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

这项研究的资金来源：特拉维夫大学的技术转移公司，拉莫特和示巴医学中心（M. CA和SI），ICRF - 以色列癌症研究基金会（M. CA）和以色列科学基金会的联合基金（ SI）。

1。细胞培养制备 MDA-MB-231细胞购自美国典型培养物保藏中心 （ATCC），并保存于液氮中。 <ol><li>种子10 6个MDA-MB-231细胞在92毫米直径的陪替氏培养皿在10毫升完全培养基中Dulbeco改进的Eagle培养基（DMEM），10％马血清，1％L-谷氨酰胺和1％两性霉素B。Penstrep允许细胞的激增至约80-100％汇合。 </li><li>从盘取出培养基并丢弃。 </li><li>简单细胞层洗净，用0.25％的胰蛋白?...

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实时共焦成像提供了一种实时PJ-34的细胞毒作用在活的多中心体的细胞在有丝分裂过程中（ 图3和补充信息）的文档。这是第一次现场文档PJ-34在人类癌细胞中的细胞毒性归因额外中心体聚类和细胞死亡，表明诱导细胞死亡的有丝分裂灾难PJ-34 5-9。与此相反，双焦聚类数目的超中心体中观察到活的未处理的MDA-MB-231细胞进行正常的有丝分裂与双向focali的群集的多余的中心体（ <str...

菲衍生PARP1抑制剂，包括PJ-34，旨在保护耗能PARP1介导的DNA修复应力条件下（中风或心肌梗死）1静止期细胞诱导细胞凋亡。然而，最近我们发现，PJ-34，比诱导PARP1抑制浓度高出一倍，完全可以导致细胞死亡在人类癌细胞中2,3。更快速的细胞增殖，细胞更有效地消灭了。 PJ-34的细胞毒活性归因于额外中心体聚集在有丝分裂2。许多人类癌细胞海港multicentrosomes 4,5。孵化人类乳腺癌细胞MDA-MB-231，怀有编外中心体，为20μmPJ-34有效地根除这些细胞在72-96小时内不损害静止细胞或一些良性增生的细胞，窝藏两个中心在有丝分裂<suP&gt; 2,3。良性的细胞包括人类乳腺上皮细胞MCF-10，人类内皮细胞（HUVEC），从人胸腺制备的原代骨髓间充质细胞。这些细胞耐PJ-34的细胞毒活性。 PJ-34没有干扰他们的细胞周期过程中培养96小时，或影响其中心体和双焦主轴形成2,3。 双极双极4,5在有丝分裂纺锤体的形成是至关重要的中心体组装。因此，有两个以上的中心体的细胞已经开发了一种几乎不理解的分子机制，多余的中心体，在两极4-9聚类。双极其中心体组装的失败可能会导致多极化扭曲纱锭和异常染色体分离逮捕细胞周期G2 / M期阻滞，并导致细胞死亡归因于有丝分裂失败4,5。额外中心体聚类的分子机制的深入研究&lt;SUP&gt; 10。了解这个死亡机制将启用独占消灭癌细胞的同时保留健康组织5,10。 因此，激活有丝分裂灾难细胞死亡的化合物有选择性的癌症治疗提供了一种新的模式，这可能是有效率的在广泛的人类固体cancers.Our结果的建议聚焦成像，可用于识别分子影响额外中心体聚类2,3有丝分裂，使这些化合物癌症靶向药物候选人。 我们已经证明通过扫描固定的和活的人体癌症细胞（额外中心体在有丝分裂的发生率较高），相对于正常细胞的细胞毒活性对菲啶PJ 34。一步一步描述用于识别PJ-34在人类癌症细胞的细胞毒活性的成像程序包括下面。

<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

菲衍生物作为有力PARP1抑制剂阻止编外中心体多中心体的人类癌症细胞有丝分裂的双焦聚类。菲啶PJ-34是最有力的分子。额外中心体的分簇多中心体细胞有丝分裂失败的原因和细胞死亡。最坚实的人力癌症有额外中心体的发生率较高。 PJ-34的活性共聚焦成像活的人乳腺癌MDA-MB-231细胞转染载体编码荧光γ-微管蛋白，这是非常丰富的中心体和荧光蛋白H2B目前在实时记录染色体。转染与PJ-34处理后的MDA-MB-231细胞中检测到异常的染色体安排和γ-微管蛋白聚集疫源代表declustered中心体。非集群额外两个纺锤体两极的中心体细胞死亡之前。这些结果与FOr的第一次检测独家额外中心体的聚类在有丝分裂，有丝分裂失败导致细胞死亡在人类癌细胞中的PJ-34的细胞毒活性。根据以前的研究结果固定细胞共聚焦成像，PJ-34独家消灭癌细胞的多中心体，而不损害正​​常细胞发生有丝分裂，两个中心和双焦主轴的观察。这PJ-34的细胞毒活性不共享等烈性PARP1抑制剂，观察，表明其独立性PARP1抑制PARP1缺失的MEF窝藏extracentrosomes。现场共聚焦成像提供了一个有用的工具，用于识别新的分子，消除细胞在有丝分裂过程中。

PJ-34是一种稳定的水溶性菲啶1（ 图1）。我们先前的研究发现细胞死亡和集群额外中心体在几种类型的固定的多中心体肿瘤细胞，分别用PJ-34。相比之下，正常增殖细胞没有受损2,3。针对centrine1在固 ​​定的额外中心体细胞2和γ-微管蛋白的抗体双标记进行鉴定中心体。 在这里，记录在这些现场额外中心体细胞的杀伤活性PJ-34实时通过现场...

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

菲PJ-34在肿瘤细胞进行有丝分裂的细胞毒活性，实时记录现场共聚焦成像。 PJ-34根除人类乳腺癌MDA-MB-231细胞有丝分裂窝藏额外中心体。额外中心体不像正常的双向焦点有丝分裂，不聚集在两个纺锤体的两极存在的PJ-34。

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