A Substrate-Independent TR-FRET Histone Deacetylase Inhibitor Assay

Marks, Bryan D.; Fakhoury, Stephen A.; Frazee, William J.; Eliason, Hildegard C.; Riddle, Steven M.

doi:10.1177/1087057111422102

Cited by 20 publications

(17 citation statements)

References 15 publications

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“…Treatment with HDACi which are not specific for HDAC1 were less effective in inducing the expression of vimentin and BRM. BML-210 (reported to have the highest affinity for HDAC3 [ 25 , 26 ]), and MC1293 (reported to bind HDAC6 and HDAC10 and to HDAC1 with lower affinity [ 26 ]) are minimally effective or ineffective in inducing vimentin expression. Depudecin treatment led to minimal expression of vimentin and BRM but this HDAC inhibitor has only been tested against recombinant HDAC1 [ 27 ] so that its specificity has not been determined.…”

Section: Resultsmentioning

confidence: 99%

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition

et al. 2016

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BackgroundThe BRM and BRG1 tumor suppressor genes are mutually exclusive ATPase subunits of the SWI/SNF chromatin remodeling complex. The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses these subunits, SW13+, and one that expresses neither subunit, SW13-. Loss of BRM expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, most previously used HDAC inhibitors are toxic and broad-spectrum, providing little insight into the mechanism of the switch between subtypes. In this work, we explore the mechanisms of HDAC inhibition in promoting subtype switching and further characterize the oncogenic potential of the two epigenetically distinct SW13 subtypes.MethodsSW13 subtype morphology, chemotaxis, growth rates, and gene expression were assessed by standard immunofluorescence, transwell, growth, and qPCR assays. Metastatic potential was measured by anchorage-independent growth and MMP activity. The efficacy of HDAC inhibitors in inducing subtype switching was determined by immunofluorescence and qPCR. Histone modifications were assessed by western blot.ResultsTreatment of SW13- cells with HDAC1 inhibitors most effectively promotes re-expression of BRM and VIM, characteristic of the SW13+ phenotype. During treatment, hyperacetylation of histone residues and hypertrimethylation of H3K4 is pronounced. Furthermore, histone modification enzymes, including HDACs and KDM5C, are differentially expressed during treatment but several features of this differential expression pattern differs from that seen in the SW13- and SW13+ subtypes. As the SW13- subtype is more proliferative while the SW13+ subtype is more metastatic, treatment with HDACi increases the metastatic potential of SW13 cells while restoring expression of the BRM tumor suppressor.ConclusionsWhen compared to the SW13- subtype, SW13+ cells have restored BRM expression, increased metastatic capacity, and significantly different expression of a variety of chromatin remodeling factors including those involved with histone acetylation and methylation. These data are consistent with a multistep mechanism of SW13- to SW13+ conversion and subtype stabilization: histone hypermodification results in the altered expression of chromatin remodeling factors and chromatin epigenetic enzymes and the re-expression of BRM which results in restoration of SWI/SNF complex function and leads to changes in chromatin structure and gene expression that stabilize the SW13+ phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2353-7) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition

et al. 2016

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“…As catalytically functional protein is not required, there is no requirement for a substrate and instead an Alexa Fluor® 647-labelled HDAC inhibitor is used as a tracer (acceptor in the TR-FRET assay). This can bind with specific HDAC enzymes tagged with GST in the presence of Europium anti-GST tag antibody (donor in the TR-FRET assay) and if the tracer is displaced by an appropriate compound, a decrease in signal would be observed [134]. …”

Section: The Histone Deacetylase (Hdac) Target Class and Relevant Scrmentioning

confidence: 99%

Epigenetic assays for chemical biology and drug discovery

Gul

2017

Clin Epigenet

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The implication of epigenetic abnormalities in many diseases and the approval of a number of compounds that modulate specific epigenetic targets in a therapeutically relevant manner in cancer specifically confirms that some of these targets are druggable by small molecules. Furthermore, a number of compounds are currently in clinical trials for other diseases including cardiovascular, neurological and metabolic disorders. Despite these advances, the approved treatments for cancer only extend progression-free survival for a relatively short time and being associated with significant side effects. The current clinical trials involving the next generation of epigenetic drugs may address the disadvantages of the currently approved epigenetic drugs.The identification of chemical starting points of many drugs often makes use of screening in vitro assays against libraries of synthetic or natural products. These assays can be biochemical (using purified protein) or cell-based (using for example, genetically modified, cancer cell lines or primary cells) and performed in microtiter plates, thus enabling a large number of samples to be tested. A considerable number of such assays are available to monitor epigenetic target activity, and this review provides an overview of drug discovery and chemical biology and describes assays that monitor activities of histone deacetylase, lysine-specific demethylase, histone methyltransferase, histone acetyltransferase and bromodomain. It is of critical importance that an appropriate assay is developed and comprehensively validated for a given drug target prior to screening in order to improve the probability of the compound progressing in the drug discovery value chain.

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“…HDAC5 and HDAC11 did not reveal any difference and the activities of other members were not in the detectable range. Comparison of the half maximal effective concentration (EC50) against the HDAC inhibitor, Trichostatin A (TSA) that exhibits more potency toward class I family HDACs (HDAC1, 2, 3) than class IIa (HDAC4, 5) [17], [18] revealed an EC50 of 16.44 nM in tumors and of 31.11 nM in wild-type cerebellum (Fig. S2) suggesting that HDAC1 and 2 might be the predominant contributors to the increased HDAC activity observed in Smo/Smo tumors.…”

Section: Resultsmentioning

confidence: 99%

Sonic Hedgehog-Induced Histone Deacetylase Activation Is Required for Cerebellar Granule Precursor Hyperplasia in Medulloblastoma

et al. 2013

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Medulloblastoma, the most common pediatric brain tumor, is thought to arise from deregulated proliferation of cerebellar granule precursor (CGP) cells. Sonic hedgehog (Shh) is the primary mitogen that regulates proliferation of CGP cells during the early stages of postnatal cerebellum development. Aberrant activation of Shh signaling during this time has been associated with hyperplasia of CGP cells and eventually may lead to the development of medulloblastoma. The molecular targets of Shh signaling involved in medulloblastoma formation are still not well-understood. Here, we show that Shh regulates sustained activation of histone deacetylases (HDACs) and that this activity is required for continued proliferation of CGP cells. Suppression of HDAC activity not only blocked the Shh-induced CGP proliferation in primary cell cultures, but also ameliorated aberrant CGP proliferation at the external germinal layer (EGL) in a medulloblastoma mouse model. Increased levels of mRNA and protein of several HDAC family members were found in medulloblastoma compared to wild type cerebellum suggesting that HDAC activity is required for the survival/progression of tumor cells. The identification of a role of HDACs in the early steps of medulloblastoma formation suggests there may be a therapeutic potential for HDAC inhibitors in this disease.

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A Substrate-Independent TR-FRET Histone Deacetylase Inhibitor Assay

Cited by 20 publications

References 15 publications

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition

Epigenetic assays for chemical biology and drug discovery

Sonic Hedgehog-Induced Histone Deacetylase Activation Is Required for Cerebellar Granule Precursor Hyperplasia in Medulloblastoma

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