Quantifying cell division with D2O and multi-isotope imaging mass spectrometry (MIMS)

M.L. Steinhauser1,2, C. Guillermier1,3, M. Wang3 and C.P. Lechene1,3 (1) Division of Genetics, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA USA

(2) Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston MA USA (3) National Resource for Imaging Mass Spectrometry, Cambridge, MA USA

ABSTRACT: Quantifying cell division has traditionally relied upon incorporation of label in newly synthesized DNA by nucleotide salvage, including labeled thymidine or thymidine analogs[1]. An alternate approach is to use precursors of the de novo nucleotide synthesis pathway, such as glucose or water[2,3]. Because such precursors are not specific for DNA synthesis, studies utilizing this approach have analyzed isolated genomic DNA to exclude background incorporation in other molecules. Due to the rapid rate of DNA synthesis during S-phase of mitosis and to the inherent stability of DNA, we hypothesized that pulse-chase administration of stable isotope labeled water would result in sufficient nuclear labeling to enable discrimination of recently divided cells by NanoSIMS ion microscopy. We administered deuterated (D)-water and 15N-thymidine to mice concurrently, guided by the rationale that 15N-thymidine incorporation would serve as a “gold standard” to identify dividing cells[1]. Mice were labeled for 3 days, followed by a 3-day label-free chase. We analyzed the small intestine, due to its recognized propensity for cell turnover. After fixation, small intestinal samples were embedded in LR white, sectioned to 0.5µm, and analyzed with a NanoSIMS 50L (Cameca). The instrument was carefully tuned to study the D:H ratio in two ways: by sequentially recording D- and H- followed by 12C15N- and 12C14N- or by simultaneously capturing C2D- and C2H- along with 12C15N- and 12C14N- [4]. We show both qualitatively and quantitatively that dividing cells in the small intestine (15N-labeled) reveal a clearly discernable 2H signal in the nucleus that was not observed in undivided cells (15N-unlabled). Correlation with 31P- and 12C15N-/12C14N- images demonstrate preferential localization of 2H labeling in regions of the nucleus with high DNA content as expected of labeling being incorporated during DNA synthesis and cell division. These data support the concept that stable isotope tagged precursors of the de novo nucleotide synthesis pathway can be used in concert with NanoSIMS to study cell division in vivo. This approach is particularly relevant to study tissues characterized by low labeling frequency, where the application of MIMS is limited by the throughput of the NanoSIMS instrument. A major implication of this study then is the possibility of using stable isotope tagged water and MIMS to study human cell turnover, where the required analysis time to achieve statistical power may be reduced by long-term labeling aimed at increasing labeling frequency.

REFERENCES: [1] M.L. Steinhauser, A.P. Bailey, S.E. Senyo, C. Guillermier, T.S. Perlstein, A.P. Gould, R.T. Lee, C.P. Lechene, Nature 481(7382), 2012, 516. [2] H. Mohri, A.S. Perelson, K. Tung, R.M. Ribeiro, B. Ramratnam, M. Markowitz, R. Kost, A. Hurley, L. Weinberger, D. Cesar, M.K. Hellerstein, D.D. Ho, J Exp Med 194(9), 2001, 1277. [3] R.A. Neese, L.M. Misell, S. Turner, A. Chu, J. Kim, D. Cesar, R. Hoh, F. Antelo, A. Strawford, J.M. McCune, M. Christiansen, M.K. Hellerstein, PNAS 99(24), 2002, 15345. [4] C. Guillermier, M.L. Steinhauser, C.P. Lechene, SIMS 19 Abstract, 2013.

C.P.L. is funded by the NIH (5P41EB001974-13, AG034641,R01 AG040019, R21AG034641-01, AG-SS-2215-08, RGP0048, R01 AG040209), HumanFrontier Science Program and the Ellison Medical Foundation. M.L.S. Is funded by Watkins Cardiovascular Leadership Award, the NIH (K08 DK090147-02) and a Harvard Stem Cell Institute Seed Grant.

SUMMARY:  MIMS distinguishes dividing cells from quiescent cells after in vivo D-

water labeling.  Labeled nuclei become more evident during a label-free chase period

as background labeling of non-genomic molecules fades due to ongoing turnover.

 The use of labeled precursors to the de novo nucleotide synthesis pathway represents a practical approach to study slowly dividing cell populations, due to the ease of long-term label administration.

 Because stable isotopes are non-toxic, the use of D-water to study cell turnover is eminently translatable to humans.

(a) Schematic of contrasting DNA labeling strategies. The most common approach to tracking DNA synthesis is by accessing the nucleotide salvage pathway with labeled thymidine (e.g. 15N-thymidine, 3H-thymidine) or halogenated nucleotide analogues (e.g. bromodeoxyuridine-BrdU). An alternative approach involves administration of labeled precursors to the de novo nucleotide synthesis pathway, such as the H-water approach demonstrated here. (b) During cell division, rapid DNA synthesis coincides with complete replication of the genome, which should result in a rapid rate of D incorporation, exceeding the incorporation rate due to other dominant biosynthetic processes, such as protein synthesis. During chase, however, D contained in protein will be diluted by ongoing turnover, whereas signal contained in genomic DNA remains relatively stable until the next cell division.

Figure 1. Labeling DNA synthesis and cell division by the de novo nucleotide synthesis pathway.

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Figure 2. D-water labels dividing cells in the mouse small intestine.

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Mice pulsed for 3d with D-water (daily i.p. injection), equivalent to approximately 1% of circulating total body water, while simultaneously receiving 15N-thymidine (500µg bolus i.p.). Small intestine was harvested after 3d pulse and again after 3d label-free chase. (a) Small intestinal crypt. The field was first analyzed for CN-, P-, S- then for C2H-, D-, and H-. Nuclei are seen brightly in the P- image. Sulfur rich granules contained in Paneth cells are seen brightly in the S- image. The 12C15N-/12C14N- HSI image shows dividing crypt cells. The D-/H- HSI image shows higher D signal in the cells that have divided (15N+, nuclear ROI traced) compared to the undivided cells (arrows). Paneth cell granules show intense D labeling. Raster=28µm, 85 planes; pixels=256 x 256, 66s/plane. (b) After 3-day chase, divided cells have migrated to the tip of the intestinal villus. Divided epithelial cells (15N+) also show D label (nuclear ROI traced); whereas undivided (15N-) interstitial cells on the interior of the villus (arrows) show low D labeling. Raster=60µm, 481 planes; pixels=256 x 256, 131s/plane. (c) Graphs of nuclear D-signal in divided (15N+) cells versus undivided (15N-) cells. Box and whisker plot shows max/min; statistical significance assessed by Mann-Whitney (nonparametric) test. (d) Graph of the ratio of nuclear to cytoplasmic D-signal in divided cells (15N+). With chase, the ratio of nuclear to cytoplasmic D-signal increases, consistent with stability of genomic labeling and fading of signal in non-genomic material (e.g. protein). Data expressed as mean +/- s.e.m., significance assessed by t-test.

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Figure 3. D signal correlates with both 15N-thymidine and phosphorus, consistent with incorporation in DNA.

(a) Deflection plate switching allowed measurement of C2D-/C2H- and 12C15N-/12C14N- in an alternating sequence with successive planes. Intestinal villus after 3d D-water/15N-thymidine pulse and 3d chase. P, 15N, and D are preferentially localized in the nuclei in a distribution expected of chromatin. Raster=28µm, 77 planes; pixels=256 x 256, 66s/plane. (b) Pixel-by-pixel correlation between the C2D- and P- signals. Blue line: best-fit linear regression line. (c) Pixel-by-pixel correlation between C2D- and 15N shows similar linear relationship, consistent with incorporation of D in the nuclear DNA of divided cells.

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