Xu, Guolin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.IV / LIST OF FIGURES --- p.VII / ABBBREVIATIONS --- p.X / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Antigens --- p.1 / Chapter 1.1.1 --- Preamble --- p.1 / Chapter 1.1.2 --- Types of antigen --- p.1 / Chapter 1.1.3 --- Autoantigens --- p.2 / Chapter 1.1.4 --- Telomerase is an important autoantigen --- p.6 / Chapter 1.2 --- Antibodies --- p.12 / Chapter 1.2.1 --- Preamble --- p.12 / Chapter 1.2.2 --- Ig structure --- p.12 / Chapter 1.2.3 --- Ig synthesis --- p.13 / Chapter 1.2.4 --- Immunoglobulin isotypes --- p.15 / Chapter 1.2.5 --- Monoclonal antibodies (mAb) --- p.17 / Chapter 1.2.6 --- Autoantibodies --- p.20 / Chapter 1.2.7 --- Telomerase detection and antibodies to telomerase --- p.22 / Chapter 1.3 --- Object and Scope of Study --- p.24 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Animals --- p.31 / Chapter 2.1.2 --- Antibodies --- p.31 / Chapter 2.1.3 --- Primers --- p.31 / Chapter 2.1.4 --- Culture media and reagents --- p.32 / Chapter 2.1.5 --- Chemicals and enzymes --- p.32 / Chapter 2.1.6 --- Miscellaneous chemicals --- p.33 / Chapter 2.1.7 --- Commercial kits --- p.33 / Chapter 2.1.8 --- Instruments --- p.33 / Chapter 2.1.9 --- Buffers --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Cells and cell culture --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction (PCR) --- p.36 / Chapter 2.2.3 --- Cloning of C-terminal gene fragment to pGEX cloning vector --- p.36 / Chapter 2.2.4 --- Detection of antibody activity by ELISA --- p.37 / Chapter 2.2.5 --- Histochemical Staining --- p.38 / Chapter 2.2.6 --- Hybridoma production --- p.40 / Chapter 2.2.7 --- Protein analysis --- p.43 / Chapter 2.2.8 --- Flow cytometry --- p.46 / Chapter 2.2.9 --- Animal handling --- p.47 / Chapter 2.2.10 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE --- PRELIMINARY STUDIES USING THE ANTI-N-TERT-TELOMERASE MAB DERIVED FROM HYBRIDOMA 476 --- p.49 / Chapter 3.1 --- Preamble --- p.49 / Chapter 3.2 --- Hybridoma 476 cells can be stained by the labeled recombinant N-TERT antigen --- p.50 / Chapter 3.3 --- Mouse spleen cells can also be stained by biotin-labeled N-TERT antigen --- p.52 / Chapter 3.4 --- Discussion --- p.53 / Chapter CHAPTER FOUR --- PRODUCTION OF MONOCLONAL ANTIBODIES TO C-TERT --- p.63 / Chapter 4.1 --- Preamble --- p.63 / Chapter 4.2 --- Construction of C-TERT expression vector --- p.63 / Chapter 4.3 --- Expression and purification of recombinant human C-terminal telomerase antigen --- p.64 / Chapter 4.4 --- Immunization of Balb/c mice with C-TERT-GST --- p.65 / Chapter 4.5 --- Generation of hybridomas to C-TERT --- p.65 / Chapter 4.6 --- Identification and selection of reactive clones --- p.65 / Chapter CHAPTER FIVE --- CHARACTERIZATION OF MAB A63 --- p.71 / Chapter 5.1 --- Preamble --- p.71 / Chapter 5.2 --- Characterization of mAb A63 by ELISA --- p.71 / Chapter 5.3 --- Characterization of mAb A63 by Western blotting analysis --- p.73 / Chapter 5.4 --- Characterization of mAb A63 by immuno-histochemical staining --- p.73 / Chapter 5.5 --- mAb A63 can also stain fish telomerase and human placenta --- p.75 / Chapter 5.6 --- mAb A63 can also stain telomerase in human tumors --- p.76 / Chapter 5.7 --- Hybridoma A63 can produce ascites fluid --- p.76 / Chapter 5.8 --- Discussion --- p.77 / Chapter CHAPTER SIX --- GENERAL DISCUSSION --- p.93 / Chapter 6.1 --- Why Enhancing buffer is required for the nuclear staining of hTERT when using mAb 476 or mAb A63 --- p.96 / Chapter 6.2 --- Why hybridoma 476 failed to form ascites while hybridoma A63 succeeded --- p.99 / Chapter 6.3 --- Can IL-6 be used to treat autoimmune diseases? --- p.102 / Chapter 6.4 --- Possible use of monoclonal antibodies in cancer therapy --- p.104 / Chapter 6.5 --- Prospects on study --- p.107 / REFERENCES --- p.111

Telomerase is a highly regulated ribonucleoprotein complex that stabilizes eukaryotic genomes by replenishing telomeric repeats on chromosome ends. Defects in telomerase RNP components involving the catalytic subunit TERT or the RNA template TER lead to stem cell-related diseases such as dyskeratosis congenita and idiopathic pulmonary fibrosis, while inappropriate telomerase expression is a rate-limiting step in carcinogenesis. In this study we report the discovery of a novel negative regulatory mechanism for telomerase that stems from duplication and diversification of key components of the telomerase RNP in the flowering plant Arabidopsis thaliana. We show that Arabidopsis encodes three distinct TERs: TER1, TER2 and a processed form of TER2 termed TER2S. Although all three RNAs can serve as templates for telomerase in vitro, in vivo they have different expression patterns, assemble into distinct RNPs with different protein binding partners, and play opposing roles in telomere maintenance. The TER1 RNP is analogous to the telomerase enzyme previously described in other eukaryotes, but the TER2 RNP is a negative regulator of telomerase activity and telomere maintenance in vivo. Furthermore, we demonstrate that the Protection Of Telomeres (POT1) paralogs in Arabidopsis (POT1a, POT1b and POT1c) are novel TER binding proteins. This finding is striking because in yeast and vertebrates, POT1 is an essential component of the telomere capping complex and functions to distinguish the chromosome terminus from a double-strand break. Thus, our data argue that Arabidopsis POT1 proteins have migrated off of the chromosome terminus and onto the telomerase RNP, indicating that duplication and diversification of Arabidopsis telomerase may be the end result of the co-evolution of the TER and POT1 RNP components. Additionally, given the dire consequences of misregulating telomerase in human cells, our discovery of a novel negative regulatory mechanism for telomerase in plants strongly suggests that additional modes of telomerase control remain to be elucidated in vertebrates.

Paediatric ependymomas are the third most common childhood brain cancer and represent a prognostic and therapeutic challenge. Previous evidence suggests that telomerase, a ribonucleoprotein critical in permitting limitless growth potential, may serve as both a prognostic marker and therapeutic target. Immunohistochemical analysis (n=198) and enzymatic detection (n=25) of telomerase was performed to determine prevalence and prognostic potential. The telomerase inhibitor Imetelstat was used to study telomerase inhibition in paediatric ependymoma cell lines, tumour initiating cells (TICs) and both subcutaneous and intracranial xenografts. Telomerase activity was detected in 76% of primary ependymomas and was associated with a reduced five-year progression-free survival (30% vs 75%). Telomerase inhibition in vitro resulted in shortened telomeres, increased senescence, growth inhibition and reduced self-renewal capacity. In vivo, Imetelstat shortened telomeres and reduced subcutaneous tumour volume by 40% compared to control mice. Therefore, telomerase may serve as an ideal prognostic marker and therapeutic target in paediatric ependymoma.

Paediatric ependymomas are the third most common childhood brain cancer and represent a prognostic and therapeutic challenge. Previous evidence suggests that telomerase, a ribonucleoprotein critical in permitting limitless growth potential, may serve as both a prognostic marker and therapeutic target. Immunohistochemical analysis (n=198) and enzymatic detection (n=25) of telomerase was performed to determine prevalence and prognostic potential. The telomerase inhibitor Imetelstat was used to study telomerase inhibition in paediatric ependymoma cell lines, tumour initiating cells (TICs) and both subcutaneous and intracranial xenografts. Telomerase activity was detected in 76% of primary ependymomas and was associated with a reduced five-year progression-free survival (30% vs 75%). Telomerase inhibition in vitro resulted in shortened telomeres, increased senescence, growth inhibition and reduced self-renewal capacity. In vivo, Imetelstat shortened telomeres and reduced subcutaneous tumour volume by 40% compared to control mice. Therefore, telomerase may serve as an ideal prognostic marker and therapeutic target in paediatric ependymoma.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

No description available.

Tam, Yeuk Fei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 63-74). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

O telômero é uma sequência repetitiva da dupla cadeia do DNA que protege as pontas dos cromossomos. Seu comprimento é mantido pela telomerase, cuja expressão ocorre em células de câncer, mas não em células somáticas. A célula apresenta um número definido de divisões antes do telômero sofrer erosão. A quebra do DNA ativa a p53, supressor tumoral que induz senescência e respostas de pontos de checagem. O desenvolvimento de inibidores de telomerase tem importância clínica para o câncer. Estudamos os efeitos dos inibidores de telomerase. Duas linhagens celulares LC-HK2 (NSCLC) e hTERT RPE-1 foram tratadas com os inibidores TMPyP4 (5µM) e Thymoquinone (10 e 40 µM) durante 72 e 120 h. TMPyP4 aumentou a porcentagem de células com dano na membrana, induziu mudança na morfologia da célula e diminuiu a expressão do mRNA da vimentina e vinculina. Thymoquinone aumentou a frequência de células senescentes, células com dano na membrana e induziu morte celular. Ambos os inibidores diminuíram a atividade da telomerase, afetando a proliferação e induzindo morte celular. / The telomere is a repetitive double-strand sequence of DNA that protects the chromosomes ends. Its length is maintained by telomerase, whose expression occurs in cancer cells, but not in somatic cells. The cell has a defined number of divisions before the telomere undergoes erosion. DNA break activates p53, tumor suppressor and induces senescence and checkpoint responses. The development of telomerase inhibitors is of clinical importance for cancer. We studied the effects of telomerase inhibitors. Two cell lines LC-HK2 (NSCLC) and hTERT RPE-1 were treated with inhibitors TMPyP4 (5 M) and Thymoquinone (10 and 40 M) for 72 and 120 h. TMPyP4 increased the percentage of cells with membrane damage, induced change in cell morphology, and decreased mRNA expression of vimentin and vinculin. Thymoquinone increased the frequency of senescent cells, cells with membrane damage and induced cell death. Both inhibitors decreased telomerase activity, affecting proliferation and inducing cell death.

Telomerase, a ribonucleoprotein enzyme has its own internal RNA template that elongates telomeres and stabilizes chromosome structure. The mayority of hepatoma express high levels of human telomerase template RNA ( hTR ) that is essential for cellular telomerase activity. In this study, we examined whether hTR¡Bantisence hTR and IFN£\ gene had a telomerase activity inhibitory effect on J5 hepatoma cell line, through transfection via an expression vector . The expression of mRNAs for hTERT¡BhTR¡Bc-myc¡BIFN£\¡Bp16 and p27 were also detected. The results suggested that inhibition of telomerase activity and overexpression of hTR gene were observed in FhTR-treated J5 cells. The expression of mRNAs for hTERT¡Bc-myc¡BIFN£\¡Bp16 and p27 genes were not changed whether telomerase was inhibited or not. In addition, genomic DNA fragmentation was observed in telomerase inhibited J5 cells indicating that apoptosis was undergoing in these cells.

Telomeres are critical for the integrity of eukaryotic genomes. They function to protect chromosome ends from DNA damage surveillance and inappropriate repair. Telomeres are maintained by the specialized ribonucleoprotein complex telomerase. Without telomerase, telomere shortening would ultimately lead to compromised genome stability and cellular senescence. Therefore, telomerase function is necessary for extension of the proliferative capacity of the cell. In this dissertation, we describe the characterization of core components of telomerase ribonucleoprotein complex in the flowering plant, Arabidopsis thaliana. We find that dyskerin, one of the core telomerase components in humans is also conserved in Arabidopsis telomerase. Arabidopsis dyskerin associates with the telomerase RNP in an RNA-dependent manner and is required for telomere length maintenance in this organism. We also describe the characterization of another core telomerase component, the telomerase RNA subunits (TERs). Unexpectedly, we uncovered two distinct TER subunits that share a region of high identity. The two TERs named TER1G7 and TER5G2, based on their chromosomal positions, display differences in their expression levels and their association with telomere-related proteins. Both TERs can serve as templates for telomerase in vitro. Through genetic analyses, we show a templating function for TER1G7 in vivo and a novel role for TER5G2 as a negative regulator of telomerase. Finally, the presence of TER genes in other plant species was investigated and evidence for duplication of TER genes in plants closely related to Arabidopsis was obtained. We also show evidence for a template mutation in Asparagus TER that could lead to variant repeats in this organism. In summary, the studies presented in this dissertation reveal that Arabidopsis telomerase shares both similarities and differences with other telomerase RNPs, making it an exciting model system for study of telomere biology.