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The cyclin-dependent kinase Cdk2 regulates thymocyte apoptosis.

Hakem A, Sasaki T, Kozieradzki I, Penninger JM - J. Exp. Med. (1999)

Bottom Line: Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle").Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein.These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The Amgen Institute, Department of Medical Biophysics, University of Toronto, Ontario, Canada M5G 2C1.

ABSTRACT
Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle"). Here we show that in noncycling developing thymocytes, the cyclin- dependent kinase Cdk2 is activated in response to all specific and nonspecific apoptotic stimuli tested, including peptide-specific thymocyte apoptosis. Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein. Inhibition of Cdk2 completely protected thymocytes from apoptosis, mitochondrial changes, and caspase activation. These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

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Cdk2 acts upstream of mitochondrial permeability transition, caspase activation, and Rb cleavage. (A) Mitochondrial permeability transition (ΔΨm disruption). Thymocytes were cultured for 5 h in medium alone, or in medium containing dexamethasone  (1 μM) or anti-CD95 (1 μg/ml) in the presence or absence of roscovitine (50 μM). Cells  induced to undergo apoptosis manifest an early reduction in the incorporation of ΔΨm-sensitive dyes, indicating a disruption of ΔΨm. For DiOC6(3) staining, 106 thymocytes  were incubated with DiOC6(3) (final concentration 20 nM in PBS) for 20 min at 37°C.  DiOC6(3) staining was analyzed immediately using a FACSCalibur™ flow cytometer.  One result representative of three independent experiments is shown. (B) Thymocytes  were stimulated with the indicated death stimuli for 5 h, and proteolytic activation of  caspase 3 (Cpp32) was assessed by Western blotting. The anti–caspase 3 Ab recognizes both the intact caspase 3 molecule (procaspase 3) and its cleaved  17-kD active form (p17). Addition of roscovitine inhibits caspase 3 cleavage in response to dexamethasone (right) and in response to all other death stimuli tested, except for anti-CD95. (C) Thymocytes were stimulated with the indicated stimuli, and the proteolytic activation of caspase 2 (Nedd2) was assessed by Western blotting. The addition of roscovitine (Rosco) after irradiation blocked caspase 2 cleavage into a p14 fragment. (D) The retinoblastoma  protein Rb is proteolytically processed in response to all cell death stimuli. Inhibition of Cdk2 blocked Rb cleavage. Thymocytes were stimulated with  dexamethasone and γ-irradiation in the presence or absence of roscovitine (Rosco, 50 μM). After 5 h, cells were harvested and lysed, and the status of  Rb was assessed by Western blotting. Rb-reactive Ab recognizes aa 300–380 of Rb. As previously described in cell lines (reference 53), Rb was cleaved  of the COOH-terminal end in apoptotic thymocytes in response to all apoptotic stimuli.
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Figure 4: Cdk2 acts upstream of mitochondrial permeability transition, caspase activation, and Rb cleavage. (A) Mitochondrial permeability transition (ΔΨm disruption). Thymocytes were cultured for 5 h in medium alone, or in medium containing dexamethasone (1 μM) or anti-CD95 (1 μg/ml) in the presence or absence of roscovitine (50 μM). Cells induced to undergo apoptosis manifest an early reduction in the incorporation of ΔΨm-sensitive dyes, indicating a disruption of ΔΨm. For DiOC6(3) staining, 106 thymocytes were incubated with DiOC6(3) (final concentration 20 nM in PBS) for 20 min at 37°C. DiOC6(3) staining was analyzed immediately using a FACSCalibur™ flow cytometer. One result representative of three independent experiments is shown. (B) Thymocytes were stimulated with the indicated death stimuli for 5 h, and proteolytic activation of caspase 3 (Cpp32) was assessed by Western blotting. The anti–caspase 3 Ab recognizes both the intact caspase 3 molecule (procaspase 3) and its cleaved 17-kD active form (p17). Addition of roscovitine inhibits caspase 3 cleavage in response to dexamethasone (right) and in response to all other death stimuli tested, except for anti-CD95. (C) Thymocytes were stimulated with the indicated stimuli, and the proteolytic activation of caspase 2 (Nedd2) was assessed by Western blotting. The addition of roscovitine (Rosco) after irradiation blocked caspase 2 cleavage into a p14 fragment. (D) The retinoblastoma protein Rb is proteolytically processed in response to all cell death stimuli. Inhibition of Cdk2 blocked Rb cleavage. Thymocytes were stimulated with dexamethasone and γ-irradiation in the presence or absence of roscovitine (Rosco, 50 μM). After 5 h, cells were harvested and lysed, and the status of Rb was assessed by Western blotting. Rb-reactive Ab recognizes aa 300–380 of Rb. As previously described in cell lines (reference 53), Rb was cleaved of the COOH-terminal end in apoptotic thymocytes in response to all apoptotic stimuli.

Mentions: To assess whether Cdk2 acts upstream or downstream of mitochondrial events, we examined changes in ΔΨm using cytometry and the fluorochromic dye, DiOC6(3). Thymocytes were stimulated either with dexamethasone or anti-CD95, and the mitochondria changes of ΔΨm were assessed at different time points. The first changes in thymocyte ΔΨm were observed 2 h after dexamethasone treatment, and ΔΨm was significantly disrupted after 5 h (Fig. 4 A). Addition of Cdk2 inhibitors blocked dexamethasone-induced losses of ΔΨm (Fig. 4 A). CD95-mediated ΔΨm disruption and apoptosis still occurred in the presence of Cdk2 inhibitors (Figs. 2 A and 4 A), implying that Cdk2 inhibition per se does not interfere with opening of mitochondrial pores. Since ΔΨm is regulated by Bcl-2 family members (43, 49), we also tested Cdk2 activation in Bcl-2 Tg thymocytes (31, 50). Although overexpression of Bcl-2 protected thymocytes from dexamethasone- and irradiation-induced cell death and disruption of ΔΨm (31, 50), Cdk2 was still activated in Bcl-2 Tg thymocytes in response to these apoptotic stimuli (not shown).


The cyclin-dependent kinase Cdk2 regulates thymocyte apoptosis.

Hakem A, Sasaki T, Kozieradzki I, Penninger JM - J. Exp. Med. (1999)

Cdk2 acts upstream of mitochondrial permeability transition, caspase activation, and Rb cleavage. (A) Mitochondrial permeability transition (ΔΨm disruption). Thymocytes were cultured for 5 h in medium alone, or in medium containing dexamethasone  (1 μM) or anti-CD95 (1 μg/ml) in the presence or absence of roscovitine (50 μM). Cells  induced to undergo apoptosis manifest an early reduction in the incorporation of ΔΨm-sensitive dyes, indicating a disruption of ΔΨm. For DiOC6(3) staining, 106 thymocytes  were incubated with DiOC6(3) (final concentration 20 nM in PBS) for 20 min at 37°C.  DiOC6(3) staining was analyzed immediately using a FACSCalibur™ flow cytometer.  One result representative of three independent experiments is shown. (B) Thymocytes  were stimulated with the indicated death stimuli for 5 h, and proteolytic activation of  caspase 3 (Cpp32) was assessed by Western blotting. The anti–caspase 3 Ab recognizes both the intact caspase 3 molecule (procaspase 3) and its cleaved  17-kD active form (p17). Addition of roscovitine inhibits caspase 3 cleavage in response to dexamethasone (right) and in response to all other death stimuli tested, except for anti-CD95. (C) Thymocytes were stimulated with the indicated stimuli, and the proteolytic activation of caspase 2 (Nedd2) was assessed by Western blotting. The addition of roscovitine (Rosco) after irradiation blocked caspase 2 cleavage into a p14 fragment. (D) The retinoblastoma  protein Rb is proteolytically processed in response to all cell death stimuli. Inhibition of Cdk2 blocked Rb cleavage. Thymocytes were stimulated with  dexamethasone and γ-irradiation in the presence or absence of roscovitine (Rosco, 50 μM). After 5 h, cells were harvested and lysed, and the status of  Rb was assessed by Western blotting. Rb-reactive Ab recognizes aa 300–380 of Rb. As previously described in cell lines (reference 53), Rb was cleaved  of the COOH-terminal end in apoptotic thymocytes in response to all apoptotic stimuli.
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Figure 4: Cdk2 acts upstream of mitochondrial permeability transition, caspase activation, and Rb cleavage. (A) Mitochondrial permeability transition (ΔΨm disruption). Thymocytes were cultured for 5 h in medium alone, or in medium containing dexamethasone (1 μM) or anti-CD95 (1 μg/ml) in the presence or absence of roscovitine (50 μM). Cells induced to undergo apoptosis manifest an early reduction in the incorporation of ΔΨm-sensitive dyes, indicating a disruption of ΔΨm. For DiOC6(3) staining, 106 thymocytes were incubated with DiOC6(3) (final concentration 20 nM in PBS) for 20 min at 37°C. DiOC6(3) staining was analyzed immediately using a FACSCalibur™ flow cytometer. One result representative of three independent experiments is shown. (B) Thymocytes were stimulated with the indicated death stimuli for 5 h, and proteolytic activation of caspase 3 (Cpp32) was assessed by Western blotting. The anti–caspase 3 Ab recognizes both the intact caspase 3 molecule (procaspase 3) and its cleaved 17-kD active form (p17). Addition of roscovitine inhibits caspase 3 cleavage in response to dexamethasone (right) and in response to all other death stimuli tested, except for anti-CD95. (C) Thymocytes were stimulated with the indicated stimuli, and the proteolytic activation of caspase 2 (Nedd2) was assessed by Western blotting. The addition of roscovitine (Rosco) after irradiation blocked caspase 2 cleavage into a p14 fragment. (D) The retinoblastoma protein Rb is proteolytically processed in response to all cell death stimuli. Inhibition of Cdk2 blocked Rb cleavage. Thymocytes were stimulated with dexamethasone and γ-irradiation in the presence or absence of roscovitine (Rosco, 50 μM). After 5 h, cells were harvested and lysed, and the status of Rb was assessed by Western blotting. Rb-reactive Ab recognizes aa 300–380 of Rb. As previously described in cell lines (reference 53), Rb was cleaved of the COOH-terminal end in apoptotic thymocytes in response to all apoptotic stimuli.
Mentions: To assess whether Cdk2 acts upstream or downstream of mitochondrial events, we examined changes in ΔΨm using cytometry and the fluorochromic dye, DiOC6(3). Thymocytes were stimulated either with dexamethasone or anti-CD95, and the mitochondria changes of ΔΨm were assessed at different time points. The first changes in thymocyte ΔΨm were observed 2 h after dexamethasone treatment, and ΔΨm was significantly disrupted after 5 h (Fig. 4 A). Addition of Cdk2 inhibitors blocked dexamethasone-induced losses of ΔΨm (Fig. 4 A). CD95-mediated ΔΨm disruption and apoptosis still occurred in the presence of Cdk2 inhibitors (Figs. 2 A and 4 A), implying that Cdk2 inhibition per se does not interfere with opening of mitochondrial pores. Since ΔΨm is regulated by Bcl-2 family members (43, 49), we also tested Cdk2 activation in Bcl-2 Tg thymocytes (31, 50). Although overexpression of Bcl-2 protected thymocytes from dexamethasone- and irradiation-induced cell death and disruption of ΔΨm (31, 50), Cdk2 was still activated in Bcl-2 Tg thymocytes in response to these apoptotic stimuli (not shown).

Bottom Line: Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle").Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein.These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The Amgen Institute, Department of Medical Biophysics, University of Toronto, Ontario, Canada M5G 2C1.

ABSTRACT
Aberrant activation of cell cycle molecules has been postulated to play a role in apoptosis ("catastrophic cell cycle"). Here we show that in noncycling developing thymocytes, the cyclin- dependent kinase Cdk2 is activated in response to all specific and nonspecific apoptotic stimuli tested, including peptide-specific thymocyte apoptosis. Cdk2 was found to function upstream of the tumor suppressor p53, transactivation of the death promoter Bax, alterations of mitochondrial permeability, Bcl-2, caspase activation, and caspase-dependent proteolytic cleavage of the retinoblastoma protein. Inhibition of Cdk2 completely protected thymocytes from apoptosis, mitochondrial changes, and caspase activation. These data provide the first evidence that Cdk2 activity is crucial for the induction of thymocyte apoptosis.

Show MeSH
Related in: MedlinePlus