title: The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens.
abstract: The phosphorylated form of the histone protein H2AX, called γH2AX, is recognized as a useful biomarker not only for DNA double-strand breaks but also for a wide range of other DNA damage. An increasing number of publications propose γH2AX to be measured when determining genotoxicity, phototoxicity, and the effectiveness of cancer therapy. Because γH2AX is also generated by apoptosis, a γH2AX-assay might assess genotoxic risk incorrectly. The aim of this study was to elucidate the influence of apoptosis on measurements of γH2AX by flow cytometry, with the clastogens mitomycin C (MMC) and etoposide (ETP), and the aneugens vinblastine (VB) and paclitaxel (PT), which do not react directly with DNA. TK6 human lymphoblastoid cells were treated with the clastogens and the aneugens, stained for the apoptotic biomarker caspase-3 and for γH2AX, and then analyzed by flow cytometry. All the test compounds caused a dose-dependent increase of γH2AX-positive (γH2AX+) cells. The γH2AX+ cell population included both caspase-3-positive (γH2AX+/caspase-3+) and caspase-3-negative (γH2AX+/caspase-3-) cells. The increase in γH2AX+ cells after treatment with the aneugens corresponded to the increase in caspase-3+ cells. The increase in γH2AX+/caspase-3- cells after treatment with the clastogens was significant, but there was only a slight increase after treatment with the aneugens. This reflects the fact that the apoptotic pathway of a clastogen starts from DNA damage, whereas that of an aneugen starts from cell-cycle arrest in the M-phase. Therefore, the two pathways contribute differently to apoptosis. Double staining for γH2AX and caspase-3 provided helpful information for the different mechanistic effects of aneugens and clastogens that induce γH2AX.
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Harada
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Matsuzaki
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412-8513, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Takeiri
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412-8513, Japan. Electronic address: mishimamsy@chugai-pharm.co.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Mishima
mesh_terms: D024422:Aneugens / Q000145:classification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004249:DNA Damage; D005047:Etoposide / Q000494:pharmacology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D015162:Micronucleus Tests; D016685:Mitomycin / Q000494:pharmacology; D009153:Mutagens / Q000145:classification* / Q000494:pharmacology*; D017239:Paclitaxel / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D024422:Aneugens; C468783:H2AX protein, human; D006657:Histones; D009153:Mutagens; D016685:Mitomycin; D014747:Vinblastine; D005047:Etoposide; D017239:Paclitaxel
keywords: Aneugen; Apoptosis; Caspase-3; Flow cytometry; Pan-nuclear; γH2AX