Analytical Biochemistry 359 (2006) 147–149 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.07.013 ANALYTICAL BIOCHEMISTRY Notes & Tips A method for determining the activities of cytokines based on the nuclear transport of nuclear factor-B Sung Bae Kim a,b , Yutaka Natori a , Takeaki Ozawa c,d , Yoshio Umezawa a,¤ , Hiroaki Tao b,¤ a Department of Chemistry, School of Science, University of Tokyo, Tokyo 113-0033, Japan b Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305–8569, Japan c Department of Molecular Structure, Institute for Molecular Science, Aichi 444–8585, Japan d Precursory Research for Embryonic Science and Technology (PREST), Japan Science and Technology Agency, Saitama 332–0012, Japan Received 28 June 2006 Available online 1 August 2006 Gene expressions are controlled by regulatory proteins known as transcription factors. One important transcrip- tion factor is nuclear factor- B (NF-B), 1 which is deeply related to various human diseases. These include cancers, aging, headaches, catabolic disorders, diabetes types 1 and 2, neurodegenerative disorders, neuropathological diseases, inXammatory diseases, viral diseases, ischemia/reperfusion, and pulmonary diseases [1]. Because of the close relation of NF-B with various human diseases, its nuclear transport has been targeted for the development of drugs and risk assessment of synthetic chemicals [1,2]. For instance, a number of chemical inhibitors that have activities against the NF-B–DNA binding have been examined. Cellular stimuli such as proinXammatory cytokines, including tumor necrosis factor- (TNF-) and interleuken-1 (IL- 1), and growth factors can activate NF-B through their speciWc plasma membrane receptors [3]. Therefore, NF-B is an attractive marker protein for the determination of cytokine activities. Cytokines are critical regulators for cellular prolifera- tion, survival, diVerentiation, or apoptosis in relevance to toxicology and immunology. The proinXammatory cyto- kines bind with their speciWc receptors on the plasma mem- brane of cells and Wnally activate NF-B. Expressions of cytokines have been quantiWed with enzyme-linked immu- nosorbent assays (ELISAs) and real-time polymerase chain reaction (PCR) [4,5]. However, these methods are focused on the concentration of cytokines, not on the potential activities or eYcacies of cytokines in physiological condi- tions of living cells. In the current study, we describe a method to quantita- tively determine inXammatory activities of cytokines within 3 h based on the cytokine-induced nuclear transport of NF- B. We constructed a genetically encoded bioluminescent indicator to confer HeLa cells a cytokine sensitivity. For a cytokine-dependent bioluminescence development in HeLa cells, a subunit of NF-B called p50 was fused with the C- terminal fragments of DnaE and split-Renilla luciferase (RLuc). The fusion protein is sequestered in the cytosol as a consequence of binding to a cytoplasmic inhibitor kappa B (IB). On the other hand, the N-terminal fragments of DnaE and split-RLuc are fused with a nuclear localization signal (NLS) and localized in the nucleus of cells (Fig. 1A). In response to proinXammatory cytokines, such as TNF- and interleukin-1, the IBs are rapidly phosphorylated at two speciWc serine residues and resulted in release and translocation of the NF-B with C-terminal fragment of split-RLuc to the nucleus. In the nucleus, it meets the N-ter- minal fragment of split-RLuc. As a consequence of protein splicing, a full-length RLuc is reconstituted and the lumi- nescence activity is restored. The basic reconstitution tech- nology of RLuc was proposed previously by us [6,7]. With the current method, we evaluated the activities of various cytokines based on the nuclear transport of NF-B in human cervical carcinoma-derived HeLa cells carrying the indicators. * Corresponding authors. E-mail addresses: umezawa@chem.s.u-tokyo.ac.jp (Y. Umezawa), hiro- tao@aist.go.jp (H. Tao). 1 Abbreviations used: NF-B, nuclear factor-kappa B; TNF-, tumor ne- crosis factor-; IL-1, interleuken-1; ELISA, enzyme-linked immunosor- bent assay; PCR, polymerase chain reaction; RLuc, Renilla luciferase; IB, inhibitor B; NLS, nuclear localization signal; RLU, relative luminescence units; IL-1RA, interleukin-1 receptor antagonist; PBS, phosphate-buVered saline.