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The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1.

Voets E, Marsman J, Demmers J, Beijersbergen R, Wolthuis R - Sci Rep (2015)

Bottom Line: Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry.This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression.We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Insitute (NKI-AvL), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase.

No MeSH data available.


Related in: MedlinePlus

Partial inhibition of Cdk1 results in a spindle checkpoint-dependent mitotic arrest.(A) Flow cytometry of RPE1 and U2OS cells treated with increasing concentrations of RO-3306. Cells were treated for 16 hours (Mean ± s.e.m, n = 3). (B) Cell death correlates to entering mitosis, whereas a full blockade of Cdk1 activity prevents cell death. Cell death was determined by flow cytometry analysis. (Mean ± s.e.m, n = 3). (C) Partial Cdk1 inhibition triggers PARP cleavage. Lysates of U2OS cells are shown. (D) Cyclin B1 degradation in metaphase is delayed in cells with impaired Cdk1 activity. U2OS-CCNB1-EYFP cells were followed by differential interference contrast (DIC) and fluorescence microscopy. NEB, nuclear envelope breakdown; nt, nuclear translocation; ana, anaphase. Scale bar, 10 μm. (E) Quantification of the cyclin B1-EYFP levels in cells treated with or without 3 μM RO-3306. Fluorescence intensity was plotted against time after NEB. (Mean ± s.e.m, n = 3, untreated = 20 cells analysed, 3 μM RO-3306 treated = 13 cells analysed). (F) Unaligned sister chromatids are detected by the mitotic checkpoint. U2OS cells, treated with or without 3 μM RO-3306 were fixed after 16 hours. Mad2-positive kinetochores are indicative of a functional spindle checkpoint. Insets: overlay of Mad2 and CREST stainings. Scale bar, 10 μm. (G) Partial Cdk1 inhibition results in sister chromatid attachment defects to the mitotic spindle. Treatments are performed as in (F). Sister chromatid pairs that are not aligned are negative for kinetochore-associated astrin. Insets: overlay of astrin and CREST stainings. Scale bar, 10 μm. (H) Quantification of the mitotic duration after partial Cdk1 inhibition in presence or absence of a functional checkpoint. Mitosis is plotted from NEB till anaphase. (Mean ± s.e.m, n = 3, 50 cells/experiment; ****p < 0.0001, Student’s t test). (I) Cdk1 inhibition results in the formation of micronuclei during mitotic exit. Analysis is performed using the cells from panel (H). (Mean ± s.e.m, n = 3, 50 cells/experiment; ***p < 0.001, Student’s t test). Western blots in panel (C) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.
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f1: Partial inhibition of Cdk1 results in a spindle checkpoint-dependent mitotic arrest.(A) Flow cytometry of RPE1 and U2OS cells treated with increasing concentrations of RO-3306. Cells were treated for 16 hours (Mean ± s.e.m, n = 3). (B) Cell death correlates to entering mitosis, whereas a full blockade of Cdk1 activity prevents cell death. Cell death was determined by flow cytometry analysis. (Mean ± s.e.m, n = 3). (C) Partial Cdk1 inhibition triggers PARP cleavage. Lysates of U2OS cells are shown. (D) Cyclin B1 degradation in metaphase is delayed in cells with impaired Cdk1 activity. U2OS-CCNB1-EYFP cells were followed by differential interference contrast (DIC) and fluorescence microscopy. NEB, nuclear envelope breakdown; nt, nuclear translocation; ana, anaphase. Scale bar, 10 μm. (E) Quantification of the cyclin B1-EYFP levels in cells treated with or without 3 μM RO-3306. Fluorescence intensity was plotted against time after NEB. (Mean ± s.e.m, n = 3, untreated = 20 cells analysed, 3 μM RO-3306 treated = 13 cells analysed). (F) Unaligned sister chromatids are detected by the mitotic checkpoint. U2OS cells, treated with or without 3 μM RO-3306 were fixed after 16 hours. Mad2-positive kinetochores are indicative of a functional spindle checkpoint. Insets: overlay of Mad2 and CREST stainings. Scale bar, 10 μm. (G) Partial Cdk1 inhibition results in sister chromatid attachment defects to the mitotic spindle. Treatments are performed as in (F). Sister chromatid pairs that are not aligned are negative for kinetochore-associated astrin. Insets: overlay of astrin and CREST stainings. Scale bar, 10 μm. (H) Quantification of the mitotic duration after partial Cdk1 inhibition in presence or absence of a functional checkpoint. Mitosis is plotted from NEB till anaphase. (Mean ± s.e.m, n = 3, 50 cells/experiment; ****p < 0.0001, Student’s t test). (I) Cdk1 inhibition results in the formation of micronuclei during mitotic exit. Analysis is performed using the cells from panel (H). (Mean ± s.e.m, n = 3, 50 cells/experiment; ***p < 0.001, Student’s t test). Western blots in panel (C) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.

Mentions: We compared the cell cycle effects of different concentrations of the specific Cdk1 inhibitor RO-3306 and found that 16 hours of treatment with RO-3306 at 3–5 μM increased the mitotic index significantly, while more completely blocking Cdk1 activity with 10 μM RO-3306 arrested cells in G2 phase. Similar effects were seen in non-transformed retinal pigment epithelial (RPE1) cells and osteosarcoma (U2OS) cells (Fig. 1A). Interestingly, mitotic arrest after 3–5 μM RO-3306 rapidly led to cell death, while G2 arrest after completely blocking Cdk1 activity was much less detrimental within this time window (Fig. 1B). Cleaved poly-ADP-ribose-polymerase (PARP) revealed the rapid induction of apoptosis after partial, but not after more complete, Cdk1 inhibition (Fig. 1C)18.


The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1.

Voets E, Marsman J, Demmers J, Beijersbergen R, Wolthuis R - Sci Rep (2015)

Partial inhibition of Cdk1 results in a spindle checkpoint-dependent mitotic arrest.(A) Flow cytometry of RPE1 and U2OS cells treated with increasing concentrations of RO-3306. Cells were treated for 16 hours (Mean ± s.e.m, n = 3). (B) Cell death correlates to entering mitosis, whereas a full blockade of Cdk1 activity prevents cell death. Cell death was determined by flow cytometry analysis. (Mean ± s.e.m, n = 3). (C) Partial Cdk1 inhibition triggers PARP cleavage. Lysates of U2OS cells are shown. (D) Cyclin B1 degradation in metaphase is delayed in cells with impaired Cdk1 activity. U2OS-CCNB1-EYFP cells were followed by differential interference contrast (DIC) and fluorescence microscopy. NEB, nuclear envelope breakdown; nt, nuclear translocation; ana, anaphase. Scale bar, 10 μm. (E) Quantification of the cyclin B1-EYFP levels in cells treated with or without 3 μM RO-3306. Fluorescence intensity was plotted against time after NEB. (Mean ± s.e.m, n = 3, untreated = 20 cells analysed, 3 μM RO-3306 treated = 13 cells analysed). (F) Unaligned sister chromatids are detected by the mitotic checkpoint. U2OS cells, treated with or without 3 μM RO-3306 were fixed after 16 hours. Mad2-positive kinetochores are indicative of a functional spindle checkpoint. Insets: overlay of Mad2 and CREST stainings. Scale bar, 10 μm. (G) Partial Cdk1 inhibition results in sister chromatid attachment defects to the mitotic spindle. Treatments are performed as in (F). Sister chromatid pairs that are not aligned are negative for kinetochore-associated astrin. Insets: overlay of astrin and CREST stainings. Scale bar, 10 μm. (H) Quantification of the mitotic duration after partial Cdk1 inhibition in presence or absence of a functional checkpoint. Mitosis is plotted from NEB till anaphase. (Mean ± s.e.m, n = 3, 50 cells/experiment; ****p < 0.0001, Student’s t test). (I) Cdk1 inhibition results in the formation of micronuclei during mitotic exit. Analysis is performed using the cells from panel (H). (Mean ± s.e.m, n = 3, 50 cells/experiment; ***p < 0.001, Student’s t test). Western blots in panel (C) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.
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Related In: Results  -  Collection

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f1: Partial inhibition of Cdk1 results in a spindle checkpoint-dependent mitotic arrest.(A) Flow cytometry of RPE1 and U2OS cells treated with increasing concentrations of RO-3306. Cells were treated for 16 hours (Mean ± s.e.m, n = 3). (B) Cell death correlates to entering mitosis, whereas a full blockade of Cdk1 activity prevents cell death. Cell death was determined by flow cytometry analysis. (Mean ± s.e.m, n = 3). (C) Partial Cdk1 inhibition triggers PARP cleavage. Lysates of U2OS cells are shown. (D) Cyclin B1 degradation in metaphase is delayed in cells with impaired Cdk1 activity. U2OS-CCNB1-EYFP cells were followed by differential interference contrast (DIC) and fluorescence microscopy. NEB, nuclear envelope breakdown; nt, nuclear translocation; ana, anaphase. Scale bar, 10 μm. (E) Quantification of the cyclin B1-EYFP levels in cells treated with or without 3 μM RO-3306. Fluorescence intensity was plotted against time after NEB. (Mean ± s.e.m, n = 3, untreated = 20 cells analysed, 3 μM RO-3306 treated = 13 cells analysed). (F) Unaligned sister chromatids are detected by the mitotic checkpoint. U2OS cells, treated with or without 3 μM RO-3306 were fixed after 16 hours. Mad2-positive kinetochores are indicative of a functional spindle checkpoint. Insets: overlay of Mad2 and CREST stainings. Scale bar, 10 μm. (G) Partial Cdk1 inhibition results in sister chromatid attachment defects to the mitotic spindle. Treatments are performed as in (F). Sister chromatid pairs that are not aligned are negative for kinetochore-associated astrin. Insets: overlay of astrin and CREST stainings. Scale bar, 10 μm. (H) Quantification of the mitotic duration after partial Cdk1 inhibition in presence or absence of a functional checkpoint. Mitosis is plotted from NEB till anaphase. (Mean ± s.e.m, n = 3, 50 cells/experiment; ****p < 0.0001, Student’s t test). (I) Cdk1 inhibition results in the formation of micronuclei during mitotic exit. Analysis is performed using the cells from panel (H). (Mean ± s.e.m, n = 3, 50 cells/experiment; ***p < 0.001, Student’s t test). Western blots in panel (C) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.
Mentions: We compared the cell cycle effects of different concentrations of the specific Cdk1 inhibitor RO-3306 and found that 16 hours of treatment with RO-3306 at 3–5 μM increased the mitotic index significantly, while more completely blocking Cdk1 activity with 10 μM RO-3306 arrested cells in G2 phase. Similar effects were seen in non-transformed retinal pigment epithelial (RPE1) cells and osteosarcoma (U2OS) cells (Fig. 1A). Interestingly, mitotic arrest after 3–5 μM RO-3306 rapidly led to cell death, while G2 arrest after completely blocking Cdk1 activity was much less detrimental within this time window (Fig. 1B). Cleaved poly-ADP-ribose-polymerase (PARP) revealed the rapid induction of apoptosis after partial, but not after more complete, Cdk1 inhibition (Fig. 1C)18.

Bottom Line: Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry.This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression.We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Insitute (NKI-AvL), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase.

No MeSH data available.


Related in: MedlinePlus