Induction of DNA damage response by the supravital probes of nucleic acids

Abstract

The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV), and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981), and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2, or p53 phosphorylation. However, pronounced ATM, Chk2, and p53 phosphorylation and perturbed G 2 progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2, and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2, and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G 2 M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5, and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis. ' 2009 International Society for Advancement of Cytometry

Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

“…The population of cells undergoing spontaneous apoptosis can be significant: 25% or higher (Qiao and Farrell 1999;Sapozhnikov et al 1999;Vassena et al 2007 It is known that supravital DNA dyes possess different negative effects on cells. DRAQ5 induces chromatin aggregation and selective blocking of cells in the G 2 M phase (Zhao et al 2009). Vybrant DyeCycle Orange induces significant cytotoxic responses in HL-60 and U937 cells (Wlodkowic and Darzynkiewicz 2008), whereas DyeCycle Violet suppresses progression through G 2 M and S-phases (Zhao et al 2009).…”

“…DRAQ5 induces chromatin aggregation and selective blocking of cells in the G 2 M phase (Zhao et al 2009). Vybrant DyeCycle Orange induces significant cytotoxic responses in HL-60 and U937 cells (Wlodkowic and Darzynkiewicz 2008), whereas DyeCycle Violet suppresses progression through G 2 M and S-phases (Zhao et al 2009). Thus, staining with supravital DNA dyes may have significant consequences for the functionality of sorted cells and their progeny.…”

“…These methods are also limited by the inherent toxicity of DNA viability dye (Wlodkowic and Darzynkiewicz 2008). Supravital DNA dyes, such as Hoechst 33342, DRAQ5, and DyeCycle Violet, at the concentrations generally applied to live cells induce DNA damage resulting in blockage of cell cycle progression, increased cell-cycle checkpoint kinase 2 (Chk2) and p53 phosphorylation and, consequently, perturbed G 2 M progression and changes in histone H2AX phosphorylation (Zhao et al 2009). Furthermore, cells considered "viable" by DNA staining are often heterogeneous by light scattering parameters and may include populations committed to apoptosis.…”

In Vitro Generation of Plasmodium falciparum Ookinetes

“…The finding that macrogametes could be separated from gametocytes by DNA content was surprising but consistent with previous fluorometric studies. [37][38][39][40][41] Region R6 was enriched for a mixture of zygotes and ookinetes ( Figure 3 ). The finding that the majority of zygotes and ookinetes had more than or equal to four times as much DNA as region R3 is consistent with our current understanding that meiotic division occurs shortly after gamete fusion.…”

Anterior positioning of sex chromosomes on the head of human sperm sorted using visible wavelengths

“…To circumvent the use of UV light, we have used the quantitative DNA dyes DRAQ5® and Dyecycle™ Vybrant® Violet, which are excited at visible (i.e., higher) wavelengths [Mari et al 2010;Zhao et al 2009]. …”