通过两个互补细胞同步方案研究细胞周期调控的基因表达
Summary
我们报告两个细胞同步协议，提供一个上下文，用于研究与细胞周期的特定阶段相关的事件。我们表明，这种方法可用于分析未受扰动细胞周期中特定基因的调节，或暴露于影响细胞周期的试剂。
Abstract
细胞周期的基因表达程序代表了解细胞周期依赖性过程及其在疾病如癌症中的作用的关键步骤。细胞周期调节基因表达分析取决于细胞同步到特定阶段。在这里，我们描述了一种利用两种互补同步协议的方法，通常用于研究细胞周期中基因表达的周期性变化。两种方法均基于在一个限定点瞬时阻断细胞周期。羟基脲（HU）治疗的同步方案导致晚期G1 /早期S期的细胞停滞，并且从HU介导的停止释放提供通过S和G2 / M均匀进展的细胞群体。通过胸苷和诺考达唑（Thy-Noc）治疗的同步方案阻止早期有丝分裂中的细胞，并从Thy-Noc介导的停止释放提供了适合于G1期和S期的同步细胞群体尝试学习。两种方法的应用都需要监测细胞周期分布特征，这通常在细胞碘化丙啶（PI）染色后进行，流式细胞仪介导的DNA含量分析。我们表明，两个同步协议的组合使用是一种可靠的方法来清楚地确定在细胞周期中差异调节的基因（ 即 E2F1和E2F7）的转录谱，从而更好地了解它们在细胞周期中的作用流程。此外，我们显示，这种方法对于基于药物治疗（ 即丝裂霉素C，抗癌剂）的机制的研究是有用的，因为它允许区分对基因毒性因子起反应性的基因仅受细胞周期扰动影响的基因由代理人强加。
Introduction
通过细胞周期的所有阶段的过渡与紧密调节的基因表达程序相结合。认为在整个细胞周期中协调基因转录的“开”和“关”是在复杂的转录调节系统的控制之下，不仅调节时间，而且调节基因表达的水平。已知解除关键细胞周期成分的调节有助于几种疾病的发展，并且是肿瘤发生的已知标志1,2 。在酵母和哺乳动物细胞中进行的全基因组转录组学分析显示，大量基因在细胞周期中表现出周期性的基因表达模式，表明细胞周期内的转录波动反映了给定基因产物的时间需求在精确的阶段3，4 ， </sup> 5 。
研究细胞周期调节基因表达的主要任务是将细胞同步到特定的细胞周期阶段。细胞同步有助于解释基因表达模式与特定细胞周期相变的关联，并且导致更好地了解许多基因的调节和功能。细胞同步对于研究抗癌药物的作用机制也是重要的，因为已知化学治疗剂影响基因表达以及细胞周期动力学6,7 。然而，通常难以确定由这些药剂治疗引起的基因表达差异是否是对治疗的直接反应，或者仅仅是细胞周期特征变化的结果。为了区分这些可能性，基因表达应该在已经存在的细胞中进行分析在加入化疗药物之前同步化。
除了一些原代细胞例如新鲜分离的淋巴样细胞，其构成在G0-8中同步的均质细胞群体， 体外建立的细胞系在培养物中异生生长。在常规生长条件下，这些异步循环细胞发现于细胞周期的所有阶段，但优先在G1 9中 。因此，本文不提供特定细胞周期阶段功能或基因表达分析的最佳方案（ 如 G1，S等）。未转化的永生化细胞系（ 例如成纤维细胞）可以与所谓的生理方法10同步。这些方法基于未转化细胞的保留的原代细胞特征，例如细胞接触抑制和生长因子依赖性，以便继续循环。切除的血清与接触抑制组合使得非转化细胞在G0 / G1处停滞。然而，同步的细胞周期进入和进展通常需要传代培养，这也涉及细胞的人工脱离和再镀覆10 。最重要的是，该方法不适用于转化细胞系的同步，绝大多数目前使用的已建立的细胞系，其特征在于缺乏细胞接触介导的生长抑制或对生长因子撤出的应答。因此，很明显，在细胞周期的特定阶段中需要替代方法用于有效的细胞同步。一般来说，最常用的同步方法是基于细胞周期的一个定义点的瞬时化学或药理学抑制，通常是DNA合成或有丝分裂纺锤体形成。抑制DNA合成通过在晚期G1期或早期S期停滞细胞来同步细胞。这可以是achi通过添加化合物，如拟合物，核苷酸生物合成抑制剂11,12 ， aphidicolin，DNA聚合酶抑制剂13,14 ，羟基脲，核糖核苷酸还原酶15,16抑制剂或过量的胸苷17,18 等化合物。另一方面，微管聚合抑制剂如秋水仙碱或诺考达唑能够阻止有丝分裂纺锤体形成，导致早期M期19,20,21细胞同步。
在这项工作中，我们描述了一种基于瞬时化学抑制的两个互补同步协议的方法，用于研究mRNA上的细胞周期调节基因的表达水平。这种方法是定义细胞周期基因在特定细胞周期过程中的作用的基础。此外，它为研究抗癌治疗的影响提供了一个总体框架，以准确地检测药物反应性基因，并最大限度地减少由这些药物产生的细胞周期进程扰动产生的误解。
Protocol
细胞周期进展的细胞同步，释放和监测 基于胸腺嘧啶和诺考达唑（Thy-Noc）的同步和释放的有丝分裂的U2OS细胞 准备所需的细胞培养基。 U2OS细胞通常在补充有10％（体积/体积）FBS（任选的：1％青霉素/链霉素）的DMEM-谷氨酰胺培养基中生长。在无菌条件下进行所有培养基制备和操作，并在使用前将补充培养基（从现在称为“完全培养基”）升温至37℃。 在10毫升完?…
Representative Results
用于细胞同步的Thy-Noc和HU协议的示意图。 图1总结了U2OS细胞同步和随后的样品收集所需的步骤，以验证细胞周期的进展并进行基因表达分析。 Phospho-H3和PI染色是选择同步方法的良好评估参数。 由于培养细胞的固有异质性，必?…
Discussion
在细胞周期中涉及短暂和特异性作用的微调调节基因的分析需要均匀的细胞群体。许多研究人员通常使用长期建立的肿瘤细胞系用于这些目的，并且已经开发了多种方法以获得同步（或部分同步的）细胞群体，目的是在确定的细胞周期阶段中积累尽可能多的细胞。此外，已经作出了大量努力，以改进和优化建立良好的同步方式。然而，所有同步协议都有缺点，这可能归因于细胞培养物的异质性，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Zubiaga和Altmeyer实验室的成员进行有益的讨论和技术支持。这项工作得到了西班牙部（SAF2015-67562-R，MINECO / FEDER，UE），巴斯克政府（IT634-13和KK-2015/89）和巴斯克历史大学UPV / EHU（ UFI11 / 20）。
Materials
| DMEM, high glucose, GutaMAX supplement | Thermo Fisher Scientific | 61965-059 | |
| FBS, qualified, E.U.-approved, South America origin | Thermo Fisher Scientific | 10270-106 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140-122 | |
| 0.25% Trypsin-EDTA (1X), phenol red | Thermo Fisher Scientific | 25200-072 | |
| Thymidine | SIGMA | T1895-5G | Freshly prepared. Slight warming might help dissolve thymidine. |
| Nocodazole | SIGMA | M-1404 | Stock solution in DMSO stored at -20 ºC in small aliquots |
| Hydroxyurea | SIGMA | H8627 | Freshly prepared |
| Mitomycin C from Streptomyces caespitosus | SIGMA | M4287 | 1.5mM stock solution in sterile H2O protected from light and stored at 4ºC |
| Dimethyl sulfoxide | SIGMA | D2650 | |
| Propidium iodide | SIGMA | P4170 | Stock solution in sterile PBS at 5 mg/ml, stored at 4º C protected from light. |
| PBS pH 7.6 | Home made | ||
| Ethanol | PANREAC | A3678,2500 | |
| Chloroform | SIGMA | C2432 | |
| Sodium Citrate | PANREAC | 131655 | |
| Triton X-100 | SIGMA | T8787 | |
| RNAse A | Thermo Fisher Scientific | EN0531 | |
| TRIzol Reagent | LifeTechnologies | 15596018 | |
| RNeasy Mini kit | QIAGEN | 74106 | |
| High-Capacity cDNA Reverse Transcription Kit | Thermo Fisher Scientific | 4368814 | |
| Anti-Cyclin E1 antibody | Cell Signaling | 4129 | 1:1000 dilution in 5% milk, o/n, 4 ºC |
| Anti-Cyclin B1 antibody | Cell Signaling | 4135 | 1:1000 dilution in 5% milk, o/n, 4 ºC |
| Anti-β-actin | SIGMA | A-5441 | 1:3000 dilution in 5 % milk, 1 hr, RT |
| Anti-pH3 (Ser 10) antiboty | Millipore | 06-570 | Specified in the protocol |
| Secondary anti-rabbit AlexaFluor 488 antibody | Invitrogen | R37116 | Specified in the protocol |
| Secondary anti-mouse-HRP antibody | Santa Cruz Biotechnology | sc-3697 | 1:3000 dilution in 5 % milk, 1 hr, RT |
| Forward E2F1 antibody (human) TGACATCACCAACGTCCTTGA | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Reverse E2F1 antibody (human) CTGTGCGAGGTCCTGGGTC | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Forward E2F7 antibody (human) GGAAAGGCAACAGCAAACTCT | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Reverse E2F7 antibody (human) TGGGAGAGCACCAAGAGTAGAAGA | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Forward p21Cip1 antibody (human) AGCAGAGGAAGACCATGTGGAC | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Reverse p21Cip1 antibody (human) TTTCGACCCTGAGAGTCTCCAG | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Forward TBP antibody (human) reference gene | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Reverse TBP antibody (human) | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Forward Oxa1L antibody (human) reference gene CACTTGCCAGAGATCCAGAAG | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Reverse Oxa1L antibody (human) CACAGGGAGAATGAGAGGTTTATAG | Biolegio | Designed by PrimerQuest tool (https://eu.idtdna.com/site) | |
| Power SYBRGreen PCR Master Mix | Thermo Fisher Scientific | 4368702 | |
| FACS Tubes | Sarstedt | 551578 | |
| MicroAmp Optical 96-Well Reaction Plate | Thermo Fisher Scientific | N8010560 | |
| Corning 100mm TC-Treated Culture Dish | Corning | ||
| Corning Costar cell culture plates 6 well | Corning | 3506 | |
| Refrigerated Bench-Top Microcentrifuge | Eppendorf | 5415 R | |
| Refrigerated Bench-Top Centrifuge Jouan CR3.12 | Jouan | 743205604 | |
| NanoDrop Lite Spectrophotometer | Thermo Scientific | ND-LITE-PR | |
| BD FACSCalibur Flow Cytometer | BD Bioscience | ||
| QuantStudio 3 Real-Time PCR System | Thermo Fisher Scientific | A28567 |
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Cite This Article
Apraiz, A., Mitxelena, J., Zubiaga, A. Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols. J. Vis. Exp. (124), e55745, doi:10.3791/55745 (2017).