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    • We showed that activation of the ATM ATR pathway

    2024-03-06

    We showed that activation of the ATM/ATR pathway leads to over-replication through suppression of CDK1 activity, consistent with previous findings that suppression of CDK1 activity is involved in the polyploidization of megakaryocyte and trophoblast cells [36], [37], [38], [39]. Suppression of CDK1 activity not only inhibits mitotic entry, but also allows the assembly of prereplication complexes (PreRCs) for licensing the DNA for another round of replication [40]. However, inhibitory phosphorylation of CDK1 is not fully sustained upon treatment with bleomycin (Fig. 2A), suggesting that other mechanisms are also involved in bleomycin-induced over-replication. We found that cyclin B1 levels are decreased in cells treated with bleomycin through proteasome-mediated degradation (Fig. 7). It is suggested that during bleomycin-induced over-replication, suppression of CDK1 activity is achieved by two successive mechanisms: (i) ATM/ATR-induced inhibitory phosphorylation; (ii) cyclin B1 degradation. Taken together, the inactivation of CDK1 is responsible for over-replication through the sustained inhibition of mitotic entry, and possibly through licensing the DNA replication in bleomycin-treated cells. By time-lapse recording of a D-box-GFP-expressing HeLa cell clone, we showed that cyclin B1 is degraded in G2 cells upon bleomycin treatment (Fig. 7). In previous studies to examine the degradation of cyclin B1, GFP-fused cyclin B1 was injected to cells instead of its stable expression, since expression of full-length cyclin B1 affects AZD0530 progression [11]. As the cyclin box plays an essential role in binding to CDK1 for activation [41], we used the region including D-box but not the cyclin box (Fig. 6) and expected no CDK1 activation with sufficient expression of D-box-GFP. Indeed, these cells normally grow at a similar rate to parental HeLa cells (data not shown). D-box-GFP oscillates during cell cycle similarly to endogenous cyclin B1 (Fig. 6). Although the expression of D-box-GFP is kept under the cytomegalovirus promoter, protein levels of D-box-GFP during the cell cycle are regulated by degradation. Therefore, D-box-GFP stably expressed in clone cells is useful as a non-invasive indicator for the degradation of cyclin B1 in living cells. In addition, this provides an example of GFP, which was tagged with a degradation-responsible motif, for visualizing the dynamics of protein degradation in living cells. A variety of agents have been reported to induce over-replication. In particular, γ-radiation induces over-replication in p53−/− and p21−/− cells through cytokinesis failure [3]. In this case, cells enter mitosis and progress into G1 phase without completion of cytokinesis. Since mitotic entry in over-replicating cells depends on the level of CDK1 activity [1], doses of γ-radiation capable of inducing over-replication may only partially inhibit CDK1 activity. Doses of γ-radiation that completely inhibit CDK1 activity may induce cytotoxicity. On the other hand, our results showed that mitotic entry is inhibited during bleomycin-induced over-replication. Even at low cytotoxic concentrations, bleomycin is likely to inhibit CDK1 activity, leading to over-replication due to inhibition of mitotic entry. Bleomycin causes 2–3 times fewer DNA cleavages in S phase cells than in G1 or G2/M phase cells [42]. Inhibition of cell cycle progression is likely to depend on the extent of DNA cleavage induced by bleomycin [43], [44]. These results suggest that bleomycin at low concentrations with low cytotoxicity seems to inhibit mitotic entry rather than DNA replication, thereby resulting in the induction of over-replication. We found that inhibition of the ATM/ATR pathway suppressed bleomycin-induced over-replication. As described above, decreased levels of cyclin B1 by degradation may be responsible for G2 arrest and subsequent over-replication in the late phase of treatment. This raises the possibility that the ATM/ATR pathway is involved in regulation of cyclin B1 degradation. Time-lapse recording (Fig. 7B) and flow cytometry analysis (Fig. 4) showed that cyclin B1 degraded gradually from the early phase (within 24 h) in response to bleomycin treatment, suggesting that the ATM/ATR pathway activated by bleomycin-induced DNA damage may stimulate the degradation pathway of cyclin B1 from the early phase (within 24 h). Several reports described crosstalk between the DNA damage checkpoint and the proteolysis pathway [45], [46], [47], [48], [49]. Nonperiodic activation of APC caused polyploidization [50]. In some types of cells, including human megakaryocytes, Drosophila follicle cells, and yeast, activation of APC-mediated proteolysis contributes to polyploidization [50], [51], [52], [53]. Activation of a degradation pathway in response to DNA damage is likely to contribute to the induction of over-replication. For instance, the degradation of geminin, an APC substrate and potent inhibitor of the initiation of DNA replication [54], might be related to over-replication as well as the degradation of cyclin B1.