Abstract
BackgroundDespite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation.MethodsA cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system.ResultsFollowing treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation.ConclusionsThe EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.
Highlights
Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay
Development of a cell-based assay system EPISSAY for screening epigenetic drugs The triple-mutated mammalianized version of nfsB, TMnfsB [26], was selected for developing the assay system as it showed the highest sensitivity to the lethal effect of CB1954 (Additional file 2)
EPISSAY, a cell-based assay system for screening of epigenetic drugs was developed based on the human nonmalignant breast epithelial cell line MCF10A expressing the well-characterized CMV promoter driving Red-Fluorescent Protein (RFP) fused with a mammalianized version of the bacterial nitroreductase nfs gene
Summary
Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. An abundance of hypoacetylated histones is usually associated with DNA hyper-methylation and gene silencing [4]. These findings are the basis for the development of HDAC and DNMT. Hydrophobic vorinostat (suberoylanilide hydroxamic acid, SAHA) and hydrophilic decitabine (5-aza-20-deoxycytidine, Dacogen) are US Food and Drug Administration (FDA) approved HDAC and DNMT inhibitors for the treatment of cutaneous T-cell lymphoma and myelodysplastic syndrome, respectively [10,11]. Decitabine is degraded more rapidly in vivo with a half-life of only 25 minutes [13]. Such chemical instability of decitabine has led to its administration in the clinic as a cold and continuous intravenous infusion in an effort to reach the maximal-tolerated doses required to achieve clinical response [14,15]
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