Labelling of Cells Engaged in DNA Synthesis: Autoradiography and BrdU Staining

Abstract

The cell cycle is divided in four phases: G1 phase, S phase (DNA‐synthesis), G2 phase (together termed interphase) and M phase (mitosis). Cells that have ceased proliferation enter a state of quiescence called G0. M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell's cytoplasm divides in half forming distinct new cells. It is often necessary to evaluate the proportion of DNA‐replicating nuclei (S‐phase cells) in a given population of cells. Classical methods for detection of cells in the S phase use incorporation of modified nucleosides, followed by direct visualisation of the DNA ‐replicating cells.

Key Concepts:

  • Identification of actively DNA‐replicating nuclei by incorporation of bromodeoxyuridine and detection by immunofluorescence using bromodeoxyuridine specific antibodies or by incorporation of [3H]thymidine and visualisation by autoradiography.

  • Labelling of S‐phase cells by bromodeoxyuridine or [3H]thymidine is a direct method to asses the proliferative index of tissue cell cultures.

  • Cell cycle studies using the number of S‐phase cells as index for cell proliferation are conveniently conducted by incorporation of bromodeoxyuridine or [3H]thymidine into nuclei engaged in DNA synthesis.

  • Bromodeoxyuridine and [3H]thymidine are both incorporated into DNA by eukaryotic DNA polymerases and are therefore valuable tools in studies of hyperproliferative cells as well as normal cells, thereby aiding to the understanding of the transition of normal cells to cancer cells.

Keywords: cell cycle; DNA replication; cell proliferation; S phase; DNA synthesis; bromodeoxyuridine (BrdU); [3H]thymidine

References

Baserga R (1978) Resting cells and G1 phase of the cell cycle. Journal of Cell Physiology 95: 377–386.

Gratzner HG (1982) Monoclonal antibody to 5‐bromo‐ and 5‐iododeoxyuridine: a new reagent for detection of DNA replication. Science 218: 474–475.

Howard A and Pelc SR (1953) Synthesis of deoxyribonucleic acid in normal and irradiated cells and its relationship to chromosome breakage. Heredity 6: 261–273.

Pardee AB, Dubraw R, Hamlin JL et al. (1978) Animal cell cycle. Annual Review of Biochemistry 47: 715–750.

Further Reading

Ford HL and Pardee AB (1999) Cancer and the cell cycle. Journal of Cellular Biochemistry Supplement 32–33: 166–172.

Natsume T and Tanaka TU (2010) Spatial regulation and organization of DNA replication within the nucleus. Chromosome Research 18: 7–17.

Nigg EA (1995) Cyclin‐dependent protein kinases: key regulators of the eukaryotic cell cycle. BioEssays 17: 471–480.

Segurado M and Tercero JA (2009) The S‐phase checkpoint: targeting the replication fork. Biologie Cellulaire 101: 617–627.

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How to Cite close
Madsen, Peder(Oct 2010) Labelling of Cells Engaged in DNA Synthesis: Autoradiography and BrdU Staining. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002572.pub2]