Tohoku J. Exp. Med., 2015 February, 235(2)

Hypoxia-Sensitive Reporter System for High-Throughput Screening

TADAYUKI TSUJITA,^1,2 SHIN-ICHI KAWAGUCHI,² TAKASHI DAN,² LIAM BAIRD,¹ TOSHIO MIYATA² and MASAYUKI YAMAMOTO¹

¹Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan
²Department of Molecular Medicine and Therapy, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan

The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS.

keywords —— chemical screening; high-throughput screening; hypoxia responsive element; hypoxia stress; reporter cell system

© 2015 Tohoku University Medical Press

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Tohoku J. Exp. Med., 2015, 235, 151-159

Correspondence: Masayuki Yamamoto, Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.

e-mail: masiyamamoto@med.tohoku.ac.jp