LIFE09200604007 Tabish - Homi Bhabha National Institute

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Indian J Med Res 135, June 2012, pp 820-829 Establishment & characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract & healthy individuals <strong>Tabish</strong> Hussain, Ashwin Kotnis, Rajiv Sarin & Rita Mulherkar Advanced Centre for Treatment, Research & Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, India Received July 20, 2011 Background & objectives: A major drawback for genetic studies as well as long-term genotype-phenotype correlation studies in cancer is lack of representative human cell lines providing a continuous source of basic biomolecules and a system to carry out various experimental investigations. This can be overcome to some extent by establishing lymphoblastoid cell lines (LCLs) by infecting peripheral blood lymphocytes with Epstein Barr virus (EBV) which is known to immortalize human resting B cells in vitro giving rise to actively proliferating B-lymphoblastoid cell lines. The present study involves preparation and characterization of LCLs generated from patients with multiple primary neoplasms (MPN) of upper aero-digestive tract (UADT). Methods: Thirty seven LCLs were established from UADT MPN patients and healthy age, sex and habit matched controls using EBV crude stock. Characterization was done with respect to expression of CD- 19 (Pan B-cell marker), CD3 (T cell specific marker), CD56 (NK-cell specific marker), cell morphology, ploidy analysis, genotype and gene expression comparison with the parent lymphocytes. Results: LCLs showed rosette morphology with doubling time of approximately 24 h. Ploidy analysis showed diploid DNA content which was maintained for at least 30 population doublings. When compared with parent lymphocytes there appeared no change at genetic and gene expression level. Interpretation & conclusions: Our results show that lymphoblastoid cell lines are a good surrogate of isolated lymphocytes bearing their close resemblance at genetic and phenotypic level to parent lymphocytes and are a valuable resource for understanding genotype-phenotype interactions. Key words Epstein Barr virus - lymphoblastoid cell lines - multiple primary neoplasia - ploidy analysis - population doubling Squamous cell carcinoma of the upper aerodigestive tract (UADT), comprising head, neck, oesophagus, trachea and lungs, are common cancers worldwide and one of the most common cancers found in Indian men 1 . Further, a 3-7 per cent annual risk of development of a second primary neoplasm among the survivors of early stage UADT cancer poses an additional threat in terms of morbidity and mortality 2 . Cumulative evidence from the case-control studies analyzing polymorphisms in xenobiotic metabolism, 820
HUSSAIN et al: PREPARATION & CHARACTERIZATION OF LYMPHOBLASTOID CELL LINES 821 DNA repair and other gatekeeper mechanisms suggest aberrant gene-environment interactions to be an important aetiological factor in the genesis of multiple primary neoplasm (MPN) 3,4 . Thus it is becoming increasingly important to validate the findings of huge number of genotyping studies in at least a subset of the patients by phenotypic assays. Short term studies to assay chromosomal aberrations, mutagen sensitivity assays and DNA repair kinetics have been carried out using lymphocytes or short term lymphocyte cultures derived from study population 5-7 . However, limited availability of biological material and lack of reproducibility has been the major limitation of such studies. Immortalization of human B lymphocytes by Epstein Barr virus (EBV) in vitro is used routinely to establish lymphoblastoid cell lines (LCLs). Infection by EBV transforms resting B cells from human peripheral blood into actively proliferating LCLs 8 . Unlike SV40 or human papilloma virus (HPV), EBV allows cell immortalization with minimal genetic and phenotypic aberrations, with ease of establishment and maintenance making LCLs ideal material for genotypic and phenotypic studies 8 . These provide 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 along with immunology and cellular biology studies 9-12 . Utility of LCLs is also been very well documented in various population based studies especially investigating in vitro carcinogen sensitivity and DNA damage/ repair 13-15 . Here we report generation of 37 LCLs from patients with UADT MPN and healthy individuals and characterization of a few randomly selected cell lines. Material & Methods This study was approved by the Hospital Ethics Committee, Tata Memorial Centre, Mumbai and conducted during 2005-2009 in the Mulherkar laboratory of Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Navi Mumbai, Maharashtra, India. Samples from healthy, age, sex and habit matched, control individuals were obtained from staff and students of ACTREC, Navi Mumbai. 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 and samples from healthy control individuals were obtained from Advanced Center for Treatment Research and Education in Cancer, Mumbai, India. The study was approved by the Hospital Ethics Committee, Tata Memorial Hospital, Mumbai. UADT MPN patients were recruited on the basis of criteria given by Hong et al 16 as described earlier 17 . Generation of viable EBV stock: EBV-transformed B95-8 marmoset cell line was procured from <strong>National</strong> Centre for Cell Sciences, India and the EBV stock prepared. Briefly, 0.5x10 6 cells/ml were seeded in RPMI-1640, 15 per cent foetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 200 mM glutamine (Sigma-Aldrich Co., USA) and 1X PenStrep (Sigma- Aldrich Co., USA). 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 through 0.22 μM filter (Millipore, Bedford, MA) to obtain EBV crude stock. The filtrate was aliquoted and stored at 4°C for short term or -80°C for long-term storage. Lymphoblastoid cell line preparation: For separation of peripheral blood lymphocytes (PBLs) approximately 3 ml of blood was separated on a Ficoll-Hypaque gradient (Sigma-Aldrich Co., USA). PBLs were seeded in a sterile 24 well plate at a density of 1.5-2x10 6 cells/ ml in Dulbecco’s minimal essential medium (DMEM) containing 15 per cent FBS 200 m M glutamine and IX Pen Strep. EBV crude stock at 1:1 ratio was added and placed in an incubator maintained at 37°C with 5 per cent CO 2 . After 24 h medium containing viral supernatant was aspirated without disturbing the cells and fresh complete DMEM was added. After 3-4 wk of incubation rosette morphology of cells ascertained the transformed phenotype of PBLs. Cells were mixed thoroughly to break clumps before splitting to ensure multiclonal population. All the used plasticware (Nunc, Denmark) was treated as biohazard, discarded in 1 per cent sodium hypochlorite after use and autoclaved wherever required. Characterization of LCL: Characterization of LCLs was done using standard techniques as described below. For all assays freshly grown cells with >90 per cent viability were harvested and washed in 1X PBS at 1500 rpm and used for the genotypic and phenotypic characterization. Immunophenotyping: 1x10 6 cells from LCLs were incubated with primary mouse anti-CD3 (T cell
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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
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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
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
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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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