LIFE09200604007 Tabish - Homi Bhabha National Institute

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Discussion or G2/M phase arrest. As described above, in response to carcinogen exposure, cell cycle checkpoint responses act and suppress initiation of carcinogenesis and therefore aberrant cell cycle control has been associated with neoplastic evolution. It is also been reported that BPDE induces over expression of CDC25B in cancer cells which is known for cell cycle regulation specifically essential for G2/M transition 231 . Therefore we assumed it important to assess the cell cycle profile after BPDE exposure along with gamma radiation in UADT MPN patients and compare it with controls to find any probable difference in cell cycle profile that can be attributed to MPN predisposition. Contrary to our observations following radiation exposure, there was no marked difference in S or G2/M arrest between patient and control group after BPDE exposure. One of the probable reasons for this observation could be a small sample size. As we have performed this experiment only in 20 MPN and 10 controls cell lines therefore it is possible that effect of BPDE exposure on cell cycle arrest is not so distinctive that it can significantly distinguish between smaller MPN and control groups. Another very important reason for our observation could be dependent on p53 status. In a study done by Xiao et al. 227 , it was observed that tumour suppressor p53 plays an important role in regulation of cellular responses to BPDE. p53 tumour suppressor is a well known mutational target of tobacco carcinogen BPDE 228 . They observed that exposure of p53 null H1299 human lung cancer cells to BPDE resulted in S and G2 phase cell cycle arrest. A similar BPDE exposure failed to activate either S or G2 phase checkpoint in H460 human lung cancer cell line which is wild type for p53. On the contrary H460 cell line was relatively more sensitive to BPDE mediated cell death as compared to H1299 cells. 227 . This implies that BPDE induced cell cycle arrest is probably not an appropriate marker for risk assessment in UADT MPN development as the effect is modulated by p53 status. However, as γ-radiation induced G2 delay was a significantly compromised in MPN cell lines, therefore, individuals with inherited defects in the G2 checkpoint may be predisposed to MPN development. Apoptotic response after genotoxic exposure Under normal circumstances when cell encounters DNA damage, a cascade of reactions takes place resulting first in cell cycle arrest followed by DNA repair so that the cell with unrepaired DNA is not replicated. However in cases where the damage is severe and cannot be repaired, the cells will undergo apoptosis or programmed cell 141
Discussion death which is a safe way to inhibit risk acquiring neoplastic autonomy 107 . Therefore 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 51, 53, 104, 105 . We observed that after radiation as well as BPDE exposure there was a significant difference in the apoptotic response between MPN and control cell lines. Our results reveal that UADT MPN patients have a reduced apoptotic capacity following an in vitro challenge by γ-radiation and BPDE which can be associated with risk of multiple primary cancers. 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. There are reports demonstrating a reduced apoptotic capacity in individuals affected with cancer. A study done by Zheng et al 104 showed a lower level of apoptosis, along with defects in cell cycle regulation as a biomarker of genetic susceptibility for salivary and thyroid carcinoma. Similarly, defective apoptotic response after γ-radiation has been associated with lung cancer risk and is suggested to be a susceptibility marker 53, 105 . Alteration in the apoptotic pathway as a risk factor for lung cancer has also been observed by Wang et al. where they assessed cell death after BPDE exposure 51 . Our results support the hypothesis that defects in apoptotic response can be attributed as a risk factor for UADT MPN development. Differential gene expression after γ-radiation exposure Differences in cellular responses after genotoxic exposure can be attributed to differential gene expression. Differential expression of genes in important carcinogenesis pathways can be considered as driving malignant changes 111-113 . Deregulated expression of DNA repair genes in pathways like nucleotide excision repair is found to be associated with in lung cancer 111 . A more than 2 fold increased of risk of head and neck cancer has been observed in individuals expressing lower levels of nucleotide excision repair genes 112 . Similarly, in a study done on Indian population reduced expression of DNA repair genes was observed in head and neck cancers 113 . However most of these studies were done by classical way of selecting known genes in important pathways already identified to be altered during malignant transformation. In order to explore the involvement of novel molecules in cancer predisposition there is a need to measure global gene expression alterations for which cDNA microarrays provide an obvious method of choice 109, 110 . The association of 142
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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
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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 150 and 151: Discussion H2AX is currently the mo
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
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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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LIFE09200604007 Tabish - Homi Bhabha National Institute

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