Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells.
Ultraviolet A (UVA) radiation represents more than 90% of the UV spectrum reaching Earth's surface. Exposure to UV light, especially the UVA part, induces the formation of photoexcited states of cellular photosensitizers with subsequent generation of reactive oxygen species (ROS) leading to damages to membrane lipids, proteins and nucleic acids. Although UVA, unlike UVC and UVB, is poorly absorbed by DNA, it inhibits cell cycle progression, especially during S-phase. In the present study, we examined the role of the DNA damage checkpoint response in UVA-induced inhibition of DNA replication. We provide evidence that UVA delays S-phase in a dose dependent manner and that UVA-irradiated S-phase cells accumulate in G2/M. We show that upon UVA irradiation ATM-, ATR- and p38-dependent signalling pathways are activated, and that Chk1 phosphorylation is ATR/Hus1 dependent while Chk2 phosphorylation is ATM dependent. To assess for a role of these pathways in UVA-induced inhibition of DNA replication, we investigated (i) cell cycle progression of BrdU labelled S-phase cells by flow cytometry and (ii) incorporation of [methyl-(3)H]thymidine, as a marker of DNA replication, in ATM, ATR and p38 proficient and deficient cells. We demonstrate that none of these pathways is required to delay DNA replication in response to UVA, thus ruling out a role of the canonical S-phase checkpoint response in this process. On the contrary, scavenging of UVA-induced reactive oxygen species (ROS) by the antioxidant N-acetyl-L-cystein or depletion of vitamins during UVA exposure significantly restores DNA synthesis. We propose that inhibition of DNA replication is due to impaired replication fork progression, rather as a consequence of UVA-induced oxidative damage to protein than to DNA.
Girard
Pierre-Marie
PM
Centre National de la Recherche Scientifique, UMR 2027, Centre Universitaire, Orsay, France. pierre-marie.girard@curie.u-psud.fr
Pozzebon
Mariaelena
M
Delacôte
Fabien
F
Douki
Thierry
T
Smirnova
Violetta
V
Sage
Evelyne
E
D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D002461:Cell Line, Transformed; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D016196:S Phase / Q000187:drug effects* / Q000528:radiation effects; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D017382:Reactive Oxygen Species; D025521:Tumor Suppressor Proteins; D048051:p38 Mitogen-Activated Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human