LIFE09200604007 Tabish - Homi Bhabha National Institute

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Introduction 1.1 Introduction Cancer has been rightfully crowned the „Emperor of all Maladies‟ owing to its increasing incidence, diverse forms and ability to affect various organs across different age groups. Even worse is its intractability to treatment and its irrepressible property to recur. With such dynamic efficacies this sovereign has evaded human understanding despite decades of research and ample funds being invested. This explains why cancer continues to be one of the principal causes of deaths worldwide and also in India, where it accounts for approximately 6% of total human deaths 1, 2 . One of the major cancers prevalent in the country is tobacco-related, Head and Neck Squamous Cell Carcinoma (HNSCC), of which, squamous cell carcinoma of Upper Aero-Digestive Tract (UADT) is the commonest cancer amongst Indian men 2, 3 . Early detection and advancement in diagnostic and treatment modalities has lead to improved disease management and increased survival of patients. However this improvement has not been able to mitigate the development of second/multiple primary neoplasm(s) (MPN) which now looms large as one of the major threats in early stage UADT cancer survivors 4-6 . In recent past increasing number of MPN cases in UADT cancers has become an important therapeutic concern. Survivors of early stage HNSCC have a 10-30% risk of developing second primary tumour in the same or distant region which is one of the prime reasons of increased morbidity and mortality 7, 8 . The most important risk factors for developing such cancers are tobacco habit and alcohol consumption, which have a synergistic effect. These genotoxic agents act by disrupting the genomic integrity leading to malignant transformation and subsequent cancer development. However it is well established that there is a considerable difference between individuals at the genetic level that affect their response to genotoxic insult and thus susceptibility to cancer, which is reflected by the fact that not all individuals exposed to similar type and dose of carcinogen develop cancer. There is a possibility of a different host environment interaction in individuals who develop UADT MPN. This difference could arise due to genetic variations in genes that are involved in carcinogen detoxification, repair of damaged DNA, cell cycle and cell death regulation and thereby, their ability to handle carcinogens. Convincing evidence from case-control studies analyzing cumulative polymorphisms suggests aberrant gene-environment interactions to be an important etiological factor in the genesis of MPN 7, 9-11 . 25
Introduction In previous studies from this laboratory, the ability of key single nucleotide polymorphisms (SNPs) in genes falling in major carcinogenesis pathways, combined with tobacco usage, in predicting the incidence of tobacco related MPN was demonstrated 11, 12 . However, in order to establish involvement of genetic variations, phenotypic analysis is necessary. If genetic polymorphisms in important carcinogenesis pathways like DNA damage/repair, apoptosis, carcinogen metabolism and cell cycle regulation have functional significance then there may be a correlation between genotype and intermediate phenotypes like defect in DNA repair, apoptosis or cell cycle regulation. We hypothesized that MPN is a genetically enriched source and is possibly a manifestation of differences in the polygenic susceptibility to carcinogens. This hypothesis was tested by establishing a correlation between genotype with the molecular phenotype, following in vitro genotoxic exposure. 1.2 Rationale of the study One of the keystones of epidemiological studies is to attempt to classify individuals into high or low risk groups in the context of disease onset and outcomes. This disease risk assessment becomes even more important when early detection of the disease is one the major factors for successful outcomes. Therefore identifying genetic risk factors to understand complex sporadic cancers like UADT cancers is of immense practical relevance as UADT cancers are a major cause of mortality in our country and often patients detected at an early stage return with a second primary cancer. Etiological role of genetic susceptibility in UADT squamous cell carcinomas has been supported by data from recent case control studies based on genotypic and phenotypic assays. The rationale behind gene-cancer risk association studies is that variations at genetic level may result in alterations in intermediate phenotypes leading to inefficient DNA repair or improper cell cycle or cell death control and thereby cancer development. Therefore inter-individual difference in their ability to deal with genotoxic agents can be an important determinant of UADT MPN. This study deals with correlating various gene polymorphisms with intermediate phenotypes and establishing a genotype-phenotype correlation. This may allow us to understand the genetic predisposition of these patients to cancer and understanding gene-environment interactions better at the functional level. Intriguingly, previous studies emphasizing role of SNPs in gene(s) involved in a single pathway with complex disease like cancer show limited predictive value as 26
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 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: Chapter 1 Introduction
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
Page 75 and 76: Materials and Methods Cobalt-60 iso
Page 77 and 78: Materials and Methods specific bind
Page 79 and 80: Materials and Methods h half of the
Page 81 and 82: Materials and Methods phenol and th
Page 83 and 84: Materials and Methods 7. Filter ste
Page 85 and 86: 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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