title: Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry.
abstract: Deuterated water (²H₂O), a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules), thus permitting the calculation of their synthesis rates. Here, we have combined ²H₂O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids) from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation), protein and lipid synthesis rates. We have used this approach to characterize the turnover rates and metabolism of a panel of mammalian cells in vitro (muscle C2C12 and colon cancer cell lines). Our data show that in actively-proliferating cells, biomass synthesis rates are strongly linked to the rate of cell division. Furthermore, in both proliferating and non-proliferating cells, it is the lipid pool that undergoes the most rapid turnover when compared to DNA and protein. Finally, our data in human colon cancer cell lines reveal a marked heterogeneity in the reliance on the de novo lipogenic pathway, with the cells being dependent on both 'self-made' and exogenously-derived fatty acid.
authors.affiliation: Institute for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Geelong 3216, Victoria, Australia. victoria.foletta@deakin.edu.au.
authors.forename: Victoria C
authors.initials: VC
authors.lastname: Foletta
authors.affiliation: Systems Biology and Personalised Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Victoria, Australia. palmieri.m@wehi.edu.au.|Department of Medical Biology, University of Melbourne, Parkville 3052, Victoria, Australia. palmieri.m@wehi.edu.au.
authors.forename: Michelle
authors.initials: M
authors.lastname: Palmieri
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville 3052, Victoria, Australia. j.kloehn@student.unimelb.edu.au.
authors.forename: Joachim
authors.initials: J
authors.lastname: Kloehn
authors.affiliation: Institute for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Geelong 3216, Victoria, Australia. s.mason@deakin.edu.au.
authors.forename: Shaun
authors.initials: S
authors.lastname: Mason
authors.affiliation: MRL, Merck & Co. Inc., Kenilworth, NJ 07033, USA. stephen_previs@merck.com.
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Previs
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville 3052, Victoria, Australia. malcolmm@unimelb.edu.au.
authors.forename: Malcolm J
authors.initials: MJ
authors.lastname: McConville
authors.affiliation: Systems Biology and Personalised Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Victoria, Australia. sieber.o@wehi.edu.au.|Department of Medical Biology, University of Melbourne, Parkville 3052, Victoria, Australia. sieber.o@wehi.edu.au.|Department of Surgery, University of Melbourne, Parkville 3052, Victoria, Australia. sieber.o@wehi.edu.au.|School of Biomedical Sciences, Monash University, Clayton 3168, Victoria, Australia. sieber.o@wehi.edu.au.
authors.forename: Oliver M
authors.initials: OM
authors.lastname: Sieber
authors.affiliation: Institute for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Geelong 3216, Victoria, Australia. clinton.bruce@deakin.edu.au.
authors.forename: Clinton R
authors.initials: CR
authors.lastname: Bruce
authors.affiliation: Institute for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Geelong 3216, Victoria, Australia. greg.kowalski@deakin.edu.au.
authors.forename: Greg M
authors.initials: GM
authors.lastname: Kowalski
keywords: C2C12; DNA synthesis; GC-MS; biomass; colon cancer; deuterated water; lipogenesis; protein synthesis; stable isotopes