Measuring DNA Synthesis Rates with [1-¹³C]Glycine

Abstract

We have devised and evaluated a stable-isotopic method for measuring DNA synthesis rates. The probe is [1-13C]-glycine that is incorporated into purines via de novo biosynthesis. The human hepatoma cell line HEP G2 was grown in medium containing [1-13C]glycine, the cells were harvested at various times, and the DNA was extracted. Following hydrolysis to the nucleosides, a reversed-phase HPLC separation was used to provide separate peaks for deoxythymidine (dT), deoxyadenosine (dA), and deoxyguanosine (dG). The HPLC effluent was continuously fed into a chemical reaction interface and an isotope ratio mass spectrometer (HPLC/CRI/IRMS). The isotope ratio of the CO2 produced in the CRI was used to monitor for enrichment. The cells were grown continuously for 5 days in labeled medium and also in a 1-day pulse labeling experiment where the washout of label was observed for the subsequent 9 days. As predicted from the role of glycine in de novo purine biosynthesis, the isotope ratio of the pyrimidine dT did not change. However, for the two purines, dA and dG, the characteristic log growth behavior of the cells was observed in their 13C/12C ratios and good agreement in the doubling time was obtained for each type of experiment. Parallel experiments that measured the HEP G2 doubling time in culture using tritiated thymidine incorporation and direct cell counts were carried out compare to our new method with established ones. We believe that the use of [1-13C]-glycine and the HPLC/CRI/IRMS is a highly sensitive and selective approach that forms the basis of a method that can measure DNA synthesis rates using a nonradioactive, nontoxic tracer.

Peer Reviewed: Measuring DNA Synthesis Rates with Stable Isotopes.

“…We hypothesize that the salvage pathway predominates under most conditions. Certainly, the low precursorproduct relationship we found in cells given [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glycine supports that suggestion (2). However, when G-CSF triggers a surge in white blood cell production with its attendant increase in DNA synthesis, the availability of preformed nucleosides is not sufficient to meet the body's needs, and the de novo pathway becomes more important.…”

“…In this article, we discuss three existing methods using stable isotopes to measure DNA synthesis rates (1)(2)(3). Beyond the results of these three procedures, we provide perspectives on choosing labels, preferred precursor species, and optimum instrumentation.…”

“…To increase detection of the label, Hellerstein et al changed their analytical method yet still used [6,6- 15 N]thymidine were tested using Chen and Abramson's method (2). Labeling efficiency was defined as the percent enrichment obtained per milligram of tracer.…”

“…The trick is finding a labeled DNA precursor that has meaningful bioavailability. To address this question, single intravenous and oral doses of [1,[2][3][4][5][6][7][8][9][10][11][12][13] C 2 ]glycine, [U-13 C]glucose, and [U-13 C, 15 N]thymidine were administered to rabbits. The bioavailability of glycine at 3 mg/kg in one rabbit was 21%, and the bioavailability of glucose at 10 mg/kg in another rabbit was 43%, showing that both glycine and glucose would allow oral dosing.…”

Incorporation of nitrogen isotope ¹⁵N into liver DNA during avian embryogenesis. A new approach for measuring the rate of DNA synthesis

“…Traditionally, pyrimidine nucleosides such as tritiated thymine and bromodeoxyuridine (Neese et al, 2001), which enter into DNA synthesis through salvage pathways, have been used to evaluate in vitro and in vivo rates of DNA synthesis, however, these may have adverse effects on cell function (Chen and Abramson, 1998). It is well known that stable isotopes are not harmful to the organism and recently several new techniques using stable isotopes have been successfully applied in measurements of DNA synthesis (Perez and Reeds, 1998;Nissim et al, 2000;Hellerstein, 2003;Martini et al, 2004;Zhang et al, 2004;Petzke et al, 2005;Fan et al, 2006).…”