LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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SYNOPSIS Aims and Objectives: To Study 1. Fractionated irradiation induced signaling events. The contribution of these events to the increased cell survival. 2. High LET induced signaling and variance from low LET gamma radiation. 3. Contribution of radiation induced bystander effect to cell survival and its mechanism. CHAPTER 2: MATERIALS AND METHODS The second chapter lists the reagents and describes in detail the methods employed in this study. A brief summary of the same is given below. 2.1 Chemicals and Reagents All fine chemicals used were from Sigma, USA. Primary antibodies were from Cell Signaling Technology, USA; Calbiochem, USA; Chemicon, USA; Santa Cruz Biotechnology, USA; Sigma, USA and Transduction Laboratories, USA. 2.2 Cell Lines All the cell lines used in this study were cultured in 25 cm 2 / 75 cm 2 plastic flasks in the appropriate medium supplemented with 10% fetal bovine serum (Sigma, USA.). Cells were kept at 37 0 C in humidified atmosphere with 5% CO 2 . Following are the list/table of the cell lines used in the experiments SN Cell line Description p53 status 1 A549 Human Lung Adenocarcinoma Wild type 2 MCF-7 Human breast adenocarcinoma Wild type 3 INT 407 human intestinal epithelial cell line Wild type 4 U937 Human histiocytic lymphoma (Monocyte) Negative 5 EL-4 Mouse lymphoma Wild type 6 RAW 264.7 Mouse macrophages Wild type 3
SYNOPSIS 2.3 Irradiation 2.3.1] Low LET: For gamma irradiation, cells were exposed to required dose of 60 Co γ radiation in in-house facility Gamma Cell 220 at dose rate of 3.5 Gy/min. Proton beam irradiation was carried out using the Radiation Biology beam line of in-house 4 MeV Folded Tandem Ion Accelerator (FOTIA) facility. 2.3.2] High LET: Heavy ion irradiation was carried out using the Radiation Biology beam line of 16 MV 15UD Pelletron at Inter University Accelerator Centre, New Delhi. 2.4 Clonogenic Cell Survival Assay 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. 2.5 Semiquantitative Reverse transcriptional polymerase chain reaction (RT-PCR) Total RNA from 1 x 10 6 cells was extracted after required time periods of irradiation and RT-PCR was carried out. Briefly, total RNA from A549 cells was extracted after required time periods of 1, 2, 3 and 4 hours of irradiation and eluted in 30 µl RNAase free water using RNA tissue kit (Roche). Equal amount of RNA in each group was reverse transcribed using cMaster RT kit (Eppendorf). Equal amount (2µl) of cDNA in each group was used for specific amplification of required genes using gene specific primers. The PCR condition were 94 o C, 5 min initial denaturation followed by 30 cycles of 94 o C 45 s, 55 – 64 o C 45 s (depending on the annealing temperature of specific primers) 72 o C 45 s and final extension at 72 o C for 10 min. Equal amount of each PCR product (10 µl) was run on 1.5 % agarose gel containing ethidium bromide in tris borate EDTA buffer at 60 V. The bands in the gel were visualized under UV lamp and relative intensities were quantified using Gel doc software (Syngene). 4
Page 1: RADIATION INDUCED SIGNALING IN MAMM
Page 4 and 5: DECLARATION I, hereby declare that
Page 6 and 7: CONTENTS Page No. SYNOPSIS………
Page 8 and 9: 2.11 Measurement of NO production
Page 10 and 11: PREAMBLE SYNOPSIS Ionising radiatio
Page 14 and 15: SYNOPSIS 2.6 Immunofluorescence sta
Page 16 and 17: SYNOPSIS ATM gene expression, which
Page 18 and 19: SYNOPSIS Percentage Survival 100 80
Page 20 and 21: SYNOPSIS Ratio of Rad52 to Actin Ra
Page 22 and 23: Relative Phosphorylation 45 40 35 3
Page 24 and 25: SYNOPSIS Fig. 3.3A Clonogenic Cell
Page 26 and 27: SYNOPSIS (A) Av No. of phospho BRCA
Page 28 and 29: SYNOPSIS Percentage Survival 120 10
Page 30 and 31: SYNOPSIS Fig.3.4B. Existance of cro
Page 32 and 33: SYNOPSIS Fig. 3.4EDNA damage in EL-
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Page 36 and 37: SYNOPSIS [4] Hall, E. J. Radiobiolo
Page 38 and 39: CHAPTER 1 INTRODUCTION INTRODUCTION
Page 40 and 41: CHAPTER 1 INTRODUCTION 15.8% 9.56%
Page 42 and 43: CHAPTER 1 INTRODUCTION far apart an
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Page 46 and 47: CHAPTER 1 INTRODUCTION 1.3 DNA dama
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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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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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CHAPTER 5 REFERENCES (MAP) kinase c
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CHAPTER 5 REFERENCES phosphorylatio
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CHAPTER 5 REFERENCES [211] Anderson
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CHAPTER 5 REFERENCES [229] Konca, K
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CHAPTER 5 REFERENCES [245] Kastan,
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CHAPTER 5 REFERENCES [261] Miralbel
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CHAPTER 5 REFERENCES [278] Weizman,
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CHAPTER 5 REFERENCES [294] Zhou, H.
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PUBLICATIONS AND PRESENTATIONS Publ
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Mutation Research 729 (2012) 61-72
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S. Ghosh, M. Krishna / Mutation Res
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Cancer Invest Downloaded from infor
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1568 I. J. Radiation Oncology d Bio
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