LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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Cancer Invest Downloaded from informahealthcare.com by University of Chicago Library on 06/10/11 For personal use only. cell cycle. In case of charged particle irradiation a prolonged to the primary beam, after the gold foil, served as a monitor cell cycle arrest (19) and a slower rejoining of DSB have been detector. The ratio of the monitor detector counts to that of the reported to occur (20). flux measured using SSB detector at the sample position was Apart from DNA repair pathways, ionizing radiation leads to measured by multiple trials and the calibration factor was obtained. Monitor detector counts and the measured ratio were activation of intracellular signaling pathways that play an important role in cellular decisions of death or survival. Although used for delivering the required fluence to the samples. Flux a fair amount of literature exists for the activation of signaling was calculated to be 10 8 and the fluence was kept such that each pathway after charged particle irradiation, there has been no cell is hit by at least 100 proton particles ensuring that bystander systematic comparison of photon and proton beam irradiation. effect is kept to minimum. Fluence was then used to calculate In this study, A549 cells were irradiated with either 2 Gy dose with the help of specific ionization curve constructed for proton beam- or 2 Gy γ -radiation and ensuing activation of few the given energy distribution and composition of the cell line. important components involved in DNA repair, cell cycle arrest, The average energy of the proton beam used in this study was and apoptosis pathways were followed to elucidate the mechanisms behind observed cellular response. Despite the variability 12.5 keV/µm within the cell layer. Initial portion of Bragg’s measured to be 3.2 MeV and corresponding estimated LET is in effectiveness of these two radiations, equal doses of each were curve was used for dose estimation since the cell layer used for chosen to ensure that each cell is hit by proton beam and no bystander effects are manifested. Essentially all biological differ- 10 µm, which helped to avoid steep increase in LET within the irradiation was in monolayer geometry with thickness less than ences between proton and γ -radiations arise from differences sample. in their ionization tracks. Equitoxic doses were not preferred The cells were grown in monolayer geometry on 35 mm because the probability of unhit cells would increase. How this petri dishes. Media was removed and petri dishes were mounted qualitative difference of radiation is translated into activation of on the exit window vertically for the irradiation, after which crucial intracellular pathways was the focus of the study. Only they were resupplemented with the medium. On an average one cell line was chosen in this study because the response of samples were irradiated for 1 min to get the required fluence. The different cell lines is different to proton irradiation (21). cells were exposed to 2 Gy dose with the help of monitor detector counts. MATERIALS AND METHODS Cell culture Clonogenic cell survival assay After treatment, cells were seeded out in appropriate dilutions (500 cells/60 mm petriplate) to form colonies in 14 days. Human A549 cells were cultured in Dulbecco’s modified eagles medium (Sigma) supplemented with 10% fetal calf serum (Sigma). Cells were maintained at 37 ◦ Colonies were fixed with glutaraldehyde (6.0% v/v), stained C in humidified atmosphere with 5% CO 2 . Cells were seeded at density of 5 × 10 5 with crystal violet (0.5% w/v) and counted using a stereomicroscope. Colonies containing more than 50 cells were scored as cells in small 35 mm petri dishes 24 hr before the irradiation. survivors. Absolute plating efficiency at 0 Gy (sham irradiated control) was 32.3 ± 2.4% Treatment of cells Irradiation Semiquantitative reverse transcriptional For γ -irradiation, cells were exposed to 2 Gy of 60 Co γ - polymerase chain reaction (RT-PCR) radiation in in-house facility Gamma Cell 220 at dose rate of 3 Gy/min. Total RNA from A549 cells (1 × 10 6 cells) was extracted after Proton beam irradiation was carried out using the Radiation required time periods of irradiation and RT-PCR was carried out Biology beam line of in-house 4 MeV Folded Tandem Ion Accelerator (FOTIA) facility. The primary proton beam from the was extracted after required time periods of 1, 2, 3, and 4 hr of as described earlier (22). Breifly, total RNA from A549 cells FOTIA was collimated using an adjustable slit to reduce the irradiation and eluted in 30 µL RNAase free water using RNA fluence and then diffused using a gold foil. The diffused beam tissue kit (Roche). Equal amount of RNA in each group was was channeled to the exit window made of 20 µm titanium foil reverse transcribed using cMaster RT kit (Eppendorf). Equal of 3-cm diameter to get uniformly distributed irradiation area. amount (2 µL) of cDNA in each group was used for specific The samples were thus irradiated under normal atmospheric amplification of β-actin, ATM, DNA-PK, ATR, Chk1, Chk2, pressure at 24 ◦ C. The target to be irradiated was positioned at a Bcl-2, or Bax gene using gene specific primers (Table 1). The distance of 11 mm from the exit window, that being the closest PCR condition were 94 ◦ C, 5 min initial denaturation followed possible place to mount the target. Before the irradiation, a silicon surface barrier (SSB) detector was positioned at the same annealing temperature of specific primers given in Table 1), by 30 cycles of 94 ◦ C 45 s, 55–64 ◦ C 45 s (depending on the position where samples were irradiated and the beam energy 72 ◦ C 45 s and final extension at 72 ◦ C for 10 min. Equal amount as well as the uniformity was measured. Another SSB detector of each PCR product (10 µL) was run on 1.5% agarose gel placed inside the scattering chamber at a forward angle of 40 ◦ containing ethidium bromide in tris borate EDTA buffer at 60 V. 616 S. <strong>Ghosh</strong> et al.
Cancer Invest Downloaded from informahealthcare.com by University of Chicago Library on 06/10/11 For personal use only. Table 1. The Sequences of Gene Specific Primers and Their Annealing Temperature (Tm) Used in RT-PCR Experiments Gene Primer Sequence Tm ( ◦ C) β-actin F 5 ′ -TGGAATCCTGTGGCATCCATGAAA-3 ′ 55 R5 ′ -TAAAACGCAGCTCAGTAACAGTCCG-3 ′ ATM F 5 ′ -GGACAGTGGAGGCACAAAAT-3 ′ 57 R5 ′ -GTGTCGAAGACAGCTGGTGA-3 ′ ATR F 5 ′ -CTCGCTGAACTGTACGTGGA-3 ′ 57 R5 ′ -GCATAGCTCGACCATGGATT-3 ′ DNA-PK F 5 ′ -TGCCAATCCAGCAGTCATTA-3 ′ 64 R5 ′ -GGTCCATCAGGCACTTCACT-3 ′ Chk2 F 5 ′ -GGCTTCAGGATGAAGACATGA-3 ′ 64 R5 ′ -CACAACACAGCAGCACACAC-3 ′ Chk1 F 5 ′ -TGTCAGAGTCTCCCAGTGGA-3 ′ 59 R5 ′ -AGGGGCTGGTATCCCATAAG-3 ′ Bax F 5 ′ -TCCCCCCGAGAGGTCTTT-3 ′ 56 R5 ′ -CGGCCCCAGTTGAAGTTG-3 ′ Bcl-2 F 5 ′ -GAGGATTGTGGCCTTCTTTG-3 ′ 59 R5 ′ -ACAGTTCCACAAAGGCATCC-3 ′ F indicates forward and R indicates reverse. The bands in the gel were visualized under UV lamp and relative intensities were quantified using Gel doc software (Syngene). Immunofluorescence staining Cells were grown in cover slips that were kept in 35 mm petri dishes and irradiated as mentioned above. Cell layers were washed 4 h after irradiation in PBS and fixed for 20 min in 4% paraformaldehyde at room temperature. Afterward, 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 hr with 5% BSA in PBS. Antibodies were diluted (1:200) in 1% BSA in PBS. Cells were incubated with primary antibodies for 1 hr 30 min at room temperature, washed three times in PBS, and incubated with secondary antibodies for 1 hr 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-γ -H2AX (S139), rabbit anti-pChk2 (Thr68), and mouse anti-phospho-p53 (S15) (Cell Signaling). Secondary antibodies used were Alexa Fluor 488 goat antirabbit IgG and Alexa Fluor 488 goat antimouse IgG (Molecular Probe, USA). Image analysis using ImageJ software The captured images were analyzed for relative quantification of phosphorylation using ImageJ software (23). A 25 × 25 pixel box was positioned over the fluorescent image of each cell, and the average intensity within the selection (the sum of the intensities of all the pixels in the selection divided by the number of pixels) was measured. At least 100 cells per experiment were analyzed from three independent experiments. Statistical analysis The data were imported to excel worksheets, and graphs were made using Origin version 5.0. One-way ANOVA with Tukey–Kramer Multiple Comparisons as post-test for p < .05 used to study the significant level. Data were insignificant at p > .05. Each point represented as mean ± S.E. (standard error of the mean). RESULTS Cell survival assay There were only 23 ± 2% of A549 cells that survived which had been exposed to 2 Gy of proton beam where as 56 ± 2.2% cells were survived which had been exposed to 2 Gy of γ - radiation as observed with clonogenic assay(Figure 1). This result clearly showed that proton beam is more cytotoxic than γ -radiation. Differential modulation of gene expression following proton beam and γ-irradiation Expression of ATM, DNA-PK, and Chk2 genes were significantly upregulated after 4 hr of irradiation only in A549 cells which had been exposed to proton beam (2 Gy) [Figures 2(A) and (C)], whereas only the ATR gene was significantly upregulated after 4 hr of irradiation in A549 cells which had been exposed to γ -radiation (2 Gy) [Figure 2(B)]. There was no change in the gene expression of Chk1 in A549 cells which had been exposed to either γ -radiation or proton beam (2 Gy) [Figures 2(C), and (D)]. Independent experiments were carried out for γ - and proton beam-irradiated cells, so the control group of γ - and proton-irradiated cells may have different transcript level. However, the data were normalized for the respective controls. Activation of signaling components involved in DNA damage sensing and repair following proton beam irradiation SinceATM protein plays a central role in DNA double-strand breaks (DSBs) sensing and repair and determines the cellular Figure 1. Clonogenic Cell Survival of A549 cells irradiated with 2 Gy proton beam or 2 Gy γ-radiation. Data shown from representative experiment of three independent experiments. Efficient Cell killing by Proton Beam 617
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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 [127] Frank, K
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CHAPTER 5 REFERENCES [141] Pierce,
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CHAPTER 5 REFERENCES [156] Roots, R
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CHAPTER 5 REFERENCES [171] Xia, Z.;
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