LIFE09200604007 Tabish - Homi Bhabha National Institute

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826 INDIAN J MED RES, JUNE 2012 Table II. DNA ploidy status of MPN patient and healthy control cell lines LCLs Channel ratio (LCL/PBL) E245 63.75/49.62 1.28 E246 53.83/49.62 1.08 E247 54.62/49.62 1.10 E248 55.16/50.40 1.09 E249 57.64/49.62 1.16 E250 52.55/49.62 1.06 E242 55.31/49.62 1.11 E252 53.98/49.62 1.09 E265 58.95/48.54 1.21 E267 60.93/48.54 1.26 E270 57.52/50.40 1.14 E304 47.23/46.58 1.01 E305 46.40/46.58 0.99 E306 49.37/46.58 1.05 E307 49.61/46.58 1.06 DNA index PBLs from healthy individual were used as reference. Values in the range 0.9-1.3 are considered diploid produce the B95-8 strain of EBV 22 . Average time taken for LCL establishment was 3-4 wk extending up to 5 wk in some cases. Variation in the time taken for cell line establishment and growth rate can be attributed to difference in the transformation efficiency occurring due to possible batch-wise difference in the viral titre. Once the cell lines were established, these behaved similarly showing comparable morphology, doubling time, genotypic and cell surface characters and behaviour in phenotypic assays. Successful transformation of B cells by EBV resulted in enlargement in size and development of aggregates of proliferative cells. Due to the acquired property of cell aggregation LCLs grew as clumps in suspension cultures, with a mean population doubling time of 24 h and when seeded at a density of 0.5- 1X10 6 /ml needed to be split twice every week. Early passage cells were cryopreserved immediately after transformation. Established LCLs were cryopreserved at later passages as well, but not later than 30 population doublings. EBV transformed LCLs are known to exist in two distinguishable forms pre-immortal and postimmortal. In the pre-immortal stage cells proliferate actively and maintain diploid karyotype. These cells are non tumorigenic and die before reaching 160 population doublings. On the other hand, in the post immortalization stage, EBV transformed cells develop a strong telomerase activity and are aneuploid. These also show cellular changes, gene mutations and have the ability to grow indefinitely 9 . Hence, in the present study, for phenotypic assays LCLs were used within 45-60 PD. Morphological analysis of cell lines by flow cytometry showed two distinct populations although as EBV is known to specifically transform B cell, only one population is expected. The R1 population represents B cells, based on cell morphology and granularity, and R2 population could be proliferating T/NK cells due to immune response elicited by EBV infected B cells. Conventionally the immune suppression of T cells in LCLs is done by supplementing LCL cultures with cyclosporine-A post infection to improve immortalization 8 . However, no cyclosporine A was added in the present study. To study the nature of cells in both clusters immunophenotyping was done using CD19, CD3 and CD56 antibodies in a few representative cell lines. All the cell lines tested were positive for B cell marker in both the clusters (data not shown). The possible reason for occurrence of R2 population could be differential size and granularity of the cells arising due to spontaneous differentiation into smaller lymphoid form with shrunken nucleus. These cells in usual cell culture further undergo apoptosis 20 . Table III. DNA ploidy status of a few MPN patient and healthy control cell lines at higher population doublings (PD) LCLs PD DNA index E304 30 0.97 120 1.00 150 0.93 E305 30 1.05 120 1.18 150 1.17 E311 45 0.99 60 0.93 E313 30 0.97 45 1.14 PBLs from healthy individual were used as reference. Values in the range 0.9-1.3 are considered diploid
HUSSAIN et al: PREPARATION & CHARACTERIZATION OF LYMPHOBLASTOID CELL LINES 827 Fig. 4. Flow cytometry analysis showing the diploid status of PBLs isolated from healthy subject and two representative LCLs E306 and E307. The black arrows in the histogram X- axis represent the channel number of the respective cell cycle stage. Arrow at smaller channel number corresponds to G0/G1 stage of cell cycle while the other arrow corresponds to G2/M stage which is located at approximately double position. Cell immortalization and proliferation in continuous cultures can result in aberrant DNA changes like aneuploidy or tetraploidy. Hence ploidy status of the cell lines was ascertained at both, low and high population doublings ranging from 100 PD taking normal PBL as control. DNA index (DI) ratio was calculated to establish the ploidy status. DI ratio of 1 was considered as diploid 23 while DI values ranging from 1.9-2.1 with proportion of cells greater than the G2/M fraction of normal control, after correction of the aggregates were considered as tetraploid 24 . All the cell lines studied at lower (100 PD) had a DI ratio ranging between 0.9-1.3 and were considered to be diploid. As described in earlier reports EBV remains episomal in lymphoblastoid cell lines maintaining a latent infection, although there is a small subpopulation of cells that switches spontaneously from a latent stage of infection into the lytic cycle. Induction of EBV lytic replication elicits cellular DNA damage response dependent on ATM 19 . DNA damage sensor MRN (Mre II, Rad 50 and Nbsl) complex and phosphorylated ATM are recruited to viral replication compartments, presumably recognizing newly synthesized viral DNAs as abnormal DNA structures 21 . The LCLs in the present study were established with an aim to eventually study the contribution of DNA damage repair in vitro in UADT MPN patients and to elucidate the mechanism involved. Hence it was necessary to ensure that the process of EBV transformation did not affect expression and activity of DNA repair gene ATM. It was observed that EBV transformation did not elicit DNA repair pathway dependent on ATM as there were no pATM foci seen in LCLs (data not shown). This was further confirmed by measuring γH2AX foci in cell lines which was also found to be negative (data not shown). Also there was no change in ATM gene expression in cell lines and PBLs as revealed by semiquantitative RT PCR data. This property of LCLs to be able to grow in continuous culture together with maintaining a close similarity to the parent lymphocytes has been exploited in various studies. There are numerous reports where LCLs have been used as a source of basic biomolecules Fig. 5. Expression of ATM gene in different cell lines and their respective PBLs (a) E302 (b) E303 (c) E313. RT-PCR products were run in 2 per cent agarose gel stained with ethidium bromide. β-actin gene was taken as loading control.
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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
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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
Page 132 and 133: Results Fig. 36b: Electrophoresis o
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Page 136 and 137: Results Table 13: Consolidated geno
Page 138 and 139: Results LCL Genes NAT2(1) NAT2(2) N
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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
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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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