In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.
Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs’ long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics.
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged α-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.
乳腺癌死亡率的主要原因就是通过转移1,2。紫杉烷类,如多西他赛和紫杉醇,目前用作一线转移性乳腺癌2,3,4,5,6的治疗方案。他们是一群扰乱微管动力学微管靶向代理(MTA)的一部分。然而,在治疗性治疗使用紫杉烷类的最大挑战之一是在癌细胞中紫杉烷抗性,从而导致疾病复发7的发展。耐药占转移性乳腺癌7之间所有死亡的90%以上。
微管是由α-和β微管蛋白异源二聚体的聚合形成类=“外部参照”> 8,9。微管动力学的精确调节是许多细胞功能,包括细胞极化,细胞周期进程,细胞内运输和细胞信号传导的重要。微管和它们的动态的失调会扰乱细胞的功能,导致细胞死亡10,11。取决于它们怎样导致此失调,MTA药物可归类为微管稳定剂(即紫杉烷)或微管destabalizing剂( 即,长春花生物碱或秋水仙碱点结合剂)20。尽管微管的质量的相反的效果,以足够的剂量,这两个类可以通过其对微管动力学21作用杀死癌细胞。
紫杉烷类通过稳定微管主轴12主要作用,导致染色体错位。在纺锤体装配检验点(SAC)的后续永久激活逮捕的有丝分裂细胞。延长的有丝分裂停滞然后导致凋亡13,14。紫杉烷与通过对β微管蛋白8,15,这是只有在组装的微管蛋白16存在的紫杉烷的结合位点的微管相互作用。
对紫杉烷抗性多种机制已经提出9,17。这些机制包括由于药物外排蛋白与紫杉类药物特异性抵抗力5,9,18,19的表达了一般多药耐药性。例如,耐紫杉类癌细胞可能已经改变了某些β桶的表达和功能同型球蛋白5,9,19,20,21,22,23。通过使用体内方法测量微管动态不稳定,我们表明,相对于非抗性,父母的MCF-7 的CC单元17时,耐多西紫杉醇的MCF-7 的TXT细胞的微管动力学是不敏感的多西他赛治疗。
为了更好地了解的MTA的功能,并在癌细胞紫杉烷抗性的确切机制,重要的是测量微管动力学。这里,我们报告这样的体内方法。通过使用实时成像结合GFP标记的微管蛋白在细胞中的表达,我们可以测量的MCF-7 的TXT和MCF-7 的CC细胞和Wi的微管动力学thout多西紫杉醇治疗。研究结果可以帮助我们设计出能够克服阻力紫杉更加有效的药物。
有两种主要的方法来测量微管动态不稳定性: 在体外和体内。在体外方法中,纯化的微管蛋白是用来测量与计算机增强的时间推移微分干涉反差显微镜微管动态不稳定性。在体内方法中,显微注射荧光微管蛋白,或表达GFP的微管蛋白,掺入微管。微管的动力学(生长和缩短)然后通过时间推移在间期细胞10,20,25</…
The authors have nothing to disclose.
This research is supported by funding from CBCF (to ZW).
Dulbecco's Modified Eagle's Medium (DMEM) | Sigma-Aldrich | D5796 | |
Non-essential amino acids | Life Technologies, Invitrogen | 11140-050 | |
FBS | Gibco, Invitrogen | 12483 | |
Anti-Anti (100x) | Life Technologies, Invitrogen | 15240-062 | |
docetaxel | Sigma-Aldrich | 01885-5mg-F | |
DMEM phenol red-free | Gibco, Invitrogen | 21063 | |
CellLight Reagent *BacMam 2.0* GFP-tubulin | ThermoFisher Scientific | C10613 | Key reagent for expressing GFP tubulin in cells |
CellLight Reagent *BacMam 2.0* GFP | ThermoFisher Scientific | B10383 | Control |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich+B9:AA9 | 472301 | for dissoving decetaxel |
22-mm glass coveslip | Fisher Scientifics | 12-545-101 | |
6-well culture plate | Greiner Bio-One International | 6 Well Celi Culture Plate | |
DeltaVision Microscopy Imaging Systems | GE Health | This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control. | |
Trypsin-EDTA (0.25%), phenol red | ThermoFisher Scientific | 25200056 | |
Bright-Line Hemacytometer Set, Hausser Scientific | Hausser Scientific, Distributed by VWR | Supplier No.: 1492 VWR No.:15170-172 |