Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629888
Title: The development of a differential assay for the determination of ataxia-telangiectasia heterozygosity in human subjects
Author: Savoury, Melanie
Awarding Body: University of Wales
Current Institution: Cardiff Metropolitan University
Date of Award: 2008
Availability of Full Text:
Access through EThOS:
Access through Institution:
Abstract:
Ataxia-telagiectasia (A-T) patients exhibit increased sensitivity to ionising radiation and also demonstrate a higher than normal risk of developing cancer. Heterozygote phenotypes exhibit intermediate levels of radiation sensitivity compared to A-T homozygotes and controls and also have an increased risk of cancer, but are asymptomatic. Radiation used in diagnostic procedures could predispose such individuals to the development of cancer. Classically, clinical diagnosis only discriminates between homozygote and non-homozygote individuals, necessitating the development of a routine approach that facilitates identification of A-T heterozygotes. Tissue culture patient derived cell lines, when used in combination with western blot analysis revealed it was possible to identify cell lines with ATM mutations compared to controls, but discrimination of each genotype was not possible using this methodology. Similarly, flow cytometry was used to determine the cell death profiles of the same patient-derived cell lines upon exposure to a variety of cell damaging agents (ionising radiation, chemicals or chemotherapeutic drugs), with the aim of discriminating A-T heterozygotes from homozygotes and controls. This methodology revealed that cell death profiles generated in response to hydrogen peroxide were able to discriminate each of the three genotypes in both lymphoblast and fibroblast cell lineages. In SV40 patient derived lymphoblasts, the increase in late apoptotic cells and the reduction in necrotic cells in the A-T homozygote cells was found to be statistically significant compared to controls. Likewise, the reduction in late apoptotic cells and the increase in necrotic cells in the A-T heterozygote cells was also found to be statistically significant compared to both A-T homozygotes and controls (P <0.05 one-way ANOVA). In untransformed fibroblasts, using a telomerase transformed fibroblast cell line as the control, the increase in early apoptotic cells and total apoptotic cell death (early and late apoptotic cells combined) in the A-T homozygote cells was found to be statistically significant compared to the control. The increase in early apoptotic cells and total apoptotic cell death in the A-T heterozygote cells was also found to be statistically significant compared to both the A-T homozygotes and the control (P <0.05 one-way ANOVA). It was on this basis that a clear distinction could be made between the three genotypes in two different cell lineages in response to hydrogen peroxide treatment. It had previously been reported that virus transformed cell lines produced different cell death profiles to primary or untransformed cell lines from A-T patients and carriers compared to controls following exposure to ionising radiation. Data presented here confirms these observations with additional A-T homozygote, A-T heterozygote and control cell lines in SV40 transformed lymphoblasts and untransformed fibroblasts. Data is also presented using the same patient-derived cell lines with the chemicals hydrogen peroxide and staurosporine, and the anticancer drugs etoposide and bleomycin. The differential response of SV40 transformed cell lines and untransformed cell lines to these treatments calls into question whether virally transformed cell lines should be used to predict in vivo cellular responses.
Supervisor: Rushmere, Neil; Bowen, S. V. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.629888  DOI: Not available
Share: