Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose:: Studies in vitro, in animals, and in humans
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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DOI：
10.1073/pnas.95.2.708
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发表时间：
1998-01-20
影响因子：
11.1
通讯作者：
Hellerstein, MK
中科院分区：
文献类型：
--
作者：
Macallan, DC;Fullerton, CA;Hellerstein, MK
本发明提供了一种体内测量DNA合成并由此测量细胞增殖的方法。该技术包括施用[6，6-H-2（2）]Glc或[U-C-13]Glc，分离基因组DNA，酶促水解成游离脱氧核糖核苷，衍生化用于dA或dG同位素富集或两者的GC-MS分析，dA或dG与细胞外Glc富集的比较（对细胞内稀释进行校正）通过应用底物-产物关系揭示了新合成的DNA的分数，因此，该技术与广泛使用的[H-3]胸苷或BrdUrd技术的不同之处在于，使用从头核苷酸合成途径而不是核苷补救途径来标记DNA;标记脱氧核糖而不是碱基部分;分析嘌呤而不是嘧啶脱氧核糖核苷;并且使用稳定同位素而不是放射性同位素。该方法在体外应用于培养的HepG（2）和H-9细胞的生长;在动物中应用于肠上皮、胸腺和肝脏的增殖;在所有情况下，测量的细胞增殖动力学与预期的或独立测量的动力学一致，该方法与先前可用的用于测量细胞更新的技术相比具有几个优点，不涉及放射性或潜在毒性代谢物，并且适用于人类。用于测量人类细胞增殖的可靠且安全的方法的可用性为直接实验测试打开了许多基本问题，包括与癌症、AIDS、和其他病理状态。
A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented, The technique consists of administering [6,6-H-2(2)]Glc or [U-C-13]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 [H-3]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 HepG(2) and H-9 cells 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.