The Best PhD thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing prize,2006-2007 / published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

The promyelocytic leukaemia protein (PML) is a tumour suppressor initially identified in acute promyelocytic leukaemia (APL). In APL, PML and the retinoic acid receptor alpha (RARalpha) genes are fused as a consequence of the translocation t(15;17). The product of the chimeric gene is the oncogenic PML-RARalpha protein. The PML gene encodes multiple nuclear and cytoplasmic isoforms. PML nuclear isoforms (nPML) are the main components of the PML nuclear bodies (PML-NBs), sub-nuclear structures involved in the modulation of essential cellular players including the tumour suppressor p53. Nuclear PML has been intensively studied, while, the role of cytoplasmic PML remains poorly understood. Increasing evidence indicates that PML could bear cytoplasmic functions in both physiological and pathological settings. This study aims to gain more insights into the function of PML and PML-RARalpha cytoplasmic pool of proteins. Recently, two missense mutations resulting in truncated PML cytoplasmic protein (Mut PML) have been identified in aggressive APL cases. We found that Mut PML alters the structure and the function of the PML-NB mainly through the cytoplasmic relocation of nPML. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive and apoptotic functions. In the cytoplasm, Mut PML interacts and stabilizes PML-RARalpha, thus potentiating its block of RA-induced transcription and differentiation. A mutant of PML-RARalpha (Delta2) accumulating in the cytoplasm is able to inhibit RA-dependent transcription and differentiation, suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we found that Delta2 expression blocks G-CSF-dependent myeloid differentiation and causes partial transformation of primary haematopoietic progenitor cells.

Cheng, Cho Yiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 186-217). / Abstracts in English and Chinese. / Acknowledgements --- p.1 / Abstract of thesis --- p.2 / Abstract of thesis in Chinese --- p.7 / Presentation attended --- p.9 / Chapter Chapter 1: --- Introduction and Background --- p.13 / Chapter 1.1 --- Anatomy and functions of human prostate gland --- p.13 / Chapter 1.2 --- Worldwide epidemiology of prostate cancer --- p.15 / Chapter 1.3 --- Prostate cancer stages and treatments in clinic --- p.21 / Chapter 1.4 --- Introduction to nuclear receptors --- p.23 / Chapter 1.5 --- Nuclear receptor structure --- p.24 / Chapter 1.6 --- Nuclear receptors nomenclature and classification --- p.28 / Chapter 1.7 --- Mode of action for nuclear receptors --- p.34 / Chapter 1.8 --- Co-regulators of nuclear receptors --- p.35 / Chapter 1.9 --- Nuclear receptors related to prostate cancer --- p.43 / Chapter Chapter 2: --- Aim of study and experimental design --- p.59 / Chapter 2.1 --- Aim of study --- p.59 / Chapter 2.2 --- In vitro cell lines models used in the study --- p.60 / Chapter Chapter 3: --- Materials and methods --- p.64 / Chapter 3.1 --- Apparatus and preparation throughout the study --- p.64 / Chapter 3.2 --- Cells culture --- p.64 / Chapter 3.3 --- RNA extraction --- p.67 / Chapter 3.4 --- Reverse transcription --- p.68 / Chapter 3.5 --- Primers specificity checking --- p.69 / Chapter 3.6 --- Real time quantitative polymerase chain reaction --- p.84 / Chapter 3.7 --- Data analysis --- p.90 / Chapter Chapter 4: --- Results --- p.92 / Chapter 4.1 --- Expression of nuclear receptors transcripts in each prostatic cell lines used --- p.92 / Chapter 4.2 --- Expression of nuclear receptor transcripts in immortalized prostatic epithelial BPH-1 and BPH-1 derived cell lines model --- p.116 / Chapter 4.3 --- Expression of nuclear receptor transcripts in androgen-dependent and androgen-independent classical prostatic cancer cell lines model --- p.121 / Chapter 4.4 --- Expression of nuclear receptor transcripts in androgen-independent and antiandrogen-resistant LNCaP derived cell lines model --- p.125 / Chapter Chapter 5: --- Discussion --- p.129 / Chapter 5.1 --- Special expression pattern of some nuclear receptors in the prostatic cell lines or prostatic cancer cell lines --- p.129 / Chapter 5.2 --- BPH-1 and BPH-1 derived cell lines model --- p.138 / Chapter 5.2.1 --- Prostatic cell lines model studying the transformation and invasion in prostate cancer (BPH-1 Snail & BPH-1 CAFTDs versus BPH-1) --- p.138 / Chapter 5.2.2 --- Prostatic cell lines model studying the transformation and invasion in prostate cancer (BPH-1 Snail & BPH-1 CAFTDs versus BPH-1 AR) --- p.159 / Chapter 5.3.3 --- classical prostatic cancer cell lines model --- p.162 / Chapter 5.3.1 --- Prostatic cancer cell lines model studying androgen-dependence and androgen-independence (DU145 & PC-3 versus LNCaP) --- p.163 / Chapter 5.4 --- LNCaP and LNCaP derived cell lines model --- p.170 / Chapter 5.4.1 --- Prostatic cancer cell lines model studying androgen-independence and antiandrogen-resistance (LNCaP-abl & LNCaP-BCs versus LNCaP) --- p.171 / Chapter Chapter 6: --- Conclusion --- p.179 / References --- p.186

No description available.

1,25-(OH)2 vitamin D3 inhibits cell proliferation of a variety of cancers including prostate. In the human prostate cancer cell line LNCaP, 1,25-(OH)2 vitamin D3-mediated growth inhibition is attributed to cell cycle G1 accumulation which correlates with a robust decrease of cyclin-dependent kinase 2 (CDK2) activity and pronounced relocalization of CDK2 into the cytoplasm. Nuclear targeting CDK2 blocks the 1,25-(OH)2 vitamin D3-mediated growth inhibition and cell cycle G1 accumulation. Further, the nuclear targeted CDK2 blocks 1,25-(OH)2 vitamin D3-mediated inhibition of CDK2 activity and nuclear exclusion in LNCaP cells. Therefore, CDK2 cytoplasmic relocalization is the key mechanism for 1,25-(OH)2 vitamin D3 effects. Since cyclin E is important for CDK2 nuclear localization and activation, 1,25-(OH)2 vitamin D3 may exert its effects through regulation of cyclin E. Cyclin E but not a cyclin E mutant deficient in CDK2 binding reverses 1,25-(OH)2 vitamin D3-mediated antiproliferation which suggests the involvement of cyclin E as a mechanism. However, the studies showed no effects of 1,25-(OH)2 vitamin D3 on cyclin E levels, intracellular localization or binding to CDK2. In order to develop a model for studying 1,25-(OH)2 vitamin D3-mediated antiproliferative effects, LNCaP vitD.R cell line, a vitamin D resistant LNCaP derivative, was generated by continuously culturing of LNCaP cells in medium supplemented with 10 nM 1,25-(OH)2 vitamin D3 for over 9 months. The initial characterization of this cell line showed complete resistance to 1,25-(OH)2 vitamin D3-mediated effects. Analysis of vitamin D regulation of VDR target gene expression revealed that vitamin D resistance in LNCaP vitD.R cells was not due to deregulation of VDR signaling. HDAC inhibitor Trichostatin A (TSA) did not confer sensitivity of LNCaP vitD.R cells to vitamin D treatment suggested the resistance to 1,25(OH)2 vitamin D3 effect of LNCaP vitD.R cells is not due to histone deacetylase remodeling of the chromatin structure which leads to inhibition of gene transcription. While the partial sensitization of LNCaP vitD.R cells to 1,25(OH)2 vitamin D3 effect by demethylation reagent 5-Aza-2¡¯-deoxycytidine treatment suggested a set of genes involved in 1,25(OH)2 vitamin D3-mediated antiproliferative effects is silenced via hypermethylation in LNCaP vitD.R cells. These results suggested LNCaP vitD. R cell line is a useful tool and further studies to elucidate the genes involved in this effect will help uncover the mechanisms of 1,25(OH)2 vitamin D3-mediated antiproliferative effects.

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Thesis (M.S.)--University of Missouri-Columbia, 2003. / Typescript. Includes bibliographical references (leaves 101-107). Also available on the Internet.

Thesis (M.S.)--University of Missouri-Columbia, 2003. / Typescript. Includes bibliographical references (leaves 101-107). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 138-170).

Intermediate filaments are fibrous proteins, appearing in a wide variety of tissue specific forms. The function of these proteins is poorly understood, although they are commonly believed to perform a structural role in the cell. Evidence suggests that the role these proteins play may be more dynamic than was previously believed. To gain more insight into their normal in vivo function, a single-chain monoclonal antibody has been constructed to serve as a specific reagent which can disrupt the intermediate filament network in vivo. The work presented in this thesis represents the first step in an approach which involves the use of single-chain monoclonal antibodies as specific reagents to target and disrupt the function of intracellular proteins. / The polymerase chain reaction was used for the cloning and modification of the heavy and light chain variable regions of the murine monoclonal antibody produced by the TIB 131 hybridoma. The variable regions of the light and heavy IgG chains were initially amplified from cDNA using degenerate 5$ sp prime$ primers and 3$ sp prime$ primers complementary to the constant region of the appropriate chain. The amplification products were cloned individually, sequenced, then modified to include restriction sites suitable for cloning into an expression vector. The two modified variable regions were cloned into an expression vector, and when expressed in either bacteria or in a rabbit reticulocyte lysate system, yielded a protein of the expected molecular weight.