LIFE09200604007 Tabish - Homi Bhabha National Institute

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Discussion H2AX is currently the most sensitive way to detect IR-induced DNA damage and to measure the extent of DSB repair. Foci of γ-H2AX can be quantified by immunofluorescence microscopy or flow cytometry 220, 221 . Measurement of association of cancer risk with DNA damage and repair is often done by measuring γ-H2AX following genotoxic exposure 98-100, 222 . Mutagen sensitivity and DNA repair We observed that after radiation exposure the level of DSB between the two groups was almost similar as the number of γ-H2AX foci and percent γ-H2AX positive cells at 30 min time point was approximately same. However the extent of DNA repair at 4 h time point was significantly different between the two groups as revealed by residual γ-H2AX foci in MPN cell lines as compared to control cell lines implying that DNA repair is either delayed or impaired in these cells. The result was replicated when we quantitatively measured γ-H2AX positive cells by flow cytometry. Our observations are in concert with studies done on breast cancer by Kennedy et al. 60 and Machella et al. 77 , where they observed that deficient DNA repair capacity may represent a risk factor which predisposes women to breast cancer. Similarly findings by Cheng et al. 83 , suggest that individuals with reduced DNA repair capacity may be at increased risk of developing head and neck cancer 83 . In numerous reports by Spitz et al., higher mutagen sensitivity, which is an indirect assessment of DNA repair capacity, has been found to be associated with head and neck cancers including UADT cancers as well as increased risk of developing second malignant tumours in such patients 223, 224 . Other than head and neck cancers, altered DNA repair capacity has also been associated with occurrence of multiple primary cancers in other malignancies. In their study, Cianciulli et al. 194 have established the usefulness of mutagen sensitivity as an indirect measure of DNA repair and genetic susceptibility to multiple primary cancers. In multiple primary cancers of Hereditary Non Polyposis Colorectal Cancer, genetic instability has been assessed as DNA replication errors and found to play an important role in MPN development 195 . Similarly Orlow et al. 197 measured DNA damage by the comet assay and observed its association with the development of multiple primary tumours in individuals with Non Small Cell Lung Cancer 197 . In another study Miller et al. 196 demonstrated that if abnormalities in DNA repair and mutagen sensitivity were cancer susceptibility factors, such findings would be seen with regularity in individuals with multiple primary cancers. Their main focus in the study was occurrence of triple 139
Discussion primary cancers 196 . This indicates that compromised DNA repair capacity is an important hallmark of cancer and reinforces our assumption that reduced DNA repair capacity might be a risk factor for UADT MPN development. Effect of γ-radiation exposure on cell cycle When a cell is exposed to DNA damaging agent it results in cell cycle arrest and activation of cell cycle check points which play an essential role in DNA repair 225 . A disruption in any of the cell cycle check points may result in accumulation of gene mutations and chromosomal aberrations by reducing the efficiency of DNA repair leading to genetic instability that may drive neoplastic evolution 226 . We hypothesized that individuals with inherited defects in cell cycle control probably are susceptible to UADT MPN development. G2 phase cells are extremely sensitive to ionising radiation induced DNA damage resulting in G2/M arrest and delaying entry into mitosis until the damage has been repaired 226 . In numerous reports defects in cell cycle G2 delay are strongly associated to human carcinogenesis 93, 104-106 . Our results demonstrated that after exposure to γ-radiation, G2 delay was more pronounced and significantly higher in control cell lines as compared to MPN cell lines. Similar to our results, in earlier studies as well, an abnormal cell cycle control has been associated with cancer risk. In a report by Wu X. et al. 93 , when a comparison of G2/M check point was carried out with lung cancer risk it was observed that γ- radiation induced S and G2/M phases arrest was significantly lower in cases than in controls. They also compared cell cycle check points with DNA repair capacity measured by comet assay and concluded that defects in cell cycle checkpoints and DNA damage/repair capacity were associated with elevated lung cancer risk 93 . Similar results for lung cancer patients were also observed by Zheng et al. 103 , Zhoa et al. 105 and Xing et al. 106 . Their results suggest that a less efficient DNA damage induced G2/M checkpoint is associated with an increased risk of lung cancer. Effect of BPDE exposure on cell cycle Tobacco specific carcinogen BPDE forms adducts with genomic DNA resulting in replication errors that can lead to formation of replicative gaps. These replicative gaps can be repaired during S phase by post-replicative repair pathways 226 . If these gaps remain in the genome due to inefficient DNA repair then they may get converted into DSB in G2 phase 226 . Hence BPDE exposure in normal cells results in S 140
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Genotype-Molecular Phenotype Correl
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CONTENTS Page No Synopsis 1 List of
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Synopsis
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Synopsis Genotype-Molecular Phenoty
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Synopsis Materials and Methods: 1.
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Synopsis loading control. PCR produ
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Synopsis 7.1 Percent cell death aft
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Synopsis slides were then dried and
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Synopsis range 41.77% - 90.95% at 5
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Synopsis 5. Genotyping of genes: Ge
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Synopsis G2/M arrest and delaying e
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Synopsis may have an important bear
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Synopsis References: 1. Bobba, R.;
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Abbreviations MOPS : 3-(N-morpholin
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List of Figures Fig. 28: Percent re
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Chapter 1 Introduction
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Introduction In previous studies fr
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Introduction important carcinogenes
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Review of Literature The yet undeci
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Review of Literature Fig. 2: Incide
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Review of Literature sustained angi
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Review of Literature 2.6 Genotype p
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Review of Literature 2.7.2 Biologic
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Review of Literature Table 1: Steps
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Review of Literature future analysi
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Review of Literature Inefficient ce
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Review of Literature XRCC1 (X-ray r
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Review of Literature 2.9.2 Gene inv
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Review of Literature MPO gene polym
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Review of Literature important role
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Materials and Methods Source of che
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Materials and Methods Method: EBV-t
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Materials and Methods Immunophenoty
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Materials and Methods with 500 μl
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Materials and Methods cDNA it can b
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Materials and Methods Cobalt-60 iso
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Materials and Methods specific bind
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Materials and Methods h half of the
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Materials and Methods phenol and th
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Materials and Methods 7. Filter ste
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Materials and Methods viz. EXO-SAP
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Materials and Methods Table 2: List
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Materials and Methods SMAD6 AREG HM
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Materials and Methods 3.9 Effect of
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Materials and Methods fresh eppendo
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Materials and Methods Power SYBR Gr
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Results Objective 1 To generate EBV
Page 100 and 101: Results Fig. 8: Frequency of second
Page 102 and 103: Results Table 6: Demographics of he
Page 104 and 105: Results Fig. 10: Cell population do
Page 106 and 107: Results Fig. 12: Cell surface marke
Page 108 and 109: Results 4.1.5 Expression of ATM gen
Page 110 and 111: Results Objective 2 To compare the
Page 112 and 113: Results Fig. 17: Standardization of
Page 114 and 115: Results Fig. 19: Standardisation of
Page 116 and 117: Results The cells were incubated fu
Page 118 and 119: Results 4.2.3.1 Percent G2 delay af
Page 120 and 121: Results 4.2.3.2 Effect of BPDE expo
Page 122 and 123: Results Fig. 29: Comparison of perc
Page 124 and 125: Results Fig. 31: Percent cell death
Page 126 and 127: Results normalization with baseline
Page 128 and 129: Results 4.2.5.2 Real time PCR valid
Page 130 and 131: Results homozygous wild-type, heter
Page 132 and 133: Results Fig. 36b: Electrophoresis o
Page 134 and 135: Results Fig. 37a: Representative el
Page 136 and 137: Results Table 13: Consolidated geno
Page 138 and 139: Results LCL Genes NAT2(1) NAT2(2) N
Page 140 and 141: Results Fig. 38: Distribution of Ge
Page 142 and 143: Results Fig. 39: Various combinatio
Page 144 and 145: Results Table 14: Genotype-phenotyp
Page 146 and 147: Discussion Introduction Squamous ce
Page 148 and 149: Discussion stimulated lymphocytes w
Page 152 and 153: Discussion or G2/M phase arrest. As
Page 154 and 155: Discussion differential expression
Page 156 and 157: Discussion DNA repair genes and MPN
Page 158 and 159: Chapter 6 Summary and Conclusions
Page 160 and 161: Summary and Conclusions Statistical
Page 162 and 163: Bibliography 1. Dikshit, R.; Gupta,
Page 164 and 165: Bibliography 43. Lei, D.; Sturgis,
Page 166 and 167: Bibliography 76. Fry, R. C.; Svenss
Page 168 and 169: Bibliography 115. Kote-Jarai, Z.; M
Page 170 and 171: Bibliography 149. Hashibe, M.; Bren
Page 172 and 173: Bibliography 183. Bergamaschi, D.;
Page 174 and 175: Bibliography 216. Bondy, M. L.; Wan
Page 176 and 177: Indian J Med Res 135, June 2012, pp
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Page 186 and 187: CASE REPORT Eben L. Rosenthal, MD,
Page 188 and 189: FIGURE 2. Chromosomal breakage anal
Page 190 and 191: FA along with ataxia telangiectasia
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LIFE09200604007 Tabish - Homi Bhabha National Institute

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