title: Kinetics of nuclear phosphorylation (γ-H2AX) in human lymphocytes treated in vitro with UVB, bleomycin and mitomycin C.
abstract: After double-strand break induction, formation of γ-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). γ-H2AX foci are then rapidly dephosphorylated as signal for the subsequent recruitment of effector proteins. The induction and disappearance of the foci can be, therefore, used to monitor the functioning of the DDR machinery in a cell population exposed to genotoxic stress. Here, we investigated the time-course of γ-H2AX in unstimulated or cultured peripheral lymphocytes in vitro treated with UVB, bleomycin and mitomycin C (MMC). Once the mutagen exposure was performed, cells were harvested at different interval times from 0.5 to 5h. The results show that (i) in 20-h stimulated peripheral lymphocytes, UVB irradiation caused extensive and dose-dependent increases in nuclear phosphorylation, and disappearance of γ-H2AX foci progressed, proportionally to the UV fluence, with increasing the harvesting time; (ii) UVB-exposed G0 cells cultured for 20-h post-irradiation displayed low amounts of DNA phosphorylation, depicting a time-course in which the maximum effect was reached at 0.5h and dephosphorylation started after 1h; (iii) treatment of unstimulated lymphocytes with bleomycin sulphate induced an increase in nuclear phosphorylation of several folds higher than that of untreated cells, depicting kinetics comparable to those observed for UVB-exposed G1 cells; (iv) in stimulated cells, MMC caused a severe and dose-dependent high degree of H2AX phosphorylation together with a very slower kinetic of dephosphorylation with respect to the other experimental treatments. This study confirms the feasibility of the γ-H2AX focus assay as a genotoxic end-point and supports the view that the proposed type of analysis should be introduced in biomonitoring studies of human populations. This could also represent a feasible and useful tool in the screening and diagnosis of precancerous states or very early stages of other diseases.
authors.affiliation: Dipartimento di Biologia, Unità di Genetica, Mutagenesi e Epidemiologia Ambientale, University of Pisa, Derna 1, 56100 Pisa, Italy. rscarpato@biologia.unipi.it
authors.forename: Roberto
authors.initials: R
authors.lastname: Scarpato
authors.forename: Sara
authors.initials: S
authors.lastname: Castagna
authors.forename: Rosamaria
authors.initials: R
authors.lastname: Aliotta
authors.forename: Alessia
authors.initials: A
authors.lastname: Azzarà
authors.forename: Francesco
authors.initials: F
authors.lastname: Ghetti
authors.forename: Erika
authors.initials: E
authors.lastname: Filomeni
authors.forename: Caterina
authors.initials: C
authors.lastname: Giovannini
authors.forename: Chiara
authors.initials: C
authors.lastname: Pirillo
authors.forename: Serena
authors.initials: S
authors.lastname: Testi
authors.forename: Sara
authors.initials: S
authors.lastname: Lombardi
authors.forename: Antonio
authors.initials: A
authors.lastname: Tomei
mesh_terms: D000328:Adult; D001761:Bleomycin / Q000494:pharmacology*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D002478:Cells, Cultured; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D008297:Male; D016685:Mitomycin / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D021381:Protein Transport / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects*; D055815:Young Adult
chemical_list: C468783:H2AX protein, human; D006657:Histones; D001761:Bleomycin; D016685:Mitomycin