LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

S. Ghosh et al. / Mutation Research 716 (2011) 10–19 15
Fig. 5. Phosphorylation of BRCA1 at 4 h after exposure to 1 Gy -rays or carbon irradiation in A549 cells. (A) Representative image showing p-BRCA1 foci 4 h after irradiation.
Each phospho-BRCA1 antibody was indirectly labeled with Molecular Probe 488 secondary antibody (green) and cells were mounted with ProLong Gold antifede with DAPI
(blue). All images were captured using Carl Zeiss confocal microscope with the same exposure time. (B) Graph represents average numbers of foci per cell, percentage of
cells showing the foci is marked above the bars. (C) Graph represents relative intensity of p-BRCA1 as determined by ImageJ software. At least 100 cells per experiment were
analyzed from three independent experiments. Data represents means ± SD of three independent experiments. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)
only at later time point of 4 h as comparing the activation of these
molecules at this time could highlight signaling differences with
respect to clustered damage.
ATM and ATR, the initial DNA damage sensors, are critical regulators
of cell cycle checkpoints and repair. ATM has particularly
been shown to respond to IR induced damage. Presence of few
ATM foci in -rays irradiated cells 4 h after irradiation indicates the
repair of the damage in most cells. However, in carbon irradiated
cells, although the percentage of cells showing the pATM foci (55%)
was less, the average number of foci/cell was more than -rays irradiated
cells (Fig. 3). Similar trend was also observed with ATR foci
with respect to radiation quality (Fig. 4). Higher percentage of -
rays irradiated cells showing the ATM/ATR foci 4 h after irradiation
as compared to carbon irradiated cells indicates more homogenous
nature of -rays induced responses while presence of lesser average
ATM/ATR foci per -rays irradiated cell as against carbon irradiation
indicates efficient repair after -rays irradiation. This observation
is in agreement with the previous studies where higher activation
of repair molecules had been observed few hours after heavy ion
irradiation as compared to low LET irradiation [22,34] and has been
attributed to unrepaired DNA that keeps the damage sensors in activated
state. However, these studies have shown that size of the foci
and the number of foci at 24 h is different between high and low
LET radiations.
The formation of BRCA1 foci was unlike ATM/ATR and very interesting.
BRCA1 exists in a complex containing ATM and other DSB
repair proteins and both ATM and BRCA1 have been regarded as primary
regulators of HRR [35]. Presence of BRCA1 foci in ∼55% (Fig. 5)
of the -rays irradiated cells is in agreement with the previous studies
where BRCA1 foci have been found to be activated only in cells
in S, G2/M phases of the cycle [34] and therefore is thought to play
a role in HRR which can take the advantage of the other strand that
may be intact. Unlike after -rays irradiation, presence of BRCA1
foci in all of the carbon irradiated cells was quite intriguing. Moreover,
the downstream substrates, Chk1 (Fig. 6A) and Chk2 (Fig. 6B)
also showed relatively higher activation in carbon irradiated cells.
Despite higher activation of all these repair components in carbon
irradiated cells, their DNA was still unrepaired as indicated by -
H2AX staining and survival data. The authors therefore, believe that
activated repair components might have different functionalities
after low and high LET radiations and that this functionality might
be imparted through association of proteins in different macromolecular
complexes. This was supported by larger size of -H2AX
and BRCA1 foci observed after carbon irradiation. This result is
in accordance with previous studies where authors had observed
larger foci of DNA damage response proteins in the cells that had
been exposed to high LET radiation [14,36]. This means that foci
from -rays in essentially have a distinct meaning from those from