Synchronization of HeLa Cells to Various Interphases Including G1, S, and G2 Phases.
Cell cycle, synchronization, interphase, mitosis, G1 phase, S phase, G2 phase, flow cytometry, Western blot
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
31
8
2022
pubmed:
1
9
2022
medline:
9
9
2022
Statut:
ppublish
Résumé
The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase, which allows the study of phase-specific cellular events. While interphase cells can be easily distinguished from mitotic cells by examining their chromosome morphology, it is much more difficult to separate and distinguish the interphases from each other. Here, we describe drug-derived protocols for synchronizing HeLa cells to various interphases of the cell cycle: G1 phase, S phase, and G2 phase. G1 phase synchronization is achieved through serum starvation, S phase synchronization is achieved through a double thymidine block, and G2 phase synchronization is achieved through the release of the double thymidine block followed by roscovitine treatment. Successful synchronization can be assessed using flow cytometry to examine the DNA content and Western blot to examine the expression of various cyclins.
Identifiants
pubmed: 36045200
doi: 10.1007/978-1-0716-2736-5_7
doi:
Substances chimiques
Thymidine
VC2W18DGKR
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
87-97Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Panagopoulos A, Altmeyer M (2021) The hammer and the dance of cell cycle control. Trends Biochem Sci 46(4):301–314
doi: 10.1016/j.tibs.2020.11.002
Wang Z (2021) Regulation of cell cycle progression by growth factor-induced cell signaling. Cell 10(12):3327
doi: 10.3390/cells10123327
Barnum KJ, O’Connell MJ (2014) Cell cycle regulation by checkpoints. Methods Mol Biol (Clifton, NJ) 1170:29–40
doi: 10.1007/978-1-4939-0888-2_2
Satyanarayana A, Kaldis P (2009) Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene 28(33):2925–2939
doi: 10.1038/onc.2009.170
Gao X, Leone GW, Wang H (2020) Cyclin D-CDK4/6 functions in cancer. Adv Cancer Res 148:147–169
doi: 10.1016/bs.acr.2020.02.002
Ma HT, Poon RY (2011) Synchronization of HeLa cells. Methods Mol Biol (Clifton, NJ) 761:151–161
doi: 10.1007/978-1-61779-182-6_10
Bjursell G, Reichard P (1973) Effects of thymidine on deoxyribonucleoside triphosphate pools and deoxyribonucleic acid synthesis in Chinese hamster ovary cells. J Biol Chem 248(11):3904–3909
doi: 10.1016/S0021-9258(19)43819-2
Schvartzman JB, Krimer DB, 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
doi: 10.1016/0014-4827(84)90581-0
Chen G, Deng X (2018) Cell synchronization by double thymidine block. Bio Protoc 8(17):e2994
doi: 10.21769/BioProtoc.2994
Maurer M, Komina O, Wesierska-Gadek J (2009) Roscovitine differentially affects asynchronously growing and synchronized human MCF-7 breast cancer cells. Ann N Y Acad Sci 1171:250–256
doi: 10.1111/j.1749-6632.2009.04717.x
Bostock CJ, Prescott DM, Kirkpatrick JB (1971) An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border. Exp Cell Res 68(1):163–168
doi: 10.1016/0014-4827(71)90599-4
Whittaker SR, Walton MI, Garrett MD, Workman P (2004) The cyclin-dependent kinase inhibitor CYC202 (R-roscovitine) inhibits retinoblastoma protein phosphorylation, causes loss of cyclin D1, and activates the mitogen-activated protein kinase pathway. Cancer Res 64(1):262–272
doi: 10.1158/0008-5472.CAN-03-0110
Darzynkiewicz Z, Gong J, Juan G, Ardelt B, Traganos F (1996) Cytometry of cyclin proteins. Cytometry 25(1):1–13
doi: 10.1002/(SICI)1097-0320(19960901)25:1<1::AID-CYTO1>3.0.CO;2-N
Pozarowski P, Darzynkiewicz Z (2004) Analysis of cell cycle by flow cytometry. Methods Mol Biol (Clifton, NJ) 281:301–311
Dulić V, Lees E, Reed SI (1992) Association of human cyclin E with a periodic G1-S phase protein kinase. Science (New York, NY) 257(5078):1958–1961
doi: 10.1126/science.1329201
Kraft C, Herzog F, Gieffers C, Mechtler K, Hagting A, Pines J, Peters JM (2003) Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J 22(24):6598–6609
doi: 10.1093/emboj/cdg627
Pines J, Hunter T (1990) Human cyclin a is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature 346(6286):760–763
doi: 10.1038/346760a0
Keyomarsi K, Sandoval L, Band V, Pardee AB (1991) Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res 51(13):3602–3609
pubmed: 1711413
Park SY, Im JS, Park SR, Kim SE, Wang HJ, Lee JK (2012) Mimosine arrests the cell cycle prior to the onset of DNA replication by preventing the binding of human Ctf4/And-1 to chromatin via Hif-1α activation in HeLa cells. Cell Cycle (Georgetown, Tex) 11(4):761–766
doi: 10.4161/cc.11.4.19209
Kim JH, Gelbard AS, Djordjevic B, Kim SH, Perez AG (1968) Action of daunomycin on the nucleic acid metabolism and viability of HeLa cells. Cancer Res 28(12):2437–2442
pubmed: 5728152
Pedrali-Noy G, Spadari S, Miller-Faurès A, Miller AO, Kruppa J, Koch G (1980) Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin. Nucleic Acids Res 8(2):377–387
doi: 10.1093/nar/8.2.377
Pfeiffer SE, Tolmach LJ (1967) Inhibition of DNA synthesis in HeLa cells by hydroxyurea. Cancer Res 27(1):124–129
pubmed: 6020353
Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, Heimbrook DC, Chen L (2006) Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci U S A 103(28):10660–10665
doi: 10.1073/pnas.0600447103
Dulla K, Daub H, Hornberger R, Nigg EA, Korner R (2010) Quantitative site-specific phosphorylation dynamics of human protein kinases during mitotic progression. Mol Cell Proteomics 9(6):1167–1181
doi: 10.1074/mcp.M900335-MCP200