O carcinoma oral de células escamosas (OSCC Oral Squamous Cell Carcinoma) é o câncer de cabeça e pescoço mais comum. Somente no Brasil, foram estimados 14.160 novos diagnósticos para o ano de 2009. O HPV está associado com o aumento no risco do câncer oral, mas seu papel na carcinogênese ainda é controverso. A BubR1, uma proteína importante para o checkpoint de fuso mitótico (SAC Spindle Assembly Checkpoint), tem sido associada com algumas proteínas codificadas por espécies virais e com o câncer. O objetivo do presente estudo foi avaliar a expressão de BubR1 em lesões orais benignas e amostras de OSCC com e sem metástase associadas com infecção pelo HPV. Nós realizamos imunoistoquímica para BubR1 em 16 biópsias de lesão oral benigna e em 70 biópsias de OSCC divididas em três grupos (tumores in situ, tumores invasivos sem metástase e tumores invasivos com metástase), com os respectivos linfonodos das amostras com metástase. A técnica de Nested PCR foi realizada com finalidade de detectar DNA do HPV. Nas lesões malignas, foi observada uma significante superexpressão de BubR1 associada com menor sobrevida (p = 0.0479). Houve também correlação significante (r = 1.000) de BubR1 entre as lesões com metástase e seus respectivos linfonodos. Noventa por cento dos OSCC e 100% das lesões benignas foram HPV positivos. HPV 16 e HPV 18 foram detectados em, respectivamente, 13% e 24% das amostras com OSCC HPV-positivas. O HPV teve maior prevalência (76%) nas amostras com alta expressão de BubR1 e a ausência de DNA viral não influenciou no padrão de expressão de BubR1. Esses resultados sugerem uma provável associação do HPV com a superexpressão de BubR1 em OSCC, o que não se aplica para lesões orais benignas. / Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is that 14,160 new diagnoses will be made in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BubR1, an important protein in the mitotic Spindle Assembly Checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BubR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BubR1 in 16 non-malignant oral lesion biopsies and in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis. Nested PCR was performed in order to detect HPV DNA. Significantly higher BubR1 expression associated with shorter survival (p = 0.0479) was observed in malignant lesions. There was also a significant correlation (r = 1.000) with BubR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV 16 and HPV 18 were present in 13% and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with high BubR1 expression and the absence of viral DNA had no influence on BubR1 expression. These findings suggest that HPV could be associated with overexpression of BubR1 in OSCC, but not in benign oral lesions.

O carcinoma oral de células escamosas (OSCC Oral Squamous Cell Carcinoma) é o câncer de cabeça e pescoço mais comum. Somente no Brasil, foram estimados 14.160 novos diagnósticos para o ano de 2009. O HPV está associado com o aumento no risco do câncer oral, mas seu papel na carcinogênese ainda é controverso. A BubR1, uma proteína importante para o checkpoint de fuso mitótico (SAC Spindle Assembly Checkpoint), tem sido associada com algumas proteínas codificadas por espécies virais e com o câncer. O objetivo do presente estudo foi avaliar a expressão de BubR1 em lesões orais benignas e amostras de OSCC com e sem metástase associadas com infecção pelo HPV. Nós realizamos imunoistoquímica para BubR1 em 16 biópsias de lesão oral benigna e em 70 biópsias de OSCC divididas em três grupos (tumores in situ, tumores invasivos sem metástase e tumores invasivos com metástase), com os respectivos linfonodos das amostras com metástase. A técnica de Nested PCR foi realizada com finalidade de detectar DNA do HPV. Nas lesões malignas, foi observada uma significante superexpressão de BubR1 associada com menor sobrevida (p = 0.0479). Houve também correlação significante (r = 1.000) de BubR1 entre as lesões com metástase e seus respectivos linfonodos. Noventa por cento dos OSCC e 100% das lesões benignas foram HPV positivos. HPV 16 e HPV 18 foram detectados em, respectivamente, 13% e 24% das amostras com OSCC HPV-positivas. O HPV teve maior prevalência (76%) nas amostras com alta expressão de BubR1 e a ausência de DNA viral não influenciou no padrão de expressão de BubR1. Esses resultados sugerem uma provável associação do HPV com a superexpressão de BubR1 em OSCC, o que não se aplica para lesões orais benignas. / Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is that 14,160 new diagnoses will be made in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BubR1, an important protein in the mitotic Spindle Assembly Checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BubR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BubR1 in 16 non-malignant oral lesion biopsies and in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis. Nested PCR was performed in order to detect HPV DNA. Significantly higher BubR1 expression associated with shorter survival (p = 0.0479) was observed in malignant lesions. There was also a significant correlation (r = 1.000) with BubR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV 16 and HPV 18 were present in 13% and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with high BubR1 expression and the absence of viral DNA had no influence on BubR1 expression. These findings suggest that HPV could be associated with overexpression of BubR1 in OSCC, but not in benign oral lesions.

La présence de cassures double-brin de l'ADN en mitose est problématique pour les cellules, car cette situation produit des fragments de chromosome ne possédant pas de centromères. En l'absence d'un mécanisme permettant leur prise en charge, ces fragments acentriques n'étant pas attachés au fuseau mitotique, pourraient être ségrégés aléatoirement dans les cellules filles, causant de l'instabilité génomique. Nous avons découvert un mécanisme permettant la transmission correcte des fragments acentriques dans les cellules filles via une structure faisant le lien entre les deux fragments cassés. Plusieurs protéines sont recrutées sur les cassures, comme les kinases mitotiques BubR1 et Polo, et favorisent la ségrégation correcte de ces chromosomes cassés. Cependant, les mécanismes permettant le recrutement de BubR1 et Polo sur les cassures d'ADN en mitose sont inconnus. De plus, les mécanismes moléculaires par lesquels BubR1 et Polo favorisent la ségrégation correcte des fragments acentriques restent à être identifiés. La première partie de mon projet a été d'étudier le rôle et la régulation de BubR1 sur les cassures d'ADN pendant la mitose. Nous avons montré que BubR1 requiert Bub3 pour se localiser sur les chromosomes cassés afin de favoriser leur ségrégation correcte. Nous avons également détecté l'accumulation de FizzyCDC20, un cofacteur de l'E3 ubiquitine ligase APC/C (Anaphase-Promoting- Complex/Cyclosome), sur les cassures d'ADN, et son recrutement dépend de son interaction avec la KEN Box de BubR1. De plus, l'utilisation d'un substrat synthétique de l'APC/C nous a permis de démontrer que la dégradation par l'APC/C est inhibée localement autour du chromosome cassé, de manière dépendante de BubR1. Ces résultats suggèrent fortement que le complexe BubR1/Bub3 recrut é sur les cassures d'ADN inhibe localement l'APC/C en séquestrant FizzyCDC20 et empêche ainsi la dégradation de substrats clefs impliqués dans la ségrégation correcte des chromosomes cassés. La seconde partie de mon projet a été d'étudier les relations d'interdépendance entre Polo et BubR1/Bub3/Fizzy sur les cassures d'ADN en mitose. Nous avons utilisé un laser UV pulsé pour induire des cassures dans un chromosome à un instant précis pendant la mitose, puis nous avons suivi le recrutement de protéines tagguées GFP sur les cassures de chromosome. Cette étude révèle que Polo est rapidement recrutée sur les cassures d'ADN et précède BubR1, Bub3 et Fizzy. De plus, la disparition de BubR1, Bub3 et Fizzy des cassures d'ADN coïncide avec la télophase alors que Polo disparait des cassures pendant l'interphase. Nous avons également montré que le recrutement de BubR1, Bub3 et Fizzy sur les cassures d'ADN est retardé dans les mutants polo, indiquant que Polo est requis pour un recrutement efficace de BubR1, Bub3 et Fizzy sur les cassures d'ADN. Pour finir, nous avons montré que l'accumulation de Polo et BubR1/Bub3/Fizzy sur les cassures d'ADN dépend de deux composants de la réponse aux dommages à l'ADN, le complexe MRN (Mre11-Rad50-Nbs1) et ATM (ataxia-telangiectasia mutated). Ce travail a permis d'avoir une meilleure compréhension sur la dynamique de recrutement de Polo et BubR1/Bub3/Fizzy sur les cassures d'ADN en mitose. De plus, le mécanisme moléculaire par lequel le complexe BubR1/Bub3 agit pour faciliter la ségrégation des chromosomes cassés a pu être en partie élucidé. / The presence of DNA double strand breaks (DSB) during mitosis is challenging for the cell, as it produces fragments of chromosome lacking a centromere. If not processed, this situation can cause genomic instability resulting in improper segregation of the broken fragments into daughter cells. We uncovered a mechanism by which broken chromosomes are faithfully transmitted to daughter cells via the tethering of the two broken chromosome ends. Several proteins including the mitotic kinase BubR1 and Polo are recruited to the breaks and mediate the proper segregation of the broken fragments. However, the mechanism underlying Polo and BubR1 recruitment to DNA breaks is unknown. Moreover, the molecular mechanisms by which Polo and BubR1 mediate the proper segregation of the broken fragments remain to be elucidated. We first investigated the role and regulation of BubR1 on DNA breaks during mitosis. We show that BubR1 requires Bub3 to localize on the broken chromosome fragment and to mediate its proper segregation. We also find that FizzyCdc20, a co--‐factor of the E3 ubiquitin ligase Anaphase--‐Promoting--‐Complex/Cyclosome (APC/C), accumulates on DNA breaks in a BubR1 KEN box--‐dependent manner. A biosensor for APC/C activity demonstrates a BubR1--‐dependent local inhibition of APC/C around the segregating broken chromosome. These results are consistent with a model where Bub3/BubR1 complex on DNA breaks functions to inhibit the APC/C locally via the sequestration of FizzyCdc20, thus preserving key substrates from degradation, which promotes proper transmission of broken chromosomes. In a second study, we investigated the dependency relationship between Polo and BubR1/Bub3/Fizzy on DNA breaks in mitosis. We used a pulsed UV laser to break one chromosome at a define time during mitosis. We immediately follow the recruitment of GFP--‐tagged proteins to laser--‐induced DNA breaks. My study reveals that Polo is promptly recruited to DNA breaks and precedes BubR1, Bub3 and Fizzy. In addition, while BubR1, Bub3 and Fizzy dissociation from the breaks coincide with telophase and the nuclear envelope reformation, Polo remains on the breaks well into interphase. We further show that the appearance of BubR1, Bub3 and Fizzy on DNA breaks is delayed in polo mutant, indicating that Polo is required for the robust and efficient recruitment of BubR1, Bub3 and Fizzy to DNA breaks. Finally, the timely accumulation of Polo, BubR1 and Bub3 to DNA breaks depends on two components of the DNA Damage Response, the MRN complex (Mre11--‐Rad50--‐Nbs1) and ATM (ataxia--‐telangiectasia mutated). This work gives us a better understanding on how Polo and BubR1, Bub3 and FizzyCdc20 are recruited to DNA breaks in mitosis and how they promote broken chromosomes segregation.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by bone marrow failure, genetic malformations, and predisposition to malignancies like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations in FA genes are also found in a variety of sporadic cancers. Recently, numerous reports have discovered that the protective function of the FA pathway extends beyond its canonical role in regulation of DNA repair in interphase. In particular, the FA pathway has been shown to function in essential mitotic processes including spindle assembly checkpoint (SAC), cytokinesis, and centrosome maintenance. Understanding of the mechanistic origins of genomic instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. To this end, using a micronucleus assay, we showed that both interphase DNA damage and mitotic errors contribute to genomic instability in FA ex vivo and in vivo. Functional studies of primary FA patient cells coupled with super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in mitotic processes. Furthermore, we dissected the interactions between the FA pathway and cellular kinase networks by employing a synthetic lethality sh-RNA screen targeting all human kinases. We mapped kinases that were synthetically lethal upon loss of FANCA, particularly those involved in highly conserved signal transduction pathways governing proliferation and cell cycle homeostasis. We mechanistically show that loss of FANCA, the most abundant FA subtype, results in in premature degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1 to prevent its premature degradation. Our results deepen our understanding of the molecular functions of the FA pathway in mitosis and uncover a mechanistic connection between FANCA and SAC phosphosignaling networks. These findings support the notion that further weakening the SAC through targeting kinases like BUBR1 in FA-deficient cancers may prove to be a rational therapeutic strategy.

No description available.

Chez la femme, le risque de concevoir un embryon aneuploïde augmente de façon exponentielle dès 35 ans. La majorité de ces aneuploïdies sont dues à de mauvaises ségrégations des chromosomes lors des deux divisions de méiose dans l'ovocyte. Afin de limiter les erreurs de ségrégation, les divisions méiotiques doivent impérativement se dérouler en deux temps et de manière contrôlée. Un premier aspect de ma thèse a consisté à étudier les mécanismes qui régulent la séparation des chromosomes dans l'ovocyte de souris. J'ai montré que la Cycline A2 joue un rôle essentiel pour la séparation des chromatides s¿urs en méiose II. Au contraire, cette protéine doit impérativement être absente des régions centromériques en méiose I afin d'éviter une séparation précoce des chromatides s¿urs qui conduirait à la formation d'un ovocyte aneuploïde. Dans un seconde temps, mes travaux de thèse ont visé à étudier le mécanisme de surveillance de la première transition métaphase-anaphase appelé SAC (Spindle Assembly Checkpoint). Il a été montré que l'expression d'une protéine du SAC appelée BubR1 décroît naturellement dans les ovocytes avec l'âge maternel. Afin d'analyser les conséquences de cette diminution, j'ai utilisé une lignée de souris totalement délétée pour la protéine BubR1 spécifiquement dans les ovocytes. J'ai montré que la perte totale de BubR1 entraîne plus de 80% d'aneuploïdies dans l'ovocyte dès la première division de méiose. La méiose I est accélérée et les chromosomes homologues se séparent de façon anarchique. De plus, le fuseau méiotique devient instable. La diminution naturelle de BubR1 pourrait ainsi expliquer l'augmentation du nombre d'aneuploïdie avec l'âge maternel. / Women in industrialized countries tend to postpone childbearing, leading to a 70% increase intrisomic pregnancies over 20 years. Meiosis in females is error prone, with rates of meiotic chromosome missegregations strongly increasing towards the end of the reproductive lifespan. A strong reduction of BubR1 has been observed in oocytes of women approaching menopause and in ovaries of aged mice, which led to the hypothesis that deterioration of spindle assembly checkpoint fidelity contributes to age-related aneuploidization. However, this idea has remained controversial since transient knock-down of BubR1 was found to prevent meiotic prophase arrest and chromosome segregation in a checkpoint independent manner. We employed a conditional knockout approach in mouse oocytes to dissect the meiotic roles of BubR1. We show that BubR1 is required for diverse meiotic functions, including persistent spindle assembly checkpoint activity, timing of meiosis I, and establishment of robust kinetochore-microtubule attachments in a meiosis specific manner, but not prophase I arrest. These data reveal that BubR1 plays a multi-faceted role in chromosome segregation during the first meiotic division and suggest that age-related loss of BubR1 is a key determinant of formation of aneuploid oocytes as women approach menopause. Using mouse oocytes, a second aspect of my thesis reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role, namely, its requirement for separase- dependent sister chromatid separation in meiosis II.

No description available.