LIFE09200604007 Tabish - Homi Bhabha National Institute

More documents

Recommendations

Info

Review of Literature lymphoblastoid cell lines by infecting peripheral blood lymphocytes with EBV which is known to immortalize human resting B cells in vitro giving rise to actively proliferating B-lymphoblastoid cell lines 56 . Being a spontaneous replicating source, and exhibiting close resemblance to the parent lymphocytes, EBV transformed LCLs very well fulfil the requirement of constant supply of patient derived cells for a variety of assays sparing the need of re-sampling. LCLs, which carry the complete set of germ line genetic material, have been instrumental in general as an unlimited source of biomolecules like DNA, RNA or proteins and are a promising in vitro model system for genetic screening studies, genotype-phenotype correlation studies, a variety of molecular and functional assays related to immunology and cellular biology studies 57- 59 . Utility of LCLs in in vitro carcinogen sensitivity and DNA damage/repair studies accounts for major segment of such studies and has been very frequently documented 60-62 . This proves the usefulness of LCLs in various genetic and functional studies. 2.7.1 Generation of lymphoblastoid cell lines LCLs are developed by infecting peripheral blood lymphocytes (PBLs) with EBV which has been shown to immortalize human resting B cells in-vitro giving rise to an actively proliferating cell population with minimum genotypic and phenotypic alterations (Table 1, Fig. 4) 56, 63 . EBV is a lymphotropic DNA virus of γ-herpes virus family. Marmoset lymphoblastoid cell line B95-8, which was established by infecting marmoset B lymphocytes with EBV isolated from human patient with infectious mononucleosis, is a constant source for producing transforming virus 56 . LCLs display ease of preparation as well as effortless maintenance and exhibit minimum somatic mutation rate in continuous culture 64 . Presence of complement receptor type 2, commonly known as CR2 (CD21) on B cells creates a route for virus entry into the cell 65 . Viral envelope glycoprotein gp350 binds to CR2 and triggers endocytosis 66 . In addition a second glycoprotein gp42 binds to human leukocyte antigen HLA class II molecule as co-receptor 66 . For B cell immortalization, EBV establishes latent infection mainly existing as covalently closed circular episome with 5-800 copies per cell 56 . This latent infection is characterized by expression of limited number of viral genes. EBV encoded nuclear antigenic protein EBNA2 and latent infection membrane protein LMP1 play crucial role in cell immortalization along with other latent phase proteins, EBNA-1, EBNA-LP, EBNA-3A, EBNA-3C 66, 67 . 36
Review of Literature 2.7.2 Biological characteristics of LCLs Immunophenotyping of LCLs confirms that the cells are positive for B cell marker CD19 and negative for T cell marker CD3 as well as for NK cell marker CD56 63 . Average population doubling time of LCLs is 24 h; they grow as clusters and exhibit typical rosette morphology due to the expression of adhesion molecules leukocyte function antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) 56, 63, 68 . LCLs are commonly considered as „immortal cell lines‟ that can grow for a large number of population doublings retaining a diploid karyotype without becoming tumourigenic, however, this classification of LCLs is slightly ambiguous since most of the LCLs are mortal, named as „preimmortal‟ LCLs because of low level of telomerase activity and shortening of telomeres with each cell division. Although few LCLs truly become immortalized, named as „post immortal‟ LCLs by development of strong telomerase activity and aneuploidy accompanied with other changes including down regulation and mutation of certain genes 69 . Hence it is recommended that LCLs be used within 2-3 months, which is far less than 180 population doublings assuming cell doubling time as 24 h, so as to minimise the undesirable and questionable effects of post immortalization 57 . 2.7.3 Utility of lymphoblastoid cell lines for genetic and functional studies LCLs have been used as a source of basic biomolecules like, DNA, including mitochondrial DNA, RNA and protein 57, 70 . DNA isolated from LCLs has been widely used for mutation analysis 70, 71 , while RNA isolated from these cell lines has been commonly used for cDNA library preparation and to assess transcriptional response to genotoxins using high throughput technologies including cDNA microarray 72, 73 together with this LCL have as well been used for proteomic studies 74, 75 . In addition, in a number of other reports, LCL have been extensively used to measure DNA damage and repair as well as apoptosis 60-62 . To a great extent LCLs have also been used to assess inter-individual variation in response to DNA-damaging agents and to develop correlation between DNA repair genes and repair capacity and analysing the relationship with cancer risk 54, 76, 77 (Fig. 5). 37
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 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: Review of Literature 2.6 Genotype p
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
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
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 152 and 153:
Discussion or G2/M phase arrest. As
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
Page 180 and 181:
824 INDIAN J MED RES, JUNE 2012 (h)
Page 182 and 183:
826 INDIAN J MED RES, JUNE 2012 Tab
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
show all

LIFE09200604007 Tabish - Homi Bhabha National Institute

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?