Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 148-164). Also available in electronic version. Access restricted to campus users.

p38 mitogen-activated protein kinases (MAPKs) control the endocytic trafficking of various growth-related cell surface receptors and transporters. Herein, I demonstrate that p38 MAPKs also regulate autophagy, or the process of self-cannibalism. In my studies, inhibition of p38 MAPKs triggered rapid formation of autophagosomes in prostate cancer cells, even under nutrient-rich conditions, and remarkably, the autophagosomal membranes emanated from endoplasmic reticulum exit sites via the concerted actions of the small GTPases, ARF1 and SAR1. Once formed, the autophagosomes fused with late endosomes and/or lysosomes, in a Rab7-dependent manner, to form “hybrid organelles” that were co-labeled with ER, autophagic, late endosomal, and lysosomal markers. Unlike other inducers of autophagy, however, inhibition of p38 MAPKs suppressed the fission of hybrid organelles, resulting in a profound but reversible accumulation of large cytoplasmic vacuoles. Thus, in addition to their previously reported roles in endocytosis, p38 MAPKs appear to coordinately regulate autophagy and the downstream biogenesis and fission of hybrid organelles. / text

IKK[alpha] is a 85KD serine/threonine protein kinase and a subunit of the IKK complex, which contains IKK[alpha], IKK[beta], and IKK[gamma]. IKK[alpha] and IKK[beta] are highly conserved and they contain three functional domains of kinase domain, leucine zipper (LZ), and helix-loop-helix (HLH). Although IKK[alpha] and IKK[beta] can phosphorylate IκB proteins in vitro, IKK[alpha] and IKK[beta] have distinct physiological functions during mouse development. Genetic studies showed that IKK[alpha] is essential for embryonic skin development in mice. Mice deficient in IKK[alpha] display a hyperplastic epidermis that lacks terminal differentiation, resulting a death soon after birth because of the severely impaired skin. Recently, we reported a reduction in IKK[alpha] expression and identified somatic Ikk[alpha] mutations in a high proportion of poorly differentiated human squamous cell carcinomas (SCCs) (Liu et al., 2006). The aim of this study is to investigate the novel role of IKK[alpha] in skin carcinogenesis. We firstly examined IKK[alpha] expression and Ikk[alpha] mutations in human SCCs and found a reduction of IKK[alpha] in poorly differentiated human SCCs and identified somatic Ikk[alpha] mutations in exon 15 of Ikk[alpha] in human SCCs. We then examined the susceptibility of Ikk[alpha] hemizygotes to chemical carcinogeninduced skin carcinogenesis. In this chemical carcinogen-induced skin carcinogenesis setting, 7,12-dimethylbenz[a]anthracene (DMBA) induces Ras mutations and 12-Otetradecanoyl-phorbol-13-acetate (TPA) promotes Ras-initiated cell proliferation. We found two times more papillomas and eleven times more carcinomas in Ikk[alpha superscript +/-] mice than in Ikk [alpha] superscript +/+] mice induced by DMBA/TPA. Ikk[alpha superscript +/-] mice developed larger and earlier tumors than did Ikk[alpha superscript +/+] mice. Poorly differentiated carcinomas expressed low levels of IKK[alpha]. Ninety five percent of the Ikk[alpha superscript +/-] carcinomas and 44% of the Ikk[alpha superscript +/-] papillomas lost the remaining wild type Ikk[alpha] allele. This result indicates that the remaining one wild type Ikk[alpha] allele is important for preventing malignant carcinoma conversion. Also Ikk[alpha] mutations were detected in these skin tumors. Reduced IKK[alpha] was found to enhance TPA-induced mitogenic and angiogenic activities in mouse skin. Taken together, these results suggest that reduction of IKK[alpha] expression provides a selective growth advantage, which cooperates with DMBA-initiated Ras activity to promote skin carcinogenesis. In addition, we observed a small group of FVB female Ikk [alpha superscript +/-] mice for 1.5 years and found that 12/ 24 mice developed various spontaneous tumors including mammary gland carcinomas, uterine and ovary tumors, and dermal fibrosacomas. Somatic Ikk[alpha] mutations, elevated IKK/ NF[subscript -k]B and extracellular signal-regulated kinases (ERK) activities and elevated cyclin D1 levels were detected in these spontaneous tumors. These results suggest that these molecular alterations may contribute to the development of these tumors although the precise role of the down-regulation of IKK in the development of the tumors remains to be determined. Overall, our data and other published results suggest that IKK[alpha] is a new tumor suppressor in men and mice. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Physiology / Master / Master of Medical Sciences

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Public Health / Master / Master of Public Health

published_or_final_version / Public Health / Master / Master of Public Health

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