In vitro-Verfahren zur Bestimmung der DNS-Synthese-Dauer einzelner Zellen Biochemische Voraussetzungen und Ergebnisse

Brinkmann, W; Dormer, Peter G.

doi:10.1007/bf00279783

Cited by 17 publications

(1 citation statement)

References 21 publications

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“…Unlike amethopterin, however, dFUrd may possibly compete with thymidine for transport across the cell membrane and/or for thymidine kinase . Autoradiographic studies have indicated that after addition of dFUrd to suspensions of bone marrow cells, incorporation of labeled thymidine, after a time lag of 1-2 min, was linear for 6-10 min only (4) . After addition of amethopterin at a concentration of 10 µM, [ 3 H]thymidine incorporation into DNA increased in a linear fashion for at least 90 min with a negligible lag period .…”

Section: Discussionmentioning

confidence: 99%

Determination of Rates of Dna Synthesis in Cultured Mammalian Cell Populations

Siegers

Schaer

Hirsiger

et al. 1974

The Journal of Cell Biology

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In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [3H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 µM) in combination with hypoxanthine and glycine. If [3H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [3H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 106 cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.

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Section: Discussionmentioning

confidence: 99%

Determination of Rates of Dna Synthesis in Cultured Mammalian Cell Populations

Siegers

Schaer

Hirsiger

et al. 1974

The Journal of Cell Biology

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Thymidine labeling index of human breast carcinoma:Enhancement of in vitrolabeling by 5-fluorouracil and 5-fluoro-2′ - deoxyuridine

Meyer

Facher

1977

Cancer

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Effect of 5‐fluoro‐2′‐deoxyuridine (FdUrd) on 5‐bromo‐2′‐deoxyuridine (BrdUrd) incorporation into DNA, measured with a monoclonal BrdUrd antibody and by the BrdUrd/hoechst quenching effect

Ellwart

Dormer

1985

Cytometry

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The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed 4.0 BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (HI technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescences were measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/ H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 pM or less are applied.Key terms: Bromodeoxyuridine, fluorodeoxyuridine, DNA synthesis rate, human leukemic cell line, flow cytometry Bromodeoxyuridine (BrdUrd) is used with increasing frequency in cell kinetic studies after the introduction of the Hoechst (H) quenching effect into flow cytometry by Latt (23,241. Bohmer (2) and Kubbies and Rabinovitch (20) described methods of cell cycle analysis using the quenching of H in divided cells. Perturbations of the cell cycle of normal and tumor cells were easy to investigate with the BrdUrdM technique (1,29). Counterstaining of total DNA per cell with ethidium bromide (EB) simplified the analysis of the DNA synthesis in the first cycle (3,4,16). This two-parameter technique allows flow cytometric measurement of the DNA synthesis rate even in subcompartments of the S-phase after a few hours of BrdUrd incubation (28,151). Since it is now possible to measure DNA synthesis with the monoclonal anti-BrdUrd antibody (10,18,26), BrdUrd is being applied increasingly for cell cycle studies. It is therefore of interest to know the exact conditions of quantitative BrdUrd incorporation.The quantity of BrdUrd instead of thymidine (dThd) incorporated per unit of time into DNA may provide information on the DNA synthesis rate. For quantitative assessment of the DNA synthesis rate, it is most convenient if BrdUrd replaces dThd completely in the newly synthesized DNA strands. It is, however, to be expected that the endogenous synthesis of the DNA precursor dThd monophosphate competes with the exogenously supplied BrdUrd. In addition, an intracellular precursor pool of dThd monophosphate exists at the beginning of BrdUrd incorporation (7) and should not mix with the newly synthesized BrdUrd monophosphate. Dormer applied fluorodeoxyuridine (FdUrd) to block the endogenous synthesis of dThd monophosphate for us...

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In vitro-Verfahren zur Bestimmung der DNS-Synthese-Dauer einzelner Zellen Biochemische Voraussetzungen und Ergebnisse

Cited by 17 publications

References 21 publications

Determination of Rates of Dna Synthesis in Cultured Mammalian Cell Populations

Determination of Rates of Dna Synthesis in Cultured Mammalian Cell Populations

Thymidine labeling index of human breast carcinoma:Enhancement of in vitrolabeling by 5-fluorouracil and 5-fluoro-2′ - deoxyuridine

Effect of 5‐fluoro‐2′‐deoxyuridine (FdUrd) on 5‐bromo‐2′‐deoxyuridine (BrdUrd) incorporation into DNA, measured with a monoclonal BrdUrd antibody and by the BrdUrd/hoechst quenching effect

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