Genomic instability in cancer : the role of the mitotic spindle checkpoint gene hBUB1
In this study, a number of human cancer cell lines were characterised for their genomic instability phenotypes: using a panel of centromeric probes utilising the fluorescence in situ hybridisation (FISH) technique to detect CIN, and a microsatellite assay using radioactive PCR labelling to detect MIN. This characterisation allowed comparison of hBUB1 expression data, and assessed the differences between phenotypic cell line groups for hBUB1 mutations and polymorphisms. Having demonstrated the potential of radioactive-microsatellite PCR to display changes between drug sensitive and selected daughter drug resistant cell lines, steps to assess the actual hBUB1 2q region in primary resected human cancers, with corresponding normal tissue pairs, was taken. NSCLC and CRC tumours were used as models for evaluation of the hBUB1 locus. NSCLC and CRC subtypes were chosen due to the high incidence of CIN observed in these tumours. The hBUB1 locus on chromosome 2q was assessed for genomic instability using a panel of seven microsatellite markers. In addition, two novel CA dinucleotide repeats: BUBCA18 a 175bp CA20 product, and BUBCA19 a 119bp CA12 product each located within hBUB1 intronic sequence, were cloned and evaluated in the 32 CRC and 20 NSCLC samples. The microsatellite assessment demonstrated that the 2q locus was highly unstable in CRC, with 62.5% (n=20/32) patient sample pairs displaying MIN in one or more marker. NSCLC had no instability at any locus, suggesting that aneuploidy due to changes around the hBUB1 chromosome 2 locus was unlikely in this cancer subtype. The known hBUB1 mutations and selected polymorphisms were assessed in the two sets of patient groups, and in the human cancer cell lines characterised previously. Sequencing of the hBUB1 exons using flanking intronic primers and restriction fragment length polymorphism (RFLP) assays, developed for specific coding changes, were utilised to achieve this. No alterations were observed for the exon 4 and 13 mutations in all samples tested. Polymorphism analysis similarly revealed low frequencies in CRC and NSCLC. The novel hBUB1 microsatellite repeat, BUBCA18, displayed potential for the use as a clinical marker in detecting CRC within the general population. Results from our BUBCA18 pilot study demonstrated that heterozygosity for BUBCA18 was significantly associated with disease predisposition (OR 8.2 95%CI 28.24-2.68). This preliminary finding, and hypothesis that BUBCA18 could be used to test risk, was investigated further in a CRC case-control population: comprising 189 CRC cases and 251 matched controls. BUBCA18 profiles were assessed using the radioactive-PCR technique. The BUBCA18 case/control investigation showed that the initial hypothesis was incorrect, as heterozygosity of BUBCA18 was insignificant when used to predict CRC risk, within the larger sample group. The studies reported in this thesis have generated novel data regarding the impact of hBUB1 in human cancer. Initial evidence suggesting that hBUB1 has a significant role for CIN initiation seems unlikely.