neoplasias malignas das glândulas salivares como carcinoma adenóide cístico e adenocarcinoma polimorfo de baixo grau de malignidade. Recentemente foi sugerido que a superexpressão da galectina-3 controla alterações nos níveis de expressão de alguns reguladores do ciclo celular, dentre eles a ciclina D1. Além disso, outros estudos revelaram que a ciclina D1 é ativada pela -catenina de uma maneira dependente da galectina-3. O objetivo desse trabalho foi comparar a marcação imunoistoquímica nuclear e / ou citoplasmática da galectina-3 no carcinoma adenóide cístico e adenocarcinoma polimorfo de baixo grau tentando relacioná-la à marcação da -catenina e ciclina D1. Foram realizadas reações de imunoistoquímica para as três proteínas em 15 casos de carcinoma adenóide cístico e em 15 casos de adenocarcinoma polimorfo de baixo grau utilizando-se material parafinado. Para a galectina-3 os carcinomas adenóides císticos apresentaram marcação imunoistoquímica apenas nas células luminais, predominantemente no núcleo. Todos os casos de adenocarcinoma polimorfo de baixo grau revelaram uma marcação predominantemente citoplasmática para essa proteína. Ambos os tumores exibiram intensa marcação citoplasmática e/ou nuclear para a -catenina na maioria dos casos. Não houve imunorreatividade para a ciclina D1 em 14/15 casos de adenocarcinoma polimorfo de baixo grau. Em contraste, os carcinomas adenóides císticos revelaram marcação nuclear específica para a ciclina D1 em 10 de 15 casos estudados em mais de 5% das células neoplásicas e essa marcação estava associada à marcação citoplasmática e nuclear da galectina-3 (p<0,05). Esses resultados sugerem que nos carcinomas adenóides císticos a expressão da galectina-3 pode exercer uma função de proliferação celular e parece estar relacionada à diferenciação celular no adenocarcinoma polimorfo de baixo grau. Além disso, a perda de expressão da galectina-3 no carcinoma adenóide cístico pode estar associada a um comportamento clínico mais agressivo dessa lesão. Embora a -catenina pareça exercer algum papel no mecanismo de carcinogênese dessas duas lesões, ela não parece se ligar à galectina-3 para ativar a ciclina D1. / Salivary gland tumors are uncommon and the mechanism by which malignant tumors progresses is still undefined. In a previous study it was shown that galectin-3 is expressed in malignant salivary gland neoplasms as adenoid cystic carcinoma and polymorphous low-grade adenocarcinoma. Galectin-3 is a multifunctional protein of a group of galactoside-binding lectins expressed in a variety of normal cells, but also has been implicated in tumor progression of some malignancies as thyroid, prostate and gastric cancers. Recently, it has been suggested that galectin-3 may be an important mediator of the -catenin/Wnt pathway. Moreover, nuclear galectin-3 expression has been implicated in cell proliferation, promoting cyclin D1 activation. Thus, in the present study we aimed to correlate galectin-3 expression, either nuclear or cytoplasmic, with the expression of -catenin (nuclear/cytoplasmic) and cyclin D1 (nuclear) in 15 cases of adenoid cystic carcinoma and in 15 cases of polymorphous low-grade adenocarcinoma. For galectin-3, adenoid cystic carcinomas showed specific staining only in luminal cells, mainly in the nuclei. In the cases of polymorphous low-grade adenocarcinoma, all tumor cells revealed a positive, mostly cytoplasmic, reaction to galectin-3. Both tumors showed intense cytoplasmic/nuclear staining for -catenin in the majority of cases. Cyclin D1 immunoreactivity was not detected in 14 of the 15 polymorphous low-grade adenocarcinomas studied. In contrast, adenoid cystic carcinomas showed specific nuclear staining for cyclin D1 in 10 of 15 cases studied in more than 5% of the neoplastic cells. Cyclin D1 expression was correlated with cytoplasmic and nuclear galectin-3 expression in adenoid cystic carcinomas (p<0,05). These results suggest that in adenoid cystic carcinoma galectin-3 may play a role in cellular proliferation through cyclin D1 activation. In polymorphous low-grade adenocarcinoma gal-3 expression seems to be associated with cellular differentiation. In addition, loss of cytoplasmic expression of galectin-3 in adenoid cystic carcinomas may be related to a more aggressive behavior of these lesions. Although -catenin seems to play a role in carcinogenesis, in both lesions, it seems that it does not bind to galectin-3 for cyclin D1 stimulation.

No Brasil, como no mundo, o carcinoma epidermóide bucal está entre os dez tipos mais comum de câncer e acomete mais de 13 mil pessoas por ano. Apesar de ser um sério problema devido a sua morbidade e mortalidade, alguns casos desta doença têm um comportamento biológico menos agressivo. A proteína ciclina D1, depois que forma complexos com as proteínas CDK4 e CDK6, tem como principal função fosforilar a proteína Retinoblastoma. Após sua fosforilação, a proteína libera um fator de transcrição, o E2F, que leva a célula à progressão da fase G1 para fase S do ciclo celular. A proteína c-jun, que faz parte do fator de transcrição AP-1, tem participação ativa no ciclo celular, principalmente durante a transcrição da fase G0 a G1. O gene retinoblastoma é um supressor de tumor. Este gene codifica uma fosfoproteína nuclear, que recebe o mesmo nome. Essa proteína regula o ciclo celular através de múltiplas funções. Também regula outros processos que afetam a proliferação celular, a diferenciação terminal e a apoptose. O HPV é um vírus de DNA que é encontrado em vários tipos de câncer e é o principal agente etiológico do carcinoma de colo uterino. Este trabalho comparou a expressão das proteínas ciclina D1, c-jun e do retinoblastoma em carcinomas epidermóides de baixo e alto grau de malignidade e tentou analisar se o HPV é um fator etiológico desta neoplasia. Apesar das lesões de baixo grau de malignidade expressarem as proteínas num maior número de células que as lesões de alto grau, só houve diferença estatística, entre os dois grupos estudados, para a proteína do retinoblastoma. Não foi encontrado o DNA do HPV em nenhum dos casos estudados. De acordo com este trabalho e com a literatura, a proteína do retinoblastoma é expressa em um número menor de células em carcinomas epidermóides bucais mais agressivos e o HPV não é um agente etiológico de todos os casos desta doença / In Brazil, as in the world, the oral squamous cell carcinoma is among the ten more common types of cancer e affects more than 13 thousand of people by year. Even though it is a serious problem due to its morbidity and mortality, some cases of this disease have a less aggressive biological behavior. The cyclin D1 protein after it forms complexes with the CDK4 and CDK6 proteins has as main function phosphorylate the Retinoblastoma protein. After its prosphorylation, the protein releases a transcription factor, the E2F, that leads the cell to the progression from the phase G1 to the phase S of the cell cycle. The c-jun protein, that is part of the transcription factor AP-1, has active participation in the cell cycle, mainly during the transcription from the phase G0 to G1. The retinoblastoma gene is a tumour suppressor. This gene codifies a nuclear phosphoprotein that receives the same name. This protein regulates the cell cycle through multiple functions. It also regulates other processes that affect the cell proliferation, the terminal differentiation and apoptosis. The HPV is a DNA virus that is found in many types of cancer and is the main etiological agent of the cervical cancer. This study compared the protein expression of the cilin D1, c-jun and retinoblastoma in low and high grade squamous cell carcinoma and tried to analyze if the HPV is a etiological factor for this neoplasm. In spite of the low grade of malignancy lesions express the protein in a greater number of cells than in the high grade lesion, there only was statistical difference, among the two studied groups, for the retinoblastoma protein. It was not found DNA of the HPV in any of the studied cases. According with this study and with the literature the retinoblastoma protein is expressed in a lower number of cells in the more aggressive oral squamous cell carcinomas and the HPV is not the etiological agent in all of the cases of this disease.

No description available.

Ischemic stroke occurs as a result of a lack or severe reduction of blood supply to the brain. Presently therapeutic interventions are limited and there is a need to develop new and efficacious stroke treatments. To this end, a great deal of research effort has been devoted to studying the potential molecular mechanisms involved in ischemic neuronal death. Correlative evidence demonstrated a paradoxical activation of the cell cycle machinery in ischemic neurons. The levels and activity of key cell cycle regulators including cyclin D1, Cdk2 and Cdk4 are upregulated following ischemic insults. However, the functional relevance of these various signals following ischemic injury was unclear. Accordingly, the research described in this thesis address the functional relevance of the activation of the cell cycle machinery in ischemic neuronal death. The data indicate that the inhibition of Cdk4 protects neurons from ischemia-induced delayed death, whereas abrogation of Cdk5 activity prevents excitotoxicity-induced damage in vitro and in vivo. Examination of upstream activators of mitotic-Cdks showed that Cdc25A is a critical mediator of delayed ischemic neuronal death. Investigation of the potential molecular mechanism by which cell cycle regulators induced neuronal death revealed perturbations in the levels and activity of key downstream targets of Cdk4. The retinoblastoma protein family members, pRb and p130 are increasingly phosphorylated following ischemic stresses. Importantly, p130 and E2F4 proteins are drastically reduced following ischemic insults. Additionally, E2F1 association with promoters of pro-apoptotic genes are induced while that of E2F4 is reduced. These changes appear to be important determinants in ischemic neuronal death. Cumulatively, the data supports the activation of the cell cycle machinery as a pathogenic signal contributing to ischemic neuronal death. The development of neuroprotectant strategies for stroke has been hampered in part by its complex pathophysiology. Previous research indicated that flavopiridol, a general CDK-inhibitor, is unable to provide sustained neuroprotection beyond one week following cerebral ischemia. The potential benefit of combining flavopiridol with another neuroprotectant, minocycline, was explored. The data indicate that while this approach provided histological protection 10 weeks after insult, the protected neurons are not functional due to progressive dendritic degeneration. This evidence indicates that targeting cell cycle pathways in stroke while important must be combined with other therapeutic modalities to fully treat stroke-induced damage.

Chez la femme, le cancer du sein est le cancer le plus fréquemment diagnostiqué. Différents traitements sont disponibles selon le sous-type tumoral. Cependant, certaines patientes sont réfractaires à ces thérapies et restent vulnérables lors de récidives. Le cancer a longtemps été défini par une division aberrante des cellules, mais aujourd'hui, il est évident que la résistance à la mort cellulaire programmée est un paramètre majeur dans l'étiologie de la maladie.Les cyclines de type D régulent le cycle cellulaire en permettant la transition de la phase G1 à la phase S. Pour cela, elles activent les kinases dépendantes des cyclines 4/6 (CDK4/6) qui phosphorylent les protéines du rétinoblastome ce qui libère le facteur de transcription E2F. La Cycline D1 (CycD1) nucléaire est donc centrale dans le contrôle du cycle. Son gène est amplifié dans les cancers humains et la moitié des patientes atteintes d'un cancer du sein ont une surexpression de CycD1. Par l’activation de CDK4, CycD1 est essentielle à l'apparition et à la progression tumorale. Ainsi, des inhibiteurs spécifiques de CDK4/6 ont été développés contre le cancer du sein. Malheureusement, certaines patientes restent insensibles à ce traitement. À ce titre, le ciblage spécifique de CycD1 pourrait représenter une alternative clinique. En effet, en plus de la régulation du cycle, CycD1 est également impliquée, indépendamment de CDK4, dans la survie des cellules cancéreuses. Cependant, aucun mécanisme de l'impact de CycD1 dans le maintien tumoral n'a été établi pour démontrer ce potentiel thérapeutique. En outre, CycD1 a été décrite dans les organes à l’âge adulte pour réguler le métabolisme du glucose et l'hématopoïèse. Par conséquent, pour éviter tout effet secondaire indésirable, nous avons décidé d’évaluer l’implication potentielle de CycD1 dans les organes adultes. Grâce au Tandem-HTRF, basé sur le transfert d'énergie entre deux anticorps, nous avons révélé la dynamique inattendue de CycD1 dans chaque organe adulte. De plus, nous avons montré que l’altération de l'expression de CycD1 conduit à une diminution des capacités de survie des cellules saines post-mitotiques.Au vu de ces limitations, nous avons développé une nouvelle approche d'ARN interférence spécifique des cellules cancéreuses appelée TAG-RNAi. Cette technologie permet de cibler CycD1 uniquement dans la tumeur afin d'épargner les cellules saines. Cette approche innovante consiste à cibler un tag présent uniquement sur l’ARNm de CycD1 des cellules cancéreuses. Ainsi, nous avons découvert que le ciblage spécifique de CycD1 induit une régression rapide et spontanée des tumeurs dépendantes des oncogènes RAS ou ERBB2. Par protéomique in vivo, j'ai découvert que lors de stress pro-apoptotiques, CycD1 cytoplasmique interagit avec la procaspase-3 et bloque son activation pour empêcher l'apoptose des cellules. Ces travaux démontrent la valeur clinique du ciblage spécifique de CycD1 dans les cancers afin d'améliorer l'efficacité des chimiothérapies.Par conséquent, il restait à déterminer comment appliquer le TAG-RNAi contre CycD1 uniquement dans les cellules cancéreuses des patientes. Puisque le tag exotique présent sur le gène Ccnd1 chez la souris nous a permis de cibler spécifiquement les cellules cancéreuses, nous avons pensé que des mutations retrouvées dans les cancers humains représentaient une option de ciblage. Ainsi, nous avons étendu le concept TAG-RNAi aux mutations somatiques caractéristiques des cancers pour cibler avec succès l'expression des mutants KRAS-G12V ou BRAF-V600E comme exemples. L'idée est donc d'identifier les mutations de Ccnd1 chez les patientes afin d'appliquer le TAG-RNAi comme une thérapie personnalisée afin d’éviter les effets secondaires. Enfin, l'expression de CycD1 représente un nouveau biomarqueur pour le cancer et les troubles liés à l'âge: de faibles taux prédisposent aux maladies dégénératives tandis que des taux élevés indiquent une susceptibilité accrue au cancer. / Breast cancer is the most frequently diagnosed cancer in women. This cancer is the leading cause of death in women aged from 35 to 65 years old. Different treatments are now available depending on tumor subtypes. However, some patients are still refractory to these therapies and are at risk of disease relapse. Cancer research has long focused on aberrant cancer cell division but today it is evident that the resistance to programmed cell death is also a major characteristic of the disease.D-type cyclins regulate cell cycle by allowing the transition from the G1-phase to the S-phase. These regulatory subunits activate the Cyclin-Dependent Kinases 4/6 (CDK4/6) that phosphorylate the retinoblastoma proteins which then release the E2F transcription factors. Nuclear Cyclin D1 (CycD1) is therefore central in the control of division. The Ccnd1 gene is amplified in human cancers and half of breast cancer patients bare an overexpression of CycD1. CycD1 is required for mammary carcinoma onset and progression in a CDK4 kinase-dependent manner. Hence, specific CDK4/6 inhibitors have been developed and authorized in the clinics against breast cancer. Unfortunately, some patients remain insensitive to this treatment. In this frame, the specific targeting of CycD1 could represent a strategic alternative in clinics to overcome these pitfalls. Indeed, in addition to cell cycle regulation with CDK4, CycD1 is also involved in CDK4-independent features of cancer cells like cell survival. However, to date, no clear mechanism for the impact of CycD1 in tumor maintenance is established to demonstrate the therapeutic value of its targeting.Moreover, recent studies have demonstrated the participation of CycD1 in adult organs to regulate glucose metabolism and hematopoiesis. As a consequence, to avoid any undesirable side effects, we decided to gauge the potential CycD1 implication in post-mitotic organs body-wide. We set up a new hypersensitive technology named Tandem-HTRF based on the energy transfer between two antibodies to reveal the unexpected dynamics of CycD1 expression in adult organ. Then, we discovered that alterations of CycD1 expression induced dramatic functional consequences on the survival capacities of healthy adult post-mitotic cells.Based on these limitations, we developed a novel RNAi approach specific to cancer cells named TAG-RNAi. This technology allows the silencing of CycD1 in cancer cells only to spare healthy cells. This innovative approach consists in the targeting of a mRNA tag only present on CycD1 from cancer cells. Using this technique, we found that the specific silencing of CycD1 induces a rapid and spontaneous regression of tumors driven by the RAS or ERBB2 oncogenes. Then, thanks to a proteomics screening in vivo, I discovered that under pro-apoptotic stresses the cytoplasmic CycD1 interacts with the procaspase-3 protein and blocks its activation to prevent cancer cell apoptosis. Altogether, my work demonstrates the clinical value of the specific targeting of CycD1 in cancers to increase the efficacy of chemotherapeutic treatments.Hence, it remained to be determined how to apply in patients RNAi against CycD1 only in cancer cells. Because the exotic tagging of its gene was instrumental in mice cancer models, we reasoned that human cancer mutations could represent such a specific tag. We have extended the concept of TAG-RNAi to somatic mutations characteristic of human cancers to successfully target the expression of KRAS-G12V or BRAF-V600E mutants as examples. The idea is therefore to identify Ccnd1 mutations in cancer patients in order to apply TAG-RNAi as a custom therapeutic approach that will manage side effects. More unanticipated, CycD1 expression represents a new biomarker for both cancer and age-related disorders: low CycD1 levels predispose to degenerative complications while high CycD1 levels indicate increased susceptibility to cancer and resistance to treatment.

La dérégulation du système de surveillance qui bloque la prolifération lorsque l'intégrité du génome est compromise fait partie intégrante de la cancérogenèse. Nous cherchons à décortiquer les mécanismes qui, en phase G2, orchestrent l'arrêt du cycle cellulaire, irréversible, en présence des lésions de l'ADN (sénescence) ou réversible (quiescence), en absence de signaux mitogéniques ou confluence. L'objectif du premier volet fut d'élucider les rôles respectifs de l'inhibiteur de CDK (CKI) p21Waf1 et des kinases Chk1 et Chk2 dans l'arrêt en G2 dû au stress génotoxique menant à la sénescence. Nous avons montré que dans les cellules humaines normales cet arrêt nécessite l'action de p21 et Chk1 tandis que Chk2 n'est pas requise. Au contraire, dans plusieurs lignées cancéreuses, malgré la présence de p53, ce rôle de p21 est compromis à cause d'une activation inefficace de la kinase ATM. Par conséquent, en dépit d'une forte activation de Chk1 bloquant la mitose, ces cellules ne parviennent pas à initier la sénescence (Lossaint et al., soumis). L'objectif du deuxième volet fut de mettre en évidence le programme déclenchant la quiescence lors de confluence ou en absence de sérum. Les travaux antérieurs de l'équipe ont montré que cette décision pouvait être prise avant la mitose même si l'arrêt du cycle n'a lieu qu'en phase G1 suivante. En étudiant les fibroblastes synchronisés nous avons trouvé que la quiescence est précédée par l'inhibition pré-mitotique de la phosphorylation de pRb due à une diminution de cycline D1 et une stabilisation du CKI p27Kip1 (Chassot et al., 2008). De plus, nos résultats récents montrent que la présence de sérum entre le point R et la mitose est requise pour initier la réplication de l'ADN au cycle suivant. Les travaux futurs devraient élucider comment différentes voies de signalisation, via la voie cycline D-pRb, affectent divers composants de l'appareil de réplication de l'ADN pour inhiber la progression du cycle de façon réversible ou irréversible. / Cancer is a multi-step process resulting from abrogation of several barriers to uncontrolled proliferation. They include inhibitory pathways with appropriate checkpoints that lead to reversible (quiescence) or irreversible (senescence, apoptosis) block of cell proliferation. We are especially interested in pathways orchestrating cell cycle exit that operate in the G2 phase. The first objective of this thesis was to decipher mechanisms that prevent mitosis in response to DNA damage. We found that Cdk inhibitor p21Waf1 plays a crucial role in blocking mitotic onset in normal cells; acting in tandem with checkpoint kinase Chk1, p21 inactivates mitotic Cdks and inhibits pRb phosphorylation, thereby irreversibly blocking mitotic entry. In contrast, in p53-proficient transformed cells, the induction of p21 in G2 is impaired, most likely because of deficient ATM activation. While, in some cases, Chk1 hyper-activation prevents mitosis, the absence of p21 compromises the senescence program from G2. Finally, we showed that Chk2 is dispensable for G2 arrest in both non-transformed and transformed cells (Lossaint et al., submitted). Our second objective was to elucidate the pathways that induce quiescence (G0). This reversible cell cycle exit occurs in G1, requires pRb family members and p27Kip1-dependent Cdk inactivation. Based on observations obtained in our team and the data in the literature, we hypothesized that reversible cell cycle exit program might be launched before mitosis. By using an in vitro wounding model, we showed that confluence-driven quiescence is preceded by pre-mitotic CDK inhibition by p27, cyclin D1 downregulation and reduced pre-mitotic pRb phosphorylation (Chassot et al., 2008). Moreover, our results obtained in synchronized fibroblasts that were serum-starved after release from G1/S block suggest that cyclin D1 might stimulate proliferation by keeping pocket proteins phosphorylated during G2/M progression (Lossaint et al., in preparation).

Ischemic stroke occurs as a result of a lack or severe reduction of blood supply to the brain. Presently therapeutic interventions are limited and there is a need to develop new and efficacious stroke treatments. To this end, a great deal of research effort has been devoted to studying the potential molecular mechanisms involved in ischemic neuronal death. Correlative evidence demonstrated a paradoxical activation of the cell cycle machinery in ischemic neurons. The levels and activity of key cell cycle regulators including cyclin D1, Cdk2 and Cdk4 are upregulated following ischemic insults. However, the functional relevance of these various signals following ischemic injury was unclear. Accordingly, the research described in this thesis address the functional relevance of the activation of the cell cycle machinery in ischemic neuronal death. The data indicate that the inhibition of Cdk4 protects neurons from ischemia-induced delayed death, whereas abrogation of Cdk5 activity prevents excitotoxicity-induced damage in vitro and in vivo. Examination of upstream activators of mitotic-Cdks showed that Cdc25A is a critical mediator of delayed ischemic neuronal death. Investigation of the potential molecular mechanism by which cell cycle regulators induced neuronal death revealed perturbations in the levels and activity of key downstream targets of Cdk4. The retinoblastoma protein family members, pRb and p130 are increasingly phosphorylated following ischemic stresses. Importantly, p130 and E2F4 proteins are drastically reduced following ischemic insults. Additionally, E2F1 association with promoters of pro-apoptotic genes are induced while that of E2F4 is reduced. These changes appear to be important determinants in ischemic neuronal death. Cumulatively, the data supports the activation of the cell cycle machinery as a pathogenic signal contributing to ischemic neuronal death. The development of neuroprotectant strategies for stroke has been hampered in part by its complex pathophysiology. Previous research indicated that flavopiridol, a general CDK-inhibitor, is unable to provide sustained neuroprotection beyond one week following cerebral ischemia. The potential benefit of combining flavopiridol with another neuroprotectant, minocycline, was explored. The data indicate that while this approach provided histological protection 10 weeks after insult, the protected neurons are not functional due to progressive dendritic degeneration. This evidence indicates that targeting cell cycle pathways in stroke while important must be combined with other therapeutic modalities to fully treat stroke-induced damage.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Diversos estudos epidemiológicos mostram que nutrientes e outros compostos bioativos presentes nos alimentos (CBA) apresentam atividade quimiopreventiva contra o câncer. Assim, destaca-se o estudo dos isoprenóides devido a sua ação promissora tanto na prevenção quanto na terapia do câncer. Todavia, apesar dessas evidências, pouco se sabe a respeito da ação dessas substâncias nos processos de proliferação celular e apoptose in vivo. Assim, 141 ratos Wistar foram tratados durante duas semanas consecutivas com farnesol (grupo FR, 25 mg/100 g de peso corporal) ou óleo de milho (grupo OM; controle, 0,25 mL/100 g de peso corporal) e sacrificados em diferentes momentos após a hepatectomia parcial (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h e 24 h). Os parâmetros hepáticos analisados foram a proliferação celular (núcleos marcados para PCNA/mm2), apoptose (corpúsculos apoptóticos [CA\'s] por mm2) e expressão de p65, ciclina D1 (\"western blot\") e HMG-CoA redutase (\"dot-blot\"). Os animais tratados com o isoprenóide, assim como o grupo controle, apresentaram reduzida taxa de proliferação celular até 8h após a cirurgia. No entanto, a partir desse momento, o grupo FR passou a apresentar taxa de proliferação celular inferior ao grupo OM, diferença esta que atingiu significância estatística (p<0,05) 24h após a HP. Com relação a apoptose, animais tratados com FR apresentaram maior número de CA\'s (p<0,05) do que o grupo OM 30 min após a HP. Já em relação à ação do FR em âmbito molecular, houve uma redução de 40% e 50% na expressão de p65 e ciclina D1 30min e 24h após a HP, respectivamente, embora essas diferenças não tenham atingido significância estatística (p>0,05). Além disso, animais tratados com o isoprenóide apresentaram maior (p<0,05) expressão do gene que codifica para HMG-CoA redutase 2 h e 12 h após a cirurgia. Assim, tanto a inibição da proliferação celular quanto a indução de apoptose podem ser reflexo das alterações da expressão hepática dos genes para HMG-CoA redutase, p65 e ciclina D1 por parte do isoprenóide. / Epidemiological data have shown that nutrients and others bioactive compounds in food have chemopreventive activities against cancer. Among these compounds, isoprenoids are suggested either as a chemopreventive or chemotherapy agents. However, despite these evidences, studies focused on the isoprenoids activities on cell proliferation and apoptosis in vivo are rare. Thus, the effect of the 15-carbon isoprenoid farnesol on liver regeneration after partial hepatectomy was evaluated. Wistar rats were treated for two consecutive weeks with farnesol (FR group, 25 mg/100 g body weight) or corn oil (OM group, control, 0,25 mL/100 g body weight) and killed at different time points after partial hepatectomy (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h and 24 h). Still, hepatic cell proliferation (PCNA lebeled nuclei), apoptosis (quantification of apoptotic bodies), p65 and cyclin D1 protein expression (western blot) and HMG-CoA reductase mRNA expression (dot blot) were also evaluated. Comparing to OM group, farnesol treatment significantly inhibited (p<0,05) hepatic cell proliferation 24 h after HP. Regarding apoptosis, also compared to controls, farnesol treated rats presented more (p<0,05) apoptotic bodies at 30 min. Besides, there were a suggestion of a higher number of apoptotic bodies 2 and 12 hours after HP in FR group comparing to OM group. According to western blot analysis, comparing to controls, this 15-carbon isoprenoid reduced 40% and 50% p65 and cyclin D1 hepatic protein expression, 30 min and 24 h after partial hepatectomy, respectively, although the differences did not also reach the statistical significance. Furthermore, farnesol treated rats had higher (p<0,05) HMG-CoA reductase mRNA levels than controls 2 h and 12 h after the surgery. Theses data suggest that the alterations on p65, cyclin D1 and HMG¬-CoA reductase gene expression observed in FR group might be associated with the inhibition of cell proliferation and the induction of apoptosis by farnesol.