title: Perfusion-culture-based secreted bioluminescence reporter assay in living cells.
abstract: Bioluminescence reporter proteins have been widely used in the development of tools for monitoring biological events in living cells. In this study, we describe the development of a reporter system with secreted Cypridina noctiluca luciferase (CLuc) for a pharmacological assay that is based on targeted promoter activity. A model cell line was established with Rat-1 fibroblasts expressing CLuc driven by the promoter of a circadian clock gene, Bmal1. To accurately assay for temporally secreted CLuc activity, a perfusion culture in which the promoter activity was sequentially monitored by the reporter activity in the perfusate was adopted. By pulsing with dexamethasone (DEX), a glucocorticoid (GC) analog, the profile of the reporter activity successfully showed diurnal fluctuation, which is a canonical expression pattern of the Bmal1 gene. Trial studies illustrated that the DEX-pulsed circadian oscillation was reasonably attenuated by RU486, a GC receptor antagonist. Moreover, SP600125, a c-Jun N-terminal kinase inhibitor, caused phase shifting of the rhythmicity. We conclude that the CLuc reporter assay in combination with perfusion culture is a suitable pharmacological tool for drug discovery.
authors.affiliation: Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Midorigaoka, Ikeda, Osaka 563-8577, Japan. kazutoshi@yahoo.co.jp
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Yamagishi
authors.forename: Toshiteru
authors.initials: T
authors.lastname: Enomoto
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
mesh_terms: D056930:ARNTL Transcription Factors; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002940:Circadian Rhythm / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008156:Luciferases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D015735:Mifepristone / Q000494:pharmacology; D010477:Perfusion / Q000379:methods; D011401:Promoter Regions, Genetic; D051381:Rats
chemical_list: D056930:ARNTL Transcription Factors; D000873:Anthracenes; D051792:Basic Helix-Loop-Helix Proteins; D003907:Dexamethasone; D004791:Enzyme Inhibitors; D048031:JNK Mitogen-Activated Protein Kinases; D008156:Luciferases; D015735:Mifepristone; C432165:pyrazolanthrone