LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

S. Ghosh, M. Krishna / Mutation Research 729 (2012) 61–72 63
Table 1
The sequences of gene specific primers and their annealing temperature (T m) used
in RT-PCR experiments.
Gene Primer sequence T m ( ◦ C)
-Actin F 5 ′ -TGGAATCCTGTGGCATCCATGAAA-3 ′ 55
R 5 ′ -TAAAACGCAGCTCAGTAACAGTCCG-3 ′
ATM F 5 ′ -GGACAGTGGAGGCACAAAAT-3 ′ 57
R 5 ′ -GTGTCGAAGACAGCTGGTGA-3 ′
DNA-PK F 5 ′ -TGCCAATCCAGCAGTCATTA-3 ′ 64
R 5 ′ -GGTCCATCAGGCACTTCACT-3 ′
Rad52 F 5 ′ -AGTTTTGGGAATGCACTTGG-3 ′ 50
R 5 ′ -TCGGCAGCTGTTGTATCTTG-3 ′
MLH1 F 5 ′ -GAGGTGAATTGGGACGAAGA-3 ′ 52
R 5 ′ -TCCAGGAGTTTGGAATGGAG-3 ′
CDKN1A (p21) F 5 ′ -CAGCAGAGGAAGACCATGTG-3 ′ 59
R 5 ′ -GGCGTTTGGAGTGGTAGAAA-3 ′
GADD45 F 5 ′ -TGCGAGAACGACATCAACAT-3 ′ 58
R 5 ′ -TCCCGGCAAAAACAAATAAG-3 ′
TLR3 F 5 ′ -GCCTCTTCGTAACTTGACCA-3 ′ 57
R 5 ′ -AAGGATGTGGAGGTGAGACA-3 ′
FDXR F 5 ′ -AGAGAACGGACATCACGAAG-3 ′ 57
R 5 ′ -GTCCTGGAGACCCAAGAAAT-3 ′
F, forward and R, reverse.
Percentage Survival
100
80
60
40
20
0
0Gy 2Gy 5X2Gy 10Gy
Radiation Dose
Fig. 1. Clonogenic cell survival of A549 cells irradiated with 2 Gy, 5 fractions of 2 Gy
or 10 Gy -radiation. Data represent means ± SE of three independent experiments;
*P < 0.05, **P < 0.01 compared with 10 Gy acute dose.
**
2.8. Semiquantitative reverse transcriptional polymerase chain reaction (RT-PCR)
Total RNA from 1 × 10 6 cells was extracted 4 h after irradiation and RT-PCR was
carried out as described earlier [44]. Primer sequence and annealing temperature of
specific primers are given in Table 1.
2.9. Transfection with ShRNA
MLH1, Rad52 and Control shRNA plasmids were procured from Santa cruz
biotechnology (Cat No. sc-35943-SH, sc-37399-SH and sc-108060 respectively). In
a six well tissue culture plate, A549 cells were grown to 50–70% confluency in
antibiotic-free normal growth medium supplemented with FBS and transfection
was carried out as per supplier’s instruction. 48 h post-transfection, medium was
aspirated and replaced with fresh medium containing puromycin (2 g/ml) for
selection of stably transfected cells. Semi-quantitative RT-PCR was performed to
monitor MLH1 and Rad52 gene expression knockdown using gene specific primers
(Table 1).
2.10. Statistical analysis
The data were imported to excel work sheets, and graphs were made using
Origin version 5.0. One-way ANOVA with Tukey–Kramer Multiple Comparisons as
post-test for P < 0.05 used to study the significant level. Data were insignificant at
P > 0.05. Each point represented as mean ± SE (standard error of the mean).
3. Results
3.1. Clonogenic cell survival
To determine the sensitivity of cells to different doses of radiation,
their ability to form colonies after irradiation was observed.
There was 60 ± 2.5% survival of A549 cells that had been exposed
to 2 Gy acute dose where as there was 48 ± 2.8% of cells survived
that had been exposed to 5 fractions of 2 Gy radiations and 4 ± 0.5%
of cells survived that had been exposed to 10 Gy acute dose (Fig. 1).
3.2. Human genome U-133 Plus 2.0 microarray data analysis
Global expression analysis of 54,675 probe set using Human
Genome U-133 Plus 2.0 microarray was performed in A549 cells.
On comparison of control vs 5X2 Gy (henceforth called fractionated
dose), 461 genes were identified as differentially expressed;
312 genes up-regulated (defined as a 2.0-fold or greater increase,
Supplementary data) and 149 down-regulated (defined as a 2.0-
fold or greater decrease, Supplementary data). Up-regulated genes
were associated with p53 signaling pathway, cytokine–cytokine
receptor interaction, hematopoietic cell lineage, Toll-like receptor
signaling pathway, Jak-STAT signaling pathway, MAPK signaling
pathway, cell-cycle check point, B cell receptor signaling pathway.
Down-regulated genes were associated with TGF-beta signaling
pathway and tyrosine metabolism.
In the second group (2 Gy vs 5X2 Gy), 234 genes were identified
as differentially expressed; 172 genes as up-regulated
(Supplementary data) and 62 as down-regulated (Supplementary
data). Genes involved in cytokine–cytokine receptor interaction,
toll-like receptor signaling pathway, hematopoietic cell lineage,
Jak-STAT signaling pathway, MAPK signaling pathway were upregulated.
Gene involved in folate biosynthesis, glycine, serine and
threonine metabolisms were down-regulated.
In the 10 Gy vs fractionated group, 289 genes were identified
as differentially expressed; 211 genes as up-regulated
(Supplementary data) and 78 as down-regulated (Supplementary
data). Genes involved in cytokine–cytokine receptor interaction,
toll-like receptor signaling pathway, cell cycle, DNA replication,
mismatch repair, homologous recombination were up-regulated.
Genes involved in regulation of autophagy, base excision repair,
folate biosynthesis were down-regulated.
Cluster analysis of the expression of these genes showed that
the fractionated group differed most in gene expression compared
to the other three groups (control, 2 Gy or 10 Gy) (Supplementary
data, TCS2 means 2 Gy treatment group, TCS3 means fractionated
group, TCS4 means 10 Gy treatment group).
3.3. Validation of microarray data
In order to verify the microarray data, semi-quantitative RT-PCR
was performed for four genes from control and fractionated group
of A549 cells. These results corresponded very well with those for
the microarray data for all four genes, strongly supporting the reliability
and rationale of our strategy (Fig. 2A and B).
3.4. Activation of repair related genes
When compared to the controls there was a significant upregulation
of ATM, DNA-PK, Rad52 and MLH1 genes in A549 cells
that had been exposed to fractionated irradiation (Fig. 3A–D, Lane
3). However, in A549 cells that had been exposed to 10 Gy acute
dose there was marginal up-regulation of ATM, DNA-PK and Rad52
genes although it was significantly lower than the fractionated
group (Fig. 3A–D, Lane 4). In A549 cells which had been exposed
to 2 Gy acute dose there was marginal up-regulation of Rad52 and