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Cell death during crisis is mediated by mitotic telomere deprotection

Abstract

Tumour formation is blocked by two barriers: replicative senescence and crisis1. Senescence is triggered by short telomeres and is bypassed by disruption of tumour-suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbour unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remains unexplained. Here we show that human cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. This phenotype is induced by loss of p53 function, and is suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating that such fusions are the underlying cause of cell death. Exacerbation of mitotic telomere deprotection by partial TRF2 (also known as TERF2) knockdown2 increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population.

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Figure 1: Spontaneous mitotic arrest upon bypass of senescence.
Figure 2: Telomere fusions induce mitotic arrest.
Figure 3: Cell fate decision during telomere crisis.
Figure 4: Mitotic telomere deprotection dictates cellular fate upon mitotic arrest.

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Acknowledgements

All data are archived at the Salk Institute. We thank the Salk Institute's J. Fitzpatrick of the Waitt Advanced Biophotonics Center and members of GT3 Core, C. O’Shea, G. Wahl, F. Zhang, and D. Sabatini for support and Karlseder laboratory members for comments. M.T.H. was supported by the Human Frontier Science Program and the Japan Society for the Promotion of Science Postdoctoral Fellowships for Research Abroad. A.J.C. was supported by a NIH NRSA T32 Fellowship (5T32CA009370). T.R. was supported by the Glenn Center for Research on Aging and CIRM training grant TG2-01158. The Salk Institute Cancer Center Core Grant (P30CA014195), the NIH (R01GM087476, R01CA174942), the Donald and Darlene Shiley Chair, the Highland Street Foundation, the Fritz B. Burns Foundation, the Emerald Foundation and the Glenn Center for Research on Aging support J.K.

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Authors

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M.T.H. and A.J.C. designed and performed experiments, and wrote the manuscript, T.R. performed experiments, J.K. designed experiments and wrote the manuscript.

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Correspondence to Jan Karlseder.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Mitotic duration in pre-crisis cells is elongated.

a, Percentage of cells that spend more than 2 h in mitosis (prolonged mitosis), shown in Fig. 1b. b, Effect of indicated oncogenes on p53 expression. c, Scatter plots show mean mitotic duration ± s.e.m. of individual IMR-90 derivative cells analysed in Fig. 1d. d, Scatter plots with bars show mean percentage of prolonged mitosis analysed in Fig. 1d (1–4 independent experiments). The dots represent independent experiments and the bars the mean. e, Scatter plots show mean (± s.e.m.) mitotic duration of IMR-90 E6E7 PD95 cells exposed to reversine and hesperadin (***P < 0.0001, Mann–Whitney test). The result was reproduced in two independent experiments.

Extended Data Figure 2 Telomere elongation abrogates extended mitotic duration.

a, Telomere elongation by hTert in IMR-90 E6E7 and p53dd cells shown in Fig. 1e, f. IMR-90 E6E7 and p53dd cells were infected at PD73 and PD82, respectively, and analysed at the indicated PD. b, c, Scatter plots show mean mitotic duration ± s.e.m. of individual IMR-90 E6E7 (b) and p53dd (c) cells expressing hTert at indicated PD shown in Fig. 1e, f. d, Effect of sgTRF2 on TRF2 expression 7 days after infection. e, f, Scatter plots with bars show mean percentage of cells with telomeric fusion (e) and prolonged mitosis (f) in IMR-90 E6E7 derivatives shown in Fig. 2a, b (1–3 independent experiments). ***P < 0.0001. Mann–Whitney test.

Extended Data Figure 3 Extended mitotic duration depends on telomere fusion.

a, Effect of sgTRF2 on TRF2 expression in cells expressing sgTRF2-resistant TRF2 (TRF2RsgRNA) 9 days after infection with CRISPR/Cas9. b, c, Scatter plots with bars show mean percentage of cells with telomeric fusion (b) and prolonged mitosis (c) in IMR-90 E6E7 expressing sgTRF2 in the presence of TRF2RsgRNA shown in Fig. 2c, d (two independent experiments). d, Schematic of 53BP1 or ligase IV suppression experiment in the presence of sgEMPTY or sgTRF2-2. e, Western analysis of IMR90 E6E7 cells expressing Cas9, Cas9-sg53BP1 or Cas9-sgLig4 in the background of sgEMPTY or sgTRF2-2. f, Representative meta-TIF images of cells suppressed for 53BP1 or LIG4 in the presence of sgEMPTY or sgTRF2-2 as described in d. g, h, Percentage of telomeric fusion (g) and mitotic duration (h) in IMR-90 E6E7 expressing sgEMPTY and sgTRF2 in the presence of DMSO or ATM inhibitor (mean ± s.e.m.). i, j, Scatter plots with bars showing mean percentage of cells with telomeric fusion (i) and prolonged mitosis (j) in IMR-90 E6E7 expressing sgTRF2 in the presence of ATM inhibitor shown in g, h (two independent experiments). k, Mean mitotic duration ± s.e.m. of IMR-90 E6E7 sgEMPTY cells exposed to 500 nM Taxol in the presence of DMSO or ATMi. l, Scatter plots show mean mitotic duration of individual cells shown in j. NS, not significant; *P < 0.05, **P < 0.005, ***P < 0.0001, Mann–Whitney tests. Results were reproduced in at least two independent experiments.

Extended Data Figure 4 Telomere fusions lead to multipolar mitosis.

ac, Ratio of anaphase chromosome with or without anaphase bridge (a), pericentrin foci in pro-, prometa- and metaphase (b) and metaphase chromosome with or without unaligned chromosome (c) in IMR-90 E6E7 expressing sgEMPTY and sgTRF2-2 7 days after infection (Fisher's exact test, for pericentrin foci, 1 and 2 foci versus ≥3 foci). Representative images from sgEMPTY cells are shown below (b, c). Results were reproduced in two independent experiments. d, Scatter plots with bars show mean percentage of cells that possess tetraploidy (FACS analysis) and multipolarity (≥3 pericentrin foci as in j) in IMR-90 E6E7 cells expressing sgEMPTY and sgTRF2-2 7 days after infection (two independent experiments). **P < 0.005, ***P < 0.0001. Fisher's exact test. Scale bars, 10 μm.

Extended Data Figure 5 Mitotic arrest occurs in near-diploid cells.

a, Bars show mean of three independent experiments of average number (± s.e.m.) of telomeric γ-H2AX foci in IMR-90 E6E7 at PD42, 72 and 105 as analysed in Fig. 4d (n = 25 per experiment, P = 0.0008, one-way ANOVA). b, Bars show the percentage of young IMR90 E6E7 cells with separated sister chromatids expressing sgEMPTY or sgTRF2-1/2 6 or 7 days post infection with CRISPR/Cas9. Results from two independent experiments except for sgEMPTY 6 days post infection (at least 31 metaphases per experiment) are shown. c, Ratio of ploidy of mitotic IMR-90 E6E7 cells at PD106. Cells with attached and separated sister chromatids are plotted separately. d, FACS analysis of cells shown in Fig. 1b. e, Bars show percentage of prolonged mitosis and tetraploid cells (4N) in IMR-90 E6E7 cells at indicated PD. Percentage of prolonged mitosis are the same data sets as Extended Data Fig. 1a.

Extended Data Figure 6 TRF2 suppression and overexpression affects cell fate.

a, Effect of shTRF2-F on TRF2 expression 7 days after infection. b, c, Telomeric and non-telomeric γ-H2AX foci in individual pre-crisis IMR-90 E6E7 cells expressing shScramble and shTRF2-F shown in Fig. 4b, c. Data in b and c were analysed as in Fig. 4d (n > 16, mean ± s.e.m.). Metaphases with separated chromatids are shown in magenta. d, Effect of Myc–TRF2 on TRF2 expression 7 days after infection. e, Ratio of mitotic cell fate in pre-crisis IMR-90 E6E7-expressing TRF2 cells, analysed as in Fig. 3f (Fisher's exact test, death versus non-death). f, Meta-TIF analysis of pre-crisis IMR-90 E6E7 cells expressing a control vector or Myc–TRF2. Bars show mean (± s.e.m.) telomeric and non-telomeric γ-H2AX foci (n = 25, Mann–Whitney tests). g, Growth curve of IMR-90 E6E7 cells expressing a control vector or infected with Myc–TRF2-F at PD90. h, i, Mean (± s.e.m.) mitotic duration of IMR-90 E6E7 cells expressing shScramble and shTRF2-F (h) and Myc–TRF2 (i) analysed in Fig. 4e and Extended Data Fig. 6e, respectively (Mann–Whitney test). j, Mean (± s.e.m.) percentage of telomere fusion of pre-crisis IMR-90 E6E7 expressing shScramble and shTRF2-F analysed in Fig. 4a–c (Mann–Whitney test). *P < 0.05, **P < 0.005, ***P < 0.0001. NS, not significant.

Extended Data Figure 7 Amplified telomere deprotection during mitotic arrest in crisis causes cell death.

a, Ratio of mitotic slippage and cell death in IMR-90 E6E7 Cas9-sg53BP1 cells expressing sgEMPTY and sgTRF2-2 in the presence of colcemid, analysed as in Fig. 3f (Fisher's exact test, death versus slippage). b, Scatter plots show mean mitotic duration ± s.e.m. before cell death of individual IMR-90 E6E7 Cas9-sg53BP1 cells expressing sgEMPTY and sgTRF2-2 in the presence of colcemid (Mann–Whitney test). c, Bars show mean (±s.d.) of three independent experiments of average telomeric and non-telomeric γ-H2AX foci in IMR-90 E6E7 cells at PD45 exposed to colcemid in the presence of DMSO or hesperadin at indicated concentrations for 24 h analysed as in Fig. 4d (50 metaphase per experiment). For one-way ANOVA telomeric foci, P < 0.0001; non-telomeric foci, not significant. d, Scatter plots show mean (± s.e.m.) mitotic duration of IMR-90 E6E7 cells at PD27 exposed to 100 ng ml−1 colcemid in the presence of DMSO or 40 nM hesperadin (Mann–Whitney test). e, Ratio of mitotic cell fate in IMR-90 E6E7 cells around PD45 exposed to 100 ng ml−1 colcemid in the presence of DMSO or 40 ng ml−1 hesperadin (Fisher's exact test, death versus slippage). **P < 0.005, ***P < 0.0001. NS, not significant.

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Hayashi, M., Cesare, A., Rivera, T. et al. Cell death during crisis is mediated by mitotic telomere deprotection. Nature 522, 492–496 (2015). https://doi.org/10.1038/nature14513

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