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Vol. 84, No. 3, 2009
Issue release date: September 2009
Pharmacology 2009;84:135–144
(DOI:10.1159/000235158)

Validation of a HeLa Mx2/Luc Reporter Cell Line for the Quantification of Human Type I Interferons

Seo Y.-J. · Kim G.-H. · Kwak H.-J. · Nam J.-S. · Lee H.J. · Suh S.-K. · Baek K.-M. · Sohn Y. · Hong S.-H.
aAdvanced Therapy Products Research Division, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration and bEwha Womans University, Pharmacy College, Seoul, Republic of Korea

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Abstract

Although antiviral assays have been the most widely available biological assays for interferons (IFNs), they are less sensitive and provide considerable interassay variation. In this study, we demonstrate a new reporter cell line, which is based on HeLa cells transfected with a plasmid containing a human Mx2 promoter driving a luciferase (Luc) cDNA. To characterize the specific gene expression profiles induced by interferon alpha, we analyzed the microarray results of interferon response gene expression induced by IFN-α2a or IFN-α2b treatment with HeLa cells. We found that the Mx2 gene increased the most by treatment with both IFN-α2a and IFN-α2b. Based on this result, we designed a reporter cell line, HeLa-Mx2, suitable for determination of IFN-α. HeLa cells were stably transfected with the luciferase gene under the control of Mx2 promoter. The expression of luciferase can be easily measured for luminescence using a 96-well luminometer and has been correlated with the concentration of added IFN and cell density. In the validation results, our reporter cell line had specificity for type I IFN, but the significant effects of a number of other cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-γ) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa-Mx2 cell culture’s age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-α2a in the range of 1–10,000 IU/ml. The 95% confidential limit and total coefficient of variation estimates ranged between 96 and 116 and 10.51% in the reducible range mentioned above, respectively. In conclusion, we established a stable IFN-responsible HeLa-Mx2 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-α activity.



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References

  1. Isaacs A, Lindenmann J: Virus interference. I. The interferon. Proc R Soc Lond B Biol Sci 1957;147:258–267.
  2. Pestka S, Langer JA, Zoon KC, Samuel CE: Interferons and their actions. Annu Rev Biochem 1987;56:727–777.
  3. Pestka S, Krause CD, Walter MR: Interferons, interferon-like cytokines, and their receptors. Immunol Rev 2004;202:8–32.
  4. LaFleur DW, Nardelli B, Tsareva T, Mather D, Feng P, Semenuk M, Taylor K, Buergin M, Chinchilla D, Roshke V, Chen G, Ruben SM, Pitha PM, Coleman TA, Moore PA: Interferon-kappa, a novel type i interferon expressed in human keratinocytes. J Biol Chem 2001;276:39765–39771.
  5. Oritani K, Medina KL, Tomiyama Y, Ishikawa J, Okajima Y, Ogawa M, Yokota T, Aoyama K, Takahashi I, Kincade PW, Matsuzawa Y: Limitin: an interferon-like cytokine that preferentially influences b-lymphocyte precursors. Nat Med 2000;6:659–666.
  6. Roberts RM, Ealy AD, Alexenko AP, Han CS, Ezashi T: Trophoblast interferons. Placenta 1999;20:259–264.
  7. Pestka S: The interferons: 50 years after their discovery, there is much more to learn. J Biol Chem 2007;282:20047–20051.
  8. Pestka S: The human interferon alpha species and receptors. Biopolymers 2000;55:254–287.
  9. Hilkens CM, Schlaak JF, Kerr IM: Differential responses to IFN-alpha subtypes in human T cells and dendritic cells. J Immunol 2003;171:5255–5263.
  10. Ortaldo JR, Herberman RB, Harvey C, Osheroff P, Pan YC, Kelder B, Pestka S: A species of human alpha interferon that lacks the ability to boost human natural killer activity. Proc Natl Acad Sci USA 1984;81:4926–4929.
  11. Meager A: Biological assays for interferons. J Immunol Methods 2002;261:21–36.
  12. Meager A: Assays for antiviral activity. Methods Mol Biol 2004;249:121–134.
  13. Lleonart R, Näf D, Browning H, Weissmann C: A novel, quantitative bioassay for type I interferon using a recombinant indicator cell line. Biotechnology (NY) 1990;8:1263–1267.
  14. Canosi U, Mascia M, Gazza L, Serlupi-Crescenzi O, Donini S, Antonetti F, Galli G: A highly precise reporter gene bioassay for type I interferon. J Immunol Methods 1996;199:69–76.
  15. Hammerling U, Bongcam-Rudloff E, Setterblad N, Kroon R, Rehnstrom AK, Viitanen E, Andersson G, Sjodin L: The beta-gal interferon assay: a new, precise and sensitive method. J Interferon Cytokine Res 1998;18:451–460.
  16. Bollati-Fogolin M, Muller W: Virus free, cell-based assay for the quantification of murine type I interferons. J Immunol Methods 2005;306:169–175.
  17. Smilovic V, Caserman S, Fonda I, Gaberc-Porekar V, Menart V: A novel reporter gene assay for interferons based on cho-k1 cells. J Immunol Methods 2008;333:192–196.
  18. Taylor MW, Grosse WM, Schaley JE, Sanda C, Wu X, Chien SC, Smith F, Wu TG, Stephens M, Ferris MW, McClintick JN, Jerome RE, Edenberg HJ: Global effect of PEG-IFN-alpha and ribavirin on gene expression in PBMC in vitro. J Interferon Cytokine Res 2004;24:107–118.
  19. McClintick JN, Jerome RE, Nicholson CR, Crabb DW, Edenberg HJ: Reproducibility of oligonucleotide arrays using small samples. BMC Genomics 2003;4:4.
  20. (COE) CoEE, (EDQM) EDftQoM: 5.3. Statistical Analysis of Results of Biological Assays and Tests. European Pharmacopoeia, ed 6. 2007.
  21. Son YW, Shin W, Jung JY, Oh IW, Jin JH, Park SY, Park TS: Study on recombinant interferon-alpha standardization. Annu Rep KFDA 2000;4:598–606.
  22. Pfeffer LM, Dinarello CA, Herberman RB, Williams BR, Borden EC, Bordens R, Walter MR, Nagabhushan TL, Trotta PP, Pestka S: Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons. Cancer Res 1998;58:2489–2499.
  23. Haller O, Stertz S, Kochs G: The Mx GTPase family of interferon-induced antiviral proteins. Microbes Infect 2007;9:1636–1643.
  24. Platanias LC: Mechanisms of type-i- and type-ii-interferon-mediated signalling. Nat Rev Immunol 2005;5:375–386.
  25. Sanda C, Weitzel P, Tsukahara T, Schaley J, Edenberg HJ, Stephens MA, McClintick JN, Blatt LM, Li L, Brodsky L, Taylor MW: Differential gene induction by type I and type II interferons and their combination. J Interferon Cytokine Res 2006;26:462–472.
  26. Oritani K, Kincade PW, Tomiyama Y: Limitin: an interferon-like cytokine without myeloerythroid suppressive properties. J Mol Med 2001;79:168–174.


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