Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Apr;10(4):1584–1592. doi: 10.1128/mcb.10.4.1584

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

C A Hoy 1, E D Lewis 1, R T Schimke 1
PMCID: PMC362263  PMID: 2181279

Abstract

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.

Full text

PDF
1584

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beck H. P. Radiotoxicity of incorporated [3H]thymidine as studied by autoradiography and flow cytometry. Consequences for the interpretation of FLM data. Cell Tissue Kinet. 1981 Mar;14(2):163–177. doi: 10.1111/j.1365-2184.1981.tb00521.x. [DOI] [PubMed] [Google Scholar]
  2. Doida Y., Okada S. Radiation-induced mitotic delay in cultured mammalian cells (L5178Y). Radiat Res. 1969 Jun;38(3):513–529. [PubMed] [Google Scholar]
  3. Ehmann U. K., Williams J. R., Nagle W. A., Brown J. A., Belli J. A., Lett J. T. Perturbations in cell cycle progression from radioactive DNA precursors. Nature. 1975 Dec 18;258(5536):633–636. doi: 10.1038/258633a0. [DOI] [PubMed] [Google Scholar]
  4. Hoy C. A., Thompson L. H., Mooney C. L., Salazar E. P. Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents. Cancer Res. 1985 Apr;45(4):1737–1743. [PubMed] [Google Scholar]
  5. JOHNSON H. A., CRONKITE E. P. The effect of tritiated thymidine on mouse spermatogonia. Radiat Res. 1959 Dec;11:825–831. [PubMed] [Google Scholar]
  6. Kohn K. W., Erickson L. C., Ewig R. A., Friedman C. A. Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry. 1976 Oct 19;15(21):4629–4637. doi: 10.1021/bi00666a013. [DOI] [PubMed] [Google Scholar]
  7. Kohn K. W., Friedman C. A., Ewig R. A., Iqbal Z. M. DNA chain growth during replication of asynchronous L1210 cells. Alkaline elution of large DNA segments from cells lysed on filters. Biochemistry. 1974 Sep 24;13(20):4134–4139. doi: 10.1021/bi00717a011. [DOI] [PubMed] [Google Scholar]
  8. Maurer H. R. Potential pitfalls of [3H]thymidine techniques to measure cell proliferation. Cell Tissue Kinet. 1981 Mar;14(2):111–120. doi: 10.1111/j.1365-2184.1981.tb00516.x. [DOI] [PubMed] [Google Scholar]
  9. Setlow R. B., Setlow J. K. Effects of radiation on polynucleotides. Annu Rev Biophys Bioeng. 1972;1:293–346. doi: 10.1146/annurev.bb.01.060172.001453. [DOI] [PubMed] [Google Scholar]
  10. Thompson L. H., Brookman K. W., Dillehay L. E., Mooney C. L., Carrano A. V. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions. Somatic Cell Genet. 1982 Nov;8(6):759–773. doi: 10.1007/BF01543017. [DOI] [PubMed] [Google Scholar]
  11. Thompson L. H., Fong S., Brookman K. Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells. Mutat Res. 1980 Feb;74(1):21–36. doi: 10.1016/0165-1161(80)90188-0. [DOI] [PubMed] [Google Scholar]
  12. Whitmore G. F., Gulyas S. Synchronization of mammalian cells with tritiated thymidine. Science. 1966 Feb 11;151(3711):691–694. doi: 10.1126/science.151.3711.691. [DOI] [PubMed] [Google Scholar]
  13. YAMADA M., PUCK T. T. Action of radiation on mammalian cells. IV. Reversible mitotic lag in the S3 HeLa cell produced by low doses of x-rays. Proc Natl Acad Sci U S A. 1961 Aug;47:1181–1191. doi: 10.1073/pnas.47.8.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES