Current Chemical Genomics and Translational Medicine

2009, 3 : 1-6
Published online 2009 February 05. DOI: 10.2174/1875397300903010001
Publisher ID: CCGTM-3-1

RESEARCH ARTICLE
 HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway

Michael K Hancock1 , Menghang Xia2 , Elizabeth S Frey1 , Srilatha Sakamuru2 and Kun Bi, *,1
1 Invitrogen Corporation, Discovery Assays and Services, 501 Charmany Drive, Madison, WI 53719, USA
2 NIH Chemical Genomics Center, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD 20893, USA

* Address correspondence to this author at the Invitrogen Corporation, Discovery Assays and Services, 501 Charmany Drive, Madison, WI 53719, USA; E-mail: kun.bi@invitrogen.com

ABSTRACT

Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR.