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

Objetivo: avaliar a concordância da expressão imuno-histoquímica (IMH) da proteína p53 (p53) na amostra endometrial e na peça cirúrgica de histerectomia, associando-a a fatores como tipo histológico, grau tumoral e estadiamento, bem como analisar a concordância interobservador para expressão e intensidade de expressão da p53 entre biópsia diagnóstica e peça cirúrgica. Métodos: estudo transversal retrospectivo, no qual foram incluídas pacientes com carcinoma endometrial submetidas à biópsia de endométrio e tratamento cirúrgico primário na Unidade de Oncologia Genital do HCPA. As lâminas foram lidas por dois patologistas. O percentual de expressão da p53 foi avaliado em três categorias (<10%, 10-50% e >50%). A intensidade foi avaliada como fraca, média ou forte. Foram extraídos dados do prontuário eletrônico das pacientes. A concordância foi avaliada através do coeficiente Kappa ponderado. Resultados: 72 pacientes foram incluídas, com idade média de 65,5 anos. O percentual de expressão da p53 entre biópsia e peça cirúrgica apresentou uma taxa de concordância de 70,8%, com um índice de Kappa ponderado de 0,64 e a intensidade de expressão apresentou uma taxa de concordância de 69,4%, com Kappa ponderado de 0,65. A avaliação da concordância do percentual de expressão da p53 conforme tipo histológico e grau tumoral apresentou coeficientes de Kappa ponderado de 0,64 e 0,72, nos carcinomas não endometrioides e G3, respectivamente. Tais dados, com diferença estatística significativa. A concordância interobservador para a expressão e intensidade da p53 na biópsia apresentou índices de Kappa 9 ponderado de 0,77 e 0,75, respectivamente. Na peça cirúrgica, os índices de kappa ponderado foram de 0,85 e 0,88, para a expressão e intensidade, respectivamente. Conclusões: as taxas de concordância para a expressão e intensidade de expressão IMH da p53 entre biópsia endometrial e peça cirúrgica foram boas, podendo ser útil em tempo pré-operatório, para a seleção de pacientes candidatas a uma cirurgia mais extensa, principalmente quando os dados anatomopatológicos da biópsia forem insuficientes para tal decisão. Devemos, no entanto, levar em consideração que existem muitos outros marcadores moleculares para a neoplasia endometrial, sendo provável futuramente o estudo da concordância deles em associação ao da p53. / Objective: To evaluate the agreement of the immunohistochemical (IMH) expression of the p53 protein (p53) in preoperative endometrial tissues and in surgical specimens obtained during hysterectomy, to determine the association of this agreement with factors such as histological type, tumor grade and stage and to evaluate interobserver agreement for the expression and intensity of p53 between the diagnostic biopsy and the surgical specimen. Methods: Retrospective cross-sectional study. Patients with endometrial carcinoma who submitted to endometrial biopsy and primary surgical treatment at the HCPA Genital Oncology Unit were included. The slides were assessed by two pathologists. The percentage of p53 expression was evaluated and categorized into one of three groups (<10%, 10-50% and >50%). The intensity was evaluated as weak, medium or strong. The agreement was assessed by the weighted Kappa coefficient. Results: 72 patients with a mean age of 65.5 years were included. The percentage of p53 expression between the biopsy and the surgical specimen presented an agreement rate of 70.8%, with a weighted Kappa index of 0.64. The intensity of expression had an agreement rate of 69.4% with a weighted Kappa of 0.65. When evaluated according to histological type and tumor grade, the agreement for p53 expression showed weighted Kappa indexes of 0.64 and 0.72 in non-endometrioid and G3 carcinomas, respectively (p<0.001). With respect to the biopsy, the interobserver agreement for the expression and intensity of p53 had weighted Kappa indexes of 0.77 and 0.75, respectively. With respect to the surgical specimen, the weighted Kappa indexes were 0.85 and 0.88 for expression and intensity, respectively. 11 Conclusions: This study found good agreement rates for the expression and intensity of p53 IMH expression between the endometrial biopsy specimen and the surgical specimen, which may be useful in a preoperative setting for the selection of patients who are candidates for more extensive surgery, especially when anatomopathological biopsy data are insufficient for such a decision.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.