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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.

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Knockdown of CDK1 in hESC results in PARP1 activation, but impaired apoptosis. (a) Western blot analysis showing downregulation of pro-survival proteins and activation of pro-apoptotic markers at 2 days post transfection of hESC with control and CDK1 siRNAs in the absence and presence of IR (extracts were collected 16 h post IR). Images are representative of at least three independent experiments. β-Actin served as the loading control. (b) Graphical representation of flow cytometric analysis for caspase 9 and 3 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. Results are presented as mean±S.E.M. (n=3). (c) Graphical representation of flow cytometric analysis for PARP1 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. 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. (d) Graphical representation of TUNEL analysis at each stage of cell cycle at 2 days post transfection with control and CDK1 siRNA under normal culture conditions as well as IR. Results are presented as mean±S.E.M. (n=3), t-test analysis was carried out to assess differences in gene expression between the control and CDK1 siRNA group, *P<0.05
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fig7: Knockdown of CDK1 in hESC results in PARP1 activation, but impaired apoptosis. (a) Western blot analysis showing downregulation of pro-survival proteins and activation of pro-apoptotic markers at 2 days post transfection of hESC with control and CDK1 siRNAs in the absence and presence of IR (extracts were collected 16 h post IR). Images are representative of at least three independent experiments. β-Actin served as the loading control. (b) Graphical representation of flow cytometric analysis for caspase 9 and 3 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. Results are presented as mean±S.E.M. (n=3). (c) Graphical representation of flow cytometric analysis for PARP1 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. 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. (d) Graphical representation of TUNEL analysis at each stage of cell cycle at 2 days post transfection with control and CDK1 siRNA under normal culture conditions as well as IR. Results are presented as mean±S.E.M. (n=3), t-test analysis was carried out to assess differences in gene expression between the control and CDK1 siRNA group, *P<0.05

Mentions: The CDK1/CYCLIN B1 complex is able to interact and phosphorylate both pro- and anti-apoptotic proteins such as Bad, Caspase 9, Caspase 8, Caspase 2, Caspase3, Bcl-2, Bcl-xl, Mcl-1 and Survivin.39, 40, 41, 42, 43 We performed western blotting analysis and observed that the expression of anti-apoptotic/survival proteins (such as Survivin, BCL2, BCL-xL) was downregulated in the CDK1 siRNA group (Figure 7a). In addition, the expression of pro-apoptotic protein BAD was increased upon CDK1 downregulation. At the same time, the expression of active BAX (pro-apoptotic protein) was downregulated (Figure 7a); however, this is more likely to reflect the loss of pluripotency in hESC upon CDK1 downregulation, as it has recently been shown that active BAX is only detected in undifferentiated hESC.44 We also performed flow cytometric analysis for expression of CASPASE 3 and 9, because it has been shown that the loss of phosphorylation on Thr34 of SURVIVIN can result in the disassociation of the Survivin-Caspase 9 complex and initiation of caspase 9-dependent apoptosis; however, we were unable to detect significant changes between the control and CDK1 siRNA groups (Figure 7b). Another important apoptosis executor is PARP1 (poly ADP ribose polymerase 1), whose activity increases substantially in response to cellular stress.45 Flow cytometric analysis indicated significant upregulation of active PARP1 upon CDK1 downregulation (Figure 7c); however, TUNEL analysis did not reveal significant differences in the number of apoptotic cells, suggesting an inability to commit to apoptosis in the absence of CDK1 (Figure 7d). Similar data were obtained in hiPSCs (Supplementary Figure 5).


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 in hESC results in PARP1 activation, but impaired apoptosis. (a) Western blot analysis showing downregulation of pro-survival proteins and activation of pro-apoptotic markers at 2 days post transfection of hESC with control and CDK1 siRNAs in the absence and presence of IR (extracts were collected 16 h post IR). Images are representative of at least three independent experiments. β-Actin served as the loading control. (b) Graphical representation of flow cytometric analysis for caspase 9 and 3 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. Results are presented as mean±S.E.M. (n=3). (c) Graphical representation of flow cytometric analysis for PARP1 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. 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. (d) Graphical representation of TUNEL analysis at each stage of cell cycle at 2 days post transfection with control and CDK1 siRNA under normal culture conditions as well as IR. Results are presented as mean±S.E.M. (n=3), t-test analysis was carried out to assess differences in gene expression between the control and CDK1 siRNA group, *P<0.05
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fig7: Knockdown of CDK1 in hESC results in PARP1 activation, but impaired apoptosis. (a) Western blot analysis showing downregulation of pro-survival proteins and activation of pro-apoptotic markers at 2 days post transfection of hESC with control and CDK1 siRNAs in the absence and presence of IR (extracts were collected 16 h post IR). Images are representative of at least three independent experiments. β-Actin served as the loading control. (b) Graphical representation of flow cytometric analysis for caspase 9 and 3 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. Results are presented as mean±S.E.M. (n=3). (c) Graphical representation of flow cytometric analysis for PARP1 activation in hESC at 2 days post transfection with control and CDK1 siRNAs. 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. (d) Graphical representation of TUNEL analysis at each stage of cell cycle at 2 days post transfection with control and CDK1 siRNA under normal culture conditions as well as IR. Results are presented as mean±S.E.M. (n=3), t-test analysis was carried out to assess differences in gene expression between the control and CDK1 siRNA group, *P<0.05
Mentions: The CDK1/CYCLIN B1 complex is able to interact and phosphorylate both pro- and anti-apoptotic proteins such as Bad, Caspase 9, Caspase 8, Caspase 2, Caspase3, Bcl-2, Bcl-xl, Mcl-1 and Survivin.39, 40, 41, 42, 43 We performed western blotting analysis and observed that the expression of anti-apoptotic/survival proteins (such as Survivin, BCL2, BCL-xL) was downregulated in the CDK1 siRNA group (Figure 7a). In addition, the expression of pro-apoptotic protein BAD was increased upon CDK1 downregulation. At the same time, the expression of active BAX (pro-apoptotic protein) was downregulated (Figure 7a); however, this is more likely to reflect the loss of pluripotency in hESC upon CDK1 downregulation, as it has recently been shown that active BAX is only detected in undifferentiated hESC.44 We also performed flow cytometric analysis for expression of CASPASE 3 and 9, because it has been shown that the loss of phosphorylation on Thr34 of SURVIVIN can result in the disassociation of the Survivin-Caspase 9 complex and initiation of caspase 9-dependent apoptosis; however, we were unable to detect significant changes between the control and CDK1 siRNA groups (Figure 7b). Another important apoptosis executor is PARP1 (poly ADP ribose polymerase 1), whose activity increases substantially in response to cellular stress.45 Flow cytometric analysis indicated significant upregulation of active PARP1 upon CDK1 downregulation (Figure 7c); however, TUNEL analysis did not reveal significant differences in the number of apoptotic cells, suggesting an inability to commit to apoptosis in the absence of CDK1 (Figure 7d). Similar data were obtained in hiPSCs (Supplementary Figure 5).

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