Tumorzellen können vielfältige Resistenzmechanismen gegenüber Zytostatika entwickeln. Untersuchungen an drei humanen Tumorzellinien und ihren cisplatinresistenten Varianten zeigten eine Assoziation von erhöhter MRP2-Expression und dem Auftreten von Cisplatinresistenz. Darüberhinaus waren die cisplatinresistenten Zellinien gegenüber Carboplatin kreuzresistent. Um weitere Faktoren im Zusammenhang mit der Cisplatinresistenz zu untersuchen, wurde der Mutationsstatus von p53 und der zelluläre Glutathiongehalt in den Zellinien bestimmt. Der offene Leserahmen von MRP2 aus der cisplatinresistenten Ovarialkarzinomzellinie A2780RCIS wurde für die Transfektion in die cisplatinsensitive Zellinie A2780 genutzt. Die Transfektanten zeigten eine Überexpression von MRP2 und wiesen eine Resistenz gegenüber Cisplatin und Carboplatin auf. Dies konnte in Zellzyklus- und Apoptose-Untersuchungen unter Cisplatinbehandlung gestätigt werden. Durch computergestützte Faltungsanalysen von Abschnitten der MRP2-mRNA wurden zwei potentielle Ribozymschnittstellen ausgewählt. Die konstruierten Anti-MRP2-Hammerhead-Ribozyme RzM1 und RzM2 wurden im zellfreien System auf ihre Schnittaktivität getestet und erwiesen sich als katalytisch aktiv. Es wurden verschiedene kinetische Parameter für RzM1 und RzM2 ermittelt und mit anderen Ribozymen verglichen. Die Ribozyme zeigten eine gute Effektivität bei der Spaltung ihres Zielmoleküls, wobei RzM1 die höhere Effektivität aufwies. Beide Ribozyme wurden auf ihre Wirksamkeit durch Transfektion in die Zellinie A2780RCIS getestet. Die untersuchten Transfektanten zeigten eine geringere Expression von MRP2 auf mRNA- und Proteinebene und wiesen eine verminderte Resistenz gegenüber Cisplatin, Carboplatin, Daunorubicin und Etoposid auf. Die Ribozyme RzM1 und RzM2 waren gleichermaßen für die Expressionsregulierung von MRP2 in Tumorzellen geeignet. In Zellzyklus- und Apoptose-Untersuchungen wurde funktionell bestätigt, daß die A2780RCIS-Anti-MRP2-Ribozym-Transfektanten auf eine Cisplatinbehandlung stärker ansprechen als die cisplatinresistente Ausgangszellinie A2780RCIS. Die Anwendung der Ribozyme RzM1 und RzM2 zur Unterstützung der Chemotherapie von Tumorzellen scheint daher vielversprechend. / Tumour cells can develop a lot of resistance mechanisms against cytostatic drugs. Examinations of three human tumour cell lines and their cisplatinresistant variants showed an association of elevated MRP2 expression and the occurrance of cisplatinresistance. Moreover, the cisplatinresistant cell lines were crossresistant against carboplatin. To examine further factors in context of cisplatinresistance the mutation status of p53 and the cellular glutathione content of the cell lines were determined. The MRP2-open reading frame of the cisplatinresistant ovarian carcinoma cell line A2780RCIS was used for transfection into the cisplatinsensitive cell line A2780. The transfectants showed an overexpression of MRP2 and a resistance against cisplatin and carboplatin. This could be confirmed with analysis of the cell cycle and apoptosis induction after treatment with cisplatin. Using computer aided folding analysis of MRP2 mRNA parts two possible ribozyme cleavage sites were selected. The constructed anti-MRP2 hammerhead ribozymes RzM1 and RzM2 were tested in a cell-free system with respect to their cleavage activities and were found to be catalytic active. Various kinetic parameters of RzM1 and RzM2 were determined and compared with other ribozymes. The ribozymes showed a good effectivity for the substrate cleavage, although RzM1 had the better effectivity. Both ribozymes were tested for their effectiveness after transfection into the cell line A2780RCIS. The transfectants showed a lower MRP2 expression on mRNA and protein level and also a reduced resistance against cisplatin, carboplatin, daunorubicin, and etoposide. The ribozymes RzM1 and RzM2 were both equally suitable for the regulation of MRP2 expression in tumour cells. Analysis of cell cycle and apoptosis induction could confirm functionally the higher sensitivity of the A2780RCIS-anti-MRP2 ribozyme transfectants after treatment with cisplatin in comparison to the cisplatinresistant cell line A2780RCIS. Therefore, the application of the ribozymes RzM1 and RzM2 for the support of cancer chemotherapy seems to be promising.

Yan, Changqing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-97). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.iii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGMENTS --- p.v / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.x / Chapter CHAPTER ONE: --- BACKGROUND INFORMATION --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Deoxyribonucleic Acid --- p.2 / Chapter 1.3 --- DNA Studies --- p.9 / Chapter 1.4 --- Cisplatin Studies --- p.11 / Chapter 1.5 --- Scope of the Thesis --- p.13 / Chapter CHAPTER TWO: --- METHODOLOY AND COMPUTATION --- p.16 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Molecular Dynamics Simulation --- p.16 / Chapter 2.3 --- Quantum Mechanics Calculation --- p.23 / Chapter 2.4 --- Verification of Methodology --- p.25 / Chapter 2.4.1 --- Backbone Torsion Angles --- p.25 / Chapter 2.4.2 --- N7-N7 Distance --- p.30 / Chapter 2.4.3 --- Location of HOMO --- p.33 / Chapter 2.5 --- Summary --- p.35 / Chapter CHAPTER THREE: --- UNDERSTANDING OF THE CISPLATIN-DNA CROSSLINKS --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- MO Analysis --- p.37 / Chapter 3.3 --- Potential Binding Products with the Ligand --- p.37 / Chapter 3.3.1 --- "1,2-d(GpG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.2 --- "l,2-d(ApG) Intrastrand Crosslink" --- p.43 / Chapter 3.3.3 --- "l,3-d(GpXpG) Intrastrand Crosslink" --- p.44 / Chapter 3.3.4 --- d(GpC)d(GpC) Interstrand Crosslink --- p.44 / Chapter 3.3.5 --- d(GpXpC)d(GpXpC) Interstrand Crosslink --- p.44 / Chapter 3.3.6 --- Summary --- p.45 / Chapter 3.4 --- Potential Binding Products Analysis --- p.47 / Chapter 3.4.1 --- Site Identification Convention --- p.47 / Chapter 3.4.2 --- Potential Binding Products Analysis --- p.48 / Chapter 3.4.3 --- Applications --- p.53 / Chapter 3.5 --- Cisplatin-DNA Crosslink Products Analysis --- p.56 / Chapter 3.5.1 --- "1,2-d(GpG) and l,2-d(ApG) Intrastrand Crosslinks" --- p.61 / Chapter 3.5.2 --- "l,3-d(GpXpG) Intrastrand and d(GpXpC)d(GpXpC) Interstrand Crosslinks" --- p.62 / Chapter 3.5.3 --- d(GpC)d(GpC) Interstrand Crosslinks --- p.63 / Chapter 3.5.4 --- Platination at Terminal Positions --- p.65 / Chapter 3.6 --- Summary --- p.65 / Chapter CAHPTER FOUR: --- CONCLUDING REMARKS --- p.67 / APPENDIX I: BACKBONE TORSION ANGLES AND SUGAR RING CONFORMATIONS OF THE OPTIMIZED GEOMETRIES --- p.69 / APPENDIX II: BACKBONE TORSION ANGLES OF THE EXPERIMENTAL SEQUENCES FROM NUCLEIC ACID DATABASE (NDB) --- p.77 / REFERENCES --- p.92

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The childhood cancer neuroblastoma mostly affects children under the age of 2 and comprises 6% of all childhood cancers. Neuroblastoma has very diverse phenotypes caused by both inter- and intra-tumour heterogeneities. The phenotypes are classified as being either low- or high-risk. This project focuses on high-risk NB cell lines with various chemotherapy sensitivity. Titration studies with chemotherapy agents cisplatin or doxorubicin showed a proneness of p53 mutated cell lines to arrest in either the S- and/or the G2/M-phase, depending on the drug and the drug dosage, indicating on a dose-dependent cell cycle response. To potentially inhibit the cells from arresting a treatment assay with 3 cell cycle key-components, pATM, Chk1 and Wee1 inhibitors was done. An initial immunocytochemistry staining of the expression levels of pATM and Wee1 showed that pATM was upregulated for 5 out 7 tested cell lines, namely SK-N-SH, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C, upon chemotherapy treatment with doxorubicin. Wee1 was however only upregulated for 3 out 7 cell lines; Kelly, SK-N-DZ and BE(2)-C. The upregulation of pATM and Wee1 showed a potential confirmation of their involvement in CT induced cell cycle arrest. Upon inhibition of pATM, Chk1 and Wee1 diverse effects were observed for each cell line (SK-N-SH, SK-N-AS, SK-N-FI, Kelly, SK-N-DZ and BE(2)-C). Wee1 showed the most promising results were the cell viability decreased for all 5 p53 mutated cell lines and the confluency over time decreased for 4 out 5 p53 mutated cell lines. The p53 wild type cell line SK-N-SH was less sensitive towards Chk1 and Wee1 inhibition indicating that cell lines with functional p53 might not be as dependent on the Chk1 and Wee1 pathways compared to cell lines with non-functional p53. Thus, targeting the cell cycle arrest might be a promising therapeutic target for high-risk neuroblastoma. / Barndomscancern neuroblastom utgör 6% av all barncancer. Majoriteten av de drabbade är under 2 år. Neuroblastom har en stor mångfald av fenotypiska utryck som orsakas av dess inter- och intra-tumör heterogenitet. Fenotyperna klassificeras antigen som låg- eller högrisk. Här har 7 högrisks-neutoblastom cellinjer med varierande grad av känslighet mot kemoterapi analyserats. Titreringsstudier med kemoterapierna cisplatin och doxorubicin påvisade en benägenhet för de p53 muterade cellinjerna att arrestera i S- och/eller i G2/M-fasen, beroende på behandlingen samt behandlingsdosen, vilket indikerar på en dos-beroende cellcykel respons. En behandlingsanalys med de 3 nyckelkomponenterna fosforylerat ATM, Chk1 samt Wee1 gjordes för att potentiellt inhibera cellerna från att arrestera. Efter en initial immunocytokemi infärgning av pATM samt Wee1 visade 5 av 7 cellinjer (SK-N-SH, SK-N-FI, Kelly, SK-N-DZ samt BE(2)-C) en uppreglering av pATM-uttryck till följd av doxorubicin behandling. Däremot var Wee1 endast uppreglerat för 3 av 7 cell linjer (Kelly, SK-N-DZ samt BE(2)-C). Uppregleringen av pATM och Wee1 påvisar ett potentiellt samband mellan kemoterapi-inducerad cellcykelarrest och ökat utryck av pATM och Wee1. Vid inhibering av pATM, Chk1 samt Wee1 gav Wee1 de mest lovande resultaten där cellviabiliteten minskade för samtliga 5 p53-muterade cellinjer och där konfluensen över tid minskade för 4 av 5 p53-muterade cellinjer. SK-N-SH med funktionerande p53 var mindre känslig gentemot Chk1 och Wee1 inhibering, vilket indikerar att cellinjer med funktionerande p53 inte är lika beroende av reaktionsvägarna för Chk1 och Wee1 jämfört med cellinjer som har icke-funktionerande p53. Därmed kan riktad behandling mot cellcykelarrest vara en lovande behandling för högrisks-neuroblastom.

Cancer is a disease characterized by complexity and unpredictability. Consequently, its treatment is difficult and all too often unsuccessful. Almost all cancers are treated with some combination of the traditional anti-cancer armamentarium: surgery, chemotherapy, and radiotherapy. Recently, however, gene therapy has emerged as a promising addition to this existing repertoire. Its application as a single agent, or in combination with other anti-cancer treatments is proving successful in both pre-clinical and clinical settings. In this work I have investigated the combination of a conventional chemotherapy drug, cisplatin, with a type of cancer gene therapy known as cytosine deaminase + 5-fluorocytosine suicide gene therapy. Suicide gene therapy is the intracellular conversion of non-toxic prodrug to its active form by a metabolic enzyme of non-mammalian origin. There are many established enzyme/prodrug combinations, but here the bacterial enzyme cytosine dearninase (CDA) was used to convert inert 5-fluorocytosine (5FC) to highly toxic 5-fluorouracil (5FU). Of the various vector systems for therapeutic gene delivery, adenoviral (Ad) vectors have proven particularly suitable for application to cancer. This work used a first generation adenovirus type 5 vector expressing the enzyme cytosine deaminase (AdCDA) cloned from E. coli. The combination of AdCDA/5FC with cisplatin was chosen because the combination of 5FU and cisplatin, both of which are used extensively in cancer treatment, has proven effective clinically and demonstrates synergy in vitro. This combination was evaluated in murine mammary carcinoma MTIA2 cells, human colorectal carcinoma HT29 cells, HT29pl4 cells, the photofrin resistant sub-line of HT29 cells, and murine melanoma Bl6/FIO cells. The classical clonogenic assay was used to evaluate this combination treatment since it provides an accurate indication of the effectiveness a cancer treatment will have in vivo. AdCDA infected MTIA2, HT29, and HT29pl4 cell lines exhibited a dose response to increasing concentrations of SFC that was significantly different from control vector infected cells. Similarly, uninfected cells demonstrated a dose response to increasing concentrations of cisplatin. The effect of the combination on clonogenic survival, administered in the sequence of a 48 h exposure to SFC followed by 1 h exposure to cisplatin, was greater than additive compared to the effect of the two treatments alone. F10 cells exhibited a dose response to increasing concentrations of cisplatin. However, it could not be shown reproducibly that AdCDA infected FlO cells exhibited a dose response to SFC that differed significantly from control vector infected cells. Work with the FlO cells was inconclusive regarding the combination treatment, but it rendered information regarding the sensitivity of these cells to what is hypothesized to be an unidentified component present in some preparations of 5FC. Evaluation of this treatment in vivo, using both murine and human tumor cell lines, will further define the potential of AdCDA/5FC + cisplatin as a clinically relevant cancer treatment. / Thesis / Master of Science (MSc)

Bladder cancer is a potentially curable malignancy; however in regards to the state of current therapy regimens, a plateau has been reached in both the non-muscle and muscle invasive types. To obtain effective treatment, and consequently a decreased mortality, it has become imperative to test and understand aspects affecting therapy response. The aim of this thesis is to illustrate a better understanding of clinical factors affecting therapy response using new drug combinations and new tumor markers alongside established risk criteria. In Paper I we reported the 5 year follow up from a multicenter, prospectively randomized study and we evaluated the 5-year outcomes of BCG alone compared to a combination of epirubicin and interferon-a2b in the treatment of patients with T1 bladder cancer. Treatment, tumor size and tumor status at second resection were independent variables associated with recurrence. Concomitant Cis was not predictive of failure of BCG therapy. Independent factor for treatment failure was remaining T1 stage at second resection. In Paper II &III we investigated the validity of emmprin, survivin and CCTα proteins as biomarkers for response and survival before neoadjuvant cisplatin chemotherapy. Bladder tumor specimens were obtained before therapy from a total of 250 patients with T1-T4 bladder cancer enrolled in 2 randomized trials comparing neoadjuvant chemotherapy before cystectomy with a surgery only arm. Protein expression was determined by immunohistochemistry (IHC). Patients in the chemotherapy cohort with negative emmprin and CCTα expression had significantly better overall survival (OS) than those with positive expression. In Paper IV primary end point was examining STMN1 as prognostic factor in bladder cancer.  Analysis was performed on three bladder cancer patient cohorts using IHC, western blot and a bladder cancer cell line. High levels of STMN1, expression correlated to shorter disease-specific survival and the growth and migration of the cells were significantly reduced when transfecting the cells with STMN1 siRNA. Conclusion Risk assessment and predictors of outcomes could help in individualized treatment and follow up.  Biomarkers will become more important for treatment choices in bladder cancer management.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy