Pharmaceutical Research

, Volume 19, Issue 8, pp 1236–1243 | Cite as

Relative Quantification of mRNA Levels in Jurkat T Cells with RT-Real Time-PCR (RT-rt-PCR): New Possibilities for the Screening of Anti-Inflammatory and Cytotoxic Compounds

  • Jürg Gertsch
  • Martin Güttinger
  • Otto Sticher
  • Jörg Heilmann
Article

Abstract

Purpose. Quantification of the pro-inflammatory action of mitogens on mRNA levels of growth-related genes, transcription factors, and cytokines in T cells as markers for the screening of compounds with immunomodulatory, anti-inflammatory or cytotoxic potential.

Method. A reverse transcription-real time-polymerase chain reaction assay with TaqMan probes was developed. Jurkat T cells were treated with cyclosporin A, hypericin, capsaicin, and catechin before phorbol 12-myristate 13-acetate stimulation, and their effects on the relative mRNA levels were determined. A cell viability assay was performed in parallel.

Results. Cyclosporin A and capsaicin were potent inhibitors of PMA-induced cytokine transcription. Cyclosporin A further targeted cyclin D1 transcription. Capsaicin exhibited no effects on the cell viability at low concentrations, whereas cyclosporin A did. Hypericin down-regulated nearly all investigated mRNAs, resulting in a strong time-dependent cytotoxicity. Catechin showed no effects on mRNA levels and cell viability.

Conclusions. The inhibition of the up-regulation of mRNA levels of cytokines points to a specific anti-inflammatory potential of capsaicin. Hypericin showed no specific effects on the mRNA expression. The overall decrease of mRNA levels is probably an early indication of the strong cytotoxic effect observed after 48 h. Therefore, quantification of mRNA levels by reverse transcription-real time-polymerase chain reaction is, in combination with the monitoring of cell viability, a valuable tool to distinguish between specific immunomodulatory and cytotoxic effects in vitro.

RT-real time-PCR cytokines Jurkat T cells NF-κB capsaicin hypericin 

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REFERENCES

  1. 1.
    A. J. Lewis and A. M. Manning. New targets for anti-inflammatory drugs. Curr. Opin. Chem. Biol. 3:489–494 (1999).Google Scholar
  2. 2.
    M. J. Suto and L. Ransone. Novel approaches for the treatment of inflammatory diseases: inhibitors of NF-κB and AP-1. Current Pharm. Design 3:515–528 (1997).Google Scholar
  3. 3.
    A. O'Garra and N. Arai. The molecular basis of T helper 1 and T helper 2 cell differentiation. Rev. Trends Cell. Biol. 10:542–550 (2000).Google Scholar
  4. 4.
    A. Tsuboi, M. Muramatsu, A. Tsusumi, K. Arai, and N. Arai. Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells. Biochem. Biophys. Res. Commun. 199:1064–1072 (1994).Google Scholar
  5. 5.
    S. Gerondakis, M. Grossmann, Y. Nakamura, T. Pohl, and R. Grumont. Genetic approaches in mice to understand Rel/NFkappaB and IkappaB function: transgenics and knockouts. Oncogene 18:6888–6895 (1999).Google Scholar
  6. 6.
    K. K. Mann, S. Doerre, J. J. Schlezinger, D. H. Sherr, and S. Quadri. The role of NF-kappaB as a survival factor in environmental chemical-induced pre-B cell apoptosis. Mol. Pharmacol. 59:302–309 (2001).Google Scholar
  7. 7.
    C. A. Heid, J. Stevens, K. J. Livak, and P. M. Williams. Real time quantitative PCR. Genome Res. 6:986–994 (1996).Google Scholar
  8. 8.
    R. Luthra, J. A. McBridge, F. Cabanillas, and A. Sarris. Novel 5' exonuclease-based real-time PCR assay for the detection of t(14;18)(q32;q21) in patients with follicular lymphoma. Am. J. Pathol. 153:63–68 (1998).Google Scholar
  9. 9.
    T. Takenouchi and E. Munekata. Trophic effects of substance P and beta amyloid peptide on dibutyryl cyclic AMP-differentiated human leukemic (HL60) cells. Life Sci. 56:479–484 (1995).Google Scholar
  10. 10.
    V. F. Quesniaux. Immunosuppressants; tools to investigate the physiological role of cytokines. Bioassays 15:731–739 (1993).Google Scholar
  11. 11.
    D. Piomelli. The ligand that came from within. Trends Pharmacol. Sci. 22:17–19 (2001).Google Scholar
  12. 12.
    Y. Surh. Molecular mechanism of chemopreventive effects of selected dietary and medicinal phenolic substances. Mutat. Res. 428:305–327 (1999).Google Scholar
  13. 13.
    Y. J. Surh, S. S. Han, Y. S. Keum, H. J. Seo, and S. S. Lee. Inhibitory effects of curcumin and capsaicin on phorbol esterinduced activation of eukaryotic transcription factors, NFkappaB and AP-1. Biofactors 12:107–112 (2000).Google Scholar
  14. 14.
    J. Barnes, L. A. Anderson, and J. D. Phillipson. St John's wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. J. Pharm. Pharmacol. 53:583–600 (2001).Google Scholar
  15. 15.
    C. M. Schempp, B. Simon-Haarhaus, C. C. Termeer, and J. C. Simon. Hypericin photo-induced apoptosis involves the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and activation of caspase-8. FEBS Lett. 493:26–30 (2001).Google Scholar
  16. 16.
    P. M. Bork, S. Bacher, M. L. Schmitz, U. Kaspers, and M. Heinrich. Hypericin as a non-antioxidant inhibitor of NF-kappa B. Planta Med. 65:297–300 (1999).Google Scholar
  17. 17.
    R. F. Anderson, L. J. Fisher, Y. Hara, T. Harris, W. B. Mak, L. D. Melton, and J. E. Packer. Green tea catechins partially protect DNA from OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals. Carcinogenesis 22:1189–1193 (2001).Google Scholar
  18. 18.
    K. M. Riedl and A. E. Hagerman. Tannin-protein complexes as radical scavengers and radical sinks. J. Agric. Food Chem. 49: 4917–4923 (2001).Google Scholar
  19. 19.
    U. E. Gibson, C. A. Heid, and P. M. Williams. A novel method for real time quantitative RT-PCR. Genome Res. 6:995–1001 (1996).Google Scholar
  20. 20.
    T. Lindl. Zell-und Gewebekultur, 4. Edition, Spektrum Akademischer Verlag, Berlin, 2000.Google Scholar
  21. 21.
    C. S. Sun, P. A. Ganchi, D. W. Ballard, and W. C. Greene. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science 259:1912–1915 (1993).Google Scholar
  22. 22.
    O. Silvennoinen. Immunodeficiencies and haematological disorders, direct connections to cellular signalling pathways. Rev. Ann. Med. 29:519–529 (1997).Google Scholar
  23. 23.
    K. J. Hardy, B. Manger, M. Newton, and J. D. Stobo. Molecular events involved in regulating human interferon-gamma gene expression during T cell activation. J. Immunol. 138:2353–2358 (1987).Google Scholar
  24. 24.
    A. Weiss, R. L. Wiskocil, and J. D. Stobo. The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL-2 production reflects events occurring at a pretranslational level. J. Immunol. 133:123–128 (1984).Google Scholar
  25. 25.
    R. L. Wiskocil, A. Weiss, J. Imbodden, R. Kamin-Lewis, and J. D. Stobo. Activation of a human T cell line: a two-stimulus requirement in the pretranslational events involved in the coordinate expression of interleukin 2 and gamma-interferon genes. J. Immunol. 134:1599–1603 (1985).Google Scholar
  26. 26.
    O. Bill, C. G. Garlisi, D. S. Grove, G. E. Holt, and A. M. Maestro. IL-2 mRNA levels and degradation rates change with mode of stimulation and phorbol ester treatment of lymphocytes. Cytokine 6:102–110 (1994).Google Scholar
  27. 27.
    V. Depraetere and P. Goldstein. Fas and other cell death signaling pathways. Review. Semin. Immunol. 9:93–107 (1997).Google Scholar
  28. 28.
    R. Wang, L. Zhang, D. Yin, R. A. Mufson, and Y. Shi. Protein kinase C regulates Fas (CD95/APO-1) expression. J. Immunol. 161:2201–2207 (1998).Google Scholar
  29. 29.
    C. C. Wilhide, C. van Dang, J. Dispersio, A. A. Kenedy, and P. F. Bray. Overexpression of cyclin D1 in the Dami megakaryocytic cell line causes growth arrest. Blood 86:294–304 (1995).Google Scholar
  30. 30.
    F. Ajchenbaum, K. Ando, J. A. De Caprio, and J. D. Griffin. Independent regulation of human D-type cyclin gene expression during G1 phase in primary human T lymphocytes. J. Biol. Chem. 268:4113–4119 (1993).Google Scholar
  31. 31.
    A. K. Khanna and J. D. Hosenpud. In vitro and in vivo transfection of p21 gene enhances cyclosporin A-mediated inhibition of lymphocyte proliferation. J. Immunol. 165:1882–1888 (2000).Google Scholar
  32. 32.
    A. K. Khanna and J. D. Hosenpud. Cyclosporine induces the expression of the cyclin inhibitor p21. Transplantation 67:1262–1268 (1999).Google Scholar

Copyright information

© Plenum Publishing Corporation 2002

Authors and Affiliations

  • Jürg Gertsch
  • Martin Güttinger
  • Otto Sticher
  • Jörg Heilmann

There are no affiliations available

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