细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定
Summary
该方法结合了DNA双链断裂（DSB）、细胞循环分布和凋亡的定量分析，实现了对DSB诱导和修复以及修复失败后果的细胞周期特定评估。
Abstract
提出的方法或稍作修改的版本已经设计，以研究特定的治疗反应和副作用的各种抗癌治疗，用于临床肿瘤学。它能够定量和纵向分析基因毒性应激后DNA损伤反应，如放射治疗和多种抗癌药物引起的。该方法涵盖DNA损伤反应的所有阶段，为诱导和修复DNA双链断裂（DSBs）、细胞周期抑制和在修复失败时细胞凋亡导致细胞死亡提供终点。结合这些测量提供了有关细胞周期依赖治疗效果的信息，从而可以深入研究细胞增殖和应对DNA损伤的机制之间的相互作用。由于许多癌症治疗（包括化疗剂和电离辐射）的作用仅限于或根据特定的细胞周期阶段而产生强烈变化，相关分析依赖于一种可靠和可行的方法来评估治疗效果以细胞周期特定的方式在DNA上。单端点检测不可能实现这一点，而且该方法具有重要优势。该方法不局限于任何特定的细胞系，并已在众多肿瘤和正常组织细胞系中进行了彻底测试。除放射肿瘤学外，在肿瘤学的许多领域，包括环境危险因素评估、药物筛选和肿瘤细胞遗传不稳定性评价，可广泛应用。
Introduction
肿瘤学的目标是在不损害正常细胞的情况下杀死或使癌细胞灭活。许多疗法要么直接或间接地诱导癌细胞中的基因毒性应激，但也在正常细胞中有些延伸。化疗或靶向药物通常与放射治疗相结合，以提高辐照肿瘤1、2、3、4、5的放射敏感性，从而减少辐射剂量，以尽量减少正常的组织损伤。
电离辐射和其他基因毒性剂会诱发不同类型的DNA损伤，包括基体修饰、链交联和单股或双链断裂。脱氧核糖核酸双链断裂（DSBs）是最严重的DNA病变，其诱导是电离辐射和各种细胞静血药物在放射化疗中细胞杀灭作用的关键。DSB不仅会损害基因组的完整性，而且促进突变6、7的形成。因此，不同的DSB修复途径，以及消除不可修复的细胞（如凋亡）的机制在进化过程中已经形成。整个DNA损伤反应（DDR）由复杂的信号通路网络调节，从DNA损伤识别和细胞周期抑制到允许DNA修复，到程序化细胞死亡或失活的情况下修复失败8。
已开发流式细胞测定法，用于在一次涵盖 DSB 感应和修复的综合测定中，对基因毒性应力后的 DDR 进行调查，以及修复失败的后果。它结合广泛应用的DSB标记βH2AX的测量与细胞周期和凋亡诱导的分析相结合，使用经典的subG1分析和更具体的卡巴塞-3活化评估。
将这些端点组合在一个测定中不仅减少了时间、人工和成本支出，还实现了对DSB感应和修复以及caspase-3活化的细胞周期特定测量。这种分析不可能与独立进行的分析，但它们对于全面了解基因毒性压力后DNA损伤反应具有高度相关性。许多抗癌药物，如细胞静态化合物，针对分裂细胞，其效率在很大程度上取决于细胞周期阶段。不同DSB修复过程的可用性也取决于细胞周期阶段和路径选择，这对修复精度至关重要，进而决定细胞9、10、11的命运。 12.此外，对DSB水平的细胞周期特定测量比集中分析更准确，因为DSB水平不仅取决于基因毒性化合物或辐射的剂量，还取决于细胞的DNA含量。
该方法已用于比较不同放射疗法的疗效，以克服胶质细胞瘤13的抗药性机制，并剖析电离辐射和骨肉瘤14、15中靶向药物的相互作用和非典型畸状类人横纹癌16。此外，所述方法已被广泛用于分析无线电和化疗对介体干细胞17、18、19、20、21的副作用。 22，23，24，这是修复治疗引起的正常组织损伤，并在再生医学中具有潜在的应用。
Protocol
1. 准备 在任何类型的培养容器中制备 ±1 x 105细胞/样品作为起始材料。 例如，在将U87胶质细胞细胞暴露于电离辐射后进行时间过程实验：在每个时间点以三联成的T25瓶中辐照亚汇U87细胞。选择早期时间点（辐照后 15 分钟至 8 小时），跟随 DSB 修复动力学（+H2AX 级别）和后期时间点（24 小时至 96 小时），以评估残留 DSB 水平、细胞周期效应和凋亡。注?…
Representative Results
人类U87或LN229胶质细胞细胞受到4Gy光子或碳离子电池辐射的照射。使用此处介绍的流式细胞测定方法（图3），在辐照后48小时的不同时间点测量细胞周期特异性βH2AX水平和凋亡。在两种细胞系中，碳离子诱导更高的βH2AX峰值水平，下降速度较慢，在24至48小时时与相同物理剂量的光子辐射相比显著升高（图4A）。这表明，与修复效率较低的光…
Discussion
该特色方法易于使用，并提供快速、准确和可重复的DNA损伤响应测量，包括双链断裂（DSB）诱导和修复、细胞周期效应和凋亡细胞死亡。与单个测定相比，这些端点的组合提供了更完整的两性关系图。该方法可广泛应用于放射生物学、治疗和保护领域的综合基因毒性测定，更普遍地应用于肿瘤学（例如，环境风险因素评估、药物筛选和基因评估）肿瘤细胞的不稳定性）。
该?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢德国癌症研究中心（DKFZ）的流式细胞测定设施团队的支持。
Materials
| 1000 µL filter tips | Nerbe plus | 07-693-8300 | |
| 100-1000 µL pipette | Eppendorf | 3123000063 | |
| 12 x 75 mm Tubes with Cell Strainer Cap, 35 µm mesh pore size | BD Falcon | 352235 | |
| 15 mL tubes | BD Falcon | 352096 | |
| 200 µL filter tips | Nerbe plus | 07-662-8300 | |
| 20-200 µL pipette | Eppendorf | 3123000055 | |
| 4’,6-Diamidin-2-phenylindol (DAPI) | Sigma-Aldrich | D9542 | Dissolve in water at 200 µg/ml and store aliquots at -20 °C |
| Alexa Fluor 488 anti-H2A.X Phospho (Ser139) Antibody, RRID: AB_2248011 | BioLegend | 613406 | Dilute 1:20 |
| Alexa Fluor 647 Rabbit Anti-Active Caspase-3 Antibody, AB_1727414 | BD Pharmingen | 560626 | Dilute 1:20 |
| BD FACSClean solution | BD Biosciences | 340345 | For cytometer cleaning routine after measurement |
| BD FACSRinse solution | BD Biosciences | 340346 | For cytometer cleaning routine after measurement |
| Dulbecco’s Phosphate Buffered Saline (PBS) | Biochrom | L 182 | Dissolve in water to 1x concentration |
| Dulbecco's Modified Eagle's Medium with stable glutamin | Biochrom | FG 0415 | Routine cell culture material for the example cell line used in the protocol |
| Ethanol absolute | VWR | 20821.330 | |
| Excel software | Microsoft | ||
| FBS Superior (fetal bovine serum) | Biochrom | S 0615 | Routine cell culture material for the example cell line used in the protocol |
| FlowJo v10 software | LLC | online order | |
| Fluoromount-G | SouthernBiotech | 0100-01 | Embedding medium for optional preparation of microscopic slides from stained samples |
| folded cellulose filters, grade 3hw | NeoLab | 11416 | |
| LSRII or LSRFortessa cytometer | BD Biosciences | ||
| MG132 | Calbiochem | 474787 | optional drug for apoptosis positive control |
| Multifuge 3SR+ | Heraeus | ||
| Paraformaldehyde | AppliChem | A3813 | Prepare 4.5% solution fresh. Dilute in PBS by heating to 80 °C with slow stirring under the fume hood. Cover the flask with aluminium foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a cellulose filter. |
| Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor® 555 Conjugate) RRID: AB_10694639 | Cell Signaling Technology | #3475 | Dilute 3:200 |
| PIPETBOY acu 2 | Integra Biosciences | 155 016 | |
| Serological pipettes, 10 mL | Corning | 4488 | |
| Serological pipettes, 25 mL | Corning | 4489 | |
| Serological pipettes, 5 mL | Corning | 4487 | |
| SuperKillerTRAIL (modified TNF-related apoptosis-inducing ligand) | Biomol | AG-40T-0002-C020 | optional drug for apoptosis positive control |
| T25 cell culture flasks | Greiner bio-one | 690160 | Routine cell culture material for the example cell line used in the protocol |
| Trypsin/EDTA | PAN Biotech | P10-025500 | Routine cell culture material for the example cell line used in the protocol |
| U87 MG glioblastoma cells | ATCC | ATCC-HTB-14 | Example cell line used in the protocol |
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Cite This Article
Lopez Perez, R., Münz, F., Kroschke, J., Brauer, J., Nicolay, N. H., Huber, P. E. Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry. J. Vis. Exp. (151), e59968, doi:10.3791/59968 (2019).