LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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Mutation Research 723 (2011) 190–198 Contents lists available at ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis j o ur nal homep ag e: www.elsevier.com/locate/gentox Co mm unit y add re ss: www.elsevier.com/locate/mutres Activation of DNA damage response signaling in lung adenocarcinoma A549 cells following oxygen beam irradiation Somnath Ghosh a,∗ , Himanshi Narang a , Asiti Sarma b , Harminder Kaur b , Malini Krishna a a Radiation Biology and Health Science Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India b Radiation Biology Laboratory, Inter University Accelerator Centre, Aruna Asaf Ali Marg, New Delhi 110 067, India a r t i c l e i n f o Article history: Received 1 December 2010 Received in revised form 5 April 2011 Accepted 9 May 2011 Available online 14 May 2011 Keywords: Oxygen Gamma -H2AX ATM ATR a b s t r a c t Oxygen beams are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between - and oxygen ion-irradiation. Activation of various signaling molecules was looked in A549 lung adenocarcinoma cells irradiated with 2 Gy oxygen, 2 Gy or 6 Gy -radiation. Oxygen beam was found to be three times more cytotoxic than -radiation. By 4 h there was efficient repair of DNA in A549 cells exposed to 2 Gy or 6 Gy gamma radiation but not in cells exposed to 2 Gy oxygen beam as determined by -H2AX counting. Number of ATM foci was found to be significantly higher in cells exposed to 2 Gy oxygen beam. Percentage of cells showing ATR foci were more with gamma however number of foci per cell were more in case of oxygen beam. Oxygen beam irradiated cells showed phosphorylation of Chk1, Chk2 and p53. Many apoptotic nuclei were seen by DAPI staining in cells exposed to oxygen beam. The noteworthy finding of this study is the activation of the sensor proteins, ATM and ATR by oxygen irradiation and the significant activation of Chk1, Chk2 and p53 only in the oxygen beam irradiated cells. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Exposure of mammalian cells to ionizing radiation leads to the activation of several signaling pathways that result in apoptosis. Although, the end point, apoptosis is effectively activated by conventional X-ray treatment, the development of radio resistance following therapy remains a major cause for concern. Clinicians are now favoring charged particle therapy to avoid the issue [1–4]. However, the mechanism of cell killing by oxygen beam is not yet well delineated. High LET radiations like oxygen have an increased biological effectiveness compared to X-rays for gene mutation, genomic instability, and carcinogenesis [5]. This is because charged particle tracks show a high density of ionizing events along the center of particle path, resulting in spatially localized energy deposition within the cellular target. This high relative biological effectiveness has been imparted to difficult to repair clustered DNA damage that is generated in cells after high LET irradiation. Lung cancer is a frequently occurring, mostly lethal disease in all countries world wide [6]. It is one of the most commonly diagnosed types of cancer and has the highest death rates [7,8]. Overall, fewer than 10% of people with primary lung cancer are alive 5 ∗ Corresponding author. Tel.: +91 22 2559 0415; fax: +91 22 2550 5151. E-mail addresses: somnath@barc.gov.in, ghosh.barc@gmail.com (S. Ghosh). years after diagnosis. Depending on tumor size, location and histology, several treatment options are available, including surgery and radiotherapy (RT), both often combined with chemotherapy [9]. A major problem remains local tumor control [10,11]. Therefore, new ways to deliver radiotherapy beyond photons have been sought for, including oxygen and carbon ions [12]. Furthermore, treatment with charged particle radiation has several potential advantages over treatment with gamma or X-ray radiation such as the Bragg peak enables precise localization of the radiation dose, an inverted depth-dose distribution, a higher relative biological effectiveness (RBE), and a lower cellular capability for repair of radiation injury. Because of their superior dose-distribution, a therapeutic gain can be expected with charged particles [1–4]. Although charged particle therapies are currently being used for the treatment of many cancers, the mechanism of cell killing and its variance from gamma irradiation is not well understood. Understanding the specific biological effects of charged particle radiation on cancer cells could also provide valuable insights for the design of novel therapeutic applications for the treatment of cancers which are resistant to many types of therapies. Moreover, therapies can be designed only if the signaling pathway is understood. ATM (ataxia telangiectasia mutated), a protein kinase, is the major mediator of DSB responses. The kinase is activated by autophosphorylation when DSBs are formed and it phosphorylates a number of proteins involved in the repair and damage signaling pathways after exposure to ionizing radiation [13,14]. Many 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.05.002
S. Ghosh et al. / Mutation Research 723 (2011) 190–198 191 proteins such as ATM, ATR (ATM and Rad3-related) and H2AX are part of irradiation-induced foci (IRIF), which are at the core of DNA damage signaling apparatus [15–17]. These foci contain hundreds to thousands of proteins and are believed to represent sites with ongoing repair or to be an indication of a checkpoint mechanism. All these proteins form foci at different time points after irradiation. In this study we have looked at the foci at 4 h after irradiation. This time point was chosen since by this time most of the repairable damage is repaired and hence the foci formation is no longer observed if DNA repair has been completed. In this study, A549 cells were irradiated with either 2 Gy oxygen beam or 2 Gy and 6 Gy -radiation and ensuing activation of few important components involved in DNA repair, cell cycle arrest, and apoptosis pathways were followed to elucidate the mechanisms behind observed cellular response. n Fractio viving F Surv 1 0.1 0.01 γ ray Oxygen 2. Materials and methods 2.1. Cell culture Human A549 cells were cultured in Dulbecco’s modified eagles medium (Sigma) supplemented with 10% fetal calf serum (Sigma). Cells were maintained at 37 ◦ C in humidified atmosphere with 5% CO 2. Cells were seeded at density of 5 × 10 5 cells in small 35 mm petri plates [NUNC] 24 h before the irradiation. 1E-3 0 1 2 Dose (Gy) 3 2.2. Irradiation of cells For the experiments, exponentially growing A549 cells were irradiated with 60 Co gamma rays and 16 O ions from 15 UD Pelletron accelerator at the Inter University Accelerator Centre, New Delhi. During oxygen irradiation, 35 mm petri plates [NUNC] with cellular monolayers were kept perpendicular to the particle beam coming out of the exit window. The petri dishes were kept in the serum free medium in a plexiglass magazine during the irradiation. The computer controlled irradiation system is such that during the actual irradiation the dish was removed from medium. At the cell surface the energy of O ions was 55.8 MeV [LET = 614 keV/m]. Both the energy and LET were calculated using the Monte Carlo Code SRIM code [18,19]. The corresponding fluence for the dose of 2 Gy was 2.02 × 10 6 particles cm −2 , thus each cell nuclei can be expected to be physically traversed at least once by oxygen ion. Fluence was calculated using the relation given below [20], approximating the density of cellular matrix as 1.0: Dose [Gy] = 1.6 × 10 −9 × LET (keV/m) × particle fluence (cm −2 ) × 1 (cm3 /g) For gamma irradiation, cells were exposed to 1–3 and 6 Gy of 60 Co radiation in in-house facility Gamma Cell 220 [AECL, Canada] at dose rate of 3 Gy/min and same experimental conditions were kept as far as possible. In all experiments, controls were sham irradiated. Fig. 1. Clonogenic cell survival of A549 cells irradiated with 1, 2 or 3 Gy Oxygen beam or -radiation. Data represents means ± SD of three independent experiments. rabbit anti-pChk1 (Ser296), rabbit anti-pChk2 (Thr68) and mouse anti-phosphop53 (S15) (all primary antibodies were from Cell Signaling). Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG (Molecular Probe, USA). 2.5. Image analysis using ImageJ software The captured images were analyzed for relative quantification of phosphorylation using ImageJ software [22]. DAPI stained nucleus of each cell was selected and the same frame was used for relative quantification of phosphorylation using ImageJ software as described earlier [21]. All the foci were counted manually; at least 100 cells per experiment were analyzed from three independent experiments. 2.6. 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 significance level. Data were insignificant at P > 0.05. Each point represented as mean ± S.D. (standard deviation). 2.3. Clonogenic cell survival assay After irradiation, clonogenic assay was done as described earlier [21]. Briefly, after treatment, cells were seeded out in appropriate dilutions (500 cells/60 mm petriplate) to form colonies in 14 days. Colonies were fixed with glutaraldehyde (6.0%, v/v), stained with crystal violet (0.5%, w/v) and counted using a stereomicroscope. Colonies containing more than 50 cells were scored as survivors. Absolute plating efficiency at 0 Gy (sham irradiated control) was 32.3 ± 2.4%. 2.4. Immunofluorescence staining Unirradiated and irradiated cells were fixed at different time periods and immunofluorescence staining was done as described earlier [21]. Briefly, cells were grown in cover slips which were kept in 35 mm petri plates and irradiated as mentioned above. Cell layer were washed at different time period after irradiation in PBS and fixed for 20 min in 4% paraformaldehyde at room temperature. Afterwards, the cells were washed twice in PBS. For immunofluorescence staining, cells were permeabilized for 3 min in 0.25% Triton X-100 in PBS, washed two times in PBS and blocked for 1 h with 5% BSA in PBS. Antibodies were diluted (1:200) in 1% BSA in PBS. Cells were incubated with primary antibodies for 1 h 30 min at room temperature, washed three times in PBS and incubated with secondary antibodies for 1 h at room temperature. Finally, cells were rinsed and mounted with ProLong Gold antifede with DAPI mounting media (Molecular Probe, USA). Images were captured using Carl Zeiss confocal microscope. Acquisition settings were optimized to obtain maximal signal in immunostained cells with minimal background. The primary antibodies used were mouse anti-pATM (S1981), rabbit anti-pATR (Ser428), rabbit anti--H2AX (S139), 3. Results and discussion 3.1. Oxygen ions were three times more cytotoxic than gamma To determine the sensitivity of cells to similar dose of high and low LET radiation, their ability to form colonies after irradiation was observed. Irradiation of exponentially growing A549 cells with equal doses of gamma and oxygen beam (614 keV/m) revealed that at same dose oxygen ions were much more cytotoxic than gamma rays as assessed by the clonogenic cell survival assay. The calculated RBE (relative biological effectiveness) for A549 cells irradiated with 1 Gy oxygen ions particles relative to rays was found to be nearly 3 at 20% survival. Thus, oxygen ions were found to be three times more toxic than gamma rays (Fig. 1). The calculated D 0 value from the plot curve for A549 cells was found to be 0.6 Gy for oxygen ions and 2.5 Gy for rays. Our results therefore further support previous studies where high LET oxygen beam have been found to be much more efficient in killing the tumor cells and hence may be a preferred mode of treatment for radio resistant tumors. However, mechanisms of cell death caused by oxygen treatment and the signaling pathways that ensue have not yet been investi-
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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 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 [98] Liu, Y.;
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