Ovarian cancer accounts for more deaths than all other gynaecological cancers taken together. The 5 year survival rate can be as high as 80% for cases diagnosed early, but the asymptomatic nature of the disease means that it is most frequently detected in the later stages. By this time, disease has invariably spread beyond the ovaries and the survival rate drops to around 30%. Treatment of ovarian cancer often fails due to a high rate of chemoresistance and novel methods of treatment and detection are required to increase the survival chances of patients. This study sought to determine whether gene therapy for sporadic ovarian cancer could offer a novel and more successful treatment option for the disease. Mutation or abnormal expression of the p53 gene has already been shown to be the most common genetic even in ovarian cancer, being involved in up to 70% of cases. Wild-type p53 was delivered, using liposomes, into p53 mutant ovarian cancer cell lines and this resulted in a restoration of the wild-type functions of the gene, namely cell cycle arrest and apoptosis. The results from the cell line studies suggested that restoration of the wild-type p53 function limit or reduce tumour progression and increase the sensitivity of the tumour to chemotherapy. A mouse model of human peritoneal ovarian cancer was then constructed and the wild-type p53 gene was administered in liposomes into the peritoneum. The results suggested that p53 gene therapy prevents tumours from growing in the mice, when compared to a control gene. It is now known that p53 gene therapy for humans is being clinically assessed. There are a proportion of tumours that do not harbour an abnormal p53 gene, raising the possibility that other tumour suppressor gene mutations may play a role in the molecular genetic control of growth arrest and apoptosis. P53-dependent, apoptosis-regulating family members bcl-2 and bax were analysed immunohistochemically to determine their involvement in ovarian cancer. Both proteins were significantly associated with malignancy and also with overall length of survival, but not associated with the various prognostic factors such as stage and differentiation of tumour. It is unlikely that these genes will become targets for gene therapy in ovarian cancer. Mutation, deletion and hypermethylation of the p53-independent pi6 gene, alter its function, resulting in loss of G1 cell cycle arrest control. The status of methylation of the pi 6 promoter in ovarian tumours was determined and combined with mutation data, resulting in the conclusion that abnormal pi 6 was not a common event in ovarian cancer and is therefore not a likely candidate for gene therapy. This study has contributed to the evergrowing wealth of knowledge on the molecular genetic events of ovarian cancer, and has shown that gene therapy for sporadic ovarian cancer as a clinical application is feasible.

Studies have shown that gain-of- function mutant p53, AKT, and NFκB promote invasion and metastasis in tumor cells. Signals transduced by AKT and p53 are integrated via negative feedback between the two pathways. Tumor derived p53 was also indicated to induce NFκB gene expression. Due to the close relationship between p53/AKT and p53/NFκB, we hypothesized that AKT and NFκB can enhance motility in cells expressing mutant p53. Effects on cell motility were determined by scratch assays. CXCL5- chemokine is also known to induce cell motility. We hypothesized that enhanced cell motility by AKT and NFκB is mediated, in part, by CXCL5. CXCL5 expression levels in the presence and absence of inhibitors were determined by qRT-PCR. We also hypothesized that gain-of-function mutant p53 contributes to the activation of AKT. The effect of mutant p53 on AKT phosphorylation was investigated with a Ponasterone A- inducible mutant cell line (H1299/R175H) and vector control. These results indicated that AKT and NFκB enhance motility in cells expressing mutant p53 and this enhanced motility is, in part, mediated by CXCL5. However, AKT phosphorylation was independent of mutant p53.

p53 is a critical tumour suppressor which acts to repair or remove abnormal cells and thus prevent cancer. Aberrant function of p53 is therefore a critical step in tumourigenesis and p53 is mutated in half of all cancers. Mutation of p53 leads to both a loss of normal wildtype function as well as the gain of oncogenic function. p53 is considered to be a promising therapeutic target and therapeutic strategies for targeting of the p53 pathway include: 1. Activation of wild-type p53 (wtp53) protein function, 2. Refolding of mutant p53 (mtp53) into the wtp53 conformation, 3. Reduction of mtp53 protein levels. In this work a number of small molecule screening assays were used to identify potentially novel regulators of both wtp53 and mtp53. Screening of a protein kinase inhibitor library for small molecules which can stimulate wtp53 activity identified the GSK3 pathway and a CDK pathway as dominant suppressors of wtp53 function. Screening of the library for inhibitors which reduce mtp53 protein levels led to the identification of two IKKβ inhibitors. The work then focused on investigating the effects of one of these compounds, IMD0354, on the mutant p53 pathway; with a specific focus on MDM2 as the most rapidly responding biomarker. IMD0354 is a well characterised inhibitor which has been shown to specifically inhibit IKKβ leading to the repression of the Nf-κB pathway. This study shows that IKKβ inhibition leads to the loss of a number of oncogenic proteins including mtp53, MDM2 and cyclin D. Mass-spectrometry based (ITRAQ) proteomic analysis was then employed to identify potential mediators and/or co-regulated factors in response to IKKβ-inhibition via IMD0354 treatment. This led to the identification of RPS3 as a potential negative regulator of MDM2 protein expression; the reduction in MDM2 protein in response to IMD0354 treatment is shown to be partially dependent on RPS3. Together this data has identified, using small molecule kinase inhibitor libraries: (i) dominant kinase signalling pathways that suppress wt-p53 and (ii) a dominant kinase signalling pathway that sustains expression of mutant p53 and MDM2 in cancer cell lines. This latter data supports further investigation to aid understanding of how the IKK signalling pathway cross-talks to the p53-MDM2 axis.

La protéine suppresseur de tumeur p53 est soumise à de complexes régulations transcriptionnelles et posttraductionnelles. La découverte d’isoformes de p53 a introduit un degré de complexité supplémentaire auxmécanismes de régulation des fonctions de p53. On dénombre à ce jour douze isoformes qui diffèrent de p53dans leurs domaines N- et C-terminal. Cependant, les modes d’expression et de fonction de ces isoformes restentà être clarifiés.Dans cette thèse, nous nous sommes intéressés aux deux isoformes Δ40p53 et Δ133p53, en analysant leurinteraction avec p53 et en mesurant leur expression dans les mélanomes, un type de cancer où p53 est trèsrarement mutée. Nous montrons que Δ40p53 peut moduler l’activité de p53 avec un effet bi-phasique, tantôtactivateur ou répresseur du niveau d’expression et des fonctions de p53. Δ133p53 est produite par un promoteurP2 localisé dans le gène TP53. Nous avons montré qu’en réponse à un stress génotoxique, l’expression de Δ133p53 est régulée par p53, qui se lie au promoteur P2. Ceci suggère une boucle d’auto-régulation par p53, quiest capable de contrôler l’expression d’une isoforme inhibant ses propres fonctions. Enfin, les isoformes Δ40p53 et Δ133p53 sont surexprimées dans les tumeurs métastatiques de mélanomes comparées aux tumeurs noninvasives,suggérant à ces isoformes un rôle dans l’inactivation de p53 dans les cancers.Ainsi, Δ40p53 et Δ133p53 interagissent avec p53 de façon complexe, avec des effets plus contrastés que lasimple inhibition de l’activité suppressive de p53. Les isoformes de p53 jouent ainsi un rôle majeur dans lesactivités basales de p53, ainsi que dans l’inactivation fonctionnelle de p53 dans les cancers. / The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms.In this Thesis, we have focused on the two major p53 N-terminal isoforms, Δ40p53 and Δ133p53. We haveanalysed their patterns of interactions with the full-length p53 and we have investigated whether their expressioncould be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that Δ40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control itslevels and activity. Moreover, we demonstrate that the internal P2 promoter produces Δ133p53 and is regulatedby p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control theexpression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminalisoforms are often over-expressed in highly metastatic melanoma when compared to non-invasiveforms, suggesting that N-terminal isoforms contribute to functionally inactivate p53. Thus, we propose that Δ40p53 and Δ133p53 modulate p53 functions within dynamic fluctuations of aprotein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functionalinactivation of p53 in cancer cells.

RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts with both p53 and pRb and has been implicated in mRNA processing. It has also been identified as an E3 ubiquitin ligase due to the presence of a RING finger domain and also assumed to have a regulatory role of p53 due to the presence p53BD through MdM2, although no substrate has been identified up to now. RBBP6 gene mutants are reported to be resistant to apoptosis inducers, which led to a belief that mutation of this gene might result in the development of lung cancer. Earlier localization and expression studies have shown that RBBP6 expression and apoptosis levels are indirectly proportional. The purpose of this study is to establish the expressional pattern of the RBBP6 gene in lung cancer at both mRNA and protein levels. The objective is also to characterize the role of this gene and apoptosis in diverse lung diseases. An understanding of the role of RBBP6 in the development of lung diseases may lead to insights into developing new therapeutic measures for those lung diseases in which apoptosis plays a prominent part. This thesis elucidate the possible role of RBBP6 in lung cancer and apoptosis, to establish tissue distribution and expression levels of RBBP6 at protein and mRNA levels in lung cancer by immunocytochemistry (ICC), in situ hybridization (ISH) and confirm findings by quantitative RT-PCR. RBBP6 mRNA transcripts were expressed in the cytoplasm of normal and tumour lung epithelium. In general, expression was highest in the cytoplasm of welldifferentiated carcinoma and invasive carcinoma that showed signs of cells undergoing mitosis. Immunolabelling results further showed high level of expression in all lung cancer types except in Small and large cell carcinomas. The immunolabeling were confirmed by ISH experiments and RT-PCR. In relation to p53, RBBP6 mRNA expression was higher in lung cancer cell lines that had p53 silenced and apoptosis induced by TRAIL and camptothecin. There was no notable change in the levels of p53 expression following RBBP6 silencing and apoptosis induction. However, there was a little correlation between RBBP6 expression and apoptosis levels in both lung cancer tissues by TUNEL and lung cancer cell line following apoptosis induction by TRAIL. The ratio of Bax/Bcl-2 was seen to be upregulated following p53 and RBBP6 silencing after apoptosis induction. The most common mutation notable after RBBP6 DNA sequencing was point mutations where only single nucleotide was mutated and mostly they were observed in lung cancer tissues. This was the first demonstration that RBBP6 is expressed in lung cancers. Because of the ubiquitin-like nature of the protein and its localized up-regulation and corresponding proapoptotic activity in lung cancer cells, it is possible that further characterization of this gene could lead to its manipulation as a diagnostic marker and a potential therapeutic target for cancer treatment.

Submitted by admin tede (tede@pucgoias.edu.br) on 2017-06-01T14:10:20Z No. of bitstreams: 1 MAGDA HELENA LAGARES.pdf: 1059348 bytes, checksum: 8bd1fe90af646043bcc1603109b51dab (MD5) / Made available in DSpace on 2017-06-01T14:10:20Z (GMT). No. of bitstreams: 1 MAGDA HELENA LAGARES.pdf: 1059348 bytes, checksum: 8bd1fe90af646043bcc1603109b51dab (MD5) Previous issue date: 2017-03-02 / Atherosclerosis is a multifactorial pathological disease that alter the morphology and function of arterial walls. The atheroma growth leads to vessel hardening and lumen narrowing, limiting the blood flow. The atheroma plaque can eventually break, exposing highly thrombogenic material and leading to platelet activation and subsequent formation of thrombus that may block blood flow in loco, or even leading to obstruction of other vessels with a smaller diameter. This process is one of the main determinants of the clinical manifestations of atherosclerosis such as coronary artery disease (CAD), ischemic stroke, and peripheral arterial disease. Although the inflammatory theory about atherosclerosis is the most renowned one, observations point to common biological characteristics between cancer and atherosclerosis suggesting a possible association between p53 and atherosclerotic diseases. We collected peripheral blood samples from 200 individuals with clinical manifestations of atherosclerotic disease and 100 individuals without manisfestation of the disease to form the control group. DNA was subjected to molecular analysis (PCR) in order to identify the polymorphism of the p53 gene. We have not find any relationship between the polymorphism of the p53 gene and atherosclerosis in the population studied (p=0.36). There was no relationship among AD, polymorphism of the p53 gene and variants studied: gender (p = 0.78 male and 0.37 female), smoking (p = 0.72, 0.51 and 0.62 for smokers, (P = 0.17 for alcoholic and 0.38 for non-alcoholic), systemic arterial hypertension (p = 0.60), diabetes mellitus (p = 0.34), and dyslipidemia (p = 0.34). = 0.89). Our population has a high rate of miscegenation and heterozygotes and, according to studies, the arginine variant is more related to plaque formation because it induces apoptosis more frequently when compared to the proline variant. According to our results there is no association between the polymorphism of the p53 gene, atherosclerosis and its risk factors in the population studied. / A aterosclerose é um processo patológico multifatorial, durante o qual a morfologia e função das paredes arteriais são alteradas. O crescimento do ateroma, leva ao endurecimento dos vasos e ao estreitamento do lúmen, limitando o fluxo sanguíneo. Essa placa pode se romper levando à exposição de material altamente trombogênico, com a ativação plaquetária e posterior formação de trombos que podem bloquear o fluxo de sangue in loco, ou romper e entrar na corrente sanguínea, obstruindo outros vasos com diâmetro menor. Este processo é um dos principais determinantes das manifestações clínicas da aterosclerose: como por exemplo doença arterial coronariana, AVC isquêmico e doença arterial periférica. Embora a teoria inflamatória da aterosclerose é o mais proeminente, observações apontam para características biológicas comuns entre câncer e aterosclerose, essas características sugerem possível associação de p53 com doenças ateroscleróticas. Foram Coletadas amostras de sangue periférico de 200 indivíduos portadores de algum tipo de doença aterosclerótica e 100 indivíduos livres da doença para formar o grupo controle. DNA foi submetido a análise molecular (PCR) para pesquisa do polimorfismo do gene p53. Não foi encontrado nenhuma relação entre o polimorfismo do gene p53 e a aterosclerose na população estudada (p=0,36). Não houve relação entre a DA, o polimorfismo do gene p53 e as variantes estudas: gênero (p=0,78 masculino e 0,37 feminino), tabagismo (p= 0,72, 0,51 e 0,62 para fumantes, não fumantes e ex fumantes respectivamente), etilismo (p=0,17 para etilista e 0,38 para não etilista), hipertensão arterial sistêmica (p=0,60), diabetes mellitus (p=0,34) e dislipidemia (p=0,89). Acredita-se que esses resultados seja em função da miscigenação da população estudada, o que favorece uma alta taxa de heterozigotos e, segundo estudos, a variante arginina está mais relacionada com a formação da placa por ter maior poder de apoptose quando comparada a variante prolina.

A síndrome de Li-Fraumeni (SLF) é uma síndrome rara de predisposição a câncer associada a mutações germinativas no gene supressor tumoral TP53. As vias de sinalização da proteína p53 estão envolvidas na regulação da apoptose, das paradas do ciclo celular, da senescência e do reparo de danos no DNA. As mutações em p53 mais comumente encontradas em tumores estão distribuídas ao longo do domínio de ligação ao DNA, incluindo a mutação G245S associada à SLF. No entanto, a mutação mais frequentemente associada à SLF nas regiões Sul e Sudeste do Brasil é a mutação R337H, que afeta o domínio de oligomerização de p53. Assim, o objetivo deste estudo foi analisar os efeitos em células de p53 mutantes associadas à síndrome de SLF na viabilidade em condições basais e na sobrevivência celular sob estresse genotóxico. Células p53 null da linhagem NCI-H1299 foram transfectadas com vetores para a expressão de p53 wt e das mutantes G245S e R337H, e ensaios celulares foram realizados. A mutante R337H inibiu a formação de colônias e diminuiu a viabilidade celular de forma similar ao observado para células com expressão p53 wt, enquanto G245S demonstrou menor influência sobre a viabilidade e sobre a proliferação das células. Após submetidas a estresse genotóxico induzido por meio de exposições à radiação UVC, células com expressão de R337H mostraram-se mais sensíveis à morte celular mesmo quando expostas à baixa dose de UVC. Já as células com a expressão de G245S apresentaram aumento nas taxas de apoptose tardia somente quando submetidas a altas doses de radiação de UVC, assim como nas células com expressão de p53 wt. Dessa forma, foram observadas atividades funcionais similares entre R337H e p53 wt quanto à influência sobre a viabilidade e sobre a proliferação celular, enquanto células com expressão de G245S apresentaram fenótipo celular mais próximo ao p53-null. Todavia, G245S demonstrou atividade próxima a de p53 wt ao conferir proteção às células contra morte induzida pela radiação UVC, e a mutante R337H gerou maior sensibilidade para morte celular em condições de estresse genotóxico. / Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition disorder associated with germline mutations in the TP53 tumor suppressor gene. The p53 signaling pathways are involved in the regulation of apoptosis, cell cycle arrest, senescence and DNA repair. The p53 mutations found in tumors are commonly distributed along the DNA binding domain, including the G245S mutation associated with LFS. However, the most frequent p53 mutation associated with LFS in Southeast and Southern Brazil is the R337H mutation, which affects the oligomerization domain of p53. Thus, the aim of this study is to analyze the effects of mutant p53 associated with LFS on cell viability at basal conditions and on cell survival in genotoxic stress. Null-p53 NCI-H1299 cell line were transfected with vectors for the expression of wild-type, G245S and R337H p53, and cell assays were performed. The R337H mutant inhibited the colony formation and decreased the cell viability similar to that observed in cells with wt p53 expression, while G245S demonstrated less influence on cell viability. After undergoing genotoxic stress induced by UVC radiation exposures, cells with R337H expression were more sensitive to cell death when exposed to low UVC dose. Cells with G245S expression showed an increase in late apoptosis rates only when subjected to high doses of UVC radiation, as well as cells with wt p53 expression. Thus, similar and functional activities were observed between R337H and wt p53 concerning influence on cell viability and proliferation, with the expression of G245S presented cellular phenotype closer to p53-null. However, G245S demonstrated to confer protection for cell death as seen for wt p53, whereas R337H generated increased of sensitivity to cell death under conditions of genotoxic stress.

Protein function and dysfunction, and their intimate ties to protein structure, has been a core focus of research for several decades. More recently, research into the lack of structure in proteins has reached fever pitch. Intrinsically disordered proteins (IDPs) are proteins (or protein regions) that exist as collapsed or extended, dynamically mobile conformational ensembles, either at secondary or tertiary level, whilst remaining biologically active. The properties of IDPs can impede their study; they are often inherently unstable, are vastly wide-ranging in molecular weight and often difficult to express in large quantities. Mass spectrometry (MS) has evolved into a tool for the study of dynamic systems such as IDPs due to its large dynamic range, high sensitivity, low sample consumption and its lack of bias towards the folded state of a protein. The addition of ion mobility separation to mass spectrometry analysis (IM-MS) provides insight into the conformations adopted by proteins and their complexes, measuring their rotationally averaged collision cross section which can be compared with coordinates from other biophysical techniques such as X-ray crystallography, NMR and to molecular modelling. The work presented in this thesis uses both MS and IM-MS, along with several other biophysical techniques, to interrogate a number of IDPs which are implicated in cancer. Firstly, variable temperature IM-MS is used to probe several proteins of increasing disorder; structured protein cytochrome c, the tumour suppressor protein p53 and the oncoprotein Murine Double Minute 2 (Mdm2), performing IM-MS measurements at a range of temperature from 200 K to 571 K to elucidate the gas-phase unfolding behaviour of each protein. The interaction between p53 and Mdm2 is a current target for cancer drug therapy. Hence MS and IM-MS, alongside circular dichroism and hydrogen-deuterium exchange are next employed to determine the effect of several known small molecule ligands on the conformations adopted by these disordered domains. The significant structuring of both of these disordered proteins upon binding to their respective ligands can be observed using IM-MS, but is not apparent when using other biophysical techniques, highlighting the ability of IM-MS to capture conformational changes occurring in solution on a short timescale. The regulation of disorder in cells is postulated to be mediated by proline residues. I investigate the impact of proline replacement on the populations of conformers presented by p53 using a range of mutants and then go on to study how these mutations impact upon the binding stoichiometry, affinity and conformational preference of p53 for its interaction partner Mdm2. Finally, the disordered melanoma associated antigen 4 MAGE-A4, and its ability to bind to p53 and block its transcriptional activity is probed using MS and IM-MS.

Wong Pak Cheung Ronald. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 121-142). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / Abbreviation --- p.VI / List of figures --- p.IX / List of tables --- p.XI / Content --- p.XII / Content / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The p53 tumor suppressor gene --- p.2 / Chapter 1.1.1 --- Structure and function --- p.2 / Chapter 1.1.2 --- Regulation of p53 stability and activity --- p.3 / Chapter 1.1.3 --- p53 and cell cycle arrest --- p.4 / Chapter 1.1.4 --- p53 and apoptosis --- p.4 / Chapter 1.2 --- Mutation in p53 gene --- p.9 / Chapter 1.2.1 --- Loss of function through dominant negative effect --- p.9 / Chapter 1.2.2 --- Gain-of-function through transactivation by mutant p53 --- p.10 / Chapter 1.2.3 --- Mutation in p53 and resistance to cancer therapy --- p.10 / Chapter 1.3 --- Objective of the study --- p.14 / Chapter Chapter 2: --- Mutant p53 induced interferon resistance and its regulation of the Jak/Stat pathway --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- IFN classification and biological activities --- p.16 / Chapter 2.1.2 --- IFN signaling --- p.17 / Chapter 2.1.3 --- IFN direct antitumor effect: cell cycle arrest and apoptosis --- p.18 / Chapter 2.1.4 --- IFN in immunotherapy --- p.20 / Chapter 2.1.5 --- Resistance to IFN therapy --- p.21 / Chapter 2.2 --- Materials and Methods --- p.24 / Chapter 2.2.1 --- Cell lines --- p.24 / Chapter 2.2.2 --- Drugs and antibodies --- p.24 / Chapter 2.2.3 --- Cell Proliferation assay- MTT assay --- p.24 / Chapter 2.2.4 --- Cell cycle analysis --- p.25 / Chapter 2.2.5 --- DNA fragmentation assay --- p.25 / Chapter 2.2.6 --- Western blot analysis --- p.26 / Chapter 2.2.7 --- "Combined treatment of IFNs and Jak inhibitors in MTT assay, DNA fragmentation assay and Western blot analysis" --- p.26 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Mutant p53-V143A and p53-R273H induced IFN resistance: the role of IFN induced apoptosis and cell cycle arrest --- p.27 / Chapter 2.3.2 --- IFN induction of apoptosis: a p53-independent and caspase-dependent pathway --- p.28 / Chapter 2.3.3 --- Mutant p53 regulation of Jak/Stat pathway --- p.36 / Chapter 2.3.4 --- Janus kinases (Jaks) and IFN-alpha sensitivity in Saos-2 cells --- p.41 / Chapter 2.3.5 --- Janus kinases (Jaks) and IFN-gamma sensitivity --- p.49 / Chapter 2.4 --- Discussion --- p.56 / Chapter 2.4.1 --- Mutant p53-V143 and p53-R273H induced IFN resistance in Saos-2 cells --- p.56 / Chapter 2.4.2 --- Role of Jaks in IFN sensitivity in Saos-2 cells --- p.57 / Chapter 2.4.3 --- IFN signaling in Saos-2 cells --- p.57 / Chapter 2.4.4 --- Jak2 and IFN induced apoptosis --- p.58 / Chapter Chapter 3: --- Mutant p53 induced drug resistance --- p.60 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.1.1 --- The multidrug resistance (MDR) --- p.61 / Chapter 3.1.2 --- Anticancer drugs used in the study: action mechanisms and resistance --- p.67 / Chapter 3.1.3 --- Jak/Stat pathway and MDR --- p.68 / Chapter 3 .2 --- Materials and Methods --- p.72 / Chapter 3.2.1 --- Cell lines --- p.72 / Chapter 3.2.2 --- Drugs and antibodies --- p.72 / Chapter 3.2.3 --- Caspase 3 activity assay --- p.72 / Chapter 3.2.4 --- Cell Proliferation assay- MTT assay --- p.73 / Chapter 3.2.5 --- Cell cycle analysis --- p.73 / Chapter 3.2.6 --- DNA fragmentation assay --- p.73 / Chapter 3.2.7 --- Reverse transcription polymerase chain reaction --- p.73 / Chapter 3.2.8 --- Western blot analysis --- p.74 / Chapter 3.2.9 --- "Combined treatment of IFNs and Jak inhibitors in MTT assay, DNA fragmentation assay and Western blot analysis" --- p.74 / Chapter 3.3 --- Results --- p.75 / Chapter 3.3.1 --- Mutant p53 and drug sensitivity --- p.75 / Chapter 3.3.2 --- Mutant p53 and drug induced apoptosis and cell cycle arrest --- p.75 / Chapter 3.3.3 --- Classical drug resistance factors in mutant p53 induced drug resistance --- p.87 / Chapter 3.3.4 --- The role of Jaks in drug sensitivity of Saos-2 cells --- p.89 / Chapter 3.3.5 --- The role of Jaks in drug induced DNA fragmentationin Saos-2 cells --- p.89 / Chapter 3.3.6 --- Jak signaling and caspase activation in MTX induced apoptosis in Saos-2 cells --- p.100 / Chapter 3.3 --- Discussion --- p.108 / Chapter 3.3.1 --- Mutant p53-V143A and p53-R273H induced drug resistance in Saos-2 cells --- p.108 / Chapter 3.3.2 --- Role of Jaks in drug sensitivity in Saos-2 cells --- p.109 / Chapter 3.3.3 --- Jak/Stat signaling in Saos-2 cells --- p.109 / Chapter 3.3.4 --- Jak2 and MTX induced apoptosis --- p.110 / Chapter Chapter 4: --- General discussion --- p.112 / Chapter 4.1 --- Mutant p53 induced immunotherapy and chemotherapy resistance --- p.113 / Chapter 4.2 --- Gain of new function of mutant p53-V143A and p53-R273H in regulating Jak/Stat pathway leading to resistance to IFN and chemotherapeutic drugs --- p.114 / Chapter 4.3 --- The role of Jaks in MTX sensitivity --- p.114 / Chapter 4.4 --- Future work --- p.115 / Chapter 4.5 --- Perspective --- p.120 / References --- p.121

Chi Tian-yi. / "July 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 213-257). / 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.