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Nucleic Acids Res. 1980 January 25; 8(2): 377–387.
PMCID: PMC327273

Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.

Abstract

Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Pedrali-Noy G, Spadari S. Effect of aphidicolin on viral and human DNA polymerases. Biochem Biophys Res Commun. 1979 Jun 27;88(4):1194–1202. [PubMed]
  • Coyle MB, Strauss B. Cell killing and the accumulation of breaks in the DNA of HEp-2 cells incubated in the presence of hydroxyurea. Cancer Res. 1970 Sep;30(9):2314–2319. [PubMed]
  • Galavazi G, Schenk H, Bootsma D. Synchronization of mammalian cells in vitro by inhibition of the DNA synthesis. I. Optimal conditions. Exp Cell Res. 1966 Feb;41(2):428–437. [PubMed]
  • XEROS N. Deoxyriboside control and synchronization of mitosis. Nature. 1962 May 19;194:682–683. [PubMed]
  • Yang SJ, Hahn GM, Bagshaw MA. Chromosome aberrations induced by thymidine. Exp Cell Res. 1966 Apr;42(1):130–135. [PubMed]
  • Lambert WC, Studzinski GP. Thymidine as a synchronizing agent. II. Partial recovery of HeLa cells from unbalanced growth. J Cell Physiol. 1969 Jun;73(3):261–266. [PubMed]
  • Thomas DB, Lingwood CA. A model of cell cycle control: effects of thymidine on synchronous cell cultures. Cell. 1975 May;5(1):37–42. [PubMed]
  • Lewis WH, McNaughton DR, Goh SH, LéJohn HB, Wright JA. Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells. J Cell Physiol. 1977 Dec;93(3):345–352. [PubMed]
  • Ikegami S, Taguchi T, Ohashi M, Oguro M, Nagano H, Mano Y. Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-alpha. Nature. 1978 Oct 5;275(5679):458–460. [PubMed]
  • Ohashi M, Taguchi T, Ikegami S. Aphidicolin: a specific inhibitor of DNA polymerases in the cytosol of rat liver. Biochem Biophys Res Commun. 1978 Jun 29;82(4):1084–1090. [PubMed]
  • Hübscher U, Kuenzle CC, Limacher W, Scherrer P, Spadari S. Functions of DNA polymerases alpha, beta, and gamma in neurons during development. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):625–629. [PubMed]
  • Hübscher U, Kuenzle CC, Spadari S. Functional roles of DNA polymerases beta and gamma. Proc Natl Acad Sci U S A. 1979 May;76(5):2316–2320. [PubMed]
  • Noy GP, Weissbach A. HeLa cell DNA polymerases: the effect of cycloheximide in vivo and detection of a new form of DNA polymerase alpha. Biochim Biophys Acta. 1977 Jul 5;477(1):70–83. [PubMed]
  • Miller AO. Etude par sédimentation en gradient de sucrose de la ferritine marquée au 59Fe, extraite de cellules de HeLa. Biochim Biophys Acta. 1968 Jan 29;155(1):262–270. [PubMed]
  • Vindelov LL. Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei. Virchows Arch B Cell Pathol. 1977 Aug 10;24(3):227–242. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Knopf KW, Yamada M, Weissbach A. HeLa cell DNA polymerase gamma: further purification and properties of the enzyme. Biochemistry. 1976 Oct 5;15(20):4540–4548. [PubMed]
  • Tyrsted G. The pool size of deoxyguanosine 5'-triphosphate and deoxycytidine 5'-triphosphate in phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Exp Cell Res. 1975 Mar 15;91(2):429–440. [PubMed]
  • Skoog L. An enzymatic method for the determination of dCTP and dGTP in picomole amounts. Eur J Biochem. 1970 Dec;17(2):202–208. [PubMed]
  • Bucknall RA, Moores H, Simms R, Hesp B. Antiviral effects of aphidicolin, a new antibiotic produced by Cephalosporium aphidicola. Antimicrob Agents Chemother. 1973 Sep;4(3):294–298. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press