A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree Master of Science. July, 2014 / 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.

Thesis (M.Sc.(Biochemistry)) -- University of Limpopo, 2019. / Retinoblastoma binding protein 6 (RBBP6) is the protein encoded by the Retinoblastoma Binding Protein 6 (RBBP6) gene that is located in chromosome 16p12.2. There is a growing list of newly discovered RBBP6 hypothetical splice variants but there are only three RBBP6 splice variants that are well documented. RBBP6 has been previously implicated in the regulation of cell cycle and apoptosis but little is known about the expression and regulation of the human RBBP6 splice variants during cell cycle progression and breast cancer development. This study was aimed at determining the expression pattern of RBBP6 alternatively spliced variants during arsenic trioxide-induced cell cycle arrest and apoptosis in breast cancer MCF-7 cells. It was also aimed at determining RBBP6 specific microRNAs and how they are regulated in MCF-7 breast cancer cells. MCF-7 cells were maintained and subjected to arsenic trioxide-induced cell cycle arrest and apoptosis. The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and the Muse™ Count & Viability assays were used to evaluate the effect of arsenic trioxide on the viability of MCF-7 cells. Cell cycle arrest using 11 μM arsenic trioxide and apoptosis using 32 μM arsenic trioxide were analysed using the MUSE® Cell Analyzer, light and fluorescence microscopy. Arsenic triode-induced apoptosis was analysed using the Muse™ Annexin V & Dead Cell Kit, MultiCaspase and MitoPotential assays using the Muse™ MultiCaspase Kit and Muse™ MitoPotential Kit. Arsenic trioxide-induced cell cycle arrest was analysed using the Muse™ Cell Cycle Kit. Semi-quantitative analysis of RBBP6 variants was carried out using the conventional Polymerase Chain Reaction (PCR), while the quantitative analysis was done using the Real-Time Quantitative PCR. The localization of RBBP6 isoforms was done using Immunocytochemistry (ICC). Web based Bioinformatics tools were used to identify RBBP6-specific microRNAs. The MTT results showed that arsenic trioxide decreased the viability of the MCF-7 cells in a dose-dependent manner. The Muse™ Cell Cycle analysis showed that 11 μM of arsenic trioxide induced G2/M cell cycle arrest in MCF-7 cells, while the Muse™ Annexin V & Dead Cell assay showed that 32 μM of arsenic trioxide induced the extrinsic apoptotic pathway in MCF-7 breast cancer cells. Using the conventional PCR, the MCF-7 cells were found to express the RBBP6 variant 1 transcript but lacks the expression of variant 2 and 3 transcripts, contrary to the kidney embryonic Hek 293 cells that exhibited the expression of RBBP6 variant 1, 2 and 3. Additionally, arsenic trioxide downregulated RBBP6 variant 1 in breast cancer cells during cell cycle arrest and apoptosis. The Real-Time PCR confirmed that MCF-7 cells lowly express RBBP6 variant 3. On the other hand, the ICC analysis showed that RBBP6 isoform 1 is localized and highly expressed in MCF-7 breast cancer cells. The Web based Bioinformatics tools showed that RBBP6 variant 1 specific microRNAs are down regulated in MCF-7 breast cancer cells. These results together showed that As2O3 is effective against MCF-7 cells and also regulated the expression of RBBP6 variants, especially, variant 1. This study showed that there are RBBP6 variants that are involved in breast cancer progression and there are those that may be involved in breast cancer suppression. Targeting these RBBP6 variants for therapeutic development is a promising strategy. In conjunction with RBBP6 expression, arsenic trioxide should be further explored as a breast cancer drug.

Abstract not available

The ability of low, clinically relevant levels of the chemotherapeutic drugs epirubicin and vinblastine to induce drug resistance was examined in the K562. U937, KG-la and HEL human leukaemia cell lines. Treatment with epirubicin and vinblastine induced the MDR phenotype and P-glycoprotein expression in K562 and U937 cells. However this treatment had no effect on drug resistance in the P-glycoprotein expressing KG-la and HEL cells. In the U937 cells, drug resistant cells were not only MDR but were also resistant to other drugs including cisplatinum and chlorambucil which are not normally associated with MDR. The drug resistant U937 sublines were also sensitised to doxorubicin, cisplatinum and chlorambucil by buthionine sulphoximine (BSO), suggesting that glutathione-related mechanisms also contributed to resistance in these sublines. The U937 sublines also had an increased DNA content and an increased ability to recover from DNA damage, as determined by cell cycle analysis, indicating that the broad cross-resistance exhibited by these cells was due to the co-existence of multiple resistance mechanisms. Drug treatment induced changes in expression of differentiation associated antigens in all four cell lines. Treatment with inducers of differentiation (TPA, sodium butyrate, granulocyte-macrophage colony-stimulating factor; GM-CSF). Treatment of K562 and K562/E15B cells with TPA induced megakaryocytic differentiation, with increases in drug resistance, and increased P-glycoprotein expression in the K562/E15B subline. TPA induced monocytic differentiation in the U937 cells but not the U937/EIS subline, with increased P-glycoprotein expression and function in the U937/E15 cells but not the U937 cells. Staurosporine, an inhibitor of PKC, inhibited differentiation in these cell lines, but did not inhibit increases in P-glycoprotein expression, suggesting drug resistance was not mediated by PKC. Sodium butyrate induced erythroid differentiation, and increased P-glycoprotein expression in the K562/E15B cells. However at a higher concentration (15 mM) this was not accompanied by increased drug resistance. Granulocyte monocyte colony stimulating factor (GM-CSF) did not induce differentiation in the K562 cells or K562/E15B subline, although the K562/E15B cells became more drug resistant after treatment with GM-CSF. Treatment with GM-CSF induced differentiation in the U937/E15 subline but did not change drug resistance in either the U937 cells or the U937/EI5 subline. Therefore the P-glycoprotein expressing K562/E15B and U937/E15 sublines were more responsive to inducers of differentiation than the parental cell lines. Induction of differentiation therefore induced increases in P-glycoprotein expression and drug resistance, suggesting that expression of P-glycoprotein or a multidrug resistance phenotype was associated with differentiation.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Also available online.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 122-140).

This thesis examines some of the challenges involved with using FTIR spectroscopy for cancer detection including sample preparation and correcting for distortion from cell scattering. Sample preparation affects the spectra differently depending on the cell type, and can lead to significant changes in cancer biomarkers for a given cell type. Biomarkers derived from specific cancer types under one sample preparation are not reliable for other cancer types, and may not be suitable for the same cancer type using a different sample preparation. Cell scattering can also significantly affect the cell spectra, and as a result, correcting for the cell scattering distortion leads to changes in the biomarkers. For reliable cancer detection controlling variability is critical, especially in the complex spectra of biological samples. Standard sample preparation methods and scattering correction post-processing could improve comparison of cancer detection methods.