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Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells

Nature Genetics volume 39, pages 391–396 (2007)Cite this article

Abstract

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice1,2,3. The role of DNMT1 in human cancer cells, however, remains highly controversial4,5,6,7. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase–mediated disruption of this allele results in hemimethylation of ∼20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.

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Figure 1: Targeted disruption of DNMT1 in HCT116 cells.
Figure 2: Inactivation of DNMT1 in HCT116 cells results in cell-cycle arrest and cell death.
Figure 3: DNMT1 mutant cells undergo mitotic catastrophe.
Figure 4: The G2/M checkpoint is activated in DNMT1 mutant cells.
Figure 5: DNMT1 mutant cells show hemimethylation of CpG sites.
Figure 6: Model of the effects of DNMT1 loss on the proliferation and survival of human cancer cells.

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Acknowledgements

We thank colleagues at Novartis Institutes for Biomedical Research, particularly Y. Kurash, C. Timmers, F. Gaudet, S. Kadam, K. Wang and Y.-N.P. Chen for discussions; W. Marston, A. Ho and A. Szilvasi for technical assistance with flow cytometric analyses; and Y. Liu for statistical analysis. We also thank T. Kanda, G. Wahl and H. Kimura for the human H2B-GFP plasmid; and P. Chambon for the Cre-ERT2 plasmid.

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Author notes

  1. Yoshihide Ueda

    Present address: Present address: Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.,

Authors and Affiliations

  1. Epigenetics Program, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Taiping Chen, Sarah Hevi, Frédérique Gay, Naomi Tsujimoto & En Li

  2. Central Technologies, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Timothy He & Bailin Zhang

  3. Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown, 02129, Massachusetts, USA

    Yoshihide Ueda

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Contributions

T.C. designed the experiments, performed DNA methylation analyses, and wrote the paper; S.H., F.G. and N.T. carried out most of the experiments; F.G. also helped to write the paper; T.H. and B.Z. measured the 5-methylcytosine content by LC-MS/MS; Y.U. generated the targeting vectors; and E.L. conceived the study.

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Correspondence to En Li.

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All authors except Y.U. are employees of Novartis Institutes for Biomedical Research.

Supplementary information

Supplementary Fig. 1

DNMT1 mutant cells lack DNMT1 protein products. (PDF 117 kb)

Supplementary Fig. 2

Loss of DNMT1 impairs cell cycle progression. (PDF 19 kb)

Supplementary Fig. 3

DNMT1 mutant cells display nuclear abnormalities. (PDF 16 kb)

Supplementary Fig. 4

Loss of DNMT1 induces chromosome instability. (PDF 147 kb)

Supplementary Table 1

Oligonucleotides used in this study. (PDF 25 kb)

Supplementary Video 1

Mitosis of wild-type HCT116 cells. HCT116 cells stably expressing H2B-GFP were cultured in the presence of 4-HT for 24 h and then visualized and photographed at 6-min intervals for another 24 h. The video, which was generated by combining the first 100 images, shows the normal progression of mitosis (1 s corresponds to 30 min of real time). (MOV 746 kb)

Supplementary Video 2

Mitotic catastrophe in DNMT1 mutant HCT116 cells. DNMT12loxP/KO (#201) cells stably expressing H2B-GFP were cultured in the presence of 4-HT for 24 h and then visualized and photographed at 6-min intervals for another 24 h. The video, which was generated by combining the first 175 images, shows various defects characteristic of mitotic catastrophe, including broken chromosomes, chromosome misalignment/missegregation, cell death during mitosis, formation of a binucleated cell, and apoptosis of a multinucleated cell (1 s corresponds to 30 min of real time). Note that many cells did not enter mitosis within the course of visualization, consistent with G2 arrest. (MOV 1345 kb)

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Chen, T., Hevi, S., Gay, F. et al. Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells. Nat Genet 39, 391–396 (2007). https://doi.org/10.1038/ng1982

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