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

Forfatterne anerkender COST Action CM1406 (epigenetiske kemisk biologi). Forskning rapporteret i denne publikation blev støttet af instituttets Pierre Mercier.

1. cellekultur
Bemærk: Følgende trin er udført i en standard vævskultur hætte. Fortrolighed med steril teknik forventes.
<ol>
	<li>Kultur HeLa celler til 80% confluency i en T75 celle kultur kolbe i MEM suppleret med 10% føtal bovint serum, penicillin G (100 U/mL), og streptomycin (100 mg/mL). Bemærk: Celle kultur, behandling og lysis trin er gennemført under laminar flow, med celle inkubation trin ved 37 ° C udført i en fugtig atmosfære i 5% CO2 under sterile forhol...

<ol>
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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. 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HDAC-hæmning er et varmt emne i drug discovery, med aktuelle fokus på HDAC6-selektiv hæmmere for kræft terapi21. HDAC selektivitet vurderes normalt af en serie af høj overførselshastighed, celle-gratis assays, som har til hensigt at fastslå de hæmmende potens mod individuelle HDAC isoformer21. Dog af en inhibitor selektivitet skal desuden bekræftes i levende celler ved at evaluere acetylation status af endogene protein substrater, såsom histoner (for klasse I HDAC...

HDAC'er tilhører en familie af enzymer, at deacetylate histoner kromatin struktur. De også har andre protein substrater i cytosol og er placeret i forskellige celle rum. Ialt 18 HDAC isoformer er konstateret indtil nu og har været relateret til flere celle mekanismer, herunder regulering af transkriptionsfaktorer og Gen-ekspression, samt celle signalering og transportere1,2,3,4,5,6,7. Katalytisk HDAC-hæmmere er dukket op som potentielle terapeutiske lægemidler for kræftbehandling. De fleste HDAC-hæmmere i øjeblikket godkendt af FDA er til behandling af T-celle lymfom og myelomatose, og uspecifikke HDAC-hæmmere, såsom vorinostat (SAHA), belinostat og panobinostat8,9. Imidlertid en række bivirkninger er blevet forbundet med pan-hæmmere, og Søg efter isoform-specifikke små molekyler er et varmt emne i medicinsk kemi og drug discovery. Derfor, klassen jeg (HDAC1-3 og HDAC8) selektive hæmmer romidepsin er et allerede godkendt lægemiddel10, mens HDAC6-specifikke hæmmere er i øjeblikket under kliniske forsøg, med øget terapeutiske potentiale i myelomatose11,12,13,14,15.
Screening assays for at karakterisere HDAC er hæmmere baseret på dyrkning af en HDAC bærematerialet med en enzymatisk kilde (enkelt isoform, nukleare ekstrakt eller celle lysate). Underlaget er normalt en lille peptid sekvens indeholdende en acetyl lysin rest koblet til en cleavable fluorophore (fx cumarin), såsom N-(4-methyl-7-aminocoumarinyl)-Nα-(t-butoxycarbonyl)-Nω-acetyllysineamide (MAL)16. For at skelne mellem isoform-specifikke aktiviteter, separat celle-fri assays involverer hver isoform er nødvendigt og kan ikke afspejle den reelle isoform aktivitet i levende celler. Isoform-specifikke substrater er kommercielt tilgængelige, såsom benzyl (S)-[1-(4-methyl-2-oxo-2H-chromen-7-ylcarbamoyl)-5-propionylaminopentyl]carbamate (MOCPAC, HDAC1 særlige substrat) og (S)-[5-acetylamino-1-(2-oxo-4-trifluoromethyl-2H-chromen-7-ylcarbamoyl)pentyl]carbamic syre tert-butyl ester (BATCP, HDAC6 særlige substrat) (figur 1B). Dog vil en multi substrat blanding indeholdende MAL, MOCPAC og BATCP givet til levende celler ikke tillade påvisning af de enkelte deacetylated produkter ved fluorometriske måling, eftersom at de er forsynet med den samme cleavable fluorophore.
Metoden beskrevet her giver mulighed for påvisning og relative kvantificering af hver substrat og dens deacetylated produkt i HeLa celler ved hjælp af en multi substrat assay efterfulgt af UHPLC-ESI-MS/MS analyse17. En HDAC assay er udført på HeLa celler at aktivere direkte identifikation af HDAC hæmmende aktivitet og specificitet test tinforbindelser på endogene HDAC'er. Der er fokus på HDAC1 og HDAC6, som evalueres samtidig. For at opnå disse enzymatisk målinger i et enkelt inkubation assay, tilføjes en blanding af uspecifikke og specifikke HDAC substrater behandlede og ubehandlede HeLa celler belagt på en 96-brønd plade. Efter en inkubation trin mængden celler til at frigive substraterne og deres respektive reaktionsprodukter, som er adskilt og påvises ved hjælp af en UHPLC-MS metoden (figur 1 c). De deacetylated produkter MAL, MOCPAC og BATCP underlag er deacetylated MAL (dMAL), deacetylated MOCPAC (dMOCPAC) og deacetylated BATCP (dBATCP), henholdsvis. Dosis-respons-kurver kan bygges med aktive forbindelser.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55878/55878fig1.jpg"> 
Figur 1: generel ordning for celle-baserede HDAC analysen til at identificere HDAC1 og HDAC6 specifikke hæmmere af UHPLC-MS analyse af flere substrater. (A) ordning af en typisk 96-brønd plade der indeholder behandlet (test forbindelser) og ubehandlet (kontrol) HeLa celler samt celle-fri blanks. (B) kemiske struktur af substrater tilføjet som en blanding (21 µM) til at være deacetylated af endogene HDAC'er. (C) typisk UHPLC-MS kromatogrammet viser toppene af de tilføjede substrater (MAL, MOCPAC og BATCP) og deres deacetylated produkter (dMAL, dMOCPAC, og dBACTP, henholdsvis). <a href="http://ecsource.jove.com/files/ftp_upload/55878/55878fig1large.jpg" target="_blank">Venligst klik her for at se en større version af dette tal.</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

Søgning efter nye Histon deacetylase (HDAC) hæmmere er af stigende interesse for drug discovery. Isoform selektivitet har været i søgelyset siden vedtagelsen af romidepsin, en klasse jeg HDAC inhibitor for kræftbehandling, og den kliniske undersøgelse af HDAC6-specifikke hæmmere for myelomatose. Den nuværende metode bruges til at bestemme den hæmmende aktivitet test tinforbindelser på HDAC1 og HDAC6 i celler. Isoform aktivitet måles ved hjælp af ultra-high-performance væskekromatografi-massespektrometri (UHPLC-MS) analyse af specifikke substrater inkuberes med behandlede og ubehandlede HeLa celler. Metoden har fordelen, afspejler den endogene HDAC aktivitet inden for celle-miljøet i modsætning til celle-fri biokemiske analyser udført på isolerede isoformer. Endvidere, fordi den er baseret på kvantitativ bestemmelse af syntetiske substrater, metoden kræver ikke antistof anerkendelse af endogene acetyleret proteiner. Det er let at tilpasse til flere cellelinjer og en automatiseret proces. Metoden har allerede vist sig nyttige i at finde HDAC6-selektiv forbindelser i neuroblasts. Repræsentative resultater er vist her med standard HDAC-hæmmere trichostatin en (ikke-specifik), MS275 (HDAC1-specifik), og tubastatin en (HDAC6-specifik) ved hjælp af HeLa celler.

At dokumentere metoden anvendes til identifikation af non-selektive og selektive hæmmere for HDAC1 (klasse I) og HDAC6 (klasse IIb), HeLa celler blev behandlet med kendte standard forbindelser: trichostatin A (non-selektive mellem HDAC1 og HDAC6)18, MS275 (HDAC1-selektiv hæmmer)19og tubastatin en (HDAC6-selektiv hæmmer)20. Ved hjælp af den nuværende metode (figur 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

Den nuværende metode tjener til at identificere isoform-specifikke hæmmere af Histon deacetyltransferase (HDAC) i HeLa celler af UHPLC-MS analysen af flere substrater. Dette er et antistof-fri metode udviklet for at afspejle HDAC1 og HDAC6 aktivitet i levende celle miljø, i modsætning til single-isoform celle-fri assays.

Samtidig måling af HDAC1 og HDAC6 aktivitet i HeLa celler ved hjælp af 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 ...

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Chemistry

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