Paediatrics (RCH) - Theses
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ItemThe role of centromere defects in cancer formation and progression( 2016)The centromere plays a crucial role in genome inheritance — ensuring proper segregation of sister chromatids into each daughter cell. In cancer, especially in later stages of solid tumours, cells are often presented with extensive chromosomal abnormalities. However, the involvement of defective centromeres in the disease formation and progression has not been carefully studied. Hence, my PhD project aimed to investigate the role of defective centromeres in cancer progression using the NCI-60 panel of cancer cell lines. The spectrum of centromere-related abnormalities were first characterised with pan-centromeric FISH probes and then with the addition of CENP-A immunofluorescence. For HOP-92 and SN12C, cell lines showing high prevalence of functional dicentric chromosomes, expansion of single cell clones from the initial heterogeneous population was carried out to further study the involvement of dicentric chromosomes in cancer genomic instability. Neocentromere formation sites in cancer cell lines were investigated using cell lines with high prevalence of neocentric chromosomes. A neocentromere was found in T-47D which marked the first report of a neocentromere discovered in a long-established and widely used cell line, and also the first neocentromere to be reported in breast cancer. Separately, an antibody screen to identify antibodies recognising components of an active centromere in methanol-acetic acid stored cells was performed because thus far, antibodies that are widely used for functional centromere detection mainly worked on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. I found a commercially available rabbit monoclonal anti-CENP-C that worked on fixed samples and in addition, I combined three methods (FISH, immunofluorescence and mFISH) to obtain more information from the same metaphase spread. I then proceeded to test anti-CENP-C in my method, CENP-IF-cenFISH-mFISH, on dicentric- and neocentric-containing cancer cell lines and proposed other utilities for this method which I have developed.
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ItemHuman centromeric and neocentromeric chromatin( 2000-09)Human centromeres contain large arrays of α-satellite DNA that are thought to provide centromere function. These arrays show size and sequence variations. However, the lower limit of the sizes of these DNA arrays in normal centromeres is unknown. Using a set of chromosome-specific α-satellite probes for each of the human chromosomes, interphase Fluorescence In Situ Hybridisation (FISH) was performed in a population screening study. This study demonstrated that extreme reduction of chromosome-specific α-satellite is unusually common in chromosome 21 (screened with the αRI probe), with a prevalence of 3.70%, compared to <=.12 % for each of chromosomes 13 and 17, and 0 % for the other chromosomes. No analphoid centromere was identified in over 17,000 morphologically normal chromosomes studied. All the low-alphoid centromeres are fully functional as indicated by their mitotic stability and binding to centromere proteins including CENtromere Protein-A (CENP-A), CENtromere Protein-B (CENP-B), CENtromere Protein-C (CENP-C), and CENtromere Protein-E (CENP-E). Sensitive metaphase FISH analysis of the low-alphoid chromosome 21 centromeres established the presence of residual αRI as well as other non-αRI α-satellite DNA suggesting that centromere function may be provided by (i) the residual αRI DNA, (ii) other non-αRI a-satellite sequences, (iii) a combination of i and ii, or (iv) an activated neocentromere DNA. (For complete abstract open document)