LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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1568 I. J. Radiation Oncology d Biology d Physics Volume 72, Number 5, 2008 supplemented with 10% fetal calf serum (Sigma, St. Louis, Mo). Cells were maintained at 37 C in humidified atmosphere with 5% CO 2 . EL-4 and RAW 264.7 cells were resuspended in RPMI medium at a density of 1 10 6 cells/mL and exposed to 5 Gy g irradiation using Gamma Cell 220 irradiator (Atomic Energy of Canada Ltd), at a dose rate of 4.74 Gy/min. Medium from irradiated cells was transferred to unirradiated cells (1 h postirradiation). Some group of RAW 264.7 cells were stimulated with 500 ng/mL of bacterial lipopolysaccharide (LPS, Sigma, St. Louis, Mo) for 3 h and then the medium was replaced before irradiation. Before designing the experiment, there were two aspects that had to be taken care of. One was the likelihood that the replacement of the medium itself caused transient expression of certain signaling factors and second that the components of the medium, which include proteins such as growth factors, which are unavoidably exposed to radiation, contribute to the changes in the bystander cell. The experimental design, therefore, consisted of the following sets: (a) control unirradiated cells, (b) g-irradiated cells, (c) EL-4 cells receiving medium from unirradiated EL-4 cells or RAW 264.7 cells or LPS-stimulated RAW 264.7 cells, (d) EL-4 cells receiving medium from g-irradiated EL-4 cells or irradiated RAW 264.7 cells or LPS-stimulated and irradiated RAW 264.7 cells, and (e) EL-4 cells receiving g-irradiated medium. The cells (EL-4 and RAW 264.7) were also divided into two groups; one was treated with 20 mM cPTIO (NO scavenger; Molecular Probe, Invitrogen, Carlsbad, CA) and the other with 1 mmol L-NAME (NOS inhibitor; Sigma, St. Louis, MO). L-NAME was washed off after 30 min so that it is not present in the bystander cells during incubation. Measurement of DNA damage DNA damage in all the groups of EL-4 cells after exposure to different conditioned media was measured after 2 h by alkaline single cell gel electrophoresis (comet assay) (13). Semiquantitative reverse transcriptional polymerase chain reaction Total RNA from all the groups of EL-4 cells (1 10 6 cells) was extracted 2 h after exposure to different conditioned media and eluted in 30 mL RNAase free water using RNA tissue kit (Roche, Mannheim, Germany). Equal amount of RNA in each group was reverse transcribed using cMaster RT kit (Eppendorf, Hamburg, Germany). Equal amount (2 mL) of cDNA in each group was used for specific amplification of b-actin, NF-kB (p65), iNOS, p21, or p53 gene using gene specific primers (Table 1). The polymerase chain reaction (PCR) condition were 94 C, 5 min initial denaturation followed by 30 cycles of 94 C 45 s, 55–62 C 45 s (depending on the annealing temperature of specific primers given in Table 1), 72 C 45 s, and final extension at 72 C for 10 min. Equal amount of each PCR product (10 mL) 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 an ultraviolet lamp and relative intensities were quantified using Gel doc software (Syngene, Nuffied Road, Cambridge, UK). NO production in EL-4 cells All the groups of EL-4 cells after exposure to different conditioned media were cultured for 24 h. The production of NO in the culture supernatants was measured by assaying NO 2 – using colorimetric Griess reaction (14). Table 1. The sequences of gene specific primers and their annealing temperature (Tm) used in reverse transcriptasepolymerase chain reaction experiments Gene Primer sequence Apoptosis in EL-4 cells Apoptosis in control unirradiated cells, cells exposed to radiation, and the cells exposed to different conditioned media as mentioned previously was measured by DNA fragmentation assay (15). The characterization and quantification of apoptosis was further confirmed by confocal microscopy (Carl Zeiss, LSM 510 META, Carl Zeiss Micro Imaging GMBH, Jena, Germany) using an annexin V/PI-staining kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Five fields of cells for each group (1,000 cells) were counted to generate apoptotic index. Annexin-V binds to phosphatidylserine is a marker of early apoptosis and binding of PI indicates necrosis. Statistical analysis The data were imported to Excel work sheets, and graphs were made using Origin version 5.0. One-way analysis of variance with Tukey-Kramer multiple comparisons as posttest for p < 0.05 used to study the significant level. Data were insignificant at p > 0.05. Each point represented as mean standard error of the mean. RESULTS Tm ( C) 55 b-Actin Forward 5 0 - TGGAATCCTGTGGCATCCATGAAAC-3 0 Reverse 5 0 - TAAAACGCAGCTCAGTAACAGTCCG-3 0 iNOS Forward 5 0 -CTCTGACAGCCCAGAGTTCC-3 0 62 Reverse 5 0 -AGGCAAAGGAGGAGAAGGAG-3 0 p21 Forward 5 0 -GCCTTAGCCCTCACTCTGTG-3 0 58 Reverse 5 0 -GGTTGGGAGGGGCTTAAATA-3 0 p53 Forward 5 0 -CGGGTGGAAGGAAATTTGTA-3 0 58 Reverse 5 0 -CTTCTGTACGGCGGTCTCTC -3 0 p65 Forward 5 0 -ACAACTGAGCCCATGCTGAT-3 0 50 Reverse 5 0 -GAGAGGTCCATGTCCGCAAT-3 0 Bystander effect in similar cells (EL-4 Vs EL-4) The expression of NF-kB (p65), iNOS, p53, and p21 genes were significantly upregulated in irradiated as well as in bystander cells (Fig. 1 A–D, Lanes 2, 3). However, in the cells cultured in the presence of only irradiated medium, there was a marginal upregulation of p53 but no upregulation of p21 (data not shown). The expression of p53 gene in these cells was significantly lower than irradiated cells as well as bystander cells. Because the expressions of iNOS gene was significantly upregulated in irradiated and bystander cells (Fig. 1B, Lanes 2, 3), it was of interest to look for NO production in these cells and expectedly NO production was also found to be significantly enhanced in irradiated and bystander cells (Fig. 2, Lanes 2, 3). Damage to cellular DNA, expressed as tail length (Fig. 3A), percent DNA in tail (Fig. 3B), Olive tail moment (Fig. 3C) and tail moment (Fig. 3D) was significantly higher in irradiated EL-4 cells and bystander cells as compared with the unirradiated control cells (Fig. 3A–D, Lanes 2, 3).
Bystander effect: role of iNOS d S. GHOSH et al. 1569 Fig. 1. Gene expression of inducible nitric oxide synthase (iNOS), p21, p53, and NF-kB (p65) in EL-4 cells cultured for 2 h in presence of different conditioned media. Total RNA from EL-4 cells was isolated and reverse transcribed. Reverse transcriptase-polymerase chain reaction (PCR) analysis of iNOS, p21, p53, and NF-kB (p65) genes was carried out as described in materials and methods. PCR products were resolved on 1.5% agarose gels containing ethidium bromide. b-Actin gene expression in each group was used as an internal control. Ratio of intensities of (A) NF-kB (p65), (B) iNOS, (C) p53, and (D) p21band to that of respective b-actin band as quantified from gel pictures are shown above each gel picture. Key: Lane 1, control unirradiated EL-4 cells; Lane 2, g irradiated EL-4 cells; Lane 3, unirradiated EL-4 cells receiving medium from g-irradiated EL-4 cells. Data represent means SE of three independent experiments; significantly different from unirradiated controls: *p < 0.05, **p < 0.01. There was a clear DNA ladder formation (a hallmark of apoptosis) in irradiated and bystander EL-4 cells. Apoptosis was found to be higher in irradiated cells than bystander cells (Fig. 4, Lanes 2, 3). Apoptosis in irradiated and bystander EL-4 cells was further confirmed by annexin V/PI assay (Fig. 5). EL-4 cells that had received only irradiated medium or medium from unirradiated control cells did not show any upregulation of NF-kB and iNOS or NO production or DNA damage or apoptosis (data not shown). Bystander effect in dissimilar cells (EL-4 Vs RAW 264.7) Having confirmed the existence of bystander signaling between similar cells (EL-4 Vs EL-4), the work was extended to determine if bystander effects can be demonstrated between different cell type (RAW 264.7 and EL-4) and whether the Fig. 2. Nitric oxide (NO) production from EL-4 cells cultured for 24 h in the presence of different conditioned media. The culture supernatant from each group of EL-4 cells was used for determination of NO 2 – with Griess reagent. Key: Lane 1, control unirradiated EL-4 cells; Lane 2, g irradiated EL-4 cells; Lane 3, unirradiated EL-4 cells receiving medium from g irradiated EL-4 cells. Data represent means SE of three independent experiments; significantly different from unirradiated controls: *p < 0.05, **p < 0.01. state of stimulation of macrophages (RAW 264.7) affects bystander response. The expression of NF-kB was not altered in EL-4 cells that had received medium from either unstimulated RAW 264.7 cells or irradiated RAW 264.7 cells, but the expression of iNOS was downregulated (Fig. 6, Lanes 3, 5). In EL-4 cells that had received medium from LPS stimulated RAW 264.7 cells there was an upregulation of iNOS gene (Fig. 6, Lane 4). The expression of both the genes (NF-kB and iNOS) was significantly upregulated in EL-4 cells that had received medium from LPS stimulated and irradiated RAW 264.7 cells (Fig. 6, Lane 2). The EL-4 cells that had received medium from unstimulated RAW 264.7 cells or LPS-stimulated RAW 264.7 cells or irradiated RAW 264.7 cells did not show any increase in DNA damage (Fig. 7, Lanes 3–5). But there was significantly higher DNA damage in EL-4 cells that had received medium from LPS stimulated and irradiated RAW 264.7 cells (p < 0.01) (Fig. 7, Lane, 2). Effect of L-NAME and cPTIO on Bystander response Because the expressions of NF-kB and iNOS gene were significantly upregulated in irradiated and bystander cells (Fig. 8, Lanes 2, 3), it was of interest to determine what
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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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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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