LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

196 S. Ghosh et al. / Mutation Research 723 (2011) 190–198
Fig. 6. Phosphorylation of Chk2 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-Chk2 4 h
after irradiation. Each 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. (B) Graph represents relative intensity of p-Chk2
as determined by ImageJ software. (C) Graph represents relative intensity of p-Chk1 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 < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
cells indicates its specific role in high LET induced complex damage.
3.4. Activation of downstream substrates of ATR and ATM: Chk1,
Chk2 and p53
Increased activation of ATM and ATR in irradiated cells led
us to investigate the activation of further downstream components
including p53, Chk1 and Chk2 which are actual effectors
of the cell cycle arrest or apoptosis [30,31]. There was significantly
higher phosphorylation of p53 at serine 15 in A549
cells that had been exposed to 2 Gy of oxygen beam, but
not after 2 Gy -radiation (Fig. 5). Cells exposed to 6 Gy -
radiation showed marginally higher phosphorylation of p53,
but it was significantly lower than 2 Gy oxygen irradiated
cells (Fig. 5). This along with survival data supports the activation
of p53 dependent apoptotic responses after high LET
radiation.