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Cytotechnology

, Volume 33, Issue 1–3, pp 71–81 | Cite as

Improvement of a method to reproducibly immortalize human T cells by oncogene transfection

  • Shahabuddin Alam
  • Yoshinori Katakura
  • Hiroshi Yoshida
  • Eun-Ho Kim
  • Sanetaka ShirahataEmail author
Article
  • 140 Downloads

Abstract

The method to immortalize human T cells efficiently and reproduciblyby oncogene transfection was improved. T cells were first grown selectively from peripheralblood lymphocytes population of healthy donors andatopic asthma patients, and from lymph nodelymphocytes population of lung cancer patients byactivating with mitogens (phytohemagglutinin andconcanavalin A) and recombinant human interleukin-2(rhIL-2) for five days. Plasmids expressingoncogenes, such as c-Ha-ras, c-myc,c-fos, v-myb and v-jun under the controlof human cytomegalovirus promoter, were then introducedinto these stimulated lymphocytes either separately orin various combinations by electropolation. Afterculturing these transfected lymphocytes for recoveryfor 1 day, they were fed every 3–4 days. Although all the control cells died within one month,oncogene-transfected lymphocytes continued toproliferate actively even for more than severalmonths, indicating that oncogene-transfectedlymphocytes were successfully immortalized. Flowcytometric analyses revealed that most of theimmortalized lymphocytes were T cells expressingCD3+ surface antigen. The ratios of CD4+and CD8+ subpopulations in immortalized T cellsderived from healthy donors varied, depending onthe kinds of oncogenes used. However, CD8+subpopulation in immortalized T cells derived fromcancer patients and atopic asthma patients weredominant, independent of the kinds of oncogenes. These immortalized T cells showed differentproliferative responses in the presence or absence ofexogenous human rhIL-2, depending on their origin ofdonors. Furthermore, immortalized T cells derivedfrom healthy donors showed stronger cytotoxicityagainst K562 cells, suggesting that MHC-nonrestrictedkiller T cells in T cell population were alsoimmortalized. Immortalized T cell lines, whichproliferate continuously without stimulation of amitogen or antigen in medium containing a lowconcentration of rhIL-2, have been maintained for morethan 2 years without any growth rate decrease.

human T cells immortalization oncogenes 

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References

  1. Alam S, Katakura Y and Shirahata S (1999) Establishment of human T cell clones exhibiting natural killer-like activity. Cytotechnology 31: 171–176.Google Scholar
  2. Alam S, Katakura Y, Yoshida H, Kim E-H and Shirahata S (1997) Functional characterization of human T cells immortalized by oncogene transfection. Cytotechnology 23: 185–192.Google Scholar
  3. Borek C (1980) X-ray-induced in vitro neoplastic transformation of human diploid cells. Nature 283: 776–778.Google Scholar
  4. Hastie ND and Alshire RC (1989) Human telomeres: fusion and interstitial sites. Trends Genet 5: 326–331.Google Scholar
  5. Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyzek MA, Shay JW, Ishioka S and Yamakido M (1995) Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 155: 3711–3715.Google Scholar
  6. Jones CL and Kane MA (1996) Oncogenic signaling. Curr Opin Oncol 8: 54–59.Google Scholar
  7. Joneson T and Bar-Saqi D (1997) Ras effectors and their role in mitogenesis and oncogenesis. J Mol Med 75: 587–593.Google Scholar
  8. Murakami H, Masui H, Sato GH, Sueoka N, Chow TP and Kano-Sueoka T (1982) Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proc Natl Acad Sci USA 79: 1158–1162.Google Scholar
  9. Murakami H, Okazaki Y, Yamada K and Omura H (1988) Egg yolk lipoprotein, a new supplement for the growth of mammalian cells in serum-free medium. Cytotechnology 1: 159–169.Google Scholar
  10. Okano F, Tachibana H, Akiyama K and Murakami H (1993a) The detection of a breast cancer antigen on MCF-7 cells reactive with the TCR (alpha) of a specific killer T cell line. Biotherapy 6: 195–203.Google Scholar
  11. Okano F, Tachibana H, Akiyama K, Shirahata S and Murakami H (1993b) Immortalization of human T lymphocytes by oncogenes. Cytotechnology 11: 205–211.Google Scholar
  12. Pereira-Smith OM and Smith JR (1988) Genetic analysis of indefinite division in human cells: Identification of four complementation groups. Proc Natl Acad Sci USA 85: 6042–6046.Google Scholar
  13. Rohme D (1981) Evidence for a relationship between longevity of mammalian species and life spans of normal fibroblasts in vitro and erythrocytes in vivo. Proc Natl Acad Sci USA 78: 5009–5013.Google Scholar
  14. Russo J, Reina D, Frederick J and Russo IH (1988) Expression of phenotypical changes by human breast epithelial cells treated with carcinogens in vitro. Cancer Res 48: 2837–2857.Google Scholar
  15. Shay JW and Wright WE (1989) Quantitation of the frequency of immortalization of normal human diploid fibroblasts by SV40 large T-antigen. Exp Cell Res 184: 109–118.Google Scholar
  16. Smith JR and Pereira-Smith OM (1996) Replicative senescence: implications for in vivo aging and tumor suppression. Science 273: 63–67.Google Scholar
  17. Smith KA (1980) T cell growth factor. Immunol Rev 51: 337–357.Google Scholar
  18. Stevenson M, Volsky B, Hedenskog M and Volsky DJ (1986) Immortalization of human T lymphocytes after transfection of Epstein-Barr virus DNA. Science 233: 981–983.Google Scholar
  19. Troidl B, Simmer B, Fickenscher H, Muller-Fleckenstein I, Emmrich F, Fleckenstein B and Gebhart E (1994) Karyotypic characterization of human T cell lines immortalized by Herpes virus saimiri. Intl. J Cancer 56: 433–438.Google Scholar
  20. Weinberg RA (1985) The action of oncogenes in the cytoplasm and nucleus. Science 230: 770–776.Google Scholar
  21. Wright WE, Pereira-Smith OM and Shay JW (1989) Reversible cellular senescence: Implication for immortalization of normal human diploid fibroblasts. Mol Cell Biol 9: 3088–3092.Google Scholar
  22. Wright WE and Shay JW (1992) The two-stage mechanism controlling cellular senescence and immortalization. Exp Gerontol 27: 383–389.Google Scholar
  23. Yano T, Teruya K, Shirahata S, Watanabe J, Osada K, Tachibana H, Ohashi H, Kim E-H and Murakami H (1994) Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells. Cytotechnology 16: 167–178.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Shahabuddin Alam
    • 1
  • Yoshinori Katakura
    • 1
  • Hiroshi Yoshida
    • 1
  • Eun-Ho Kim
    • 2
  • Sanetaka Shirahata
    • 3
    Email author
  1. 1.Laboratory of Cellular Regulation Technology, Graduate School of Genetic Resources TechnologyKyushu UniversityFukuokaJapan
  2. 2.Department of BiotechnologyKorea UniversityChung NamKorea
  3. 3.Laboratory of Cellular Regulation Technology, Graduate School of Genetic Resources TechnologyKyushu UniversityFukuokaJapan

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