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.

There remains a pressing need for the development of effective drugs that meet the clinical needs for cancer treatment, and inhibition of telomere length maintenance by disrupting human telomerase is a proven and tractable target for suppression of cancer cell growth. In response to the lack of currently available small molecules with efficacy against human telomerase, we developed a genetically and chemically tractable cell-based system in which S. cerevisiae is used to streamline the search for novel human telomerase inhibitors. Our results confirmed that yeast cell growth was rapidly inhibited upon induction of functional human telomerase at the telomere. This inducible growth arrest was used as a read-out for a high-throughput chemical screen for human telomerase inhibitors based on their ability to restore growth in the yeast system. From a library consisting of small, bioactive and cell-permeable compounds of diverse structure, we identified three novel “drug-like” compounds that inhibited the activity of native and recombinant telomerase complexes in vitro. “Validation assays” also confirmed the novel inhibitors were free of uncharacterized adverse effects against yeast and human cell models, thus confirming the specificity of these novel inhibitors against human telomerase target. This surrogate yeast model has therefore proven to be a cost-effective alternative to accelerate the search for human telomerase inhibitors, which we hope will serve to streamline the identification of further lead compounds effective against human cancer.

癌病是人類的主要死亡原因之一，所以有必要研發出一個有效的癌症治療辦法。大多數癌病是由細胞無限增殖所引起，而端粒酶活性和端粒長度的維持是細胞永生化和轉型的關鍵。超過 85的永生化腫瘤細胞株表達高水平端粒酶。因此，端粒酶的調控機理成為研究和治療癌病的一個主要目標。 / 這項端粒酶的調控機制研究，集中在調查端粒酶抑製蛋白PinX1及其相互作用的蛋白質。透過牽出試驗和質譜鑑定發現45個潛在與PinX1有相互作用的蛋白。其中Nucleophosmin（NPM）被選定作為進一步研究的對象。通過牽出試驗與免疫共沉澱的方法證明NPM與PinX1可在细胞内和外作直接的相互作用。NPM、PinX1和hTERT在細胞內形成復合體，而PinX1是連接NPM和hTERT之間的連接蛋白。PinX1招聘NPM至端粒酶可以減輕 PinX1對端粒酶的抑制作用，表明PinX1/NPM的相互作用可能參與端粒酶的激活過程。此外，NPM和hTERT被發現在細胞周期的S-早期共定位於核仁，而此發現與以往研究中的端粒酶激活的時間相匹配。所有提供的證據表明，PinX1/NPM相互作用在端粒酶激活過程中扮演重要角色。 / 此外，研究證明PinX1參與在端粒酶的募集過程，通過siRNA下調PinX1的表達導致在細胞週期的不同階段中減少端粒酶在端粒的定位。這項研究顯示出PinX1在端粒酶激活和募集過程方面的重要性。 / Cancer is always one of the leading causes of death in humankind and an effective approach for cancer therapy is needed. Most cancers are caused by unlimited proliferation of cells. Telomerase activation and telomere maintenance are found to be critical in cellular immortalization and transformation. Over 85% of the immortal cancer cell lines express high level of telomerase which is essential for telomere maintenance. Therefore, studies on the telomerase regulatory pathway become one of the major targets in cancer research for cancer therapy. / This study focused on investigating a telomerase inhibitor, PinX1 and its interacting proteins for understanding the telomerase regulation. 45 potential PinX1 interacting proteins were identified by pull-down assay coupled with mass spectrometry. Out of these potential partners, Nucleophosmin (NPM) was chosen for further studies and confirmed to have direct interaction with PinX1 through in vitro pull down assay and co-immunoprecipitation. NPM, PinX1 and hTERT form complex inside the cell and PinX1 acts as the linker to bridge the association between NPM and hTERT. The recruitment of NPM by PinX1 to the telomerase can attenuate the PinX1 inhibition on telomerase activity, indicating that PinX1/NPM interaction may involve in telomerase activation. Moreover, NPM and hTERT were found to co-localize in nucleolus during early S-phase which matched the timing of telomerase activation in previous studies. All these provided evidence that PinX1/NPM interaction is implicated in telomerase activation. / Besides, PinX1 was shown to be involved in the telomerase recruitment to telomere, as down-regulation of PinX1 led to reduction of hTERT localization to telomere at different stages of cell cycle. This study revealed the importance of PinX1 in telomerase regulation in terms of its activation and recruitment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Hang Cheong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 119-135). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Contents --- p.vi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of Telomere and Telomerase in Cancer --- p.1 / Chapter 1.2 --- Introduction to Telomere / Chapter 1.2.1 --- General function and structure of telomere --- p.2 / Chapter 1.2.2 --- Role of shelterin complex in telomere maintenance / Chapter 1.2.2.1 --- TRF1 and TRF2 --- p.4 / Chapter 1.2.2.2 --- Pot1 --- p.5 / Chapter 1.2.2.3 --- TPP1 --- p.5 / Chapter 1.2.2.4 --- TIN2 --- p.6 / Chapter 1.2.2.5 --- RAP1 --- p.6 / Chapter 1.2.3 --- Telomere shortening and replicative senescence in human cell / Chapter 1.2.3.1 --- End replication problem of DNA polymerase --- p.6 / Chapter 1.2.3.2 --- Replicative senescence pathway --- p.7 / Chapter 1.2.4 --- Telomere shortening and cancer formation --- p.7 / Chapter 1.3 --- Introduction to Telomerase / Chapter 1.3.1 --- Function and organization of telomerase --- p.9 / Chapter 1.3.2 --- Telomerase expression in normal cells --- p.9 / Chapter 1.3.3 --- Role of telomerase in cancer cells --- p.10 / Chapter 1.3.4 --- Other roles of telomerase in cells --- p.12 / Chapter 1.3.5 --- Regulation and Recruitment of telomerase / Chapter 1.3.5.1 --- Protein counting model on telomerase regulation --- p.12 / Chapter 1.3.5.2 --- Evidences of telomerase activation on short telomere --- p.13 / Chapter 1.3.5.3 --- Telomerase regulation by shelterin and its associate factors --- p.14 / Chapter 1.3.5.4 --- Cell cycle dependent trafficking of telomerase --- p.15 / Chapter 1.4 --- Introduction to PinX1 / Chapter 1.4.1 --- Discovery of PinX1 as telomerase inhibitor --- p.16 / Chapter 1.4.2 --- Role of PinX1 in telomerase and telomere regulation / Chapter 1.4.2.1 --- Interaction between PinX1 and telomerase --- p.17 / Chapter 1.4.2.2 --- PinX1 mediates nucleolar localization of hTERT --- p.17 / Chapter 1.4.2.3 --- Interaction between PinX1 and TRF1 --- p.17 / Chapter 1.4.2.4 --- Dual role of PinX1 in telomere maintenance --- p.18 / Chapter 1.4.3 --- PinX1 expression in Cancer cells / Chapter 1.4.3.1 --- Genetic analysis of PinX1 in cancers --- p.19 / Chapter 1.4.3.2 --- Treating of cancer through PinX1 manipulation --- p.19 / Chapter 1.5 --- Introduction to Nucleophosmin / Chapter 1.5.1 --- Nucleophosmin (NPM) as a multi-functional protein / Chapter 1.5.1.1 --- NPM is a molecular chaperone --- p.21 / Chapter 1.5.1.2 --- Involvement of NPM in ribosome biogenesis --- p.21 / Chapter 1.5.1.3 --- NPM maintains genomic stability --- p.22 / Chapter 1.5.2 --- Role of Nucleophosmin in Cancer cell / Chapter 1.5.2.1 --- NPM as an oncogene? --- p.22 / Chapter 1.5.2.2 --- NPM as a tumor-suppressor gene? --- p.23 / Chapter 1.6 --- Long term impact and objectives of the study --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Plasmids --- p.26 / Chapter 2.1.2 --- Bacterial Cells --- p.26 / Chapter 2.1.3 --- Mammalian Cells --- p.26 / Chapter 2.1.4 --- Serum and Antibodies --- p.27 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Molecular cloning / Chapter 2.2.1.1 --- Basic scheme of cloning --- p.28 / Chapter 2.2.1.2 --- Cloning of PinX1 constructs --- p.29 / Chapter 2.2.1.3 --- Cloning of Nucleophosmin (NPM) constructs --- p.29 / Chapter 2.2.1.4 --- Cloning of hTERT constructs --- p.29 / Chapter 2.2.2 --- Preparation of the competent cells --- p.30 / Chapter 2.2.3 --- Chemical Transformation of competent cells --- p.30 / Chapter 2.2.4 --- Expression of recombinant protein in bacteria --- p.31 / Chapter 2.2.5 --- Purification of GST-PinX1 and GST-PinX1-N --- p.31 / Chapter 2.2.6 --- Purification of PinX1-M and PinX1-C --- p.32 / Chapter 2.2.7 --- Purification of Sumo-NPM and its truncations --- p.33 / Chapter 2.2.8 --- In vitro Pull Down Assay of PinX1-C against HepG2 Lysate / Chapter 2.2.8.1 --- Immobilization of PinX1-C to NHS-column --- p.33 / Chapter 2.2.8.2 --- Preparation of nuclear fraction of HepG2 Lysate --- p.33 / Chapter 2.2.8.3 --- In vitro Pull Down Assay by NHS-column --- p.34 / Chapter 2.2.9 --- 2D-gel electrophoresis --- p.35 / Chapter 2.2.10 --- Mass Spectrommetry --- p.35 / Chapter 2.2.11 --- In-vitro pull down assay --- p.36 / Chapter 2.2.12 --- Plasmid Transfection into mammalian cells --- p.36 / Chapter 2.2.13 --- Co-immunoprecipitation --- p.37 / Chapter 2.2.14 --- Immunofluorescence / Chapter 2.2.14.1 --- Immunostaining of PinX1 and NPM --- p.37 / Chapter 2.2.14.2 --- Immunostaining of hTERT and TRF2 --- p.38 / Chapter 2.2.15 --- TRAP Assay / Chapter 2.2.15.1 --- Basic Scheme of TRAP Assay --- p.39 / Chapter 2.2.15.2 --- TRAP Assay with exogenous purified proteins --- p.40 / Chapter 2.2.16 --- Immunoprecipitation-TRAP Assay --- p.41 / Chapter 2.2.17 --- Transient knock-down of PinX1 or NPM by siRNA --- p.42 / Chapter 2.2.18 --- Synchronization of HeLa cells --- p.42 / Chapter 2.2.19 --- Cell cycle analysis of HeLa cells by flow cytometry --- p.42 / Chapter Chapter 3 --- Identification of PinX1-interacting partners / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Results / Chapter 3.2.1 --- Purification of PinX1 constructs --- p.52 / Chapter 3.2.2 --- Identification of PinX1 interacting partners by Pull Down assay --- p.55 / Chapter 3.2.3 --- Mass spectrometry analysis of potential PinX1 partners --- p.55 / Chapter 3.3 --- Discussion --- p.64 / Chapter Chapter 4 --- Role of PinX1/NPM interaction on telomerase regulation / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results / Chapter 4.2.1 --- Confirmation of PinX1/NPM interaction / Chapter 4.2.1.1 --- Association of PinX1 and NPM inside the cell --- p.70 / Chapter 4.2.1.2 --- Direct interaction between PinX1 and NPM in vitro --- p.70 / Chapter 4.2.1.3 --- Co-localization of NPM and PinX1 within the nucleus --- p.73 / Chapter 4.2.2 --- PinX1/NPM/hTERT associated as a complex inside the cell --- p.73 / Chapter 4.2.3 --- Characterization of PinX1/NPM/hTERT interaction / Chapter 4.2.3.1 --- Nucleophosmin interacts with the C-terminal region of PinX1 --- p.76 / Chapter 4.2.3.2 --- PinX1 interacts with the N-terminal region of Nucleophosmin and E56, E61 and E63 of Nucleophosmin are critical for the interaction --- p.78 / Chapter 4.2.3.3 --- Nucleophosmin associates with hTERT through the interaction with PinX1 --- p.83 / Chapter 4.2.4 --- PinX1 recruits NPM to telomerase and attenuates the PinX1 inhibition on telomerase activity --- p.89 / Chapter 4.2.5 --- Nucleophosmin co-localize with hTERT in nucleolus during early S-phase of cell-cycle --- p.91 / Chapter 4.3 --- Discussion --- p.97 / Chapter Chapter 5 --- Importance of PinX1 in telomerase recruitment / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Results and Discussion / Chapter 5.2.1 --- Synchronization and silencing of PinX1 in HeLa cells --- p.103 / Chapter 5.2.2 --- Reduced telomerase localization to telomere in PinX1 down-regulated HeLa cells --- p.103 / Chapter 5.3 --- Discussion --- p.110 / Chapter Chapter 6 --- Discussion / Chapter 6.1 --- Concluding Remarks --- p.113 / Chapter 6.2 --- PinX1/NPM interaction as a potential target for cancer treatment --- p.115 / Chapter 6.3 --- Future Prospects / Chapter 6.3.1 --- Studies on other potential PinX1 interacting partners --- p.116 / Chapter 6.3.2 --- Cell-cycle dependent interaction between PinX1, NPM and hTERT --- p.116 / Chapter 6.3.3 --- Designation of inhibitory peptide to disrupt PinX1/NPM interaction --- p.117 / Chapter 6.3.4 --- Importance of PinX1/NPM interaction on tumor growth --- p.117 / Chapter 6.3.5 --- Interaction between NPM and other shelterin proteins --- p.118 / Literature Cited --- p.119

No / Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C→A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells. / CAEB, Balearic Islands and Yorkshire Cancer Research

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.