HeLa TI cell-based assay as a new approach to screen for chemicals able to reactivate the expression of epigenetically silenced genes

Abstract

Chemicals reactivating epigenetically silenced genes target diverse classes of enzymes, including DNMTs, HDACs, HMTs and BET protein family members. They can strongly influence the expression of genes and endogenous retroviral elements with concomitant dsRNA synthesis and massive transcription of LTRs. Chemicals reactivating gene expression may cause both beneficial effects in cancer cells and may be hazardous by promoting carcinogenesis. Among chemicals used in medicine and commerce, only a small fraction has been studied with respect to their influence on epigenetic silencing. Screening of chemicals reactivating silent genes requires adequate systems mimicking whole-genome processes. We used a HeLa TSA-inducible cell population (HeLa TI cells) obtained by retroviral infection of a GFP-containing vector followed by several rounds of cell sorting for screening purposes. Previously, the details of GFP epigenetic silencing in HeLa TI cells were thoroughly described. Herein, we show that the epigenetically repressed gene GFP is reactivated by 15 agents, including HDAC inhibitors–vorinostat, sodium butyrate, valproic acid, depsipeptide, pomiferin, and entinostat; DNMT inhibitors–decitabine, 5-azacytidine, RG108; HMT inhibitors–UNC0638, BIX01294, DZNep; a chromatin remodeler–curaxin CBL0137; and BET inhibitors–JQ-1 and JQ-35. We demonstrate that combinations of epigenetic modulators caused a significant increase in cell number with reactivated GFP compared to the individual effects of each agent. HeLa TI cells are competent to metabolize xenobiotics and possess constitutively expressed and inducible cytochrome P450 mono-oxygenases involved in xenobiotic biotransformation. Thus, HeLa TI cells may be used as an adequate test system for the extensive screening of chemicals, including those that must be metabolically activated. Studying the additional metabolic activation of xenobiotics, we surprisingly found that the rat liver S9 fraction, which has been widely used for xenobiotic activation in genotoxicity tests, reactivated epigenetically silenced genes. Applying the HeLa TI system, we show that N-nitrosodiphenylamine and N-nitrosodimethylamine reactivate epigenetically silenced genes, probably by affecting DNA methylation.

Insights into the Mechanism of Curaxin CBL0137 Epigenetic Activity: The Induction of DNA Demethylation and the Suppression of BET Family Proteins

“…Kantidze et al demonstrated that CBL0137 possesses chromatin remodeling activity [ 18 ]. This result is in agreement with the ability of CBL0137 to activate epigenetically silenced genes, which was shown in our previous studies on HeLa TI (HeLa Trichostatin-Induced) cells with the epigenetically silenced reporter gene GFP [ 19 , 20 ]. HeLa TI cell treatment with CBL0137 caused the significant activation of GFP expression similar to 5-azacytidine (5-Aza) or Trichostatin A (TSA).…”

Pharmaceutical Approach to Develop Novel Photosensitizer Nanoformulation: An Example of Design and Characterization Rationale of Chlorophyll α Derivative

“…We analyzed the ability of XL and NP–XL to reactivate epigenetically repressed genes with HeLa TI cell-based test system, representing HeLa cells harboring the epigenetically silent GFP reporter gene. Previously, we verified the possibility of using this cell population as a model for the detection of epigenetically active xenobiotics [ 64 , 65 ]. Under an epigenetically active compound’s influence, GFP gene expression is reactivated, and GFP-positive (GFP+) cells can be easily detected by flow cytometry.…”

Molecular Mechanisms of Anticancer Activity of N-Glycosides of Indolocarbazoles LCS-1208 and LCS-1269

“…To determine the epigenetic activity of LCS-1208 and LCS-1269, the HeLa TI test system was used. HeLa TI is a polyclonal population of HeLa cells containing a genome-integrated vector (derived from avian sarcoma retrovirus) carrying epigenetically repressed GFP and capable of detecting inhibitors of histone deacetylases, DNA methyltransferases, bromodomains and some histone methyltransferases [ 41 , 42 ]. The baseline level of GFP-positive cells in the population ranges from 3.5 to 5%.…”