Measurement of Cell Proliferation by Labeling of DNA with Stable Isotope-Labeled Glucose: Studies in vitro, in Animals, and in Humans

A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrich...

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Veröffentlicht in:Proceedings of the National Academy of Sciences - PNAS 1998-01, Vol.95 (2), p.708-713
Hauptverfasser: Macallan, Derek C., Fullerton, Catherine A., Neese, Richard A., Haddock, Katherine, Park, Sunny S., Hellerstein, Marc K.
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container_title Proceedings of the National Academy of Sciences - PNAS
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creator Macallan, Derek C.
Fullerton, Catherine A.
Neese, Richard A.
Haddock, Katherine
Park, Sunny S.
Hellerstein, Marc K.
description A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H] thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2and H9cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.
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The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H] thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2and H9cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>9435257</pmid><doi>10.1073/pnas.95.2.708</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record>
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subjects Animals
B lymphocytes
Biological Sciences
Cell Division
Cell growth
Cell lines
Cellular biology
Cytological Techniques
Deoxyribonucleic acid
DNA
DNA - analysis
DNA - biosynthesis
DNA Replication
Glucose
Hep G2 cells
Hepatocytes
Humans
Isotope Labeling
Isotopes
Isotopic labeling
Lymphocytes
Radioisotopes
Stem cells
T lymphocytes
Weights & measures
title Measurement of Cell Proliferation by Labeling of DNA with Stable Isotope-Labeled Glucose: Studies in vitro, in Animals, and in Humans
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