Made available in DSpace on 2016-08-10T10:39:21Z (GMT). No. of bitstreams: 1 CIRCONCISTO LAURENTINO RIBEIRO JUNIOR.pdf: 2083186 bytes, checksum: bfcfede933b252abac1e5de63aa561f2 (MD5) Previous issue date: 2009-03-13 / Endometriosis it is considered as the presence of ectopic endometrial tissue, with similar histology and function to the endometrium usually located. However, the explanation for the implantation of the endometrial tissue in certain women is still unknown. The determination of the degree of compromising of the endometriosis is based on a system of points proposed by American Fertility Society finds of the laparoscopy. Endometriosis is a disease that occurs mainly in women in reproductive age. The disease can be related with infertility in 30% to 40% of the cases. The possible mechanisms as the endometriosis as cause of the infertility are: (a) interference with the sexual function (dyspareunia, reduction of the frequency of sexual intercourse). (b) Interference with the ovulation: (anovulation; deficient luteal phase and luteinization phase. The aim of this study was to determine the frequency of p53 polymorphism codon 72, in two groups of patients with endometriosis and other symptoms of the disease. The study included 38 peripheral blood samples from women submitted to videolaparoscopy that confirmed endometriosis. The patients were divided into two groups of 19 women each, one with infertility and the other not. The polymorphism was evaluated by PCR. The data were submitted to statistical analysis using the chi-square and odds ratio tests. Of the patients who only had pain and/or bleeding without infertility, 09 (47.3%) were arginine homozygous and 10 (52.6%) were proline homozygote or heterozygote. Of those who presented with infertility associated with pain and/or bleeding, 12 (100%) were proline homozygous or heterozygous, and in those with only complaint of infertility, 05 (71.4%) were arginine homozygous and 02 (28.6%) proline homozygous or heterozygous). In correlating pain intensity, 04 (23.5%) women with the proline allele (homozygous or heterozygous) reported slight or moderate pain and 20 (95.5%) intense pain. All these differences were statistically significant (p= 0.003). In relating pain intensity to laparoscopic classification, the following was found: 08 (50%) patients who showed slight or moderate pain had a classification I or II and 08 (50%) III or IV. Of those with intense pelvic pain, 08 (36.4%) were included in classification I or II and 14 (63.6%) in III or IV. The proline allele (homozygous or heterozygous) is linked more to patients with infertility and with a more severe clinical picture. / A endometriose é conceituada como a presença de tecido endometrial ectópico, com histologia e função semelhante ao endométrio normalmente situado. No entanto, a explicação para a implantação do tecido endometrial em determinadas mulheres ainda é desconhecida. O grau do comprometimento da endometriose baseia-se num sistema de pontos proposta pela American Fertility Society (1978), hoje denominada American Society for Reproductive Medicine com base nos achados de laparoscópica. A intenção deste estudo foi determinar a freqüência do polimorfismo do p53, códon 72, em dois grupos de pacientes com endometriose e outros sintomas da doença. O estudo incluiu 38 amostras de sangue periférico de mulheres submetidas à videolaparoscopia com diagnóstico confirmado de endometriose. As pacientes foram divididas em dois grupos de 19 mulheres cada, um com infertilidade e o outro não. O polimorfismo foi avaliado por PCR. Os dados foram submetidos a análise estatística sendo usado o teste de qui-quadrado e Odds Ratio. Dos pacientes que só tiveram dor e/ou sangramento sem infertilidade, 09(47,3 %) eram arginina homozigoto e 10(52,6%) eram prolina homozigoto ou heterozigoto. Das pacientes que apresentaram infertilidade associado à dor e/ou sangramento 12(100%) era prolina homozigoto ou heterozigoto. Das pacientes que queixavam apenas de infertilidade, 05 (71,4%) eram arginina homozigoto e 02 (28,6%) prolina homozigoto ou heterozigoto. Correlacionando intensidade da dor, 04(23,5%) com alelo prolina (homozigoto ou heterozigoto) informaram dor leve e/ou moderada e 20(95,5%) dor intensas. Todas essas diferenças foram estatisticamente significativas (p=0,003). Em intensidade da dor relacionado com a classificação laparoscópica foi encontrado o seguinte: 08(50%) pacientes que tiveram dor leve e/ou moderada tiveram classificação I ou II e 08(36,4%) foram incluídos na classificação III ou IV. Das pacientes com dor intensa, 08(36,4%) foram incluídas na classificação I ou II e 14 (63,6%) em III ou IV. Conclui-se que a presença do alelo prolina (homozigoto ou heterozigoto) está mais relacionado com pacientes com endometriose e com quadro clínico mais severo da doença.

Zhang, Zhiyi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves ). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

L’apoptose, ou mort cellulaire programmée, joue un rôle majeur au cours du développement embryonnaire et dans le maintien de l’homéostasie tissulaire chez l’adulte. La voie mitochondriale de l’apoptose est principalement activée par la protéine oncosuppressive p53. Le FGF1 est un facteur de croissance atypique, majoritairement intracellulaire et nucléaire qui induit la prolifération, la différenciation et la survie cellulaires. Dans les cellules PC12, le FGF1 présente des activités neurotrophique et anti-apoptotique vis-à-vis de l’apoptose dépendante de p53. De plus, il interagit avec la protéine p53. La localisation nucléaire du FGF1 semble nécessaire à ses activités intracellulaires ainsi qu’à son interaction avec p53.Au cours de ma thèse, j’ai étudié les activités neurotrophique et anti-apoptotique de différentes formes mutantes du FGF1. J’ai entrepris l’étude de l’activité intracellulaire de la forme FGF1K132E, forme mutante dont les activités extracellulaires sont inhibées. La mutation du résidu lysine 132 pourrait modifier la phosphorylation du résidu sérine 130 du FGF1, j’ai donc également étudié deux autres formes mutantes : le FGF1S130A, dont la phosphorylation est inhibée et le FGF1S130D dont la phosphorylation est mimée. Les résidus mutés (K132 et S130) sont situés dans le domaine C-terminal du FGF1.Cette étude nous a permis de montrer que la phosphorylation du FGF1 inhibe son activité anti-apoptotique mais ne modifie pas son activité neurotrophique, et que le domaine C-terminal du FGF1 est fortement impliqué dans la régulation de ses activités intracellulaires. Toutes ces formes mutantes sont capables d’être transloquées dans le noyau ce qui suggère que la localisation nucléaire du FGF1 soit nécessaire mais insuffisante pour ses activités intracellulaires. Par ailleurs, p53 peut interagir avec le FGF1WT et certaines formes mutantes, toutefois cette interaction n’est pas strictement corrélée à l’activité anti-apoptotique du FGF1, ce qui suggère l’existence d’autres régulations nucléaires qui restent à caractériser.Mes travaux ont permis pour la première fois de mettre en évidence le rôle de la phosphorylation du FGF1 et de son domaine C-terminal dans la régulation de ses activités intracellulaires. La poursuite de cette étude permettra de mieux caractériser le rôle nucléaire de ce facteur de croissance et de caractériser ses éventuelles interactions avec des protéines nucléaires comme p53 / Apoptosis, a form of programmed cell death, is required for embryonic development and tissue homeostasis. The mitochondrial pathway of apoptosis is mainly induced by p53, an oncosuppressor that acts as a transcription factor. FGF1 is one of the two prototypic members of the FGF family. Contrarily to most FGFs, FGF1 lacks a secretion peptide signal and acts mainly in an intracellular and nuclear manner. Intracellular FGF1 induces cell proliferation, differentiation and survival. In PC12 cells, FGF1 inhibits p53-induced apoptosis and interacts with p53. FGF1 nuclear localization seems to be required for its intracellular activities and its interaction with p53.To better characterize the FGF1 intracellular pathway, we studied neurotrophic and anti-apoptotic activities of several mutant forms of FGF1: the FGF1K132E that could affect FGF1 phosphorylation, and two phosphorylation-site mutant forms, i.e. the FGF1S130A (preventing phosphorylation) and the FGF1S130D (mimicking phosphorylation). All these mutations are localized in the C-terminal domain of FGF1. This study showed that phosphorylation inhibits FGF1 anti-apoptotic activity but not its neurotrophic activity in PC12 cells and that the FGF1 C-terminal domain is strongly involved in the regulation of its intracellular activities. Despite their different activities, all mutant forms are localized both in the cytosol and the nucleus. Therefore, nuclear localization is required but insufficient for FGF1 to display its intracellular activities.Besides, p53 can interact with wild-type and some of the mutant forms of FGF1. This interaction does not strictly correlate with FGF1 anti-apoptotic activity. Thus, the nuclear mechanisms regulating FGF1 intracellular activities remain to be characterized.Our study highlights for the first time the role of the phosphorylation and the C-terminal domain of FGF1 on the regulation of its intracellular activities. This work must continue on to further characterize FGF1 nuclear activities and its interactions with nuclear proteins such as p53

L’épithélium mammaire est organisé en bicouche : une couche de cellules luminales sécrétrices et une couche de cellules basales myoépithéliales. Des cellules souches multipotentes capables de régénérer l’épithélium mammaire après transplantation résident dans le compartiment basal tandis que les deux couches épithéliales mammaires contiennent des cellules souches/progénitrices à propriétés clonogéniques. De nombreuses études suggèrent que les cellules souches et progénitrices de la glande mammaire pourraient être les cibles d’une transformation oncogénique menant à des cancers du sein, justifiant l’attention particulière portée à ces populations cellulaires mineures.Pour étudier les rôles du proto-oncogène Myc, ou du suppresseur de tumeurs Trp53, dans le contrôle des fonctions des cellules souches et progénitrices, nous avons généré des souris transgéniques présentant une invalidation de Myc ou Trp53 dans la couche basale de l’épithélium mammaire. Les souris déficientes en Myc présentent des glandes hypoplasiques et les cellules basales dépourvues de Myc perdent complètement leur capacité régénérative. En revanche, la perte de p53 induit une augmentation des populations de cellules souches et progénitrices avec un potentiel d’autorenouvellement accru, suggérant que p53 restreint la propagation des cellules souches/progénitrices dans l’épithélium mammaire. Ces résultats montrent que Myc et p53 jouent un rôle essentiel dans le contrôle des fonctions des cellules souches et progénitrices mammaires.Les souris transgéniques K5Ncat générées précédemment dans notre laboratoire, développent des carcinomes mammaires de type basal avec des composants métaplastiques, induits par l’expression d’une forme activée de la -caténine dans la couche basale mammaire. Nous avons trouvé que la population de cellules souches fonctionnelles est augmentée dans l’épithélium pré-néoplasique des souris K5Ncat. Pour étudier les mécanismes cellulaires et moléculaires du développement de ces tumeurs, les souris déficientes en Myc ou Trp53 ont été croisées avec des souris K5Ncat. Nous avons constaté une inhibition complète de la tumorigenèse induite par la -caténine en absence de Myc et une accélération de celle-ci en absence de p53. Nos résultats suggèrent que les cellules souches/progénitrices basales pourraient être à l’origine des tumeurs mammaires de type basal avec des caractéristiques métaplastiques et que les rôles joués par Myc et p53 dans la tumorigenèse sont liés à leurs fonctions régulatrices des cellules souches mammaires. / Numerous studies suggested that mammary stem and progenitor cells could be targets of the oncogenic transformation in breast cancer, attracting a particular attention to these cell populations. Mammary epithelium is organized as bilayer, with a layer of luminal secretory cells and a basal myoepithelial layer. Multipotent stem cells able to regenerate mammary epithelium upon transplantation reside in the basal compartment, whereas both mammary epithelial cell layers contain clonogenic stem/progenitor cells.To study the roles played by a proto-oncogene Myc and a tumor suppressor p53 in the control of mammary stem and progenitor cell function, we generated mouse mutants presenting conditional deletion of Myc and Trp53 genes in the basal layer of the mammary epithelium. The Myc-mutants presented hypoplastic glands, and basal cells depleted of Myc were unable to regenerate mammary epithelium. In contrast, deletion of p53 led to increased stem and progenitor cell activity with enhanced capacity to self-renew suggesting that p53 restricts the propagation of the stem/progenitor cell populations in the mammary epithelium. These data clearly demonstrate that Myc and p53 play a central role in the control of the mammary stem cell function. Transgenic mice K5Ncat obtained previously by our team develop basal-type mammary carcinomas with metaplastic component, induced by the expression of activated (N-terminally truncated) -catenin in mammary epithelial basal layer. We found that stem cell activity is increased in preneoplastic K5Ncat mammary epithelium. To study molecular and cellular mechanisms underlying tumor development, Myc- and Trp53-mutants were bred to K5Ncat mice. The deletion of Myc completely inhibited tumorigenesis, whereas in the absence of p53, tumor development was significantly accelerated. Our data suggest that basal stem/progenitor cells might be at the origin of basal-type breast tumors with metaplastic characteristics and that roles played by Myc and p53 in the tumorigenesis are associated with their regulatory functions in stem cells.

Les résultats insuffisants de la radiochimiothérapie conventionnelle ont motivé l’évaluation de nouvelles cibles afin de moduler la radiosensibilité tumorale: voies intrinsèques impliquées dans la réponse aux rayonnements ionisants, vascularisation tumorale, stroma non vasculaire. A travers cette thèse, nous avons évalué trois nouvelles stratégies de radiosensibilisation pharmacologique. Nous avons d’abord étudié en association à la radiothérapie l’intérêt de la modulation de l’axe p53/Mdm2 par le JNJ26854165, un inhibiteur de la dégradation de p53 par le protéasome. Les résultats in vitro et in vivo dans des xénogreffes sous-cutanées de cancers bronchiques non à petites cellules (CBNPC) montrent que cette stratégie permet d’améliorer significativement l’efficacité de la radiothérapie. Nous avons également rapporté des résultats encourageants in vitro dans plusieurs lignées cellulaires tumorales avec un nouvel agent antivasculaire ciblant la tubuline, l’EHT 6706. Cette stratégie augmentait l’efficacité de l’irradiation et potentialisait l’effet antiprolifératif de certains agents de chimiothérapie conventionnelle. Enfin, le développement le plus abouti a consisté en l’évaluation de l’association d’un triple inhibiteur de MET/AXL/FGFR en association à l’irradiation in vitro et dans des modèles de CBNPC implantés en xénogreffes sous-cutanées, mais également sous forme de tumeurs pulmonaires orthotopiques. Cet agent pharmacologique potentialisait l’efficacité de la radiothérapie dans des lignées ne surexprimant pas MET. Il est apparu que l’activité de la drogue faisait intervenir, au moins partiellement, l’inhibition de l’activité d’acteurs de la cytocinèse. Ces trois évaluations, qui s’inscrivent dans la recherche translationnelle, montrent l’importance de la recherche préclinique pour les études d’association aux rayonnements ionisants. Seul un développement préclinique rationnel permettra de faire émerger de nouveaux standards dans le domaine de la biomodulation pharmacologique de la radiosensibilité tumorale. / Insufficient results of conventional chemoradiation have encouraged assessment of new targets for radiosensitization: intrinsic cellular pathways involved in radiation response, tumor angiogenesis, and nonvascular stroma. We have investigated these three strategies for pharmacological radiosensitization. First, we examined the usefulness of targeting p53/Mdm2 pathway in combination with irradiation. In vitro and in vivo results obtained in non-small cell lung carcinoma (NCSLC) showed that this strategy was promising for enhancing radiation efficacy. We also found encouraging results within several cell lines with a novel vascular disrupting agent targeting tubulin. This strategy enhanced radiation effects and also increased the antiproliferative effects of various chemotherapeutics. Finally, the most advanced preclinical development was obtained with a novel MET/AXL/FGFR inhibitor, which improved effectiveness of radiation therapy in vitro and in subcutaneous and orthotopic models of non MET-dependent cell cancer lines. This effect was not only related to an inhibition of stroma/cancer cell interactions, as it probably involved activity toward actors of cytocinesis. These studies, which are part of translational research, highlight the importance of preclinical investigations in the area of radiation research. Only rationale preclinical development will allow new standards to emerge for pharmacological modulation of tumor radiosensitivity.

P53 est mutée dans plus de la moitié des cancers humains et son inactivation est souvent associée à une résistance à la thérapie anti-cancer. Notre équipe a montré que dans le cas de tumeurs où p53 est inactive, la surexpression de la phosphatase Wip1 permettait la restauration de l’efficacité de la chimiothérapie, et une protection des tissus sains face aux effets secondaires du traitement. Afin d’améliorer cette stratégie, deux objectifs principaux ont été étudiés : trouver une protéine qui peut interagir avec la voie de signalisation de Wip1 et potentialiser son action dans cette stratégie thérapeutique, et trouver des molécules chimiques pouvant activer Wip1. Nous avons réalisé un criblage d’interférence à ARN de l’intégralité du kinome humain afin d’identifier plusieurs kinases, dont l’inhibition potentialise l’action anti-tumorale de Wip1. Cela a ainsi mis en évidence l’action de Wee1 et Hipk2, dont des inhibiteurs existent, sur la voie de signalisation de Wip1. Inhiber Wee1 avec une faible dose de MK-1775, un inhibiteur spécifique de cette kinase, a permis de réduire la concentration efficace de cisplatine, en entraînant une apoptose caspase-3-dépendante. De plus, combiner MK-1775 avec la surexpression de Wip1 n’a pas d’impact sur l’effet protecteur de cette phosphatase envers les tissus sains. Nous avons ensuite montré que le Vorinostat, un inhibiteur des histone-déacétylases, permettait une augmentation de la transcription de Wip1 dans des cellules de cancer du sein mutées pour p53. Ce travail de thèse a permis de mettre en évidence un moyen de potentialiser la stratégie de traitement des tumeurs p53-négatives basée sur la phosphatase Wip1. / P53 is mutated in more than half of human cancers and when inactivated is often associated with a resistance to anti-cancer therapy. Our team has hown that in the case of p53-negative tumors, overexpression of Wip1 phosphatase sensitizes tumor cells to chemotherapy, while protecting normal tissues from the side effects of the treatment. To improve this strategy, two main objectives were studied. Firstly, to find a protein which can interact with Wip1 pathway and potentiate its action in this therapeutic strategy. Secondly, to find a molecule which can activate Wip1. We realized a siRNA screening of the whole human kinome to identify several kinases, whose inhibition could potentiate anti-tumoral action of Wip1. We have shown that Wee1 and Hipk2, which both have available inhibitors, have an action on Wip1 pathway. Inhibiting Wee1 with a low dose of MK-1775, a specific inhibitor of this kinase, allowed us to decrease effective cisplatin concentration, inducing a caspase-3-dependent apoptosis. Moreover, the combination of MK-1775 with Wip1 overexpression does not impair the protective effect that this phosphatase provides towards normal tissues. We then showed that the Vorinostat, an HDAC inhibitor, induces an increase in Wip1 transcription in breast cancer cells with inactive p53. This work uncovered a way to potentiate the Wip1-based therapeutic strategy of p53-negative tumors.

The p53 tumor suppressor protein has been well-characterized for its role in inducing growth arrest, senescence, and apoptosis upon various types of stresses. Recently, however, roles of p53 have expanded beyond the canonical functions, and now include cellular processes such as metabolism, oxidative balance, and ferroptosis. Through RNA-seq screening, we first identified phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in the serine biosynthesis pathway, as a novel metabolic target of p53. p53 suppresses PHGDH expression and inhibits de novo serine biosynthesis. Notably, upon serine starvation, p53-mediated cell death is significantly enhanced in response to Nutlin-3 treatment. Moreover, PHGDH has been demonstrated to be frequently amplified in human melanomas. We found that PHGDH overexpression significantly suppresses the apoptotic response, whereas RNAi-mediated knock-down of endogenous PHGDH promotes apoptosis under the same treatment. Together, our findings demonstrate an important role of p53 in regulating serine biosynthesis through suppressing PHGDH expression, and reveal serine deprivation as a novel approach to sensitize p53-mediated apoptotic responses in human melanoma cells. In addition, we also identified spermidine/spermine N1-acetyltransferase 1 (SAT1) as a novel metabolic target of p53. SAT1 is a rate-limiting enzyme in polyamine catabolism critically involved in the conversion of spermidine and spermine back to putrescine. Surprisingly, we found that activation of SAT1 expression induces lipid peroxidation and sensitizes cells to undergo ferroptosis upon reactive oxygen species (ROS)-induced stress, which also leads to suppression of tumor growth in xenograft tumor models. Notably, SAT1 expression is down-regulated in human tumors, and CRISPR-cas9-mediated knockout of SAT1 partially abrogates p53-mediated ferroptosis. Moreover, SAT1 induction is correlated with the expression levels of arachidonate 15-lipoxygenase (ALOX15), and SAT1-induced ferroptosis is significantly abrogated in the presence of PD146176, a specific inhibitor of ALOX15. Together, these data indicate a novel regulatory role of p53 in polyamine metabolism and provide insight into the regulation of p53-mediated ferroptotic responses. Our studies on PHGDH and SAT1 led us to the question of whether these unconventional functions of p53 contribute to its role as a tumor suppressor. In fact, previous view regarding the mechanism of p53-mediated tumor suppression, which was long thought to be growth arrest, apoptosis, and senescence, has recently been challenged by several knockout and knock-in mouse studies. Previously, we established mice (p533KR/3KR) in which p53 acetylation at lysine residues K117, K161, and K162 were abolished by replacing lysine with arginine. p533KR/3KR mice completely lost p53-mediated cell cycle arrest, apoptosis, and senescence functions in response to stresses. However, unlike p53-null mice which rapidly develop spontaneous thymic lymphomas, all of the p533KR/3KR mice remain tumor-free, indicating that other aspects of p53 functions are sufficient to prevent tumor formation. Notably, p533KR retains the ability to regulate metabolic targets including TIGAR and SAT1, as well as ferroptosis regulator SLC7A11. In this study, we have identified two novel acetylation sites- K98 and K136, in the mouse p53 DNA-binding domain. Whereas loss of K98 or K136 acetylation (p53K98R, p53K136R) alone has modest effect on p53 transcriptional activity, simultaneous mutations at all of these acetylation sites (p534KR98: K98R+3KR, p534KR136: K136R+3KR, p535KR: K98R+K136R+3KR) completely abolish the ability of p53 to regulate TIGAR, SAT1, and SLC7A11. In addition, p534KR98, p534KR136, and p535KR are defective in Erastin-induced ferroptosis. Notably, p534KR98/4KR98, p534KR136/4KR136, and p535KR/5KR knock-in mice lost intact tumor suppression and developed spontaneous tumors. This suggests that p53-mediated ferroptosis may function as a critical barrier to prevent tumor formation independently from growth arrest, apoptosis, and senescence. Interestingly, both p534KR98/4KR98 and p534KR136/4KR136 mice displayed significantly delayed tumorigenesis comparing with p53-null and p535KR/5KR mice. We found that unlike p535KR, p534KR98 retains the capacity to inhibit mammalian target of rapamycin (mTOR) signaling pathway through activating the expression of two mTOR negative regulators, Sestrin2 and DDIT4. Altogether, our findings underscore the extensive scope of p53 functions in metabolic regulation, oxidative stress response, and ferroptosis, and provide novel insights into the tumor suppression mechanism of p53.

研究背景和目的 / 肝癌是非常高死亡率的恶性肿瘤之一。由于传统化疗方式的局限性，表观遗传治疗方法可能成为肝癌治疗的替代方法。研究报道ZBP-89诱导肝癌细胞Bak的表达，表观调控是否参与该诱导作用，目前仍然不清楚。 / HDAC3被认为是化疗靶点和肝癌复发的肿瘤标记物。它常常在肝癌组织中高表达，对HDAC3的抑制作用可以增加肝癌的化疗效果。我们的研究表明ZBP-89可以降低肝癌细胞HDAC3的表达，但机制未明。蛋白的翻译后调控是细胞生化过程的重要调节因素。所以，研究调节HDAC3的降低途径对肝癌的发生和复发具有非常重要的研究意义。 / 本研究旨在研究ZBP-89调控Bak表达的表观遗传机制。同时，弄清楚DNA甲基化转移酶和组蛋白去乙酰化酶是否参与ZBP-89对Bak的调控作用，进一步阐明ZBP-89对HDAC3降低通路的机制。 / 方法和结果 / 肝癌病人组织蛋白分析表明，相对于癌旁组织，肝癌组织Bak和ZBP-89蛋白表达降低，而DNMT1和HDAC3表达升高。免疫共沉淀技术显示ZBP-89与HDAC3、 DNMT1结合，但不与HDAC4， DNMT3a和DNMT3b结合。相应地，HDAC3和 DNMT1免疫沉淀分析也显示三者形成免疫复合物。我们在肝癌细胞中过表达ZBP-89，验证它会不会影响HDACs和DNMTs的活性。实验结果表明过表达的ZBP-89抑制HDACs和DNMTs的活性。进一步发现ZBP-89调节的Bak表达可能是通过抑制HDACs活性和维持组蛋白H3和H4乙酰化水平实现的。另一方面，我们同样证明HDAC的抑制剂（HDACi）VPA和TSA可以诱导肝癌细胞Bak表达，此外，siRNA干扰HDAC3的表达同样可以诱导Bak表达。 / 对DNMT1表达的抑制和使用DNMT抑制剂（DNMTi）Zebularine也可以诱导Bak的表达。染色质免疫沉淀结果显示ZBP-89结合于Bak的启动子区域，从-3188bp到-3183bp，从-275到-49。 ZBP-89可以抑制DNMT的活性，那么ZBP-89是否会影响DNA中CpG岛甲基化状态和甲基化结合蛋白（MeCP2）的结合能力，这一点仍需要进一步证实。结果表明ZBP-89可以抑制MeCP2结合基因组DNA。为进一步揭示MeCP2是否由于启动子区域CpG岛去甲基化影响其结合能力，我们采用亚硫酸盐测序方法。测序结果显示ZBP-89过表达可以影响Bak启动子CpG岛的甲基化状态，并促进其去甲基化。 / 腺病毒介导的ZBP-89过表达降低HDAC3表达呈现剂量依赖性，然而HDAC3 的mRNA水平并没有受到ZBP-89的表达。免疫共沉淀方法和蛋白免疫印迹实验用于分析Pin1和HDAC3复合物，磷酸化IκB和HDAC3复合物的结合情况。结果表明Pin1结合HDAC3并促进HDAC3的减少。同时，HDAC3与磷酸的IκB结合并进入蛋白减少途径。 / 构建的mU6-siPin1表达质粒用于敲除肝癌细胞Pin1的表达，方法检测基因表达水平。Pin1的缺失表达阻碍ZBP-89介导的HDAC3降低。在Pin1 敲除细胞系 JB6 C141 Pin1⁻/⁻ 和Pin1过表达细胞系的研究，ZBP-89更加能促进Pin1⁺/⁺细胞中HDAC3减少，而对Pin1⁺/⁺的细胞则没那么明显。由此肯定了Pin1在ZBP-89介导的HDAC3降低中的重要作用。进一步研究发现， IκB激酶 (IKK)抑制剂，CAY10576，能抑制 ZBP-89介导的HDAC3的降低；而SN50, p65/p50人核抑制多肽，则不影响HDAC3的降低。研究结果证明HDAC3的降低依赖IκB通路，而不是NF- κB活性。 / 我们用人肝癌细胞的裸鼠移植瘤模型研究ZBP-89调控Bak表达的表观遗传机制，及其对肝癌的治疗效果。研究结果表明ZBP-89蛋白和组蛋白抑制剂VPA和DNA甲基化抑制zebularine都能抑制肿瘤的生长，并诱导肿瘤组织Bak表达及细胞凋亡。VPA和zebularine联合治疗的效果更好。研究也表明ZBP-89可以在体内降低HDAC3蛋白水平。 / 结论 / 本研究揭示了ZBP-89调节Bak蛋白表达和肝癌细胞凋亡的表观遗传机制。同时，进一步揭示ZBP-89联合Pin1经由IκB通路调节HDAC3降低的机制. 本研究为肝癌表观遗传学的治疗提供研究基础和科学依据。 / Background / Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, epigenetic therapy may represent an alternative for HCC management. ZBP-89 is known to induce Bak in HCC. However, it is unclear whether epigenetic mechanisms contribute to ZBP-89-mediated Bak. / Histone acetylase 3 (HDAC3) is realized as a chemotherapy target and a biomarker of recurrence in HCC. HDAC3 is frequently overexpressed in HCC and its inhibition enhances the efficacy of anti-HCC chemotherapy. The pilot data have indicated that ZBP-89 reduced HDAC3 in HCC but the mechanism responsible was unknown. The post-translational modification of proteins functions as a key regulatory factor in cellular physiological procedures, such as ubiquitinoylation degradation. As a biomarker of HCC development and recurrence, it is important to understand how ZBP-89 mediates the reduction of HDAC3. / This study focuses on if ZBP-89 regulates Bak expression through epigenetic mechanisms. It is designed to investigate whether DNA methyltransferases (DNMTs), histone acetylases (HDACs) are involved in regulation of ZBP-89-induced Bak expression. The study also elucidates the mechanism how ZBP-89 reduces the level of HDAC3 protein. / Methods and Results / The levels of Bak and ZBP-89 as shown on western blots were reduced but DNMT1 and HDAC3 were increased in HCC cancer tissues compared to the corresponding non-cancer tissues. Co-immunoprecipitation experiments showed that ZBP-89 bound to HDAC3 and DNMT1 but not other epigenetic enzymes, such as HDAC4, DNMT3a and DNMT3b. To clarify if ZBP-89 affects the activities of HDACs and DNMTs, ZBP-89 was overexpressed in HCC cells. Enzyme activities of HDACs and DNMTs were determined using relevant assay kits. Results showed that overexpressed ZBP-89 inhibited the activities of HDACs and DNMTs. Further experiments indicated that ZBP-89-mediated Bak up-regulation might contribute to maintenance of histone H3 and H4 acetylation through inhibition of HDACs activity. In another set of experiments, we also found an increased Bak expression in HCC cells when the cells were treated with HDAC inhibitors (HDACi) VPA and TSA. HDAC3 siRNA also increased Bak expression. / Both knockdown of DNMT1 expression and administration of DNMTs inhibitors (zebularine) induced Bak expression. Chromatin immunoprecipitation (ChIP) showed that ZBP-89 bound to Bak promoter at the region from -3188bp to -3183bp and from -275 to -49. As ZBP-89 inhibits DNMT activity, it is essential to know whether its inhibition affectes DNA CpG methylation status and methyl-CpG binding protein (MeCP) binding. The results showed that ZBP-89 overexpression inhibited MeCP2 binding to genomic DNA. The finding indicated that decreased MeCP2 binding to DNA might be due to decreased methyl-CpG number in Bak promoter, suggesting that ZBP-89 might affect CpG island methylation status. Therefore, the bisulfite modified DNA sequencing method was used to clarify if Bak promoter CpG island methylation status was altered after ZBP-89 overexpression. Results revealed that ZBP-89 overexpression could demethylate the CpG islands in Bak promoter. / ZBP-89 overexpression dose-dependently reduced the expression of HDAC3 at protein level but not at mRNA level. Co-immunoprecipitation and western blot methods were used to analyze Peptidyl-prolyl cis/trans isomerase 1 (Pin1) and HDAC3, phospho-I kappa B (pIκB), and the result revealed that HDAC3 could bound with either Pin1 or pIκB to promote the reduced expression of HDAC3. / Constructed mU6-siPin1 vector was used to knockdown Pin1 expression in HCC cells. We found that knockdown of Pin1 expression blocked ZBP-89-mediated HDAC3 reduction. Experiments performed in Pin1 allele-knockdown JB6 C141 Pin1⁻/⁻ and Pin1⁺/⁺ cells showed that the reduction of HDAC3 by ZBP-89 was greater in Pin1⁺/⁺ cells than in Pin1⁻/⁻ cells, confirming the role of Pin1 in ZBP-89-mediated HDAC3 reduction. Furthermore, the ZBP-89-mediated HDAC3 reduction was suppressed by CAY10576, an IκB kinase (IKK) activation inhibitor but not by SN50, a p65/p50 translocation inhibitor, suggesting that HDAC reduction may depend on IκB kinase rather than NF-κB activity. / HCC xenograft mouse model was used to support the involvement of epigenetic mechanism in ZBP-89-induced Bak expression and its therapeutic effects against HCC. Results showed that ZBP-89 as well as HDAC inhibitor valproic acid (VPA) or/and DNMT inhibitor zebularine stimulated Bak expression and induced apoptosis of tumor cells in an HCC xenograft mouse model, arresting tumor growth. In HCC xenografe model, treatment by injection of Ad-ZBP-89 viral expression vector mediated ZBP-89 expression decreased HDAC3 expression, but not HDAC4. / Conclusions / In conclusion, the study demonstrates a novel mechanism through which ZBP-89 mediates an epigenetic pathway to promote Bak expression, and induce apoptosis in HCC cells. It also reveals the mechanism of HDAC3 reduction by ZBP-89 is dependent on IκB, which requires the presence of Pin1. This pathway may help develop future epigenetic therapy against HCC. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ye, Caiguo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 123-140). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Publications --- p.viii / Acknowledgements --- p.ix / Abbreviations --- p.xi / List of Tables --- p.xiii / List of figures --- p.xiv / Chapter Chapter One: --- General Introduction --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- The complexity of HDAC family and functions --- p.3 / Chapter 1.2.1 --- HDAC family --- p.4 / Chapter 1.2.2 --- Multifunction of HDACs --- p.6 / Chapter 1.3 --- HDACs and apoptosis --- p.6 / Chapter 1.3.1 --- HDAC regulates apoptotic-related gene expression --- p.9 / Chapter 1.3.2 --- HDACs regulate apoptosis through protein complexes --- p.18 / Chapter 1.3.3 --- HDACs mediates non-histone deacetylation and apoptosis --- p.21 / Chapter 1.3.4 --- HDACs degradation deficiency and apoptosis --- p.24 / Chapter 1.4 --- DNMTs and epigenetic modification --- p.25 / Chapter 1.4.1 --- DNMT family --- p.25 / Chapter 1.4.2 --- CpG islands methylation and HCC --- p.26 / Chapter 1.5 --- Perspectives --- p.28 / Chapter Chapter Two: --- ZBP-89 up-regulates Bak expression through inhibition the activity of HDACs and DNMTs --- p.30 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Materials and Methods --- p.33 / Chapter 2.2.1 --- Hepatocellular carcinoma patient samples and cell lines --- p.33 / Chapter 2.2.2 --- Chemicals and reagents --- p.34 / Chapter 2.2.3 --- Cell proliferation --- p.34 / Chapter 2.2.4 --- Adenovirus infection of cells --- p.35 / Chapter 2.2.5 --- Apoptosis detection --- p.36 / Chapter 2.2.6 --- Transfection of siRNA and plasmid --- p.36 / Chapter 2.2.7 --- Co-immunoprecipitation (co-IP) --- p.37 / Chapter 2.2.8 --- Western blotting --- p.37 / Chapter 2.2.9 --- Immunohistochemistry and Immunofluorescence --- p.38 / Chapter 2.2.10 --- Chromatin immunoprecipitation --- p.38 / Chapter 2.2.11 --- Sodium bisulfite modified sequencing of Bak promoter --- p.40 / Chapter 2.2.12 --- Histone deacetylase activity assay --- p.41 / Chapter 2.2.13 --- DNA methyltransferases enzyme activity --- p.42 / Chapter 2.2.14 --- Xenograft animal model --- p.43 / Chapter 2.2.15 --- Statistical analysis --- p.43 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- ZBP-89 interacts with DNMT1 and HDAC3 --- p.45 / Chapter 2.3.2 --- DNA methyltransferase-1 and histone deacetylase 3 are overexpressed in cancer tissues --- p.48 / Chapter 2.3.3 --- Inhibition of HDACs and DNMTs induces Bak expression and apoptosis --- p.58 / Chapter 2.3.4 --- Adenovirus mediated ZBP-89 expression inhibits HDACs activity --- p.65 / Chapter 2.3.5 --- ZBP-89 suppresses DNMTs activity --- p.67 / Chapter 2.3.6 --- Overexpressed ZBP-89 demethylates methyl-CpG islands --- p.69 / Chapter 2.3.7 --- Downregulation of HDAC3 and DNMT1 enhances Bak expression --- p.74 / Chapter 2.3.8 --- Xenograft nude mouse model reveals that Ad-ZBP-89 adenovirus diminishes tumor volume and induces Bak expression and apoptosis --- p.75 / Chapter 2.4 --- Discussion --- p.81 / Chapter Chapter Three: --- ZBP-89 targets IkappaB to reduce HDAC3 via a Pin1-dependent pathway --- p.86 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Materials and Methods --- p.89 / Chapter 3.2.1 --- Cell lines, chemicals and reagents --- p.89 / Chapter 3.2.2 --- Transfection of siRNA plasmid --- p.89 / Chapter 3.2.3 --- Plasmid extraction by mini-prep --- p.90 / Chapter 3.2.4 --- Co-immunoprecipitation (co-IP) and Western blotting --- p.91 / Chapter 3.2.5 --- Total RNA extraction --- p.92 / Chapter 3.2.6 --- Reverse transcription and real-time PCR --- p.93 / Chapter 3.2.7 --- Immunohistochemistry and Immunofluorescence --- p.94 / Chapter 3.2.8 --- Xenograft animal model --- p.95 / Chapter 3.2.9 --- Statistical analysis --- p.95 / Chapter 3.3 --- Results --- p.97 / Chapter 3.3.1 --- ZBP-89 overexpression diminishes HDAC3 expression but not HDAC4 --- p.97 / Chapter 3.3.2 --- Knockdown of Pin1 blocks ZBP-89-mediated HDAC3 reduction --- p.99 / Chapter 3.3.3 --- ZBP-89 reduces the level of IκB --- p.103 / Chapter 3.3.4 --- IκB degradation inhibitors suppresses ZBP-89-meditaed HDAC3 reduction --- p.105 / Chapter 3.3.5 --- ZBP-89 decreases HDAC3 but increases Bak in xenograft tumor tissues --- p.111 / Chapter 3.4 --- Discussion --- p.115 / Chapter Chapter Four: --- Conclusions and Future Perspectives --- p.119 / Chapter 4.1 --- Summary of results --- p.120 / Chapter 4.2 --- Conclusions --- p.121 / Chapter 4.3 --- Future Perspectives --- p.121 / References --- p.123

Mutação 249Ser no TP53 no Carcinoma Hepatocelular (CHC), frequente em países da África e Ásia, é uma evidência molecular de exposição à aflatoxina. O objetivo deste estudo é analisar a freqüência de 249Ser em 74 amostras de CHC no Brasil. A mutação foi analisada por RFLP e sequenciamento. A presença de vírus da hepatite B (VHB) foi analisada por PCR em tempo real. 249Ser foi encontrada em 13/74 (28%) e VHB em 13/74 (16%). A mutação foi encontrada em maior freqüência em tumores indiferenciados (OR = 2,415, IC = 1,001 - 5,824). O tamanho médio de tumores com 249Ser foi de 9,4 cm contra 5,5 cm de amostras sem a mutação (p=0,012). Não foi encontrada relação entre VHB e 249Ser. Os resultados indicam que 249Ser é um fator importante na carcinogênese do CHC no Brasil sendo associada à uma forma maior e menos diferenciada de tumor. / TP53 249Ser mutation has been proved a molecular evidence for aflatoxin-related Hepatocellular Carcinoma (HCC) and is frequent in Africa and Asia. The aim of our study was to analyze the frequency of 249Ser mutation in HCC from Brazil. We studied 74 samples of paraffin embedded HCC. 249Ser mutation was analyzed by RFLP and sequencing. Presence of HBV DNA was determined by Real-Time PCR. 249Ser was found in 21/74 (28%) samples while HBV DNA was found in 13/74 (16%). Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, IC = 1.001 - 5.824). The mean tumor size of 249Ser HCC was 9.4 cm versus 5.5cm on wild type (p=0.012). HBV DNA was not related with 249Ser. Results indicate that 249Ser is an important factor of HCC carcinogenesis in Brazil and is associated with large and poorly differentiated tumors.

O carcinoma epidermoide de lábio é precedido por uma desordem potencialmente maligna, a queilite actínica. Histologicamente o epitélio da lesão é caracterizado pela presença de hiperqueratose ou hiperparaqueratose, presença de displasia epitelial e, muitas vezes, pela presença de variações de espessura de epitélio, sendo estas a acantose e a atrofia. O desenvolvimento da queilite actínica está ligado a exposição excessiva a luz solar que contém radiação UV, esta pode produzir mutações com assinatura UV no DNA através de um fenômeno conhecido como fotocarcinogênese. As mutações com assinatura UV estão presentes no gene supressor de tumor TP53, quando este gene se encontra mutado, muitas vezes, ele codifica uma proteína que sofre um dobramento e exibe uma conformação específica. Esta pesquisa pretendeu determinar, à partir de 458 casos de queilite actínica, a porcentagem de casos que apresentaram espessura normal de epitélio, acantose ou atrofia, relacionando os graus de displasia epitelial, segundo o sistema da OMS e o sistema binário e também relacionar estas variações de espessura com a presença de proteína p53 mutada através de análise imuno-histoquímica. O que se observou foi que 20,31% dos casos apresentavam espessura normal de epitélio, 38,65% apresentavam acantose e 41,05% apresentavam atrofia. Não houve relevância estatística quando a variação de espessura foi comparada com a proteína p53 mutada. Concluiu-se que há uma dificuldade real em determinar quais queilites actínicas evoluirão para uma lesão maligna, por tanto, o acompanhamento clínico desses pacientes tem caráter essencial. / Squamous cell carcinoma of the lip is preceded by a potentially malignant disorder, the actinic cheilitis. Histologically the lesion is characterized by the presence of hyperkeratosis or hyperparakeratosis, the presence of dysplasia, and often, the presence of variations in thickness of the epithelium, which is acanthosis or atrophy. The development of actinic cheilitis is linked to the excessive exposure to sunlight containing UV radiation, this can produce UV signature mutations through the photocarcinogenesis. UV signature mutations are present in the tumor supressor gene TP53, when this gene is mutated, often it encodes a protein that undergoes bending and displays a specific conformation. This research intended to determine, from 458 cases of actinic cheilitis, the percentage of the cases whitch had normal thickness of the epithelium, acanthosis or atrophy and relate to the degree of dysplasia, and relates them to the presence of mutated p53 protein through immunohistochemical analysis. There was noted that 20.31% of cases showed normal thickness of the epithelium, 38.65% had acanthosis and 41.05% had atrophy. There was no statistical significance when the thickness variation was compared to the mutated p53, it was concluded that there is a real difficulty to determining which actinic cheilitis evolve to a malignant lesion, and therefore, clinical monitoring of these patients is of essential nature.