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 Abstract 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. Purpose. 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. Method. 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. Results. 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 Conclusions. in vitro. RT-real time-PCR cytokines Jurkat T cells NF-κB capsaicin hypericin REFERENCES
A. J. Lewis and A. M. Manning. New targets for anti-inflammatory drugs.
Curr. Opin. Chem. Biol.
M. J. Suto and L. Ransone. Novel approaches for the treatment of inflammatory diseases: inhibitors of NF-κB and AP-1.
Current Pharm. Design
A. O'Garra and N. Arai. The molecular basis of T helper 1 and T helper 2 cell differentiation.
Rev. Trends Cell. Biol.
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.
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.
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.
C. A. Heid, J. Stevens, K. J. Livak, and P. M. Williams. Real time quantitative PCR.
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.
T. Takenouchi and E. Munekata. Trophic effects of substance P and beta amyloid peptide on dibutyryl cyclic AMP-differentiated human leukemic (HL60) cells.
V. F. Quesniaux. Immunosuppressants; tools to investigate the physiological role of cytokines.
D. Piomelli. The ligand that came from within.
Trends Pharmacol. Sci.
Y. Surh. Molecular mechanism of chemopreventive effects of selected dietary and medicinal phenolic substances.
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.
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.
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.
P. M. Bork, S. Bacher, M. L. Schmitz, U. Kaspers, and M. Heinrich. Hypericin as a non-antioxidant inhibitor of NF-kappa B.
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.
K. M. Riedl and A. E. Hagerman. Tannin-protein complexes as radical scavengers and radical sinks.
J. Agric. Food Chem.
: 4917–4923 (2001).
U. E. Gibson, C. A. Heid, and P. M. Williams. A novel method for real time quantitative RT-PCR.
T. Lindl. Zell-und Gewebekultur, 4. Edition, Spektrum Akademischer Verlag, Berlin, 2000.
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.
O. Silvennoinen. Immunodeficiencies and haematological disorders, direct connections to cellular signalling pathways.
Rev. Ann. Med.
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.
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.
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.
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.
V. Depraetere and P. Goldstein. Fas and other cell death signaling pathways. Review.
R. Wang, L. Zhang, D. Yin, R. A. Mufson, and Y. Shi. Protein kinase C regulates Fas (CD95/APO-1) expression.
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.
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.
A. K. Khanna and J. D. Hosenpud. In vitro and in vivo transfection of p21 gene enhances cyclosporin A-mediated inhibition of lymphocyte proliferation.
A. K. Khanna and J. D. Hosenpud. Cyclosporine induces the expression of the cyclin inhibitor p21.
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