LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

194 S. Ghosh et al. / Mutation Research 723 (2011) 190–198
Fig. 4. Phosphorylation of ATR at 4 h after exposure to 2 Gy and 6 Gy gamma or 2 Gy oxygen irradiation in A549 cells. (A) Representative image showing p-ATR foci 4 h after
irradiation. Each phospho-ATR 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-ATR 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; significantly different from unirradiated controls: *P < 0.05,
**P < 0.01. Significantly different from 6 Gy gamma: ## P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
lowed only at later time point of 4 h. Moreover, by this time low
LET induced damage is mostly repaired and the activation of these
factors is reduced to control levels [28] thereby sealing the fate of
the cell. Comparing the activation of these molecules at this time
could therefore give important insight into differences in damage
response signaling with respect to radiation quality. Phosphorylation
of H2AX even at 4 h indicates the presence of large number
of double strand breaks at that time indicating very slow repair in
A549 cells exposed to oxygen beam.
3.3. Radiation induced foci of DNA damage response proteins
ATM and ATR
ATM is an important DNA damage response protein to ionizing
radiation and a part of irradiation induced foci (IRIF). It gets activated
after DNA DSB by phosphorylation at ser 1981 and activates
many key cellular proteins that include H2AX, BRCA1, Chk1/2 and
p53 [14,29]. Phospho ATM foci and intensity were looked at 4 h
after gamma or oxygen irradiation. At 4 h after 2 Gy gamma irradiation,
most of the cells (93%) cells showed ATM foci (5.9 ± 3.7)
as against 25% unirradiated cells (3.4 ± 1.6) (Fig. 3). Like -H2AX
foci, lesser number of ATM foci 4 h after 2 Gy gamma irradiation
indicates that by 4 h most of the repair due to damage by low LET
irradiation would have taken place. 4 h after 6 Gy gamma irradiation,
100% of the cells showed the foci and average ATM foci per
cell were 14.5 ± 6.05. However, at 4 h after oxygen ion irradiation,
100% of the cells still showed the foci and average ATM foci per cell
(18.6 ± 8.7) were higher than gamma irradiation. Total intensity
levels of phospho ATM matched with the number of foci observed
(Fig. 3). ATM is major mediator of DSB response and is involved
in cell cycle arrest, repair and apoptosis. Persistence of significant
pATM foci till 4 h after oxygen ion irradiation indicates the presence
of unrepaired DNA, which is still keeping the damage sensor
protein ATM in activated form. The slower disappearance of pATM