LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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192 S. <strong>Ghosh</strong> et al. / Mutation Research 723 (2011) 190–198 Fig. 2. Phosphorylation of H2AX (-H2AX) at different time points after exposure to 2 Gy and 6 Gy gamma or 2 Gy oxygen irradiation in A549 cells. Representative image 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; significantly different from unirradiated controls: *P < 0.05, **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) gated in detail although heavy ion beam therapy is in use for many cancers [1–4]. 3.2. Numbers of initial -H2AX foci formed were almost same for both types of radiation Since DNA double strand breaks are the primary toxic lesions induced by radiation, the number of DSBs were scored by examining the foci of phosphorylated H2AX in irradiated cells. It has been established recently that H2AX at the DNA DSB sites is immediately phosphorylated upon irradiation, and the phosphorylated H2AX (-H2AX) can be visualized in situ by immunostaining with a - H2AX specific antibody [23,24]. -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 [16,23]. After 15 min of either radiation, -H2AX foci, visualized as bright spots, were present in all the cells. The average numbers of foci after 15 min of 2 Gy gamma irradiation were 24 ± 5, while after 2 Gy oxygen irradiation; the foci were 28 ± 4.7 (Fig. 2(A and C)). Our result on -H2AX foci detected in cells irradiated with 2 Gy oxygen ion is contradictory to the fact that number of ion that hits on nucleus and number of -H2AX foci formed agrees well in heavy-ion irradiated cells, because heavy-ion densely deposits energy along their trajectories. Calculating from LET of oxygen ion, it is estimated that the number of ion hit on each cell nucleus by irradiation of 2 Gy oxygen ions was approximately 3 particles. However, nearly 10 times of foci were detected in the cells irradiated with 2 Gy of oxygen ions. This discrepancy between observed -H2AX foci and estimated -H2AX foci after high LET radiation has also been reported previously [25]. Thus unlike the cell survival, the difference in induction of -H2AX foci 15 min after 2 Gy of either radiation was not as profound. We looked at activation of signaling molecules at dose of 2 Gy of gamma radiation because the initial response manifested as double strand breaks, as indicated by -H2AX foci was found to be almost same at 2 Gy of either radiation. Since the RBE of oxygen ions was nearly 3, it would be more logical to compare 2 Gy of oxygen ion with 6 Gy of gamma ray. The average numbers of foci after 15 min of 6 Gy gamma irradiation were 35 ± 3.5. Thus, 6 Gy gamma ray induced 1.25 times more foci than 2 Gy oxygen ions. The cell irradiated with
S. <strong>Ghosh</strong> et al. / Mutation Research 723 (2011) 190–198 193 Fig. 3. Phosphorylation of ATM at 4 h after exposure to 2 Gy and 6 Gy gamma or 2 Gy oxygen 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; significantly different from unirradiated controls: *P < 0.05, **P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6 Gy of gamma-ray exhibited more -H2AX foci than 2 Gy gammaray sample. However the increased ratio is only 1.45, and far from 3. This result is contradictory to the fact that induction of DNA double strand break is physical process, not biological process. Thus it is expected to increase in proportion with irradiated physical dose if the source of radiation is same. We have used immunofluorescent staining assay for -H2AX, one of the limitation of this assay is the exposures of cells higher than 4 Gy result in foci saturation, reducing the useful range of the assay to doses less than 4 Gy. However, major advantage of this assay, it is more sensitive than other methods for detecting -H2AX [26]. Many downstream signaling molecules are known to get activated from these irradiation induced -H2AX foci [27]. The -H2AX foci were also counted 4 h after irradiation to compare the repair status at that time with respect to radiation quality. 53% of 2 Gy gamma irradiated cells still showed -H2AX foci but the average foci per cell had reduced to only 4.3 ± 2.2, where as 100% of 6 Gy gamma irradiated cells showed -H2AX foci and the average foci per cell had reduced to only 8 ± 2.5. In oxygen ion irradiated cells, although bright green -H2AX intensity was observed till 4 h in 60% of cells, there was a significant reduction in appearance of distinct foci like structures (10 ± 2) (Fig. 2(B and C)). Disappearance of -H2AX foci indicates DNA repair/rejoining. Instead of counting number of foci, the intensity of -H2AX in cells showing the foci was estimated by ImageJ software in the same images. In case of 2 Gy or 6 Gy gamma irradiation, both -H2AX foci and its total intensity levels had reduced at 4 h (Fig. 2(D)). However, in case of oxygen irradiation, the total intensity of -H2AX at 4 h was yet very high (Fig. 2(D)) though foci were not properly visible. This could indicate diffusion of -H2AX foci rather than its disappearance due to actual dephosphorylation of -H2AX. Presence of -H2AX even 4 h after oxygen ion irradiation suggests that DNA had not been repaired. Similar number of -H2AX at 15 min after irradiation irrespective of the radiation quality indicated that initial events might not differ much with the radiation quality. Activation of other molecules involved in DNA damage response was therefore fol-
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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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CHAPTER 5 REFERENCES [52] McKay, B.
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CHAPTER 5 REFERENCES [69] Cary, R.
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CHAPTER 5 REFERENCES [84] Uziel, T.
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CHAPTER 5 REFERENCES [98] Liu, Y.;
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CHAPTER 5 REFERENCES [111] Fei, P.;
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