No description available.

²Ä¤@³¡¥÷ Nasopharyngeal carcinoma¡]NPC¡^ is a malignant tumor which occurs at high incidence in southern China. Several risk factors have now been recognized, but the molecular mechanism of this disease is not well understood. To investigate the c-myc¡Bcyclin D1¡Bp16¡Bp27 and Rb gene expression in NPC at protein level, 46 cases of nasopharyngeal carcinoma in southern Taiwan were detected by immunohistochemistry. There was no detectable p16 in 31/45 cases¡]69¢M¡^¡F 34/46 cases¡]73.9¢M¡^had intense staining for the Rb protein¡F 29¡]70.7¢M¡^of 41 cases had c-myc protein expression¡Fcyclin D1 was not overexpression in nasopharyngeal carcinoma¡F 32¡]69.6¢M¡^of 46 cases had high level expression of p27, which was inverse correlation with other tumors. No expression of c-myc protein correlated with higher neck metastasis¡]P¡Õ0.05¡^. No correlation was found between other proteins and any of the clinicopathological parameters. ²Ä¤G³¡¥÷ To better understand nasopharyngeal carcinoma¡]NPC¡^, we have newly established two NPC cell lines. Biopsy specimens from NPC patients were collected, primary culture were set up. Two NPC cell lines were established¡GNPCGK 01 was derived from differentiated carcinoma and NPCGK 02 was derived from undifferentiated carcinoma. Two cell lines have been passaged for more than 25 times. Two cell lines had telomerase activity¡Fstrong expression of hTERT gene and keratin-19 gene were also observed. TGF£] RI protein expression of these NPC cell lines is higher than normal epithelial cell.The oncogenes, c-myc¡Bc-fos and cyclin D1 were overexpressed. The Rb protein was expressed stronger than normal epithelial cells. NPCGK 01 that was derived from differentiated carcinoma had p16 down-regulation and p27 gene not expression, but p21 protein had excess expression. In short, two cell lines had cancer cell characteristics, oncoproteins were overexpression and tumor suppressor proteins were abnormal expression. This result may lead to tumorigenesis of nasopharyngeal carcinoma.

NK cell lymphoma is one of the cellular malignancies that arise from lymphocytes. Due to its rarity and aggressiveness the detailed molecular pathogenesis of NK cell lymphoma remains to be discovered. There are recent studies showing that the master regulator of B-cell differentiation into plasma cells, the Positive Regulatory Domain containing 1, with ZNF domain(PRDM1) has tumor suppressive function not only in diffuse large B-cell lymphoma (DLBCL), but also in NK cell lymphoma. The PRDM1 has two isoforms, αand β, where the former one is a functional isoform and the latter is a defective isoform with shortened and disrupted positive regulatory domain formed from transcription of internal promoter. By semi -quantitative RT-PCR, PRDM1-αexpression was found to be absent in 80% (4/5) NK cell lines while present in the normal NK cells. Loss of PRDM1 expression suggests its role as tumor suppressor. In order to study the tumor suppressive role of the αisoform of PRDM1, short-hairpin RNA (shRNA) with isoform specific sequence is used to knockdown the expression of PRDM1-αin NK cell lines. Western blot result showed about 40% decrease of PRDM1-αprotein after knockdown. Retroviral infection of the NK cell lines, NKYS and YT which have endogenous α-isoforms of expression, for the delivery of the shRNA was done and were subsequently subjected to in vitro functional analyses including MTS assay, colony formation assay, cell viability test and cell cycle analysis to determine potential effect of the loss of PRDM1-αon the NK cell lines. The PRDM1-αprotein isoform is expected to be able to repress excessive growth of NK cell line. When this isoform is inactivated, the NK cell lines are expected to proliferate significantly than the negative control counterpart in functional analyses. However in this study only YTcell line showed significant proliferation advantage in MTS and colony formation assay after the knockdown of PRDM1-α by shRNA. Cell viability assays and cell cycle analyses failed to show significant changes in both NK cell lines and yet even showed inhibitory effect after the knockdown of the gene. Ectopic expression of PRDM1-αby retroviral infection was done in KHYG cell line to further evaluate its tumor suppressive function. Apoptotic assay on the KHYG cells with ectopic expression of PRDM1-αwas performed and percentage of cells with late apoptosis was found to be significantly higher in this cell line. This suggests that one of the mechanisms for PRDM1-αto act as tumor suppressor is via the apoptosis pathway which in turn promotes the cell death. Future studies will be made to further investigate the effects of knockdown of PRDM-1αby designing another shRNA sequence which knockdown the expression of gene by at least 50% and to further investigate the role of PRDM1-αinthe pathogenesis NK cell lymphomaby proliferation assays, colony formation assay and cell cycle analysis. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Cutaneous melanoma is an increasingly common skin cancer characterized by aggressive metastatic growth and poor prognosis. The mechanisms behind melanoma progression are not fully understood, but emerging evidence suggests that a group of newly discovered small regulatory RNAs, named microRNAs (miRNAs), plays an important role. miRNAs are ~ 22 nucleotide single strand non-coding RNAs that post-transcriptionally regulate gene expression by binding to target messenger RNAs (mRNAs), leading to mRNA degradation and translation inhibition. Abnormal expression of miRNAs has been observed in human malignancies and is associated with tumorigenesis. The main goals of this thesis are to investigate miRNA dysregulation in melanoma and to identify potential miRNAs involved in melanoma pathogenesis. Initially, the expression of 470 miRNAs was profiled in 8 metastatic melanoma and 8 benign nevus tissue samples. We discovered unique miRNA expression profiles and identified differentially expressed miRNAs in melanomas as compared to nevi. miR-193b was one of the most significantly downregulated miRNAs in melanoma, and its function and regulatory targets were unknown. Subsequently, in vitro functional studies revealed that ectopic expression of miR-193b in melanoma cells drastically repressed cell proliferation and migration. Although it does not directly induce apoptosis in melanoma cells, miR-193b does sensitize these cells to ABT-737-mediated cell death. In concert with functional studies, gene expression analysis and in silico target prediction were performed to globally screen for mRNA targets of miR-193b. We identified eighteen genes as candidates in that they were downregulated by miR-193b and contained predicted miR-193b binding sites. Based on their known biological functions, three genes were particularly interesting: cyclin D1 (CCND1), myeloid cell leukemia sequence 1 (Mcl-1), and stathmin 1 (STMN1). CCND1 and Mcl-1 are two well-known melanoma oncogenes, and we validated their role in cell proliferation and apoptosis respectively. Furthermore, using similar approach, we were the first to identify STMN1 as a novel melanoma oncogene. We demonstrated that CCND1, Mcl-1, and STMN1 were directly regulated by miR-193b. During melanoma progression, reduced expression of miR-193b may promote cell proliferation, migration and survival. Taken together, this thesis describes the dysregulation of miRNAs in melanoma and demonstrates that miR-193b functions as a tumor suppressor. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2012-05-31 15:27:01.707

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph.D.)--Georgetown University, 2008. / Includes bibliographical references.

Thesis (M.Med.Sc.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 56-60). Also available in print.

No description available.

Yuen, Ka Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 160-185). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENTS --- p.VI / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Pseudomalignant nature of the placenta --- p.2 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- "Proliferation, migration and invasion behaviour" --- p.3 / Chapter 1.3 --- Gene expression --- p.4 / Chapter 1.3.1 --- Angiogenic factors --- p.5 / Chapter 1.3.2 --- Growth factors --- p.5 / Chapter 1.3.3 --- Proto-oncogenes --- p.6 / Chapter 1.3.4 --- Tumour suppressor genes --- p.8 / Chapter CHAPTER 2: --- Epigenetics --- p.10 / Chapter 2.1 --- Overview --- p.10 / Chapter 2.2 --- DNA methylation in mammals --- p.11 / Chapter 2.3 --- Regulation of DNA methylation machinery --- p.12 / Chapter 2.4 --- Role of DNA methylation --- p.13 / Chapter 2.5 --- Aberrant DNA methylation --- p.16 / Chapter 2.6 --- DNA methylation in normal cells --- p.17 / Chapter 2.6.1 --- X-chromosome inactivation --- p.17 / Chapter 2.6.2 --- Genomic imprinting --- p.18 / Chapter 2.6.3 --- Cell-type-specific methylation --- p.19 / Chapter 2.6.4 --- Placental-specific methylation --- p.20 / Chapter 2.7 --- Aim of Thesis --- p.21 / Chapter SECTION II: --- MATERIALS AND METHODOLOGY --- p.23 / Chapter CHAPTER 3: --- Materials and methods --- p.24 / Chapter 3.1 --- Preparation of samples --- p.24 / Chapter 3.1.1 --- Collection of placental tissues --- p.24 / Chapter 3.1.2 --- Preparation of blood cells --- p.25 / Chapter 3.1.3 --- Preparation of cell lines --- p.25 / Chapter 3.1.4 --- Treatment of JAR and JEG3 with 5-aza-2'-deoxycytidine (5-aza-CdR) and Trichostatin A (TSA) --- p.26 / Chapter 3.2 --- Nucleic acid extraction --- p.26 / Chapter 3.2.1 --- DNA extraction from tissue samples --- p.26 / Chapter 3.2.2 --- DNA extraction from blood cells --- p.29 / Chapter 3.2.3 --- RNA extraction from cell lines --- p.30 / Chapter 3.3 --- Methylation analysis --- p.31 / Chapter 3.3.1 --- Principles of bisulfite modification --- p.31 / Chapter 3.3.2 --- Bisulfite Conversion --- p.32 / Chapter 3.3.3 --- Primer design for methylation-specific polymerase chain reaction / Chapter 3.3.4 --- Methylation-specific polymerase chain reaction (MSP) --- p.33 / Chapter 3.3.5 --- Primer design for bisulfite sequencing --- p.34 / Chapter 3.3.6 --- Cloning and bisulfite genomic sequencing --- p.35 / Chapter 3.4 --- Quantitative measurements of nucleic acids --- p.39 / Chapter 3.4.1 --- Principles of real-time quantitative PCR --- p.39 / Chapter 3.4.2 --- Real-time quantitative MSP --- p.42 / Chapter 3.4.3 --- Real-time reverse transcriptase (RT)-PCR --- p.42 / Chapter 3.5 --- MALDI-TOF mass spectrometry (MS) --- p.43 / Chapter 3.5.1 --- Principle of homogeneous MassEXTEND assay and MALDI-TOF MS --- p.43 / Chapter 3.5.2 --- Methylation-sensitive restriction enzyme digestion and homogeneous MassEXTEND assay for APC and H19 --- p.46 / Chapter SECTION III: --- A SEARCH FOR HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE HUMAN PLACENTA --- p.48 / Chapter CHAPTER 4: --- Screening on TSGs and non TSGs --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Materials and methods --- p.50 / Chapter 4.2.1 --- Sample collection --- p.50 / Chapter 4.2.2 --- Sample processing and DNA extraction --- p.50 / Chapter 4.2.3 --- Experimental Design --- p.51 / Chapter 4.3 --- Results --- p.63 / Chapter 4.3.1 --- Identification of hypermethylated TSGs by methylation-specific PCR screening --- p.63 / Chapter 4.3.2 --- Validation of hypermethylated TSGs by bisulfite sequencing --- p.69 / Chapter 4.4 --- Discussion --- p.77 / Chapter CHAPTER 5: --- Methylation status of TSGs in different tissues --- p.80 / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and methods --- p.81 / Chapter 5.2.1 --- Sample collection --- p.81 / Chapter 5.2.2 --- Sample processing and DNA extraction --- p.81 / Chapter 5.2.3 --- Experimental design --- p.81 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- Methylation patterns of TSGs in non-placental fetal tissues --- p.86 / Chapter 5.4 --- Discussion --- p.90 / Chapter SECTION IV: --- FUNCTIONAL IMPLICATION OF HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE PLACENTA --- p.94 / Chapter CHAPTER 6: --- Imprinting checking --- p.95 / Chapter 6.1 --- Introduction --- p.95 / Chapter 6.2 --- Materials and methods --- p.96 / Chapter 6.2.1 --- Sample collection --- p.96 / Chapter 6.2.2 --- Sample processing and DNA extraction --- p.97 / Chapter 6.2.3 --- Experimental design --- p.97 / Chapter 6.3 --- Results --- p.100 / Chapter 6.3.1 --- Imprinting checking of H19 by enzyme digestion on placental tissues --- p.100 / Chapter 6.3.2 --- Imprinting checking of　APC by enzyme digestion on placental tissues --- p.101 / Chapter CHAPTER 7: --- CORRELATION OF HYPERMETHYLATION AND GENE EXPRESSION --- p.107 / Chapter 7.1 --- Introduction --- p.107 / Chapter 7.2 --- Materials and methods --- p.108 / Chapter 7.2.1 --- Sample preparation and processing --- p.108 / Chapter 7.2.2 --- DNA and RNA extraction from cell lines --- p.108 / Chapter 7.2.3 --- Experimental design --- p.108 / Chapter 7.3 --- Results --- p.111 / Chapter 7.3.1 --- Methylation status of APC in choriocarcinoma cell lines --- p.111 / Chapter 7.3.2 --- Demethylation of APC in choriocarcinoma cell lines --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Chapter SECTION V: --- CONSERVATION OF METHYLATION IN PLACENTA ACROSS DIFFERENT SPECIES --- p.118 / Chapter CHAPTER 8: --- Methylation analysis of hypermethylated TSG homologues in the placentas of the mouse and rhesus monkey --- p.119 / Chapter 8.1 --- Introduction --- p.119 / Chapter 8.2 --- Materials and methods --- p.120 / Chapter 8.2.1 --- Sample collection --- p.120 / Chapter 8.2.2 --- Sample processing and DNA extraction --- p.120 / Chapter 8.2.3 --- Experimental design --- p.120 / Chapter 8.3 --- Results --- p.124 / Chapter 8.3.1 --- Methylation status of TSGs in rhesus monkey and murine placental tissues --- p.124 / Chapter 8.4 --- Discussion --- p.136 / Chapter SECTION VI: --- CONCLUDING REMARKS --- p.138 / Chapter CHAPTER 9: --- Conclusion and future perspectives --- p.139 / Chapter 9.1 --- Pseudomalignant nature of placenta at the epigenetic level --- p.139 / Chapter 9.2 --- Functional implication of TSG hypermethylation --- p.140 / Chapter 9.3 --- Significance of hypermethylated TSGs in the placental evolution --- p.142 / Chapter 9.4 --- Clinical implication of TSG hypermethylation --- p.143 / Chapter 9.5 --- Future perspectives --- p.145 / APPENDIX I COMPLETE BISULFITE SEQUENCING DATA FOR HYPERMETHYLATED TSGS --- p.147 / APPENDIX II BISULFITE SEQUENCING DATA FOR PTEN --- p.156 / APPENDIX III BISULFITE SEQUENCING DATA OF LOCI NOT SHOWING HYPERMETHYLATION --- p.158 / REFERENCES --- p.160