Abstract
Genotoxic treatments, such as UV light, camptothecin, and adozelesin, stall DNA replication and subsequently generate DNA strand breaks. Typically, DNA breaks are reflected by an increase in ataxia and Rad-related kinase (ATR)-regulated phosphorylation of H2AX (gammaH2AX) and require replication fork movement. This study examined the potential of the monofunctional DNA alkylating agent hedamycin, a powerful inhibitor of DNA replication, to induce DNA strand breaks, phosphorylated H2AX (gammaH2AX) foci, and chromosome aberrations. Hedamycin treatment of HCT116 carcinoma cells resulted in a rapid induction of DNA strand breaks accompanied by increasing H2AX phosphorylation and focalization. Unlike many other treatments that also stall replication, such as UV, camptothecin, and adozelesin, gammaH2AX formation was not suppressed in ATR-compromised cells but actually increased. Similarly, hedamycin induction of gammaH2AX is not dependent on ataxia telangiectasia mutated or DNA-protein kinase, and pretreatment of cells with the phosphatidylinositol 3-kinase-related kinase inhibitor caffeine did not substantially reduce induction of H2AX phosphorylation by hedamycin. Furthermore, the DNA replication inhibitor aphidicolin only modestly depressed hedamycin-induced gammaH2AX formation, indicating that hedamycin-induced DNA double-strand breaks are not dependent on fork progression. In contrast, camptothecin- and adozelesin-induced gammaH2AX was strongly suppressed by aphidicolin. Moreover, after 24 hours following a short-term hedamycin treatment, cells displayed high levels of breaks in interphase nuclear DNA and misjoined chromosomes in metaphase cells. Finally, focalization of a tightly bound form of Ku80 was observed in interphase cells, consistent with the subsequent appearance of chromosomal aberrations via abnormal nonhomologous end joining. Overall, this study has revealed a disparate type of DNA damage response to stalled replication induced by a bulky DNA adduct inducer, hedamycin, that seems not to be highly dependent on ATR or DNA replication.
Highlights
Treatments that induce replication stalling, albeit by diverse mechanisms, such as UV [1], hydroxyurea [2], or camptothecin [3], typically activate an ataxia and Radrelated kinase (ATR) to induce a phosphatidylinositol 3-kinase – related kinase (PIKK) – regulated checkpoint response [4] and induce DNA breaks [5]
double-strand breaks (DSB) stemming from replication arrest, such as those generated by treatments that induce bulky DNA lesions, such as UV and camptothecin, are generally detected by their ability to trigger H2AX phosphorylation and focalization at DNA damage sites (17, 30 – 32)
Recent studies suggest that PIKK-regulated DNA damage checkpoint responses to DNA adducts may be triggered by the subsequent generation of single-strand DNA or DNA breaks resulting from sustained DNA replication arrest [7, 40]
Summary
Treatments that induce replication stalling, albeit by diverse mechanisms, such as UV [1], hydroxyurea [2], or camptothecin [3], typically activate an ataxia and Radrelated kinase (ATR) to induce a phosphatidylinositol 3-kinase – related kinase (PIKK) – regulated checkpoint response [4] and induce DNA breaks [5]. DNA lesions, whether induced by depletion of deoxynucleotide triphosphate pools or photoproducts, activate an intra-S-phase checkpoint response, resulting in the blockage of late-firing origins and stabilization of stalled replication forks [14]. This intra-S-phase checkpoint is unable to stop fork progression of early-firing origins, which were triggered before checkpoint activation [15, 16]. Collisions between early-firing replication forks and hydroxyurea or UV-induced DNA lesions can result in DSBs and gH2AX formation and its nuclear foci [17]. H2AX phosphorylation is regulated prominently by ATR, consistent with its role in sensing stalled replication forks, such as those generated by UV type lesions [17, 18]
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