title: Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine.
abstract: BACKGROUND
  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. Here, we describe a cell-based assay system for screening gene reactivation.
  
  
  METHODS
  A 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.
  
  
  RESULTS
  Following 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.
  
  
  CONCLUSIONS
  The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.
authors.affiliation: Cancer Therapeutics Laboratory, Centre for Personalized Cancer Medicine, The University of Adelaide, Adelaide, South Australia, Australia. sue.lim@adelaide.edu.au
authors.forename: Sue Ping
authors.initials: SP
authors.lastname: Lim
authors.forename: Raman
authors.initials: R
authors.lastname: Kumar
authors.forename: Yamini
authors.initials: Y
authors.lastname: Akkamsetty
authors.forename: Wen
authors.initials: W
authors.lastname: Wang
authors.forename: Kristen
authors.initials: K
authors.lastname: Ho
authors.forename: Paul M
authors.initials: PM
authors.lastname: Neilsen
authors.forename: Diego J
authors.initials: DJ
authors.lastname: Walther
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Suetani
authors.forename: Clive
authors.initials: C
authors.lastname: Prestidge
authors.forename: David F
authors.initials: DF
authors.lastname: Callen
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001940:Breast / Q000166:cytology*; D002352:Carrier Proteins / Q000235:genetics; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D019175:DNA Methylation; D000077209:Decitabine; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D044127:Epigenesis, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D053835:Unilamellar Liposomes; D000077337:Vorinostat; D000097573:Red Fluorescent Protein
chemical_list: D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D001374:Azacitidine; D002352:Carrier Proteins; D000077209:Decitabine; D006877:Hydroxamic Acids; D008164:Luminescent Proteins; D009601:Nitroreductases; D012333:RNA, Messenger; D012097:Repressor Proteins; D053835:Unilamellar Liposomes; D000077337:Vorinostat; D000097573:Red Fluorescent Protein; C492515:ANKRD11 protein, human; C486715:TXNIP protein, human