Limits...
CDK1 plays an important role in the maintenance of pluripotency and genomic stability in human pluripotent stem cells.

Neganova I, Tilgner K, Buskin A, Paraskevopoulou I, Atkinson SP, Peberdy D, Passos JF, Lako M - Cell Death Dis (2014)

Bottom Line: Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process.On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation.Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne NE1 3BZ, UK.

ABSTRACT
Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are characterised by an unusual and tightly regulated cell cycle that has been shown to be important for the maintenance of a pluripotent phenotype. Cyclin-dependant kinase 1 (CDK1) is a key player in cell cycle regulation and particularly mitosis; however, its role has not been studied previously in hESC and hiPSC. To investigate the impacts of CDK1 downregulation, we performed RNA interference studies which in addition to expected mitotic deficiencies revealed a large range of additional phenotypes related to maintenance of pluripotency, ability to repair double strand breaks (DSBs) and commitment to apoptosis. Downregulation of CDK1 led to the loss of typical pluripotent stem cell morphology, downregulation of pluripotency markers and upregulation of a large number of differentiation markers. In addition, human pluripotent stem cells with reduced CDK1 expression accumulated a higher number of DSBs were unable to activate CHK2 expression and could not maintain G2/M arrest upon exposure to ionising radiation. CDK1 downregulation led to the accumulation of cells with abnormal numbers of mitotic organelles, multiple chromosomal abnormalities and polyploidy. Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process. On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation. Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.

Show MeSH

Related in: MedlinePlus

Knockdown of CDK1 induces the activation of γH2.AX and downregulation of CHK2 expression in hESC. (a) Confocal microscopy analysis showing the presence of γH2 A.X foci in hESC at 1, 2 and 3 days post transfection of control and CDK1 siRNAs. Phosphorylated histone H2A.X (γ -H2A.X foci) is shown as white dots. Chromatin is stained with DAPI (blue). Scale bar=10 μm, D=day. Images are representative of at least three independent experiments. Percentage of γ-H2A.X-positive cells is shown at the bottom. (b) Graphic representation of the average number of γH2A.X foci per nucleus in hESC during 3-day time course post CDK1 and control siRNAs transfections. Data areshown as average±S.E.M., n=3. (c) Flow cytometric analysis of γ-H2A.X on the second day post transfection and 16 h after administration of IR. Data are shown as average±S.E.M., n=3. (d) Representative images of four repeats of western blot analysis for CHK1 and CHK2 expression up to 96 h post transfection of siRNAS. β-Actin was used as the loading control. (e) Impacts of CDK1 inhibition on the regulation of key factors involved in G2 checkpoint activation analysed by western blotting at day 2 post transfection and after 6 h post IR on the same day (shown in the figure as IR group). β-Actin served as the loading control. The data represent at least three independent experiments. (f) Representative flow cytometric histograms at 2 days post transfection+6 h post IR. The percentage of cells in each stage of cell cycle was calculated using ModFit. Graphic representation of these data is shown on the right hand panel. Results are presented as mean±S.E.M. (n=3). T-test analysis was carried out to assess the differences in gene expression between the control and CDK1 siRNA group, *P<0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4260724&req=5

fig4: Knockdown of CDK1 induces the activation of γH2.AX and downregulation of CHK2 expression in hESC. (a) Confocal microscopy analysis showing the presence of γH2 A.X foci in hESC at 1, 2 and 3 days post transfection of control and CDK1 siRNAs. Phosphorylated histone H2A.X (γ -H2A.X foci) is shown as white dots. Chromatin is stained with DAPI (blue). Scale bar=10 μm, D=day. Images are representative of at least three independent experiments. Percentage of γ-H2A.X-positive cells is shown at the bottom. (b) Graphic representation of the average number of γH2A.X foci per nucleus in hESC during 3-day time course post CDK1 and control siRNAs transfections. Data areshown as average±S.E.M., n=3. (c) Flow cytometric analysis of γ-H2A.X on the second day post transfection and 16 h after administration of IR. Data are shown as average±S.E.M., n=3. (d) Representative images of four repeats of western blot analysis for CHK1 and CHK2 expression up to 96 h post transfection of siRNAS. β-Actin was used as the loading control. (e) Impacts of CDK1 inhibition on the regulation of key factors involved in G2 checkpoint activation analysed by western blotting at day 2 post transfection and after 6 h post IR on the same day (shown in the figure as IR group). β-Actin served as the loading control. The data represent at least three independent experiments. (f) Representative flow cytometric histograms at 2 days post transfection+6 h post IR. The percentage of cells in each stage of cell cycle was calculated using ModFit. Graphic representation of these data is shown on the right hand panel. Results are presented as mean±S.E.M. (n=3). T-test analysis was carried out to assess the differences in gene expression between the control and CDK1 siRNA group, *P<0.05

Mentions: Under normoxic culture conditions, hESC accumulate a small number of DSBs (Figures 4a and b;3). However, upon CDK1 knockdown, we observed a significant increase both in the percentage of hESC with DSBs (Figure 4a) and the number of DSB foci per cell (Figures 4a and b), corroborating data previously published in mammalian somatic cells.24, 25 To further confirm this, we carried out γ-H2A.X detection by flow cytometric analysis (Figure 4c). It is evident that upon CDK1 downregulation, there is a significant increase in the number of accumulated DSBs (Figures 4a and c); however, this is not accompanied by increased apoptosis in the CDK1 siRNA group when compared with the control (Figure 7d). When analysed under the context of cell cycle regulation, 59% of S-phase cells were positive for γ-H2A.X, in contrast to control cells which showed only 6.7% of S-phase cells with γ-H2A.X foci (Figure 4c). We repeated the same analysis at 16 h after administrating ionising radiation (IR) (2Gy) to hESC (Figure 4c). Although there is a slightly higher DSBs accumulation in hESC with reduced CDK1 expression under IR when compared with the same group under non-IR conditions (Figure 4c), the pattern is similar with the majority of cells with γ-H2A.X in the S phase of the cell cycle.


CDK1 plays an important role in the maintenance of pluripotency and genomic stability in human pluripotent stem cells.

Neganova I, Tilgner K, Buskin A, Paraskevopoulou I, Atkinson SP, Peberdy D, Passos JF, Lako M - Cell Death Dis (2014)

Knockdown of CDK1 induces the activation of γH2.AX and downregulation of CHK2 expression in hESC. (a) Confocal microscopy analysis showing the presence of γH2 A.X foci in hESC at 1, 2 and 3 days post transfection of control and CDK1 siRNAs. Phosphorylated histone H2A.X (γ -H2A.X foci) is shown as white dots. Chromatin is stained with DAPI (blue). Scale bar=10 μm, D=day. Images are representative of at least three independent experiments. Percentage of γ-H2A.X-positive cells is shown at the bottom. (b) Graphic representation of the average number of γH2A.X foci per nucleus in hESC during 3-day time course post CDK1 and control siRNAs transfections. Data areshown as average±S.E.M., n=3. (c) Flow cytometric analysis of γ-H2A.X on the second day post transfection and 16 h after administration of IR. Data are shown as average±S.E.M., n=3. (d) Representative images of four repeats of western blot analysis for CHK1 and CHK2 expression up to 96 h post transfection of siRNAS. β-Actin was used as the loading control. (e) Impacts of CDK1 inhibition on the regulation of key factors involved in G2 checkpoint activation analysed by western blotting at day 2 post transfection and after 6 h post IR on the same day (shown in the figure as IR group). β-Actin served as the loading control. The data represent at least three independent experiments. (f) Representative flow cytometric histograms at 2 days post transfection+6 h post IR. The percentage of cells in each stage of cell cycle was calculated using ModFit. Graphic representation of these data is shown on the right hand panel. Results are presented as mean±S.E.M. (n=3). T-test analysis was carried out to assess the differences in gene expression between the control and CDK1 siRNA group, *P<0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260724&req=5

fig4: Knockdown of CDK1 induces the activation of γH2.AX and downregulation of CHK2 expression in hESC. (a) Confocal microscopy analysis showing the presence of γH2 A.X foci in hESC at 1, 2 and 3 days post transfection of control and CDK1 siRNAs. Phosphorylated histone H2A.X (γ -H2A.X foci) is shown as white dots. Chromatin is stained with DAPI (blue). Scale bar=10 μm, D=day. Images are representative of at least three independent experiments. Percentage of γ-H2A.X-positive cells is shown at the bottom. (b) Graphic representation of the average number of γH2A.X foci per nucleus in hESC during 3-day time course post CDK1 and control siRNAs transfections. Data areshown as average±S.E.M., n=3. (c) Flow cytometric analysis of γ-H2A.X on the second day post transfection and 16 h after administration of IR. Data are shown as average±S.E.M., n=3. (d) Representative images of four repeats of western blot analysis for CHK1 and CHK2 expression up to 96 h post transfection of siRNAS. β-Actin was used as the loading control. (e) Impacts of CDK1 inhibition on the regulation of key factors involved in G2 checkpoint activation analysed by western blotting at day 2 post transfection and after 6 h post IR on the same day (shown in the figure as IR group). β-Actin served as the loading control. The data represent at least three independent experiments. (f) Representative flow cytometric histograms at 2 days post transfection+6 h post IR. The percentage of cells in each stage of cell cycle was calculated using ModFit. Graphic representation of these data is shown on the right hand panel. Results are presented as mean±S.E.M. (n=3). T-test analysis was carried out to assess the differences in gene expression between the control and CDK1 siRNA group, *P<0.05
Mentions: Under normoxic culture conditions, hESC accumulate a small number of DSBs (Figures 4a and b;3). However, upon CDK1 knockdown, we observed a significant increase both in the percentage of hESC with DSBs (Figure 4a) and the number of DSB foci per cell (Figures 4a and b), corroborating data previously published in mammalian somatic cells.24, 25 To further confirm this, we carried out γ-H2A.X detection by flow cytometric analysis (Figure 4c). It is evident that upon CDK1 downregulation, there is a significant increase in the number of accumulated DSBs (Figures 4a and c); however, this is not accompanied by increased apoptosis in the CDK1 siRNA group when compared with the control (Figure 7d). When analysed under the context of cell cycle regulation, 59% of S-phase cells were positive for γ-H2A.X, in contrast to control cells which showed only 6.7% of S-phase cells with γ-H2A.X foci (Figure 4c). We repeated the same analysis at 16 h after administrating ionising radiation (IR) (2Gy) to hESC (Figure 4c). Although there is a slightly higher DSBs accumulation in hESC with reduced CDK1 expression under IR when compared with the same group under non-IR conditions (Figure 4c), the pattern is similar with the majority of cells with γ-H2A.X in the S phase of the cell cycle.

Bottom Line: Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process.On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation.Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetic Medicine, Newcastle University, International Centre for Life, Newcastle upon Tyne NE1 3BZ, UK.

ABSTRACT
Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are characterised by an unusual and tightly regulated cell cycle that has been shown to be important for the maintenance of a pluripotent phenotype. Cyclin-dependant kinase 1 (CDK1) is a key player in cell cycle regulation and particularly mitosis; however, its role has not been studied previously in hESC and hiPSC. To investigate the impacts of CDK1 downregulation, we performed RNA interference studies which in addition to expected mitotic deficiencies revealed a large range of additional phenotypes related to maintenance of pluripotency, ability to repair double strand breaks (DSBs) and commitment to apoptosis. Downregulation of CDK1 led to the loss of typical pluripotent stem cell morphology, downregulation of pluripotency markers and upregulation of a large number of differentiation markers. In addition, human pluripotent stem cells with reduced CDK1 expression accumulated a higher number of DSBs were unable to activate CHK2 expression and could not maintain G2/M arrest upon exposure to ionising radiation. CDK1 downregulation led to the accumulation of cells with abnormal numbers of mitotic organelles, multiple chromosomal abnormalities and polyploidy. Furthermore, such cells demonstrated an inability to execute apoptosis under normal culture conditions, despite a significant increase in the expression of active PARP1, resulting in tolerance and very likely further propagation of genomic instabilities and ensuing of differentiation process. On the contrary, apoptosis but not differentiation, was the preferred route for such cells when they were subjected to ionising radiation. Together these data suggest that CDK1 regulates multiple events in human pluripotent stem cells ranging from regulation of mitosis, G2/M checkpoint maintenance, execution of apoptosis, maintenance of pluripotency and genomic stability.

Show MeSH
Related in: MedlinePlus