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Genome stability is crucial to the very existence of organisms. DNA-strand breaks [1] increase the occurrence of cancer and developmental defects. Thus, the advance in methodologies allowing for detailed study of DNA-behavior, especially under metabolically stressful conditions is crucial to the very progress of our understanding of processes guarantying DNA-stability. The labeling of newly synthesized DNA in cells is undoubtedly an experimental technique of great importance widely used in biology. So far the most common methods allowing detection of proliferating cells are based on treatment with tritium-labeled thymidine or 5-bromo-2′-deoxyruidine (BrdU) followed by autoradiographic or immunohistochemical detection methods, respectively. Both techniques, despite their sensitivity and usefulness, are associated with several imperfections that users should be aware of. The first method is based on radioactivity, whereas the second one requires DNA denaturation by heat or acid treatment to expose BrdU to the antibody [2]. For instance, it was demonstrated that BrdU is capable of passing to daughter cells upon replication and affects cell cycle progression of some human cancer cell lines [3, 4].

Quite recently, a new agent 5-ethynyl-2′deoxyuridine (EdU) has emerged as an interesting alternative to H3-tymidinethymidine and BrdU, allowing detection and quantification of newly synthesized DNA [5]. In contrast to 5-bromo-2′-deoxyruidine, EdU is incorporated during DNA synthesis in a quick “click” chemistry reaction and does not require harsh, chemical, or enzymatic disruption of DNA structure [6]. “Click” chemistry (Huisgen's 1,3-dipolar cycloaddition) is a type of chemical reaction that occurs at room temperature and is catalyzed by copper Cu(I), resulting in the formation of a strong covalent bond between an azide (present in a structure of fluorescent dye) and an alkyne group (present in EdU). The alkyne group is rather un-reactive in biological systems, thus EdU gives a great opportunity for using it inside living cells [7]. Although EdU has greater sensitivity of detection than BrdU [7] and seems to be less harmful to cells, it has some drawbacks as well. The fact that EdU has cytotoxic properties has been lately elucidated by several groups, which demonstrated that this DNA precursor may be responsible for cell cycle arrest and/or cell death [8, 9].

Using a novel methodology that involves the “click-chemistry,” the report by Zhao and colleagues in this issue (page 979) describes a combined cytometric approach to investigate the influence of EdU on cell cycle progression, DNA damage, and induction of apoptosis in A549 cells and cells differing in p53 status: TK6 and WKT1 cells. Using laser scanning cytometry and flow cytometry the authors have shown that EdU incorporated into DNA induces DNA damage signaling manifested mainly by phosphorylation of histone H2AX and activation of ATM (Ataxia-Teleangiectasia Mutated).

Despite highlighted above drawbacks, the relatively “gentle,” non-disruptive properties of EdU-click staining may create less staining-based artifacts. Furthermore, in combination with gentler methods of cell fixation, like for example by using zinc salt-based fixatives [10], such staining protocols may prove particularly useful to study nuclear architecture, and other structural phenomena in cell nuclei, mitochondria, and other cellular compartments, especially in relation to DNA-damage and related events.

The appearance of γ-H2AX (phosphorylated-H2AX) foci is considered as a hallmark of DNA double-strand breaks (DSBs) which are the most dangerous of other types of DNA damages [11]. DSBs may be a result of both exogenous (ionizing radiation, chemical agents) and endogenous factors (reactive oxygen species) but also may arise in biological processes such as V(D)J recombination or simply during the replication or DNA damage repair [12]. In the discussed manuscript in this issue (page 979) the authors have shown that induction of γ-H2AX and activation of ATM after exposure to EdU are also accompanied by phosphorylation of p53 and Chk2. This led in turn to inhibition of cell cycle progression by cell cycle arrest in G2 phase and lately to induction of apoptosis. The effects of EdU on DNA damage signaling and cell cycle progression were more clearly visible when cells were advancing through S-phase (e.g., after longer exposure to DNA precursor). Interestingly, some results seemed to be specific only to EdU, since exposure of cells to BrdU did not result in γ-H2AX appearance or ATM activation. The difference in cellular response after treatment with EdU was also observed between cells with different p53 status.

Most likely, the observed EdU-induced DNA damage is triggered by defective DNA-replication. Artificial nucleosides upon incorporation to DNA may slow down the replication process (as compared to reaction rate with “natural” nucleosides) leading to temporary formation of double strand breaks that serve as signal for activation of ATM and phosphorylation of H2AX. These events in turn lead to the activation of DNA repair machinery.

With all its advantages, EdU-staining requires some individual optimization for different cell types. As shown by Sun and colleagues [13], EdU concentration, incubation time, and the volume of “Click” reaction solution, even the way and order, the permeabilization and fixation are performed, may lead to differences in the staining intensity, and positivity. In their hands, 100 microliter volume of click-reaction, and incubation time of 8–12 hr, worked best for the labeling of 106 cells. They also recommend fixing cells before permeabilization, rather than doing both procedures simultaneously.

Taken together, detection of cell proliferation by compounds like EdU is a fundamental method for assessing cell health and evaluating stress responses. As signalized in the discussed manuscript, the researchers should however analyze their experimental results with the full awareness that 5-ethynyl-2'deoxyuridine is not an inert indicator in their experimental settings. As shown by Zhao and coworkers (in this issue (page XXX), EdU may slow down the replication process, trigger DNA damage signaling, result in G2 cell cycle arrest, and can lead to apoptosis. These findings however do not decrease the enormous potential of the“click-chemistry” compatible dye EdU, and related molecules in biology and medicine. The presented data provides new insight into this novel DNA-labeling tool, and could serve as powerful instrument for the assessment of the extent of DNA damage especially in proliferating cells. Besides, once again new applications for flow cytometry or laser scanning cytometry, modifications of the dye may find their ways into other powerful techniques, like quantitative PCR or other genetic methods.

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