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S. <strong>Ghosh</strong>, M. Krishna / Mutation Research 729 (2012) 61–72 69 Fig. 7. Response of A549 and MCF-7 cells exposed to fractionated irradiation. (A) Clonogenic cell survival of A549 and MCF-7 cells exposed to fractionated irradiation. Data represent means ± SE of three independent experiments; **P < 0.01 compared with MCF-7. (B) Representative image of three independent experiments showing gene expression of ATM, DNA-PK, Rad52 and MLH1 in A549 and MCF-7 cells exposed to fractionated irradiation. -Actin gene expression in each group was used as an internal control. (C) Phosphorylation of ATM, BRCA1 and p53 in A549 and MCF-7 cells exposed to fractionated irradiation. (D) Repair kinetics as determined by -H2AX foci in A549 and MCF-7 cells exposed to fractionated irradiation at 15 min and 4 h after irradiation. Each phospho-site antibody was indirectly labeled with Molecular Probe 488 secondary antibody (green) and cells were mounted with ProLong Gold antifede with DAPI (blue). The encircled area roughly represents cell nuclei as seen by DAPI staining. All the images were taken using a 100× objective lens except the images of p53 phosphorylation. The images of p53 phosphorylation were taken using 40× objective lens for A549 cells and MCF-7 cells. All images were captured using Carl Zeiss confocal microscope with the same exposure time. Representative images of three independent experiments are shown here; at least 100 cells per experiment were analyzed from three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
70 S. <strong>Ghosh</strong>, 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
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RADIATION INDUCED SIGNALING IN MAMM
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DECLARATION I, hereby declare that
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CONTENTS Page No. SYNOPSIS………
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2.11 Measurement of NO production
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PREAMBLE SYNOPSIS Ionising radiatio
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SYNOPSIS Aims and Objectives: To St
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SYNOPSIS 2.6 Immunofluorescence sta
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SYNOPSIS ATM gene expression, which
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SYNOPSIS Percentage Survival 100 80
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SYNOPSIS Ratio of Rad52 to Actin Ra
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Relative Phosphorylation 45 40 35 3
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SYNOPSIS Fig. 3.3A Clonogenic Cell
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SYNOPSIS (A) Av No. of phospho BRCA
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SYNOPSIS Percentage Survival 120 10
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SYNOPSIS Fig.3.4B. Existance of cro
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SYNOPSIS Fig. 3.4EDNA damage in EL-
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SYNOPSIS subsequent cytotoxicity ca
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SYNOPSIS [4] Hall, E. J. Radiobiolo
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CHAPTER 1 INTRODUCTION INTRODUCTION
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CHAPTER 1 INTRODUCTION 15.8% 9.56%
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CHAPTER 1 INTRODUCTION far apart an
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CHAPTER 1 INTRODUCTION and are more
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CHAPTER 1 INTRODUCTION 1.3 DNA dama
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CHAPTER 1 INTRODUCTION associates w
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CHAPTER 1 INTRODUCTION are unable t
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CHAPTER 1 INTRODUCTION Perhaps one
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CHAPTER 1 INTRODUCTION occurs at DS
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CHAPTER 1 INTRODUCTION related prot
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CHAPTER 1 INTRODUCTION damage must
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CHAPTER 1 INTRODUCTION At the prese
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CHAPTER 1 INTRODUCTION predominant
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CHAPTER 1 INTRODUCTION provide a me
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CHAPTER 1 INTRODUCTION hypersensiti
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CHAPTER 1 INTRODUCTION cycle-specif
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CHAPTER 1 INTRODUCTION 1.4 Overview
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CHAPTER 1 INTRODUCTION 1.4.1 Radiat
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CHAPTER 1 INTRODUCTION interaction
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CHAPTER 1 INTRODUCTION p90S6 kinase
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CHAPTER 1 INTRODUCTION CD4 (formerl
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CHAPTER 1 INTRODUCTION unrepaired d
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CHAPTER 1 INTRODUCTION well to the
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CHAPTER 1 INTRODUCTION 1.6 Radiatio
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CHAPTER 1 INTRODUCTION becomes pote
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CHAPTER 2 MATERIALS AND METHODS MAT
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CHAPTER 2 MATERIALS AND METHODS 2.2
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CHAPTER 2 MATERIALS AND METHODS res
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CHAPTER 2 MATERIALS AND METHODS Arr
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CHAPTER 2 MATERIALS AND METHODS PBS
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CHAPTER 2 MATERIALS AND METHODS the
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CHAPTER 2 MATERIALS AND METHODS 2.1
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CHAPTER 3 RESULTS RESULTS 93
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CHAPTER 3 RESULTS fractionated regi
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CHAPTER 3 RESULTS On comparison of
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CHAPTER 3 RESULTS Table 3.1.2A List
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CHAPTER 3 RESULTS 80 NM_002185 IL7R
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CHAPTER 3 RESULTS SAA2 149 AU134977
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CHAPTER 3 RESULTS 221 LOC643167 RNA
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CHAPTER 3 RESULTS 299 BF244402 FBXO
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CHAPTER 3 RESULTS CDK4) 57 AU152107
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CHAPTER 3 RESULTS 134 AL045793 METT
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CHAPTER 3 RESULTS 58 AF237813 ABAT
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CHAPTER 3 RESULTS 127 AI809749 ORMD
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CHAPTER 3 RESULTS 31 BE615699 UNC84
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CHAPTER 3 RESULTS 47 AF026303 SULT1
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CHAPTER 3 RESULTS 117 BF062193 CYor
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CHAPTER 3 RESULTS 187 AL036662 ---
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CHAPTER 3 RESULTS 52 AV686514 EMP2
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CHAPTER 3 RESULTS exposed to 10 Gy
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CHAPTER 3 RESULTS Fig.3.1.4 Gene ex
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CHAPTER 3 RESULTS Fig. 3.1.6 Radiat
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CHAPTER 3 RESULTS Fig. 3.1.7 Transl
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CHAPTER 3 RESULTS 3.1.9 Stabilizati
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CHAPTER 3 RESULTS 23±1.5% (Fig. 3.
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CHAPTER 3 RESULTS whereas only the
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CHAPTER 3 RESULTS with equal doses
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CHAPTER 3 RESULTS Fig.3.3.1.2 Forma
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CHAPTER 3 RESULTS Fig.3.3.1.3 Phosp
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CHAPTER 3 RESULTS (7.5±0.64) (Fig.
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CHAPTER 3 RESULTS Fig.3.3.1.7b Phos
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CHAPTER 3 RESULTS 3.3.2 Oxygen beam
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CHAPTER 3 RESULTS in all the cells.
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CHAPTER 3 RESULTS 3.3.2.3 Radiation
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CHAPTER 3 RESULTS Fig.3.3.2.7 Apopt
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CHAPTER 3 RESULTS PMA stimulated U9
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CHAPTER 3 RESULTS Fig.3.4.2. Exista
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CHAPTER 3 RESULTS up-regulation of
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CHAPTER 3 RESULTS Fig.3.4.3.2 NO pr
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CHAPTER 3 RESULTS 3.4.3.4 Apoptotic
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CHAPTER 3 RESULTS 3.4.4 Bystander e
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CHAPTER 3 RESULTS Fig.3.4.4b DNA da
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CHAPTER 3 RESULTS Fig.3.4.5a Gene e
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CHAPTER 4 DISCUSSION DISCUSSION 185
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CHAPTER 4 DISCUSSION directly expos
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CHAPTER 4 DISCUSSION dose is delive
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CHAPTER 4 DISCUSSION A clear cause
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CHAPTER 4 DISCUSSION 4.2 Proton bea
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CHAPTER 4 DISCUSSION Although the e
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CHAPTER 4 DISCUSSION Similar number
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CHAPTER 4 DISCUSSION the mechanism
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CHAPTER 4 DISCUSSION Thus unlike th
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CHAPTER 4 DISCUSSION different from
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CHAPTER 4 DISCUSSION upregulates ma
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CHAPTER 4 DISCUSSION Numerous studi
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CHAPTER 4 DISCUSSION inhibition of
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CHAPTER 4 DISCUSSION The survival o
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