LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

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SYNOPSIS subsequent cytotoxicity caused by the same [15-17]. However the mechanism and the factors that contribute to radioresistance of an irradiated cell are yet not known. It was of interest to study the signaling factors that contribute to this phenomenon. A549 cells were found to be relatively more radioresistant if the 10Gy dose was delivered as a fractionated regimen. Microarray analysis showed upregulation of DNA repair and cell cycle arrest genes in fractionated regimen. We have also observed intense activation of DNA repair pathway (DNA-PK, ATM, Rad52, MLH1 and BRCA1), efficient DNA repair and phospho-p53 was found to be translocated to the nucleus of A549 cells (5X2 Gy treatment group). When we silenced the Rad52 gene in A549 cells, the sensitivity of the cells (5X2 Gy treatment group) increased by more than 50%. The above results indicate that fractionated dose led to increased radioresistance in A549 cells and Rad52 gene is one of the factors responsible for increased radioresistance in A549 cells. 4.2 Proton beam induced signaling in mammalian cells and its comparison with Gamma irradiation. Exposure of mammalian cells to ionising 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 radioresistance following therapy remains a major cause for concern. Clinicians are now favoring proton beam therapy to avoid the issue [18-23]. However, the mechanism of cell killing by proton beam is not yet well delineated. In this study, A549 cells were irradiated with either 2Gy proton beam or 2Gy γ-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. Increased phosphorylation of p53, upregulation of Bax and down-regulation of Bcl-2 in proton beam irradiated A549 cells as compared to γ- irradiated cells indicate the activation of apoptotic pathways. The noteworthy finding of this study is the biphasic activation of the sensor proteins, ATM and DNA-PK and no activation of ATR by proton irradiation and the significant activation of Chk2 even at the gene level only in the proton beam irradiated cells. Unlike gamma irradiation, the biphasic induction of ATM and DNA-PK following proton beam irradiation could be responsible for the activation of apoptotic machinery, however, further studies in this direction will reveal the importance of such biphasic response. 4.3 High LET irradiation induced signaling in mammalian cells and its comparison with Gamma irradiation. Because of the effective cell killing clinicians now favor charged particle beam therapy over conventional photons for tumors that are radioresistant and when the survival of nearby cells cannot be compromised [24-26]. Although charged particle beams like carbon etc are currently being used for the treatment of many cancers [25-27], the signaling pathways activated and their variance from γ -irradiation is not well understood. These results suggest that while ATM has been shown to be the critical regulator of low LET radiation induced responses, BRCA1 seems to dominate the high LET induced response. The subtle differences leading to different pathways of initiation of downstream responses with respect to radiation quality seem to lie in pattern of the phosphorylationdephosphorylation of early response proteins such as H2AX rather than their immediate phosphorylation per se. 25
SYNOPSIS 4.4 Radiation induced bystander signaling in mammalian cells. It is now apparent that the target for biological effects of ionizing radiation is not solely the irradiated cell but also the surrounding cells and tissues. Preliminary evidence indicates that similar signal transduction pathways are activated in directly irradiated cells and the bystander cells and contribute to the induction of bystander DNA damage [28]. Many genes have been shown to be activated in bystander cells [29]but evidence for genes that are involved in the signaling pathways is lacking. The present study was carried out to investigate what genes are activated in the bystanding cell and whether the state of activation of the irradiated cell alters the bystander response. Bystander signaling was found to exist between U937 cells (human monocyte) and Lung carcinoma A549 cells. The mechanism of such a cross bystander signaling was investigated in mouse cell line. Bystander signaling was also found to exist between RAW 264.7 (macrophage) and EL-4 (lymphoma) cells. Unstimulated and/or irradiated RAW 264.7 cells did not induce bystander effect in unirradiated EL-4 cells but LPS stimulated and irradiated RAW 264.7 cells induced an upregulation of NF-κB and iNOS genes and increased the DNA damage in bystander EL-4 cells. Treatment of EL-4 or RAW 264.7 cells with L-NAME (NOS inhibitor) significantly reduced the induction of gene expression and DNA damage in the bystander EL-4 cells while treatment with cPTIO (NO scavenger) only partially reduced the induction of gene expression and DNA damage in the bystander EL-4 cells. These results showed that active iNOS in the irradiated cells was essential for bystander response and some long-lived bioactive factors downstream of NO are most likely involved in this bystander response. Summary: The irradiation of cells with fractionated doses led to a signaling response that was different from a single dose of irradiation. The reasons for the radioresistance of the A549 cells lay in the repair pathway, the Rad52, the inhibition of which could revert the cells to radiosensitivity leading to a decrease in survival. The contribution of the bystander effect to the decrease in survival of A549 cells cannot be ruled out. There was a significant bystander response by unirradiated A549 cells. The mechanism of which may be via NO generation. The effectiveness of heavy ions (carbon and oxygen) in decreasing the survival of A549 cells could be due to the lack of efficient repair despite the activation of the repair pathways (HRR). The pathway to apoptosis seems to be p53 independent. Conclusion: The lung carcinoma cell line A549, a highly radioresistant cell line could be effectively made radiosensitive by inhibiting Rad52, one of the components of its repair pathway. The survival of the cells decreased significantly after doing so. The survival of the A549 cell line could also be significantly decreased by heavy ion irradiation. The mechanism of which seems to be a lack of repair, despite the activation of some of the component of the repair pathway. CHAPTER 5: REFERENCES [1] Rontgen, W. C. On a New Kind of Rays. Science 3:227-231; 1896. [2] Becquerel, H. Emission of New Radiations by Metallic Uranium. Compt. Ren. 122:1086-1088; 1896. [3] Alpen, E. L. Radiation Biophysics. Prentice Hall, New Jersey; 1990. 26
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
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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
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Page 36 and 37: SYNOPSIS [4] Hall, E. J. Radiobiolo
Page 38 and 39: CHAPTER 1 INTRODUCTION INTRODUCTION
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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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