Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis fails to consistently induce sufficient reperfusion to facilitate repair, targeted manipulation of angiogenesis is clinically desirable. The Wilms' tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that WT1 in the endothelium influences the angiogenic function of endothelial cells. WT1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from WT1-GFP reporter mice. Inducible deletion of WT1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p < 0.05 relative to controls). In vivo, WT1 expression was evident in vascular endothelial and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia, often in cells expressing epithelial and mesenchymal markers simultaneously. However, VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density or expression of angiogenic factors (qRT-PCR) in sponges inserted subcutaneously in mice (20 days). To further understand the role of WT1 in angiogenesis, transcriptomic RNA expression analysis was performed in WT1+ and WT1- cells isolated (FACs) from sponges after implantation in WT1-GFP mice. WT1+ cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including angiogenesis (p=3.11x10-8), wound healing (p=3.45x10-7) and epithelial-to-mesenchymal transition (EMT) (p=5.86x10-4). These results shed new light on the role of WT1 in ischaemic angiogenesis. In concurrence with previously published work, we show that deletion of endothelial WT1 can delay angiogenesis however, WT1 is not just instrumental in endothelial cells in this context. WT1 has a broader role in tissue repair in ischaemia, in part through regulation of cell transition (EMT). This work has improved our understanding of the regulatory role of WT1 in angiogenesis and repair, while revealing a number of novel insights into the function of WT1. This highlights WT1 as a potentially beneficial therapeutic target to facilitate regeneration in cardiovascular disease.

腫瘤發生過程中，遺傳和/或表觀遺傳異常都可導致抑癌基因(TSG)的失活。新TSG的鑒定和研究對理解癌症發展至關重要，并能提供潛在的治療靶點和腫瘤標誌物。本研究的目標是在人類腫瘤中鑒定新的被表觀遺傳異常沉默的TSG并進一步研究其抑癌的分子機理。 / 表觀遺傳修飾因子是重要的腫瘤相關基因。這裡，我研究了兩個作為功能性TSG的表觀遺傳修飾基因。首先，我發現一個在腫瘤中被甲基化和下調的候選TSG, PRDM5, 可通過抑制TCF/LEF依賴的轉錄和引起多個癌基因的表觀遺傳下調而抑制癌細胞增殖。其次，通過檢測24個表觀遺傳修飾基因在癌細胞系中的表達，我發現一個頻繁下調的新候選TSG，TUSC12。TUSC12在正常組織中普遍表達，但在腫瘤細胞系中被啟動子區甲基化下調，且原發腫瘤中也存在高頻TUSC12甲基化。TUSC12顯著抑制癌細胞克隆形成能力，但這種抑制效果因其PHD功能域的失活被部份削弱。TUSC12與轉錄抑制因子KAP1在細胞核中共定位，并可能通過招募HDAC相關複合體而抑制基因轉錄。 / 另外，我研究了一個通過以前鼻咽癌(NPC) aCGH鑒定得到的新3p14.2 TSG，ZNF312。啟動子區甲基化導致ZNF312在NPC細胞系和原發癌中的沉默。恢復ZNF312表達可抑制NPC細胞生長，并引起細胞週期阻滯和凋亡。作為一個轉錄抑制因子，ZNF312靶向EZH2和MDM2的啟動子區并下調它們的表達。 / 之前的基因數字表達分析已篩選出一些可能在腫瘤中低表達的基因。我研究了一個通過這種方法分離出的新TSG，TUSC45。相比正常組織，TUSC45表達在癌組織中顯著下降，而且TUSC45低表達與病人的低存活相關。TUSC45在多個腫瘤細胞系中也被下調，但它的基因組缺失卻不多見。啟動子區甲基化導致TUSC45在多數細胞系中的沉默，而且藥物或遺傳去甲基化能顯著激活它的表達。特別的，TUSC45在腫瘤組織而不在正常組織中被高頻率甲基化。誘導TUSC45表達可抑制細胞增殖，引起細胞凋亡、週期阻滯、和衰老，並上調一個關鍵抑癌基因p53。TUSC45以p53依賴的方式激活p53的靶基因，而TUSC45對p53缺失的H1299和HCT116/p53KO細胞沒有生長抑制作用。TUSC45與p53/MDM2複合物結合并正向調節p53蛋白穩定性。 TUSC45表達導致MDM2蛋白半衰期縮短，提示一種可能的TUSC45調節p53通路的機制。 / 本研究表明癌變過程中四個抑癌基因腫瘤特異的甲基化和沉默引起了多個細胞信號通路紊亂，並可作為潛在的腫瘤檢測標誌物。 / Tumor suppressor genes (TSGs) can be inactivated by genetic and/or epigenetic alterations during carcinogenesis. Identification and characterization of novel TSGs are critical for the understanding of cancer development, and provide potential targets for clinical treatment and biomarkers for tumor diagnosis. In this study, I aimed to identify novel TSGs epigenetically silenced in human cancers and further characterize the molecular basis of their anti-tumorigenic functions. / Emerging evidence highlights the importance of epigenetic modifiers as cancer genes. Here, I characterized two epigenetic modifier genes as functional TSGs. First, I found PRDM5, a candidate TSG methylated and downregulated in multiple cancers, suppressed tumor cell proliferation through inhibiting TCF/LEF-dependent transcription and inducing epigenetic repression of multiple oncogenes. Second, through expression profiling of 24 epigenetic modifiers, I identified a novel candidate TSG, TUSC12, showing frequent silencing in tumor cell lines. TUSC12 was broadly expressed in human normal tissues, but downregulated by promoter CpG methylation in tumor cell lines. Frequent TUSC12 methylation was detected in primary tumors as well. TUSC12 dramatically inhibited tumor cell clonogenicity, but this growth inhibitory effect was partially impaired by disrupting its PHD domain. TUSC12 colocalized with the transcription corepressor KAP1 in the nucleus and is likely to repress gene transcription through recruit HDAC-associated complex. / I also studied a novel 3p14.2 TSG, ZNF312, identified from previous aCGH profiling of nasopharyngeal carcinoma (NPC). Frequent promoter methylation silenced ZNF312 in NPC cell lines and tissues. Restored ZNF312 expression strongly suppressed NPC cell growth through inducing cell cycle arrest and apoptosis. Further, ZNF312 acted as a transcription repressor targeting the promoter regions of EZH2 and MDM2 and downregulating their expression. / Moreover, previous digital gene expression subtraction from cDNA libraries revealed a list of genes possibly downregulated in tumors compared to normal tissues. I characterized a novel TSG, TUSC45, initially isolated from this strategy. The expression of TUSC45 was significantly reduced in tumor tissues compared to normal tissues, with lower TUSC45 expression associated with poorer patient survival. Downregulation of TUSC45 in multiple tumor cell lines was also observed, while only infrequent genomic deletion was detected. In contrast, promoter methylation was responsible for TUSC45 silencing in most cell lines, in which pharmacologic or genetic demethylation can dramatically restore its expression. Remarkably, TUSC45 was frequently methylated in primary tumors but not in normal tissues. Further, TUSC45 suppressed anchorage-dependent and -independent tumor cell growth. Induced TUSC45 expression inhibited cell proliferation, induced apoptosis, cell cycle arrest and senescence, and lead to the upregulation of a key tumor suppressor p53. Moreover, TUSC45 activated p53 target genes in a p53-dependent manner. Forced TUSC45 expression in p53-null H1299 and HCT116/p53KO (knock out) cells showed no inhibitory effect on cell growth. Finally, TUSC45 interacted with p53/MDM2 complex and positively regulated p53 protein stability. The protein half-life of MDM2 was shortened by TUSC45, indicating a possible mechanism for TUSC45 modulation on p53 signaling. / In conclusion, this study showed the tumor-specific methylation and silencing of the four TSGs lead to the epigenetic disruption of multiple cell signalings during tumorigenesis and could potentially be used as biomarkers for cancer detection. / 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. / Shu, Xingsheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 121-140). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Publications --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction and Literature Reviews --- p.1 / Chapter 1.1 --- Tumor suppressor genes (TSGs) and the pathways they control --- p.2 / Chapter 1.1.1 --- Basic concepts about TSG --- p.2 / Chapter 1.1.1.1 --- The discovery of TSG --- p.2 / Chapter 1.1.1.2 --- Knudson’s “two-hit“ hypothesis --- p.3 / Chapter 1.1.1.3 --- New insights of the “two-hit“ model --- p.4 / Chapter 1.1.2 --- Key TSGs and their cellular pathways --- p.5 / Chapter 1.1.2.1 --- p53 pathway --- p.6 / Chapter 1.1.2.2 --- Rb pathway --- p.9 / Chapter 1.1.2.3 --- APC and Wnt/β-catenin pathway --- p.10 / Chapter 1.1.2.4 --- Chromatin regulators with tumor suppressive properties --- p.12 / Chapter 1.1.3 --- Methods for TSG identification --- p.17 / Chapter 1.2 --- The epigenetic abnormalities of TSGs in cancer --- p.19 / Chapter 1.2.1 --- Promoter CpG methylation --- p.20 / Chapter 1.2.1.1 --- Molecular basis of DNA methylation --- p.20 / Chapter 1.2.1.2 --- Silencing of TSGs by promoter methylation --- p.22 / Chapter 1.2.1.3 --- Mechanism of methylation-induced transcription repression --- p.23 / Chapter 1.2.1.4 --- Abnormal DNA methylation contributes to early stages of tumorigenesis --- p.25 / Chapter 1.2.2 --- Aberrant histone modification and chromatin remodeling --- p.26 / Chapter 1.2.3 --- Clinical applications of epigenetic biomarkers and therapeutic targets --- p.28 / Chapter 1.2.3.1 --- DNA methylation and histone modification as biomarkers for cancer diagnosis and prognosis --- p.28 / Chapter 1.2.3.2 --- Epigenetic targets for cancer treatment --- p.29 / Chapter Chapter 2 --- Aims and Design of This Study --- p.32 / Chapter Chapter 3 --- Materials and Methods --- p.34 / Chapter 3.1 --- Cell lines --- p.34 / Chapter 3.2 --- Human normal and cancer tissues --- p.35 / Chapter 3.3 --- DNA and RNA extraction --- p.35 / Chapter 3.4 --- Semi-quantitative and quantitative RT-PCR --- p.36 / Chapter 3.5 --- DNA methylation analysis --- p.37 / Chapter 3.5.1 --- Bisulfite modification of genomic DNA --- p.37 / Chapter 3.5.2 --- CpG island analysis --- p.37 / Chapter 3.5.3 --- Methylation-specific PCR (MSP) --- p.38 / Chapter 3.5.4 --- Bisulfite genomic sequencing (BGS) --- p.38 / Chapter 3.5.5 --- Pharmacologic demethylation treatment of cell lines --- p.38 / Chapter 3.6 --- Luciferase assay of gene promoter activity --- p.39 / Chapter 3.7 --- Multiplex genomic-DNA PCR --- p.39 / Chapter 3.8 --- Construction of expression plasmids and PCR-mediated mutagenesis --- p.40 / Chapter 3.9 --- Plasmid mini- and midi-preparation --- p.41 / Chapter 3.10 --- Creation of stable cell line for inducible gene expression --- p.42 / Chapter 3.11 --- Monolayer-culture and soft-agar colony formation assay --- p.42 / Chapter 3.12 --- Cell proliferation assay --- p.43 / Chapter 3.13 --- Flow cytometry analysis of cell cycle --- p.43 / Chapter 3.14 --- Apoptosis assay --- p.44 / Chapter 3.15 --- Senescence cell staining --- p.44 / Chapter 3.16 --- Western blotting --- p.45 / Chapter 3.17 --- Co-immunoprecipitation (Co-IP) --- p.46 / Chapter 3.18 --- Transcription factor activity assay --- p.46 / Chapter 3.19 --- Immunofluorescence microscopy --- p.47 / Chapter 3.20 --- Chromatin Immunoprecipitation (ChIP) --- p.47 / Chapter 3.21 --- Gene expression and copy number analysis using Oncomine database --- p.48 / Chapter 3.22 --- Protein half-life assay --- p.48 / Chapter 3.23 --- In vivo ubiquitination assay --- p.48 / Chapter 3.24 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Two Epigenetic Modifying Genes, PRDM5 and TUSC12, Suppress Tumor Cell Growth and are Silenced by Promoter Methylation in Human Cancers --- p.50 / Chapter 4.1 --- PRDM5 --- p.50 / Chapter 4.1.1 --- PRDM5 is a candidate TSG downregulated and methylated in multiple carcinomas --- p.50 / Chapter 4.1.2 --- PRDM5 is a stress responsive gene but its response is disrupted by promoter methylation --- p.53 / Chapter 4.1.3 --- PRDM5 suppresses cancer cell growth and proliferation --- p.54 / Chapter 4.1.4 --- PRDM5 inhibits TCF/LEF-dependent transcription and induced epigenetic repression of multiple oncogenes --- p.55 / Chapter 4.2 --- TUSC12 --- p.59 / Chapter 4.2.1 --- mRNA expression profiling of epigenetic modifying genes in tumor cell lines --- p.59 / Chapter 4.2.2 --- Promoter CpG methylation contributes to TUSC12 silencing in tumor cells --- p.61 / Chapter 4.2.3 --- Demethylation of TUSC12 promoter restores its expression --- p.63 / Chapter 4.2.4 --- TUSC12 methylation in primary tumor tissues --- p.65 / Chapter 4.2.5 --- TUSC12 expression inhibits anchorage-dependent and -independent tumor cell growth --- p.66 / Chapter 4.2.6 --- TUSC12 is an epigenetic modifier repressing transcription --- p.68 / Chapter 4.3 --- Discussion --- p.69 / Chapter Chapter 5 --- The Human Zinc Finger Protein 312 is a Novel Tumor Suppressor for Nasopharyngeal Carcinoma --- p.73 / Chapter 5.1 --- Identification of ZNF312 as a candidate 3p14.2 TSG --- p.74 / Chapter 5.2 --- Silencing of ZNF312 by promoter methylation in NPC cell lines --- p.75 / Chapter 5.3 --- ZNF312 is frequently downregulated and methylated in primary NPC tissues --- p.78 / Chapter 5.4 --- ZNF312 suppresses tumor cell clonogenicity --- p.79 / Chapter 5.5 --- ZNF312 is a transcription repressor --- p.80 / Chapter 5.6 --- ZNF312 regulates cell cycle progression, induces apoptosis, and inhibits cell stemness --- p.83 / Chapter 5.7 --- ZNF312 represses oncogene expression --- p.85 / Chapter 5.8 --- Discussion --- p.87 / Chapter Chapter 6 --- Identification of a Novel Tumor Suppressor Regulating p53 Signaling and Frequently Methylated in Multiple Tumors --- p.91 / Chapter 6.1 --- TUSC45 is broadly expressed in human normal tissues but frequently downregulated in tumor cell lines --- p.91 / Chapter 6.2 --- Reduced TUSC45 expression in primary tumors is associated with poor survival of patients --- p.94 / Chapter 6.3 --- TUSC45 is mainly silenced by promoter CpG methylation in tumor cell lines --- p.96 / Chapter 6.4 --- Pharmacologic or genetic demethylation reactivates TUSC45 in silenced cell lines --- p.98 / Chapter 6.5 --- TUSC45 is frequently methylated in primary tumors --- p.99 / Chapter 6.6 --- TUSC45 suppresses anchorage-dependent and -independent tumor cell growth --- p.101 / Chapter 6.7 --- Induction of TUSC45 expression inhibits tumor cell growth through inducing apoptosis, cell cycle arrest and senescence --- p.104 / Chapter 6.8 --- TUSC45 tumor-suppressive function is dependent on p53 signaling --- p.108 / Chapter 6.9 --- TUSC45 positively regulates p53 protein stability --- p.110 / Chapter 6.10 --- Discussion --- p.112 / Chapter Chapter 7 --- General Discussion --- p.116 / Chapter 7.1 --- General discussion --- p.116 / Chapter 7.2 --- Future perspectives --- p.118 / Reference List --- p.121

Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 164-183). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 164-183). Also available on the Internet.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

TAX1 Binding Protein 2 (TAX1BP2) has been found to be a centrosome duplication regulating protein. Previous findings have demonstrated that over-expression of TAX1BP2 suppresses centrosome over-duplication. Recently, our lab has revealed that TAX1BP2 is a novel tumor suppressor in hepatocellular carcinoma (HCC) regulated by cyclin-dependent protein kinase 2 (CDK2), nevertheless, the molecular mechanism of how TAX1BP2 regulates centrosome duplication and the link between its centrosome duplication regulatory ability and the tumor suppressing property remain elusive. With the aim to understand the roles of TAX1BP2 in HCC, the present study intended to investigate the link between centrosome duplication regulating ability and tumor suppressing property. Polo-like kinase 4 (PLK4) is a special member of the Polo-like kinase family as its structure is diverged from other family members. Instead of having two Polo-boxes, it carries one Polo-box and one cryptic Polo-box. It has been shown that PLK4 is involved in the formation of centrioles, an important component of centrosome, and is a key regulator of centrosome duplication. Based on the functional similarity, it was hypothesized that PLK4 may function as a regulator of TAX1BP2. To define if PLK4 regulate TAX1BP2, the interaction between PLK4 and TAX1BP2, both in vivo and in vitro, was first confirmed using affinity pulldown and co-immunoprecipitation assays. To understand the significance of the physical interaction, in vitro and in vivo kinase assay were used to study the phosphorylation activity between PLK4 and TAX1BP2. It was demonstrated that TAX1BP2 is a potential substrate of PLK4. Centrosome duplication assay was also performed to investigate if over-expression of PLK4 abolished the centrosome over-duplication suppressing ability of TAX1BP2. In order to delineate the signaling pathway of TAX1BP2, the interaction between TAX1BP2 and its cellular interacting partners was investigated in this study. Ten proteins were isolated as potential interacting partners of TAX1BP2 using Tandem affinity purification (TAP) coupled with Mass Spectrometry protein fingerprinting. Two of the ten proteins, the Ezrin and Mortalin, were confirmed to be binding partners of TAX1BP2 using affinity pull-down assay and TAP, respectively. The identification of the interacting partners suggested that TAX1BP2 may modulate centrosome duplication via alteration of the subcellular localization of Mortalin. These findings helped to delineate the signaling pathway of TAX1BP2 and enabled the better understanding of the roles of TAX1BP2 in tumor suppressor function of HCC. In summary, we demonstrated that TAX1BP2 contains a centrosome duplication regulatory domain (CDRD) and its centrosome duplication regulating ability is critical for its tumor suppressing property. Moreover, three novel interacting partners of TAX1BP2, including Ezrin, PLK4 and Mortalin, are identified. Our findings provide a new insight into the roles of TAX1BP2 in centrosome duplication, hepatocarcinogenesis and metastasis. / published_or_final_version / Anatomy / Master / Master of Philosophy

Non-Hodgkin’s lymphoma is the fifth most common cancer in the UK, accounting for 4% of all new cases. The control of lymphomagenesis still remains a challenge. Early growth response gene (Egr) 2 and 3 are zinc finger transcription factors. Egr2 plays an important role in the development of both central nervous system and lymphocytes. However the mechanism of action in lymphocytes is still unknown. In order to fully understand the function of Egr2, in lymphocytes, we developed Egr2 and 3 double knockout mice (Egr2-/-Egr3-/-) by crossbreeding lymphocyte specific Egr2 knockout mice (CD2-Egr2-/-) with Egr3 knockout mice (Egr3-/-), as previous reports suggested that Egr3 compensates for the role of Egr2. In the absence of Egr2 and 3, the homeostasis of T cells is dysregulated with hyper-homeostatic proliferation of effector like phenotype cells. More importantly the development of spontaneous B and T cell lymphoma was found in more than 70% of Egr2-/-Egr3-/- mice. The lymphoma cells from Egr2-/-Egr3-/- mice were highly proliferative and metastatically spread into other non-lymphoid organs, such as lung, liver and kidney. In additional to this lymphoma development the Egr2-/-Egr3-/- mice showed signs of chronic inflammatory disorder. This inflammatory disorder was characterised by glomerulonephritis and an increase in serum cytokines, which may provide the microenvironment for the lymphoma development. To explore the molecular mechanism of tumour development in Egr2-/-Egr3-/- mice, the transcriptional profile of Egr2 was studied by microarray and ChIP-on-chip. We found firstly that Egr2 directly binds to the promoter regions of Ikaros and FOXO3. The deletion of Egr2 and 3 in lymphocytes led to the downregulation of Ikaros, Aiolos and FOXO3 expression. The impaired expression was found to be associated with proliferative disorder and the development of T and B cell lymphoma. Secondly Egr2 strongly inhibits STAT3 transcriptional activity by regulating SOCS3, which is a known inhibitor of STAT3. The breakdown of this regulation could be an important mechanism in lymphomagenesis. A model is proposed which defines Egr2 and Egr3 as the backbone of important tumour suppressor genes that control cell fate decision and regulates homeostasis in the lymphoid system. Thus, our results suggest that Egr2 and 3 are important regulators of lymphocyte function by their involvement in multiple cell signalling pathways, which could potentially be key genes for future cancer therapy.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

HACE1 is a tumour suppressor gene located at human chromosome 6q21. HACE1 is downregulated in Wilms’ tumour as well as several other human cancers. Its role in normal development remains unknown. The zebrafish has established itself as a robust model for studying vertebrate development and human cancers. A zebrafish hace1 homologue has been identified. Whole mount in situ hybridization (WISH) assays and colocalization studies demonstrate conserved hace1 expression. Moreover, morpholino knockdown of hace1 reveals perturbed cardiac development and function. Transgenic zebrafish harboring either wild type or dominant negative mutated C876S (C876S DN) human HACE1 genes have been generated. DN zebrafish display increased apoptosis, both untreated and following irradiation-induced cellular damage. There was no difference in cell cycle progression between wild type embryos and C876S DN. Further characterization of the HACE1 transgenic zebrafish model will serve to better our understanding of the role of human HACE1 in normal development and tumourigenesis.