LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis starting material we successfully generated continuously growing LCLs following infection of isolated PBLs with EBV-containing supernatants. LCLs showed expression of B cell surface marker (CD19) while markers for T cell (CD3) and NK cells (CD56) were absent. DNA ploidy status of the LCLs demonstrated that they were diploid in nature. Generation of EBV transformed cell lines has proven to be cost effective, rapid and reliable method. A comparison with normal PBLs revealed that these cell lines exhibit minimal deviation from normal phenotype and genotype and can be used as an ideal surrogate of lymphocytes. It is apparent from our results using LCLs that there is a marked difference in γ-radiation and BPDE induced phenotypic responses between MPN patient and healthy controls. Exposure to ionising radiations results in DNA double strand breaks (DSB) and phosphorylation of histone H2AX at ser139 (γH2AX), which is a sensitive marker for measuring DSB. 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 were approximately same. But the extent of DNA repair at 4 h time point was significantly different between the two groups as revealed by residual γ-H2AX foci and percent positive cells at both the radiation doses. This indicates that DNA repair is compromised in UADT MPN patients resulting in accumulation of more DNA damage than healthy individuals after genotoxic exposure and suggests that DNA repair capacity might be a risk factor for UADT MPN development. It is well proven that when a cell is exposed to DNA damaging agents it results in cell cycle arrest and activation of cell cycle check points which play an essential role in DNA repair 10 . The checkpoints provide time for DNA repair before entering into the next phase of cell cycle and prevent cell multiplication with damaged DNA. 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. 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 17 . If these gaps remain in the genome due to inefficient DNA repair then they may get converted into DSB in G2 phase 17 . Hence BPDE exposure in normal cells results in S or G2/M phase arrest. Similarly G2 phase cells are extremely sensitive to ionising radiation induced DNA damage resulting in 15
Synopsis G2/M arrest and delaying entry into mitosis until the damage has been repaired 17 . 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 patients‟ cell lines. Defects in cell cycle G2 delay are strongly associated to human carcinogenesis. Consequently we speculate that individuals with inherited defects in the G2 checkpoint may be predisposed to MPN development. Contrary to the observations following radiation exposure, there was no marked difference in S or G2/M arrest between patient and control group after BPDE exposure. Under normal circumstances when DNA damage is severe and cannot be repaired, an apoptotic response is elicited to eliminate the damaged cell. A failure in proper apoptotic response after extreme genotoxic exposure may also contribute to MPN development. There are reports available where disruption of apoptosis is associated with cancer 11, 14, 18 . We found that after radiation as well as BPDE exposure there was remarkable difference in the apoptotic response between MPN patient and control cell lines which was statistically significant. We believe that low apoptotic response in MPN cases could enable cells with DNA damage to accumulate, possibly conserving mutations in critical genes and inducing carcinogenesis. Differences in cellular responses can be traced back to differential gene expression. In order to find any association between varied phenotypic response after genotoxic exposure and gene expression in our cell lines we carried out expression profiling. Although there was no marked difference in expression of genes falling in important pathways related to cancer development but we indeed observed a variation in expression of genes like MAFB, ZNF429 and HMGA2 that are involved in transcription regulation, along with MAPK10, ARHGAP25, and GNG12 signal transduction genes which may in turn have an effect on downstream molecules. While few other differentially expressed genes like TMEFF1, AREG and SMAD6 are known to be associated with cancers. It is unlikely that such microarray studies will identify the etiology of UADT MPN but we expect that it may help in highlighting pathway defects playing crucial role related to pathogenesis. The differences that were observed in phenotypic response after genotoxic exposure between patient and controls can be attributed to differences in the polygenic susceptibility to genotoxins between the two groups. Genetic variations present in patients may give rise to phenotypes leading to multiple cancers on genotoxic exposure. Therefore we considered it important to compare phenotypic findings with the genotype 16
Page 1: Genotype-Molecular Phenotype Correl
Page 7 and 8: CONTENTS Page No Synopsis 1 List of
Page 9 and 10: Synopsis
Page 11 and 12: Synopsis Genotype-Molecular Phenoty
Page 13 and 14: Synopsis Materials and Methods: 1.
Page 15 and 16: Synopsis loading control. PCR produ
Page 17 and 18: Synopsis 7.1 Percent cell death aft
Page 19 and 20: Synopsis slides were then dried and
Page 21 and 22: Synopsis range 41.77% - 90.95% at 5
Page 23: Synopsis 5. Genotyping of genes: Ge
Page 27 and 28: Synopsis may have an important bear
Page 29 and 30: Synopsis References: 1. Bobba, R.;
Page 31 and 32: Abbreviations MOPS : 3-(N-morpholin
Page 33 and 34: List of Figures Fig. 28: Percent re
Page 35 and 36: Chapter 1 Introduction
Page 37 and 38: Introduction In previous studies fr
Page 39 and 40: Introduction important carcinogenes
Page 41 and 42: Review of Literature The yet undeci
Page 43 and 44: Review of Literature Fig. 2: Incide
Page 45 and 46: Review of Literature sustained angi
Page 47 and 48: Review of Literature 2.6 Genotype p
Page 49 and 50: Review of Literature 2.7.2 Biologic
Page 51 and 52: Review of Literature Table 1: Steps
Page 53 and 54: Review of Literature future analysi
Page 55 and 56: Review of Literature Inefficient ce
Page 57 and 58: Review of Literature XRCC1 (X-ray r
Page 59 and 60: Review of Literature 2.9.2 Gene inv
Page 61 and 62: Review of Literature MPO gene polym
Page 63 and 64: Review of Literature important role
Page 65 and 66: Materials and Methods Source of che
Page 67 and 68: Materials and Methods Method: EBV-t
Page 69 and 70: Materials and Methods Immunophenoty
Page 71 and 72: Materials and Methods with 500 μl
Page 73 and 74: 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
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Results Fig. 8: Frequency of second
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Results Table 6: Demographics of he
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Results Fig. 10: Cell population do
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Results Fig. 12: Cell surface marke
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Results 4.1.5 Expression of ATM gen
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Results Objective 2 To compare the
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Results Fig. 17: Standardization of
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Results Fig. 19: Standardisation of
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Results The cells were incubated fu
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Results 4.2.3.1 Percent G2 delay af
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Results 4.2.3.2 Effect of BPDE expo
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Results Fig. 29: Comparison of perc
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Results Fig. 31: Percent cell death
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Results normalization with baseline
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Results 4.2.5.2 Real time PCR valid
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Results homozygous wild-type, heter
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Results Fig. 36b: Electrophoresis o
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Results Fig. 37a: Representative el
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Results Table 13: Consolidated geno
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Results LCL Genes NAT2(1) NAT2(2) N
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Results Fig. 38: Distribution of Ge
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Results Fig. 39: Various combinatio
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Results Table 14: Genotype-phenotyp
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Discussion Introduction Squamous ce
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Discussion stimulated lymphocytes w
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Discussion H2AX is currently the mo
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Discussion or G2/M phase arrest. As
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Discussion differential expression
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Discussion DNA repair genes and MPN
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Chapter 6 Summary and Conclusions
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Summary and Conclusions Statistical
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Bibliography 1. Dikshit, R.; Gupta,
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Bibliography 43. Lei, D.; Sturgis,
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Bibliography 76. Fry, R. C.; Svenss
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Bibliography 115. Kote-Jarai, Z.; M
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Bibliography 149. Hashibe, M.; Bren
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Bibliography 183. Bergamaschi, D.;
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Bibliography 216. Bondy, M. L.; Wan
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Indian J Med Res 135, June 2012, pp
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822 INDIAN J MED RES, JUNE 2012 mar
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824 INDIAN J MED RES, JUNE 2012 (h)
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826 INDIAN J MED RES, JUNE 2012 Tab
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828 INDIAN J MED RES, JUNE 2012 lik
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CASE REPORT Eben L. Rosenthal, MD,
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FIGURE 2. Chromosomal breakage anal
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FA along with ataxia telangiectasia
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LIFE09200604007 Tabish - Homi Bhabha National Institute

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