Elsevier

Experimental Cell Research

Volume 315, Issue 15, 10 September 2009, Pages 2515-2528
Experimental Cell Research

Research Article
Bleomycin-induced over-replication involves sustained inhibition of mitotic entry through the ATM/ATR pathway

https://doi.org/10.1016/j.yexcr.2009.06.007Get rights and content

Abstract

Polyploid cells result in aneuploidy through aberrant chromosome segregation, possibly leading to tumorigenesis. Although polyploid cells are induced through over-replication by a variety of agents, including DNA-damaging drugs, the mechanisms that induce polyploidy have been hitherto unknown. Here, we show that treatment with bleomycin, a glycopeptide anticancer drug, induces over-replication at low cytotoxic doses. During bleomycin-induced over-replication, mitotic entry is inhibited through tyrosine phosphorylation of CDK1 along the ATM/ATR pathway in the early phase of treatment. Bleomycin-induced over-replication is inhibited by the inhibitors of the ATM/ATR pathway through abrogation of bleomycin-induced G2 arrest, and the ATM/ATR inhibitors promote cell death instead of over-replication. Following the phosphorylation of CDK1, the level of cyclin B1 is decreased in the late phase of treatment. Time-lapse imaging of clone cells that express a live cell marker of endogenous cyclin B1 revealed that cyclin B1 is degraded in G2-arrested cells upon bleomycin treatment. Our findings lead to a model of how the ATM/ATR pathway acts as a molecular switch for regulating cell fates, flipping between cell death via progress into mitosis, and over-replication via sustained G2 arrest upon DNA damage, where cyclin B1 degradation is an important factor for inducing over-replication.

Introduction

Polyploidy is the condition of cells exhibiting the presence of more than two homologous sets of chromosomes. Polyploidy is observed in plants and in some types of animal cells, liver cells, trophoblasts, and megakaryocytes [1]. Abrogation of cell division accompanying over-replication of DNA is thought to result in polyploidization. Some polyploid cell types do not express mitotic regulators, CDK1, Cyclin B, Cyclin A, and Cdc25C, and bypass mitosis, suggesting that decreasing levels of mitotic regulators activate over-replication through abrogation of mitosis in these cell types [1].

A variety of agents, such as microtubule poison, actin depolymerizing agents, membrane traffic inhibitors, and topoisomerase inhibitors, have been reported to induce over-replication by disrupting cytokinesis or karyokinesis [2]. These agents are thought to directly act on cytokinesis machineries or chromosome segregation machineries. DNA-damaging agents have also been reported to induce over-replication by disrupting cytokinesis [3]. However, DNA damage is unlikely to be linked directly with disruption of cytokinesis. DNA damage is known to inhibit CDKs, retarding cell cycle progression at G1 or at G2, dependent on cell types [4]. DNA damage activates the ATM/ATR pathway, inhibiting CDK1 activity through the stabilization of phosphorylation at Thr14/Tyr15 of CDK1, resulting in abrogation of cell division [5]. While the ATM/ATR pathway plays a crucial role in abrogation of mitosis in response to DNA damage, whether the ATM/ATR pathway triggered by DNA damage is responsible for DNA-damage-induced over-replication has not been investigated.

The DNA-damaging anticancer drugs known as bleomycins, a family of glycopeptides, are an important component in a number of combination chemotherapies [6]. The cytotoxicity of bleomycin is related to its ability to induce single- and double-strand DNA breaks [7]. The extent of these DNA breaks depends on the concentration and time of incubation [8]. Low concentrations of bleomycin caused G2 arrest, and produced enlarged and binucleated cells [8], suggesting that the DNA damage caused by low concentrations of bleomycin may induce over-replication. We wished, therefore, to explore the mechanisms that induce over-replication upon bleomycin treatment.

In this study, we show that treatment with bleomycin at low cytotoxic doses induces over-replication after sustained inhibition of mitotic entry in the ATM/ATR pathway-dependent manner. Treatment of cells with an inhibitor of the ATM/ATR pathway abrogates bleomycin-induced over-replication, and in turn promotes cell death. Time-lapse analysis of clone cells that express a live cell marker of cyclin B1 revealed that cyclin B1 degradation is induced in the G2 phase upon bleomycin treatment. Our findings lead to a model of how the ATM/ATR pathway plays a role in bleomycin-induced over-replication, where cyclin B1 degradation is an important factor for inducing over-replication.

Section snippets

Plasmid constructs

To generate a fusion protein of a modified form of green fluorescent protein (MmGFP) [9] with the destruction-box (D-box)-containing an N-terminal fragment of human cyclin B1 (D-box-GFP), the KpnI–EcoRI fragment of human cyclin B1-MmGFP [10] (provided by J. Pines) was replaced with the KpnI–EcoRI fragment of pBluescript as a spacer, which encodes 14-amino acids (GPPSRSTVSISLIS). (R42A)D-box-GFP, which is a nondegradable mutant of D-box-GFP, was generated in the same way, as D-box-GFP, from

Induction of over-replication by DNA-damaging anticancer agents

To investigate whether treatment with bleomycin, a DNA-damaging anticancer agent, induces over-replication, HeLa cells treated with bleomycin were examined for morphology and DNA ploidy by microscopy and flow cytometry. Enlarged cells having a single giant nucleus were induced after 3-day treatment (Fig. 1A), in agreement with previous observations [8]. It is evident that treatment with 1–10 μg/ml bleomycin induced over-replicated cells through inhibition of cell division, although bleomycin at

Discussion

In the present study, we show that low concentrations of bleomycin induce over-replication in a manner dependent on the ATM/ATR pathway. Upon treatment with bleomycin, cells are over-replicated following G2 arrest induced through phosphorylation of CDK1 and degradation of cyclin B1. Abrogation of bleomycin-induced G2 arrest by inhibition of the ATM/ATR pathway promotes cell death instead of over-replication. Our results suggest that in response to bleomycin-induced DNA damage, the ATM/ATR

Acknowledgments

We would like to thank Dr. Jonathon Pines (Wellcome Trust/Cancer Research UK Gurdon Institute), Dr. Jun-ichi Miyazaki (Osaka University), Dr. Takeshi Fujiyasu (The Chemo-Sero-Therapeutic Research Institute, Kumamoto), and Dr. Hiroyuki Miyoshi (Riken BRC) for their invaluable plasmids. This work was supported in part by grants-in-aid for Scientific Research and Global COE program (Global Center for Education and Research in Immune System Regulation and Treatment) from the Japanese Ministry of

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