Global Epigenetic Changes Induced by SWI2/SNF2 Inhibitors Characterize Neomycin-Resistant Mammalian Cells

Abstract

BackgroundPreviously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes.MethodologyUsing fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase A Domain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized.SignificanceThe use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation

“…This observation was reproduced in other triple negative breast cancer lines (MDA-MB-468 and HDQ-P1) that were treated with a previously validated pool of siRNAs targeting BRG1 [22, 27] (Figure 1G–1H). ADAADi (Active DNA-dependent ATPase A Domain inhibitor), a minor product generated by the bacterial APH (3′)-III enzyme that encodes for aminoglycoside resistance, inhibits the ATPase activity of the SWI2/SNF2 family of ATPases [28, 29] and increases the chemosensitivity of triple negative breast cancer cells to clinically relevant therapeutic drugs [30]. Pharmacological inhibition of the BRG1 ATPase domain by ADAADi in MDA-MB-231cells also decreased de novo lipid synthesis (Figure 1I).…”

“…D- 14 C-Glucose, 3 H-Leucine, 35 S-Methionine and [2– 14 C] acetic acid were purchased from PerkinElmer (PerkinElmer Life Sciences, Waltham, MA, USA). ADAADi was prepared as described [28]. …”

BRG1 is a prognostic indicator and a potential therapeutic target for prostate cancer

“…However, promising results for inhibition of cancer cell proliferation have resulted from studies of an as yet structurally undefined inhibitor of the SNF2 family of ATPases called ADAADi (Active DNA-dependent ATPase A Domain inhibitor). ADAADi is a chromatographically separable byproduct of the bacterial aminoglycoside-phosphotransferase (APH) action upon aminoglycosides (40,41). ADAADi has largely been used as a biochemical probe to help define enzymatic activities of SNF2 ATPases in vitro (40)(41)(42), and it therefore functions as an inhibitor of other ATP-dependent activities of these enzymes, such as chromatin remodeling (41).…”

Mammalian SWI/SNF Enzymes and the Epigenetics of Tumor Cell Metabolic Reprogramming

“…The natural product ADAADi (Active DNA-dependent ATPase A Domain inhibitor) inhibits the ATPase activity of the SWI2/SNF2 family of ATPases (88, 89). Enzymes from other families of DNA-dependent ATPases have no or greatly reduced sensitivity to ADAADi, and DNA-independent or RNA-dependent ATPases are not affected (88).…”