DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5‐ethynyl‐2′‐deoxyuridine incorporated into DNA

Biomarkers of cellular function applied as supravital probes should have minimal effect on the interrogated cell. This is of particular importance in studies involving cell sorting for evaluation of their functional properties, cloning, or propagation as it is in the case of analysis of stem cells. Several biomarkers used as supravital probes however interact with different cell constituents affecting the studied cells and perturbing their growth. Among such biomarkers are probes of DNA replication. Radioactive precursors of DNA such as tritium [3H]-thymidine or [14C]-thymidine, used in the early studies utilizing autoradiography, have been shown to induce DNA radiation damage, including formation of DNA double-strand breaks (DSBs), and to perturb the cell cycle progression (1–4). The most widely used DNA precursor 5-bromo-2′-deoxyuridine (BrdU) introduced with the down of flow cytometry was also shown to induce cytostatic and cytotoxic effects (5–9). The BrdU-labeled DNA in cells exposed to visible or UV light undergoes photolysis (10) resulting in the formation of DNA double-strand breaks (DSBs) and DNA damage signaling (DDS) (11). Likewise, another halogenated DNA precursor 5-iodo-2′-deoxyuridine is cytotoxic and once incorporated into DNA increases cell sensitivity to ionizing radiation and also to light (11,12). The “click chemistry” approach utilizing 5-ethynyl-2′-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication (13) and adapted to flow and imaging cytometry (14–20). Compared with BrdU, the method offers several advantages such as simplicity and rapidity as well as no need for partial DNA denaturation as the latter is incompatible with immunocytochemical detection of other cell constituents. Given its advantages, it is expected that EdU will be widely used as DNA precursor in many areas of cell biology and medicine. While EdU is an excellent marker of DNA replication, the evidence is forthcoming that its applicability for long-term cell labeling, at least in some cell types, may be problematic because of the effect on cell cycle progression and cytotoxicity (20–22). The findings by Diermeier-Daucher et al. (20) revealed impairment of the cell cycle progression of cells that incorporated EdU that varied significantly depending on the cell type. Ross et al. (21) observed perturbation of the cell cycle progression and cytotoxic effects following incorporation of EdU. Interestingly, in the in vivo studies on mice, administration of EdU was shown to reduce growth of the subcutaneous grafts of human glioblastoma and increased animal survival, without apparent significant toxicity. In light of the evidence that EdU crosses the blood–brain barrier, these findings prompted the authors to propose investigation of EdU as potential therapy for malignant brain tumors (21). Most recently, when this article was in preparation, Kohlmeier et al. (22) reported that depending on the cell type EdU can grossly perturb the cell cycle progression and induce cell death. The most sensitive were mouse embryonic stem cells which become arrested in G2/M phase and underwent apoptosis (22). These authors also observed that incorporation of EdU triggers DDS, manifested as histone H2AX Ser139 phosphorylation (induction of γH2AX) and formation of 53BP1 foci (22). We previously reported that several“supravital” DNA probes, commonly used to label DNA in live cells, induce DDS detected by activation of Ataxia Telangiectasia mutated (ATM), Chk2 and p53 as well as induction of γH2AX (23). These phosphorylation events detected by cytometry using phosphospecific Abs are considered to be indicative of damage to DNA (24,25). Phosphorylation of H2AX, especially manifested by formation of characteristic γH2AX foci, combined with activation of ATM are likely the reporters of DSBs (26,27). In the present study, we explored whether incorporation of EdU into DNA can also induce DDS that can be detected by cytometry. Specifically, using the multiparameter flow and laser scanning-cytometry combined with confocal microscopy, we attempted to observe a possible correlation between the incorporated EdU and cell cycle phase(s) at which the EdU-induced cell cycle progression was impaired. We have tested the EdU effects on the non-small cell pulmonary adenocarcinoma A549 cells that express wt p53, used previously by us to assess induction of DDS by oxidative stress or by DNA topoisomerase inhibitors in relation to DNA replication (28,29). In addition, the effect of EdU incorporation was also tested on human B-cell lymphoblastoid leukemic cells using two sister cell lines TK6 and WTK1 derived from the same WIL2 cell line, the TK6 having wt p53 while WTK1 expressing spontaneously mutated p53 (30,31).