LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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12 S. <strong>Ghosh</strong> et al. / Mutation Research 716 (2011) 10–19 Fig. 2. Phosphorylation of H2AX (-H2AX) at different time points after exposure to 1 Gy -rays or carbon irradiation in A549 cells. Representative images showing -H2AX foci (A) 15 min and (B) 4 h after irradiation. Each phospho-H2AX antibody was indirectly labeled with Molecular Probe 488 secondary antibody (green) and cells were mounted with ProLong Gold antifede with DAPI (blue). All images were captured using Carl Zeiss confocal microscope with the same exposure time. (C) Graph represents average numbers of foci per cell, percentage of cells showing the foci is marked above the bars. (D) Graph represents relative intensity of -H2AX as determined by ImageJ software. At least 100 cells per experiment were analyzed from three independent experiments. Data represents means ± SD of 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.) bon irradiation, the total intensity of -H2AX at 4 h was yet high (Fig. 2D). Since initial response as observed from -H2AX foci at 15 min was similar with both types of radiation, irradiation induced foci of downstream signaling components was followed at 4 h to highlight the signaling differences at that time period. 3.3. ATM/ATR foci in gamma and carbon irradiated cells ATM, an important DNA damage response protein to ionizing radiation and a part of radiation induced foci (RIF), is mainly involved in cell cycle arrest and repair. In response to DSBs, ATM is activated and phosphorylates key players in various branches of the
S. <strong>Ghosh</strong> et al. / Mutation Research 716 (2011) 10–19 13 Fig. 3. Phosphorylation of ATM at 4 h after exposure to 1 Gy -rays or carbon irradiation in A549 cells. (A) Representative image showing p-ATM foci 4 h after irradiation. Each phospho-ATM antibody was indirectly labeled with Molecular Probe 488 secondary antibody (green) and cells were mounted with ProLong Gold antifede with DAPI (blue). All images were captured using Carl Zeiss confocal microscope with the same exposure time. (B) Graph represents average numbers of foci per cell, percentage of cells showing the foci is marked above the bars. (C) Graph represents relative intensity of p-ATM as determined by ImageJ software. At least 100 cells per experiment were analyzed from three independent experiments. Data represents means ± SD of 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.) DNA damage response network that include H2AX, BRCA1, Chk1/2 and p53. Phospho ATM foci and intensity were scored at 4 h after - rays or carbon irradiation. At 4 h after gamma irradiation, 88% cells showed that pATM foci with the average foci per cell was 6.5 ± 4.4, as against 25% control cells (3.4 ± 1.67) (Fig. 3). Like -H2AX foci, small number of ATM foci 4 h after -rays irradiation indicates that by 4 h most of the repair has taken place. However, at 4 h after carbon ion irradiation only 52% of the cells showed the foci but the average pATM foci per cell (21 ± 10) was much higher as compared to 4 h after -rays (Fig. 3). Total intensity levels of phospho ATM matched with the number of foci observed (Fig. 3C). ATR, another important DNA damage sensor, which is known to respond late to the IR induced damage, was also looked for its activation at 4 h. After -rays irradiation, 53% of the cells showed ATR foci (2.8 ± 1.64), as against no foci in any of the control-unirradiated cells. On the other hand, 4 h after carbon ion irradiation although only 10% of cells showed the foci, the average foci per cell (7 ± 4.5) were thrice that of -rays irradiated cells (Fig. 4). Total intensity levels of phospho ATR matched with the number of foci observed (Fig. 4C). 3.4. BRCA1 foci Another important protein involved in DSB repair is the tumor suppressor protein BRCA1 which is phosphorylated by ATM and is also a part of RIF. 4 h after -rays irradiation, 55% cells displayed BRCA1 foci (13 ± 8) while 100% of carbon irradiated cells displayed BRCA1 foci (7.5 ± 0.64) (Fig. 5) and the foci, although less in number were larger in size than -ray induced foci. The intensity of phosphorylation of BRCA1 as determined by ImageJ software was higher in carbon irradiated cells (Fig. 5C). Presence of BRCA1 foci in all cells irradiated after carbon irradiation indicates that BRCA1 might be the important player in response to complex DNA damage induced by high LET radiation. 3.5. Activation of downstream effectors, Chk1 and Chk2 Activation of ATM, ATR and BRCA1 after DNA damage leads to the activation of further downstream components including Chk1 and Chk2. After activation in the nuclei, Chk1 and Chk2 move to cytoplasm. Phospho Chk1, the major target of ATR, was found to increase in nuclei of carbon-irradiated cells but not in -rays irradiated cells (Fig. 6A). Marginal activation of Chk2, the preferred substrate of ATM, was observed in the nucleus in all the cells irrespective of radiation quality (Fig. 6B). 3.6. Activation of intracellular MAPKs, ERK and JNK ERK1/2 showed significant activation 1 h after -rays irradiation and thereafter the increase was persistent till 4 h (Fig. 7A), while
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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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CHAPTER 5 REFERENCES [1] Khan, F. T
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CHAPTER 5 REFERENCES [19] Woo, R. A
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CHAPTER 5 REFERENCES [35] Nghiem, P
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