LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

16 S. Ghosh et al. / Mutation Research 716 (2011) 10–19
Fig. 6. Phosphorylation of Chk1 and Chk2 at 4 h after exposure to 1 Gy -rays or carbon irradiation in A549 cells. (A) Representative image showing p-Chk1 4 h after
irradiation. Graph represents relative phosphorylation of Chk1 as determined by ImageJ software. (B) Representative image showing p-Chk2 4 h after irradiation. Graph
represents relative phosphorylation of Chk2 as determined by ImageJ software. Each phospho-Chk1 or phospho-Chk2 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. 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.)
high LET ions. Presence of ATM foci in only half of carbon irradiated
cells while BRCA1 foci in all the cells also suggest that BRCA1
is associated with different proteins and may not require activated
ATM. Further study on co-localization experiment for BRCA1 with
ATM by immunofluorescence would provide better understanding
of repair responses elicited by low and high LET radiations. Number
of Chk2 foci, another important regulator of HRR, also did not
seem to parallel ATM foci. This, along with the fact that the survival
of the cells is only 20% (Fig. 1) and many apoptotic nuclei are
present at 4 h (Fig. 8) indicates that these macromolecular complexes
containing BRCA1 may be involved in apoptotic responses
after a complex damage to the DNA. Recent studies suggest multifunctional
nature of BRCA1 in maintaining genome integrity. It has
been shown to play an important role in apoptosis and cell cycle
control and does so by associating in distinct protein complexes
[37]. However, further studies on BRCA1 and apoptosis again need
to be experimentally addressed.
The cytoplasmic MAPK pathways, ERK and JNK, that feed into
and are fed upon by DNA damage repair signaling also play an
equally important role in deciding the fate of the irradiated cell.
Reactive oxygen species generated after -rays irradiation has been
thought to be one of the mechanisms for early activation of these
kinases [18]. ATM has been shown to enhance repair via activation
of ERK pathway [35] and inhibition of JNK [21]. Observed activation
of ERK (Fig. 7A) but not JNK (Fig. 7B) in -rays irradiated cells
indicated the dominance of pro-survival pathways.