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. Figure 4. Gene expression of Bax and Bcl-2 12 hr after irradiation in A549 cells irradiated with 2 Gy proton beam or 2 Gy γ-radiation. Total RNA from A549 cells was isolated and reverse transcribed. RT-PCR analysis of Bax and Bcl-2 genes was carried out as described in materials and methods. PCR products were resolved on 1.5% agarose gels containing ethidium bromide. β-actin gene expression in each group was used as an internal control. Ratio of intensities of Bax and Bcl-2 band to that of respective β-actin band as quantified from gel pictures are shown above each gel picture. Con means unirradiated control, H means proton. Data represents means ± SE of three independent experiments, significantly different from unirradiated controls. ∗ p < .05, ∗∗ p < .01. DISCUSSION More than two-fold decrease in survival of the A549 cells as assessed by the clonogenic cell survival assay is indicative of the fact that the proton beam is more efficient in killing the tumor cells and may be a more preferred mode of treatment. The mechanisms of cell death caused by proton treatment and the signaling pathways that ensue have not yet been investigated in detail although proton beam therapy is in use for many cancers (1–6). The massive activation of DNA-PK and ATM after proton irradiation when compared with γ [Figures 2, (A) & (B)] was more or less expected and may be due to the fact that DNA damage activates phosphatidylinositol 3-kinase-like kinases (DNA- PK, ATM), which then amplify and channel the signal by activating downstream kinases (Chk1 and Chk2). These, in turn, phosphorylate target proteins such as p53, Cdc25A, and Cdc25C (25) to delay the cell cycle, a process called the DNA damage checkpoint. In addition to checkpoint control, the downstream kinases also modulate DNA repair and trigger apoptosis (26). The lack of activation of ATR after proton irradiation was however intriguing and was found to be reflecting no activation of Chk1 (Figure 2). The phosphorylation of H2AX was found to significantly increase 4 hr after proton beam irradiation (Figure 3). This time period of 4 hr was chosen for looking at the activation of important components involved in repair because by 4 hr after γ -irradiation most of the repair is complete and the activation of these factors is reduced to control levels (27). Phosphorylation of H2AX even at 4 hr indicates the presence of large number of DSBs at that time indicating very slow repair. It has been established recently that H2AX at the DNA DSB sites is immediately phosphorylated upon irradiation, and the phosphorylated H2AX (γ -H2AX) can be visualized in situ by immunostaining with a γ -H2AX specific antibody (28, 29). γ -H2AX induction can be measured quantitatively at physiological doses, and the numbers of residual γ -H2AX foci can be used to estimate the kinetics of DSB rejoining. This has become the gold standard for the detection of DSB (17, 28). Since ATM protein plays a central DNA DSBs sensing and repair and determines the cellular response in eukaryotes it was of interest to look for its phosphorylation at 4 hr after irradiation. There was significantly higher phosphorylation of ATM at serine 1981 in A549 cells, which had been exposed to 2 Gy of proton beam (Figure 3). This indicates that despite significant activation of ATM till 4 hr after irradiation much of the damage still persisted as indicated by the presence of significant levels of γ -H2AX. The activated/phosphorylated ATM further phosphorylates various downstream target proteins such as H2AX, Chk2, p53 leading to opening of the chromatin and recruitment of other proteins involved in DNA repair or cell cycle arrest. Chk2 which is a another substrate of ATM and is involved in cell cycle arrest was also found to be phosphorylated at threonine 68 in A549 cells which had been exposed to 2 Gy of proton as compared to controls (Figure 3), however the activation was not as intense as observed with ATM or H2AX. This indicates that small component of ATM activation is also diverted to cell cycle arrest pathways involving Chk2, CDC25 phosphatase, and CDKs. Chk2 could also be involved in activation of other proteins such as BRCA1 and p53. Phosphorylation of p53 at serine 15 was then observed in unirradiated and irradiated A549 cells at 2 Gy of proton beam (Figure 3). Whether DNA-PK signals DSBs to the checkpoint machinery remains controversial, but the emerging consensus is that it does not (30). DNA-PK may, however, be involved in signalling DNA damage to the apoptosis machinery (31). The degree of radiation-induced apoptosis has been shown to correlate with the p53 wild-type status (32). In addition, apoptosis is induced when wild-type p53 is transfected into certain cell lines lacking p53 (33). This indicates that p53 not only plays a role in regulating the progression through the cell cycle, it can also induce apoptosis in cells. p53 is not an essential component of the machinery that carries out apoptosis; however, 620 S. Ghosh et al.
Cancer Invest Downloaded from informahealthcare.com by University of Chicago Library on 06/10/11 For personal use only. it appears to be an activator of apoptosis. Although the exact means by which p53 activates apoptosis is unclear, evidence has shown that p53 mediates apoptosis by way of transcriptionindependent and transcription-dependent mechanisms (34, 35). p53 is known to regulate the expression of several proteins involved in the apoptotic pathway and also interacts with BAX, BCL-XL, and Bcl-2 to exert a direct apoptotic effect at the level of the mitochondria (36–38). Increased phosphorylation of p53, upregulation of Bax and downregulation of Bcl-2 in proton beam irradiated A549 cells as compared to γ -irradiated cells (Figures 3 and 4) 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 beamirradiated cells (Figure 2). Such biphasic response after irradiation with proton or other heavy ions has been observed by us earlier and may be characteristics of particle irradiation (39). Unlike γ -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. The use of proton beam therapy has definite advantage over γ therapy. The mechanism of apoptosis will give the clinician a handle to manipulate therapy for enhanced cell killing. DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES 1. Krengli, M.; Hug, E.B.; Adams, J.A.; Smith, A.R.; Tarbell, N.J.; Munzenrider, J.E. Proton radiation therapy for retinoblastoma: comparison of various intraocular tumor locations and beam arrangements. Int J Radiat Oncol Biol Phys 2005, 61, 583–593. 2. St Clair, W.H.; Adams, J.A.; Bues, M.; Fullerton, B.C.; La Shell, S.; Kooy, H.M.; Loeffler, J.S.; Tarbell, N.J. Advantage of protons compared to conventional X-ray or IMRT in the treatment of a pediatric patient with medulloblastoma. Int J Radiat Oncol Biol Phys 2004, 58, 727–734. 3. Miralbell, R.; Crowell, C.; Suit, H.D. Potential improvement of three dimension treatment planning and proton therapy in the outcome of maxillary sinus cancer. Int J Radiat Oncol Biol Phys 1992, 22, 305–310. 4. Jones, B.; Dale, R.G.; Cárabe-Fernández, A. Charged particle therapy for cancer: the inheritance of the Cavendish scientists? Appl Radiat Isot 2009, 67, 371–377. 5. Chang, J.Y.; Zhang, X.; Wang, X.; Kang, Y.; Riley, B.; Bilton, S.; Mohan, R.; Komaki, R.; Cox, J.D. Significant reduction of normal tissue dose by proton radiotherapy compared with three-dimensional conformal or intensity-modulated radiation therapy in Stage I or Stage III non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 2006, 65, 1087–1096. 6. Fukumitsu, N.; Sugahara, S.; Nakayama, H.; Fukuda, K.; Mizumoto, M.; Abei, M.; Shoda, J.; Thono, E.; Tsuboi, K.; Tokuuye, K. A prospective study of hypofractionated proton beam therapy for patients with hepatocellular carcinoma. Int J Radiat Oncol Biol Phys 2009, 74, 831–836. 7. Shibuya, K.; Mathers, C.D.; Boschi-Pinto, C.; Lopez, A.D.; Murray, C.J. Global and regional estimates of cancer mortality and incidence by site: II. Results for the global burden of disease 2000. BMC Cancer 2002, 2, 37. 8. Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.; Thun, M.J. Cancer statistics, 2008. CA Cancer J Clin 2008, 58, 71–96. 9. Ferlay, J.; Autier, P.; Boniol, M.; Heanue, M.; Colombet, M.; Boyle, P. Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol 2007, 18, 581–592. 10. Jassem J. The role of radiotherapy in lung cancer: where is the evidence? Radiother Oncol 2007, 83, 203–213. 11. Kim, Y.S.; Yoon, S.M.; Choi, E.K.; Yi, B.Y.; Kim, J.H.; Ahn, S.D.; Lee, S.W.; Shin, S.S.; Lee, J.S.; Suh, C.; Kim, S.W.; Kim, D.S.; Kim, W.S.; Park, H.J.; Park, C.I. Phase II study of radiotherapy with three-dimensional conformal boost concurrent with paclitaxel and cisplatin for Stage IIIB non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 2005, 62, 76–81. 12. Farray, D.; Mirkovic, N.; Albain, K.S. Multimodality therapy for stage III non-small-cell lung cancer. J Clin Oncol 2005, 23, 3257–3269. 13. Bush, D.A.; Slater, J.D.; Bonnet, R.; Cheek, G.A.; Dunbar, R.D.; Moyers, M.; Slater, J.M. Proton-beam radiotherapy for early-stage lung cancer. Chest 1999, 116, 1313–1319. 14. Shioyama, Y.; Tokuuye, K.; Okumura, T.; Kagei, K.; Sugahara, S.; Ohara, K.; Akine, Y.; Ishikawa, S.; Satoh, H.; Sekizawa, K. Clinical evaluation of proton radiotherapy for non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 2003; 56: 7–13. 15. Koto, M.; Miyamoto, T.; Yamamoto, N.; Nishimura, H.; Yamada, S.; Tsujii, H. Local control and recurrence of stage I non-small cell lung cancer after carbon ion radiotherapy. Radiother Oncol 2004, 71, 147–156. 16. Maser, R.S.; Monsen, K.J.; Nelms, B.E.; Petrini, J.H. hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol Cell Biol 1997, 17, 6087–6096. 17. Fernandez-Capetillo, O.; Lee, A.; Nussenzweig, M.; Nussenzweig, A. H2AX: the histone guardian of the genome. DNA Repair 2004, 3, 959–967. 18. Bekker-Jensen, S.; Lukas, C.; Kitagawa, R.; Melander, F.; Kastan, M.B.; Bartek, J.; Lukas, J. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 2006, 173, 195–206. 19. Goto, S.; Watanabe, M.; Yatagai, F. Delayed cell cycle progression in human lymphoblastoid cells after exposure to high-LET radiation correlates with extremely localized DNA damage. Radiat Res 2002, 158, 678–686. 20. Stenerlow, B.; Hoglund, E.; Carlsson, J.; Blomquist, E. Rejoining of DNA fragments produced by radiations of different linear energy transfer. Int J Radiat Biol 2000, 76, 549–557. 21. Lee, K.B.; Lee, J.S.; Park, J.W.; Huh, T.L.; Lee, Y.M. Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases. Exp Mol Med 2008, 40, 118–129 22. Ghosh, S.; Maurya, D.K.; Krishna, M. Role of iNOS in bystander signaling between macrophages and lymphoma cells. Int J Radiat Oncol Boil Phy 2008, 72, 1567–1574 23. Rasband, W.S. and Image, J.U.S. National Institutes of Health, Bethesda, USA, http://rsb.info.nih.gov/ij/ (1997–2006). 24. Khanna, K.K.; Lavin, M.F.; Jackson, S.P.; Mulhern, T.D. ATM, a central controller of cellular responses to DNA damage. Cell Death Differ 2001, 8(11), 1052–1065. 25. Lakin, N.D.; Jackson, S.P. Regulation of p53 in response to DNA damage. Oncogene 1999, 18, 7644–7655. 26. Rich, T.; Allen, R.L.; Wyllie, A.H. Defying death after DNA damage. Nature 2000, 407, 777–783. Efficient Cell killing by Proton Beam 621
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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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