Thesis (M.Sc.)--York University, 2008. Graduate Programme in Kinesiology and Health Science. / Typescript. Includes bibliographical references (leaves 71-78). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38823

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

No description available.

Approximately 40% of men and women in the United States will be diagnosed with at least one form of cancer in their lifetime, with cancer being implicated in one in four deaths. While great strides have been made in early diagnosis and treatment using standard regimens of chemotherapy and radiation, resulting in an overall decrease in cancer mortality, tumor initiation, growth and metastasis continue to evade control. The continued search for effective and targeted drugs has been hindered by the high failure rate of costly clinical trials, highlighting a need for more accurate preclinical models of disease, not only for pharmaceutical testing, but also biological research and assay development. The dominant role of the tumor microenvironment in regulating tumor initiation, progression, and metastasis has been well documented, driving the application of tissue engineering strategies in cancer biology. In vitro models that recapitulate clinically-relevant features of native tumors with greater fidelity than monolayer tissue cultures have the potential to yield discovery of novel therapeutic targets and regimens while also providing critical insights into mechanisms of tumor resistance. This thesis describes a tissue engineering strategy for generating an in vitro tumor model of human conventional chondrosarcoma using a custom biomimetic scaffold, and characterizes the effect of the biomaterial on cancer cell phenotype. Together with a previously validated and published in vitro model of human Ewing’s sarcoma tumors, we further investigated the effect of microenvironmental factors including matrix stiffness, niche composition, and three-dimensionality on the expression of a key cell cycle regulator and tumor suppressor mutated or lost in a wide variety of cancers, p53. A transcription factor nicknamed the “guardian of the genome,” p53 is activated in normal tissues in response to stress and triggers cellular responses including cell cycle arrest and apoptosis, or induces transcription of DNA repair enzymes to promote cell survival. The unifying hypothesis of this thesis was that the tumor microenvironment does in fact influence expression of tumor suppressors like p53, ultimately contributing to the progression of tumors toward metastasis and chemoresistance, and that these effects can be probed in vitro using disease-specific engineered tumor models to identify novel druggable targets and biomarkers with prognostic significance.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 97-108). Also available in electronic version. Access restricted to campus users.

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Ovarian cancer is the most lethal gynecological malignancy. Most of ovarian cancer patients relapse and subsequently die due to the development of resistance to chemotherapy. P73 belongs to the tumor suppressor p53 family. Like p53, the transcriptionally active TAp73 can bind specifically to p53 responsive elements and transactivates some of the p53 target genes, and finally leads to cell cycle arrest and apoptosis. TAp73 can be induced by DNA damage to enhance cellular sensitivity to anticancer agents in human cancer cells. However, the functions of TAp73 in ovarian cancer cells and the role in the regulation of cellular response to commonly used chemotherapeutic agents cisplatin are still poorly understood. The aims of this study were to examine the functions of TAp73 in ovarian cancer cells and its role in cellular response to cisplatin, as well as the relationship between TAp73 and p53 in ovarian cancer cells. Functional studies showed that over-expression of TAp73alpha (TAp73α) inhibited cell proliferation, colony formation ability and anchorage-independent growth of ovarian cancer cells, and this was irrespective of p53 expression status. In addition, TAp73α inhibited cell growth by arresting cell cycle at G2/M phase and up-regulating the expressions of G2/M regulators of p21, 14-3-3sigma and GADD45α. TAp73α enhanced the cellular sensitivity to cisplatin through the activation of JNK signaling pathway, at least partially, in ovarian cancer cells. TAp73α activated the JNK pathway through the up-regulation of its target gene GADD45α and subsequent activation of MKK4, the JNK up-stream kinase. Inhibition of JNK activity by a specific inhibitor (SP600125) or small interfering RNAs (siRNAs) significantly abrogated TAp73-mediated apoptosis induced by cisplatin. Moreover, the activations of MKK4, JNK and c-Jun were abolished when GADD45α was knocked down by siRNAs, and the JNK-dependent apoptosis was not observed. Collectively, these results supported that TAp73α was able to mediate apoptotic response to cisplatin through the GADD45α/MKK4/JNK signaling pathway, which was respective of p53 expression status. Further investigation on the relationship between TAp73α and p53 demonstrated that TAp73α increased p53 protein, but not mRNA expression by attenuating p53 protein degradation in wild-type p53 ovarian cancer cells. TAp73α could directly interact with p53 protein, which might interfere with the binding ability of MDM2 to p53, and consequently block the p53 protein degradation. In addition, TAp73α inactivated the Akt and ERK pathways and activated the p38 pathway in response to cisplatin in wild-type p53 OVCA433, but not in null-p53 SKOV3 cells, suggesting that the effect of TAp73α on these pathways might be p53-dependent. These results indicated that a functional cooperation of TAp73α and p53, to some extent, existed in ovarian cancer cells. In conclusion, this study demonstrated that TAp73α acted as a tumor suppressor in ovarian carcinogenesis. It promoted the cellular sensitivity to cisplatin via, at least partially, the activation of JNK signaling pathway. These TAp73α functions were irrespective of p53 expression. In addition, TAp73α was able to bind to p53 and increase p53 expression. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Ovarian cancer is a leading cause of death among of gynecological cancers. Current therapies are ineffective with a poor 5-year survival of only ~25%. p70 S6 kinase (p70 S6K) is a downstream target of the phosphatidylinositol 3-kinase pathway and is frequently activated in human ovarian cancer. However, the molecular targets and signaling pathways by which p70 S6K may affect tumor development and progression are poorly understood. Interestingly, in the laboratory, Mdm2, an important negative regulator of the p53 tumor suppressor, was identified in a yeast two hybrid screening of potential interacting partners for p70 S6K. In this study, I aimed to investigate the specific interaction of p70 S6K and Mdm2 and determine how this may contribute to ovarian tumorigenesis. Using a co-immunoprecipitation assay, the in vivo interaction of p70 S6K and Mdm2 in human ovarian cancer cells was confirmed. Upon UV-induced genotoxic stress, p70 S6K activation was associated with Mdm2 phosphorylation on S166 and subsequent p53 accumulation. This could be reversed by the use of rapamycin and p70 S6K siRNA to inhibit its kinase activity and expression respectively, confirming that the effect was p70 S6K specific. Conversely, ectopic expression of wildtype p70 S6K or a constitutively active mutant of p70 S6K, D3E-E389 (D3E) was sufficient to induce phosphorylation of Mdm2. Moreover, the p70 S6K mediated activation of Mdm2 was independent of p53 mutations. Similar results were observed upon other stress challenges such as hypoxia using hypoxia mimicking agent desferrioxamine (DFX). These findings identify Mdm2 as a new target of p70 S6K and reveal that p70 S6K intervenes the Mdm2-p53 regulatory loop in ovarian cancer, which may provide a survival advantage to cancer cells under stress conditions. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Endoplasmic Reticulum (ER) stress signal is a cellular response to various insults including abnormal protein folding load, activating the unfolded protein response. Under severe ER stress, apoptosis will occur in most cell types. Interestingly, this does not happen in a disease model for Metaphyseal chondrodysplasia type Schmid (MCDS), where ER stress was activated in the hypertrophic zone of the growth plate where mutant collagen X proteins that cannot be folded correctly is expressed. Instead of normal progression from proliferating chondrocytes (PCs) to hypertrophic chondrocytes (HCs) and conversion to bone, HCs in MCDS mice undergo re-differentiation to PCs as a survival strategy due to an activation of ER stress. Transcription factors are known to be important in regulating differentiation. p53 family members, as transcription factors, are known to play important roles in developmental processes including cellular reprogramming, thus, we hypothesize that the ectopic expression of key transcription factors, p53 and TAp63, which are activated by ER stress is involved in HC re-differentiation. p53 is normally expressed in late PCs, Pre-HCs, and upper HCs, while TAp63 is expressed in PCs and Pre-HCs suggesting they may have roles in chondrocyte differentiation. p53 activated under ER stress in HCs are nuclear localized in MCDS mice, but did not invoke the apoptotic programme. In this project, using quantitative analyse to study the expression level of p53 and p63 isoforms, it was confirmed that p53 and TAp63γ are in part transcriptionally activated upon ER stress. From functional study by inactivating p53 in MCDS mice, it was shown that p53 alone was not sufficient to mediate re-differentiation. Given that TAp63γ isoforms is also highly upregulated upon ER stress, and the negative regulator, ΔNp63, is downregulated, this combination of change in gene expression also need to be considered. Furthermore, known regulators of p53 and p63 activity such as ASPP1 and iASPP are also differentially expressed in HCs, and are altered upon activation of ER stress favouring cell survival. Thus, it would be important to evaluate the combination of TAp63 in the re-differentiation process from conditional inactivation of p63 or in combination with p53 to gain a clearer understanding of the contribution and relationship of these transcription factors in the survival strategy of stressed HCs. / published_or_final_version / Biochemistry / Master / Master of Philosophy

The human wildtype p53 tumor suppressor gene is found in two different forms, p53 Arginine and p53 Proline. This difference results in a substitution of a proline for an arginine at codon 72 producing the polymorphism. Knowing that apoptosis and cell cycle arrest are the two main functions of p53, the objective of this project was to determine the difference in the capacity of these allelic variants of p53 to induce apoptosis and/or cell cycle arrest in two different experimental model systems. The first experimental system was composed of non-transformed 10(1) cells and the second one was transformed Saos-2 cells. In the first experimental system, the two wildtype forms of p53 induced cell cycle arrest at the same level and did not induce apoptosis. On the other hand, in transformed cells, both p53Arg and p53Pro induced apoptosis at similar levels. No cell cycle arrest activity has been detected in Saos-2 cells. In conclusion, this study suggests that the induction of cell cycle arrest or apoptosis depends more on the cell type than on the type of the p53 protein. Also, the intensity of cell cycle arrest or apoptosis is independent of which allelic variant of p53 is present under the experimental conditions used in this study.