LIFE09200604007 Tabish - Homi Bhabha National Institute

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Materials and Methods 3.5 Measurement of DNA damage and repair Formation of DNA double strand breaks triggers phosphorylation of the histone variant H2AX, producing γH2AX. Phosphorylation of H2AX plays a key role in damage repair and is required for the assembly of DNA repair proteins at the sites of DNA damaged as well as for activation of checkpoints proteins required for cell cycle arrest. Analysis of γ-H2AX expression can be used to detect the genotoxic effect of various toxic agents. To assess DNA repair capacity between MPN patient and control groups, LCLs were treated with γ-radiation. For qualitative analysis the experiment was performed by immunofluorescence foci formation assay for γ-H2AX by confocal microscopy and for quantitative measurement the experiment was done by flow cytometry for γ-H2AX positive cells. 3.5.1 Immunofluorescence foci formation assay for γ-H2AX after γ-radiation exposure: Materials: 1. Cobalt-60 isotope source (Bhabhatron-II) 2. 0.1% lysine (Sigma) coated cover slips 3. 4% paraformaldehyde 4. 0.3% TritonX-100 in PBS 5. BSA 6. Anti γ-H2AX primary antibody 7. Anti mouse Alexa-546 secondary antibody 8. DABCO Method: 0.3x10 6 cells were seeded in 1ml complete DMEM in 35 mm culture plates and irradiated with 2 Gy and 5 Gy γ-radiation dose in two sets using a Cobalt-60 isotope source (Bhabhatron-II) at RT. Unirradiated cells treated in the same way served as control. Cells were collected following 30 min incubation (maximum γ-H2AX are seen after 30 min) from one set and 4 h incubation (time taken to repair the DNA) from the other set. Cells were attached, fixed, permeabilized and treated with blocking solution in the same way as described in section 4.4. Cells were incubated with γ- H2AX primary antibody at 1:150 dilution in blocking buffer overnight at 4 o C. Next day cells were washed with PBS and PBST alternately thrice to get rid of any non 63
Materials and Methods specific binding. Cells were further incubated with Alexa-546 secondary antibody at 1:200 dilution in blocking buffer for 1 h at RT. Cells were washed for the second time with PBS and PBST thrice alternately to get rid of nonspecific antibody binding. Nuclei were counterstained with DAPI and to remove excess DAPI cells were washed twice with PBS. Coverslips were mounted on glass slide using Dabco as anti-quenching agent. Edges of the coverslip were sealed with nail paint. γ-H2AX was visualized as red foci on DAPI stained nuclei and acquired on confocal microscope. 3.5.2 Measurement of γ-H2AX by flow cytometry after γ-radiation exposure: Materials: 1. Cobalt-60 isotope source (Bhabhatron-II) 2. 1% PFA 3. FACS buffer (PBS containing 2% FBS and 0.02% Na-azide) 4. 0.3% TritonX-100 in FACS buffer 5. BSA 6. Anti γ-H2AX primary antibody 7. Anti mouse Alexa-488 secondary antibody Method: 0.5x10 6 cells were seeded in 1ml complete DMEM in 35 mm culture plates and irradiated with 2 Gy and 5 Gy γ-radiation in two sets. Unirradiated cells treated in the same way served as control. Following 30 min incubation from one set and 4 h incubation from the other set, cells were collected and washed with FACS buffer and fixed with 1% PFA for 15 min at RT. Cells were washed with PBS and permeabilized by addition of 0.3% TritonX-100 in FACS buffer for 15 min at RT. Cells were again washed with FACS buffer followed by blocking in 3% BSA in FACS buffer for 1 h. Next the cells were collected by centrifuging at 1500 rpm/ 10 min/ RT and incubated with 50 μl γ-H2AX primary antibody at 1:250 dilution in blocking buffer overnight at 4 o C. Next day the cells were washed with FACS buffer and incubated with 50 μl Alexa- 488 secondary antibody at 1:300 dilution in blocking buffer. Cells were once again washed with FACS buffer to get rid of any unbound antibody and further diluted with 200 μl FACS buffer and acquired on flow. Data analysis was done using CellQuest Pro software. 64
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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
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
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: Materials and Methods Cobalt-60 iso
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
Page 88 and 89: Materials and Methods Table 2: List
Page 90 and 91: Materials and Methods SMAD6 AREG HM
Page 92 and 93: Materials and Methods 3.9 Effect of
Page 94 and 95: Materials and Methods fresh eppendo
Page 96 and 97: 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
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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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