LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

14 S. Ghosh et al. / Mutation Research 716 (2011) 10–19
Fig. 4. Phosphorylation of ATR at 4 h after exposure to 1 Gy -rays or carbon 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. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
JNK did not show any activation after -rays irradiation (Fig. 7B).
Interestingly, after carbon irradiation the activation pattern of these
kinases was exact opposite. ERK was not activated at all after carbon
irradiation (Fig. 7A) while JNK showed marginal activation at
15 min that remained so till 1 h (Fig. 7B). The expression of total
ERK did not change during the same period in the cells that had
been exposed to either -rays or carbon beam.
3.7. Apoptosis
At 4 h after irradiation, morphological features of DAPI stained
apoptotic nuclei were scored and many apoptotic nuclei were
observed in cells irradiated with carbon ion but not in -rays irradiated
cells (Fig. 8A and B).
Upregulation of Bax expression was also observed as early as
2 h after carbon ion irradiation indicating apoptotic tendency of the
cells (Fig. 8C). -Rays irradiated cells did not show any upregulation
of Bax (data not shown).
4. Discussion
Although 1 Gy dose of carbon ions was three times more toxic to
the cells than -rays, both produced same number of -H2AX foci,
indicators of DNA double strand breaks. This discrepancy between
observed cytotoxicity and estimated -H2AX foci after high LET
radiation has also been reported previously [28]. Since our aim
was to find out signaling differences arising from DNA damaged
by either of the radiations, we chose 1 Gy of both radiations rather
than their equitoxic doses. The observed decrease in number of -
H2AX foci 4 h after -rays irradiation indicates repair of damage
and is supported by nearly 100% survival. However, the decrease in
-H2AX foci after carbon ion irradiation was definitely not indicative
of repair because the cell survival data contradicts the fact
(Fig. 2). Foci formation is a biochemical kinetic process involving
both formation and loss of foci [29], there is never an exact one-toone
correspondence between the number of DSB and foci [30]. In
addition, multiple DSB may be visible as a single focus due to DNA
supercoiling [31]. For heavy ions, clustering of damage including
DSB along the trajectory of the ion leads to further complexities
in relating foci counts to damage numbers [14]. However, since
total intensity of -H2AX was yet high at 4 h in carbon irradiated
cells, indicating its phosphorylated state, it may represent sites of
increased DNA damage after carbon irradiation, arising from misrepair
or incomplete repair [32,33].
Similar number of -H2AX foci at 15 min after both high and low
LET radiations indicated that initial events might not differ much
with the radiation quality. Activation of other molecules including
ATM, ATR, BRCA1, Chk1, Chk2 and Bax was therefore followed