γ-H2AX Expression Pattern in Non-Irradiated Neonatal Mouse Germ Cells and after Low-Dose γ-Radiation: Relationships Between Chromatid Breaks and DNA Double-Strand Breaks1

Abstract

The DNA double-strand breaks (DSBs) are considered to be the most relevant lesions for the deleterious effects of ionizing radiation exposure. The discovery that the induction of DSBs is rapidly followed by the phosphorylation of H2AX histone at Ser-139, favoring repair protein recruitment or access, opens the possibility for a wide range of research. This phosphorylated histone, named gamma-H2AX, has been shown to form foci in interphase nuclei as well as megabase chromatin domains surrounding the DNA lesion on chromosomes. Using detection of gamma-H2AX on germ cell mitotic chromosomes 2 h after gamma-irradiation, we studied radiation-induced DSBs during the G(2)/M phase of the cell cycle. We show that 1) non-irradiated neonatal germ cells express gamma-H2AX with variable patterns at metaphase, 2) gamma-irradiation induces foci whose number increases in a dose-dependent manner, 3) some foci correspond to visible chromatid breaks or exchanges, 4) sticky chromosomes characterizing cell radiation exposure during mitosis are a consequence of DSBs, and 5) gamma-H2AX remains localized at the sites of the lesions even after end-joining has taken place. This suggests that completion of DSB repair does not necessarily imply disappearance of gamma-H2AX.

Delayed and stage specific phosphorylation of H2AX during preimplantation development of γ-irradiated mouse embryos

“…Furthermore, metaphase chromosomes of one-cell stage embryos were stained for g-H2AX even without irradiation. Such staining for g-H2AX of metaphase chromosomes without external DNA damage was reported to occur in meiotic cells as well as in HeLa cells (Fernandez-Capetillo et al 2004, Forand et al 2004, Turner et al 2004, Ichijima et al 2005, and this damage independent phosphorylation required ATM activity (Ichijima et al 2005). Taken together, these results indicate that ATM kinase is present in one-cell and two-cell stage embryos.…”

“…The phosphorylated form of H2AX is designated as g-H2AX, and forms foci at the site of DNA damage which then serve as a platform to recruit various repair and cell cycle checkpoint proteins (Fernandez-Capetillo et al 2004). The g-H2AX focus formation is used as a marker of DNA double strand breaks in irradiated cells, and they are visible even at break and exchange points of metaphase chromosomes (Rogakou et al 1998, Rothkamm et al 2003, Forand et al 2004. g-H2AX is most likely to play important roles in DNA repair and chromatin remodeling during DNA damage response.…”

Acute Overexpression of Myc in Intestinal Epithelium Recapitulates Some but Not All the Changes Elicited by Wnt/β-Catenin Pathway Activation

“…Myc activation rapidly induced accumulation of both p53 and p21 cip1 . In contrast, anti-phospho-H2.AX staining (a marker of DNA damage) showed only very occasional foci following Myc activation (yellow arrowheads) that may be attributable merely to normal mitosis (14). Hence, widespread p53 and p21 cip1 induction occurs without concomitant evidence of widespread DNA damage.…”

Checkpoint adaptation in human cells

“…As the number of g-H2AX foci correlates with unrepaired DSBs in IR-treated human cells , each single g-H2AX focus in the mitotic osteosarcoma cells may represent one unrepaired double-strand break. However, g-H2AX may persist on rejoined chromosomes (Forand et al, 2004;Suzuki et al, 2006) and even became duplicated during replication in progenies of IR-treated cells (Klokov et al, 2006), suggesting that g-H2AX may also represent other lesions related to aberrant chromatin structure or improper repair. Furthermore, g-H2AX staining was increased during apoptosis in a Jun N-terminal kinasedependent fashion (Lu et al, 2006).…”