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Jan 2018
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Cell Synchronization by Double Thymidine Block
用胸腺嘧啶核苷双阻断法同步细胞   

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Abstract

Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at G1/S boundary by using double thymidine block. After release into normal medium, cell population at distinct cell cycle phase could be collected at different time points.

Keywords: Cell synchronization (细胞同步化), Cell cycle (细胞周期), Thymidine (胸腺嘧啶核苷), DNA synthesis (DNA合成), DNA replication (DNA复制)

Background

Cell cycle and cell division lie at the heart of cell biology. To build multicellular organism, cell duplication is necessary to generate specialized cells, which can execute particular function. The normal cell cycle is composed of interphase (G1, S and G2 phase) and mitotic (M) phase (Rodríguez-Ubreva et al., 2010; Léger et al., 2016). During interphase, the genetic materials are duplicated and make everything ready for mitosis. Whereas, during mitotic phase, the duplicated chromosomes are segregated and distributed into daughter cells (Sakaue-Sawano et al., 2008).

To precisely preserve genetic information, cell cycle progression must be tightly regulated. Cyclin/CDK complexes control the cell cycle progression through rapidly promoting activities at their respective stages, and are quickly inactivated when their stages are completed (Graña and Reddy, 1995).

Cell synchronization is particularly useful for investigating a cell-cycle regulated event. Using different methods, cells could be synchronized at different cell cycle stage. Treatment of nocodazole, which is an inhibitor of microtubule formation, could synchronize cells at G2/M phase (Ho et al., 2001), while, hydroxyurea, a dNTP synthesis inhibitor, synchronize cells at early S phase (Koç et al., 2004). As an Inhibitor of DNA synthesis (Schvartzman et al., 1984), thymidine can arrest cell at G1/S boundary. Here, we describe a detail method to synchronize cells at G1/S boundary by thymidine (Chen et al., 2018).

Materials and Reagents

  1. 10 cm culture dish (Corning, catalog number: 430167 )
  2. Gloves (VWR International, catalog number: 82026 )
  3. Protective clothing (VWR International, catalog number: 414004-444 )
  4. Eyewear (VWR International, catalog number: 89187-984 )
  5. Human tumor cell lines: H1299 (ATCC, catalog number: ATCC® CRL-5803TM )
  6. Dulbecco's Modified Eagle's Medium (DMEM) (high glucose with L-glutamine) (Corning, catalog number: 10-013-CV )
  7. Phosphate-Buffered Saline (PBS) (Corning, catalog number: 21-040-CV )
  8. Fetal bovine serum (FBS) (ATLANTA BIOLOGICALS, catalog number: S11150 )
  9. Thymidine (Sigma-Aldrich, catalog number: T9250 )
  10. Propidium Iodide (PI) (Thermo Fisher Scientific, catalog number: P3566 )
  11. Antibodies
    1. Anti-Cyclin A (Abcam, catalog number: ab38 )
    2. Anti-Cyclin D (Santa Cruz Biotechnology, catalog number: sc-753 )
    3. Anti-β-Actin (Santa Cruz Biotechnology, catalog number: sc-58673 )
  12. Tris-HCl, pH 8.0 (Thermo Fisher Scientific, catalog number: 15568025 )
  13. NaCl (Sigma-Aldrich, catalog number: S9888 )
  14. NP-40 (Abcam, catalog number: ab142227 )
  15. EDTA (Thermo Fisher Scientific, catalog number: 15576028 )
  16. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  17. EBC cell lysis buffer (see Recipes)
  18. Electrophoresis running buffer (see Recipes)
  19. Transfer buffer (see Recipes)

Equipment

  1. Cell culture incubator (VWR International, model: 98000-368 )
  2. Flow cytometry system (BD, model: FACSLyric )
  3. X-RAY Film processor (Konica Minolta Healthcare Americas, model: SRX-101A )

Procedure

  1. Plate H1299 cells at 20-30% confluence in a 10 cm culture dish (2 x 106-3 x 106 cells per dish) containing 10 ml of Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% Fetal Bovine Serum (FBS).
  2. Incubate cells at 37 °C overnight.
  3. Add thymidine to a final concentration of 2 mM.
  4. Culture cells in a tissue culture incubator at 37 °C for 18 h.
  5. Remove thymidine by washing cells through addition of 10 ml pre-warmed 1x PBS and discard PBS.
  6. Add 10 ml of pre-warmed fresh medium and incubate for 9 h in a tissue culture incubator at 37 °C.
  7. Add second round of thymidine to a final concentration of 2 mM.
  8. Culture cells at the tissue culture incubator for another 18 h at 37 °C.
  9. Cells are now in G1/S boundary.
  10. Release cells by washing with pre-warmed 1x PBS and incubating cells in pre-warmed fresh media. Cells are collected at 0, 2, 6, 8, 10, 12, 14, 24 h for analysis of cell cycle by DNA staining using PI, or analysis of protein by Western blot using cyclin A, cyclin D and β-Actin antibodies (Figure 1).


    Figure 1. G1/S phase synchronized H1299 cells enter into normal cell cycle progression after release into fresh medium. A. Cell cycle profiles at indicated time points after release following double thymidine block. B. Expression levels of Cyclin A, Cyclin B and β-actin in cells at indicated time points after release.

Data analysis

Cell cycle was analyzed by flow cytometry with Flowjo software (Figure 1A). Cyclin A, Cyclin B and β-actin were detected by Western blotting (Figure 1B). Data are the representative of three independent experiments.

Notes

  1. Dissolve thymidine in PBS and make 100 mM stock solution.
  2. The time points for distinct cell cycle phase are dependent on the cell cycle progression time of different cell lines.
  3. Propidium Iodide (PI) is a mutagen. Gloves, protective clothing, and eyewear should be worn.

Recipes

  1. EBC cell lysis buffer
    50 mM Tris-HCl pH 7.6-8.0
    120 mM NaCl
    0.5% NP-40
    1 mM EDTA
    1 mM Na3VO4
    50 mM NaF
    1 mM β-Mercaptoethanol
  2. Electrophoresis running buffer
    25 mM Tris-HCl pH 8.3
    192 mM glycine
    0.1% SDS
  3. Transfer buffer
    25 mM Tris-HCl pH 8.3
    192 mM glycine
    10% methanol
  4. Cell culture medium
    Roswell Park Memorial Institute (RPMI) 1640 Medium
    10% Fetal Bovine Serum (FBS)

Acknowledgments

This work was supported by NIH/NCI grants R01CA193828, R01CA136534 and R01CA200905 (to X. Deng), by the Winship Research Pathology and Integrated Cellular Imaging shared resource and the Emory Comprehensive Glycomics Core (ECGC) supported by the Winship Cancer Institute of Emory University (P30CAJ 38292), by the Winship Fashion a Cure Research Scholar Award (to X. Deng), a philanthropic award provided by the Winship, and by the Winship Endowment Fund (to XD). This protocol was adapted from our previous work (Chen et al., 2018).

Competing interests

The authors have declared that no conflict of interest exists.

References

  1. Chen, G., Magis, A. T., Xu, K., Park, D., Yu, D. S., Owonikoko, T. K., Sica, G. L., Satola, S. W., Ramalingam, S. S., Curran, W. J., Doetsch, P. W. and Deng, X. (2018). Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy. J Clin Invest 128(1): 500-516.
  2. Graña, X. and Reddy, E. P. (1995). Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11(2): 211-219.
  3. Ho, Y. S., Duh, J. S., Jeng, J. H., Wang, Y. J., Liang, Y. C., Lin, C. H., Tseng, C. J., Yu, C. F., Chen, R. J. and Lin, J. K. (2001). Griseofulvin potentiates antitumorigenesis effects of nocodazole through induction of apoptosis and G2/M cell cycle arrest in human colorectal cancer cells. Int J Cancer 91(3): 393-401.
  4. Koç, A., Wheeler, L. J., Mathews, C. K. and Merrill, G. F. (2004). Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. J Biol Chem 279(1): 223-230.
  5. Léger, K., Hopp, A. K., Fey, M. and Hottiger, M. O. (2016). ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells. Cell Cycle 15(15): 2042-2052.
  6. Rodríguez-Ubreva, F. J., Cariaga-Martinez, A. E., Cortés, M. A., Romero-De Pablos, M., Ropero, S., López-Ruiz, P. and Colás, B. (2010). Knockdown of protein tyrosine phosphatase SHP-1 inhibits G1/S progression in prostate cancer cells through the regulation of components of the cell-cycle machinery. Oncogene 29(3): 345-355.
  7. Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S., Fukami, K., Miyata, T., Miyoshi, H., Imamura, T., Ogawa, M., Masai, H. and Miyawaki, A. (2008). Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132(3): 487-498.
  8. Schvartzman, J. B., Krimer, D. B. and Van't Hof, J. (1984). The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment. Exp Cell Res 150(2): 379-389.

简介

细胞同步广泛用于研究涉及细胞周期进程调节的机制。 通过同步,可以获得不同细胞周期阶段的细胞。 胸苷是一种DNA合成抑制剂,可在DNA复制前阻止细胞在G1 / S边界。 在这里,我们提出了使用双胸苷阻滞同步G1 / S边界细胞的协议。 释放到正常培养基中后,可以在不同的时间点收集不同细胞周期阶段的细胞群。

【背景】细胞周期和细胞分裂是细胞生物学的核心。为了构建多细胞生物体,细胞复制对于产生可以执行特定功能的特化细胞是必需的。正常细胞周期由间期(G1期,S期和G2期)和有丝分裂期(M期)组成(Rodríguez-Ubreva et al。,2010;Léger et al。 ,2016)。在间期期间,遗传物质被复制并使一切为有丝分裂做好准备。然而,在有丝分裂期,重复的染色体被分离并分配到子细胞中(Sakaue-Sawano 等人,,2008)。

为了精确保存遗传信息,必须严格控制细胞周期进程。细胞周期蛋白/ CDK复合物通过在各自阶段快速促进活动来控制细胞周期进展,并且当它们的阶段完成时迅速失活(Graña和Reddy,1995)。

细胞同步对于研究细胞周期调节事件特别有用。使用不同的方法,细胞可以在不同的细胞周期阶段同步。治疗作为微管形成抑制剂的诺考达唑可以使细胞处于G2 / M期同步(Ho et al。,2001),而羟基脲,一种dNTP合成抑制剂,在早期S同步细胞阶段(Koç et al。,2004)。作为DNA合成的抑制剂(Schvartzman et al。,1984),胸苷可以阻止细胞在G1 / S边界。在这里,我们描述了一种通过胸苷同步G1 / S边界细胞的详细方法(Chen et al。,2018)。

关键字:细胞同步化, 细胞周期, 胸腺嘧啶核苷, DNA合成, DNA复制

材料和试剂

  1. 10厘米培养皿(康宁,目录号:430167)
  2. 手套(VWR International,目录号:82026)
  3. 防护服(VWR International,目录编号:414004-444)
  4. 眼镜(VWR International,目录号:89187-984)
  5. 人肿瘤细胞系:H1299(ATCC,目录号:ATCC ® CRL-5803 TM )
  6. Dulbecco改良Eagle's培养基(DMEM)(含L-谷氨酰胺的高葡萄糖)(Corning,目录号:10-013-CV)
  7. 磷酸盐缓冲盐水(PBS)(康宁,目录号:21-040-CV)
  8. 胎牛血清(FBS)(亚特兰大生物学,目录号:S11150)
  9. 胸苷(Sigma-Aldrich,目录号:T9250)
  10. 碘化丙啶(PI)(赛默飞世尔科技,目录号:P3566)
  11. 抗体
    1. 抗Cyclin A(Abcam,目录号:ab38)
    2. 抗Cyclin D(Santa Cruz Biotechnology,目录号:sc-753)
    3. 抗β-肌动蛋白(Santa Cruz Biotechnology,目录号:sc-58673)
  12. Tris-HCl,pH 8.0(Thermo Fisher Scientific,目录号:15568025)
  13. NaCl(Sigma-Aldrich,目录号:S9888)
  14. NP-40(艾博抗(Abcam),目录号:ab142227)
  15. EDTA(赛默飞世尔科技,目录号:15576028)
  16. β-Mercaptoethanol(Sigma-Aldrich,目录号:M6250)
  17. EBC细胞裂解缓冲液(见食谱)
  18. 电泳运行缓冲液(见食谱)
  19. 转移缓冲区(见食谱)

设备

  1. 细胞培养箱(VWR International,型号:98000-368)
  2. 流式细胞仪系统(BD,型号:FACSLyric)
  3. X-RAY胶片处理器(柯尼卡美能达医疗保健美洲,型号:SRX-101A)

程序

  1. 在含有10毫升罗斯威尔公园纪念碑的10厘米培养皿(每皿2 x 10 6 -3 x 10 6 细胞)中以20-30%汇合的平板H1299细胞Institute(RPMI)1640培养基补充有10%胎牛血清(FBS)。
  2. 将细胞在37°C孵育过夜。
  3. 加入胸苷至终浓度为2mM。
  4. 在组织培养箱中于37℃培养细胞18小时。
  5. 通过加入10ml预热的1x PBS洗涤细胞并弃去PBS来除去胸苷。
  6. 加入10ml预热的新鲜培养基,在37℃的组织培养箱中孵育9小时。
  7. 加入第二轮胸苷至终浓度为2mM。
  8. 在37℃下在组织培养箱中培养细胞另外18小时。
  9. 细胞现在处于G1 / S边界。
  10. 通过用预热的1x PBS洗涤并在预热的新鲜培养基中孵育细胞来释放细胞。在0,2,6,8,10,12,14,24小时收集细胞用于通过使用PI的DNA染色分析细胞周期,或通过使用细胞周期蛋白A,细胞周期蛋白D和β-肌动蛋白抗体的蛋白质印迹分析蛋白质(图1)。


    图1. G1 / S期同步H1299细胞在释放到新鲜培养基后进入正常细胞周期进展。 A.双胸苷阻断后释放后指定时间点的细胞周期谱。 B.释放后指定时间点细胞中细胞周期蛋白A,细胞周期蛋白B和β-肌动蛋白的表达水平。

数据分析

用Flowjo软件通过流式细胞术分析细胞周期(图1A)。通过蛋白质印迹检测细胞周期蛋白A,细胞周期蛋白B和β-肌动蛋白(图1B)。数据是三个独立实验的代表。

笔记

  1. 将胸苷溶解在PBS中并制备100mM储备溶液。
  2. 不同细胞周期阶段的时间点取决于不同细胞系的细胞周期进展时间。
  3. 碘化丙啶(PI)是一种诱变剂。应穿戴手套,防护服和眼镜。

食谱

  1. EBC细胞裂解缓冲液
    50mM Tris-HCl pH 7.6-8.0
    120 mM NaCl
    0.5%NP-40
    1 mM EDTA
    1mM Na 3 VO 4
    50 mM NaF
    1mMβ-巯基乙醇
  2. 电泳运行缓冲液
    25mM Tris-HCl pH 8.3
    192 mM甘氨酸
    0.1%SDS
  3. 转移缓冲区
    25mM Tris-HCl pH 8.3
    192 mM甘氨酸
    10%甲醇
  4. 细胞培养基
    罗斯威尔公园纪念研究所(RPMI)1640中等
    10%胎牛血清(FBS)

致谢

NIH / NCI拨款R01CA193828,R01CA136534和R01CA200905(X. Deng),Winship研究病理学和综合细胞成像共享资源以及埃默里大学Winship癌症研究所支持的Emory Comprehensive Glycomics Core(ECGC)支持这项工作。 (P30CAJ 38292),由Winship Fashion颁发的治愈研究奖学金奖(给X. Deng),Winship提供的慈善奖,以及Winship捐赠基金(XD)。该协议改编自我们以前的工作(Chen et al。,2018)。

利益争夺

作者宣称不存在利益冲突。

参考

  1. Chen,G.,Magis,AT,Xu,K.,Park,D.,Yu,DS,Owonikoko,TK,Sica,GL,Satola,SW,Ramalingam,SS,Curran,WJ,Doetsch,PW和Deng,X (2018年)。 靶向Mcl-1可增强DNA复制应激对癌症治疗的敏感性。 J Clin Invest 128(1):500-516。
  2. Graña,X。和Reddy,E。P.(1995)。 哺乳动物细胞的细胞周期控制:细胞周期蛋白,细胞周期蛋白依赖性激酶(CDKs),生长抑制基因的作用和细胞周期蛋白依赖性激酶抑制剂(CKIs)。 Oncogene 11(2):211-219。
  3. Ho,Y.S。,Duh,J。S.,Jeng,J。H.,Wang,Y.J.,Liang,Y.C.,Lin,C.H。,Tseng,C.J。,Yu,C.F.,Chen,R.J。和Lin,J.K。(2001)。 灰黄霉素通过诱导人结直肠癌细胞凋亡和G2 / M细胞周期阻滞增强诺考达唑的抗肿瘤发生作用细胞。 Int J Cancer 91(3):393-401。
  4. Koç,A.,Wheeler,LJ,Mathews,CK和Merrill,GF(2004)。 Hydroxyurea通过保留基础dNTP池的机制阻止DNA复制。 J Biol Chem 279(1):223-230。
  5. Léger,K.,Hopp,A.K.,Fey,M。和Hottiger,M。O.(2016)。 ARTD1调节细胞周期蛋白E的表达,从而调节细胞周期再入和T24膀胱的G1 / S进展癌细胞。 细胞周期 15(15):2042-2052。
  6. Rodríguez-Ubreva,F。J.,Cariaga-Martinez,A。E.,Cortés,M。A.,Romero-De Pablos,M.,Ropero,S.,López-Ruiz,P。andColás,B。(2010)。 敲除蛋白酪氨酸磷酸酶SHP-1通过调节前列腺癌细胞抑制G1 / S进展细胞周期机制的组成部分。 Oncogene 29(3):345-355。
  7. Sakaue-Sawano,A.,Kurokawa,H.,Morimura,T.,Hanyu,A.,Hama,H.,Osawa,H.,Kashiwagi,S.,Fukami,K.,Miyata,T.,Miyoshi,H 。,Imamura,T.,Ogawa,M.,Masai,H。和Miyawaki,A。(2008)。 可视化多细胞细胞周期进展的时空动态。 细胞 132(3):487-498。
  8. Schvartzman,J.B.,Krimer,D.B。和Van't Hof,J。(1984)。 不同胸苷浓度对豌豆根细胞DNA复制的影响同步延长的5-氟脱氧尿苷治疗。 Exp Cell Res 150(2):379-389。
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引用:Chen, G. and Deng, X. (2018). Cell Synchronization by Double Thymidine Block. Bio-protocol 8(17): e2994. DOI: 10.21769/BioProtoc.2994.
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Barry Barclay
Barry Barclay
This is a very nice protocol that induces a very high degree of cell synchrony and useful for many studies. My only concern is that high levels of dThd are intensely mutagenic for both nuclear and mitochondrial genomes. I wonder if this might be a problem for certain studies.
2018/11/20 9:03:39 回复