Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on January 7, 2009). Research advisor: Gabor Tigyi, M.D., Ph.D. Document formatted into pages (xiv, 105 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 90-105).

No description available.

Few weeks after birth, a switch in peptide elongation factor 1As from EF-1alpha/EF1A-1 to S1/EF1A-2 occurs in brain neurons, heart and skeletal muscles of mammalians. In order to elucidate the reason behind this switch, I studied the expression of both homologous proteins during muscle differentiation and apoptosis and, documented the relation between peptide elongation factors and caspase-3 activation. I found that during in vitro muscle differentiation of L6 myoblasts, a switch in peptide elongation factors 1A occurs as physiologically observed in skeletal muscles. While EF-1alpha/EF1A-1 is expressed in replicating myoblasts, S1/EF1A-2 is solely found in differentiated myotubes where it replaces EF-1alpha/EFIA-1 as the major elongation factor. Similarly, upon serum deprivation-induced apoptosis, a reversion in peptide elongation factors 1A is observed: EF-1alpha/EF1A-1 replaces S1/EF1A-2 and becomes the major form of elongation factor 1A present in dying myotubes. This switch correlates in myotubes with the activation of caspase-3 protein, a cysteine protease involved in apoptosis. When L6 myotubes constitutively express S1/EF1A-2 as caused by adenoviral gene transfer, they become resistant to serum deprivation-induced apoptosis. In contrast, when L6 myotubes are transfected with EF-1alpha/EF1A-1 gene, they die more rapidly from serum deprivation-induced apoptosis than control cells. Transfection using anti-sense EF-1alpha/EF1A-1 gene protects myotubes from apoptotic cell death. Thus, both elongation factor 1As exert opposing effect on muscle survival: while EF-1alpha/EF1A-1 accelerates apoptotic cell death, S1/EF1A-2 protects muscles against apoptosis. / I found that skeletal muscles are the only tissues where, despite the constitutive expression of caspase-3 mRNA, the protein can be absent. Furthermore, I found that while immediately after birth, caspase-3 protein is present in skeletal muscles, a few weeks afterwards, the protein cannot be detected by Western blotting. In skeletal muscle, this change correlates with the observed switch in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 and suggests that caspase-3 is translationally regulated in skeletal muscles. The laboratory previously reported that while EF-1alpha/EF1A-1 protein reappears; S1/EF1A-2 protein becomes absent from regenerating muscles. However, once tissue regeneration is completed, the situation returns to normal as EF-1alpha/EF1A-1 disappears and S1/EF1A-2 reappears to become the only type 1A elongation factor expressed in muscle. / In conclusion, I found that the developmental switch observed in peptide elongation factors from EF-1alpha/EF1A-1 to S1/EF1A-2 partly serves to protect muscle cells from apoptosis. Thus, I am the first to identify a noncanonical function for S1/EF1A-2. (Abstract shortened by UMI.)

by Lai Bo San Paul. / "April 1997." / "Revised May 1998." / Thesis (M.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 175-228). / 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. / Abstract in Chinese.

by Lau Kwok Wing, Wilson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 166-188). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 : --- GENERAL INTRODUCTION --- p.1 / Chapter (1.1) --- Hematopoiesis : An Overview --- p.2 / Chapter (1.1.1) --- Development of Blood Cells and Sites of Hematopoiesis --- p.2 / Chapter (1.1.2) --- Hematopoietic Cytokine Network --- p.4 / Chapter (1.1.3) --- Molecular Control of Hematopoietic Cell Development --- p.5 / Chapter (1.2) --- Leukemia : An Overview --- p.8 / Chapter (1.2.1) --- Leukemia : Abnormalities in Blood Cell Formation --- p.8 / Chapter (1.2.2) --- Pathophysiology and Etiology of Leukemia --- p.10 / Chapter (1.2.3) --- New Avenues for Therapy : Induction of Differentiation and Apoptosis --- p.12 / Chapter (1.3) --- Induction of Differentiation in Myeloid Leukemia Cells --- p.14 / Chapter (1.3.1) --- Inducers of Leukemic Cell Differentiation --- p.14 / Chapter (1.3.2) --- Cytokines as Inducers of Myeloid Leukemic Cell Differentiation --- p.17 / Chapter (1.3.3) --- Phenotypic Changes and Functional Characterizations --- p.20 / Chapter (1.3.4) --- Modulation of Gene Expression in Myeloid Leukemic Cell Differentiation --- p.22 / Chapter (1.4) --- Apoptosis and Leukemic Cell Death --- p.23 / Chapter (1.4.1) --- Apoptosis : An Overview --- p.23 / Chapter (1.4.2) --- Cytokines and Apoptosis in Myeloid Leukemia --- p.26 / Chapter (1.5) --- Objectives and Research Strategy --- p.27 / Chapter (1.5.1) --- The Murine Myeloid Leukemia Cell Line (Ml) as an Experimental Cell Model for Acute Myeloid Leukemia --- p.27 / Chapter (1.5.2) --- Leukemia Inhibitory Factor (LIF) as a Differentiation Inducer --- p.28 / Chapter (1.5.3) --- Aims and Scopes of This Investigation --- p.31 / Chapter CHAPTER 2 ： --- MATERIALS AND METHODS --- p.33 / Chapter (2.1) --- Materials --- p.34 / Chapter (2.1.1) --- Mice --- p.34 / Chapter (2.1.2) --- Cell Lines --- p.34 / Chapter (2.1.3) --- Recombinant Cytokines --- p.34 / Chapter (2.1.4) --- Monoclonal Antibodies --- p.36 / Chapter (2.1.5) --- Oligonucleotide Primers and Internal Probes --- p.37 / Chapter (2.1.6) --- "Buffers, Culture Medium and Other Reagents" --- p.39 / Chapter (2.1.7) --- Reagents and Solutions for Gene Expression Study --- p.41 / Chapter (2.2) --- Methods --- p.46 / Chapter (2.2.1) --- Culture of Myeloid Leukemia Cell Lines --- p.46 / Chapter (2.2.2) --- Induction of Leukemic Cell Differentiation --- p.46 / Chapter (2.2.3) --- Determination of Cell Growth and Proliferation --- p.46 / Chapter (2.2.4) --- Cell Morphological Study --- p.47 / Chapter (2.2.5) --- Assessment of Differentiation-Associated Characteristics --- p.48 / Chapter (2.2.5.1) --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter (2.2.5.2) --- Phagocytosis Assay --- p.48 / Chapter (2.2.5.3) --- Assay of Plastic Adherence --- p.49 / Chapter (2.2.6) --- Flow Cytometric Analysis --- p.49 / Chapter (2.2.6.1) --- Surface Antigen Immunophenotyping --- p.49 / Chapter (2.2.6.2) --- Assay of Endocytic Activity --- p.50 / Chapter (2.2.6.3) --- Assay of Non-specific Esterase Activity --- p.50 / Chapter (2.2.6.4) --- Cell Cycle / DNA Content Evaluation --- p.51 / Chapter (2.2.7) --- Gene Expression Analysis --- p.52 / Chapter (2.2.7.1) --- Preparation of Cell Lysate --- p.52 / Chapter (2.2.7.2) --- RNA Isolation --- p.52 / Chapter (2.2.7.3) --- Reverse Transcription --- p.53 / Chapter (2.2.7.4) --- Polymerase Chain Reaction (PGR) --- p.54 / Chapter (2.2.7.5) --- Agarose Gel Electrophoresis --- p.55 / Chapter (2.2.7.6) --- 3' End Labelling of Oligonucleotide Probes --- p.56 / Chapter (2.2.7.7) --- Dot Blot Hybridization --- p.56 / Chapter (2.2.7.8) --- Digoxigenin (DIG) Chemiluminescent Detection --- p.57 / Chapter (2.2.8) --- DNA Fragmentation Analysis --- p.58 / Chapter (2.2.9) --- Statistical Analysis --- p.59 / Chapter CHAPTER 3 ： --- "EFFECTS OF LEUKEMIA INHIBITORY FACTOR ON THE PROLIFERATION, DIFFERENTIATION, AND APOPTOSIS OF MURINE MYELOID LEUKEMIA Ml CELLS" --- p.60 / Chapter (3.1) --- Introduction --- p.61 / Chapter (3.2) --- Results --- p.63 / Chapter (3.2.1) --- Induction of Growth Arrest in rmLIF-Treated Ml Cells --- p.63 / Chapter (3.2.2) --- Induction of Monocytic Differentiation of Ml cells by rmLIF --- p.66 / Chapter (3.2.2.1) --- Morphological Changes --- p.66 / Chapter (3.2.2.2) --- Induction of Plastic Adherence --- p.70 / Chapter (3.2.2.3) --- Surface Antigen Immunophenotyping --- p.70 / Chapter (3.2.2.4) --- NBT-Reducing Activity of rmLIF-Treated Ml Cells --- p.76 / Chapter (3.2.2.5) --- Non-specific Esterase Activity of rmLIF-Treated Ml Cells --- p.77 / Chapter (3.2.2.6) --- Endocytic Activity of rmLIF-Treated Ml Cells --- p.78 / Chapter (3.2.2.7) --- Phagocytic Activity of rmLIF-Treated Ml Cells --- p.79 / Chapter (3.2.3) --- Induction of Differentiation-Associated DNA Fragmentation --- p.80 / Chapter (3.2.4) --- Production of Differentiation-Inducing Factors --- p.84 / Chapter (3.3) --- Discussion --- p.88 / Chapter CHAPTER 4 : --- CYTOKINE INTERACTIONS IN REGULATING THE PROLIFERATION AND DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.94 / Chapter (4.1) --- Introduction --- p.95 / Chapter (4.2) --- Results --- p.97 / Chapter (4.2.1) --- Synergistic Effect of LIF and IL-6 on the Proliferation and Differentiation of Ml Cells --- p.97 / Chapter (4.2.2) --- Regulation of Proliferation and Differentiation of Ml Cells by LIF and OSM --- p.101 / Chapter (4.2.3) --- Effects of LIF and TNF-α on the Proliferation and Differentiation of Ml Cells --- p.104 / Chapter (4.2.4) --- Synergistic Effect of LIF and IL-1 on the Proliferation and Differentiation of Ml Cells --- p.107 / Chapter (4.3) --- Discussion --- p.115 / Chapter CHAPTER 5 ： --- MODULATION OF CYTOKINE AND CYTOKINE RECEPTOR GENE EXPRESSION IN LIF- INDUCED DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.120 / Chapter (5.1) --- Introduction --- p.121 / Chapter (5.2) --- Results --- p.123 / Chapter (5.3) --- Discussion --- p.152 / Chapter CHAPTER 6 ： --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.158 / REFERENCES --- p.166

根據文獻研究記載，化療藥物可誘導癌細胞的凋亡，這是公認的化療療法的主要治療效果。作為一種程式性細胞死亡，積累的實驗證據表明，誘導所致的細胞凋亡是可逆轉的。這就引出了對於細胞凋亡恢復及其調節機制的相關問題。 / 在這項研究中，我們證明了在質膜不對稱的散失和半胱天冬酶（caspase）啟動後，HeLa細胞的凋亡的啟動可逆轉。我們發現，除了被廣泛研究的抗增殖作用外，金雀異黃素(genistein)可抑制細胞凋亡的復蘇。即時定量PCR發現抗凋亡基因MDM2和XIAP在凋亡逆轉過程中表達水準上調，金雀異黃素可抑制其表達水準的上調。金雀異黃素，MDM2蛋白抑制劑和XIAP抑制劑的利用，造成復原細胞內持續的半胱天冬酶活性和增強的細胞死亡效果。然而，半胱天冬酶抑制劑並不能挽救金雀異黃素的抑制作用。流式細胞儀的研究表明，金雀異黃素可以導致凋亡逆轉細胞持久磷脂醯絲氨酸（PS）外化和逆轉細胞的細胞壞死。抑制半胱天冬酶活性將金雀異黃素的主要作用轉移到壞死效果。這些結果揭示了金雀異黃素抑制細胞凋亡逆轉的兩個可能的機制。 / 金雀異黃素能維持現有的細胞凋亡信號從而增強細胞凋亡。它也可以破壞凋亡恢復過程，導致繼發性壞死。金雀異黃素對於細胞凋亡逆轉的抑制可與常規化療相結合，以提高治療結果. / It is well documented that chemotherapeutical agents could induce apoptosis of cancer cells, which is recognized as a major treatment effect of chemotherapy. Accumulating evidence indicates that chemopreventive agents like soybean isoflavone genistein could potentiate the antitumor effect of chemotherapeutic drugs both in vivo and in vitro. The mechanistic basis of this augmentation effect by genistein remains to be fully elucidated. / In this study, we demonstrated while low-concentration ethanol stressed cancer cells could recover, the presence of genistein promoted the cell death of stressed cancer cells that displayed apoptotic features. In HeLa cells, quantitative real-time PCR revealed the up-regulation of anti-apoptotic genes MDM2 and XIAP during the recovery process, and genistein suppressed their expression. The application of genistein, MDM2 inhibitor and XIAP inhibitor to the recovering HeLa cells caused persistent caspase activity and enhanced cell death. However, the death-promoting effect of genistein was not rescued by caspase inhibitor. Flow cytometry study indicated that genistein treatment could lead to persistent phosphatidylserine (PS) externalization and necrotic events in the recovering HeLa cells. Caspase activity inhibition shifted the major effect of genistein to secondary necrosis. / These results suggested two possible mechanisms through which genistein promoted cell death in stressed HeLa cells. Genistein could maintain the existing apoptotic signal to enhance apoptotic cell death. It could also disrupt the recovering process in caspase-independent manner, which lead to secondary necrosis. These effects may account for the enhanced antitumor effect of chemotherapeutic drugs when they were combined with genistein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Xin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 79-90). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in also in Chinese. / Cover Page / Statement --- p.i / Thesis Committee members --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / Table of contents --- p.vi / List of abbreviations --- p.ix / List of figures and tables --- p.xi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction to general cancer biology --- p.1 / Chapter 1.1.1 --- Overview of cancer --- p.1 / Chapter 1.1.1.1 --- Classification of cancer --- p.1 / Chapter 1.1.1.2 --- Risk factors of carcinogenesis --- p.2 / Chapter 1.1.1.3 --- Cancer prevention and therapies --- p.4 / Chapter 1.1.2 --- Models of cancer development --- p.6 / Chapter 1.1.2.1 --- Multistage model of carcinogenesis --- p.6 / Chapter 1.1.2.2 --- Colorectal cancer as an example of multistep / multigene carcinogenesis --- p.7 / Chapter 1.1.2.3 --- Driving force for cancer development --- p.9 / Chapter 1.1.3 --- Properties of cancer cells --- p.11 / Chapter 1.2 --- Apoptosis and its roles in cancer development and treatment --- p.14 / Chapter 1.2.1 --- Overview of apoptosis --- p.14 / Chapter 1.2.2 --- Molecular mechanism of apoptosis --- p.15 / Chapter 1.2.3 --- Positive and negative regulation of apoptosis --- p.18 / Chapter 1.2.4 --- Apoptotic defects in cancer development --- p.20 / Chapter 1.2.5 --- Apoptosis in cancer treatment --- p.23 / Chapter 1.3 --- The reversibility of apoptotic events --- p.25 / Chapter 1.4 --- Genistein and its relevance to cancer therapy --- p.27 / Chapter 1.5 --- Objectives of the study --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Cancer cell lines --- p.30 / Chapter 2.1.2 --- Cell culture media and additives --- p.30 / Chapter 2.1.3 --- Biochemical kits --- p.30 / Chapter 2.1.4 --- Chemicals and reagents --- p.30 / Chapter 2.1.5 --- Antibodies --- p.31 / Chapter 2.1.6 --- Primers used for quantitative real-time PCR --- p.32 / Chapter 2.1.7 --- Buffers and solutions --- p.32 / Chapter 2.2 --- Methods and procedures --- p.33 / Chapter 2.2.1 --- Cell culture establishment and cryopreservation --- p.33 / Chapter 2.2.2 --- Living cell staining and imaging --- p.34 / Chapter 2.2.3 --- MTT cell viability assay --- p.34 / Chapter 2.2.4 --- BrdU cell proliferation assay --- p.35 / Chapter 2.2.5 --- LDH cytotoxicity assay --- p.35 / Chapter 2.2.6 --- Quantitative real-time PCR --- p.36 / Chapter 2.2.7 --- Western blotting --- p.37 / Chapter 2.2.8 --- Annexin V/ Propidium Iodide Assay --- p.38 / Chapter 2.2.9 --- Trypan Blue Dye Exclusion Assay --- p.39 / Chapter 2.2.10 --- Cleaved-Caspase 3 Immunostaining --- p.39 / Chapter 2.2.11 --- Statistical Analysis --- p.39 / Chapter Chapter 3 --- Results --- p.40 / Chapter 3.1 --- Low concentration ethanol stressed cancer cells displayed apoptotic features and the stressed cell could recover after stress removal --- p.40 / Chapter 3.1.1 --- Morphological changes and apoptotic marker activation in low concentration ethanol stress --- p.40 / Chapter 3.1.2 --- In situ study of morphological changes and caspase 3 activation in HeLa --- p.44 / Chapter 3.2 --- Genistein promoted the cell death of stressed cancer cells at non-cytotoxic concentration towards unstressed cells --- p.46 / Chapter 3.2.1 --- Dose-dependent response of genistein on stressed and unstressed cells --- p.46 / Chapter 3.2.2 --- In HeLa cells, genistein suppressed the recovery from stress treatment at non-cytotoxic concentration --- p.48 / Chapter 3.2.3 --- Genistein promoted both apoptosis and necrosis in stressed cells. . --- p.49 / Chapter 3.3 --- Genes involved in the recovery from stress treatment were influenced by genistein --- p.53 / Chapter 3.3.1 --- Stressed HeLa cells were more sensitive to the inhibition of de novo synthesis --- p.53 / Chapter 3.3.2 --- Expression profiles of genes involved in recovery and the influence of genistein --- p.55 / Chapter 3.4 --- Like genistein, MDM2 and XIAP inhibitor potentiated the cell death and caused persistent caspase-3 activity in stressed cells --- p.58 / Chapter 3.4.1. --- Stressed HeLa cells were much more sensitive to the inhibition of XIAP and MDM2 --- p.58 / Chapter 3.4.2 --- The presence of inhibitor at non-cytotoxic concentration to unstressed cells suppressed the recovery of the stressed cells --- p.60 / Chapter 3.4.3 --- Genistein, MDM2 inhibitor and XIAP inhibitor caused persistent apoptotic signals in recovering cells. --- p.61 / Chapter 3.5 --- The death-promoting effect by genistein could be caspase-independent --- p.64 / Chapter 3.6 --- Caspase activity abrogation shifted genistein’s action profile --- p.66 / Chapter Chapter 4 --- Discussion and prospect --- p.70 / Chapter 4.1 --- The apoptotic features were induced by low concentration ethanol stress --- p.70 / Chapter 4.2 --- The apoptotic features caused by ethanol stress were reversible --- p.71 / Chapter 4.3 --- Genistein showed death-promoting effects on the recovering cells --- p.72 / Chapter 4.4 --- The genes (XIAP and MDM2) that were involved in the recovery process may function to terminate apoptotic signal --- p.73 / Chapter 4.5 --- Genistein suppressed the upregulation of anti-apoptotic genes and promoted the expression of pro-apoptotic genes --- p.74 / Chapter 4.6 --- The XIAP and MDM2 activity were essential for the recovery from stress --- p.75 / Chapter 4.7 --- Caspase inhibition increased the secondary necrosis in recovering cells with genistein treatment --- p.76 / Chapter 4.8 --- Hypothetic mechanism of genistein’s inhibitory effect on the recovery of stressed cells --- p.77 / Chapter 4.9 --- Summary and prospects --- p.78 / Reference list --- p.79

First study determined the effects of genetic deletion of nNOS on the levels of spontaneous apoptosis in brain of young-adult (2-3 months) and aged (12-18 months) mice, using nNOS-knockout mice with age-matched B6129SF2/J mice as wild-type control. The results indicate that aging resulted in 11-fold increase in levels of apoptotic-DNA-fragmentation in B6129SF2/J mouse brain. nNOS-knockout mice demonstrated dramatic (72-fold) increases in levels of apoptotic-DNA-fragmentation in young-adult, but not aged, brains. Aging resulted in decreased number of nNOS-positive cells, increased number of iNOS-positive cells and no change of eNOS-positive cells in control mice. The data suggest that nNOS may serve an anti-apoptotic/neuroprotective role in young-adult mouse brain. However, because of diminished nNOS and increased iNOS with aging, this neuroprotective effect may become less effective in aged mice. / Fourth study showed that new microchip-electrophoresis-technology can be successfully used to identify and quantify levels of apoptosic-DNA-fragments in brain slice cultures, similar to our previous studies with CE-LIF. Because of the much greater throughput of microchip-electrophoresis-system, compared to CE-LIF, this new technology should help accelerate the progress of apoptosis research. / In second study, apoptotic effects of genetic deletion of either eNOS or iNOS were studied using young-adult (1-4 months) and aged (12-24 months) eNOS- or iNOS-knockout mice with age-matched C57BL/6J wild-type control mice. The data show that both young-adult and aged iNOS-knockout mice had dramatically (8- to 36-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially noticeable in hippocampus and medulla oblongata. Both young-adult and aged eNOS-knockout mice also had dramatically (18- to 35-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially in cerebral cortex, hippocampus and medulla oblongata. The data suggest that both iNOS and eNOS provide neuroprotective effects, helping to limit the extent of spontaneous apoptosis in brain of young-adult and aged mice. / Nitric oxide (NO) has either pro-apoptotic or anti-apoptotic effects on neuronal cells, depending on concentration of NO produced by different source of NO synthases (NOSs) including neuronal-NO-synthase (nNOS/NOS-1), inducible-NO-synthase (iNOS/NOS-2) or endothelial-NO-synthase (eNOS/NOS-3) and possibly age of the individual. The present study determines if genetic deletion of nNOS, iNOS or eNOS alters levels of aging-induced apoptosis in vivo and hydrogen peroxide (H2O2)-induced-apoptosis in organotypic brain slice cultures using NOS-knockout mice. The quantitative ultrasensitive techniques using capillary-electrophoresis with laser-induced-fluorescent detector (CE-LIF) and Cell-Death---Detection-ELISA were used as novel ways to accurately measure the levels of apoptotic-DNA-fragmentation. Expressions of different forms of NOSs were determined by immunohistochemical-staining. / Third study determined H2O2-induced apoptosis in hippocampal and cerebellar slices from young-adult (8-10 weeks) and aged (12-24 months) C57BL/6J control mice, as well as iNOS- and eNOS-knockout mice (determined by Cell-Death-Detection-ELISA measuring levels of apoptotic-DNA-fragmentation). The data show spontaneous onset of apoptosis occurred in both hippocampal and cerebellar slices during culturing, beginning at 24 hours and progressively increasing for 48--72 hours. Staurosporine (positive-control) and H2 O2 both caused time-dependent increases in apoptosis in both hippocampal and cerebellar slices, compared to time-matched controls. Lastly, genetic deletion of iNOS greatly reduced levels of spontaneous apoptosis in young hippocampus and aged cerebellum, suggesting iNOS had contributed to induction of spontaneous apoptosis. / Chow Wing Han Vivian. / "Dec 2005." / Advisers: Siew Boon Chew Cheng; Ray Ronald Fiscus. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6218. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 144-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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.

Apart from CAD, the transient liberation of AIF during day 6 of TF-1 differentiation could pose another threat to the genomic DNA in cells. We have demonstrated the absence of AIF in the nucleus of TF-1 cells despite its release from the mitochondria by using confocal studies. Moreover, the expression of heat shock protein 70 kDa (Hsp70), a well-known antagonist of AIF, was found to be temporarily increased at day 6. Taken together, our results implied a plausible retention of AIF in the cytoplasm by Hsp70. Although Hsp70 is commonly utilized by many cancer cells to counteract AIF and avoid DNA fragmentation, we are the first to demonstrate its role in suppressing AIF during normal erythroid maturation. / As a whole, we have illustrated that the activated caspase-3, mediated most likely by the mitochondrial pathway, is an essential component in the differentiation of TF-1 cells. Its activation was nevertheless not coupled with DNA fragmentation due to some protective mechanisms such as CAD downregulation, Hsp70 upregulation and overexpression of Bcl-XL. Our study therefore provides some insights in the understanding of the relationship between human erythropoiesis and apoptosis and a better understanding in this regard will undoubtedly facilitate the development of new drugs in the treatment of different hematopoietic diseases. / Caspases play a central role in apoptosis. Their activations during the process are accounted for different biochemical and morphological changes in apoptotic cells. Yet in recent years, increasing studies had shown that caspases were also involved in some non-apoptotic cellular events, including T and B-lymphocytes activation, as well as the terminal differentiation of lens cells, megakaryocytes and erythrocytes. / In order to find out other unknown cellular mechanisms in erythropoiesis, mRNA differential display was employed to compare the gene expression pattern of TF-1 cells at different stages of differentiation. Several differentially expressed genes were identified and subsequently confirmed by RT PCR. These genes include formin binding protein 3, destrin and T-complex protein-1 (TCP-1). Their involvement in erythroid differentiation was still not clear at the moment but would be investigated in the near future. Furthermore, aiming at identifying the interacting proteins or inhibitors of caspase-3 in the system, a pull down assay was developed by means of the bacterial expression of a recombinant human caspase-3 mutant protein. With the mutation in the active site, the binding of our recombinant caspase-3 mutant with two known partners ICAD and BIRII (Baculovirus Inhibitor of apoptosis protein Repeat II) domain has been demonstrated. We hope in the near future that it can be employed to fish out some novel caspase-3 substrates from the differentiating TF-1 cell lysate. / In the present study, the participation of caspase in in vitro erythropoiesis was investigated using a human erythroleukemia cell line TF-1. Erythropoietin (EPO) induced erythroid maturation of TF-1 as indicated by the expression of erythroid-lineage markers like glycophorin A (GPA), transferrin receptors (CD71) and synthesis of hemoglobin (Hb). Activation of caspase-3 was observed from day 6 to day 12 during TF-1 differentiation after EPO treatment. With the administration of caspase-3 specific inhibitor, expressions of GPA and CD71 were partially blocked, suggesting that caspase-3 activation is essential in erythropoiesis in our TF-1 model. / Possible involvement of the intrinsic and extrinsic apoptotic pathways was studied by investigating respectively the activation of pro-caspase-9 and -8. It was found that caspase-9, but not -8, was activated at the corresponding time point when caspase-3 was activated. Besides, a transient mitochondrial depolarization coupled with the release of cytochrome c and apoptosis inducing factor (AIF) were detected on day 6, strongly implying a role of mitochondria in triggering the activation of executioner caspase-3. On the other hand, GPA and CD71 expressions were blocked by the application of mitochondrial depolarization inhibitor cyclosporin A (CyA). Also, the recovery of mitochondrial membrane potential was found to be correlated with an overexpression of Bcl-XL at a late stage of TF-1 differentiation, and the role of Bcl-XL was subsequently manifested further by a significant retardation of erythroid differentiation in the siRNA Bcl-XL knocked down TF-1 cells. / The exact role of caspase-3 in erythroid differentiation is far from clear at this moment. Yet, its regulation in the process is equally intriguing. On the course of TF-1 maturation, activated caspase-3 was able to cleave and de-localize the Inhibitor of Caspase-activated DNase (ICAD) from the nucleus, but at the same time DNA fragmentation was not detected by TUNEL assay nor agarose electrophoresis. Furthermore, protection against DNA fragmentation was observed in the EPO-treated TF-1 cells when challenged with a potent apoptotic inducer staurosporine (STS). These observations are in contrast to our understanding that DNA is fragmented by CAD (Caspase-activated DNase) when ICAD in the ICAD-CAD complex is cleaved by caspase-3. For these apparently contradictory observations, we demonstrated that downregulation of CAD occurred at the mRNA and protein levels during the erythroid differentiation in TF-1. This provides a cell rescuing mechanism in non-apoptotic cells with activated caspases. / Lui Chun Kin Julian. / "September 2006." / Adviser: Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1620. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 239-253). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In addition, we investigated the cytotoxic effect of a widely used chemotherapeutic agent 5Fu on laryngeal squamous cell cancer cell lines and evaluated the role of p53 in 5Fu treatment. We found that the apoptosis and G1/S cell arrest mediated by 5Fu in laryngeal cancers is p53-independent but p21 WAF1/CIP1-dependent. We further demonstrated the effect of 5Fu on HPV16-associated laryngeal cancer cells. Using cytotoxicity assay and Annexin V staining, we proved that 5Fu induces apoptosis in all of the transfected cells in a dose- and time-dependent manner, suggesting that the process was not prevented by HPV16 E6 or E7. 5Fu induced the accumulation of active pRb and cyclin dependent kinase inhibitor p21WAF1/CIP1 together with an increase in Bak and Bax expression and a decrease in Bcl-2 levels in all the transfected cells. In addition, G1/S phase cell cycle arrest was associated with the antiproliferation activity of 5Fu in all cell lines. Through RT-PCR, 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in transfected UMSCC 12 cells. / Our results suggest that HPV16 E6 and E7 oncoproteins do not prevent 5Fu medicated apoptosis and G1/S cell arrest in laryngeal cancers. The anti-cancer effect of 5Fu is probably decided by the level of p21 WAF1/CIP1 while the sensitivity of laryngeal cancer cells responded to 5Fu treatment is associated with the increase of Bak or/and the decrease in Bcl-2, not with the HPV16 viral proteins and p53 status. 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in laryngeal cancer. However, the anti-viral effect of 5Fu still needs further investigation. / Our study indicated that (1) the evasion of apoptosis mediated by HPV16 E6 and E7 plays a critical role in laryngeal carcinogenesis; (2) HPV16 E6 or E7 plays an important role in regulating the expression of Bak, Bax and Bcl-2; (3) The degradation of Bak by HPV16 E6 is not caused by interacting with the promoter of Bak; (4) The induction of Bcl-2 is mediated through HPV16 E7; (5) HPV16 transfection does not interfere with the apoptosis and cell cycle arrest mediated by 5Fu in human laryngeal squamous cancer cells. / There is a growing body of evidence that human papillomavirus type 16 (HPV16) is involved in the development of human laryngeal cancer, especially in Chinese population. The two oncoproteins, HPV16 E6 and E7 that target host cell tumor suppressor proteins p53 and Rb respectively, may generate antiapoptotic effects and induce cell immortalization. However, the effect of both oncoproteins on apoptosis in laryngeal cancers is not completely clear. In this study, we demonstrated the possible mechanism of high risk HPV16 in laryngeal carcinogenesis and evaluated the effect of 5Fu on HPV16-positive laryngeal cancer cells. / We employed two human laryngeal cancer cell lines---UMSCC12 (with truncated p53) and UMSCC11A (with mutant but functional p53) in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1, which provided a good foundation for further study on the carcinogenic mechanism of HPV16 E6 or E7 in human laryngeal cancers. Through Annexin V staining and protein stability assay, we found that the transfection of HPV16 E6 and E7 induced fewer spontaneous apoptosis in both UMSCC11A and UMSCC12 cells accompanied with enhanced protein stability of Bcl-2 and increased protein degradation of Bak. Similar results were obtained when E6- and E7-transfected cells exposed to apoptosis stimuli---TNF-alpha/CHX. These results indicate that stable transfection of E6 and E7 in human laryngeal cancer cells on one hand shortened the half-life of Bak protein, and on the other hand, enhanced the steady-state levels of Bcl-2 protein. In order to gain insight into the role of Bak and Bcl-2 in regulating apoptosis in HPV-associated laryngeal cancer cells, we performed transient transfection of Bcl-2 into E6- and E7-transfected cells. It is found that HPV16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer, indicating that the induction of Bcl-2 require the transfection of HPV16 E7. Furthermore, Luciferase assay was performed to investigate whether the viral proteins E6 and E7 altered the stability of Bak through interaction with the promoter of Bak. Negative results were obtained, suggesting that E6 or E7 do not alter the transcription activity of Bak, indicating the degradation of Bak by E6 or E7 may be mediated through other mechanisms. / Liu Han-ching. / "August 2006." / Advisers: C. A. van Hasselt; George G. Chen. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1569. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 245-274). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.