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Mutation of NIMA-related kinase 1 (NEK1) leads to chromosome instability.

Chen Y, Chen CF, Chiang HC, Pena M, Polci R, Wei RL, Edwards RA, Hansel DE, Chen PL, Riley DJ - Mol. Cancer (2011)

Bottom Line: These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice.Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates.NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Endocrinology, University of California at Irvine, 1130 Gross Hall, Irvine, CA 92697, USA. yumayc@uci.edu

ABSTRACT

Background: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage.

Results: We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates.

Conclusion: NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

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Related in: MedlinePlus

Aneuploidy in NEK1 -/- cells. (A, B). Passage 4 RTEs cultured from NEK1 -/- mice and from wild type littermates were treated with colchicine and hypotonically lysed. Free chromosomes were dispersed and stained with Giemsa. A. Representative images of chromosome spreads. Note the more variable sister chromatids observed in the spread prepared from NEK1 -/- cells. Bar = 50 μM. B. Numbers of chromosomes per cell were quantified (more than 200 spreads from at least two unique cell lines for each genotype). (C, D) Wild type or NEK1 -/- RTEs at passage 4 were harvested during exponential phase growth, stained with propidium iodide, and quantified by FACS for DNA content. C. Plots for wild type cells show sharp 2n and 4n peaks. One NEK1 -/- line (#432) shows a substantial fraction of cells with >4n DNA content (yellow). In another NEK1 -/- line (#436), the majority of the cells have >2n DNA content, and a broad peak is seen, representing a range of different DNA content in individual cells. D. Histograms showing 5-13% of cells with >4n DNA content and 6-9% with DNA content intermediate between 2n and 4n, specifically in NEK1 -/- RTEs.
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Figure 3: Aneuploidy in NEK1 -/- cells. (A, B). Passage 4 RTEs cultured from NEK1 -/- mice and from wild type littermates were treated with colchicine and hypotonically lysed. Free chromosomes were dispersed and stained with Giemsa. A. Representative images of chromosome spreads. Note the more variable sister chromatids observed in the spread prepared from NEK1 -/- cells. Bar = 50 μM. B. Numbers of chromosomes per cell were quantified (more than 200 spreads from at least two unique cell lines for each genotype). (C, D) Wild type or NEK1 -/- RTEs at passage 4 were harvested during exponential phase growth, stained with propidium iodide, and quantified by FACS for DNA content. C. Plots for wild type cells show sharp 2n and 4n peaks. One NEK1 -/- line (#432) shows a substantial fraction of cells with >4n DNA content (yellow). In another NEK1 -/- line (#436), the majority of the cells have >2n DNA content, and a broad peak is seen, representing a range of different DNA content in individual cells. D. Histograms showing 5-13% of cells with >4n DNA content and 6-9% with DNA content intermediate between 2n and 4n, specifically in NEK1 -/- RTEs.

Mentions: We have previously shown that NEK1 -/- cells suffer from numerous chromosome breaks, and that NEK1 has an important role in DNA damage checkpoint control [14]. The array of abnormalities we observed in mitotic NEK1 -/- cells, especially the lagging chromosomes and bizarre incomplete cytokinesis, suggested a potential role for NEK1 in the spindle checkpoint. To explore the extent of chromosomal abnormalities further, we analyzed chromosome spreads. In wild type cells, 40 normal mitotic chromosomes were observed in >90% of the spreads. In contrast, only 25% of the mitotic spreads from NEK1 -/- cells contained 40 chromosomes (Figure 3A, B). While the vast majority of wild type cells had diploid (2n) chromosome copy number, NEK1 -/- cells had variable numbers of chromosomes (median = 62) (Figure 3B). Hypoploid spreads were relatively rare, especially in late passage cells, but a significant fraction (74%) of NEK1 -/- cells had chromosome numbers greater than 4n. These findings suggest that NEK1 -/- cells fail to undergo cytokinesis properly. Giemsa staining showed that chromosomes from NEK1 -/- cells were more variable than wild type chromosomes, and that sister chromatids (Figure 3A, arrows) were often unequal in length. This latter finding suggests chromosome rearrangements.


Mutation of NIMA-related kinase 1 (NEK1) leads to chromosome instability.

Chen Y, Chen CF, Chiang HC, Pena M, Polci R, Wei RL, Edwards RA, Hansel DE, Chen PL, Riley DJ - Mol. Cancer (2011)

Aneuploidy in NEK1 -/- cells. (A, B). Passage 4 RTEs cultured from NEK1 -/- mice and from wild type littermates were treated with colchicine and hypotonically lysed. Free chromosomes were dispersed and stained with Giemsa. A. Representative images of chromosome spreads. Note the more variable sister chromatids observed in the spread prepared from NEK1 -/- cells. Bar = 50 μM. B. Numbers of chromosomes per cell were quantified (more than 200 spreads from at least two unique cell lines for each genotype). (C, D) Wild type or NEK1 -/- RTEs at passage 4 were harvested during exponential phase growth, stained with propidium iodide, and quantified by FACS for DNA content. C. Plots for wild type cells show sharp 2n and 4n peaks. One NEK1 -/- line (#432) shows a substantial fraction of cells with >4n DNA content (yellow). In another NEK1 -/- line (#436), the majority of the cells have >2n DNA content, and a broad peak is seen, representing a range of different DNA content in individual cells. D. Histograms showing 5-13% of cells with >4n DNA content and 6-9% with DNA content intermediate between 2n and 4n, specifically in NEK1 -/- RTEs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Aneuploidy in NEK1 -/- cells. (A, B). Passage 4 RTEs cultured from NEK1 -/- mice and from wild type littermates were treated with colchicine and hypotonically lysed. Free chromosomes were dispersed and stained with Giemsa. A. Representative images of chromosome spreads. Note the more variable sister chromatids observed in the spread prepared from NEK1 -/- cells. Bar = 50 μM. B. Numbers of chromosomes per cell were quantified (more than 200 spreads from at least two unique cell lines for each genotype). (C, D) Wild type or NEK1 -/- RTEs at passage 4 were harvested during exponential phase growth, stained with propidium iodide, and quantified by FACS for DNA content. C. Plots for wild type cells show sharp 2n and 4n peaks. One NEK1 -/- line (#432) shows a substantial fraction of cells with >4n DNA content (yellow). In another NEK1 -/- line (#436), the majority of the cells have >2n DNA content, and a broad peak is seen, representing a range of different DNA content in individual cells. D. Histograms showing 5-13% of cells with >4n DNA content and 6-9% with DNA content intermediate between 2n and 4n, specifically in NEK1 -/- RTEs.
Mentions: We have previously shown that NEK1 -/- cells suffer from numerous chromosome breaks, and that NEK1 has an important role in DNA damage checkpoint control [14]. The array of abnormalities we observed in mitotic NEK1 -/- cells, especially the lagging chromosomes and bizarre incomplete cytokinesis, suggested a potential role for NEK1 in the spindle checkpoint. To explore the extent of chromosomal abnormalities further, we analyzed chromosome spreads. In wild type cells, 40 normal mitotic chromosomes were observed in >90% of the spreads. In contrast, only 25% of the mitotic spreads from NEK1 -/- cells contained 40 chromosomes (Figure 3A, B). While the vast majority of wild type cells had diploid (2n) chromosome copy number, NEK1 -/- cells had variable numbers of chromosomes (median = 62) (Figure 3B). Hypoploid spreads were relatively rare, especially in late passage cells, but a significant fraction (74%) of NEK1 -/- cells had chromosome numbers greater than 4n. These findings suggest that NEK1 -/- cells fail to undergo cytokinesis properly. Giemsa staining showed that chromosomes from NEK1 -/- cells were more variable than wild type chromosomes, and that sister chromatids (Figure 3A, arrows) were often unequal in length. This latter finding suggests chromosome rearrangements.

Bottom Line: These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice.Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates.NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Endocrinology, University of California at Irvine, 1130 Gross Hall, Irvine, CA 92697, USA. yumayc@uci.edu

ABSTRACT

Background: NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage.

Results: We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates.

Conclusion: NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

Show MeSH
Related in: MedlinePlus