Kinetics of gamma-H2AX focus formation upon treatment of cells with UV light and alkylating agents.
Histone H2AX is rapidly phosphorylated in response to DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). Here we show that DNA damage induced by alkylating agents [methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] and ultraviolet light (UV-C) leads to a dose and time dependent accumulation of phosphorylated H2AX (gamma-H2AX). Time course experiments revealed that the number of gamma-H2AX foci reached peak levels 8 hr after MMS or MNNG treatment and declined to almost control values within 24 hr after exposure. Upon UV-C treatment, a biphasic response was observed with a maximum 12 hr after treatment. In 43-3B cells deficient in nucleotide excision repair (NER) the number of gamma-H2AX foci increased steadily. gamma-H2AX foci were preferentially formed in BrdU labeled cells. In proliferation compromised cells, the gamma-H2AX level was significantly reduced, indicating that most of the gamma-H2AX foci induced by UV-C and alkylating agent treatments were replication dependent. The data are in line with the view that DNA damage induced by UV-C light and simple alkylating agents, leads to the formation of DSBs during DNA replication giving rise to H2AX phosphorylation. In replicating NER defective cells, DSBs accumulate due to nonrepaired primary DNA lesions that produce a high level of DSBs during replication. The data support that gamma-H2AX foci are a useful marker of DSBs that are induced by S-phase dependent genotoxins during replication.
Staszewski
Ori
O
Department of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
Nikolova
Teodora
T
Kaina
Bernd
B
D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004260:DNA Repair; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism*; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D013997:Time Factors; D014466:Ultraviolet Rays
D000477:Alkylating Agents; C468783:H2AX protein, human; D006657:Histones; D008769:Methylnitronitrosoguanidine; D008741:Methyl Methanesulfonate