LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

Cancer Invest Downloaded from informahealthcare.com by University of Chicago Library on 06/10/11
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Figure 4. Gene expression of Bax and Bcl-2 12 hr after irradiation
in A549 cells irradiated with 2 Gy proton beam or 2 Gy γ-radiation.
Total RNA from A549 cells was isolated and reverse transcribed.
RT-PCR analysis of Bax and Bcl-2 genes was carried out as described
in materials and methods. PCR products were resolved on
1.5% agarose gels containing ethidium bromide. β-actin gene expression
in each group was used as an internal control. Ratio of
intensities of Bax and Bcl-2 band to that of respective β-actin band
as quantified from gel pictures are shown above each gel picture.
Con means unirradiated control, H means proton. Data represents
means ± SE of three independent experiments, significantly different
from unirradiated controls. ∗ p < .05, ∗∗ p < .01.
DISCUSSION
More than two-fold decrease in survival of the A549 cells
as assessed by the clonogenic cell survival assay is indicative
of the fact that the proton beam is more efficient in killing the
tumor cells and may be a more preferred mode of treatment.
The mechanisms of cell death caused by proton treatment and
the signaling pathways that ensue have not yet been investigated
in detail although proton beam therapy is in use for many
cancers (1–6).
The massive activation of DNA-PK and ATM after proton
irradiation when compared with γ [Figures 2, (A) & (B)] was
more or less expected and may be due to the fact that DNA damage
activates phosphatidylinositol 3-kinase-like kinases (DNA-
PK, ATM), which then amplify and channel the signal by activating
downstream kinases (Chk1 and Chk2). These, in turn,
phosphorylate target proteins such as p53, Cdc25A, and Cdc25C
(25) to delay the cell cycle, a process called the DNA damage
checkpoint. In addition to checkpoint control, the downstream
kinases also modulate DNA repair and trigger apoptosis (26).
The lack of activation of ATR after proton irradiation was however
intriguing and was found to be reflecting no activation of
Chk1 (Figure 2).
The phosphorylation of H2AX was found to significantly
increase 4 hr after proton beam irradiation (Figure 3). This
time period of 4 hr was chosen for looking at the activation of
important components involved in repair because by 4 hr after
γ -irradiation most of the repair is complete and the activation of
these factors is reduced to control levels (27). Phosphorylation
of H2AX even at 4 hr indicates the presence of large number
of DSBs at that time indicating very slow repair. It has been
established recently that H2AX at the DNA DSB sites is immediately
phosphorylated upon irradiation, and the phosphorylated
H2AX (γ -H2AX) can be visualized in situ by immunostaining
with a γ -H2AX specific antibody (28, 29). γ -H2AX induction
can be measured quantitatively at physiological doses, and the
numbers of residual γ -H2AX foci can be used to estimate the
kinetics of DSB rejoining. This has become the gold standard
for the detection of DSB (17, 28).
Since ATM protein plays a central DNA DSBs sensing and
repair and determines the cellular response in eukaryotes it was
of interest to look for its phosphorylation at 4 hr after irradiation.
There was significantly higher phosphorylation of ATM at
serine 1981 in A549 cells, which had been exposed to 2 Gy of
proton beam (Figure 3). This indicates that despite significant
activation of ATM till 4 hr after irradiation much of the damage
still persisted as indicated by the presence of significant levels
of γ -H2AX. The activated/phosphorylated ATM further phosphorylates
various downstream target proteins such as H2AX,
Chk2, p53 leading to opening of the chromatin and recruitment
of other proteins involved in DNA repair or cell cycle arrest.
Chk2 which is a another substrate of ATM and is involved in
cell cycle arrest was also found to be phosphorylated at threonine
68 in A549 cells which had been exposed to 2 Gy of proton as
compared to controls (Figure 3), however the activation was not
as intense as observed with ATM or H2AX.
This indicates that small component of ATM activation is also
diverted to cell cycle arrest pathways involving Chk2, CDC25
phosphatase, and CDKs. Chk2 could also be involved in activation
of other proteins such as BRCA1 and p53. Phosphorylation
of p53 at serine 15 was then observed in unirradiated and irradiated
A549 cells at 2 Gy of proton beam (Figure 3).
Whether DNA-PK signals DSBs to the checkpoint machinery
remains controversial, but the emerging consensus is that it does
not (30). DNA-PK may, however, be involved in signalling DNA
damage to the apoptosis machinery (31).
The degree of radiation-induced apoptosis has been shown
to correlate with the p53 wild-type status (32). In addition,
apoptosis is induced when wild-type p53 is transfected into
certain cell lines lacking p53 (33). This indicates that p53 not
only plays a role in regulating the progression through the cell
cycle, it can also induce apoptosis in cells. p53 is not an essential
component of the machinery that carries out apoptosis; however,
620 S. Ghosh et al.