Replication Stress Induces Micronuclei Comprising of Aggregated DNA Double-Strand Breaks

Abstract

BackgroundMicronuclei (MN) in mammalian cells serve as a reliable biomarker of genomic instability and genotoxic exposure. Elevation of MN is commonly observed in cells bearing intrinsic genomic instability and in normal cells exposed to genotoxic agents. DNA double-strand breaks are marked by phosphorylation of H2AX at serine 139 (γ-H2AX). One subclass of MN contains massive and uniform γ-H2AX signals. This study tested whether this subclass of MN can be induced by replication stress.Principal FindingsWe observed that a large proportion of MN, from 20% to nearly 50%, showed uniform staining by antibodies against γ-H2AX, a marker of DNA double-strand breaks (DSBs). Such micronuclei were designated as MN-γ–H2AX (+). We showed that such MN can be induced by chemicals that are known to cause DNA replication stress and S phase arrest. Hydroxyurea, aphidicolin and thymidine could all significantly induce MN-γ–H2AX (+), which were formed during S phase and appeared to be derived from aggregation of DSBs. MN-γ–H2AX (−), MN that were devoid of uniform γ-H2AX signals, were induced to a lesser extent in terms of fold change. Paclitaxel, which inhibits the disassembly of microtubules, only induced MN-γ–H2AX (−). The frequency of MN-γ–H2AX (+), but not that of MN-γ–H2AX (−), was also significantly increased in cells that experience S phase prolongation due to depletion of cell cycle regulator CUL4B. Depletion of replication protein A1 (RPA1) by RNA interference resulted in an elevation of both MN-γ–H2AX (+) and MN-γ–H2AX (−).Conclusions/SignificanceA subclass of MN, MN-γ–H2AX (+), can be preferentially induced by replication stress. Classification of MN according to their γ-H2AX status may provide a more refined evaluation of intrinsic genomic instabilities and the various environmental genotoxicants.

Oxidative stress preferentially induces a subtype of micronuclei and mediates the genomic instability caused by p53 dysfunction

“…We treated U2OS cells with increasing concentrations of H 2 O 2 (50 µM, 100 µM, 150 µM) for different durations (2 h, 6 h, 12 h, 24 h), and examined the frequencies of the MN-γ-H2AX (+) at 48 h after H 2 O 2 was washed out. We previously showed that the frequency of MN-γ-H2AX (+) usually peaked at 48 h after drug removal [27]. As shown in Figure 1A, at the lowest concentration tested (50 µM), H 2 O 2 treatment led to a significant increase in the frequency of MN-γ-H2AX (+) in all the treatment durations.…”

“…MN and MN-γ-H2AX (+) were scored as previously reported [27]. Briefly, nuclei were scored first for MN using a 100× objective under DAPI filter.…”

“…We observed that a large proportion of MN showed uniform and pan-staining by antibodies against γ-H2AX. We designated them as a MN-γ-H2AX (+), to distinguish them from the MN-γ-H2AX (−) that lack uniform labeling of γ-H2AX in the MN [27]. We had shown that the formation of MN-γ-H2AX (+) probably involves active clustering and disposal of DNA DSBs before the onset of mitosis, and demonstrated that MN-γ-H2AX (+) can be preferentially induced by DNA replication stress.…”

Short- and long-term effects of chromosome mis-segregation and aneuploidy

“…These micronuclei are not just miniature nuclei, as they are not fully functional 39 . DNA replication proceeds slowly in micronuclei and results in abnormal and stalled replication forks 39,43 . Given the importance of a functional nuclear envelope for efficient DNA replication 44 , defects in nuclear import [39][40][41] or irreversible nuclear envelope collapse 45 could be responsible for this inability of micronuclei to properly replicate their DNA (FIG.…”

Utilization of CDKN1A/p21 gene for class discrimination of DNA damage-induced clastogenicity

“…ETOP is a topoisomerase II inhibitor that produces DSBs throughout the cell cycle (Liu et al, 1980;Olive and Banáth, 1993). HU inhibits ribonucleotide reductase (Hendricks and Mathews, 1998), and induce DSBs during S phase (Xu et al, 2011). Robust expression of CDKN1A is caused by a 4-h treatment with DNA-damaging compounds-including adriamycin, MMS, CP, or CAMP (Amundson et al, 2005).…”