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Alternative Chk1-independent S/M checkpoint in somatic cells that prevents premature mitotic entry

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Abstract

Genomic instability is the hallmark of cancer. Checkpoint kinase-1 (Chk1) is required for cell cycle delay after DNA damage or blocked DNA replication. Chk1-depleted tumor cells undergo premature mitosis and apoptosis. Here we analyzed the depletion of Chk1 in normal somatic cells in the absence of DNA damage in order to investigate alternative cell cycle checkpoint mechanism(s). By means of adenoviruses, flow cytometry, immunofluorescence and Western blotting, Chk1-depleted mouse embryonic fibroblasts (MEFs) were investigated. Chk1−/− MEFs arrested at the S/G2 boundary of the cell cycle with decreased protein levels of many cell cycle key players. Cyclin B1 was predominantly cytoplasmic. Interestingly, overexpression of nuclear dominant Cyclin B1 leads to nuclear translocation and premature mitosis. Chk1−/− MEFs exhibited the absence of double-strand breaks, yet cells showed delayed DNA damage recovery with pan-nuclear immunostaining pattern of Histone H2AX. Activation of this checkpoint would elicit a senescent-like phenotype. Taken together, our elaborated data revealed the existence of an additional S/M checkpoint functioning via γH2AX signaling and cytoplasmic retention of Cyclin B1 in somatic cells.

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Abbreviations

Chk1:

Checkpoint kinase 1

MEF:

Mouse embryonic fibroblast

Cdk1:

Cyclin-dependent kinase 1

DSB:

Double-strand break

Lac-z:

Gene encoding β-galactosidase

ATM:

Ataxia telangiectasia mutated

ATR:

Ataxia telangiectasia mutated and Rad3 related

SAHF:

Senescence-associated heterochromatin foci

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Acknowledgements

We are greatly thankful for Prof. Nakanishi M. (Tokyo University, Japan) for providing Chk1 MEFs, adenoviruses and antibodies and Prof. Noeman S. (Faculty of Medicine, Tanta University, Egypt) for critical reading of the manuscript and helpful discussion.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Tanta University, El-Geish Street, Tanta, El-Gharbia, Egypt

    Doaa Hussein Zineldeen & Noha Mohamed Shafik

  2. Affiliated Zhongshan Hospital, Dalian University, Dalian, China

    Sheng Fan Li

  3. Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan

    Sheng Fan Li

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  1. Doaa Hussein Zineldeen
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  2. Noha Mohamed Shafik
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Contributions

All authors contributed sufficiently to this work. DHZ designed the research protocol, conducted experimental work, performed statistical analysis and drafted the manuscript, NMS helped in experimental work and data analysis, and SFL participated in molecular experiments. All authors read and approved the final manuscript.

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Correspondence to Doaa Hussein Zineldeen.

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All applicable international, national and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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Supplementary Fig. 1

(A) Chk1flox/− MEFs were seeded into 10-cm dishes, serum starved in 0.5% FBS-containing medium at 50% for 2 days, then infected with Adeno-Cre, Adeno-Lac-Z viruses for 2 days, 15 h before the end of the second day of infection cells were released into 15% FBS medium containing 10uM Aphidicholine‬‏ (to synchronize cells at S phase), on the end of the second day of infection (15 h after Aphidicholine‬‏ block), NLS Cyclin B1, and tTA adenoviruses transduction was performed either alone or with Adeno-Cre (to deplete Chk1); 3 h after Cyclin B1/tTA transduction samples were collected at 3–9, 12, 24, 30 h post-transduction and trypsinized at the indicated time points and double stained with PI to visualize DNA content and FITC to detect pHH3 (mitotic index) by FACS, where a is Lac-Z, b; Cre, c; tTA/NLS alone and d; tTA/NLS/Cre. Percentages of pre-mitotic cells are encircled. B; Graph representing the mean percentages of pHH3 positive cells; error bars are ± SD. Data are representative of 3 independent experiments (TIFF 12130 kb)

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Zineldeen, D.H., Shafik, N.M. & Li, S.F. Alternative Chk1-independent S/M checkpoint in somatic cells that prevents premature mitotic entry. Med Oncol 34, 70 (2017). https://doi.org/10.1007/s12032-017-0932-3

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