Initiation of DNA Fragmentation during Apoptosis Induces Phosphorylation of H2AX Histone at Serine 139

Abstract

Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif, -KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as ␥-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. ␥-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. ␥-H2AX formation is inhibited by N-benzyloxycarbonyl-Val-AlaAsp-fluoromethyl ketone and the inhibitor of caspaseactivated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce ␥-H2AX formation. These results indicate that ␥-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis.

p21WAF1/CIP1 induction by 5-azacytosine nucleosides requires DNA damage

“…Genotoxic stress response may upregulate p21 WAF1/CIP1 expression to induce cell cycle arrest and allow for DNA repair before cell division. Expression of phosphorylated histone H2AX (g-H2AX) is a sensitive marker of double-strand breaks in DNA (Rogakou et al, 2000). ML-1 cells were treated with increasing concentrations of DAC for 48 h prior to immunoblotting for g-H2AX (Figure 1d).…”

F-actin-binding domain of c-Abl regulates localized phosphorylation of C3G: role of C3G in c-Abl-mediated cell death

“…Ectopic expression of c-Abl has earlier been shown to induce apoptosis, and we confirmed this by counter-staining using TUNEL reagent and cleaved caspase-3 antibody ( Figure 1f). As a marker for early apoptotic changes in cells, gH2AX (Ragakou et al, 2000) was also used to show that on c-Abl expression, p-C3G-positive cells were indeed undergoing apoptosis (Figure 1f). Confocal sections through the z axis from the adherent surface of a c-Ablexpressing apoptotic cell (Supplementary Video 1) and 3D reconstruction using IMARIS software showed restricted pattern of p-C3G staining relative to that of c-Abl (Figure 1g).…”

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

“…The recently introduced technique to visualise sites of histone g-H2AX phosphorylation (Rogakou et al, 1998) needs much lower doses than gel electropheresis to monitor dsb repair (Rothkamm and Löbrich, 2003) and may thus reduce apoptosis and cell cycle pertubation, although this technique is generally also sensitive to DNA degradation (Rogakou et al, 2000) and stalled replication (Ward and Chen, 2001). However, it needs to be shown whether recording of g-H2AX will be an advantage over conventional gel electrophoresis for monitoring residual damage in tumour cells.…”