LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

SYNOPSIS
Comparison of carbon and oxygen beam irradiation induced signaling
The differences in activation of signaling components with respect to LET and fluence of
radiation can be very helpful in elucidating repair mechanisms in cells. Foci formation of
pH2AX, pATM and pATR was also compared between carbon and oxygen at 1 Gy,
which differ on the basis of LET by a factor of 2. The dose was kept constant since LET
of oxygen is double of carbon, the fluence of oxygen should be half that of carbon ions.
The probability is that with carbon irradiation one cell may be hit by 2.8 carbon ions and
with oxygen irradiation one cell may be hit by 1.3 oxygen ions. Thus the probability that
a cell is not being hit by oxygen increases the bystander responses with oxygen ion
irradiation. pH2AX foci were observed in 60% of the cells at 15 minutes indicating that
only 60% of the cells might have been hit by oxygen (Fig. 3.3I). However, presence of
significant number of pATM foci in 96% of the cells indicates activation of ATM in
bystander cells. pATR foci were also significantly high in oxygen irradiated cells and was
found in 36% of the cells unlike in carbon irradiated cells where only10% cells showed
the foci. This could indicate that complex damage may lead to increased activation of
ATR (Fig. 3.3I).
3.4 Radiation induced bystander signaling in mammalian cells.
To establish the contribution of the bystander effect in the survival of the
neighbouring cells, the lung carcinoma A549 cells were exposed to 2 Gy γ-irradiation.
The medium from irradiated cells was transferred to unirradiated cells. Irradiated A549
cells as well as those cultured in the presence of medium from γ-irradiated A 549 cells
showed decrease in survival, increase in γ-H2AX and pATM foci (Fig. 3.4A). Bystander
signaling was also found to exist between U937 cells (human monocyte) and Lung
carcinoma A549 cells. PMA stimulated and irradiated U937 cells induced bystander
response in unirradiated A549 cells. The latter showed a decrease in survival, increase in
γ-H2AX and pATM foci (Fig. 3.4B). The unstimulated and/or irradiated U937 cells did
not induce bystander effect in unirradiated A549 cells. The mechanism of such a cross
bystander effect was investigated in mouse cell line.
EL-4 and RAW 264.7 cells were exposed to 5 Gy γ-irradiation. The medium from
irradiated cells was transferred to unirradiated cells. Irradiated EL-4 cells as well as those
cultured in the presence of medium from γ-irradiated EL-4 cells showed an upregulation
of NF-κB, iNOS, p53 and p21/waf1 genes (Fig. 3.4C). The directly irradiated and the
bystander EL-4 cells showed an increase in NO production (Fig. 3.4D), DNA damage
(Fig. 3.4E) and apoptosis. Bystander signaling was also found to exist between RAW
264.7 (macrophage) and EL-4 (lymphoma) cells. Unstimulated and/or irradiated RAW
264.7 cells did not induce bystander effect in unirradiated EL-4 cells but LPS stimulated
and irradiated RAW 264.7 cells induced an upregulation of NF-κB and iNOS genes (Fig.
3.4F) and increased the DNA damage in bystander EL-4 cells. Treatment of EL-4 or
RAW 264.7 cells with L-NAME significantly reduced the induction of gene expression
and DNA damage in the bystander EL-4 cells while treatment with cPTIO only partially
reduced the induction of gene expression and DNA damage in the bystander EL-4 cells
(Fig. 3.4G). It was concluded that active iNOS in the irradiated cells was essential for
bystander response.
8