Thesis (Ph. D.)--University of Sydney, 2007. / Title from title screen (viewed 10 June 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Medicine, Faculty of Medicine. Degree awarded 2007; thesis submitted 2006. Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--Northeastern University, 2008. / Bouvé College of Health Sciences, School of Pharmacy. Includes bibliographical references (p. 181-188).

Thesis (Ph.D.)--Northeastern University, 2008. / Bouvé College of Health Sciences, School of Pharmacy. Includes bibliographical references (p. 181-188).

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

Colon cancer, a disease in which malignant tumors form in the tissues of colon, is the first commonest cancer and the second leading cause of cancer-related deaths in Hong Kong. The standard treatment options for colon cancer include surgery and chemotherapy. However, multidrug resistance (MDR) develops in nearly all patients with colon cancer. In fact, most of the cancer-related deaths are due to chemotherapy failure caused by MDR, which occurs during the course of cancer progression and chemotherapy. Thus, the reversal of MDR plays an important role in the successful chemotherapy for colon cancer. This study investigated such a pharmacological action in reversing MDR in colon cancer cells by tanshinones, targeting the two common mechanisms responsible for MDR, i.e. overexpression of ATP-binding cassette (ABC) transporters and suppression of apoptosis. / Overexpression of P-glycoprotein (P-gp), one of the most important ABC transporters, can mediate the efflux of drugs out of cancer cells, leading to MDR and chemotherapy failure. The reversal of P-gp-mediated MDR by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone was evaluated in colon cancer cells. Bi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased the P-gp-mediated digoxin efflux in Caco-2 cells. The two tanshinones potentiated the cytotoxicities of doxorubicin and irinotecan in P-gp overexpressing colon cancer SW620 Ad300 cells. Moreover, these two tanshinones also increased intracellular accumulation of P-gp substrate in SW620 Ad300 cells, presumably by down-regulating P-gp mRNA and protein levels, as well as inhibiting P-gp ATPase activity. / Suppression of apoptosis can lead to MDR in cancer cells to anticancer agents with pro-apoptotic property. Hence, this study also investigated the circumvention of resistance to apoptosis in drug resistant colon cancer cells by cryptotanshinone and dihydrotanshinone, two potential MDR-reversing tanshinones. The drug resistant SW620 Ad300 cells were still sensitive to both cryptotanshinone and dihydrotanshinone in the promotion of cell death. When compared with the parental SW620 cells, the two tanshinones induced less apoptosis but more autophagy in the drug resistant cells. Further studies showed that cell viability was increased after inhibition of autophagy by siRNA interference or autophagy inhibitor. Thus, autophagy induced by the two tanshinones was pro-cell death in SW620 Ad300 cells, which could overcome resistance to apoptosis. / In addition, suppression of apoptosis can be caused by p53 defects/mutations, which were found in more than 50% of all human cancers. Our results also showed that apoptosis and autophagy induced by cryptotanshinone and dihydrotanshinone were independent of the status of p53 in colon cancer cells. The p53-independent cytotoxic actions of the two tanshinones could be useful in overcoming resistance to apoptosis in cancer cells caused by p53 defects/mutations. / Taken together, the current findings indicate a great potential of cryptotanshinone and dihydrotanshinone in the reversal of MDR caused by P-gp overexpression and suppression of apoptosis. They are promising candidates to be further developed as therapeutic agents in the adjuvant therapy for colon cancer, especially for the multidrug resistant cancer types. / 結腸癌是指形成在結腸組織的惡性腫瘤，在香港常見的癌症中排第一位，亦是香港排第二位的致死癌症。結腸癌的標準治療方案主要包括手術和化療。然而，多藥耐藥是結腸癌成功化療的一個障礙。事實上，大多數癌症引起的死亡都和在癌症的發展和化療的過程中產生的多藥耐藥有關。因此，多藥耐藥的逆轉對於結腸癌的成功化療非常重要。本研究旨在通過針對多藥耐藥兩種常見的機制ABC跨膜蛋白的過表達和抑制的細胞凋亡來探討丹參酮對結腸癌細胞多藥耐藥的逆轉。 / P-gp的過表達可介導藥物排出癌細胞，從而導致多藥耐藥和化療失敗。本研究評價了tanshinone I，tanshinone IIA，cryptotanshinone，dihydrotanshinone和miltirone對P-gp介導的結腸癌細胞多藥耐藥的逆轉。雙向轉運實驗表明，只有cryptotanshinone和dihydrotanshinone可以減少P-gp介導的digoxin外排。這兩個丹參酮可以增加doxorubicin和irinotecan在P-gp過表達的結腸癌SW620 Ad300細胞中的毒性。此外，這兩個丹參酮也增加P-gp底物在SW620 Ad300細胞內的積累，推測是通過下調P-gp的mRNA和蛋白水平，以及抑制P-gp的ATP酶活性。 / 抑制的細胞凋亡可導致腫瘤細胞對促凋亡的抗癌藥物产生多藥耐藥。因此，本研究也探討了cryptotanshinone和dihydrotanshinone能否克服結腸癌細胞的凋亡耐受。結果表明cryptotanshinone和dihydrotanshinone仍然能够杀死耐藥的SW620 Ad300細胞。當與SW620細胞相比，這兩個丹參酮在耐藥細胞中誘導的細胞凋亡較少，但自噬增多。進一步研究表明，這兩個丹參酮誘導的自噬是促進細胞死亡的，從而可以克服細胞的凋亡耐受。 / 此外，p53的缺陷/突變存在於50%以上的人類癌症中，并可以抑制細胞產生凋亡。結果表明，cryptotanshinone和dihydrotanshinone誘導的凋亡和自噬與p53在結腸癌細胞中的表達無關。這兩個丹參酮不依賴於p53的細胞毒性可以用於克服p53缺陷/突變引起的凋亡耐受。 / 綜上所述，本研究結果表明cryptotanshinone和dihydrotanshinone在逆轉P-gp的過表達和抑制的細胞凋亡引起的多藥耐藥中具有巨大潛力。它們可以進一步發展為有前途的治療劑并用於結腸癌的輔助治療，尤其是用於多藥耐藥的結腸癌。 / Hu, Tao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 163-182). / Abstracts also in Chinese. / Title from PDF title page (viewed on 06, December, 2016). / Hu, Tao. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Fung Ka Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 159-179). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.II / ABBREVIATIONS --- p.III / ABSTRACTS --- p.V / LIST OF FIGURES --- p.VIII / LIST OF TABLES --- p.IX / CONTENTS --- p.X / CONTENTS / Chapter CHAPTER ONE: --- GENERAL INTRODUCTION / Chapter 1.1. --- Review: Cellular mechanisms of drug resistance in cancer cells --- p.2 / Chapter 1.1.1. --- Drug resistance by decrease of drug accumulation --- p.2 / Chapter 1.1.2. --- Drug resistance conferred by detoxification of drug in cells --- p.5 / Chapter 1.1.3. --- Drug resistance conferred by alteration of drug targets or by enhancement of target repair --- p.6 / Chapter 1.1.4. --- Drug resistance conferred by genes alteration in apoptotic pathways --- p.9 / Chapter 1.2. --- Objective and review --- p.11 / Chapter CHAPTER TWO: --- PROFILING AND IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES OF HUMAN SQUAMOUS CARCINOMA A431 CELLS BY mRNA DIFFERENTIAL DISPLAY AND cDNA MICROARRAY / Chapter 2.1. --- Introduction / Chapter 2.1.1. --- Changes in gene expression and drug resistance --- p.16 / Chapter 2.1.2. --- Current states of technologies in gene expression analysis --- p.17 / Chapter 2.1.3. --- Monitoring the gene expression profile in the field of drug resistance by cDNA microarray --- p.18 / Chapter 2.1.4. --- Monitoring the gene expression profile in the field of drug resistance by differential display --- p.22 / Chapter 2.2. --- Materials and Methods / Chapter 2.2.1. --- Materials --- p.27 / Chapter 2.2.2. --- Methods / Chapter 2.2.2.1. --- Cell culture and cell lines --- p.29 / Chapter 2.2.2.2. --- cDNA microarray / Chapter 2.2.2.2.1. --- RNA preparation --- p.29 / Chapter 2.2.2.2.2. --- Differential hybridization of ResGen GenFilters Mammalian micro array --- p.30 / Chapter 2.2.2.3. --- mRNA differential display / Chapter 2.2.2.3.1. --- RNA preparation --- p.31 / Chapter 2.2.2.3.2. --- RT-PCR based mRNA differential display --- p.31 / Chapter 2.2.2.3.3. --- Reamplification of cDNA probes --- p.32 / Chapter 2.2.2.3.4. --- Subcloning of differentially expressed cDNAs / Chapter A. --- Preparation of ultra-competent E.coli cells for transformation --- p.33 / Chapter B. --- Preparation of cloning vector and DNA transformation --- p.33 / Chapter 2.2.2.3.5. --- Verification of cDNA expression by colony-PCR and cDNA probes / Chapter A. --- colony-PCR --- p.34 / Chapter B. --- Bacterial glycerol stock and plasmid preparation --- p.34 / Chapter C. --- cDNA probes preparation for Northern blot analysis --- p.35 / Chapter 2.2.2.3.6. --- Northern blot analysis --- p.35 / Chapter 2.2.2.3.7. --- Sequencing of cDNA cloned inserts --- p.36 / Chapter 2.3. --- Results / Chapter 2.3.1. --- Differential gene expression profile of A431 cells --- p.43 / Chapter 2.3.2. --- Identification of differentially expressed genes by mRNA differential display --- p.53 / Chapter 2.4. --- Discussion --- p.60 / Chapter CHAPTER THREE: --- CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES.。PART 1: THE ROLE OF VACUOLAR PROTON PUMP IN REGULATION OF DRUG SENSITIVITY AND APOPTOSIS IN A431 CELLS / Chapter 3.1. --- Introduction / Chapter 3.1.1. --- An overview of vacuolar proton pump/vacuolar-H+-ATPase (V-ATPase): Structure and function --- p.66 / Chapter 3.1.2. --- V-ATPases and drug resistance mechanisms --- p.69 / Chapter 3.1.3. --- Objective --- p.70 / Chapter 3.2. --- Materials and Methods / Chapter 3.2.1. --- Materials --- p.73 / Chapter 3.2.2. --- Methods / Chapter 3.2.2.1. --- Cell culture and cell lines --- p.74 / Chapter 3.2.2.2. --- "RNA isolation, mRNA differential display, cDNA reamplification and colony-PCR verification" --- p.74 / Chapter 3.2.2.3. --- Cellular pH measurement by flow cytometry --- p.75 / Chapter 3.2.2.4. --- MTT Drug Sensitivity Assay --- p.76 / Chapter 3.2.2.5. --- DNA fragmentation assay --- p.77 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Identification of cDNA band encoding vacuolar proton pump subunit c by differential display --- p.78 / Chapter 3.3.2. --- Effect of bafilomycin A1 (BAF) and concanamycin A (CON) on DOX- and CP-induced cytotoxicity and apoptosis ' --- p.79 / Chapter 3.3.3. --- Elevation of cellular pH in cisplatin-resistant cells correlated with cisplatin resistance mechanism of A431 cells --- p.94 / Chapter 3.4. --- Discussion --- p.98 / Chapter CHAPTER FOUR: --- CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES。PART 2: IDENTIFICATION OF A NOVEL cDNA OVEREXPRESSED IN HUMAN SQUAMOUS CARCINOMA A431 PARENT CELLS / Chapter 4.1. --- Introduction --- p.102 / Chapter 4.2. --- Materials and Methods / Chapter 4.2.1. --- Materials --- p.105 / Chapter 4.2.2. --- Methods / Chapter 4.2.2.1. --- Cell culture and cell lines --- p.105 / Chapter 4.2.2.2. --- "RNA isolation, mRNA differential display, cDNA reamplification and colony-PCR verification" --- p.105 / Chapter 4.2.2.3. --- Northern blot analysis --- p.106 / Chapter 4.2.2.4. --- Human tissue distribution of clone PA-8P19 --- p.107 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Identification of novel cDNA clone PA-8P19 in A431 cells by differential display --- p.108 / Chapter 4.3.2. --- Gene expression profile of clone PA-8P19 in human tissues and other tumor cell lines --- p.113 / Chapter 4.3.3. --- Regulation of PA-8P19 expression by DNA damaging agents --- p.117 / Chapter 4.4. --- Discussion --- p.122 / Chapter CHAPTER FIVE: --- PROFILING OF DIFFERENTIAL EXPRESSED PROTEINS BY TWO-DIMENSIONAL GEL ELECTPOPHORESIS / Chapter 5.1. --- Introduction / Chapter 5.1.1. --- Protein analysis on proteomic scale --- p.125 / Chapter 5.1.2. --- Protein expression profiling for studying drug resistance by Two- dimensional gel electrophoresis --- p.127 / Chapter 5.2. --- Materials and Methods / Chapter 5.2.1. --- Materials --- p.130 / Chapter 5.2.2. --- Methods / Chapter 5.2.2.1. --- Cell culture and cell lines --- p.132 / Chapter 5.2.2.2. --- Two-dimensional gel electrophoresis / Chapter I. --- Sample preparation --- p.132 / Chapter II. --- First-dimensional gel: isoelectric focusing (IEF) --- p.133 / Chapter III. --- Second-dimensional gel: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.134 / Chapter IV. --- Protein visualization and image analysis --- p.134 / Chapter V. --- In-gel digestion --- p.135 / Chapter VI. --- Matrix-assisted laser desorption ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and database search --- p.136 / Chapter 5.2.2.3. --- Western blot analysis --- p.137 / Chapter 5.3. --- Results / Chapter 5.3.1. --- Identification of differentially expressed proteins in A431 cells by two-dimensional gel electrophoresis --- p.140 / Chapter 5.4. --- Discussion / Chapter 5.4.1. --- Two-dimensional gel electrophoresis as a powerful tool for identification of differential protein expression in A431 cells --- p.150 / Chapter CHAPTER SIX: --- GENERAL CONCLUSION AND PERSPECTIVES --- p.155 / REFERENCES --- p.159

Choi, Siu Cheong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 132-148). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abbreviation --- p.v / List of Figures --- p.viii / List of Tables --- p.xii / Table of Content --- p.xiii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- Treatment resistance in cancer --- p.1 / Chapter 1.1.1.1 --- Surgery --- p.2 / Chapter 1.1.1.2 --- Chemotherapy --- p.3 / Chapter 1.1.1.3 --- Radiotherapy --- p.3 / Chapter 1.1.1.4 --- Hormonal therapy --- p.4 / Chapter 1.1.2 --- Hypoxia/reoxygenation and its correlation with treatment resistance --- p.5 / Chapter 1.1.3 --- Aim of the study --- p.6 / Chapter Chapter 2: --- The drug sensitivity in HepG2 cells and A431 cells / Chapter 2.1 --- Introduction --- p.8 / Chapter 2.1.1 --- Treatment of cancer --- p.8 / Chapter 2.1.2 --- Drug resistance --- p.9 / Chapter 2.2 --- Materials and Methods --- p.10 / Chapter 2.2.1 --- Cell culture --- p.10 / Chapter 2.2.2 --- Drugs --- p.10 / Chapter 2.2.3 --- MTT assay --- p.11 / Chapter 2.3 --- Results --- p.12 / Chapter 2.3.1 --- The drugs to which G10HR and G20HR cells were more resistant --- p.12 / Chapter 2.3.2 --- "The drugs of which GP, G10HR and G20HR cells have similar response" --- p.12 / Chapter 2.3.3 --- The drugs to which A10HR and A20HR cells were more resistant --- p.17 / Chapter 2.3.4 --- The drugs to which A10HR and/or A20HR cells were more sensitive --- p.17 / Chapter 2.3.5 --- "The drugs which AP, A10HR and A20HR cells have similar response" --- p.18 / Chapter 2.4 --- Discussion --- p.24 / Chapter 2.4.1 --- Camptothecin and 10-hydroxy camptothecin --- p.27 / Chapter 2.4.2 --- Etoposide --- p.30 / Chapter 2.4.3 --- Hydrogen peroxide --- p.32 / Chapter 2.4.4 --- Interferons --- p.32 / Chapter 2.4.4.1 --- Interferon alpha --- p.33 / Chapter 2.4.4.2 --- Interferon gamma --- p.34 / Chapter 2.4.5 --- Methotrexate --- p.35 / Chapter 2.4.6 --- Vincristine --- p.36 / Chapter Chapter 3: --- The resistance mechanism of doxorubicin in A431 cells / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.1.1 --- Chemotherapeutic resistance --- p.38 / Chapter 3.1.2 --- Tumor hypoxia --- p.39 / Chapter 3.1.3 --- Structure and function of doxorubicin --- p.39 / Chapter 3.1.4 --- Clinical use of doxorubicin --- p.40 / Chapter 3.1.5 --- Mechanisms of doxorubicin resistance --- p.41 / Chapter 3.1.6 --- Structure and function of P-glycoprotein --- p.42 / Chapter 3.1.7 --- Drug resistance contributed by P-glycoprotein and the solution --- p.43 / Chapter 3.1.8 --- Epigenetic modulation of mdr1 --- p.45 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Cell culture --- p.47 / Chapter 3.2.2 --- MTT assay --- p.47 / Chapter 3.2.3 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.48 / Chapter 3.2.5 --- Doxorubicin efflux assay --- p.50 / Chapter 3.2.6 --- Drug sensitivity of A431 cells treated with verapamil --- p.50 / Chapter 3.2.7 --- Treatment with DNA methyltransferase inhibitor --- p.51 / Chapter 3.2.8 --- Drug sensitivity of A431 cells treated with 5-Aza-dC --- p.51 / Chapter 3.2.9 --- Methylation-specific PCR (MSP) --- p.51 / Chapter 3.2.10 --- Bisulfite genomic DNA sequencing --- p.52 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- Drug sensitivity of A431 cells to doxorubicin --- p.54 / Chapter 3.3.2 --- Expression profile of mdrl and P-glycoprotein in A431 cells --- p.54 / Chapter 3.3.3 --- Dox efflux-pump activity in A431 cells --- p.57 / Chapter 3.3.4 --- Drug sensitivity of A431 cells in the presence of verapamil --- p.59 / Chapter 3.3.5 --- Expression profile of mdrl in A431 cells in the presence of 5- Aza-dC --- p.59 / Chapter 3.3.6 --- Drug sensitivity of A431 cells in the presence of 5-Aza-dC --- p.62 / Chapter 3.3.7 --- Methylation status of mdrl promoter region --- p.64 / Chapter 3.3.8 --- Bisulfite genomic DNA sequencing of the mdrl promoter --- p.64 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4: --- The resistance mechanism of cisplatin in HepG2 cells / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.1.1 --- Tumor hypoxia and chemotherapeutic resistance --- p.70 / Chapter 4.1.2 --- Cisplatin and its action mechanism --- p.71 / Chapter 4.1.3 --- Mechanisms of cisplatin resistance --- p.74 / Chapter 4.1.4 --- Mismatch repair genes --- p.79 / Chapter 4.1.5 --- Epigenome and drug resistance in cancer --- p.80 / Chapter 4.2 --- Materials and Methods --- p.84 / Chapter 4.2.1 --- Cell culture --- p.84 / Chapter 4.2.2 --- MTT assay --- p.84 / Chapter 4.2.3 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.84 / Chapter 4.2.4 --- Oligonucleotide transfection --- p.85 / Chapter 4.2.5 --- Treatment with DNA methyltransferase inhibitor --- p.86 / Chapter 4.2.6 --- Drug sensitivity of HepG2 cells treated with 5-Aza-dC --- p.87 / Chapter 4.2.7 --- Treatment with histone deacetylase inhibitor --- p.87 / Chapter 4.2.8 --- Drug sensitivity of HepG2 cells treated with TSA --- p.87 / Chapter 4.3 --- Results --- p.89 / Chapter 4.3.1 --- Drug sensitivity of HepG2 cells to cisplatin --- p.89 / Chapter 4.3.2 --- Expression profile of the MMR genes in HepG2 cells --- p.89 / Chapter 4.3.3 --- Drug sensitivity of HepG2 cells to cisplatin after the knock- down of PMS2 --- p.91 / Chapter 4.3.4 --- Expression profile of MMR genes in the presence of 5-Aza-dC --- p.95 / Chapter 4.3.5 --- Drug sensitivity of HepG2 cells to cisplatin after the addition of 5-Aza-dC --- p.95 / Chapter 4.3.6 --- Expression profile of MMR genes in the presence of trichostatin A --- p.98 / Chapter 4.3.7 --- Sensitivity of HepG2 cells to cisplatin after the addition of trichostatin A --- p.98 / Chapter 4.4 --- Discussion --- p.101 / Chapter Chapter 5: --- The role of PMS2 in cisplatin-induced apoptosis / Chapter 5.1 --- Introduction --- p.105 / Chapter 5.1.1 --- Apoptosis --- p.105 / Chapter 5.1.2 --- Extrinsic pathway of apoptosis --- p.106 / Chapter 5.1.3 --- Intrinsic pathway of apoptosis --- p.106 / Chapter 5.1.4 --- Cisplatin-induced apoptosis --- p.107 / Chapter 5.1.5 --- MMR and apoptosis --- p.109 / Chapter 5.2 --- Materials and Methods --- p.111 / Chapter 5.2.1 --- Cell culture --- p.111 / Chapter 5.2.2 --- Flow cytometric analysis of apoptosis --- p.111 / Chapter 5.2.3 --- Oligonucleotide transfection --- p.111 / Chapter 5.2.4 --- Western blot analysis --- p.111 / Chapter 5.2.5 --- Drug and antibodies --- p.112 / Chapter 5.3 --- Results --- p.113 / Chapter 5.3.1 --- Cisplatin induced apoptosis --- p.113 / Chapter 5.3.2 --- Knockdown of PMS2 by siRNA --- p.113 / Chapter 5.3.3 --- Cisplatin-induced apoptosis involved caspases --- p.115 / Chapter 5.3.4 --- Protein expressions of anti-apoptotic genes --- p.119 / Chapter 5.3.5 --- Protein expressions of pro-apoptotic genes --- p.119 / Chapter 5.3.6 --- Protein expressions of apoptotic proteins after knockdown of PMS2 --- p.122 / Chapter 5.4 --- Discussion --- p.124 / Chapter Chapter 6: --- General discussion and conclusion / Chapter 6.1 --- Diverse sensitivity for hypoxia/reoxygenation treated cells to anticancer drugs --- p.128 / Chapter 6.2 --- Resistance mechanism of doxorubicin in A10HR and A20HR cells --- p.129 / Chapter 6.3 --- Resistance mechanism of cisplatin in G10HR and G20HR cells --- p.129 / Chapter 6.4 --- The role of PMS2 as a direct signaling molecule and the alteration of apoptotic proteins in cisplatin-induced apoptosis --- p.130 / Chapter 6.5 --- Future work --- p.131 / References --- p.132

The development of resistance to chemotherapeutic drugs is the main obstacle to the successful treatment of many cancers, including childhood neuroblastoma, the most common solid tumour of infants. One factor that may play a role in determining response of neuroblastoma tumours to therapeutic agents is the p53 tumour suppressor gene. A number of previous studies have suggested that this tumour suppressor protein is inactive in neuroblastoma due to its cytoplasmic sequestration. This thesis therefore has examined the functionality of p53 and its role in determining drug response of neuroblastoma cells. An initial study was undertaken that characterised an unusually broad multidrug resistance (MDR) phenotype of a neuroblastoma cell line (IMR/KAT100). The results demonstrated that the MDR phenotype of the IMR/KAT100 cells was associated with the acquisition of mutant p53. To explore the role of p53 in drug resistance further, p53-deficient variants in cell lines with wild-type p53 were generated by transduction of p53-suppressive constructs encoding either shRNA or a dominant-negative p53 mutant. Analysis of these cells indicated that: (i) in contrast to previous reports, wild-type p53 was fully functional in all neuroblastoma lines tested, as evidenced by its activation and nuclear translocation in response to DNA damage, transactivation of target genes and control of cell cycle checkpoints; (ii) inactivation of p53 in neuroblastoma cells resulted in establishment of an MDR phenotype; (iii) knockdown of mutant p53 did not revert the drug resistance phenotype, suggesting it is determined by loss of wild-type function rather than gain of mutant function; (iv) p53-dependent cell senescence, the primary response of S-type neuroblastoma cells to DNA damage, is replaced, after p53 inactivation, by mitotic catastrophe and subsequent apoptosis. In contrast to neuroblastoma, p53 suppression had no effect or increased drug susceptibility in several other tumour cell types, indicating the importance of tissue context for p53- mediated modulation of tumour cell sensitivity to treatment. Taken together, these data provide strong evidence for p53 having a role in mediating drug resistance in neuroblastoma and suggest that p53 status may be an important prognostic marker of treatment response in this disease.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Medical Sciences - Medical Genetics. Title from pdf file main screen (viewed on March 19, 2010). Includes bibliographical references.