LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis cycle regulation and carcinogen metabolism and created a Genotype Score (G Score) from the number of variant alleles. Intermediate phenotypes including transient arrest in cell cycle, proper function of DNA damage repair machinery and activation of cell death program upon exposure to genotoxic agents are known to be disrupted in variety of cancers and are associated with neoplastic evolution 10-14 . Hence we assumed that individuals with inherited defects in cell cycle control, apoptosis, and/or DNA repair, owing to interindividual difference at the genetic level, might be susceptible to UADT MPN development. Hence we have correlated these genotypic and phenotypic measurements to investigate their potential relationships to have a better understanding of pathogenesis of UADT MPN. Aim: To develop a correlation between genotype (polymorphisms in candidate genes involved in DNA repair, apoptosis, cell cycle regulation and carcinogen metabolism) with the phenotype (defect in DNA repair, apoptosis or cell cycle regulation) using lymphoblastoid cell lines established from MPN patients and healthy controls following genotoxic exposure in vitro. Objectives: 1. To generate EBV LCLs from peripheral blood lymphocytes (PBLs) isolated from MPN patients and healthy controls-an established in vitro model for genetic studies. 2. To compare the response of MPN patients with tobacco habits and appropriate controls in vitro, to the exposure to DNA damaging agents such as γ-radiations and Benzo[a]pyrene-diol-epoxide (BPDE, a tobacco specific carcinogen), by assessing DNA damage and repair, cell cycle profiling, apoptosis and global gene expression profiling. 3. To genotype selected candidate genes involved in DNA repair, carcinogen metabolism, apoptosis and cell cycle regulation. 4. To establish a correlation between the phenotype (e.g., poor DNA repair capacity, apoptosis) with the genotype (e.g., polymorphisms in the genes). 3
Synopsis Materials and Methods: 1. Collection of samples: After obtaining IRB approval and patient informed consent, 3 ml whole blood was collected in an EDTA vacutainer from patients with MPN and cancer free healthy individuals by venipuncture. Patients with MPN were accrued from the Cancer Genetics Clinic in Tata Memorial Hospital (TMH) and samples from healthy control individuals were obtained from Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Mumbai, India. The study was approved by the Hospital Ethics Committee, TMH. UADT MPN patients were recruited on the basis of criteria given by Hong et al. where they met one of the following standards 15 : (i) The second primary tumour (SPT) should not be the recurrence and regional or distant metastasis of the first primary tumour, (ii) the histology of SPT should be different from the first primary tumour, (iii) if the histology is same the SPT should be separated from first primary by more than 2 cm of normal epithelium, (iv) or has to occur at least three years after the diagnosis of the first primary tumour. Individuals were accrued as controls if they had no personal history of cancer and consented to participate in the study. 2. Generation of viable EBV stock: EBV-transformed B95-8 marmoset cell line was procured from <strong>National</strong> Centre for Cell Science (NCCS), India and the EBV stock prepared. Briefly, 0.5x10 6 cells/ml were seeded in RPMI-1640, 15% FBS, 200 mM glutamine and 1X PenStrep. After 7 days confluent cultures of B95-8 appearing straw yellow in colour were lysed by freeze thawing at -80 °C and 37 °C and filtered to obtain EBV crude stock. The filtrate was stored at 4 C for short term or -80 C for long-term storage. 3. Lymphoblastoid cell line preparation: For separation of PBLs blood was separated on a Ficoll-Hypaque gradient. PBLs were seeded in a 24 well plate in complete medium (DMEM containing 15% FBS, 200 mM glutamine and 1X PenStrep). Crude stock of EBV at 1:1 ratio was added to the PBLs and incubated at 37 C with 5% CO 2 . After 24 h, medium containing viral supernatant was aspirated without disturbing the cells and fresh complete DMEM was added. After 3-4 weeks of incubation, rosette morphology of cells ascertained the transformed phenotype of PBL. Cells were mixed thoroughly to break clumps before splitting to ensure multiclonal population. 4
Page 1: Genotype-Molecular Phenotype Correl
Page 7 and 8: CONTENTS Page No Synopsis 1 List of
Page 9 and 10: Synopsis
Page 11: Synopsis Genotype-Molecular Phenoty
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 and 24: Synopsis 5. Genotyping of genes: Ge
Page 25 and 26: Synopsis G2/M arrest and delaying e
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
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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
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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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