Year: 2010
Type: dissertation
Abstract: Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in \nthe radiation-response of cells as measured by frequencies of chromosomal aberrations. \nDifferent frequencies of IR-induced aberrations can be conveniently observed in \nphytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals \nand sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the \nfollowing cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid \ndiscontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been \nshown that the frequency of chromatid breaks in cells from approximately 40% of sporadic \nbreast cancer patients, are significantly higher than in groups of normal individuals. This \nsuggests that elevated radiation-induced chromatid break frequency may be linked with \nsusceptibility to breast cancer. \nIt is known that chromatid breaks are initiated by a double strand break (DSB), but it appears \nthat the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not \nmatch. Therefore, the underlying causes of the wide variation in frequencies of chromatid \nbreaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast \ncancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. \nMy research has focused on the mechanism through which chromatid breaks are formed \nfrom initial DSBs. The lack of a direct association suggested that a signalling process might \nbe involved, connecting the initial DSB and resulting chromatid break. The signal model, \nsuggested that the initial DSB is located within a chromatin loop that leads to an intra- or \ninterchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the \ndecatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha \n(topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister \nchromatid decatenation. \nDuring my PhD research I have used a strategy of altering topo II activity or expression and \nstudying whether this alters IR-induced chromatid break frequency. The first approach \ninvolved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid \nbreaks was found to correlate positively with topo IIα expression level, as measured in three \ndifferent cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression \nexhibited lower chromatid break frequency. Topo II activity in these three cell lines was also \nestimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this \nrelated well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα \nprotein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by \nimmunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression \ncorrelated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that \ninhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed \nthat lowered chromatid break frequency was not due to cells with high chromatid break \nfrequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with \nlowered topo IIα expression or activity. These experiments show that topo IIα is involved in \nIR-induced chromatid break formation. \nThe final experiments reported here attempted to show how topo II might be recruited in the \nprocess of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of \nreactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under \nthese conditions is involved in the entanglement of metaphase chromosomes and formation \nof chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy \nattempted to confirm these dots as excised chromatin loops. \nThe possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA \ndamage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, \neven though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen \nperoxide-induced DNA damage at low doses. The results support the idea that topo IIα is \ninvolved in the conversion of DSBs to chromatid breaks after both irradiation and treatment \nwith hydrogen peroxide at a low concentrations. \nI have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most \nlikely by converting the initial DSBs into chromosomal aberrations as suggested by the signal \nmodel.
Author: Samantha Y.A. Terry
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: University of St Andrews
OpenAlex ID: W1770696066