LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

S. Ghosh et al. / Mutation Research 716 (2011) 10–19 13
Fig. 3. Phosphorylation of ATM at 4 h after exposure to 1 Gy -rays or carbon 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. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)
DNA damage response network that include H2AX, BRCA1, Chk1/2
and p53. Phospho ATM foci and intensity were scored at 4 h after -
rays or carbon irradiation. At 4 h after gamma irradiation, 88% cells
showed that pATM foci with the average foci per cell was 6.5 ± 4.4,
as against 25% control cells (3.4 ± 1.67) (Fig. 3). Like -H2AX foci,
small number of ATM foci 4 h after -rays irradiation indicates that
by 4 h most of the repair has taken place. However, at 4 h after carbon
ion irradiation only 52% of the cells showed the foci but the
average pATM foci per cell (21 ± 10) was much higher as compared
to 4 h after -rays (Fig. 3). Total intensity levels of phospho ATM
matched with the number of foci observed (Fig. 3C).
ATR, another important DNA damage sensor, which is known to
respond late to the IR induced damage, was also looked for its activation
at 4 h. After -rays irradiation, 53% of the cells showed ATR
foci (2.8 ± 1.64), as against no foci in any of the control-unirradiated
cells. On the other hand, 4 h after carbon ion irradiation although
only 10% of cells showed the foci, the average foci per cell (7 ± 4.5)
were thrice that of -rays irradiated cells (Fig. 4). Total intensity
levels of phospho ATR matched with the number of foci observed
(Fig. 4C).
3.4. BRCA1 foci
Another important protein involved in DSB repair is the tumor
suppressor protein BRCA1 which is phosphorylated by ATM and is
also a part of RIF. 4 h after -rays irradiation, 55% cells displayed
BRCA1 foci (13 ± 8) while 100% of carbon irradiated cells displayed
BRCA1 foci (7.5 ± 0.64) (Fig. 5) and the foci, although less in number
were larger in size than -ray induced foci. The intensity of
phosphorylation of BRCA1 as determined by ImageJ software was
higher in carbon irradiated cells (Fig. 5C). Presence of BRCA1 foci
in all cells irradiated after carbon irradiation indicates that BRCA1
might be the important player in response to complex DNA damage
induced by high LET radiation.
3.5. Activation of downstream effectors, Chk1 and Chk2
Activation of ATM, ATR and BRCA1 after DNA damage leads to
the activation of further downstream components including Chk1
and Chk2. After activation in the nuclei, Chk1 and Chk2 move to
cytoplasm. Phospho Chk1, the major target of ATR, was found to
increase in nuclei of carbon-irradiated cells but not in -rays irradiated
cells (Fig. 6A). Marginal activation of Chk2, the preferred
substrate of ATM, was observed in the nucleus in all the cells irrespective
of radiation quality (Fig. 6B).
3.6. Activation of intracellular MAPKs, ERK and JNK
ERK1/2 showed significant activation 1 h after -rays irradiation
and thereafter the increase was persistent till 4 h (Fig. 7A), while