CUHK electronic theses & dissertations collection

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.iAbstract --- p.iii摘要 --- p.vTable of Contents --- p.viChapter Chapter 1 --- General IntroductionChapter 1.1 --- Overview of Telomere and Telomerase in Cancer --- p.1Chapter 1.2 --- Introduction to TelomereChapter 1.2.1 --- General function and structure of telomere --- p.2Chapter 1.2.2 --- Role of shelterin complex in telomere maintenanceChapter 1.2.2.1 --- TRF1 and TRF2 --- p.4Chapter 1.2.2.2 --- Pot1 --- p.5Chapter 1.2.2.3 --- TPP1 --- p.5Chapter 1.2.2.4 --- TIN2 --- p.6Chapter 1.2.2.5 --- RAP1 --- p.6Chapter 1.2.3 --- Telomere shortening and replicative senescence in human cellChapter 1.2.3.1 --- End replication problem of DNA polymerase --- p.6Chapter 1.2.3.2 --- Replicative senescence pathway --- p.7Chapter 1.2.4 --- Telomere shortening and cancer formation --- p.7Chapter 1.3 --- Introduction to TelomeraseChapter 1.3.1 --- Function and organization of telomerase --- p.9Chapter 1.3.2 --- Telomerase expression in normal cells --- p.9Chapter 1.3.3 --- Role of telomerase in cancer cells --- p.10Chapter 1.3.4 --- Other roles of telomerase in cells --- p.12Chapter 1.3.5 --- Regulation and Recruitment of telomeraseChapter 1.3.5.1 --- Protein counting model on telomerase regulation --- p.12Chapter 1.3.5.2 --- Evidences of telomerase activation on short telomere --- p.13Chapter 1.3.5.3 --- Telomerase regulation by shelterin and its associate factors --- p.14Chapter 1.3.5.4 --- Cell cycle dependent trafficking of telomerase --- p.15Chapter 1.4 --- Introduction to PinX1Chapter 1.4.1 --- Discovery of PinX1 as telomerase inhibitor --- p.16Chapter 1.4.2 --- Role of PinX1 in telomerase and telomere regulationChapter 1.4.2.1 --- Interaction between PinX1 and telomerase --- p.17Chapter 1.4.2.2 --- PinX1 mediates nucleolar localization of hTERT --- p.17Chapter 1.4.2.3 --- Interaction between PinX1 and TRF1 --- p.17Chapter 1.4.2.4 --- Dual role of PinX1 in telomere maintenance --- p.18Chapter 1.4.3 --- PinX1 expression in Cancer cellsChapter 1.4.3.1 --- Genetic analysis of PinX1 in cancers --- p.19Chapter 1.4.3.2 --- Treating of cancer through PinX1 manipulation --- p.19Chapter 1.5 --- Introduction to NucleophosminChapter 1.5.1 --- Nucleophosmin (NPM) as a multi-functional proteinChapter 1.5.1.1 --- NPM is a molecular chaperone --- p.21Chapter 1.5.1.2 --- Involvement of NPM in ribosome biogenesis --- p.21Chapter 1.5.1.3 --- NPM maintains genomic stability --- p.22Chapter 1.5.2 --- Role of Nucleophosmin in Cancer cellChapter 1.5.2.1 --- NPM as an oncogene? --- p.22Chapter 1.5.2.2 --- NPM as a tumor-suppressor gene? --- p.23Chapter 1.6 --- Long term impact and objectives of the study --- p.25Chapter Chapter 2 --- Materials and MethodsChapter 2.1 --- MaterialsChapter 2.1.1 --- Plasmids --- p.26Chapter 2.1.2 --- Bacterial Cells --- p.26Chapter 2.1.3 --- Mammalian Cells --- p.26Chapter 2.1.4 --- Serum and Antibodies --- p.27Chapter 2.2 --- MethodsChapter 2.2.1 --- Molecular cloningChapter 2.2.1.1 --- Basic scheme of cloning --- p.28Chapter 2.2.1.2 --- Cloning of PinX1 constructs --- p.29Chapter 2.2.1.3 --- Cloning of Nucleophosmin (NPM) constructs --- p.29Chapter 2.2.1.4 --- Cloning of hTERT constructs --- p.29Chapter 2.2.2 --- Preparation of the competent cells --- p.30Chapter 2.2.3 --- Chemical Transformation of competent cells --- p.30Chapter 2.2.4 --- Expression of recombinant protein in bacteria --- p.31Chapter 2.2.5 --- Purification of GST-PinX1 and GST-PinX1-N --- p.31Chapter 2.2.6 --- Purification of PinX1-M and PinX1-C --- p.32Chapter 2.2.7 --- Purification of Sumo-NPM and its truncations --- p.33Chapter 2.2.8 --- In vitro Pull Down Assay of PinX1-C against HepG2 LysateChapter 2.2.8.1 --- Immobilization of PinX1-C to NHS-column --- p.33Chapter 2.2.8.2 --- Preparation of nuclear fraction of HepG2 Lysate --- p.33Chapter 2.2.8.3 --- In vitro Pull Down Assay by NHS-column --- p.34Chapter 2.2.9 --- 2D-gel electrophoresis --- p.35Chapter 2.2.10 --- Mass Spectrommetry --- p.35Chapter 2.2.11 --- In-vitro pull down assay --- p.36Chapter 2.2.12 --- Plasmid Transfection into mammalian cells --- p.36Chapter 2.2.13 --- Co-immunoprecipitation --- p.37Chapter 2.2.14 --- ImmunofluorescenceChapter 2.2.14.1 --- Immunostaining of PinX1 and NPM --- p.37Chapter 2.2.14.2 --- Immunostaining of hTERT and TRF2 --- p.38Chapter 2.2.15 --- TRAP AssayChapter 2.2.15.1 --- Basic Scheme of TRAP Assay --- p.39Chapter 2.2.15.2 --- TRAP Assay with exogenous purified proteins --- p.40Chapter 2.2.16 --- Immunoprecipitation-TRAP Assay --- p.41Chapter 2.2.17 --- Transient knock-down of PinX1 or NPM by siRNA --- p.42Chapter 2.2.18 --- Synchronization of HeLa cells --- p.42Chapter 2.2.19 --- Cell cycle analysis of HeLa cells by flow cytometry --- p.42Chapter Chapter 3 --- Identification of PinX1-interacting partnersChapter 3.1 --- Introduction --- p.50Chapter 3.2 --- ResultsChapter 3.2.1 --- Purification of PinX1 constructs --- p.52Chapter 3.2.2 --- Identification of PinX1 interacting partners by Pull Down assay --- p.55Chapter 3.2.3 --- Mass spectrometry analysis of potential PinX1 partners --- p.55Chapter 3.3 --- Discussion --- p.64Chapter Chapter 4 --- Role of PinX1/NPM interaction on telomerase regulationChapter 4.1 --- Introduction --- p.68Chapter 4.2 --- ResultsChapter 4.2.1 --- Confirmation of PinX1/NPM interactionChapter 4.2.1.1 --- Association of PinX1 and NPM inside the cell --- p.70Chapter 4.2.1.2 --- Direct interaction between PinX1 and NPM in vitro --- p.70Chapter 4.2.1.3 --- Co-localization of NPM and PinX1 within the nucleus --- p.73Chapter 4.2.2 --- PinX1/NPM/hTERT associated as a complex inside the cell --- p.73Chapter 4.2.3 --- Characterization of PinX1/NPM/hTERT interactionChapter 4.2.3.1 --- Nucleophosmin interacts with the C-terminal region of PinX1 --- p.76Chapter 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.78Chapter 4.2.3.3 --- Nucleophosmin associates with hTERT through the interaction with PinX1 --- p.83Chapter 4.2.4 --- PinX1 recruits NPM to telomerase and attenuates the PinX1 inhibition on telomerase activity --- p.89Chapter 4.2.5 --- Nucleophosmin co-localize with hTERT in nucleolus during early S-phase of cell-cycle --- p.91Chapter 4.3 --- Discussion --- p.97Chapter Chapter 5 --- Importance of PinX1 in telomerase recruitmentChapter 5.1 --- Introduction --- p.101Chapter 5.2 --- Results and DiscussionChapter 5.2.1 --- Synchronization and silencing of PinX1 in HeLa cells --- p.103Chapter 5.2.2 --- Reduced telomerase localization to telomere in PinX1 down-regulated HeLa cells --- p.103Chapter 5.3 --- Discussion --- p.110Chapter Chapter 6 --- DiscussionChapter 6.1 --- Concluding Remarks --- p.113Chapter 6.2 --- PinX1/NPM interaction as a potential target for cancer treatment --- p.115Chapter 6.3 --- Future ProspectsChapter 6.3.1 --- Studies on other potential PinX1 interacting partners --- p.116Chapter 6.3.2 --- Cell-cycle dependent interaction between PinX1, NPM and hTERT --- p.116Chapter 6.3.3 --- Designation of inhibitory peptide to disrupt PinX1/NPM interaction --- p.117Chapter 6.3.4 --- Importance of PinX1/NPM interaction on tumor growth --- p.117Chapter 6.3.5 --- Interaction between NPM and other shelterin proteins --- p.118Literature Cited --- p.11