LIFE09200604007 Tabish - Homi Bhabha National Institute

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Discussion stimulated lymphocytes which consists mainly of T cell population. A variation in carcinogen sensitivity mainly to γ-radiation between B-lymphocytes compared to T- lymphocytes has been reported 208, 209 . Moreover, differences in the mutagenic response can occur as LCL are exposed in the G1/S/G2 phase of the cell cycle as they are continuously proliferating and undergo continuous cell division; whereas stimulated T lymphocytes remain quiescent. Difference in the efficiency of pathways, owing to differential expression of genes, activated on genotoxic exposure in quiescent and proliferating lymphocytes are known to contribute to the dissimilar sensitivity 210, 211 . Furthermore the type of assay used to compare the sensitivity of LCL with lymphocytes can also give rise to variation due to varied sensitivity of the assays 78 . Therefore care must be taken to ascertain the suitability of using LCL in certain experiments and selecting the type of assay. Despite the studies where the utility of LCL in place of isolated lymphocytes has been shown to be compromised, there are reports which provide evidence for the relevance of their use in biological research. Talebizadeh et al. 212 have assessed the feasibility of using LCL to study the role of miRNAs in the etiology of autism. To test for genotypic errors potentially caused by EBV transformation, Herbeck et al. 213 compared SNP using Affymetrix GeneChip Human Mapping 500k array set, in peripheral blood mononuclear cells (PBMCs) and LCL from the same individuals. They reported that genotypic changes found in PBMCs and LCLs were not significantly different and hence LCL constitute a reliable DNA source for host genotype analysis. Whole-exome sequencing of DNA from PBMCs and EBV transformed lymphocytes from the same donor demonstrated that there occur very minor changes in EBV LCL and that too at a higher population doubling, thus providing evidence that LCL are an appropriate representation of the donor 214 . LCLs as an in vitro model Comparison and characterisation of representative EBV LCLs generated in our study with the respective parent lymphocytes revealed comparable genotypic and phenotypic characteristics in terms of DNA ploidy, population doubling time, expression and activity of ATM gene and cell surface markers. Apart from this, using LCLs generated as a part of previous study in the lab, a comparison was done for the genotype of genes including GSTT1 and GSTM1 between DNA isolated from LCLs and respective PBLs. No difference in the genotype of parent lymphocyte and 137
Discussion respective cell line genotype was observed. All this emphasizes the utility of LCLs as a surrogate for isolated lymphocytes. The cell lines developed in the present study thus provide a valuable, cost effective, in vitro model system for genotypic and phenotypic assays ensuring adequate starting material for current and future analysis. Genotype – phenotype correlation using MPN derived LCLs In order to elucidate a combination of genetic alterations that drive tobacco carcinogenesis, in a previous study from our laboratory, we have explored a unique model system and analytical method for an unbiased qualitative and quantitative assessment of gene-gene and gene-environment interactions 11 . This was the first Indian study associating multiple SNPs in tobacco carcinogenesis in patients with MPN 11 . Current study was undertaken to investigate implications of the same gene polymorphisms on intermediate phenotypes, after γ-radiation and BPDE exposure in vitro and to develop a genotype-phenotype correlation. Both, γ-radiation and BPDE are well known genotoxic and carcinogenic agents 215, 216 . In our study after genotoxic exposure we analyzed phenotypes that are frequently reported to be altered in cancer including DNA damage and repair, cell cycle regulation, percent cell death and gene expression profile. It is evident from our results using LCLs that there is a marked difference in γ-radiation and BPDE induced phenotypic responses between MPN patient and healthy control cell lines. DNA damage and repair after genotoxic exposure Inter-individual differences in DNA repair capacity have been suggested to be an important source of variability in cancer risk 82 . Deficiency in DNA repair capacity contributes to the accumulation of DNA damage and accelerates genetic variations involved in human carcinogenesis. A number of epidemiologic studies, primarily in lung, breast and head and neck cancer suggest association of variation in extent of DNA damage and repair with cancer risk 60, 83-86, 197, 217, 218 . We therefore hypothesized that deficiency in DNA repair capacity following exposure to γ-radiation would be more apparent in patients with UADT than in control subjects. Exposure to ionising radiation results in DNA double strand breaks (DSB) and one of the first cellular responses is the rapid phosphorylation of histone H2AX at ser139 (γ-H2AX), which is a sensitive marker for measuring DNA damage 96, 219 . Measurements of γ-H2AX phosphorylation using fluorescently labelled antibodies to γ- 138
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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
Page 98 and 99: 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 150 and 151: Discussion H2AX is currently the mo
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
Page 178 and 179: 822 INDIAN J MED RES, JUNE 2012 mar
Page 180 and 181: 824 INDIAN J MED RES, JUNE 2012 (h)
Page 182 and 183: 826 INDIAN J MED RES, JUNE 2012 Tab
Page 184 and 185: 828 INDIAN J MED RES, JUNE 2012 lik
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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