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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2002 as doi:10.1096/fj.02-0142fje. |
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Departments of
* Biochemistry and Molecular Biology and
Pharmacology, The George Washington University Medical Center, Washington, DC, USA
2Correspondence: Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, 2300 Eye St., N.W., Washington, DC 20037, USA. E-mail: bcmvwh{at}gwumc.edu
SPECIFIC AIMS
Metabolic incorporation of tritiated thymidine (3H-TdR) into cellular DNA is still a widely used protocol to monitor rates of DNA synthesis and cell proliferation despite accumulating evidence demonstrating the ability of this radiochemical to induce cell cycle arrest and apoptosis. In this study, we used stable isotope-labeled thymidine and chemical reaction interface mass spectrometry (CRIMS) to 1) determine whether 3H-TdR inhibits the rate of DNA synthesis in mouse 3T3 fibroblasts and 2) monitor the time course of any possible inhibition. Our findings not only urge caution in interpreting data from experiments using the radioactive tracer to determine the rates of DNA synthesis and cell replication, but also demonstrate the superiority of stable isotopic methods in analyzing these processes.
PRINCIPAL FINDINGS
1. Dose-dependent inhibition of cell proliferation and DNA synthesis
Figure 1
A demonstrates that incorporation of 3H-TdR inhibits cell proliferation. To determine whether this inhibition reflects a decrease in the rate of DNA synthesis, 3T3 cells were labeled with a stable isotopic form of TdR before incorporation of 3H-TdR and monitored by CRIMS to assess the effect of the radiolabel on washout of the stable isotope over 3 days (
3.5 doubling periods). Inhibition of DNA synthesis is measured by a decreased rate of washout of 13C and 15N-enriched thymidine ([U-13C, 15N]-TdR) from cellular DNA. Once cells are removed from media containing the stable isotope-enriched TdR and grown in media containing only unenriched TdR, any newly synthesized DNA dilutes the original 13C enrichment and lowers the 
13C value3
observed in the DNA-derived nucleosides. Inhibition of cell growth or cell cycle progression by 3H-TdR reduces this isotopic dilution and maintains the preexisting level of enrichment.
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Figure 1B
shows that 3H-TdR significantly inhibits the washout of [U-13C, 15N]-TdR at 1 and 10 µCi/ml, concentrations often used in cell proliferation assays. This in turn decreases the calculated rate constant, k, for DNA synthesis (Fig. 1C
) and increases the corresponding doubling time for these 3T3 cells from 0.93 days (control cells) to 1.38 ± 0.02 (SD) days and 2.28 ± 0.06 (SD) days for samples labeled with 1 and 10 µCi/ml, respectively. The lowest dose tested (0.1 µCi/ml) had no effect on the rate of DNA synthesis despite a reduction in cell count relative to control after 3 days. This apparent discrepancy may be due to a temporary cell cycle arrest in response to radiolabel-induced DNA fragmentation, which we determined to be significant even at 0.1 µCi/ml. Such a transient arrest would be expected to delay cell division relative to the control cells, which could explain a lower cell count in the radiolabeled sample on day 3 despite no difference in the extent of [U-13C, 15N]-TdR washout. We noted that the degree of DNA fragmentation induced by 3H-TdR at 0.1 and 1 µCi/ml even in the presence of 10–100-fold excess unlabeled TdR was still significantly higher than that of the control sample.
2. Time dependence of DNA synthesis inhibition
To determine the time course of inhibition, cells were cultured with [U-13C, 15N]-TdR followed by 3H-TdR at 1 µCi/ml; washout of the stable isotope was determined 24, 48, and 72 h after addition of the radioisotope. Figure 2
shows the decline in the enrichment of the stable isotope as a function of time over 3 days. 3H-TdR-labeled cells exhibited a significantly reduced rate of [U-13C, 15N]-TdR depletion starting at the first time point, indicating that the inhibition of DNA synthesis occurs within the first replication period. This suggests that the use of the radiolabel would compromise rate determinations for DNA synthesis and the corresponding cell doubling times even in relatively short-term assays. The decreased rate of DNA synthesis is manifested over 3 days inasmuch as the retention of stable isotope enrichment in cellular DNA per day is always greater in the samples exposed to 3H-TdR than in the controls. However, the lower number of cells in radiolabeled samples at day 3 (Fig. 1)
is not simply the result of a decreased rate of DNA synthesis, as discussed below.
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3. Effect of 3H-thymidine on cell proliferation kinetics
Cell growth was monitored over 3 days for cells radiolabeled with 3H-TdR and for control cells. Control cells (prelabeled with stable isotope labeled TdR) grew exponentially at first, leveling off in culture around day 3. In contrast, radiolabeled cells first declined in number to 60% of the initial number within the first 24 h after 3H-TdR was added, indicating that a substantial amount of cell death occurred during this period. The remaining cells, however, were still capable of DNA replication as indicated by partial washout of cell-incorporated [U-13C, 15N]-TdR during the first 24 h (see Fig. 2
). The surviving 3H-TdR-labeled cells apparently entered and passed through at least one mitosis inasmuch as cell number doubled between days 1 and 2, a phenomenon not uncommon with irradiated cells that eventually die or become permanently growth arrested. By day 3, cell number again declined in the radiolabeled samples, suggestive of further radiation-induced cell death.
4. Effect of 3H-thymidine on cell morphology
Aside from the prominent effects of 3H-TdR on DNA synthesis and cell proliferation, we noted that metabolic labeling with this radioactive tracer induces dramatic dose-dependent changes in cell morphology from spindle-shaped fibroblastoid morphology to enlarged cells with more dendritic morphology (Fig. 3
). Such morphological changes (which we also noticed in [35S]methionine-labeled smooth muscle cells) are generally characteristic of cells damaged by ionizing radiation. Thus, it is likely that low-energy ß-irradiation affects cytoskeletal elements and/or arrangements in a manner not yet understood.
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CONCLUSIONS AND SIGNIFICANCE
Although the damaging effects of cell-incorporated, low-energy ß-emitting compounds such as 3H-TdR and [35S]methionine have been documented sporadically over the years, these radioactive metabolic tracers continue to be widely used in the biological sciences to study dynamic cellular processes. Using a stable isotopic form of thymidine and mass spectrometry, we now show that 3H-TdR significantly inhibits the rate of DNA synthesis at all but the lowest conventional doses even in a short-term assay covering only one round of replication. This effect is due to the radioisotope, not the thymidine itself, as unlabeled thymidine has no inhibitory effect at the concentrations used. Thus, nonradioactive, stable isotopic precursors of DNA in combination with mass spectrometry provide a more accurate and reliable measurement of DNA synthesis rates than the traditional radioisotopic labeling method, which we have shown markedly alters the very process it was designed to measure.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0142fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.02-0142fje 
3 13C = 1000 [(IRX/IRS) -1], where IR = 13C/12C isotope ratio, X = sample, and S = standard.
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±SD) after 3 days in culture after initial incubation with varying doses of 3H-TdR. The initial value of 
