T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid disorder that results from an over proliferation of immature lymphocytes in the blood and bone marrow. It has been determined that 60% of patients stricken with T-ALL aberrantly express TAL-1 and have been shown to respond poorly to chemotherapy. This research sought to determine if TAL-1 influences the expression of the Bcl-2 family members Bcl-2 (anti-apoptotic), Bad and Bax (pro-apoptotic). TAL-1 and Bcl-2 levels were elevated while Bad and Bax levels were lower in etoposide-treated Jurkat cells as compared to TRAIL-treated and dual-treated Jurkat cells in which TAL-1 and Bcl-2 levels were lower while Bad and Bax levels were elevated. These results suggest TAL-1 up-regulates Bcl-2 and suppress Bad and Bax expression in response to etoposide treatment, thus inducing an anti-apoptotic response in the cell. These results also suggest that TRAIL and the dual treatment of etoposide and TRAIL down-regulates TAL-1 and Bcl-2 expression while up-regulating Bad and Bax, thus inducing a pro-apoptotic response in the cell. / Department of Biology

I have used a molecular genetic approach to study early events in the gene network that precede apoptotic commitment in glucocorticoid-induced thymocyte apoptosis. A panel of recessive, apoptotic-deficient (Apt⁻) mutants were isolated that are cross resistant to several diverse apoptotic treatments. These results indicated that the signal pathways initiated by glucocorticoids, gamma radiation, and c-AMP analog treatment converge to a common apoptotic pathway. Complementation analysis of Apt⁻ cell lines has defined five independent complementation groups that appear to represent mutations in genes that are required for apoptotic commitment. In addition, I have characterized induced gene expression patterns characteristic of dexamethasone (dex)-induced apoptosis and have found that glutathione-s-transferase (GST), Dag8 (a gene of unknown function) and calmodulin (Cam) transcript levels are elevated following dex treatment. Dex-treatment of Apt⁻ cell lines does not change GST or Cam transcript levels which suggests that these cell lines are blocked in early steps of the apoptotic pathway. In contrast, the dominant oncogene, Bcl-2, blocks apoptosis and appears to affect a relatively late event in the apoptotic pathway since the pattern of dex-induced gene expression is normal in cells that express this protein. Since the Apt⁻ cells contain wild type levels of functional glucocorticoid receptor (GR), GST and Cam do not appear to be primary GR target genes, but seem to respond to cellular events that occur prior to apoptotic commitment. In support of this conclusion, it was found that GST transcript levels increase in calcium ionophore-induced apoptotic cells. In contrast, Dag8, transcript levels increased in dex-treated Apt⁻ cells indicating that Dag8 is most likely a primary GR target gene. Furthermore, Dag8 expression was found to be restricted to thymocyte containing tissues and its locus was mapped to the H2 complex of chromosome 17, a region that is known to contain many immunologically important genes. Finally, a model is presented to describe a common apoptotic pathway in murine thymocytes and proposes that an increase in oxidative stress precedes calcium mobilization in response to glucocorticoid treatment.

Yao, Yao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 131-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

T-ALL accounts for 15% of childhood leukemias and approximately 60% of patients overexpress TAL1. TAL1/SCL encodes a transcription factor that regulates hematopoiesis by dimerizing with additional transcription factors including E12, E47, and GATA-1. TAL1 has also been found to repress expression of NF-κB1, potentially promoting formation of an NF-κB p65/c-Rel heterodimer that encourages cell survival by up-regulating IAPs and IκB. However, the correlation between TAL1 and p65/c-Rel expression and their effects on downstream targets like IKK, IκB, and other anti-apoptotic proteins is poorly understood. Jurkat cells, expressing TAL1, were treated with TNFα and/or etoposide to induce apoptosis and experiments were performed to assess the expression of proteins of interest. Caspase-8 activity assays were also performed to help delineate the apoptotic signal present in these cells. Determining if interactions between TAL1, NF-κB, and other downstream targets help promote apoptotic resistance will further research into better, more targeted treatments for T-ALL. / Department of Biology

T-cell acute lymphoblastic leukemia (T-ALL) is a childhood cancer created through genetic alterations; most commonly upregulation of TALI and LMOI oncoproteins. T-ALL is treated with radiation and chemotherapy, but malignant T-cells are resistant to apoptotic stimulation. To study this disorder, AKR-DP-603 cells were transduced to express both oncoproteins. Western blots verified protein expression and each population was treated with etoposide. Caspase-3 and Annexin-V/FITC apoptosis assays were performed following treatment. When the response of control cells was compared to engineered cells, no difference was observed from the Annexin-V/FITC assay, and only LM01 cells showed a difference in the caspase-3 assay. Furthermore, cells were transfected with siRNA to TALI and LM01 and the apoptotic response was re-tested. Complete silencing was verified by Western and apoptotic activity varied in the TALI population for both assays. These differences might indicate that cells resisted etoposide induction and following silencing were sensitized apoptotic induction. / Department of Biology

Tam Ka-ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 164-179). / Abstracts in English and Chinese. / Chapter Chapter one: --- Introduction --- p.4 / Chapter 1.1 --- Introduction of BRE --- p.4 / Chapter 1.1.1 --- Discovery of BRE --- p.4 / Chapter 1.1.2 --- cDNA sequence and amino acids sequence of BRE --- p.4 / Chapter 1.1.3 --- BRE expression level in Human and rat organs --- p.5 / Chapter 1.1.4 --- Expression of Human and mouse BRE in multiple isoforms --- p.6 / Chapter 1.1.4.1 --- BRE isoforms in Human --- p.6 / Chapter 1.1.4.2 --- BRE isoforms in mouse --- p.6 / Chapter 1.1.5 --- mRNA level of BRE upon stress --- p.7 / Chapter 1.1.6 --- BRE and steroidogenesis --- p.8 / Chapter 1.1.7 --- BRE and p55 tumor necrosis factor α (TNF) receptor --- p.9 / Chapter 1.1.8 --- BRE and NFkB activity --- p.9 / Chapter 1.1.9 --- Anti-apoptotic effect of BRE --- p.10 / Chapter 1.1.10 --- BRE enhances the growth of tumor cells --- p.12 / Chapter 1.1.11 --- BRE and its ubiquitination activity --- p.12 / Chapter 1.1.12 --- Regulation of Prohibitin and p53 expression and proliferation by BRE --- p.13 / Chapter 1.2 --- Regulation of transcription --- p.15 / Chapter 1.2.1 --- Cis-acting elements --- p.17 / Chapter 1.2.1.1 --- The TATA box --- p.18 / Chapter 1.2.1.2 --- The GC box and CAAT box --- p.18 / Chapter 1.2.1.3 --- The initiator (Inr) --- p.19 / Chapter 1.2.1.4 --- CpG islands --- p.20 / Chapter 1.2.2 --- Trans- acting protein factors --- p.21 / Chapter 1.2.2.1 --- Zinc finger domain --- p.21 / Chapter 1.2.2.2 --- Basic helix-turn-helix domain (bHLH) --- p.22 / Chapter 1.3 --- Hypothesis and Objectives --- p.23 / Chapter Chapter two: --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Primers used in polymerase chain reaction (PCR) and sequencing --- p.25 / Chapter 2.1.2 --- DNA clones used in the studies --- p.26 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.27 / Chapter 2.1.4 --- Materials for protein manipulation --- p.28 / Chapter 2.1.5 --- Antibodies --- p.28 / Chapter 2.1.6 --- Chemical used in treatments --- p.29 / Chapter 2.1.7 --- Kits --- p.29 / Chapter 2.1.8 --- Culture media and reagents --- p.30 / Chapter 2.1.9 --- Instrumentation --- p.30 / Chapter 2.1.10 --- Bacterial strain used for transfection and cloning --- p.31 / Chapter 2.2 --- Methodologies --- p.36 / Chapter 2.2.1 --- Cell culture --- p.36 / Chapter 2.2.1.1 --- Monolayer cells --- p.36 / Chapter 2.2.1.2 --- Suspension cell --- p.36 / Chapter 2.2.2 --- Identification of the transcriptional start site (TSS) of BRE by RNA ligase- mediated rapid amplification of 5，and 3，cDNA ends (RLM-RACE) --- p.37 / Chapter 2.2.3 --- Preparation of the 5' untranslated region (UTR) fragments of BRE --- p.39 / Chapter 2.2.3.1 --- Polymerase chain reaction (PCR) with Taq polymerase --- p.39 / Chapter 2.2.3.2 --- Polymerase chain reaction (PCR) with PhusiońёØ high-fidelity DNA.… --- p.40 / Chapter 2.2.4 --- Construction of the reporter constructs --- p.42 / Chapter 2.2.5 --- Cell transfection --- p.42 / Chapter 2.2.6 --- Dual-luciferase reporter assay --- p.43 / Chapter 2.2.7 --- Western blotting --- p.44 / Chapter 2.2.8 --- Cell cycle analysis by flow cytometry --- p.45 / Chapter 2.2.9 --- BRE antibody production --- p.43 / Chapter Chapter Three: --- Identification of transcriptional start sites and promoter region for BRE --- p.52 / Chapter 3.1 --- Identification of the transcriptional start sites for BRE --- p.52 / Chapter 3.2 --- Computational analysis of the 5' region of BRE --- p.57 / Chapter 3.2.1 --- Putative transcriptional factor binding sites --- p.57 / Chapter 3.2.2 --- CpG island --- p.58 / Chapter 3.3 --- Identification of BRE promoter --- p.64 / Chapter Chapter Four: --- Characterization of transcriptional regulation of BRE --- p.70 / Chapter 4.1 --- Regulation of BRE promoter by genotoxic stimuli and retinoic acid --- p.71 / Chapter 4.1.1 --- Etoposide --- p.71 / Chapter 4.1.2 --- 4-nitroquinoline-l -oxide (4NQO) --- p.79 / Chapter 4.1.3 --- Retinoic acid (RA) --- p.87 / Chapter 4.2 --- Regulation of BRE promoter by p53 protein and gamma irradiation --- p.90 / Chapter 4.2.1 --- Co-transfection with p53 plasmid --- p.90 / Chapter 4.2.2 --- Gamma irradiation (y irradiation) --- p.96 / Chapter 4.2.2.1 --- γ irradiation treatment of HeLa cells --- p.96 / Chapter 4.2.2.2 --- γ irradiation treatment of Balb/c 3T3 cells --- p.99 / Chapter 4.3 --- Regulation of BRE promoter by BRE --- p.103 / Chapter 4.3.1 --- Co-transfection with V5-tagged BRE (GS-BRE) --- p.103 / Chapter 4.3 2 --- Co-transfection with untagged BRE (pcDNA3-BRE) --- p.107 / Chapter 4.4 --- Regulation of BRE promoter by culture condition --- p.110 / Chapter 4.4.1 --- Cell density --- p.110 / Chapter 4.4.2 --- Serum deprivation --- p.114 / Chapter 4.5 --- Regulation of BRE promoter by kinase inhibitors --- p.120 / Chapter Chapter Five: --- BRE and cell cycle analysis --- p.127 / Chapter 5.1 --- Cell synchronization in G1 phase by aphidicolin (APC) --- p.127 / Chapter 5.1.1 --- Flow analysis --- p.128 / Chapter 5.1.2 --- Luciferase reporter assay --- p.128 / Chapter 5.1.3 --- Western blot analysis --- p.129 / Chapter 5.2 --- Cell synchronization in G2/M phase by colchicine (COL) --- p.137 / Chapter 5.2.1 --- Flow analysis --- p.137 / Chapter 5.2.2 --- Luciferase reporter assay --- p.137 / Chapter 5.2.3 --- Western blot analysis --- p.138 / Chapter 5.3 --- Cell cycle analysis of the treatments investigated by luciferase assays --- p.144 / Chapter Chapter Six: --- Discussion --- p.149 / Chapter 6.1 --- Study of BRE expression --- p.149 / Chapter 6.2 --- Study of BRE regulation --- p.154 / Chapter 6.3 --- Conclusion --- p.163 / Reference --- p.164 / Appendix (Raw data and statistical information of luciferase assays)

Wong, Wan Man. / Thesis submitted in: December 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 85-98). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iii / Acknowledgements --- p.v / Lists of Figures and Tables --- p.vi / Table of Abbreviations --- p.xi / Table of Contents --- p.xv / Chapter Chapter I --- Introduction / Chapter 1.1 --- Brain and Reproductive Organ Expressed Gene --- p.1 / Chapter 1.2 --- Programmed cell death --- p.4 / Chapter 1.3 --- Limb development in mouse --- p.8 / Chapter 1.4 --- Role of BRE in apoptosis --- p.12 / Chapter 1.5 --- Role of programmed cell death in interdigital tissue regression --- p.14 / Chapter 1.6 --- Aim of study --- p.17 / Chapter Chpater II --- Materials and methods / Chapter 2.1 --- Mice --- p.18 / Chapter 2.2 --- In-situ hybridization / Chapter 2.2.1 --- Histology --- p.18 / Chapter 2.2.2 --- Preparation of riboprobe for in-situ hybridization --- p.19 / Chapter 2.2.3 --- In-situ hybridization --- p.20 / Chapter 2.3 --- Interdigital tissue culture --- p.21 / Chapter 2.4 --- Gene interference / Chapter 2.4.1 --- Construction of Bre-siRNA --- p.22 / Chapter 2.4.2 --- siRNA transfection of cultured interdigital cells --- p.23 / Chapter 2.5 --- Semi-quantitative RT-PCR / Chapter 2.5.1 --- Sample collection of interdigital cells and explants --- p.23 / Chapter 2.5.2 --- RNA isolation and extraction --- p.24 / Chapter 2.5.3 --- Reverse-transcription and cDNA synthesis --- p.25 / Chapter 2.5.4 --- Polymerase chain reaction --- p.26 / Chapter 2.6 --- Assay of cell viability by MTT --- p.28 / Chapter 2.7 --- Comparative proteomics --- p.30 / Chapter 2.7.1 --- Collection of interdigital cells --- p.30 / Chapter 2.7.2 --- Preparation of cell lysate --- p.31 / Chapter 2.7.3 --- Assay of protein concentration in cell lysate --- p.31 / Chapter 2.7.4 --- Two-dimensional gel electrophoresis --- p.33 / Chapter 2.7.5 --- Protein identification by mass fingerprinting --- p.36 / Chapter 2.8 --- Statistical Method --- p.38 / Chapter Chapter III --- Results / Chapter 3.1 --- Spatial and temporal expression of Bre in murine embryonic hindlimbs --- p.39 / Chapter 3.2 --- Expression of Bre isoforms in interdigital tissues --- p.45 / Chapter 3.3 --- Silencing of Bre expression by siRNA in interdigital cells --- p.49 / Chapter 3.4 --- Effect on viability of Bre-silenced interdigital cells by siRNA --- p.51 / Chapter 3.5 --- Comparative proteomic profile of Bre-silenced interdigital cultured cells --- p.53 / Chapter 3.6 --- Identification of proteins that were differentially expressed by MALDI- TOF --- p.71 / Chapter 3.7 --- The mRNA levels of proteins identified that were differentially expressed --- p.74 / Chapter Chapter IV --- Discussion --- p.77 / References --- p.85 / Appendices --- p.99 / Publication --- p.108

Li Qing. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 161-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.