Abstract
DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio–mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (≥0.002% atom percent excess 2H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of 2H2O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (>∼107). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.
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Acknowledgements
We thank Chancy Fessler, Kris Prado, Daniel Holochwost, Tracy A. Gee, Iche M. Siah and Ablatt Mahsut for technical assistance and Dr. Nabil Saad for contributing analytical expertise. The work was supported by funds from KineMed Inc., NIH grants AI43866 and HD 40543-06 and University of California Discovery Program BioSTAR grant bio04-10445 to M.K.H., and an NIH grant from the National Cancer Institute (R44 CA097686) to E.J.M.
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M.K.H. is co-founder and chief scientific officer of KineMed, Inc.; the other coauthors are employees of KineMed, Inc. (J.N.V., G.M.H.) or consultants (R.B., M.A.) and hold stock options in the company.
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Voogt, J., Awada, M., Murphy, E. et al. Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio–mass spectrometric analysis. Nat Protoc 2, 3058–3062 (2007). https://doi.org/10.1038/nprot.2007.421
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DOI: https://doi.org/10.1038/nprot.2007.421
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