A role for topoisomerase II alpha in chromosome damage in human cell lines
Abstract
Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in
the radiation-response of cells as measured by frequencies of chromosomal aberrations.
Different frequencies of IR-induced aberrations can be conveniently observed in
phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals
and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the
following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid
discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been
shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic
breast cancer patients, are significantly higher than in groups of normal individuals. This
suggests that elevated radiation-induced chromatid break frequency may be linked with
susceptibility to breast cancer.
It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears
that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not
match. Therefore, the underlying causes of the wide variation in frequencies of chromatid
breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast
cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining.
My research has focused on the mechanism through which chromatid breaks are formed
from initial DSBs. The lack of a direct association suggested that a signalling process might
be involved, connecting the initial DSB and resulting chromatid break. The signal model,
suggested that the initial DSB is located within a chromatin loop that leads to an intra- or
interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the
decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha
(topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister
chromatid decatenation.
During my PhD research I have used a strategy of altering topo II activity or expression and
studying whether this alters IR-induced chromatid break frequency. The first approach
involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid
breaks was found to correlate positively with topo IIα expression level, as measured in three
different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression
exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also
estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this
related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα
protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by
immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression
correlated 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
inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed
that lowered chromatid break frequency was not due to cells with high chromatid break
frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with
lowered topo IIα expression or activity. These experiments show that topo IIα is involved in
IR-induced chromatid break formation.
The final experiments reported here attempted to show how topo II might be recruited in the
process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of
reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under
these conditions is involved in the entanglement of metaphase chromosomes and formation
of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy
attempted to confirm these dots as excised chromatin loops.
The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA
damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs,
even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen
peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is
involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment
with hydrogen peroxide at a low concentrations.
I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most
likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal
model.
Type
Thesis, PhD Doctor of Philosophy
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