Genomics & Informatics

Korea Genome Organization (한국유전체학회)
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Optimized Internal Control and Gene Expression Analysis in Epstein-Barr Virus-Transformed Lymphoblastoid Cell Lines

  • Nam, Hye-Young (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC)) ;
  • Kim, Hye-Ryun (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC)) ;
  • Shim, Sung-Mi (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC)) ;
  • Lee, Jae-Eun (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC)) ;
  • Kim, Jun-Woo (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC)) ;
  • Park, Hye-Kyung (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC)) ;
  • Han, Bok-Ghee (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC)) ;
  • Jeon, Jae-Pil (National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control & Prevention, Osong Health Technology Administration Complex (OHTAC))
  • Accepted : 2011.08.30
  • Published : 2011.09.30
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Abstract

The Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) is one of the major genomic resources for human genetics and immunological studies. Use of LCLs is currently extended to pharmacogenetic studies to investigate variations in human gene expression as well as drug responses between individuals. We evaluated four common internal controls for gene expression analysis of selected hematopoietic transcriptional regulatory genes between B cells and LCLs. In this study, the expression pattern analyses showed that TBP (TATA box-binding protein) is a suitable internal control for normalization, whereas GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is not a good internal control for gene expression analyses of hematopoiesis-related genes between B cells and LCLs at different subculture passages. Using the TBP normalizer, we found significant gene expression changes in selected hematopoietic transcriptional regulatory genes (downregulation of RUNX1, RUNX3, CBFB, TLE1, and NOTCH2 ; upregulation of MSC and PLAGL2) between B cells and LCLs at different passage numbers. These results suggest that these hematopoietic transcriptional regulatory genes are potential cellular targets of EBV infection, contributing to EBV-mediated B-cell transformation and LCL immortalization.

Keywords

References

  1. Baik, S.Y., Yun, H.S., Lee, H.J., Lee, M.H., Jung, S.E., Kim, J.W., Jeon, J.P., Shin, Y.K., Rhee, H.S., Kimm, K.C., and Han, B.G. (2007). Identification of stathmin 1 expression induced by Epstein-Barr virus in human B lymphocytes. Cell Prolif. 40, 268-281. https://doi.org/10.1111/j.1365-2184.2007.00429.x
  2. Baran-Marszak, F., Fagard, R., Girard, B., Camilleri-Broet, S., Zeng, F., Lenoir, G.M., Raphael, M., and Feuillard, J. (2002). Gene array identification of Epstein Barr virus-regulated cellular genes in EBV-converted Burkitt lymphoma cell lines. Lab. Invest. 82, 1463-1479. https://doi.org/10.1097/01.LAB.0000035025.51772.2B
  3. Cahir-McFarland, E.D., Carter, K., Rosenwald, A., Giltnane, J.M., Henrickson, S.E., Staudt, L.M., and Kieff, E. (2004). Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency III-infected cells. J. Virol. 78, 4108-4119. https://doi.org/10.1128/JVI.78.8.4108-4119.2004
  4. Carter, K.L., Cahir-McFarland, E., and Kieff, E. (2002). Epstein-barr virus-induced changes in B-lymphocyte gene expression. J. Virol. 76, 10427-10436. https://doi.org/10.1128/JVI.76.20.10427-10436.2002
  5. de Kok, J.B., Roelofs, R.W., Giesendorf, B.A., Pennings, J.L., Waas, E.T., Feuth, T., Swinkels, D.W., and Span, P.N. (2005). Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab. Invest. 85, 154-159.
  6. Filby, A.L., and Tyler, C.R. (2007). Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish. BMC Mol. Biol. 8, 10. https://doi.org/10.1186/1471-2199-8-10
  7. Haas, C.S., Creighton, C.J., Pi, X., Maine, I., Koch, A.E., Haines, G.K., Ling, S., Chinnaiyan, A.M., and Holoshitz, J. (2006). Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins. Arthritis Rheum. 54, 2047-2060. https://doi.org/10.1002/art.21953
  8. Hur, D.Y., Lee, M.H., Kim, J.W., Kim, J.H., Shin, Y.K., Rho, J.K., Kwack, K.B., Lee, W.J., and Han, B.G. (2005). CD19 signalling improves the Epstein-Barr virus-induced immortalization of human B cell. Cell Prolif. 38, 35-45. https://doi.org/10.1111/j.1365-2184.2005.00328.x
  9. Janssens, N., Janicot, M., Perera, and T., Bakker, A. (2004). Housekeeping genes as internal standards in cancer research. Mol. Diagnostics Diagn. 8, 107-113. https://doi.org/10.1007/BF03260053
  10. Janz, M., Dorken, B., and Mathas, S. (2006). Reprogramming of B lymphoid cells in human lymphoma pathogenesis. Cell Cycle 5, 1057-1061. https://doi.org/10.4161/cc.5.10.2737
  11. Kang, M.S., Lu, H., Yasui, T., Sharpe, A., Warren, H., Cahir-McFarland, E., Bronson, R., Hung, S.C., and Kieff, E. (2005). Epstein-Barr virus nuclear antigen 1 does not induce lymphoma in transgenic FVB mice. Proc. Natl. Acad. Sci. U.S.A. 102, 820-825. https://doi.org/10.1073/pnas.0408774102
  12. Kelly, G., Bell, A., and Rickinson, A. (2002). Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2. Nat. Med. 8, 1098-1104. https://doi.org/10.1038/nm758
  13. Kilger, E., Kieser, A., and Baumann, M. (1998). Hammerschmidt W. Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor. EMBO J. 17, 1700-1709. https://doi.org/10.1093/emboj/17.6.1700
  14. Landrette, S.F., Kuo, Y.H., Hensen, K., Barjesteh van Waalwijk., van Doorn-Khosrovani, S., Perrat, P.N., Van de Ven, W.J., Delwel, R., and Castilla, L.H. (2005). Plag1 and PLAGL2 are oncogenes that induce acute myeloid leukemia in cooperation with CBFB-MYH11. Blood 105, 2900-2907. https://doi.org/10.1182/blood-2004-09-3630
  15. Lossos, I.S., Czerwinski, D.K., Wechser, M.A., and Levy, R. (2003). Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. Leukemia 17, 789-795. https://doi.org/10.1038/sj.leu.2402880
  16. Massari, M.E., Rivera, R.R., Voland, J.R., Quong, M.W., Breit, T.M., van Dongen, J.J., de Smit, O., and Murre, C. (1998). Characterization of ABF-1, a novel basic helix- loop-helix transcription factor expressed in activated B lymphocytes. Mol. Cell. Biol. 18, 3130-3139. https://doi.org/10.1128/MCB.18.6.3130
  17. Miyoshi, H., Shimizu, K., Kozu, T., Maseki, N., Kaneko, Y., and Ohki, M. (1991). t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. Proc. Natl. Acad. Sci. U.S.A. 88, 10431-10434. https://doi.org/10.1073/pnas.88.23.10431
  18. Natkunam, Y., Lossos, I.S., Taidi, B., Zhao, S., Lu, X., Ding, F., Hammer, A.S., Marafioti, T., Byrne, G.E. Jr. Levy, S., Warnke, R.A., and Levy, R. (2005). Expression of the human germinal center-associated lymphoma (HGAL) protein, a new marker of germinal center B-cell derivation. Blood 105, 3979-3986. https://doi.org/10.1182/blood-2004-08-3112
  19. Ohl, F., Jung, M., Xu, C., Stephan, C., Rabien, A., Burkhardt, M., Nitsche, A., Kristiansen, G., Loening, S.A., Radonic, A., and Jung, K. (2005). Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization? J. Mol. Med. 83, 1014-1024. https://doi.org/10.1007/s00109-005-0703-z
  20. Rho, H.W., Lee, B.C., Choi, E.S., Choi, I.J., Lee, Y.S., and Goh, S.H. (2010) Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR. BMC Cancer 10, 240-252. https://doi.org/10.1186/1471-2407-10-240
  21. Saunders, C.I., Kunde, D.A., Crawford, A., and Geraghty, D.P. (2007). Expression of transient receptor potential vanilloid 1 (TRPV1) and 2 (TRPV2) in human peripheral blood. Mol. Immunol. 44, 1429-1435. https://doi.org/10.1016/j.molimm.2006.04.027
  22. Selvey, S., Thompson, E.W., Matthaei, K., Lea, R.A., Irving, M.G., and Griffiths, L.R. (2001). Beta-actin--an unsuitable internal control for RT-PCR. Mol. Cell. Probes 15, 307-311. https://doi.org/10.1006/mcpr.2001.0376
  23. Shukla, S.J., and Dolan, M.E. (2005). Use of CEPH and non-CEPH lymphoblast cell lines in pharmacogenetic studies. Pharmacogenomics 6, 303-310. https://doi.org/10.1517/14622416.6.3.303
  24. Simon-Sanchez, J., Scholz, S., Fung, H.C., Matarin, M., Hernandez, D., Gibbs, J.R., Britton, A., de Vrieze, F.W., Peckham, E., Gwinn-Hardy, K., Crawley, A., Keen, J.C., Nash, J., Borgaonkar, D., Hardy, J., and Singleton, A. (2007). Genome-wide SNP assay reveals structural genomic variation, extended homozygosity and cell-line induced alterations in normal individuals. Hum. Mol. Genet. 16, 1-14. https://doi.org/10.1093/hmg/ddm004
  25. Spender, L.C., Cornish, G.H., Sullivan, A., and Farrell, P.J. (2002). Expression of transcription factor AML-2 (RUNX3 , CBF(alpha)-3) is induced by Epstein-Barr virus EBNA-2 and correlates with the B-cell activation phenotype. J. Virol. 76, 4919-4927. https://doi.org/10.1128/JVI.76.10.4919-4927.2002
  26. Spender, L.C., Whiteman, H.J., Karstegl, C.E., and Farrell, P.J. (2005). Transcriptional cross-regulation of RUNX1 by RUNX3 in human B cells. Oncogene 24, 1873-1881. https://doi.org/10.1038/sj.onc.1208404
  27. Stranger, B.E., Forrest, M.S., Dunning, M., Ingle, C.E., Beazley, C., Thorne, N., Redon, R., Bird, C.P., de Grassi, A., Lee, C., Tyler-Smith, C., Carter, N., Scherer, S.W., Tavare, S., Deloukas, P., Hurles, M.E., and Dermitzakis, E.T. (2007). Relative impact of nucleotide and copy number variation on gene expression phenotypes. Science 315, 848-853. https://doi.org/10.1126/science.1136678
  28. Theis, T., Skurray, R.A., and Brown, M.H. (2007). Identification of suitable internal controls to study expression of a Staphylococcus aureus multidrug resistance system by quantitative real-time PCR. J. Microbio. Methods 70, 355-362. https://doi.org/10.1016/j.mimet.2007.05.011
  29. Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T., Igout, A., and Heinen, E. (1999). Housekeeping genes as internal standards: use and limits. J. Biotechnol. 7, 291-295.
  30. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research 0034.1-0034.11