published_or_final_version / abstract / Physiology / Master / Master of Philosophy

Limb-Bud and Heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of functional TCF/LEF binding sites in the LBH locus and rapid beta-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/beta-catenin signaling is inhibited by Wnt7a, which in limb development signals through a non-canonical pathway involving Lmx1b. Furthermore, we show that Lbh is aberrantly overexpressed in mammary tumors of MMTV-Wnt1 transgenic mice and in aggressive basal-subtype human breast cancers that display Wnt/beta-catenin hyperactivation. Deregulation of LBH in human breast cancer appears to be Wnt/beta-catenin dependent as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. RNAi mediated knockdown of LBH in basal breast cancer cell lines resulted in loss of CD44high/CD24low tumor cells, luminal differentiation, reduced cell growth, reduced colony forming ability, and increased apoptosis, suggesting a novel pro-survival and stem cell maintenance function of LBH in breast cancer. Reciprocal overexpression studies in the basal breast carcinoma line BT549 resulted in increased tumorigenicity in vitro, suggesting that LBH overexpression is indeed oncogenic. Finally, we further characterized LBH protein expression patterns and post-transcriptional regulation. Collectively, this thesis demonstrates that LBH is a direct Wnt target gene in both development and basal breast cancer that promotes the undifferentiated phenotype and survival of basal breast tumor cells.

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

No description available.

Notch signalling is an evolutionary highly conserved signalling pathway. It plays various important roles in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance and differentiation during embryonic and adult development. Notch signalling has a simple transduction pathway. Upon Notch ligand binding to the receptor, the Notch intracellular domain (NICD) is released into the nucleus. The nuclear NICD interacts with the DNA-binding protein Suppressor of Hairless (Su(H)) to activate the expression of target genes, which are silenced by the Su(H)-corepressor complex in the absence of Notch activity. The functions of Notch are very context-dependent, making it important to identify the Notch regulated genes in different processes. Neural stem cells (NSCs) are cells that can divide and differentiate into all kinds of cells within the brain while they self-renew. Notch signalling is one of the key regulators in maintaining NSCs and performs a similar function in both Drosophila and vertebrate NSCs. Drosophila NSCs serve as an ideal model for studying the relationship between Notch function and stem cell behaviours. Although many target genes, such as the Hes genes, have been identified, they cannot fully account for the diversity of Notch responses. Therefore, further functional study of more potential target genes is needed to gain understanding about Notch-regulated NSC maintenance. In this thesis, a group of potential direct Notch target genes are examined for their responsiveness to Notch regulation and their functions in Drosophila NSCs. Previous genome-wide study in the Bray lab has found a number of potential Notch target genes in the Drosophila larval brain, with the characteristics of Notch transcription factor Su(H) binding and mRNA upregulation by Notch over-activation (Zacharioudaki et al. 2016). I first examined the Notch responsive element (NRE) activity of these potential Notch targets and their regulation by Notch both in vivo and in cell lines. The presented findings validated path, cables and Asph as direct Notch target genes in Drosophila NSCs, while syp, lola and Fer2 do not exhibit characteristics of Notch responsive targets in NSCs. The functional roles of two of the responsive genes, path and cables, were subsequently explored in Drosophila larval brains. Firstly, I found that Path, a potential amino acid transporter, is not only important for protecting NSC proliferation under normal and abnormal conditions through integrating growth pathways, but is also required for protecting brain growth under nutrition deprivation. Secondly, the cables gene was connected to a distal NRE through knocking out the suspected NRE region and the gene itself using the CRISPR/Cas9 technique. Subsequent experiments revealed that cables is also required for NSC proliferation. In summary, a group of direct Notch target genes were validated and as a consequence two genes that are important for protecting NSC proliferation were identified.

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

The function and role of PAK6, serine/threonone kinase, in cancer progressionhas not yet been clearly identified. Several studies reveal that PAK6 may participate in key changes contributing to cancer progression such as cell survival, cell motility, and invasiveness. Basedon the membrane localization of PAK6 in prostate and breast cancer cells,we speculated that PAK6 plays a rolein cancer progression cells by localizing on the membrane and modifying proteins linked to motility and proliferation. We isolated the raft domain of breast cancer cells expressing either wild type (WT), constitutively active (SN), or kinase dead PAK6 (KM) and found that PAK6 is a membrane associated kinase which translocates from the plasma membrane to the cytosol when activated. The downstream effects of PAK6 are unknown ; however, results from cell proliferation assays suggest a growth regulatory mechanism. / by JoAnn Roberts. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Further studies were done to investigate the mechanism responsible for 1-BI-induced apoptosis in NCI-H460. It was found that 1-BI stimulated the expression of pro-apoptotic p53, Bax and cytosolic NF-kB p65 subunit but decreased pERK in NCI-H460 cells. The active forms of caspase 3 and caspase 9 were detected by Western blot, accompanied by an increase in caspase 3 activity. Reactive oxygen species (ROS) was highly generated at 24 hours after the treatment and the mitochondrial membrane potential was significantly decreased at 48 and 72 hours. The application of either N-acetyl cysteine (NAC) or glutathione (GSH) attenuated the cell growth inhibition caused by 1-BI. NCI-H460 cells pretreated with NAC showed a decrease in ROS production and p65 protein but an increase in pERK. / Taken together, these findings suggest that the inhibition of THXS suppresses lung cancer cell growth by promoting either G1 cell cycle arrest or apoptosis. The status of p53 is critical for both cell cycle arrest and apoptosis in 1-BI-mediated growth inhibition, which is evident by enhanced apoptosis detected in p53-transfected NCI-H23 and DMS 114 cells and G1 cell cycle in lung cancer cells treated with PFT-alpha. The 1-BI-induced growth-inhibitory pathway is associated with the generation of ROS, alteration of mitochondrial membrane potential, down-regulation of pERK and p65. / The result showed that THXS expressed in all of the three lung cancer cell lines (NCI-H23, DMS 114 and NCI-H460). The activity of THXS was also reflected by the presence of THXS metabolite thromobxane B2 (TXB2) in the cells, which was detected by ELISA. 1-Benzylimidazole (1-BI), a specific THXS inhibitor, suppressed the lung cancer cell proliferation measured by MTT assay. 1-BI treatment caused G1 phase arrest and enhanced the level of cyclin dependent kinase inhibitor p27 in a time-dependent manner in NCI-H23 and DMS 114 cells. It markedly increased DNA fragmentation in NCI-H460 cells. The findings suggest that 1-BI inhibits cell growth by arresting cell cycle and inducing cell death. Annexin V/PI staining revealed that the cell death induced by I-BI was mainly in the format of apoptosis. Further experiments showed that the I-BI-induced apoptosis could be enhanced by the introduction of p53 into NCI-H23 and DMS 114 cells, and such enhancement was associated with a decrease in mitochondrial membrane potential. This result suggests that the p53 may play a positive role in apoptosis induced by 1-BI through changing of the mitochondrial membrane potential. The role of p53 in I-BI-mediated apoptosis was further confirmed by the experiment of the p53 inhibition. Pifithrin-alpha hydrobromide (PFT-alpha), a p53 specific inhibitor, suppressed the 1-BI-induced p53 protein expression and increased G1 cell cycle arrest. / Thromboxane A2 (TXA2) is a potent arachidonate metabolite in the cyclooxygenase-2 (COX-2) pathway, which is produced by a member of cytochrome P450 (CYP) superfamily called thromboxane synthase (THXS). Recent studies have showed that thromboxane and THXS are associated with cancer cell migration, angiogenesis, tumor metastasis and cancer proliferation but there is limited information on their role in lung cancer development. This thesis is to test the hypothesis that inhibition of THXS could alter lung cancer cell growth. / Leung, Kin Chung. / Adviser: George G. Chen. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3319. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 130-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Abstract not available