LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

70 S. Ghosh, M. Krishna / Mutation Research 729 (2012) 61–72
Fig. 8. Effect of inhibition of Rad52 and MLH1 on the growth and radioresistance of
A549 cells which had been exposed to 5 fractions of 2 Gy radiation. A549 cells were
transfected with either MLH1 or Rad52 ShRNA as described in Section 2. (A) Transfection
efficiency as determined by semi-quantitative RT-PCR. Gene expression of
Rad52 and MLH1 in A549 cells transfected with Control ShRNA, Rad52 ShRNA or
MLH1 ShRNA. Total RNA from A549 cells was isolated and reverse transcribed 7
days after transfection. RT-PCR analysis of Rad52 and MLH1 genes were carried out
as described in Section 2. PCR products were resolved on 1.5% agarose gels containing
ethidium bromide. -Actin gene expression in each group was used as an internal
control. Ratio of intensities of Rad52 or MLH1 band to that of respective -actin band
as quantified from gel pictures are shown above each gel picture. Key: Rad52, Gene
expression of Rad52 to that of -actin in A549 cells transfected with control ShRNA;
−ve Rad52, gene expression of Rad52 to that of -actin in A549 cells transfected
with Rad52 ShRNA; MLH1, gene expression of MHL1 to that of -actin in A549 cells
transfected with control ShRNA; −ve MLH1, gene expression of MLH1 to that of -
actin in A549 cells transfected with MLH1 ShRNA. (B) Clonogenic survival assay of
A549 cells. Key: control, control unirradiated A549 cells; 5X2 Gy, A549 cells exposed
to 2 Gy of 60Co -irradiation daily over a period of 5 days; 5X2 Gy + M, A549 cells
transfected with MLH1 ShRNA and exposed to 2 Gy of 60 Co -irradiation daily over a
period of 5 days; 5X2 Gy + R, A549 cells transfected with Rad52 ShRNA and exposed
to 2 Gy of 60 Co -irradiation daily over a period of 5 days. (C) Representative image
of three independent experiments. Data represent means ± SE of three independent
experiments; *P < 0.05, **P < 0.01 compared with 5X2 Gy treatment group.
irradiation; it was our interest to look at the phosphorylation of
p53 and its translocation to the nucleus of A549 cells. Lung adenocarcinoma
A549 cells typically express wild-type p53 protein
[51] and would be expected to be sensitive to the DNA-damaging
agents used for cancer therapy; however, these cells are radioresistant
if the radiation dose is delivered as fractionated irradiation.
p53 classically considered to be a tumor suppressor protein, but in
this study the results are contradictory. Even though the p53 levels
are up-regulated following fractionated doses (Fig. 6A and B), the
apoptotic function of the p53 appears to be compromised. p53 is
considered to be a classic “gatekeeper” of cellular fate [52,53]. p53
is activated in response to genotoxic stress such as radiation. And
once activated, it initiates cell cycle arrest, senescence or apoptosis
via pathways involving the transactivation of p53 target genes
[52,53]. There was no change in the expression of pro apoptotic
targets of p53 such as Bax, Perp, Puma and Noxa in the cells that
had been exposed to fractionated irradiation as compared to control
cells (Microarray data in Supplementary Files). However, there
was higher expression of cell cycle target genes of p53 such as p21
and GADD45 in the cells that had been exposed to fractionated
irradiation as compared to control cells (Fig. 2).
Since DNA repair pathways were up-regulated in A549 cells that
had been exposed to fractionated irradiation, it was of interest to
look at the activation of genes and proteins involved in DNA repair
pathways. Our results indicate that there was intense activation of
repair genes and protein in A549 cells exposed to fractionated irradiation
(Figs. 3 and 5). Our results on DNA-PK are consistent with
earlier works where authors have also shown the role of DNA-PK in
the radioresistance of lung carcinoma cells [54,55]. However, these
authors have looked at the response only after single dose of irradiation
and it is logical to expect these pathways to get activated.
In this study we reiterate the fact that DNA-PK is also involved in
radioresistance if the cells are subjected to fractionated irradiation
but the reason for the development of radioresistance still remains
an enigma.
ATM and BRCA1 have been regarded as primary regulators of
homologous recombination repair (HRR) [56]. In this study there
was intense activation of ATM gene and ATM foci were seen in all
the cells exposed to fractionated irradiation and most of the cells
showed BRCA1 foci (Figs. 3 and 5) indicating the fact that HRR pathway
was activated in A549 cells and therefore, these proteins could
be involved in HRR which can take advantage of the other strand
that may be intact. ATM and BRCA1 reside in macromolecular complex
and form foci after irradiation. Our results are in agreement
with earlier works where authors have also shown the role ATM
in radioresistance of cancer cells [57,58]. However, these authors
have studied using single dose of irradiation.
By 4 h there was an efficient repair of DNA in A549 cells exposed
to fractionated irradiation but not in cells that had been exposed to
10 Gy acute dose as determined by the disappearance of -H2AX
foci (Fig. 4). It has been established recently that H2AX at the DNA
DSB sites are immediately phosphorylated upon irradiation, and
the phosphorylated H2AX (-H2AX) can be visualized in situ by
immunostaining with a -H2AX specific antibody [59,60]. -H2AX
induction can be measured quantitatively at physiological doses,
and the numbers of residual -H2AX foci can be used to estimate
the kinetics of DSB rejoining. This has become the gold standard for
the detection of DSB [60,61].
A clear cause and effect relationship was established between
DNA damage signaling, gene expression and efficient DNA repair
and was evident in the form of cell survival in A549 cells that had
been exposed to fractionated irradiation.
It has been reported that A549 cells are relatively more
radioresistant than MCF-7 cells if radiation dose was delivered as
fractionated regimen [62]. But the mechanism of such response has
not been studied. To the authors’ knowledge this is the first report