ETD Collection

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    Protein interactions with drosophila p53
    (2014-09-23) Cajee, Umar-Faruq
    Drosophila melanogaster, a key model organism, has cognates of over 70% of human disease genes. This has created opportunities in the development of treatments for life threatening illnesses like cancer. Mutations on the p53 tumour suppressor protein, which is an activator of apoptosis, are common in many cancers. In mammals, p53 interacts with the Retinoblastoma Binding Protein 6 (RBBP6) which enhances the activity of MDM2, the prototypical negative regulator of p53, that is absent in invertebrates. In the absence of MDM2 the Drosophila RBBP6 homolog, SNAMA, through its DWNN Catalytic Module (DCM), is suspected to play an important role in the regulation of p53, probably via the ubiquitin proteasome pathway. Through bioinformatics analyses, and experimental analysis of transcripts, this study has shown the existence of two isoforms of SNAMA named here SNAMA A and SNAMA B for the long and short isoforms, respectively. SNAMA B appears to be expressed after genotoxic stress (DNA damage) in adults as well as during embryonic development. Recombinant protein expression in bacterial and yeast systems as well as HIS-tag chromatography and Western blot analyses were used to investigate interactions with Dmp53. Due to poor expression of recombinant Dmp53 protein in both prokaryotic and eukaryotic systems and unreliable commercial antibodies, it was impossible to complete interaction studies. Overall, these studies show that the SNAMA isoforms may play important roles during development and in response to DNA damage.
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    The study of cationic amphiphilic peptides with anti-cancer selective toxicity
    (2014-09-01) Maumela, Pfariso
    The exposure of organisms to environmental stresses and pathogens results in rapid activation of a range of defensive pathways that act as part of the innate immune system. The most common innate immunity response is the activation of cationic amphiphilic peptides in response to microbial infection. Moreover, cationic amphiphilic peptides possess desirable attributes for the pharmaceutical development of cancer-selective drugs. They selectively and rapidly kill cancer cells without killing normal mammalian cells and have a broad spectrum of mechanisms of action. The aim of this exploratory study was to screen for cationic amphiphilic peptides with anti-proliferative activity that is induced by genotoxicity. GeneFishing® technology, 2-D gel analysis and bioassays were used to identify and analyse molecules induced in response to genotoxic stress in an embryonic cell line originating from the dung beetle Euoniticellus intermedius. Bioassay results revealed that the cell line has constitutive expression of probable cationic amphiphilic proteins that are further induced by camptothecin treatment. GeneFishing® and 2-D gel analysis showed changes in gene expression at both transcriptional and translational levels, respectively. Overall, the study failed to identify the involvement or induction of cationic amphiphilic peptides in response to genotoxic stress. However, gene expression analyses revealed changes in the expression of classes of proteins involved in stress response, oxidative phosphorylation, mitochondrial maintenance, protein translation, cytoskeletal proteins and immunophilins. The results show that the cell line constitutively expresses probable cationic amphiphilic peptides which are further induced by camptothecin.
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    The effect of the siRNA-mediated downregulation of the non-integrin laminin receptor on cancer cell viability
    (2013-08-08) Moodley, Kiashanee
    Cancer is a hypernym used to describe a group of diseases characterised by the uninhibited growth and spread of abnormal cells in the body. An estimated 7.6 million annual deaths are attributed to the disease while 12.7 million new cases are reported every year. The severity of this disease demonstrates the urgent requirement of novel anti-cancer therapeutic agents. The non-integrin laminin receptor, here designated the 37 kDa/67 kDa laminin receptor (LRP/LR), is a multifunctional protein located on the surface, in the cytoplasm, in the perinuclear compartment and in the nucleus of cells. While this receptor is imperative for normal cellular functioning, it has also been implicated in many diseases – it serves as a cell surface receptor for numerous viruses, infectious prion proteins as well as certain respiratory tract pathogens. Additionally, LRP/LR has been found to have some involvement in zoonotic diseases and those involving neurodegeneration, such as Alzheimer’s disease. Most importantly for this study, LRP/LR has been implicated in cancer progression, where it was found to be overexpressed on the surface of various cancer cell lines, this overexpression correlating to increased metastasis. The aim of this study was to investigate the effect of the siRNA-mediated downregulation of LRP expression on the viability of tumorigenic lung and cervical cancer cells (A549 and HeLa, respectively). The cell surface LRP/LR and total LRP levels were investigated using flow cytometry and western blotting, respectively, in A549 and HeLa cells, the results revealing high percentages of both cell lines expressing LRP/LR on their surface. Additionally, A549 and HeLa cells express similar total levels LRP. The transfectability of these cells was confirmed and siRNA-LAMR1 was shown to significantly downregulate LRP expression (80% and 60% in A549 and HeLa cells, respectively). MTT assays revealed that the significant 13% and 18% reduction in cellular viability in A549 and HeLa cells, respectively, was as a consequence of LRP downregulation. This reduction in cellular viability was found to be a consequence of induced apoptosis (identified by the visualisation of the loss in nuclear integrity, as well as the significantly increased activity of the apoptosis-associated protein caspase- 3) and inhibited cellular proliferation in the aforementioned cells. These findings suggest that siRNA targeting LRP mRNA may act as a potential alternative therapeutic tool for the teatment of cancer.
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    Molecular analysis of the domain with no name (DWNN)/RBBP6 in human cancers
    (2012-10-08) Mbita, Zukile
    Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. It is a multi-domain protein containing a Zinc finger, a RING finger, an Rb binding domain, a p53 binding domain and a novel N-terminal protein domain, the so called, Domain With No Name or DWNN. The RBBP6 gene encodes three isoforms of this particular protein. A common feature of all three isoforms of RBBP6 is the presence of the N-terminal DWNN domain. RBBP6 isoform 3 is comprised of the DWNN domain only. The DWNN itself has a ubiquitin-like fold, sharing 22% similarity with ubiquitin. It is likely that DWNN regulates intracellular levels of the two tumour suppressors, Rb and p53 through the ubiquitin-proteasome pathway and as such, DWNN may therefore play a role in the deregulation of cell cycle control in cancer cells. A mouse homologue, P2P-R of the gene has been implicated in mitotic apoptosis. The expression of DWNN, RBBP6 and their roles in the cell cycle, apoptosis and human cancer were investigated. RT-PCR and real-time PCR were used to determine the gene expression of DWNN and RBBP6 variants in human cancer cells. An anti-human DWNN antibody was characterized using both Western Blotting analysis and MALDI-TOF mass spectroscopy to determine whether the antibody specifically recognizes DWNN and RBBP6 isoforms, or if it recognizes other proteins. Western blotting was also used to determine the nature of the DWNN in human cell lines. A DWNN probe and the characterized anti-human antibody were used to localize DWNN and RBBP6 gene products at the mRNA and protein levels using ISH/FISH and Immunohistochemistry respectively. Cell labelling was also performed using this antibody to localize RBBP6 products in human cell lines. RNA interference and over-expression of DWNN and RBBP6 gene products was carried out to further investigate the role of RBBP6 products in the cell cycle, apoptosis and carcinogenesis. Cloned RT-PCR products of RBBP6 binding domains, the RING finger domain, pRb-binding and p53-binding domains in human cancers cell lines (Hek 293T, MCF7, HeLa and HepG2 cells) showed no mutations, but MCF-7 cells showed the lowest expression of the RBBP6. Real-time PCR and Western blotting analysis confirmed that MCF-7 cells express very little DWNN (RBBP6 isoform 3) and RBBP6 gene products when compared to Hek 293T, HeLa and HepG2 cells. It was also shown that the anti-human DWNN antibody recognizes the DWNN domain (RBBP6 isoform 3) and the larger RBBP6 isoforms. Using 2D gel electrophoresis and MALDI-TOF spectrometry, it was also found that DWNN is associated with other proteins namely, Recoverin and a hypothetical protein XP_002342450. This result suggested that DWNN may be a ubiquitin-like protein, which may be specific to these proteins in human cells. FISH and IHC demonstrated that the DWNN domain and its relatives are down-regulated in human cancers at both mRNA and protein levels, respectively. In contrast, however, cell staining showed that the expression of the DWNN gene products was high during the G2/Mitosis transition. Knocking-down the DWNN domain or over-expressing it did not sensitise the Hek 293T cells to Camptothecin (CPT)-induced apoptosis but rather slowed down cell growth. These results strongly suggest that the DWNN gene is likely to be involved in cell cycle control. Up-regulation in mitotic cells and down-regulation in cancers also implies that RBBP6 gene products may additionally be involved in cell cycle arrest. Moreover, down-regulation in human cancers particularly indicates that the loss of its function which correlates with loss of cell cycle control in this disease may be involved in the pathogenesis of cancer. This was confirmed by up-regulation of the DWNN in arsenic trioxide induced cell cycle arrested cells specifically at G2/M phase where a p53-dependent cell cycle arrest ensued. It is thus proposed that the DWNN may be implicated both as a p53 stabilizer and additionally as a G2/M progression regulator.