Elsevier

Analytical Biochemistry

Volume 375, Issue 2, 15 April 2008, Pages 237-248
Analytical Biochemistry

A quantitative high-throughput screen identifies potential epigenetic modulators of gene expression

https://doi.org/10.1016/j.ab.2007.12.028Get rights and content

Abstract

Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced green fluorescent protein (GFP) reporter to identify novel classes of compounds involved in epigenetic regulation. This locus derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration–response method that assays compounds at multiple concentrations. From structure–activity relationships of the 411 actives recovered from the qHTS, 6 distinct chemical series were chosen for further study. A total of 48 qHTS actives and analogs were counterscreened using the parental line of the LDR cells, which lack the GFP reporter. Three series—8-hydroxy quinoline, quinoline-8-thiol, and 1,3,5-thiadiazinane-2-thione—were not fluorescent and reconfirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS.

Section snippets

Cell culture and general reagents

C127 (a kind gift from Gordon Hager) and locus derepression (LDR) 6103 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with l-glutamine and high glucose, 1 mM sodium pyruvate, 0.1 mM minimal essential medium (MEM) nonessential amino acids, 1% (v/v) penicillin–streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). All lung cancer cells (provided by John D. Minna) were maintained in RPMI 1640 media supplemented with 10% FBS. Immortalized normal human bronchial

Results

The LDR assay detects the derepression of a GFP reporter that is stably integrated in the mouse mammary carcinoma line, C127. In the vector, GFP transcription is controlled by a cytomegalovirus (CMV) promoter, which normally is strong and constitutively active. However, this cell line (referred to as LDR cells) was selected for lack of constitutive expression of the GFP reporter [12], presumably due to epigenetic silencing of the integration locus and/or methylation of the CMV promoter. GFP

Discussion

In this study, we used a cell-based imaging assay to screen for small molecules that activate expression of a silenced locus. Using qHTS, we identified six chemically distinct series of compounds that derepress or induce the GFP–GER reporter. Follow-up experiments revealed that compounds within three series were true reproducible actives, one series was inconclusive, and two series were false positive fluorescent. Compounds from each of the three active series did not inhibit HDAC enzymatic

Acknowledgments

We gratefully acknowledge Sam Michael and Carleen Klumpp for automation assistance, Ruili Huang for informatics support, Paul Shinn and Adam Yasgar for compound management, Jennifer Wichterman and Jianjun Chang for experimental assistance, Gordon Hager and John D. Minna for provision of cells, and Craig Thomas and David Shames for helpful discussions. This research was supported by the NIH Roadmap for Medical Research (through resources awarded under an X01 mechanism), by the National Cancer

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