LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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Mutation Research 716 (2011) 10–19 Contents lists available at ScienceDirect Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis jou rnal h omepa g e: www.elsevier.com/locate/molmut Co mm unit y ad d ress: www.elsevier.com/locate/mutres DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation Somnath Ghosh a , Himanshi Narang a,∗ , Asitikantha Sarma b , Malini Krishna a a Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, 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 24 March 2011 Received in revised form 22 July 2011 Accepted 26 July 2011 Available online 3 August 2011 Key words: Carbon -Rays -H2AX ATM BRCA1 a b s t r a c t Carbon beams (5.16 MeV/u, LET = 290 keV/m) 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 -rays and carbon ion-irradiation. A549 cells were irradiated with 1 Gy carbon or -rays. Carbon beam was found to be three times more cytotoxic than -rays despite the fact that the numbers of -H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with -rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike -rays irradiated cells and prosurvival ERK pathway was activated after -rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Biological consequences of high-energy charged particle exposure are of crucial importance in the clinic, space exploration and risk assessments [1]. Track structure of charged-particle radiation is known to be a critical determinant of its biological effectiveness [2–4]. Energy deposition by HZE ions is highly heterogeneous, with a localized contribution along the trajectory of every particle and lateral diffusion of energetic electrons (i.e., -rays, the target atom electrons ionized by the incident HZE ion and emitted at high energy) many microns from the path of the ions. These particles are therefore densely ionizing (high-LET) along the primary track (e.g., the track followed by the incident heavy ion); and also they have a low-LET component because of the high-energy electrons ejected by ions as they traverse tissue. Biophysical models have shown that energy-deposition events by high-LET radiation produce different DNA lesions, including complex DNA breaks, and that qualitative difference between high-LET radiation and low- LET radiation affects both the induction and repair of DNA damage [4–8]. Because of the effective cell killing many clinicians now favor charged particle beam therapy over conventional photons for tumors that are radio resistant and when the survival of nearby ∗ Corresponding author. Tel.: +91 22 2559 5310; fax: +91 22 2550 5151. E-mail address: himinarang@gmail.com (H. Narang). cells cannot be compromised e.g. in treatment of lung cancer [9,10]. Although charged particle beams like carbon etc. are currently being used for the treatment, the signaling pathways activated and their variance from -rays irradiation is not well understood. DNA, the main target for radiation induced cell killing, upon damage, triggers a signaling network that senses different types of damage and coordinates responses which include activation of transcription, cell cycle control, apoptosis, senescence, and DNA repair processes [11]. Hundreds of proteins are recruited in time dependent manner in the vicinity of a DSB leading to formation of a signaling-repair complex which can be visualized as foci and are therefore termed as radiation induced foci (RIF). One of the components of RIF is -H2AX that is immediately phosphorylated upon irradiation, can be visualized in situ by immunostaining with a -H2AX specific antibody and measured quantitatively [12–14]. Mathematical methods has been developed to analyze flow cytometry data to describe the kinetics of DNA damage response protein activation after exposure to X-rays and high energy Iron nuclei [15]. Other proteins like ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related) and BRCA1, also function in complexes that can be seen as foci. There are reports that ATM responds differently to DSBs induced by high-LET and low-LET radiations [16,17]. The signal is transduced downstream by proteins such as Chk1/Chk2 and p53 that trigger off DNA repair, cycle arrest or apoptosis. The cytoplasmic MAPK pathways, namely ERK and JNK, have also been linked to the DNA damage response and ATM-mediated signaling 0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2011.07.015
S. Ghosh et al. / Mutation Research 716 (2011) 10–19 11 events [18]. ERK1/2 signaling has been shown to be a positive regulator of homologous recombination repair (HRR) [19] and has also been shown to be activated by ATM [20]. JNK on the other hand has been shown to be inhibited by ATM [21] and plays a role in radiation induced apoptosis. These cytoplasmic signaling pathways coordinate with DNA damage activated pathways and summation of all the pathways decides the ultimate fate of the cell [18]. Many studies on signaling pathways after low and high LET radiations have found the activation to be similar except in the intensity [22] but since the end result is different, there must be a divergence of pathways at some stage. It is this stage of signaling, that is different, that will be crucial to designing therapies that target specific molecules or pathways. The aim of the present study was to look for these subtle differences that are of important consequences. A549, a lung carcinoma cell line was chosen as lung cancer is one of the most commonly diagnosed types of cancer with the highest death rates [23] and high LET ions pose a therapeutic advantage over conventional photons for treatment of non-small-cell lung cancer (NSCLC) [10]. 2. Methods 2.1. Cell culture Human Lung Adenocarcinoma, A549 cells were cultured and seeded as described earlier [24]. 2.2. Irradiation of cells For the experiments plateau phase A549 cells were irradiated with 60 Co -rays and 12 C ions from 15 UD Pelletron accelerator at the Inter University Accelerator Centre, New Delhi. During carbon 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. Control cells were sham irradiated in similar way. At the cell surface the energy of C ions was 62 MeV [5.16 MeV/u, LET = 290 keV/ m]. Both the energy and LET were calculated using the Monte Carlo Code SRIM code [25]. The corresponding fluence for the dose of 1 Gy was 2.2 × 10 6 particles cm −2 , thus each cell nuclei can be expected to be physically traversed at least once by carbon ion. Fluence was calculated using the relation given below, 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, 2 and 3 Gy of 60 Co -rays 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. 2.3. Clonogenic cell survival assay 2.6. Image analysis using ImageJ software The captured images were analyzed for relative quantification of phosphorylation using ImageJ software [27]. 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 [24]. All the foci were counted manually; at least 100 cells per experiment were analyzed from three independent experiments. 3. Results 3.1. Carbon ions were three times more cytotoxic than gamma To determine the sensitivity of cells to similar dose of high and low LET radiations, their ability to form colonies after irradiation was observed. Irradiation of plateau phase A549 cells with equal doses of -rays and carbon (290 keV/m) revealed that at same dose carbon ions were much more cytotoxic than -rays. The calculated RBE (relative biological effectiveness) of carbon ions relative to rays for 20% survival of A549 cells was found to be nearly 3. Thus, carbon ions were found to be three times more toxic than - rays. At 2 Gy percentage survivals were 60.6 ± 4% and 4.8 ± 0.24% for -rays and carbon respectively (Fig. 1). 3.2. Number of initial -H2AX foci formed was almost same for both radiation type Since DNA double strand breaks are the primary toxic lesions induced by radiation, the number of DSBs was scored by examining the foci of phosphorylated H2AX in irradiated cells. 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 1 Gy -rays irradiation were 15.8 ± 4.8, while after 1 Gy carbon irradiation the foci were 19 ± 4.7 (Fig. 2). Thus unlike the cell survival, not much difference was observed in number of -H2AX foci after irradiation with equal doses of carbon and -rays. However, the size of -H2AX foci in carbon irradiated samples was larger than -rays irradiated samples. The foci were also counted 4 h after irradiation. 57% of -rays irradiated cells still showed -H2AX foci and the average foci per cell had reduced to only 2.9 ± 2.28. Carbon ion irradiated cells also showed a significant reduction in the number of -H2Ax foci (2 ± 2) per cell that were visible in only 22% of cells (Fig. 2B and C). When total intensity of -H2AX rather than the foci was estimated by ImageJ software in the same images, it corroborated well with the number of -H2AX foci in -ray irradiated cells at 15 min and 4 h after irradiation (Fig. 2D). However, in case of car- 1 After irradiation, clonogenic assay was done as described earlier [24]. Absolute plating efficiency at 0 Gy was 32.3 ± 2.4%. 2.4. Western blotting Unirradiated and irradiated cells were lysed at different time periods and immunoblotted, as described previously [26]. Blots were probed with anti-Bax, antiphospho ERK (Thr/Tyr) and anti-phospho JNK (Thr/Tyr). The bound HRP labeled secondary antibody was detected by Chemiluminiscence (Roche Molecular Biochemicals, Germany). The total intensity of bands obtained in ponceau staining was used as a protein loading and transfer control. All band intensities were divided by the intensity of their respective ponceau blot. Surviving Fraction 0.1 0.01 γ ray Carbon beam 2.5. Immunofluorescence staining Unirradiated and irradiated cells were fixed at different time periods and immunofluorescence staining was done as described earlier [24]. The primary antibodies used were mouse anti-pATM (S1981), rabbit anti--H2AX (S139), rabbit anti-pChk1 (Ser296), rabbit anti-pChk2 (Thr68), rabbit anti-pBRCA1 (Ser1524) and rabbit anti-pATR (Ser428) (Cell Signaling). Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG (Molecular Probe, USA). 1E-3 0 1 Dose (Gy) Fig. 1. Clonogenic cell survival of A549 cells irradiated with 1 and 2 Gy carbon beam or -rays. Data represents means ± SD of three independent experiments. 2 3
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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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