Name: cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells
Uploaded: 2013-08-21T13:00:00
Description: Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Finansieringskilder af denne forskning: en fælles fond af Tel Avivs Universitets teknologioverførsel selskab, Ramot og Sheba-Medical Center (M. CA og SI.) ICRF - Israelsk Cancer Research Foundation (M. CA.), Og Israel Science Foundation ( SI).

1.. Cell Culture Forberedelse MDA-MB-231-celler blev indkøbt fra ATCC (American Type Culture Collection) og opbevaret i flydende nitrogen. <ol><li> Seed 10 6 MDA-MB-231 celler i 92 mm diameter petriskål i 10 ml komplet medium indeholdende Dulbeco Modified Eagle Medium (DMEM), 10% hesteserum, 1% L-glutamin og 1% Penstrep-Amphotericin B. Tillad celler at formere til omkring 80-100% konfluens. </li><li> Fjern dyrkningsmedium fra fad og kassér. </li><li> Vask cellelag ko...

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	<li>Jagtap, P., 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=Novel+phananthridine+inhibitors+of+poly(adenosine+5'-diphosphate-ribose)+synthetase:+Potent+cytoprotective+and+antishock+agents.">Novel phananthridine inhibitors of poly(adenosine 5'-diphosphate-ribose) synthetase: Potent cytoprotective and antishock agents.</a> Crit. Care Med. 30, 1071-1082 (2002).</li><li>Castiel, 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=A+small+molecule+exclusively+eradicates+human+cancer+cells:+Extra-centrosomes+de-clustering+agent.">A small molecule exclusively eradicates human cancer cells: Extra-centrosomes de-clustering agent.</a> BMC Cancer. 11 (1), 412 (2011).</li><li>Inbar-Rozensal, D., 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=A+selective+eradication+of+human+nonhereditary+breast+cancer+cells+by+phenanthridine+-derived+polyADP-ribose+polymerase+inhibitors.">A selective eradication of human nonhereditary breast cancer cells by phenanthridine -derived polyADP-ribose polymerase inhibitors.</a> Breast Cancer Res. 11 (6), R78 (2009).</li><li>Gergely, F., Basto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+centrosomes:+together+they+stand,+divided+they+fall.">Multiple centrosomes: together they stand, divided they fall.</a> Genes Dev. 22, 2291-2296 (2008).</li><li>Godinho, S. A., Kwon, M., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomes+and+cancer:+how+cancer+cells+divide+with+too+many+centrosomes.">Centrosomes and cancer: how cancer cells divide with too many centrosomes.</a> Canc. Met. Rev. 28, 85-98 (2009).</li><li>Doxsey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluating+centrosome+function.">Re-evaluating centrosome function.</a> Nat. Rev. Mol. Cell Biol. 2, 688-698 (2001).</li><li>Walczak, C. E., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitotic+spindle+assembly+and+function.">Mechanisms of mitotic spindle assembly and function.</a> International Rev. of Cytology. 265, 111-158 (2008).</li><li>Ogden, A., Rida, P. C. G., Aneja, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Let's+huddle+to+prevent+a+muddle:+centrosome+declustering+as+an+attractive+anticancer+strategy.">Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy.</a> Cell Death Differ. 19, 1255-1267 (2012).</li><li>Kramer, A., Anderhub, S., Maier, B., Schatten, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+Consequences+of+centrosomes+clustering+in+cancer+cells.">Mechanisms and Consequences of centrosomes clustering in cancer cells.</a> The Centrosome: Cell and Molecular mechanisms of functions and disfunctions in disease. , 255-277 (2012).</li><li>Galimberti, F., 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=Anaphase+Catastrophe+Is+a+Target+for+Cancer+Therapy.">Anaphase Catastrophe Is a Target for Cancer Therapy.</a> Clin. Cancer Res. 17, 1218-1222 (2011).</li><li>Kanai, 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=Haploinsufficiency+of+poly(ADP-ribose)+polymerase-1-mediated+poly(ADP-ribosyl)ation+for+centrosome+duplication.">Haploinsufficiency of poly(ADP-ribose) polymerase-1-mediated poly(ADP-ribosyl)ation for centrosome duplication.</a> Biochem. Biophys. Res. Commun. 359, 426-430 (2007).</li><li>Gartner, E. M., Burger, A. M., Lorusso, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribose)+polymerase+inhibitors:+a+novel+drug+class+with+a+promising+future.">Poly(ADP-ribose) polymerase inhibitors: a novel drug class with a promising future.</a> Cancer J. 16, 83-90 (2010).</li><li>Wahlberg, E., 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=Family-wide+chemical+profiling+and+structural+analysis+of+PARP+and+tankyrase+inhibitors.">Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors.</a> Nature Biotechnology. 30, 283-288 (2012).</li><li>Rouleau, M., Patel, A., Hendze, M. J., Kaufmann, S. H., Poirier, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP+inhibition:+PARP1+and+beyond.">PARP inhibition: PARP1 and beyond.</a> Nature Rev. Cancer. 10, 293-301 (2010).</li><li>Leber, 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=Proteins+Required+for+Centrosome+Clustering+in+Cancer+Cells.">Proteins Required for Centrosome Clustering in Cancer Cells.</a> Sci. Transl. Med. 2 (33), 33-38 (2010).</li><li>Kwon, 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=Mechanisms+to+suppress+multipolar+division+in+cancer+cells+with+extra+centrosomes.">Mechanisms to suppress multipolar division in cancer cells with extra centrosomes.</a> Gene Dev. 22, 2189-2203 (2008).</li></ol>

Levende konfokal imaging forudsat en real-time dokumentation af den cytotoksiske effekt af PJ-34 i levende multi-centrosomal celler under mitose (figur 3, og supplerende oplysninger). Dette var den første levende dokumentation tilskrive cytotoksicitet PJ-34 i humane kræftceller til ekstra centrosomes de-clustering og celledød, hvilket tyder på induktion af Mitotisk Catastrophe celledød ved PJ-34 5-9. I modsætning hertil var bi-omdrejningspunkt gruppering af super-numerary centrosomes ob...

Phenanthren afledte PARP1 hæmmere, inklusive PJ-34, blev designet til at beskytte hvilende celler fra apoptotisk celledød induceret af energikrævende PARP1 medieret DNA-reparation under stress betingelser (slagtilfælde eller myokardieinfarkt) 1. Men for nylig opdagede vi, at PJ-34, på det dobbelte højere koncentration end inducerende PARP1 hæmning kan udelukkende medføre celledød i humane cancerceller 2,3. Den hurtigere spredning af cellen var, jo mere effektiv udryddelse af cellerne var. Den cytotoksiske aktivitet PJ-34 blev tilskrevet ekstra-centrosomes de-clustering i mitose 2.. Mange human cancer celler havnen multicentrosomes 4,5. Inkubation af humane brystkræftceller MDA-MB-231, som huser overtallige centrosomes, med 20 uM PJ-34 effektivt udryddet disse celler inden 72-96 timer uden at forringe hvilende celler eller nogle godartede prolifererende celler huser to centrosomes i mitose <sup&gt; 2,3. Godartede celler omfattede humane mammae epitelceller MCF-10, humane endotelceller (HUVEC) og primære mesenchymale celler fremstillet fra humane thymus. Disse celler var resistente over for den cytotoksiske aktivitet af PJ-34. PJ-34 ikke forstyrre deres cellecyklus i løbet 96 timer inkubation eller påvirker deres centrosomes og bi-omdrejningspunkt spindel dannelse 2,3. Bipolar centrosome samling er afgørende for bipolar spindel dannelse i mitose 4,5. Derfor har celler med mere end to centrosomes udviklet en næppe forstået molekylære mekanisme, clustering deres ekstra centrosomes på to poler 4-9. Manglende bipolar samling af deres centrosomes kan forårsage multipolær forvrænget spindler og afvigende kromosomer segregation, at anholdelser celle-cyklus G2 / M anholdelse, og fører til celledød tilskrives mitotisk fiasko 4,5. De molekylære mekanismer bag ekstra-centrosomes de-clustering intensivt undersøgte &lt;sup&gt; 10. Forståelse dette dødsfald mekanisme vil gøre det muligt for eksklusiv udryddelse af kræftceller, mens besparende raske væv 5,10. Således forbindelser, som aktiverer mitotisk katastrofe celledød tilbyde en ny form for en selektiv cancerterapi, som kan være effektive i en bred vifte af humane faste cancers.Our resultater antyder, at konfokal billeddannelse kan anvendes til at identificere molekyler, der påvirker ekstra-centrosomes klyngedannelse i mitose 2,3, hvilket gør disse forbindelser kræft målretning lægemiddelkandidater. Vi har dokumenteret den cytotoksiske aktivitet af phenanthridin PJ-34 ved at scanne faste og levende humane kræftceller (med høj forekomst af ekstra-centrosomes i mitosen) versus normale celler. En trin-for trin beskrivelse af billeddiagnostiske procedurer, der anvendes til at identificere den cytotoksiske aktivitet PJ-34 i humane cancerceller er medtaget nedenfor.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

Phenanthren derivater fungerer som potente PARP1 hæmmere forhindrede bi-omdrejningspunkt klyngedannelse af overtallige centrosomes i multi-centrosomal humane kræftceller i mitose. Den phenanthridin PJ-34 var den mest potente molekylet. Declustering ekstra-centrosomes forårsager mitotisk svigt og celledød i multi-centrosomal celler. Mest solide menneskelige kræftformer har høj forekomst af ekstra-centrosomes. Aktiviteten af ​​PJ-34 blev dokumenteret i realtid ved konfokal billeddannelse af levende human brystcancer MDA-MB-231-celler transficeret med vektorer, der koder for fluorescerende γ-tubulin, som er meget rigelige i centrosomes og fluorescerende histon H2b stede i kromosomerne. Afvigende kromosomer arrangementer og de-klynger γ-tubulin foci repræsenterer declustered centrosomes blev påvist i de transficerede MDA-MB-231-celler efter behandling med PJ-34. Un-klynger ekstra-centrosomes i de to spindel poler forud deres celledød. Disse resultater er forbundet for første gang den nyligt opdaget eksklusiv cytotoksiske aktivitet af PJ-34 i humane cancerceller med ekstra centrosomes de-clustering i mitose, og mitotisk svigt fører til celledød. Ifølge tidligere resultater observeret ved konfokal imaging af faste celler, PJ-34 udelukkende udryddet kræftceller med multi-centrosomes uden at forringe normale celler undergår mitose med to centrosomes og bi-knudepunkt spindler. Denne cytotoksiske aktivitet af PJ-34 deltes ikke af andre potente PARP1 hæmmere, og blev observeret i PARP1 deficiente MEF huser extracentrosomes, tyder dens uafhængighed af PARP1 hæmning. Levende konfokal imaging tilbudt et nyttigt redskab til at identificere nye molekyler udrydde celler under mitose.

PJ-34 er en stabil vandopløselig phenanthridin 1 (figur 1). Vores tidligere resultater viste celledød og de-grupperet ekstra centrosomes i flere typer af faste multi-centrosomal kræftceller, der blev behandlet med PJ-34. I modsætning hertil var normale prolifererende celler ikke værdiforringede 2,3. Centrosomes blev identificeret ved at dobbeltklikke mærkning med antistoffer rettet mod centrine1 og γ-tubulin i de faste ekstra centrosomal celler 2. <p class="jo...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Den cytotoksiske aktivitet af phenanthridin PJ-34 i cancerceller undergår mitose blev dokumenteret i realtid ved levende konfokal billeddannelse. PJ-34 udryddet human brystkræft MDA-MB-231 celler, der huser ekstra-centrosomes i mitose. I modsætning til normal bi-omdrejningspunkt mitose blev de ekstra-centrosomes ikke grupperet i de to spindel poler i tilstedeværelse af PJ-34.

Celledød forbundet med abnorme Mitosis Observeret ved konfokal Imaging i Live kræftceller

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

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