Perfusion-culture-based secreted bioluminescence reporter assay in living cells.
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.
Yamagishi
Kazutoshi
K
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
Enomoto
Toshiteru
T
Ohmiya
Yoshihiro
Y
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
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