A monoclonal antibody, mAb 203.37, raised against purified nuclear matrix proteins identified a single ~270 kDa protein that localized to the nuclear envelope. Double-label immunofluorescent microscopy using differential permeabilization protocols showed that this protein was present exclusively on the nucleoplasmic side of the nuclear envelope and that it co-localized with components of the nuclear pore complex. The nucleotide sequence of clones isolated using mAb 203.37 identified this protein as Tpr, a protein previously shown to be involved in oncogenic fusions with a number of protein kinases. Sequence analysis showed Tpr to be a 2348 amino acid protein with a predicted molecular weight of 265 kDa protein and a bipartite structure consisting of an ~1600 amino acid N-terminal domain that is almost entirely an α-helical coiled-coil followed by a highly acidic non-coiled carboxy-terminus. Ectopic expression of epitope-tagged Tpr constructs revealed two functional domains for Tpr: a nuclear pore complex binding domain and a nuclear localization sequence. The amino-terminus of Tpr, the portion of the protein shown to activate protein kinase oncogenes, did not localize to the nuclear pore complex indicating that the transforming activity of Tpr-protein kinase chimeras did not involve interactions with the nuclear pore complex. Ectopic expression of Tpr and a number of Tpr constructs resulted in the accumulation of poly (A)+ RNA in the nuclear interior but did not effect the import of a reporter protein into the nucleus indicating a role for Tpr in the export of mRNA from the nucleus.

Pancreatic cancer is the 4th leading cause of cancer related death in the United States with a median survival time of less than 6 months. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 85% of all pancreatic cancers, and is marked by early and frequent mutation of the KRAS oncogene, with activating KRAS mutations present in over 90% of PDAC. To date, though, targeting activated KRAS for cancer treatment has been very difficult, and targeted therapies are currently being sought for the downstream effectors of activated KRAS. Activation of KRAS stimulates multiple signaling pathways, including the MEK-ERK and PI3K-AKT signaling cascades, but the role of downstream effectors in pancreatic tumor initiation and progression remains unclear. I therefore used primary pancreatic ductal epithelial cells (PDECs), the putative cell of origin for PDAC, to determine the role of specific downstream signaling pathways in KRAS activated pancreatic tumor initiation. As one third of KRAS wild type PDACs harbor activating mutations in BRAF , and KRAS and BRAF mutations appear to be mutually exclusive, I also sought to determine the effect of activated BRAF (BRAF V600E ) expression on PDECs and the signaling requirements downstream of BRAF. I found that both KRAS G12D and BRAF V600E expressing PDECs displayed increased proliferation relative to GFP expressing controls, as well as increased PDEC survival after challenge with apoptotic stimuli. This survival was found to depend on both the MEK-ERK and PI3K-AKT signaling cascades. Surprisingly, I found that this survival is also dependent on the IGF1R, and that activation of PI3K/AKT signaling occurs downstream of MEK/ERK activation, and is dependent on signaling through the IGF1R. Consistent with this, I find increased IGF2 expression in KRAS G12D and BRAF V600E expressing PDECs, and show that ectopic expression of IGF2 rescues survival in PDECs with inhibited MEK, but not PI3K. Finally, I showed that the expression of KRAS G12D or BRAF V600E in PDECs lacking both the Ink4a/Arf and Trp53 tumor suppressors is sufficient for tumor formation following orthotopic transplant of PDECs, and that IGF1R knockdown impairs KRAS and BRAF-induced tumor formation in this model. In addition to these findings within PDECs, I demonstrate that KRAS G12D or BRAF V600E expressing tumor cell lines differ in MEK-ERK and PI3K-AKT signaling from PDECs. In contrast to KRAS G12D or BRAF V600E expressing PDECs, activation of AKT at serine 473 in the KRAS G12D or BRAF V600E expressing tumor cell lines does not lie downstream of MEK, and only the inhibition of PI3K alone or both MEK and the IGF1R simultaneously results in loss of tumor cell line survival. However, inhibition of MEK, PI3K, or the IGF1R in KRAS G12D or BRAF V600E expressing tumor cell lines also resulted in decreased proliferation relative to DMSO treated cells, demonstrating that all three signaling cascades remain important for tumor cell growth and are therefore viable options for pancreatic cancer therapeutics.

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

Maspina, proteína da família das serpinas, tem um único gene descrito, porém diversas funções biológicas foram observadas: modulação da adesão; inibição do crescimento, da angiogênese e da invasão tumoral; efeito pró-apoptótico entre outras. Está no núcleo, no citoplasma e na membrana plasmática. Tantas funções e localizações não podem ser justificadas apenas por sua estrutura primária. Maspina pode sofrer modificações pós-traducionais controlando sua atividade, localização subcelular e interações proteicas. Propôs-se assim, caracterizar a modificação de maspina pela adição de SUMO. Dois prováveis sítios de sumoilação, nas lisinas 47 e 277 e um provável motivo de interação com SUMO entre aminoácidos 156 e 159 foram encontrados pelos programas de predição SUMOplot, SUMOsp e GPS-SBM. A estrutura 3D de maspina dessas regiões mostra que são compatíveis e coerentes com sumoilação. Ensaio de imunoprecipitação sugere que maspina endógena é sumoilada na linhagem MCF-10A. Esses dados sugerem que sumoilação pode ser importante na regulação das funções biológicas de maspina. / Maspin, a protein from the serpin family, has only one gene described, but diverse biological functions observed: adhesion modulation; inhibition of growth, of angiogenesis and of tumoral invasion; pro-apoptotic effect and others. It is in the nucleus, the cytoplasm and the plasma membrane. Those many functions and localizations cant be justified only by its primary structure. Maspin could suffer posttranslational modifications controlling its activity, subcellular localization and protein interaction. So, we propose to characterize maspin modification by the addition of SUMO. Two possible sumoylation sites in lisines 47 and 277 and a possible SUMO interacting motif between amino acids 156 and 159 were found by prediction programs SUMOplot, SUMOsp and GPS-SBM. The 3D structure of maspin in those regions shows that they are compatible and coehrent with sumoylation. Immunoprecipitation assay suggests that endogenous maspin is sumoylated in MCF-10A cell line. This data suggest that sumoylation could be important in the regulation of maspin biological functions.

人類乳頭瘤病毒 (HPV) 是引起宮頸癌的必要條件。在高危型HPV中，以HPV16在癌症樣本中最為常見，其全球盛行率達50%以上。近年來，用以辨認HPV16變異子譜系的序列特徵已經建立。雖然這個系統建基於全球的HPV16變異株，但是它只包含了四個亞洲地區。為了改善這個系統於亞洲樣本的準確性，是次研究收集了更多亞洲地區的序列。 / 是次研究提供了在香港和韓國收集的HPV16樣本的系統發生史及序列變異 (LCR、E6 和 E7)。此外，是次研究也檢測了HPV16變異株的在兩地的分佈和致癌風險。 / 是次研究從香港和韓國收集了329個HPV16呈陽性的宮頸樣本。利用LCR、E6、E7 和整合的LCR-E6基因序列以極大似然法來構建HPV16變異株的系統發生樹。序列變異會按照系統發生樹之拓撲結構來分類並詳細描述。卡方檢驗或費雪精確性檢定用於分析HPV16變異株在兩地的分佈和致癌風險。 / 是次研究結果顯示用以辨認HPV16變異子譜系的序列特徵需加以改善。我們建議採用A7287C/T作為亞洲子譜系的序列特徵，以替代原有的A7287C。有關HPV16變異株的地理分佈，亞洲和歐洲的變異株在香港 (亞洲變異株: 70%，歐洲變異株: 25.3%) 和韓國 (亞洲變異株: 61.2%，歐洲變異株: 20.2%) 均十分普遍。另外，1和2型亞美變異株在香港和韓國的分佈有著明顯差別 (1型亞美變異株: 2% 與12.4%，P < 0.001; 2型亞美變異株: 0% 與2.8%，P = 0.04)。 / 另外，是次研究發現亞洲子譜系於韓國民族中呈較高致癌風險 [比值比 (95% 置信區間) 2.02 (1.03-3.99)]。在進化支中，E6的第五進化支[2.44 (1.27-4.74)]和E7的第三進化支[2.02 (1.03-3.99)]也於韓國民族中呈較高致癌風險。在SNP中，E6 T178G [2.17 (1.11-4.23)]、兩個E7的SNPs (A647G [1.73 (0.88-3.42)]、T846C [2.27 (1.16-4.49)]) 和9個LCR SNPs (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] 和 C24T [2.36 (1.20-4.66)])於韓國民族中也呈較高致癌風險。這些進化支和SNPs都與亞洲子譜系有關聯。在香港方面，兩個LCR SNPs (A7289C [1.89 (0.92-3.87)] 和 T7781C [2.07 (0.92-4.71)])呈較高致癌風險。 / 是次研究發現的高危SNPs和進化支需要進一步的大型流行病學研究和生物化學實驗來核實。這些序列特徵可作為生物標誌物以檢測出與HPV有關的早期宮頸病變。 / Human papillomavirus (HPV) is necessary for the development of cervical cancer. Of those high-risk HPV types, HPV16 is the most common type detected in cervical cancer and accounts for a prevalence of greater than 50% worldwide. Recently, a sequence signature-based system for identifying the sub-lineages of HPV16 variants has been established. Although this system was developed from HPV16 variants collected worldwide, only four Asian regions were included. To improve the accuracy of this sub-lineage classification system for Asian samples, more sequence data from Asian regions were included in the current study. / The current study provided data on the phylogeny and the sequence variation of Long control region (LCR), E6 and E7 open reading frames (ORFs) of HPV16 isolates collected in Hong Kong and Korea. The distribution of HPV16 variants between two regions and the risk association of HPV16 variants with cervical cancer development were also examined. / A total of 329 HPV16-positive cervical samples were collected from Hong Kong and Korea. The phylogenetic trees were constructed for the LCR, E6, E7 and concatenated LCR-E6 sequences using the maximum likelihood method. The sequence variation of each region was delineated and grouped according to the tree topology. The distribution and risk association of HPV16 variants were examined using the chi-square test or Fisher’s exact test as appropriate. / The results showed that the previously described sequence signatures for classifying sub-lineages of HPV16 variants required further improvement, especially for the Asian sub-lineage. We proposed A7287C/T as a signature SNP of the Asian sub-lineage rather than A7287C as suggested by Cornet et al. In regard to the distribution of HPV16 variants, the Asian (As) and European (Eur) variants were commonly found in Hong Kong (As: 70%, Eur: 25.3%) and Korea (As: 61.2%, Eur: 20.2%). Furthermore, Asian American-1 and 2 (AA1 and AA2) variants were found to distribute significantly different between Hong Kong and Korea (AA1: 2% versus 12.4%, P < 0.001; AA2: 0% versus 2.8%, P = 0.04). / A key finding was that variants of the Asian sub-lineage carried a higher oncogenicity among Korean population [odds ratio (95% confidence interval) = 2.02 (1.03-3.99)]. In clade level, E6 clade 5 [2.44 (1.27-4.74)] and E7 clade 3 [2.02 (1.03-3.99)] were found to carry a higher oncogenicity among Korean population. In SNP level, E6 T178G [2.17 (1.11-4.23)], two SNPs of E7 ORF (A647G [1.73 (0.88-3.42)] and T846C [2.27 (1.16-4.49)]) and nine SNPs of LCR (A7175C, T7177C, T7201C, C7270T, A7730C, G7842A [2.02 (1.03-3.99)], A7289C [2.04 (1.05-3.96)], T7781C [2.07 (1.02-4.22)] and C24T [2.36 (1.20-4.66)]) were also found to carry a higher oncogenicity among Korean population. Those clades and SNPs were linked to the Asian sub-lineage. In contrast, only two SNPs of LCR (A7289C [1.89 (0.92-3.87)] and T7781C [2.07 (0.92-4.71)]) were found to associate with a higher oncogenicity among Hong Kong population. / The risk associations of SNPs, clades of the HPV16 Asian sub-lineage revealed by the current study should be verified by large-scale epidemiological studies and biochemical experiments. These signatures may serve as biomarkers for early detection of HPV-related cervical neoplasia. / 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. / Ma, Tsz Ue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-156). / Abstracts also in Chinese. / Abstract of Thesis --- p.I / 論文摘要 --- p.V / Acknowledgements --- p.VIII / Contents --- p.X / Figures --- p.XIII / Tables --- p.XIV / Abbreviations --- p.XVI / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- History of Human Papillomavirus --- p.2 / Chapter 1.2 --- Biology of Human Papillomavirus --- p.4 / Chapter 1.2.1 --- Genome Organization and Protein Functions --- p.4 / Chapter 1.2.1.1 --- E5 Protein --- p.7 / Chapter 1.2.1.2 --- E6 Protein --- p.8 / Chapter 1.2.1.3 --- E7 Protein --- p.9 / Chapter 1.2.2 --- Life Cycle of Human Papillomavirus --- p.10 / Chapter 1.2.3 --- Taxonomy of Human Papillomavirus --- p.12 / Chapter 1.3 --- Cervical Cancer --- p.16 / Chapter 1.3.1 --- Natural History --- p.16 / Chapter 1.3.2 --- Risk Factors --- p.17 / Chapter 1.4 --- Epidemiology of Cervical Cancer --- p.19 / Chapter 1.4.1 --- Global Disease Burden --- p.19 / Chapter 1.4.2 --- Disease Burden in Hong Kong --- p.21 / Chapter 1.4.3 --- Disease Burden in South Korea --- p.22 / Chapter 1.5 --- Human Papillomavirus Type 16 --- p.23 / Chapter 1.6 --- Background and Objectives --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Study Design --- p.31 / Chapter 2.2 --- Study Samples --- p.35 / Chapter 2.2.1 --- HPV16-Positive Samples --- p.35 / Chapter 2.2.2 --- Samples with Unknown HPV Status --- p.36 / Chapter 2.3 --- Laboratory Methods --- p.39 / Chapter 2.3.1 --- DNA Extraction --- p.39 / Chapter 2.3.2 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.2.1 --- PGMY09/11 PCR --- p.40 / Chapter 2.3.2.2 --- HPV16-Specific PCR --- p.42 / Chapter 2.3.3 --- Genotyping of HPV --- p.48 / Chapter 2.3.4 --- Purification of PCR Products --- p.51 / Chapter 2.3.5 --- Sequencing Reaction --- p.52 / Chapter 2.4 --- Data Analysis --- p.54 / Chapter 2.4.1 --- Sequence Edit and Alignment --- p.54 / Chapter 2.4.2 --- Sequence Variation of HPV16 Variants --- p.56 / Chapter 2.4.3 --- Construction of Phylogenetic Tree --- p.56 / Chapter 2.4.4 --- Distribution and Comparison of HPV16 Variants in Hong Kong and Korea --- p.57 / Chapter 2.4.5 --- Distribution of HPV16 Variants in Normal and Cancer Samples and Risk Association Study --- p.58 / Chapter Chapter 3 --- Results --- p.59 / Chapter 3.1 --- Study Samples --- p.60 / Chapter 3.1.1 --- HPV16-Positive Samples --- p.60 / Chapter 3.1.2 --- Samples with Unknown HPV Status --- p.61 / Chapter 3.2 --- Sub-lineage Identification of HPV16 Variants --- p.63 / Chapter 3.2.1 --- Based on the Phylogenetic Analysis in the Current Study --- p.63 / Chapter 3.2.1.1 --- Concatenated LCR-E6 Phylogenetic Tree --- p.63 / Chapter 3.2.1.2 --- LCR Phylogenetic Tree --- p.66 / Chapter 3.2.1.3 --- E6 Phylogenetic Tree --- p.69 / Chapter 3.2.2 --- Based on the Single Nucleotide Polymorphisms Proposed by Cornet et al. --- p.74 / Chapter 3.2.2.1 --- Single Nucleotide Polymorphisms of LCR Sequence --- p.74 / Chapter 3.2.2.2 --- Single Nucleotide Polymorphisms of E6 Open Reading Frame --- p.78 / Chapter 3.3 --- Sequence Variation of HPV16 Variants --- p.82 / Chapter 3.3.1 --- LCR Sequence --- p.82 / Chapter 3.3.2 --- E6 Open Reading Frame --- p.91 / Chapter 3.3.3 --- E7 Open Reading Frame --- p.95 / Chapter 3.4 --- Distribution of HPV16 Variants in Hong Kong and Korea --- p.100 / Chapter 3.4.1 --- Sub-lineage Level --- p.100 / Chapter 3.4.2 --- Clade Level of E6 Open Reading Frame --- p.101 / Chapter 3.4.3 --- Clade Level of E7 Open Reading Frame --- p.102 / Chapter 3.4.4 --- Single Nucleotide Polymorphisms Level --- p.105 / Chapter 3.4.4.1 --- LCR Sequence --- p.105 / Chapter 3.4.4.2 --- E6 Open Reading Frame --- p.107 / Chapter 3.4.4.3 --- E7 Open Reading Frame --- p.108 / Chapter 3.5 --- Risk Association and distribution of HPV16 Variants in normal and Cancer samples --- p.112 / Chapter 3.5.1 --- Sub-lineage Level --- p.112 / Chapter 3.5.2 --- Clade Level of E6 Open Reading Frame --- p.114 / Chapter 3.5.3 --- Clade Level of E7 Open Reading Frame --- p.115 / Chapter 3.5.4 --- Single Nucleotide Polymorphisms Level --- p.122 / Chapter 3.5.4.1 --- LCR Sequence --- p.122 / Chapter 3.5.4.2 --- E6 Open Reading Frame --- p.125 / Chapter 3.5.4.3 --- E7 Open Reading Frame --- p.126 / Chapter Chapter 4 --- Discussion --- p.132 / Chapter 4.1 --- HPV16 Variant Sub-lineages --- p.133 / Chapter 4.2 --- Comparison of HPV16 variants between Hong Kong and Korea --- p.137 / Chapter 4.3 --- Risk Association of HPV16 Variants --- p.138 / Chapter 4.4 --- Strength and Weakness --- p.144 / Chapter 4.5 --- Implications for Future Work --- p.146 / References --- p.148

by Lai Wing Hong Kevin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 63-74). / Acknowledgments --- p.i / Abbreviations --- p.ii / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction --- p.3 / Chapter 1.1 --- Advantages of Antisense Oligonucleotides Inhibition --- p.4 / Chapter 1.2 --- The Uses of Antisense Oligonucleotide in Leukemic Therapy --- p.5 / Chapter 1.3 --- Oncogenes in the Pathogenesis of Leukemia --- p.6 / Chapter 1.4 --- Apoptosis and Apoptosis-Related Genes --- p.9 / Chapter 1.5 --- Protooncogene bcl-2 --- p.10 / Chapter 1.6 --- Bcl-2 Homologues --- p.11 / Chapter 1.7 --- Regulation of Apoptosis by Other Genes --- p.13 / Chapter 1.8 --- Promyelocytic Leukemia HL-60 Cell Line --- p.15 / Chapter 1.9 --- Aim of Project --- p.16 / Chapter Chapter 2 --- Chemical Synthesis of DNA Oligonucleotides / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.3 --- Results --- p.24 / Chapter 2.4 --- Discussion --- p.26 / Chapter Chapter 3 --- The Apoptotic Effects of TPA and Ouabain on the Promyelocytic Leukemic HL-60 cell line / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods --- p.33 / Chapter 3.3 --- Results --- p.40 / Chapter 3.4 --- Discussion --- p.44 / Chapter Chapter 4 --- Effect of Antisense Oligonucleotides on TPA-Induced Apoptosisin Leukemic HL-60 cells / Chapter 4.1 --- Introduction --- p.48 / Chapter 4.2 --- Materials and Methods --- p.49 / Chapter 4.3 --- Results --- p.52 / Chapter 4.4 --- Discussion --- p.54 / Chapter Chapter 5 --- General Discussion --- p.57 / References --- p.63

by Yam Hin Fai. / "June 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 118-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

Du Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 70-89). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.IV / PUBLICATIONS --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATIONS --- p.VIII / CONTENTS --- p.X / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- Laryngeal carcinoma and HPV --- p.1 / Chapter 1.2 --- HPV --- p.2 / Chapter 1.3 --- Human papillomavirus E6 protein --- p.6 / Chapter 1.3.1 --- Transformation by HPV E6 --- p.7 / Chapter 1.3.2 --- Inhibition of apoptosis by E6 --- p.8 / Chapter 1.3.3 --- Alteration of gene transcription --- p.11 / Chapter 1.3.4 --- E6 interation with other proteins --- p.12 / Chapter 1.3.5 --- E6 as a therapeutic target --- p.14 / Chapter 1.4 --- HPV E7 protein --- p.15 / Chapter 1.4.1 --- Regulation of viral life cycle by HPV E7 --- p.16 / Chapter 1.4.2 --- Degradation of retinoblastoma tumor suppressor by HPV E7 --- p.18 / Chapter 1.4.3 --- Inhibition of p53 by HPV E7 --- p.22 / Chapter 1.4.4 --- Interaction with other proteins by HPV E7 --- p.24 / Chapter 1.5 --- Objective --- p.26 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Materials for cDNA and RNA manipulation --- p.28 / Chapter 2.1.2 --- Culture media and transfection reagents --- p.28 / Chapter 2.1.3 --- Antibodies --- p.29 / Chapter 2.1.4 --- Materials for protein manipulation --- p.29 / Chapter 2.1.5 --- Kits --- p.30 / Chapter 2.1.6 --- Instrumentation --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Plasmid construction --- p.32 / Chapter 2.2.1.1 --- DNA preparation --- p.34 / Chapter 2.2.1.2 --- DNA ligation --- p.34 / Chapter 2.2.1.3 --- Transformation of competent E. coli --- p.35 / Chapter 2.2.2 --- Mini preparation --- p.35 / Chapter 2.2.3 --- Clone selection and confirmation --- p.37 / Chapter 2.2.4 --- Sequencing gel electrophoresis --- p.37 / Chapter 2.2.5 --- Cell culture and cytokine treatment --- p.39 / Chapter 2.2.6 --- Plasmid transfection --- p.39 / Chapter 2.2.7 --- Confirming construction of stable cell lines by RT-PCR --- p.40 / Chapter 2.2.7.1 --- Total cellular RNA extraction --- p.40 / Chapter 2.2.7.2 --- First strand cDNA synthesis --- p.41 / Chapter 2.2.7.3 --- Polymerase chain reaction (PCR) --- p.41 / Chapter 2.2.8 --- Fluorescence microscopy and imaging --- p.43 / Chapter 2.2.9 --- DNA fragmentation assay --- p.44 / Chapter 2.2.10 --- Protein detection --- p.46 / Chapter 2.2.10.1 --- Preparation of protein extract --- p.46 / Chapter 2.2.10.2 --- SDS-PAGE electrophoresis and protein transfer --- p.47 / Chapter 2.2.10.3 --- Immunoblotting analysis --- p.47 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE: --- RESULTS --- p.49 / Chapter 3.1 --- Plasmid construction --- p.49 / Chapter 3.2 --- Expression of HPV16 viral oncogenes in transfected UMSCC12 --- p.51 / Chapter 3.3 --- HPV16 E6 and E7 protect apoptosis induced by TNF-alpha and CHX --- p.53 / Chapter 3.4 --- Detection of apoptosis with fluorescence staining --- p.55 / Chapter 3.5 --- Regulation of the expression of apoptosis-associated proteins by E6 and E7 oncoproteins --- p.57 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.59 / Chapter CHAPTER FIVE: --- CONCLUSION AND FUTURE PERSPECTIVE --- p.68 / REFERENCES --- p.70 / APPENDIX DNA SEQUENCING RESULTS --- p.90

Background. B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) acts as an oncogene in various cancer such as non-small cell lung cancer, colon cancer, gastric cancer, bladder cancer and nasopharyngeal cancer (NPC). / Methods. Immunohistochemistry was performed to evaluate Bmi-1 expression in 252 breast cancer samples. The correlations were analyzed between Bmi-1 expression and clinicopathologic parameters, including age, tumor size, lymph nodal involvement, distant metastasis, clinical stages, hormone receptor (ER, PR) and Human Epidermal Growth Factor Receptor 2 (HER-2). The overall survivals were compared by Kaplan-Meier analysis based on Bmi-1 expression. / Results. Bmi-1 expression was significantly increased in primary cancer tissues than in matched adjacent non-cancerous tissues ( P&lt;0.001). Only 35.9% (14 of 39) of adjacent non-cancerous tissues displayed high expression compared with 72.2% (182 of 252) in primary cancer tissues. Among adjacent non-cancerous tissues, no Bmi-1 staining signal was detected in 30.8% (12 in 39) samples. Only 28.2% (11 in 39) samples showed nucleus staining and the remaining 41.0% (16 in 39) samples exhibited cytoplasm staining. Of those cancer tissues, however, 75.4% (190 in 252) was stained in the nucleus and 24.6% (62 in 252) located in the cytoplasm. The elevated Bmi-1 expression was correlated with advanced clinicopathologic classifications (T, N, M) and clinical stages (P&lt;0.001, respectively). A high level of Bmi-1 expression displayed unfavorable overall survival ( P&lt;0.001). The overall survival rate, assessed by the Kaplan-Meier method, was 85.1% (57 in 67) in low Bmi-1 expression group, whereas it was only 59.9% (103 in 172) in high Bmi-1 expression group. In addition, Bmi-1 serves as a high risk for breast cancer and the relative risk increased almost four fold in patients with high Bmi-1 expression compared with that with low Bmi-1 expression by univariate Cox regression analyses. After the adjustment of the confounding factors, Bmi-1 was still found to predict the poor survival (P=0.042), which indicated Bmi-1 was an independent prognostic factor. The overexpression of Bmi-1 increased the mobility and invasiveness in 76N-TERT and MCF-10A, concurrent EMT-like molecular changes, the stabilization of Snail protein and the activation of Akt/GSK3beta pathway. Consistent with these observations, the repression of Bmi-1 in MDA-MB-435S remarkably attenuated the cellular mobility, invasiveness and transformation, as well as tumorigenesis and spontaneous lung metastases in nude mice. In addition, the repression of Bmi-1 reversed the EMT markers and inhibited the Akt/GSK3beta/Snail pathway. However, ectopic Bmi-1 alone was not able to lead to the phenotype of HMECs. Additionally, discordant mRNA expression levels of Bmi-1 and E-cadherin were detected between primary cancer tissues and matched adjacent non-cancerous tissues. The mRNA level of Bmi-1 was strongly up-regulated in breast cancer tissues compared with paired non-cancerous tissues ( P=0.001), whereas the mRNA level of E-cadherin was markedly down-regulated (P=0.042). Furthermore, there was a converse correlation between Bmi-1 and E-cadherin expression at the transcriptional level ( P=0.041). (Abstract shortened by UMI.) / Guo, Baohong. / Adviser: Kung, Hsiang Fu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 161-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Maspina, proteína da família das serpinas, tem um único gene descrito, porém diversas funções biológicas foram observadas: modulação da adesão; inibição do crescimento, da angiogênese e da invasão tumoral; efeito pró-apoptótico entre outras. Está no núcleo, no citoplasma e na membrana plasmática. Tantas funções e localizações não podem ser justificadas apenas por sua estrutura primária. Maspina pode sofrer modificações pós-traducionais controlando sua atividade, localização subcelular e interações proteicas. Propôs-se assim, caracterizar a modificação de maspina pela adição de SUMO. Dois prováveis sítios de sumoilação, nas lisinas 47 e 277 e um provável motivo de interação com SUMO entre aminoácidos 156 e 159 foram encontrados pelos programas de predição SUMOplot, SUMOsp e GPS-SBM. A estrutura 3D de maspina dessas regiões mostra que são compatíveis e coerentes com sumoilação. Ensaio de imunoprecipitação sugere que maspina endógena é sumoilada na linhagem MCF-10A. Esses dados sugerem que sumoilação pode ser importante na regulação das funções biológicas de maspina. / Maspin, a protein from the serpin family, has only one gene described, but diverse biological functions observed: adhesion modulation; inhibition of growth, of angiogenesis and of tumoral invasion; pro-apoptotic effect and others. It is in the nucleus, the cytoplasm and the plasma membrane. Those many functions and localizations cant be justified only by its primary structure. Maspin could suffer posttranslational modifications controlling its activity, subcellular localization and protein interaction. So, we propose to characterize maspin modification by the addition of SUMO. Two possible sumoylation sites in lisines 47 and 277 and a possible SUMO interacting motif between amino acids 156 and 159 were found by prediction programs SUMOplot, SUMOsp and GPS-SBM. The 3D structure of maspin in those regions shows that they are compatible and coehrent with sumoylation. Immunoprecipitation assay suggests that endogenous maspin is sumoylated in MCF-10A cell line. This data suggest that sumoylation could be important in the regulation of maspin biological functions.