Name: assessment of resistance to tyrosine kinase inhibitors by an interrogation of signal transduction pathways by antibody arrays
Uploaded: 2018-09-19T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays at JoVE.com

我们感谢劳伦斯·埃里森变革医学研究所的慷慨财政支持 (大卫 Agus 的礼物)。我们感谢秋宝马和丽莎 Flashner 的支持, 这导致了这篇手稿的产生和出版。我们感谢劳拉. 吴的行政支持。

1. 蛋白质提取
<ol><li>板 5 x 106细胞在10毫米组织培养板 (或烧瓶) 在组织培养敞篷。使用自动单元格计数器或 hemocytometer 计算电镀时的单元格。或者, 估计单元格计数。</li>
	<li>用10毫升磷酸盐缓冲盐水 (PBS) 冲洗10毫米板 (或烧瓶) 上生长的细胞 (pH 值 = 7.4)。在添加裂解缓冲液之前, 一定要卸下所有 PBS。 注: 溶解缓冲通常与试剂盒一起提供。如果没有, 则使用 radioimmunoprecipitation 检测缓冲器 (...

<ol>
	<li>Knauer, D. J., Smith, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+biological+activity+of+multiplication-stimulating+activity+by+binding+to+its+carrier+protein.">Inhibition of biological activity of multiplication-stimulating activity by binding to its carrier protein.</a> Proceedings of the National Academy of Sciences of the United States of America. 77 (12), 7252-7256 (1980).</li><li>Glossmann, H., Presek, P., Eigenbrodt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin+inhibits+tyrosine+phosphorylation+by+the+cyclic+nucleotide-independent,+transforming+protein+kinase,+pp60src.">Quercetin inhibits tyrosine phosphorylation by the cyclic nucleotide-independent, transforming protein kinase, pp60src.</a> Naunyn-Schmiedebergs Archives of Pharmacology. 317 (1), 100-102 (1981).</li><li>Di Fiore, P. 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=Overexpression+of+the+human+EGF+receptor+confers+an+EGF-dependent+transformed+phenotype+to+NIH+3T3+cells.">Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells.</a> Cell. 51 (6), 1063-1070 (1987).</li><li>Maguire, H. C., Greene, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neu+(c-erbB-2)+oncogene.">The neu (c-erbB-2) oncogene.</a> Seminars in Oncology. 16 (2), 148-155 (1989).</li><li>Busse, 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=Reversible+G(1)+arrest+induced+by+inhibition+of+the+epidermal+growth+factor+receptor+tyrosine+kinase+requires+up-regulation+of+p27(KIP1)+independent+of+MAPK+activity.">Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity.</a> Journal of Biological Chemistry. 275 (10), 6987-6995 (2000).</li><li>Kondapaka, B. S., Reddy, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+kinase+inhibitor+as+a+novel+signal+transduction+and+antiproliferative+agent:+prostate+cancer.">Tyrosine kinase inhibitor as a novel signal transduction and antiproliferative agent: prostate cancer.</a> Molecular and Cellular Endocrinology. 117 (1), 53-58 (1996).</li><li>Cao, F., Zhang, L., Wang, S., Zhong, D., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Effectiveness+of+EGFR-TKIs+versus+chemotherapy+as+first-line+treatment+for+advanced+non-small+cell+lung+cancer:+a+meta-analysis].">[Effectiveness of EGFR-TKIs versus chemotherapy as first-line treatment for advanced non-small cell lung cancer: a meta-analysis].</a> Zhongguo Fei Ai Za Zhi. 18 (3), 146-154 (2015).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+turning+point+in+cancer+research:+sequencing+the+human+genome.">A turning point in cancer research: sequencing the human genome.</a> Science. 231 (4742), 1055-1056 (1986).</li><li>Barinaga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DoE+provides+funds+for+human+genome+sequencing.">DoE provides funds for human genome sequencing.</a> Nature. 331 (6152), 103 (1988).</li><li>Lander, E. S., 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=Initial+sequencing+and+analysis+of+the+human+genome.">Initial sequencing and analysis of the human genome.</a> Nature. 409 (6822), 860-921 (2001).</li><li>McCain, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+genome+atlas:+new+weapon+in+old+war?.">The cancer genome atlas: new weapon in old war?.</a> Biotechnology Healthcare. 3 (2), 46-51 (2006).</li><li>Weinstein, J. 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=The+Cancer+Genome+Atlas+Pan-Cancer+analysis+project.">The Cancer Genome Atlas Pan-Cancer analysis project.</a> Nature Genetics. 45 (10), 1113-1120 (2013).</li><li>Carlson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+Generation+Sequencing:+The+Next+Iteration+of+Personalized+Medicine:+Next+generation+sequencing,+along+with+expanding+databases+like+The+Cancer+Genome+Atlas,+has+the+potential+to+aid+rational+drug+discovery+and+streamline+clinical+trials.">Next Generation Sequencing: The Next Iteration of Personalized Medicine: Next generation sequencing, along with expanding databases like The Cancer Genome Atlas, has the potential to aid rational drug discovery and streamline clinical trials.</a> Biotechnology Healthcare. 9 (2), 21-25 (2012).</li><li>Huang, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+antibody+arrays:+a+promising+tool+to+identify+molecular+targets+for+drug+discovery.">Cytokine antibody arrays: a promising tool to identify molecular targets for drug discovery.</a> Combinatorial Chemistry &amp; High Throughput Screening. 6 (8), 769-775 (2003).</li><li>Kopf, E., Zharhary, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+arrays--an+emerging+tool+in+cancer+proteomics.">Antibody arrays--an emerging tool in cancer proteomics.</a> International Journal of Biochemistry and Cell Biology. 39 (7-8), 1305-1317 (2007).</li><li>Rani, S., O'Driscoll, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+changes+in+phosphorylation+of+receptor+tyrosine+kinases:+antibody+arrays.">Analysis of changes in phosphorylation of receptor tyrosine kinases: antibody arrays.</a> Methods in Molecular Biology. 1233, 15-23 (2015).</li><li>Kani, K., 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=JUN-Mediated+Downregulation+of+EGFR+Signaling+Is+Associated+with+Resistance+to+Gefitinib+in+EGFR-mutant+NSCLC+Cell+Lines.">JUN-Mediated Downregulation of EGFR Signaling Is Associated with Resistance to Gefitinib in EGFR-mutant NSCLC Cell Lines.</a> Molecular Cancer Therapeutics. 16 (8), 1645-1657 (2017).</li><li>Brivanlou, A. H., Darnell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+and+the+control+of+gene+expression.">Signal transduction and the control of gene expression.</a> Science. 295 (5556), 813-818 (2002).</li><li>Rikova, K., 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=Global+survey+of+phosphotyrosine+signaling+identifies+oncogenic+kinases+in+lung+cancer.">Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer.</a> Cell. 131 (6), 1190-1203 (2007).</li><li>Chen, V. W., 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=Analysis+of+stage+and+clinical/prognostic+factors+for+colon+and+rectal+cancer+from+SEER+registries:+AJCC+and+collaborative+stage+data+collection+system.">Analysis of stage and clinical/prognostic factors for colon and rectal cancer from SEER registries: AJCC and collaborative stage data collection system.</a> Cancer. 120, 3793-3806 (2014).</li><li>Rueden, C. T., 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=ImageJ2:+ImageJ+for+the+next+generation+of+scientific+image+data.">ImageJ2: ImageJ for the next generation of scientific image data.</a> BMC Bioinformatics. 18 (1), 529 (2017).</li><li>Ward, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+Grouping+to+Optimize+an+Objective+Function.">Hierarchical Grouping to Optimize an Objective Function.</a> Journal of the American Statistical Association. 58 (301), 236-244 (1963).</li><li>Hanahan, D., Weinberg, 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=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Griffin, 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=Array-based+quantitative,+automated+analysis+of+protein+glycosylation.">Array-based quantitative, automated analysis of protein glycosylation.</a> Glycobiology. 14 (11), 1205-1206 (2004).</li><li>Zhou, T., 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=Identification+of+ubiquitin+target+proteins+using+cell-based+arrays.">Identification of ubiquitin target proteins using cell-based arrays.</a> Journal of Proteome Research. 6 (11), 4397-4406 (2007).</li><li>McGarry, T., Biniecka, M., Veale, D. J., Fearon, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia,+oxidative+stress+and+inflammation.">Hypoxia, oxidative stress and inflammation.</a> Free Radical Biology and Medicine. , (2018).</li></ol>

将许多生物读数结合在一起进行的实验中, 是细胞机械的更准确的表示方法。磷抗体阵列的出现使对修饰模式的快速表征, 这可能比任何单一蛋白质的修饰状态更具信息量。应用磷抗体阵列的一般工作流程是基于对丝氨酸、苏氨酸或酪氨酸的修饰。这个例子集中于描述与肺癌治疗的抗性相关的变化。这个应用的主要依据是蛋白质磷酸化在许多人的癌症18的信号转导中起着中心作用,...

靶向酪氨酸激酶抑制剂 (TKI) 的临床应用, 为医生提供了有效的工具来靶向推动肿瘤转化的特定蛋白, 从而改变了癌症治疗的效果。这些化合物抑制或阻止酪氨酸激酶1,2靶蛋白的磷酸化。TKIs 的发展部分是因为各种关键信号基因的基因改变足以推动癌症的启动和进展 [如表皮生长因子受体 (EGFR), 原癌基因酪氨酸-蛋白激酶 src (src),BCR, 人表皮生长因子受体 2 (HER2)]3,4。TKIs 对细胞周期5和分子信号通路6的影响代表了从不相关到分子导向肿瘤治疗的转变。TKIs与化疗的主要优点是反应速度增加, 对健康细胞的毒性风险降低7。因此, 对新型 TKIs 的研究与开发越来越受到重视。
对基因组测序结果的访问开始于人类基因组项目8、9、10 , 并在今天继续进行各种下一代 (NextGen) 癌症测序工作 [例如, 癌症基因组阿特拉斯 (TCGA)11,12]。这启发了许多实验方法, 提供成千上万基因的同时信息和/或提供无偏见的快照的基因或蛋白质调制的生物扰动13。由于细胞功能的调节发生在多个层次, 从基因的转录到蛋白质的后转化修饰和它们的活动, 对控制细胞功能的事件的完全理解将最终需要从各种生物读数中集成数据。通过单细胞基因分辨率来监测成千上万基因的信使 RNA (mRNA) 水平, 提高了对基因功能和整个基因组的相互作用进行推断的能力。然而, 基因表达阵列的解释将永远是完全不完整的, 没有整合其他水平的调节: 即蛋白质表达水平, 蛋白质修饰状态, 和蛋白质后转化修改 (磷酸化、泛素化、甲基化等)。在这里, 我们描述的效用, 抗体阵列作为手段, 以审问后修改的重要信号元件作为一个功能的各种条件在一个实验14,15,16。
磷抗体阵列可以用来区分和分析信号传导通路16的变化。这些可能来自基因修饰或细胞系的治疗与激酶抑制剂, 化疗, 压力引起的葡萄糖剥夺, 缺氧, 或血清饥饿。注意, 耐药性或特定基因的下调也可能导致信号转导通路17的变化。
例如, 药物耐药性可以从药物靶的突变中产生, 以避免敏感。在肺癌中, 已知的 EGFR 突变使癌症不受影响到某些 TKIs, 但更容易受到其他人的影响。替代信号通路可以激活后, 突变17。作为一个更广泛的应用, 以识别的信号转导通路参与抵抗和缺氧等, 磷抗体阵列提供更多的洞察, 并因此了解, 所涉及的机制。
允许对蛋白质修饰进行评估的技术是系统生物学的一个重要组成部分, 因为它们通常服务于调节功能, 例如调节酶的活性或蛋白质之间的物理相互作用。在几乎所有细胞外触发信号传导通路18中, 蛋白质磷酸化的作用说明了转化后修饰的重要性。传统上, 蛋白激酶的鉴定或蛋白质的磷酸化状态也可以由西方的印迹分析来确定, 特别是如果研究者只对1–5目标感兴趣。然而, 西方的印迹是非常有选择性的, 可以偏向先验知识, 可能会错过重要的目标, 结果。抗体阵列通过嵌入各种捕获抗体 [泛特异磷酸化酪氨酸 (s)、反泛素等] 在固体基质 (如玻璃或硝化纤维素) 中, 提供了多目标的中等吞吐量读数。次级抗体提供了基于三明治的 ELISA 格式的特定蛋白质的信息 (图 1)。随着更多的目标感兴趣或先前的知识被限制在15, 这种分析变得更加强大和相关。磷阵列是更广泛的可就业, 因为他们可以比较的磷酸化和一般数量的蛋白质的更广泛的目标在一个实验, 并提供了显着改善量化的质谱。这种技术不适用于鉴定新的或以前未知的磷酸化地点。
基于大规模质谱的蛋白质组学可以用来识别特定的磷酸化部位的蛋白质19。虽然这项技术可以列举数以千计的翻译后事件, 它需要昂贵的仪器, 专用的实验管道, 和计算专长, 是超出了大多数研究人员。
抗体阵列提供了在不同的蛋白质读数16的同时读数。这些可能是蛋白质泛素化 (泛素阵列) 或磷酸化的变化。该阵列技术的主要优点是, 它提供了重要的反馈, 对与重要细胞参数相关的各种生物通路 (蛋白质 53 kDa, p53, 受体酪氨酸激酶, 细胞内通路) 的生物状态。同时。此外, 可以结合各种阵列类型, 以增加检测的渗透 (例如, 凋亡和泛素和磷酸化)。这种将多个阵列组合在一起以时间和成本效益的方式来评估多个样本中的各种平移后更改的能力, 在这种情况下是一个很大的优势。抗体阵列。

<table border="0" cellpadding="0" cellspacing="0" width="1045">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Odysee SA Imager</td>
			<td style="width:195px;">Li-Cor Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:364px;">Fluorescent Imager</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">1.5 ml tube</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">22363212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Cell Scraper</td>
			<td style="width:195px;">Falkon (Corning)</td>
			<td style="width:119px;">353085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Dulbeco&#39;s Phosphate buffered Saline (PBS)</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td>wash buffer for protein extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tissue Culture dish 100mm</td>
			<td style="width:195px;">TRP</td>
			<td style="width:119px;">93100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ICC Insulin syringe U100</td>
			<td style="width:195px;">Becton Dickinson</td>
			<td style="width:119px;">329412</td>
			<td>27G5/8,&#160; 1ml for needle treatment of protein samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Protein Profiler ARRAY&#160;</td>
			<td style="width:195px;">R&#38;D</td>
			<td style="width:119px;">ARY003B</td>
			<td>Human phospho MAPK array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Protein Profiler ARRAY&#160;</td>
			<td style="width:195px;">R&#38;D</td>
			<td style="width:119px;">ARY002B</td>
			<td>Human phospho kinase array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Centrifuge Eppendorf 5430R</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Table top centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Pierce BCA protein assay kit</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">SpectraMAX M2</td>
			<td style="width:195px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Absorbance reader for protein quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">IRDye 800CW Streptavidin</td>
			<td style="width:195px;">Li-Cor Biosciences</td>
			<td style="width:119px;">925-32230</td>
			<td>Streptavidin conjugate for fluorescent detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">LabGard ES Class II, Type A2 biosafety cabinet</td>
			<td style="width:195px;">NuAire</td>
			<td style="width:119px;">NU-425-400</td>
			<td>Tissue culture hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">TC20 automated cell counter</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:119px;">1450102</td>
			<td>Cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Halt Protease &#38; Phosphatase Inhibitor Cocktail (100X)</td>
			<td style="width:195px;">Thermo Fisher</td>
			<td style="width:119px;">78446</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">RIPA buffer</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">R0278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Sonic Dismembrator</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">F60</td>
			<td>sonicator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Rocking platform shaker</td>
			<td style="width:195px;">VWR</td>
			<td style="width:119px;">10860-780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">ImageJ</td>
			<td style="width:195px;">NIH open source</td>
			<td style="width:119px;"></td>
			<td>https://imagej.net/Welcome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAS Institutie JMP&#174; 12.1.0 (64-bit)</td>
			<td style="width:195px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
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assessment of resistance to tyrosine kinase inhibitors by an interrogation of signal transduction pathways by antibody arrays

癌症患者对蛋白质磷酸化网络的异常调节通常是用酪氨酸激酶抑制剂治疗的。反应率接近85% 是常见的。不幸的是, 患者往往通过改变信号转导通路而成为治疗的顽固。使用微阵列的表达式分析的实现可以识别整个 mRNA 水平的变化, 蛋白质组学可以识别蛋白质水平的总体变化, 或者可以识别所涉及的蛋白质, 但信号传导的活动途径只能通过询问翻译后的蛋白质的修改来建立。因此, 确定药物治疗是否成功或是否出现抗药性的能力, 或者是否能够表征信号通路的任何改变, 是一项重要的临床挑战。在这里, 我们提供了一个详细的解释抗体阵列作为一个工具, 可以识别全系统的变化, 在不同的翻译后修改 (例如, 磷酸化)。使用抗体阵列的优点之一包括它们的可访问性 (阵列不需要蛋白质组学或昂贵设备的专家) 和速度。针对组合后翻译修改的阵列的可用性是主要的限制。此外, 无偏方法 (phosphoproteomics) 可能更适合于新发现, 而抗体阵列是最广泛的特征目标的理想。

为探讨 TKI 抗性对细胞系信号传导通路的影响, 对四例进行了分析。一个控制样本 (H3255r1) 和 3 TKI 的细胞线 (H3255r2-4) (图 2) 与现场抗体模板 (图 3) 相关。所有4个样本都是使用本议定书编写的。六磷酸有差异活性的选择, 以证明对抗体阵列的分析 (图 4)。热图 (图 5) 提供了关于重要信号?...

Watch this Scientific Journal Video about Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays at JoVE.com

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

在这里, 我们提出了抗体阵列的协议, 以确定各种细胞模型中信号通路的变化。这些变化, 由药物/缺氧/紫外线光/辐射, 或过度表达/下调/挖空, 是重要的各种疾病模型, 并可以表明治疗是否有效或能识别药物的机制电阻。

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

此方法可以帮助回答有关信号网络中可能导致癌症生长或对特定疗法的耐药性发展的关键问题。该技术的主要优点是，它可以提供快速和可靠的反馈，对多网络的磷酸化状态，经常解除管制的癌症。该技术的含义延伸到抗药性癌症的治疗，因为改变和翻译后修饰的识别可以阐明信号转导通路活性的变化。虽然这种技术用于磷酸化，它也可以用来查看其他翻译后修饰，如糖基化和泛基化。首先彻底冲洗隔夜培养细胞三次，10毫升PBS小心切开所有PBS后最后一次洗涤。接下来，从蛋白质测定试剂盒中加入适当体积的解解缓冲液，并使用细胞刮刀分离细胞。将产生的细胞溶液三解约10次，然后将至少10倍转移到第七细胞到1.5毫升管。在4摄氏度下用震动孵育细胞30分钟。然后将落酸装入装有27表针的注射器中，吸气并分配样品10次，切入细胞膜。当细胞膜完全被破坏时，将解液离心，然后将上清液转移到新的1.5毫升管中，以便根据标准协议进行后续总蛋白质定量。要进行人类磷酸激酶测定，首先使用平尖钳子将一个阵列膜放入四孔多托盘的每个孔中，该孔包含两毫升新鲜准备的阵列缓冲器，每孔一个孔的膜数量朝上。浸水后，膜上的染料将消失。用盖子盖住托盘，在室温下在摇床摇床上孵化膜 60 分钟。在阻断孵育过程中，将每个解液样品稀释至50至100微克的蛋白质浓度，最大体积为334微升的解解缓冲液，并将最终提取的蛋白质样品量与新鲜阵列缓冲液一起稀释至1.5毫升。接下来，小心地从多托盘的每个孔中吸出阵列缓冲液，并在摇台摇床上用每孔1.5毫升蛋白质样品孵育膜，在2至8摄氏度的温度下过夜。第二天早上，小心地将每个阵列转移到单个塑料容器中，里面装着 20 毫升的洗涤缓冲液，在室温下在摇台上清洗 3 个 10 分钟。上次洗涤后，将20微升适当重组的抗体鸡尾酒加入两毫升阵列缓冲液中，每孔两升，并小心地将每膜的下边缘印在纸巾上，以去除多余的洗涤缓冲液。然后将每个阵列放回抗体托盘的适当井中，并在摇台的室温下盖住托盘进行两个小时的孵育。在孵化结束时，小心地将每个阵列转移到一个新的8乘11厘米的塑料容器中，里面装着20毫升的洗涤缓冲液，进行3次10分钟的洗涤，正如刚才所证明的。上次洗涤后，在多孔托盘的每个孔中加入一毫升适当的链球菌素马萝卜过氧化物酶溶液，并将膜返回适当的井中，在室温下孵育30分钟。在孵化结束时，将每个膜放在两块五毫米滤纸之间，以去除多余的缓冲液，用适当的马萝卜过氧化物酶检测溶液孵育干燥膜三分钟。然后将膜放入单独的透明塑料板保护器中，以便进行后续成像。要评估每个样本中的受体酪氨酸激酶表达，请在适当的图像处理程序（如图像 J）中打开图像，然后从适当的下拉菜单中选择"编辑"和"反转"，将图像白色背景上的黑点反转到黑色背景上的白色点。接下来，使用"椭圆形"工具将一对白点包围在点印上，然后选择"分析和测量"以自动打开显示选定区域值的新窗口。然后将鼠标箭头的点放入椭圆形的中心，并拖动下一个点周围的圆形区域，直到测量所有感兴趣点。在这个具有代表性的实验中，在三个酪氨酸激酶抑制剂抗性细胞系中，对酪氨酸激酶抑制剂耐药通路的影响进行了一次控制分析。选择六种具有差分活性的磷蛋白，并确定了每个细胞系中几种候选蛋白的相对强度。如热图所证明的，可以生成关于重要信号转导蛋白蛋白质磷酸化变化的半定量读出。在尝试此程序时，重要的是要记住从健康和主动分裂的细胞开始，因为压力源可能诱发信号转导通路活性的变化。按照这个程序，其他方法，如数字PCR和免疫印迹可以用来回答其他问题，如磷酸化的变化会导致转录因子的激活？该技术开发后，为癌症生物学的研究人员探索蛋白质转化后修饰的根本变化铺平了道路，导致癌症患者出现个性化药物。不要忘记，使用癌细胞系是非常危险的，所以一定要使用无菌技术，在处置利器时要小心，并时刻穿着实验室外套。

Research

JoVE Journal

Cancer Research

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

骨髓增殖性疾病的转导移植小鼠模型

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

科研

癌症研究

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

寻找获得性抗靶治疗的驱动途径: 耐药亚克隆生成与基因击倒敏感性恢复

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

球体、菌落形成测定和斑马鱼移植对抗癌香豆素 OT48 生物活性的临床前评价

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

抗肿瘤药物对抗癌抗体疗效的潜力: 用组合指数方程检测抗体-药物协同作用

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

通过 SHP2 （PTPN11） 磷酸酶抑制剂评估细胞目标参与度

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...