title: Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D, and xenograft models.
abstract: Aberrant DNA methylation that results in silencing of genes has remained a significant interest in cancer research. Despite major advances, the success of epigenetic therapy is elusive due to narrow therapeutic window. A wide variety of naturally occurring epigenetic agents and synthetic molecules that can alter methylation patterns exist, however, their usefulness in epigenetic therapy remains unknown. This underlines the need for effective tumor models for large-scale screening of drug candidates with potent hypomethylation activity. In this study, we present the development of a cell-based DNA demethylation detection system, which is amenable for high content screening of epigenetic drugs in two-dimensional and three-dimensional cell culture models. Additionally, the detection system also supports the in vivo monitoring of demethylation efficacy of potential lead compounds from in vitro screens in tumor xenografts. The described detection system not only permits the continuous monitoring of demethylation but also of the induced cytostatic/cytotoxic drug effects in live cells, as a function of time. The detection system is fluorescence based and exploits the dominant ability of DNA methylation to inhibit gene transcription, and utilizes FLJ32130 gene, which is silenced on account of promoter hypermethylation in human colorectal cancer. The described work will provide the researchers with an efficient tool for epigenetic drug screens on a high throughput platform and would therefore benefit academic and industrial drug discovery. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hněvotínská 5, 77900, Olomouc, Czech Republic.
authors.forename: Khushboo
authors.initials: K
authors.lastname: Agrawal
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hněvotínská 5, 77900, Olomouc, Czech Republic.
authors.forename: Viswanath
authors.initials: V
authors.lastname: Das
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i, Flemingovo náměstí 2, 16610, Prague 6, Czech Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Otmar
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i, Flemingovo náměstí 2, 16610, Prague 6, Czech Republic.
authors.forename: Marcela
authors.initials: M
authors.lastname: Krečmerová
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hněvotínská 5, 77900, Olomouc, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Džubák
authors.affiliation: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hněvotínská 5, 77900, Olomouc, Czech Republic.
authors.forename: Marián
authors.initials: M
authors.lastname: Hajdúch
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents
keywords: DNA methylation; DNA methylation inhibitors; demethylation detection system; epigenetic drugs; high content screening