Research ArticleCell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1
Introduction
Topoisomerases relax the superhelical tension of DNA. Type II topoisomerases are able to break and rejoin the two strands that make up duplex DNA. The activity of topoisomerase II (topo II) is essential for proliferating cell survival and participates in virtually all processes involving double-stranded DNA including replication, transcription, recombination, chromosome condensation, and the decatenation of sister chromatids prior to the anaphase of mitosis [1]. In cancer chemotherapy, topo II is one of the major targets for a variety of anticancer drugs. According to their mechanism of action, these drugs have been classified into two groups. One class of drugs termed topo II poisons, including anthracyclines (adriamycin/doxorubicin and daunorubicin), epipodophyllotoxins (etoposide/VP-16 and teniposide/VM-26), anthracenedione (mitoxantrone), isoflavonoid (genistein), and aminoacridines (amsacrine/m-AMSA), stabilizes the protein-linked DNA intermediate “cleavable complex” and produces DNA double-strand breaks (DSB) through this complex [2]. Topo II poisons are much more cytotoxic than the other class of drugs, topo II catalytic inhibitors. Topo II catalytic inhibitors that do not stabilize the cleavable complex inhibit topo II by locking topo II in a “closed clamp,” thus preventing strand passage [3]. Bis-dioxopiperazines (ICRF-154, ICRF-187, ICRF-193, etc.), fostriecin, aclarubicin, suramin, novobiocin, and merbarone all belong to this class of drugs.
DNA damage induced by ionizing radiation (IR), ultraviolet radiation (UV), or abnormal structures such as stalled replication forks generally results in the rapid activation of DNA damage signaling pathways, cell cycle arrest, and DNA repair, with the overall purpose of maintaining genome stability. In vertebrates, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), members of the phosphatidylinositol 3-kinase-related protein family, are critical checkpoint regulators which work upstream of the DNA damage response pathway [4]. In humans, ATM is mutated in the autosomal disorder, ataxia-telangiectasia (A-T) syndrome. These patients display an increased rate of chromosomal recombination and are defective in IR-induced G1/S, S phase, and G2/M checkpoints. ATM seems to be more specifically involved in responses to DSB, whereas one human disease, ATR-Seckel syndrome, has been reported to have ATR deficiency [5], and in mice, ATR disruption leads to early embryonic lethality [6]. ATR has been shown to regulate responses to a broad range of damage, including stalled replication forks, UV-induced photodimers, nucleotide depletion, polymerase arrest, interstrand crosslinks, and DSB [7], [8]. The checkpoint functions of ATR and ATM are mediated in part by a pair of checkpoint effector kinases termed CHK1 and CHK2 [9], [10]. Histone H2AX, 53BP1, BRCA1, MDC1, FANCD2, and NBS1 are all targets for ATM- or ATR-mediated phosphorylation [11], [12], [13], [14], [15], [16], [17], [18]. These molecules participate in the transmission of DNA damage signals to downstream molecules such as CHK1 and CHK2 and colocalize to foci containing the site of damaged DNA. These foci are presumed to be checkpoint/repair factories. Whereas the phosphorylation of CHK1 by ATR is induced by IR, UV, stalled replication forks, and upon activation of the mismatch repair system by 6-thioguanine or methylating agents [9], [19], CHK2 is phosphorylated by ATM in response to IR, stalled replication forks, and activation of the mismatch repair system by 6-thioguanine or methylating agents [4], [19], [20]. The topo II poisons, doxorubicin, genistein, and etoposide, induce DSB in which the signal is transduced through CHK2 in an ATM-dependent manner [21], [22].
ICRF-193 has been extensively analyzed as a topo II catalytic inhibitor to study the function of topo II [23]. ICRF-193-treated cells delay G2/M transition as well as the progression from metaphase to anaphase in mammalian cells [24]. The nature of this G2 delay by ICRF-193 treatment has been proposed as a “decatenation checkpoint,” in which cells monitor chromatid catenation status after DNA replication and inhibit progression into mitosis until the chromatids are correctly decatenated by topo II [25]. Activation of the decatenation G2 checkpoint relies on ATR activity and the subsequent nuclear exclusion of cyclin B1. However, several recent observations suggest that ICRF-193 may induce DNA damage in vivo and in vitro, although the extent of DNA damage is weak as compared to that induced by topo II poisons [26], [27], [28], [29], [30], [31].
Although several reports suggest that ICRF-193 can induce DNA damage, this issue is still controversial. Moreover, the mechanism by which ICRF-193 induces DNA damage has not been studied extensively. We initiated this study with the aim of understanding the mechanism of G2 arrest by ICRF-193 treatment. Here, we show that ICRF-193 induced DNA damage resulting in G2 arrest and that DNA damage signaling by ICRF-193 involved molecules reminiscent of those participating in DSB by IR. In addition, cell cycle-dependent DNA damage induced by ICRF-193 treatment demonstrated that topo II is essential for the progression of the cell cycle at several stages, including S, G2, and mitosis [1]. Lastly, for the first time in mammalian cells, we provide evidence that topo II is required during late mitosis and the early G1 phase, presumably for chromosome decondensation.
Section snippets
Cell lines and culture conditions
HeLa cells (purchased from ATCC) were maintained in DMEM supplemented with 10% fetal calf serum. GM16666 and GM16667 cells (purchased from Coriell Cell Repositories) were maintained in DMEM supplemented with 10% fetal calf serum, 2 mM of l-glutamine, and 100 μg/ml of hygromycin B. The ATM-deficient (A-T) fibroblasts immortalized with hTERT and normal human fibroblasts immortalized with hTERT (generously provided by Dr. Pandita at Washington University School of Medicine, St. Louis, MO) were
ICRF-193 induces DNA damage and nuclear foci formation of γ-H2AX, NBS1, BRCA1, 53BP1, MDC1, and FANCD2
To test whether ICRF-193 treatment induces DNA damage, the nuclear foci formation of proteins including γ-H2AX, NBS1, BRCA1, 53BP1, MDC1, and FANCD2 was examined in HeLa cells. Phosphorylation of histone H2AX is among the earliest responses to DNA damage [33], [34]. We found that 6 h of treatment with 10 μM ICRF-193 induced the formation of γ-H2AX, NBS1, BRCA1, 53BP1, MDC1, and FANCD2 nuclear foci (Fig. 1A). Induction of γ-H2AX foci was observed after ICRF-193 treatment, but the kinetics of the
Discussion
To explore the function of topo II, various inhibitors have been used, including poisons and catalytic inhibitors. Whereas topo II poisons induce DNA damage by forming a “cleavable complex,” catalytic inhibitors of topo II are generally regarded as not inducing DNA damage and just inhibiting the catalytic activity of the enzyme [3]. For these reasons, catalytic inhibitors of topo II are preferentially used to study the function of topo II. Whereas several recent observations suggest that
Acknowledgments
This work was supported in part by the National Institutes of Health Grants K18HL74106 (to H.A.), by CA 96805 (to H.A.), and by the Department of Defense grants W81XWH-04-1-0721 and W81XWH-04-1-0711. We thank W.K. Kaufmann for the generous gift of ATR-inducible GM847 cells, T.K. Pandita for the hTERT-immortalized normal fibroblasts and A-T fibroblasts, S.J. Elledge for polyclonal antibodies against MDC1, and T.D. Halazonetis for monoclonal antibody against 53BP1. We also thank R. Hauser and A.
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2013, Toxicology LettersCitation Excerpt :The presence of a decatenation checkpoint that is independent of DNA damage has been supported by a variety of studies (Damelin et al., 2005; Nakagawa et al., 2004). The concept is however still controversial as the topo II inhibitor ICRF-193 has been shown to activate DNA damage signaling in some cancer cell lines (Park and Avraham, 2006). Mitosis can be subdivided into karyokinesis (prophase, prometaphase, metaphase, anaphase, telophase) and cytokinesis.