Radionucleoside Incorporation by Thymidine-Synchronized Cells

HeLa S3 cells were synchronized by treatment with excess thymidine, and their capacity to incorporate H3-thymidine ( H3 TDR), ${\rm H}^{3}\text{-deoxycytidine}$ (${\rm H}^{3}{\rm CDR}$), and 5-iodo-2′-deoxyuridine (${\rm I}^{125}{\rm DU}$) into DNA was determined at various times after release from...

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Bibliographic Details
Published in:Radiation research 1967-03, Vol.30 (3), p.455-467
Main Authors: Drew, R. M., Commerford, S. L.
Format: Article
Language:English
Subjects:
Autoradiography
Cell culture techniques
Cell growth
Cell lines
Culture Techniques
Cultured cells
Deoxyuridine - metabolism
DNA
DNA - biosynthesis
HeLa Cells
Interphase
Kidney cells
L cells
Mitotic index
Space life sciences
Thymidine - metabolism
Tritium
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Summary:HeLa S3 cells were synchronized by treatment with excess thymidine, and their capacity to incorporate H3-thymidine ( H3 TDR), ${\rm H}^{3}\text{-deoxycytidine}$ (${\rm H}^{3}{\rm CDR}$), and 5-iodo-2′-deoxyuridine (${\rm I}^{125}{\rm DU}$) into DNA was determined at various times after release from thymidine blocking. H3 TDR and ${\rm H}^{3}{\rm CDR}$ incorporation was estimated radioautographically on Feulgen-stained cells. Incorporation of the thymidine analog ${\rm I}^{125}{\rm DU}$ was estimated in DNA fractions obtained by differential extraction with perchloric acid; the ${\rm I}^{125}$ activity was determined from multiple counts made in a sodium iodide well-type counter. ${\rm H}^{3}{\rm CDR}$ incorporation was shown to occur without delay, providing unequivocal evidence that 90 to 97% of the cells had been blocked in S phase (DNA synthesis). Recause H3 TDR and ${\rm I}^{125}{\rm DU}$ incorporation was suppressed, DNA synthesis could be demonstrated in only 15 to 20% of the cells at the time of release from excess thymidine treatment. ${\rm I}^{125}{\rm DU}$ incorporation into DNA was very sensitive to thymidine competition. The data are compatible with the hypothesis that an abnormally large, internal thymidine pool is established during thymidine treatment, which competes with ${\rm I}^{125}{\rm DU}$ or H3 TDR. Mitotic indices and the rate of appearance of labeled mitoses after thymidine blocking were established. No delay in the transit of cells through S phase, premitotic rest, and mitosis was found.
ISSN:0033-7587
1938-5404
DOI:10.2307/3572110