Tu Lan Chun, Matsui Sei Ichi, Beerman Terry A
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Mol Cancer Ther. 2005 Aug;4(8):1175-85. doi: 10.1158/1535-7163.MCT-05-0054.
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.
基因毒性处理，如紫外线、喜树碱和阿多来新，会使DNA复制停滞，并随后产生DNA链断裂。通常，DNA断裂表现为共济失调和Rad相关激酶（ATR）调节的H2AX（γH2AX）磷酸化增加，且需要复制叉移动。本研究检测了单功能DNA烷化剂赫达霉素（一种强大的DNA复制抑制剂）诱导DNA链断裂、磷酸化H2AX（γH2AX）灶和染色体畸变的潜力。用赫达霉素处理HCT116癌细胞导致DNA链断裂迅速诱导，同时H2AX磷酸化和灶化增加。与许多其他也使复制停滞的处理方法不同，如紫外线、喜树碱和阿多来新，γH2AX的形成在ATR功能受损的细胞中并未受到抑制，反而实际上增加了。同样，赫达霉素诱导的γH2AX不依赖于共济失调毛细血管扩张突变蛋白或DNA蛋白激酶，用磷脂酰肌醇3激酶相关激酶抑制剂咖啡因预处理细胞并不能显著降低赫达霉素诱导的H2AX磷酸化。此外，DNA复制抑制剂阿非迪霉素仅适度抑制赫达霉素诱导的γH2AX形成，表明赫达霉素诱导的DNA双链断裂不依赖于叉的进展。相比之下，喜树碱和阿多来新诱导的γH2AX被阿非迪霉素强烈抑制。此外，在短期赫达霉素处理24小时后，细胞在间期核DNA中显示出高水平的断裂，在中期细胞中显示出错接的染色体。最后，在间期细胞中观察到紧密结合形式的Ku80灶化，这与随后通过异常非同源末端连接出现的染色体畸变一致。总体而言，本研究揭示了一种对由大分子DNA加合物诱导剂赫达霉素引起的复制停滞的不同类型的DNA损伤反应，这种反应似乎不太依赖于ATR或DNA复制。
