LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 3
RESULTS
3.3.2.5. Activation of downstream substrates of ATR and ATM: Chk1, Chk2 and p53
There was significantly higher phosphorylation of p53 at serine 15 in A549 cells that had been
exposed to 2Gy of oxygen beam, but not after 2Gy γ-radiation (Fig.3.3.2.5). Cells exposed to
6Gy γ-radiation showed marginally higher phosphorylation of p53, but it was significantly lower
than 2Gy oxygen irradiated cells (Fig.3.3.2.5).
Chk2, which is activated by ATM and shares its substrates including p53, was found to be
significantly activated in oxygen irradiated cells. Activation of Chk2 is critical in regulating cell
cycle arrest and DSB repair. Presence of pChk2 foci in nuclei of oxygen irradiated cells but not
gamma indicates Chk2 as an active player in high LET radiation induced responses
(Fig.3.3.2.6A, B). Immunofluorescence of phospho Chk1, which is a major target of ATR, was
also found to increase in nuclei of oxygen-irradiated cells but not after 2Gy γ-radiation
(Fig.3.3.2.6C). Cells exposed to 6Gy γ-radiation showed marginally higher phosphorylation of
Chk1, but it was significantly lower than 2Gy oxygen irradiated cells (Fig.3.3.2.6C).
3.3.2.6 Apoptosis
At 18 hours after irradiation, morphological features of DAPI stained apoptotic nuclei were
scored and many apoptotic nuclei were observed in cells irradiated with 2Gy oxygen ion and
6Gy gamma but not in 2Gy gamma irradiated cells (Fig.3.3.2.7). Cells exposed to 2Gy oxygen
ion showed significantly higher apoptotic nuclei than 6Gy gamma (Fig.3.3.2.7).
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