Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio-mass spectrometric analysis.
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 ((2)H) from heavy water ((2)H(2)O) 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 (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O 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 (> approximately 10(7)). 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.
Voogt
Jason N
JN
KineMed Inc., 5980 Horton Street, Emeryville, California 94608, USA.
Awada
Mohamad
M
Murphy
Elizabeth J
EJ
Hayes
Gregory M
GM
Busch
Robert
R
Hellerstein
Marc K
MK
D000818:Animals; D049109:Cell Proliferation; D002849:Chromatography, Gas / Q000379:methods*; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D004261:DNA Replication; D003855:Deoxyribose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D017666:Deuterium Oxide / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008968:Molecular Conformation
D003855:Deoxyribose; D004247:DNA; D017666:Deuterium Oxide