No description available.

none

Chan Wai Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 199-210). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vi / CHINESE ABSTRACT --- p.x / TABLE OF CONTENTS --- p.xiii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- What is hematopoiesis? --- p.1 / Chapter 1.1.1 --- The development of hematopoietic progenitor cells --- p.1 / Chapter 1.1.2 --- The regulation of hematopoiesis by environmental factors --- p.4 / Chapter 1.1.3 --- The regulation of hematopoiesis by transcription factors --- p.7 / Chapter 1.2 --- Leukemia --- p.9 / Chapter 1.2.1 --- Classification of leukemia --- p.11 / Chapter 1.2.2 --- Pathology and etiology of leukemia --- p.15 / Chapter 1.2.2.1 --- Inheritance --- p.15 / Chapter 1.2.2.2 --- Environmental factors --- p.16 / Chapter 1.2.2.3 --- Virus infection --- p.17 / Chapter 1.2.3 --- Genetics of leukemia --- p.17 / Chapter 1.2.3.1 --- Point mutations --- p.17 / Chapter 1.2.3.2 --- Translocations --- p.18 / Chapter 1.2.3.3 --- Gene and chromosomal deletions --- p.18 / Chapter 1.2.3.4 --- Chromosomal duplication or gene amplification --- p.18 / Chapter 1.2.4 --- Current therapeutic strategies for leukemia --- p.20 / Chapter 1.2.4.1 --- Chemotherapy --- p.20 / Chapter 1.2.4.2 --- Stem cell transplantation --- p.21 / Chapter 1.2.4.3 --- Immunotherapy --- p.22 / Chapter 1.2.4.4 --- Gene therapy --- p.23 / Chapter 1.2.5 --- Novel approaches for the treatment of leukemia --- p.23 / Chapter 1.2.5.1 --- Differentiation therapy of leukemia --- p.24 / Chapter 1.2.5.2 --- Induction of apoptosis in the treatment of leukemia --- p.25 / Chapter 1.3 --- Topoisomerase-targeting agents --- p.27 / Chapter 1.3.1 --- What is topoisomerase? --- p.27 / Chapter 1.3.2 --- Structures and action mechanisms of Top I- and Top II-targeting agents --- p.28 / Chapter 1.3.3 --- Anti-tumor activities of topoisomerase-targeting agents --- p.37 / Chapter 1.4 --- Aims and scopes of this investigation --- p.39 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.42 / Chapter 2.1.1 --- Mice --- p.42 / Chapter 2.1.2 --- Cell lines --- p.42 / Chapter 2.1.3 --- "Cell culture medium, buffers and other reagents" --- p.43 / Chapter 2.1.4 --- Radioisotope and scintillation fluid --- p.47 / Chapter 2.1.5 --- Reagents and buffers for flow cytometry --- p.48 / Chapter 2.1.6 --- Antibodies for flow cytometry --- p.50 / Chapter 2.1.7 --- Recombinant cytokines --- p.51 / Chapter 2.1.8 --- Reagents for DNA extraction --- p.52 / Chapter 2.1.9 --- Reagents for total RNA isolation --- p.53 / Chapter 2.1.10 --- Reagents and buffers for RT-PCR --- p.54 / Chapter 2.1.11 --- Reagents and buffers for gel electrophoresis --- p.58 / Chapter 2.1.12 --- Reagents and buffers for Western blot analysis --- p.59 / Chapter 2.1.13 --- Reagents for measuring caspase activity --- p.64 / Chapter 2.2 --- Methods --- p.67 / Chapter 2.2.1 --- Culture of the leukemia cell lines --- p.67 / Chapter 2.2.2 --- Isolation and preparation of normal hematopoietic cells --- p.67 / Chapter 2.2.3 --- [3 H]-TdR proliferation assay --- p.68 / Chapter 2.2.4 --- Determination of cell viability --- p.69 / Chapter 2.2.5 --- Assay for anti-leukemic activity in vivo --- p.70 / Chapter 2.2.6 --- Assessment of differentiation-associated characteristics --- p.71 / Chapter 2.2.7 --- Assays for apoptosis --- p.73 / Chapter 2.2.8 --- Cell cycle analysis (DNA content evaluation) --- p.75 / Chapter 2.2.9 --- Gene expression study --- p.75 / Chapter 2.2.10 --- Protein expression study --- p.79 / Chapter 2.2.11 --- Measurement of caspase activity --- p.82 / Chapter 2.2.12 --- Statistical analysis 一 --- p.83 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-PROLIFERATIVE EFFECT OF TOPOISOMERASE-TARGETING AGENTS ON LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.84 / Chapter 3.2 --- Results --- p.86 / Chapter 3.2.1 --- The anti-proliferative effect of topoisomerase-targeting agents on human and murine leukemia cells in vitro --- p.86 / Chapter 3.2.2 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the clonogenicity of the murine myeloid leukemia WEHI-3B JCS cells in vitro --- p.105 / Chapter 3.2.3 --- Effects of 10-hydroxy-camptothecin and etoposide phosphate on the tumorigenicity and proliferation of the murine myeloid leukemia WEHI-3B JCS cells invivo --- p.106 / Chapter 3.2.4 --- Cytotoxic effect of 10-hydroxy-camptothecin and etoposide phosphate on normal hematopoietic cells and WEHI-3B JCS cells in vitro --- p.109 / Chapter 3.2.5 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the cell cycle kinetics of WEHI-3B JCS cells --- p.114 / Chapter 3.2.6 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the expression of cell cycle-regulatory genes in the murine myeloid leukemia WEHI-3B JCS cells --- p.116 / Chapter 3.2.7 --- Combination effect of 10-hydroxy-camptothecin or etoposide phosphate with cytokines on the proliferation of the murine myeloid leukemia WEHI-3B JCS cells --- p.123 / Chapter 3.3 --- Discussion --- p.127 / Chapter CHAPTER 4: --- STUDIES ON THE DIFFERENTIATION-INDUCING EFFECT OF 10-HYDROXY-CAMPTOTHECIN AND ETOPOSIDE PHOSPHATE ON THE MURINE MYELOID LEUKEMIA WEHI-3B JCS CELLS / Chapter 4.1 --- Introduction --- p.132 / Chapter 4.2 --- Results --- p.134 / Chapter 4.2.1 --- Morphological changes in the murine myeloid leukemia WEHI-3B JCS cells treated with 10-hydroxy-camptothecin and etoposide phosphate --- p.134 / Chapter 4.2.2 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the size and granularity of the murine myeloid leukemia WEHI-3B JCS cells --- p.138 / Chapter 4.2.3 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the plastic adhering property of the murine myeloid leukemia WEHI-3B JCS cells --- p.140 / Chapter 4.2.4 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the NBT-reducing activity of the murine myeloid leukemia WEHI-3B JCS cells --- p.142 / Chapter 4.2.5 --- Surface antigen immunophenotyping of the murine myeloid leukemia WEHI-3B JCS cells treated with 10-hydroxy- camptothecin and etoposide phosphate --- p.145 / Chapter 4.2.6 --- Induction of non-specific esterase activity in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy- camptothecin and etoposide phosphate --- p.152 / Chapter 4.3 --- Discussion --- p.154 / Chapter CHAPTER 5: --- STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF 10-HYDROXY-CAMPTOTHECIN AND ETOPOSIDE PHOSPHATE ON THE MURINE MYELOID LEUKEMIA WEHI-3B JCS CELLS / Chapter 5.1 --- Introduction --- p.157 / Chapter 5.2 --- Results --- p.160 / Chapter 5.2.1 --- Induction of nuclear disintegration in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy-camptothecin and etoposide phosphate --- p.160 / Chapter 5.2.2 --- Induction of DNA fragmentation in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy-camptothecin and etoposide phosphate --- p.162 / Chapter 5.2.3 --- Induction of phosphatidylserine translocation in murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy- camptothecin and etoposide phosphate --- p.167 / Chapter 5.2.4 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the expression of apoptosis-regulatory genes in the murine myeloid leukemia WEHI-3B JCS cells --- p.171 / Chapter 5.2.5 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on the expression of apoptosis-regulatory proteins in the murine myeloid leukemia WEHI-3B JCS cells --- p.177 / Chapter 5.2.6 --- Induction of mitochondrial membrane depolarization in the murine myeloid leukemia WEHI-3B JCS cells by 10-hydroxy- camptothecin and etoposide phosphate --- p.179 / Chapter 5.2.7 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate on caspase activity in the murine myeloid leukemia WEHI-3B JCS cells --- p.181 / Chapter 5.2.8 --- Effect of 10-hydroxy-camptothecin and etoposide phosphate intracellular Ca2+ level in the murine myeloid leukemia WEHI-3B JCS cells --- p.186 / Chapter 5.3 --- Discussion --- p.189 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.192 / REFERENCES --- p.199

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xiii, 287 p. : ill. Includes abstract. Includes bibliographical references (p. 114-123).

CPT-Derivate wie Topotecan sind zunehmend Bestandteil gegenwärtiger Studien geworden, da eine synergistische Verstärkung der Zytotoxizität der ionisierenden Strahlung durch diese belegt werden konnte. Bei dem Chemotherapeutikum CPT handelt es sich um ein pflanzliches Alkaloid aus der chinesischen Pflanze Camptotheca acuminata. Es wirkt S-Phase-spezifisch und sein Wirkmechanismus beruht auf Hemmung von TopoI, ein Enzym, das für die DNA-Replikation, -Transkription und -Reparatur eine entscheidende Rolle spielt [Morris et al., 1996; Hsiang et al., 1989; Farray et al., 2006]. Der Nachweis einer radiosensibilisierenden Wirkung von CPT stand im Mittelpunkt der vorliegenden Arbeit. Untersucht wurde dabei das Ansprechen der fünf Glioblastomzelllinien U87-MG, U373-MG, SNB19, GHE und GaMG auf CPT und ionisierende Strahlung. Im Wesentlichen wurde ein signifikanter radiosensibilisierender Effekt des CPTs in den Zelllinien U87 und SNB19 gezeigt. Weiterhin erkannte man, dass das anti-apoptotische Protein Survivin nach kombinierter CPT-IR-Behandlung beim Vergleich der Survivin-Konzentrationen der fünf Glioblastomzelllinien in den Zelllinien U87 und SNB19 die niedrigsten Werte aufzeigte, was mit den Ergebnissen des Koloniebildungstests und der Zellauszählung korreliert. Dies kann ebenfalls als Indiz für einen radiosensibilisierenden Effekt von CPT in den beiden Zelllinien U87 und SNB19 gewertet werden. Des Weiteren wurden, parallel in unserer Arbeitsgruppe, nach CPT-Behandlung mindestens zwei Subpopulationen in jeder der fünf getesteten Glioblastomzelllinien ermittelt, die sich bezüglich ihres DNA-Schadens merklich voneinander unterschieden [Djuzenova et al., 2008]. Zusätzlich wurde gezeigt, dass die Menge der DNA-Schäden nach kombinierter CPT-IR-Behandlung höher war als in den einzelnen Behandlungsmodalitäten. Die Untersuchung der Kinetik der DNA-Reparatur mittels Rad50-Foci ließ einen radiosensibilisierenden Effekt von CPT in den Zelllinien SNB19 und U87 erkennen. Diese wiesen im Vergleich zu den Zelllinien U373, GHE und GaMG eine verlangsamte Reparaturhalbwertszeit unter kombinierter CPT-IR-Behandlung auf. Der Vergleich der Expression von TopoI in Western Blot und Durchflusszytometrie [s. Doktorarbeit von Mitdoktorandin T. Güttler] zeigte die höchsten Hintergrundwerte für dieses Enzym in den Zelllinien U87 und SNB19. Die erhöhte Expression von TopoI in U87 und SNB19 korrelierte mit den verlängerten Reparaturhalbwertszeiten für diese Zelllinien, mit der Reduktion der Überlebensraten im Koloniebildungstest bzw. der Abnahme des Zellwachstums in der, parallel in unserer Arbeitsgruppe durchgeführten, Zellauszählung, sowie mit der niedrigeren Survivin-Konzentration im Survivin-ELISA. Eine Resistenz gegenüber CPT könnte folglich durch eine schnellere Reparaturhalbwertszeit aber auch durch eine reduzierte Expression von TopoI in den Zelllinien U373, GHE und GaMG begründet sein. Daher ist vorstellbar, dass durch eine schnelle Proliferation von CPT-resistenten Zellen die Apoptose- und Zellabbaurate der CPT-sensitiven Subpopulation kompensiert oder sogar übertroffen werden kann, was für eine Chemoradioresistenz der jeweiligen Zelllinien sprechen würde. Die weitere Erforschung des radiosensibilisierenden Effekts von Chemotherapeutika sollte auch weiterhin einen wichtigen Bestandteil zukünftiger Forschungsarbeiten darstellen, da sowohl in der vorliegenden als auch in einer Reihe von anderen Arbeiten Ansätze dieses Effekts bereits belegt werden konnten. Die Entwicklung von neuen TopoI-Inhibitoren, wie das Homocamptothecin und dessen Derivate, die sich durch eine verbesserte chemische Stabilität auszeichnen [Teicher, 2008], sollte weiter vorangetrieben werden, um eines Tages die mittleren Überlebensraten bei Glioblastoma multiforme weit über ein Jahr hinaus verlängern zu können. Die Resultate dieser Studie unterstützen das Konzept einer chemotherapeutischen Behandlung kurz vor Bestrahlung und sehen ein derartiges Verfahren als effiziente Methode an, um möglichst schnell das Ansprechen von intrakraniellen Tumoren auf chemotherapeutische Wirkstoffe in vitro zu untersuchen. Außerdem sollte versucht werden diese Erkenntnisse auf die klinische Ebene auszuweiten und man sollte zusätzlich herausfinden, ob weitere DNA-Schadensoder DNA-Reparatur-Proteine als Marker dienen könnten wie Darzynkiewicz [2008] in seinem Leitartikel zu unserer Publikation vorschlägt. / Influence of Camptothecin and ionizing radiation in vitro on the induction and repair of DNA-double strand breaks in glioblastoma cell lines

Camptothecin and its analogues (CPTs) represent one of the most potent classes of anticancer drugs used to treat several solid tumors. CPTs bind topoisomerase I during the replication process and cause DNA damage that results in cell death. However, its effectiveness is limited to 13-30 percent of patients. TopoI cuts and re-ligates DNA supercoiling but in the presence of CPT it fails to re-ligate DNA and collision of replication forks leads to DNA double strand break (DNA-DSB) and cell death. However, in resistant cells, due to deregulated kinase cascade, topoI is continually phosphorylated by DNA-PKcs and rapidly degraded by the ubiquitin proteasomal pathway (UPP). It has been found that BRCA1 plays a key role in imparting cellular resistance to topoI inhibitors. Importantly, BRCA1 ubiquitinates topoI in response to CPT. We hypothesize that disruption of BRCA1 binding to phosphorylated topoI would interrupt the resistance mechanism resulting in higher cellular sensitivity of CPT. Based on an in-silico drug screen, we identified a compound that inhibits topoI degradation by blocking BRCA1 binding. Imaging and survival assays findings are consistent with the hypothesis that BRCA1 plays a role in CPT resistance through its co-localization with topoI, and we speculate this role is through UPP degradation. CPTs are commonly used in combination with cytotoxic compounds, but this study focuses on discovering compounds that can overcome resistance without causing further cytotoxicity. / 2019-07-03T00:00:00Z

Diverse dendrimer peripheries are often produced through convergent synthesis with multiple protection-deprotection steps. Achieving such diversity while maintaining monodispersity, has previously proven problematic. Interception of an electrophilic poly(monochlorotriazine) dendrimer with a molecule of interest bearing a reactive, nucleophilic group presents an efficient method to achieve large quantities of dendrimers with biologically relevant peripheries. Kilogram-scale synthesis of a triazine-based dendrimer relies on reaction of the dichlorotriazine monomer with the amine terminated dendrimer to afford a poly(monochlorotriazine) dendrimer. Normally, the dendrimer is then reacted with piperidine, an inexpensive “cap” due to its chemically inert nature after reaction. The dendrimer then undergoes a global deprotection to afford an amine-terminated dendrimer. Subsequent iterations with the dichlorotriazine monomer affords higher generation architectures. Intercepting the poly(monochlorotriazine) dendrimer with biologically relevant molecules containing reactive amines enables the development of a drug delivery vehicle. Desferrioxamine B, an iron chelate, and camptothecin, and anticancer drug, are two clinically approved drugs of interest investigated for macromolecular drug delivery. Upon acylation of each drug with BOC-isonipecotic acid, substitution on the dendrimer may occur with varying levels of success depending on the drug in question. Upon successful substitution to afford the desired product,biological studies may be performed. Each synthetic approach will be discussed along with alternative routes leading to this general strategy.

The anticancer drug Camptothecin (CPT) specifically targets topoisomerase I (topoI). While this class of drug is used to treat various solid tumors, CPT is only effective in 13-30 percent of the patient population. Although the mechanism of the CPT resistance pathway is not fully understood, we found cancer cells degrade topoisomerase I (topoI) in response to CPT. The observed relationship between higher basal DNA-PKcs mediated phosphorylation of topoI, rapid degradation of topoI, and resistance to CPT suggested that DNA-PKcs is a critical regulator of the phosphorylation status of topoI and the rate of topoI degradation in response to CPT. The data shows CTDSP1 (a dual phosphatase) acts as a negative regulator of DNA-PKcs. Because CTDSP1 functionality plays a role in maintaining higher basal phosphorylation levels of topoI, CTDSP1 impairment contributes to greater CPT resistance. This renders the CPT chemotherapy treatment ineffective. We hypothesized inducing the catalytically inactive form of CTDPS1 via both knockdown and inhibition experiments would increase cancer cell resistance to CPT as a result of an overactive topoI degradation pathway. The function of CTDSP1 was impaired in the two colon cancer cell lines, HCT15 and HCT116, by silencing with siRNA and using Rabeprazole (a pharmacological agent known to inhibit CTDSP1). Inhibition of CTDSP1 phosphatase activity resulted in greater phosphorylation of DNA-PKcs and increased the rate of topoI degradation in the cells in response to CPT. Cellular resistance was also more notable in the sensitive HCT116 cell lines. HCT15 cells degrade topoI rapidly and are resistant to CPT. Thus, the effect of CPT is not as pronounced in this cell line. CTDSP1 knock down cells showed the greatest DNA-PKcs phosphorylation and topoI degradation when treated with CPT. In addition, the present study includes a clonogenic assay that shows a larger cell survival fraction in Rabeprazole treated HCT116 cells in comparison to controls after exposure to CPT. A higher survival fraction after CPT exposure is reflective of greater CPT resistance. This suggests that cell viability is enhanced during CPT chemotherapy treatment in CTDSP1 null colorectal cancer cells. Lastly, An EGFP read out experiment of topoI tagged to EGFP demonstrated CTDSP1 inhibition results in reduced topoI levels in colorectal cancer cells. The present study showed the potential link between lower topoI levels and greater resistance to CPT by showing that both effects are outcomes of silencing CTDSP1. / 2018-07-11T00:00:00Z

This study presents studies on camptothecin (CPT), a potent antitumor agent in order to improve its stability and solubility without reducing its activity. The work describes the modification of camptothecin at 20-OH position a new strategy to overcome the stability and solubility problems of the free drug. Camptothecin is conneted to linker that could be processed to a terminal thiol group and this thiol group was connected to gold surface, to obtain CPT-gold nanoparticles. In the first part of the study / undecenol was chosen as the starting material and reacted with azobisisobutylonitrile to obtain S-11-hydroxyundecyl ethanethioate. 11-hydroxyundecyl ethanethioate was reacted with NaOMe to synthesize the target linker 11, 11&rsquo / -disulfanediyldiundecan. After synthesis of the target linker, the 20- OH functional group of CPT was replaced with this linker to obtain 20- (11, 11&rsquo / -disulfanediyldiundecan) - captothecin. The second part of the study, gold nanoparticles were synthesized by using HAuCl4 solution and the camptothecin derivative containing thiol group at 20-OH position was connected to the gold surface.

This study presents synthetic studies on camptothecin, a potent antitumor agent in order to improve its stability and solubility without reducing its activity. The study consists of the modification of camptothecin at 20-OH position a new strategy for the targeted and controlled release of the drug and modification at C-7 position to overcome the stability and solubility problems of the free drug. In the first part of the study, the 20-OH functional group of camptothecin was replaced with an unsymmetrical benzoin derivative that is able to release the drug under photolysis at 350 nm. The new prodrugs synthesized possessed higher stability than the camptothecin itself. The in vitro irradiation of the prodrugs at 350 nm was satisfactory without any decomposition of the active substance. The second part of the study comprises the studies on the modification of the 7th position of camptothecin, which is the most suitable position for the modification. In this part of the study, 7-amino and silyl substituted camptothecins were synthesized.Combination of camptothecin with some other drugs such as cisplatin was also investigated in this study. The synthetic efforts showed that the reactions are very promising and the combination studies can be studied as a major subject in the future.