Establishment of a high-throughput detection system for DNA demethylating agents

ABSTRACT

Epigenetic alterations underlie various human disorders, including cancer, and this has resulted in the development of drugs targeting epigenetic alterations. Although DNA demethylating agents are one of the major epigenetic drugs, only two compounds—5-azacytidine (5-aza-CR, azacitidine) and 5-aza-2'-deoxycytidine (5-aza-dC, decitabine)—have obtained clinical approval. Here, we aimed to establish a detection system for DNA demethylating agents suitable for a high-throughput screening (HTS) in mammalian cells. We inserted luciferase and EGFP reporter genes under the UCHL1 promoter, which is methylation-silenced in human colon cancers and can be readily demethylated to drive strong expression. Methylated UCHL1 promoter was introduced into HCT116 colon cancer cells, and transfectants with methylated exogenous UCHL1 promoter were obtained. By screening subclones from each of the epigenetically heterogeneous transfectant clones, we finally obtained three optimal subclones that expressed luciferase and EGFP after 5-aza-dC treatment with high signal-to-noise ratios. Nucleosomes with H3K9me2 were present around the exogenous UCHL1 promoter in all three subclones. Using one of the subclones (HML58-3), HTS was conducted using 19,840 small molecules. Two hit compounds were obtained, and these turned out to be 5-aza-dC and 5-aza-CR. The assay system constructed here demonstrates a robust response to DNA demethylating agents, along with high specificity, and will be useful for screening and biological assays in epigenetics.

KEYWORDS:

• Epigenetics, DNA methylation
• DNA demethylating agent
• mammalian cells
• luciferase
• high-throughput screening

Acknowledgement

The authors are grateful to Dr. Hiroyuki Osada (RIKEN Center for Sustainable Resource Science) for providing the NPDepo chemical library.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the National Cancer Center Research and Development Fund [grant number 26-A-15].