LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

SYNOPSIS
ATM gene expression, which was found to significantly upregulated in A 549 cell line as
compared to the MCF-7 (Fig. 3.1B). DNA-PK gene was also significantly upregulated in
A 549 cell line which had been exposed to 5 fraction of 2 Gy γ-radiation (Fig. 3.1C). As a
consequence of this the Phospho-p53 was found to be translocated to the nucleus of A
549 cell line which had been exposed to fractionated regimen. The expression of
Phospho- p53 was confirmed by western blotting (Fig. 3.1D).
The fractionated dose regimen led to a significant upregulation of ATM and DNA-PK in
A549 cells line. The differences in the two cell lines (A549 and MCF-7) could be
summarized as significant differences in the repair capability of the two. Significant
activation of BRCA1, phospho ATM and Rad52 seems to be causative factors in A549
cells. None of this activation was apparent in MCF-7 (Fig. 3.1E).
The activation of DNA-PK was accompanied by the stabilization of its mRNA and was
regulated by the p38 MAP Kinase pathway but not by ERK or JNK MAP Kinase
pathway (Fig. 3.1F). The ATM and Rad 52 mRNA were not stabilized indicating a
specific and selective stabilization of DNA-PK mRNA. Finally the inhibition of Rad52
with its ShRNA reversed the radioresistance of the A549 cells and made it sensitive to
fractionated irradiation (Fig. 3.1G).
3.2 Proton beam induced signaling in mammalian cells and its comparison with
Gamma irradiation.
Having established that A549 cells are relatively more radioresistant. The
variance in signaling pattern and effectiveness of cell killing with the change in LET was
looked at. A549 Lung Adenocarcinoma cells were irradiated with 2 Gy proton beam or γ-
radiation. Proton beam was found to be more cytotoxic than γ-radiation (Fig. 3.2A).
Proton beam irradiated cells showed phosphorylation of H2AX, ATM, Chk2 and p53
(Fig. 3.2B). The mechanism of excessive cell killing in proton beam irradiated cells was
found to be upregulation of Bax and down-regulation of Bcl-2 (Fig. 3.2C). The
noteworthy finding of this study is the biphasic activation of the sensor proteins, ATM
and DNA-PK and no activation of ATR by proton irradiation (Fig. 3.2D).
3.3 High LET irradiation induced signaling in mammalian cells and its comparison
with Gamma irradiation.
After establishing the signaling differences between proton beam and gamma ray in lung
carcinoma cells. The variance in signaling pattern with higher LET (carbon and oxygen)
was studied.
Lung carcinoma cell line A549 were irradiated with 1 or 2 Gy doses of carbon ions,
oxygen ions or gamma rays and ensuing activation of few important components
involved in DNA repair, cell cycle arrest, apoptosis and survival pathways were
followed.
Expectedly the carbon beam was found to be three times more cytotoxic than γ-radiation
despite the fact that the number of H2AX foci was the same with both the radiations. The
repair, although activated in carbon beam-irradiated cells, was not effective in reviving
the cells. The carbon beam irradiated cells showed increased phosphorylation of primary
regulators of Homologous Recombination Repair (HRR) pathway i.e. H2AX, ATM,
BRCA1 and Chk2 as compared to gamma irradiation (Fig. 3.3). The noteworthy finding
of this study was the biphasic activation of the pH2AX and the unusually large foci of
BRCA1. Apoptosis was found to be p53 independent.
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