Name: simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms
Uploaded: 2017-08-10T12:30:00
Description: Watch this Scientific Journal Video about Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS at JoVE.com

The authors acknowledge COST Action CM1406 (Epigenetic Chemical Biology). Research reported in this publication was supported by the Pierre Mercier foundation.

1. Cell Culture
NOTE: The following steps are performed in a standard tissue culture hood. Familiarity with sterile technique is expected.
<ol>
	<li>Culture HeLa cells to 80% confluency in a T75 cell culture flask in MEM supplemented with 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 mg/mL). 
	NOTE: Cell culture, treatment, and lysis steps are conducted under laminar flow, with cell incubation steps at 37 &#176;C performed in a humidified atmosphere of 5% CO<sub...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+modifications+in+transcriptional+regulation.">Histone modifications in transcriptional regulation.</a> Curr Opin Genet Dev. 12 (2), 142-148 (2002).</li><li>Sim&#245;es-Pires, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+as+a+target+for+neurodegenerative+diseases:+what+makes+it+different+from+the+other+HDACs?.">HDAC6 as a target for neurodegenerative diseases: what makes it different from the other HDACs?.</a> Mol Neurodegener. 8 (7), 1-16 (2013).</li><li>Mottamal, M., Zheng, S., Huang, T. L., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+in+clinical+studies+as+templates+for+new+anticancer+agents.">Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents.</a> Molecules. 20 (3), 3898-3941 (2015).</li><li>Manal, M., Chandrasekar, M. J., Gomathi Priya, J., Nanjan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+histone+deacetylase+as+antitumor+agents:+A+critical+review.">Inhibitors of histone deacetylase as antitumor agents: A critical review.</a> Bioorg Chem. 67, 18-42 (2016).</li><li>Mack, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+selectivity+and+beyond.">To selectivity and beyond.</a> Nat Biotech. 28 (12), 1259-1266 (2010).</li><li>. Study of ACY-1215 alone and in combination with bortezomib and dexamethasone in multiple myeloma (ACY-1215) Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01323751">https://clinicaltrials.gov/ct2/show/NCT01323751</a> (1211)</li><li>. Study of ACY-1215 in combination with lenalidomide, and dexamethasone in multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01583283">https://clinicaltrials.gov/ct2/show/NCT01583283</a> (2012)</li><li>. 13ACY-1215 (ricolinostat) in combination with pomalidomide and low-dose dex in relapsed-and-refractory multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT01997840">https://clinicaltrials.gov/ct2/show/NCT01997840</a> (2013)</li><li>. Study of ACY-241 alone and in combination with pomalidomide and dexamethasone in multiple myeloma Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT02400242">https://clinicaltrials.gov/ct2/show/NCT02400242</a> (2015)</li><li>Yee, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ricolinostat+plus+lenalidomide,+and+dexamethasone+in+relapsed+or+refractory+multiple+myeloma:+a+multicentre+phase+1b+trial.">Ricolinostat plus lenalidomide, and dexamethasone in relapsed or refractory multiple myeloma: a multicentre phase 1b trial.</a> Lancet Oncol. 17 (11), 1569-1578 (2016).</li><li>Jung, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogenous+non-isotopic+assays+for+histone+deacetylase+activity.">Homogenous non-isotopic assays for histone deacetylase activity.</a> Expert Opin Ther Pat. 13 (6), 935 (2003).</li><li>Zwick, V., Sim&#245;es-Pires, C., Cuendet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+multi-substrate+assay+coupled+to+UHPLC-ESI-MS/MS+for+a+quick+identification+of+class-specific+HDAC+inhibitors.">Cell-based multi-substrate assay coupled to UHPLC-ESI-MS/MS for a quick identification of class-specific HDAC inhibitors.</a> J Enzyme Inhibi Med Chem. 31 (1), 209-214 (2016).</li><li>Khan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+class+and+isoform+selectivity+of+small-molecule+histone+deacetylase+inhibitors.">Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors.</a> Biochem J. 409 (2), 581-589 (2008).</li><li>Glaser, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+protein+acetylation+induced+by+novel+histone+deacetylase+inhibitors.">Differential protein acetylation induced by novel histone deacetylase inhibitors.</a> Biochem Biophys Res Commun. 325 (3), 683-690 (2004).</li><li>Butler, K. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+and+simple+chemistry+yield+a+superior,+neuroprotective+HDAC6+inhibitor,+tubastatin+A.">Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A.</a> J Am Chem Soc. 132 (31), 10842-10846 (2010).</li><li>Jung, M., Yong, K. -. J., Velena, A., Lee, S., Sippl, W., Jung, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Assays+for+HDAC+Inhibitor+Hit+Validation.">Cell-Based Assays for HDAC Inhibitor Hit Validation.</a> Epigenetic Targets in Drug Discovery. , (2010).</li><li>Milli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+cellular+response+to+novel+proapoptotic+agents+related+to+atypical+retinoids+in+human+IGROV-1+ovarian+carcinoma+cells.">Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells.</a> J Proteome Res. 10 (3), 1191-1207 (2011).</li><li>Zwick, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+selective+HDAC6+inhibitor+active+in+neuroblasts.">Synthesis of a selective HDAC6 inhibitor active in neuroblasts.</a> Bioorg Med Chem Lett. 26 (20), 4955-4959 (2016).</li><li>Ciossek, T., Julius, H., Wieland, H., Maier, T., Beckers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homogeneous+cellular+histone+deacetylase+assay+suitable+for+compound+profiling+and+robotic+screening.">A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening.</a> Anal Biochem. 372 (1), 72-81 (2008).</li><li>Heltweg, B., Dequiedt, F., Marshall, B. L., Brauch, C., Yoshida, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtype+selective+substrates+for+histone+deacetylases.">Subtype selective substrates for histone deacetylases.</a> J Med Chem. 47 (21), 5235-5243 (2004).</li><li>Copeland, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evaluation of enzyme inhibitors in drug discovery. , (2005).</li></ol>

HDAC inhibition is a hot topic in drug discovery, with a current focus on HDAC6-selective inhibitors for cancer therapy21. HDAC selectivity is usually assessed by a series of high-throughput, cell-free assays, which intend to determine the inhibitory potency towards individual HDAC isoforms21. However, the selectivity of an inhibitor must be additionally confirmed in living cells by evaluating the acetylation status of endogenous protein substrates, such as histones (for cl...

HDACs belong to a family of enzymes able to deacetylate histones within the chromatin structure. They also have other protein substrates in the cytosol and are located in various cell compartments. A total of 18 HDAC isoforms have been identified so far and have been related to several cell mechanisms, including the regulation of transcription factors and gene expression, as well as cell signaling and transport1,2,3,4,5,6,7. HDAC catalytic inhibitors have emerged as potential therapeutic drugs for cancer therapy. Most HDAC inhibitors currently approved by the FDA are for the treatment of T-cell lymphoma and multiple myeloma, and are non-specific HDAC inhibitors, such as vorinostat (SAHA), belinostat, and panobinostat8,9. However, a series of side effects have been associated with pan-inhibitors, and the search for isoform-specific small molecules is a hot topic in medicinal chemistry and drug discovery. Accordingly, the class I (HDAC1-3 and HDAC8) selective inhibitor romidepsin is an already approved drug10, while HDAC6-specific inhibitors are currently under clinical trials, with increased therapeutic potential in multiple myeloma11,12,13,14,15.
Screening assays to characterize HDAC inhibitors are based on the incubation of an HDAC substrate with an enzymatic source (single isoform, nuclear extract, or cell lysate). The substrate is usually a small peptide sequence containing an acetyl lysine residue coupled to a cleavable fluorophore (e.g., coumarin), such as N-(4-methyl-7-aminocoumarinyl)-N&#945;-(t-butoxycarbonyl)-N&#969;-acetyllysineamide (MAL)16. To distinguish between isoform-specific activities, separate cell-free assays involving each isoform are necessary and might not reflect the real isoform activity in living cells. Isoform-specific substrates are commercially available, such as benzyl (S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate (MOCPAC, HDAC1 specific substrate) and (S)-[5-acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamic acid tert-butyl ester (BATCP, HDAC6 specific substrate) (Figure 1B). However, a multi-substrate mixture containing MAL, MOCPAC, and BATCP given to living cells will not permit the detection of the individual deacetylated products by fluorometric measurement, given that they bear the same cleavable fluorophore.
The method described here allows for the detection and relative quantification of each substrate and its deacetylated product in HeLa cells using a multi-substrate assay followed by UHPLC-ESI-MS/MS analysis17. An HDAC assay is conducted on HeLa cells to enable the direct identification of HDAC inhibitory activity and of the specificity of test compounds on endogenous HDACs. There is a focus on HDAC1 and HDAC6, which are simultaneously evaluated. To achieve these enzymatic measurements in a single incubation assay, a mixture of non-specific and specific HDAC substrates is added to treated and untreated HeLa cells plated on a 96-well plate. Following an incubation step, cells are lysed to release the substrates and their respective reaction products, which are separated and detected using a UHPLC-MS method (Figure 1C). The deacetylated products of the MAL, MOCPAC, and BATCP substrates are the deacetylated MAL (dMAL), deacetylated MOCPAC (dMOCPAC), and deacetylated BATCP (dBATCP), respectively. Dose-response curves can be built with active compounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55878/55878fig1.jpg" /> 
Figure 1: General scheme for the cell-based HDAC assay to identify HDAC1- and HDAC6-specific inhibitors by the UHPLC-MS analysis of multiple substrates. (A) Scheme of a typical 96-well plate containing treated (test compounds) and untreated (control) HeLa cells, as well as cell-free blanks. (B) Chemical structure of substrates added as a mixture (21 &#181;M each) to be deacetylated by endogenous HDACs. (C) Typical UHPLC-MS chromatogram showing the peaks of the added substrates (MAL, MOCPAC, and BATCP) and their deacetylated products (dMAL, dMOCPAC, and dBACTP, respectively). <a href="http://cloudflare.jove.com/files/ftp_upload/55878/55878fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BATCP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">B4061</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAL</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>SCP0168</td>
			<td>Synonym: BOC-Ac-Lys-AMC&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MOCPAC</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td>M2195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MS275</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">EPS002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trichostatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">T8552</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tubastatin A</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">SML0044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acetonitrile, HPLC grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10660131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formic acid, LC/MS grade</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">10596814</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:271px;">H2O, HPLC grade</td>
			<td style="width:191px;"></td>
			<td style="width:152px;"></td>
			<td>distilled H2O filtered through a Milli-Q purification system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">HeLa cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:152px;">ATCC CRM-CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell dissociation reagent TrypLE Express</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">12604013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMSO, cell culture grade</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:152px;">146463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DPBS</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fetal bovine serum&#160;</td>
			<td style="width:191px;">Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">MEM</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Bioconcept</td>
			<td>4-01F00-H&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X RIPA buffer</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:152px;">ab156034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SigmaFast protease inhibitor tablets</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">S8820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, sterile, flat-bottom, tissue culture treated Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plates, non-sterile, V-bottom Corning</td>
			<td style="width:191px;">VWR</td>
			<td style="width:152px;">29442-404</td>
			<td>used in the centrifugation step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">96-well plate, conical bottom, Nunc</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:152px;">249944</td>
			<td>compatible with the Acquity UHPLC system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">T75&#160; cell culture flasks Corning</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:152px;">CLS430641&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Peelable heat sealing foil&#160;</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;">186002789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acquity UPLC system</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Eppendorf Centrifuge 5810 R</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:152px;">05-413-323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Integration software: MassLynx V4.1</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Combi thermo-sealer SP-0669/240</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Quattro micro API Tandem Quadrupole System</td>
			<td style="width:191px;">Waters</td>
			<td style="width:152px;"></td>
			<td>Catalog number not available</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of hdac1 and hdac6 activity in hela cells using uhplc-ms

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

To demonstrate the application of the method to identifying non-selective and selective inhibitors for HDAC1 (class I) and HDAC6 (class IIb), HeLa cells were treated with known standard compounds: trichostatin A (non-selective between HDAC1 and HDAC6)18, MS275 (HDAC1-selective inhibitor)19, and tubastatin A (HDAC6-selective inhibitor)20. By using the present method (Figure 1), IC50</...

Watch this Scientific Journal Video about Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS at JoVE.com

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

The overall goal of this method is to determine the HDAC inhibitory potential of compounds and to identify selectivity towards HDAC6 or HDAC1 in cells using a single ultra high performance liquid chromatography mass spectrometry assay. This method can help answer key questions in the drug discovery field such as determining whether an HDAC inhibitor is selective for HDAC6 over Class I HDACs in living cells. The main advantage of this technique is that it requires a single incubation step with cells, inhibitors, and selective substrates in order to determine potency against two HDAC isoforms simultaneously.The implication of this technique extend toward early drug discovery of selective HDAC inhibitors because it detects whether HDAC6 selectivity is maintained in living cells avoiding endogenous protein acetylation assays. Though this method assesses selectivity of HDAC inhibitors in HeLa cells, it can also be applied to other cell lines such as primary and other tumor cells including clinical specimens. Culture HeLa cells to 80%confluency in a T75 cell culture flask in minimal essential media supplemented with 10%fetal bovine serum, penicillin G, and streptomycin.Wash the cells twice with five milliliters of Dulbecco's Phosphate-Buffered Saline. Then, aspirate the buffer and add one milliliter of cell dissociation reagent. Incubate at 37 degrees Celsius for five minutes to detach the cells.After five minutes, add nine milliliters of MEM culture medium to the cells and thoroughly resuspend the cell suspension. Transfer the cells to a new 15 milliliter tube and then count the cells using a hemocytometer. Adjust the density of the cell suspension to 60, 000 cells per milliliter in MEM.Next, add 100 microliters of the cell suspension to the control and test wells of a sterile, flat bottom, tissue culture treated 96 well plate. Then, add 100 microliters of MEM culture medium to the blank wells of the 96 well plate. Incubate the 96 well plate at 37 degrees Celsius for 24 hours.Aspirate the medium of the wells of the 96 well plate. While aspirating the medium, avoid detaching and removing cells from the well. This can be achieved by using a low aspiration rate and carefully placing the aspirating tips at the bottom corner of the well surface.Then, add 25 microliters of the two times test compounds in MEM to the test wells, including Trichostatin A, MS275, and Tubastatin A.Add 25 microliters of MEM to the control and blank wells. Next, add 25 microliters of the substrate mixture to the control and test wells. Then, add 25 microliters of MEM to the blank wells.Incubate the 96 well plate at 37 degrees Celsius for eight hours in a humidified 5%carbon dioxide incubator. To perform cell lysis, first add 10 microliters of six times RIPA buffer supplemented with protease inhibitor to each well of the 96 well plate. Stop the reaction by adding 160 microliters of cold acetonitrile to each well and mix by pipetting up and down.Place the plate in a minus 80 degrees Celsius freezer for 10 minutes. Remove the plate from the freezer and transfer 220 microliters of the content of each well to a non-sterile, conical bottom 96 well plate. Centrifuge the plate at 5, 000 times g and four degrees Celsius for 10 minutes.Following centrifugation, transfer 200 microliters of supernatant into a 96 well plate compatible with the UHPLC system. Then, seal the plate with peelable heat-sealing foil using a plate sealer. Prepare the ultra high performance liquid chromatography mass spectrometry system by filling it with the mobile phase system.Place the 96 well plate into a sample manager containing a plate holder. Run the samples according to the UHPLC ESI MS/MS conditions found in the text. To perform data analysis, integrate the peak area of each substrate and its deacetylated product with the appropriate UHPLC integration software.For each substrate, calculate the peak area ratio of deacetylated to acetylated in each chromatogram. Include the test and control samples. Finally, calculate the percent HDAC inhibition of each test sample according to the equation in the text protocol.The UHPLC-MS chromatogram of a sample is presented here. The peaks corresponding to the substrates added to the cells are detected according to their mass transitions as described in table one of the written protocol. MOCPAC is deacetylated by HDAC1 to provide the deacetylated MOCPAC peak.Similarly, BATCP is deacetylated by HDAC6 to provide the deacetylated BATCP peak. Finally, MAL provides the deacetylated MAL peak which reflects the deacetylation of several endogenous HDACs unspecifically. Shown here, the figure with the chromatogram obtained with different concentrations of the HDAC inhibitor Trichostatin A.A dose response evaluation of standard HDAC inhibitors was performed.The integrated values obtained from MOCPAC and dMOCPAC were used to calculate the potency of standard inhibitors against HDAC1. Following IC 50 calculations, MS275, a selective Class I HDAC inhibitor, showed lower IC 50 values for HDAC1 than Tubastatin A, a known HDAC6 inhibitor. On the other hand, the isolated values from BATCP and dBATCP were used to calculate the potency of the standards against HDAC6.This time, Tubastatin A provided a lower IC 50 value than MS275, confirming their isoform selectivity profile. Trichostatin A is a potent, nonselective inhibitor. Once mastered, this technique can be done in 72 hours if it's performed properly including the cell treatment time and the analysis of the 96 well plate.While attempting this procedure, it's important to remember that the incubation times indicated here are for HeLa cells. If another cell line is used, this needs to be optimized. Following this procedure, selective HDAC inhibitory activity of other isoforms could be measured as long as specific substrates exist.Don't forget that working with organic solvents and other chemicals can be extremely hazardous and protection glasses, gloves, and a lab coat should always be worn while performing this procedure.

Research

JoVE Journal

Chemistry

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