Validation of a HeLa Mx2/Luc Reporter Cell Line for the Quantification of Human Type I InterferonsSeo Y.-J.a · Kim G.-H.a, b · Kwak H.-J.a · Nam J.-S.a · Lee H.J.b · Suh S.-K.a · Baek K.-M.a · Sohn Y.a · Hong S.-H.a
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
Do you have an account?
- Rent for 48h to view
- Buy Cloud Access for unlimited viewing via different devices
- Synchronizing in the ReadCube Cloud
- Printing and saving restrictions apply
Rental: USD 8.50
Cloud: USD 20.00
Article / Publication Details
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.
© 2009 S. Karger AG, Basel
- Isaacs A, Lindenmann J: Virus interference. I. The interferon. Proc R Soc Lond B Biol Sci 1957;147:258–267.
- Pestka S, Langer JA, Zoon KC, Samuel CE: Interferons and their actions. Annu Rev Biochem 1987;56:727–777.
- Pestka S, Krause CD, Walter MR: Interferons, interferon-like cytokines, and their receptors. Immunol Rev 2004;202:8–32.
- 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.
- 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.
- Roberts RM, Ealy AD, Alexenko AP, Han CS, Ezashi T: Trophoblast interferons. Placenta 1999;20:259–264.
- Pestka S: The interferons: 50 years after their discovery, there is much more to learn. J Biol Chem 2007;282:20047–20051.
- Pestka S: The human interferon alpha species and receptors. Biopolymers 2000;55:254–287.
- 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.
- 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.
- Meager A: Biological assays for interferons. J Immunol Methods 2002;261:21–36.
- Meager A: Assays for antiviral activity. Methods Mol Biol 2004;249:121–134.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- McClintick JN, Jerome RE, Nicholson CR, Crabb DW, Edenberg HJ: Reproducibility of oligonucleotide arrays using small samples. BMC Genomics 2003;4:4.
(COE) CoEE, (EDQM) EDftQoM: 5.3. Statistical Analysis of Results of Biological Assays and Tests. European Pharmacopoeia, ed 6. 2007.
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.
- 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.
- Haller O, Stertz S, Kochs G: The Mx GTPase family of interferon-induced antiviral proteins. Microbes Infect 2007;9:1636–1643.
- Platanias LC: Mechanisms of type-i- and type-ii-interferon-mediated signalling. Nat Rev Immunol 2005;5:375–386.
- 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.
- Oritani K, Kincade PW, Tomiyama Y: Limitin: an interferon-like cytokine without myeloerythroid suppressive properties. J Mol Med 2001;79:168–174.
Article / Publication Details
Copyright / Drug Dosage / DisclaimerCopyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.