LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

S. Ghosh et al. / Mutation Research 723 (2011) 190–198 193
Fig. 3. Phosphorylation of ATM 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-ATM foci 4 h
after irradiation. Each phospho-ATM 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-ATM 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
6 Gy of gamma-ray exhibited more -H2AX foci than 2 Gy gammaray
sample. However the increased ratio is only 1.45, and far from
3. This result is contradictory to the fact that induction of DNA double
strand break is physical process, not biological process. Thus
it is expected to increase in proportion with irradiated physical
dose if the source of radiation is same. We have used immunofluorescent
staining assay for -H2AX, one of the limitation of this
assay is the exposures of cells higher than 4 Gy result in foci saturation,
reducing the useful range of the assay to doses less than 4 Gy.
However, major advantage of this assay, it is more sensitive than
other methods for detecting -H2AX [26]. Many downstream signaling
molecules are known to get activated from these irradiation
induced -H2AX foci [27].
The -H2AX foci were also counted 4 h after irradiation to compare
the repair status at that time with respect to radiation quality.
53% of 2 Gy gamma irradiated cells still showed -H2AX foci but the
average foci per cell had reduced to only 4.3 ± 2.2, where as 100% of
6 Gy gamma irradiated cells showed -H2AX foci and the average
foci per cell had reduced to only 8 ± 2.5. In oxygen ion irradiated
cells, although bright green -H2AX intensity was observed till 4 h
in 60% of cells, there was a significant reduction in appearance of
distinct foci like structures (10 ± 2) (Fig. 2(B and C)). Disappearance
of -H2AX foci indicates DNA repair/rejoining. Instead of counting
number of foci, the intensity of -H2AX in cells showing the foci was
estimated by ImageJ software in the same images. In case of 2 Gy
or 6 Gy gamma irradiation, both -H2AX foci and its total intensity
levels had reduced at 4 h (Fig. 2(D)). However, in case of oxygen
irradiation, the total intensity of -H2AX at 4 h was yet very high
(Fig. 2(D)) though foci were not properly visible. This could indicate
diffusion of -H2AX foci rather than its disappearance due to actual
dephosphorylation of -H2AX. Presence of -H2AX even 4 h after
oxygen ion irradiation suggests that DNA had not been repaired.
Similar number of -H2AX at 15 min after irradiation irrespective
of the radiation quality indicated that initial events might
not differ much with the radiation quality. Activation of other
molecules involved in DNA damage response was therefore fol-