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  • Original Paper
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The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe

Abstract

Fusion between nonsynchronized cells leads to the formation of heterokarya which transiently activate Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 and enter the prophase of the cell cycle, where they arrest due to a loss of Cdk1/cyclin B1 activity, activate p53, disorganize centrosomes, and undergo apoptosis. Here, we show that the down regulation of Cdk1/cyclin B is secondary to the activation of the DNA structure checkpoint kinase Chk2. Thus, syncytia generated by the fusion of asynchronous HeLa cells contain elevated levels of active Chk2 but not Chk1. Chk2 bearing the activating phosphorylation on threonine-68 accumulates in BRCA1 nuclear bodies when the cells arrest at the G2/M boundary. Inhibition of Chk2 by transfection of a dominant-negative Chk2 mutant or a chemical inhibitor, debromohymenialdesine, stabilizes centrosomes, maintains high cyclin B1 levels, and allows for a prolonged activation of Cdk1. Under these conditions, multinuclear HeLa syncytia do not arrest at the G2/M boundary and rather enter mitotis and subsequently die during the metaphase of the cell cycle. This mitotic catastrophe is associated with the activation of the pro-apoptotic caspase-3. Inhibition of caspases allows the cells to go beyond the metaphase arrest, indicating that apoptosis is responsible for cell death by mitotic catastrophe. In another, completely different model of mitotic catastrophe, namely 14.3.3σ-deficient HCT116 colon carcinoma cells treated with doxorubicin, Chk2 activation was also found to be deficient as compared to 14.3.3σ-sufficient controls. Inhibition of Chk2 again facilitated the induction of mitotic catastrophe in HCT116 wild-type cells. In conclusion, a conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe, provided that the checkpoint kinase Chk2 is inhibited. Inhibition of Chk2 thus can sensitize proliferating cells to chemotherapy-induced apoptosis.

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Abbreviations

Casp-2a:

activated caspase-2

Casp-3a:

activated caspase-3

Cdk1:

cyclin dependent kinase-1

Cyt. c:

cytochrome c

DBH:

debromohymenialdisine

DN:

dominant negative

Env:

envelope glycoprotein complex

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

HIV:

human immunodeficiency virus

KG:

karyogamy

MMP:

mitochondrial membrane permeabilization

mTOR:

mammalian target of rapamycin

p53S15P:

p53 with phosphorylated serine 15

RNAi:

RNA interference

siRNA:

small interfering RNA

SC:

single cell

Syn:

syncytia

Z-VAD.fmk:

N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone

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Acknowledgements

We thank Dr. Bernd Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA) for cell lines, Dr. Thierry Soussi (Institut Curie, Paris, France) for cDNA constructs, Vladimir Lazar (Institut Gustave Roussy, Villejuif, France) for RT-PCR analyses; Didier Métivier, Nathanael Larochette (CNRS, Villejuif, France) for assistance, and the NIH AIDS reagents program (Bethesda, MD, USA) for cell lines. This work has been supported by a special Grant from LNC, as well as Grants from ANRS, FRM, European Commission (QLG1-CT-1999-00739 and Contract No. QLK3-CT-20002-01956) (to GK).

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Correspondence to Guido Kroemer.

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Castedo, M., Perfettini, JL., Roumier, T. et al. The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe. Oncogene 23, 4353–4361 (2004). https://doi.org/10.1038/sj.onc.1207573

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