title: Loss of histone H2AX increases sensitivity of immortalized mouse fibroblasts to the topoisomerase II inhibitor etoposide.
abstract: In mammalian cells, the H2AX histone is rapidly phosphorylated upon the induction of DNA double strand breaks and promotes their repair, which is required for preserving genomic integrity. Etoposide is an inhibitor of DNA topoisomerase II, which causes DNA breaks and induces H2AX phosphorylation. To elucidate whether H2AX may affect cellular sensitivity to etoposide, we studied the response to this agent in immortalized embryonic fibroblasts derived from H2AX knockout mice. Clonogenic assays in cells treated with the drug revealed a greater sensitivity of H2AX null cells compared to wild-type cells, possibly due to the persistence of a higher number of DNA breaks, as detected with the comet assay. In both cell lines, etoposide induced micronuclei formation and nuclear fragmentation; however, in H2AX deficient cells nuclear fragmentation was observed at a lower drug concentration. Flow cytometric analysis showed that etoposide induced a G2/M cell cycle arrest in both cell lines, which occurred at lower drug concentrations in H2AX deficient cells. G2/M arrest was paralleled by an accumulation of cyclin A and cyclin B1, suggesting that treated cells are not able to complete cell cycle correctly and undergo cell death. Taken together, our observations suggest that H2AX takes part to the cellular response to etoposide and confirm its role in the maintenance of genome stability.
authors.affiliation: Istituto di Genetica Molecolare CNR, I-27100 Pavia, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Donà
authors.forename: Ennio
authors.initials: E
authors.lastname: Prosperi
authors.forename: Monica
authors.initials: M
authors.lastname: Savio
authors.forename: Tania
authors.initials: T
authors.lastname: Coppa
authors.forename: A Ivana
authors.initials: AI
authors.lastname: Scovassi
authors.forename: Chiara
authors.initials: C
authors.lastname: Mondello
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002453:Cell Cycle; D020552:Comet Assay; D019925:Cyclin A / Q000096:biosynthesis / Q000187:drug effects; D019926:Cyclin B / Q000096:biosynthesis / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005047:Etoposide / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects*; D005434:Flow Cytometry; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000187:drug effects; D059005:Topoisomerase II Inhibitors
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D019925:Cyclin A; D019926:Cyclin B; D005047:Etoposide; D006657:Histones; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D059005:Topoisomerase II Inhibitors; C499321:Parp1 protein, mouse; C475961:gamma-H2AX protein, mouse