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Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ - J. Cell Biol. (1999)

Bottom Line: Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions.Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade.Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.

ABSTRACT
Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

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Cytochrome c initiates apoptotic changes in Jurkat  cell–free extracts. Rat liver nuclei were incubated at 37°C in postnuclear extracts of Jurkat cells, prepared as described in Materials and Methods, in the presence or absence of 50 μg/ml of bovine heart cytochrome c. (A) At the indicated times, 2-μl aliquots  of extract were removed for analysis of nuclear morphology by  Hoescht 33342 staining. Nuclei were scored as apoptotic if they  exhibited chromatin condensation and nuclear fragmentation  characteristic of apoptosis. Each data point represents counts on  300 nuclei. Triplicate determinations (± SEM) are shown from a  representative experiment. (B) At the indicated times, samples  of each cell-free reaction were taken for SDS-PAGE, followed  by Western blotting and probing for the indicated proteins.
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Figure 1: Cytochrome c initiates apoptotic changes in Jurkat cell–free extracts. Rat liver nuclei were incubated at 37°C in postnuclear extracts of Jurkat cells, prepared as described in Materials and Methods, in the presence or absence of 50 μg/ml of bovine heart cytochrome c. (A) At the indicated times, 2-μl aliquots of extract were removed for analysis of nuclear morphology by Hoescht 33342 staining. Nuclei were scored as apoptotic if they exhibited chromatin condensation and nuclear fragmentation characteristic of apoptosis. Each data point represents counts on 300 nuclei. Triplicate determinations (± SEM) are shown from a representative experiment. (B) At the indicated times, samples of each cell-free reaction were taken for SDS-PAGE, followed by Western blotting and probing for the indicated proteins.

Mentions: Addition of purified cytochrome c to postnuclear (15,000 g; S15) extracts of Jurkat T lymphoblastoid cells was sufficient to initiate the whole spectrum of events characteristic of apoptosis in these extracts. Nuclei incubated in the extracts in the presence of cytochrome c rapidly exhibited apoptotic features (chromatin margination and nuclear fragmentation; Fig. 1 A) and chromatin also underwent fragmentation into ∼200-bp multiples (data not shown). Proteolysis of several caspase substrates (α-fodrin, U1sn- RNP, PARP) was also observed in response to cytochrome c (Fig. 1 B). Interestingly, although previous reports have shown that addition of dATP (or ATP) to cell extracts is required for the proapoptotic activities of cytochrome c, many extracts did not require addition of exogenous nucleotide triphosphates, presumably due to sufficiently high levels of ATP or dATP endogenous to these extracts.


Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ - J. Cell Biol. (1999)

Cytochrome c initiates apoptotic changes in Jurkat  cell–free extracts. Rat liver nuclei were incubated at 37°C in postnuclear extracts of Jurkat cells, prepared as described in Materials and Methods, in the presence or absence of 50 μg/ml of bovine heart cytochrome c. (A) At the indicated times, 2-μl aliquots  of extract were removed for analysis of nuclear morphology by  Hoescht 33342 staining. Nuclei were scored as apoptotic if they  exhibited chromatin condensation and nuclear fragmentation  characteristic of apoptosis. Each data point represents counts on  300 nuclei. Triplicate determinations (± SEM) are shown from a  representative experiment. (B) At the indicated times, samples  of each cell-free reaction were taken for SDS-PAGE, followed  by Western blotting and probing for the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132895&req=5

Figure 1: Cytochrome c initiates apoptotic changes in Jurkat cell–free extracts. Rat liver nuclei were incubated at 37°C in postnuclear extracts of Jurkat cells, prepared as described in Materials and Methods, in the presence or absence of 50 μg/ml of bovine heart cytochrome c. (A) At the indicated times, 2-μl aliquots of extract were removed for analysis of nuclear morphology by Hoescht 33342 staining. Nuclei were scored as apoptotic if they exhibited chromatin condensation and nuclear fragmentation characteristic of apoptosis. Each data point represents counts on 300 nuclei. Triplicate determinations (± SEM) are shown from a representative experiment. (B) At the indicated times, samples of each cell-free reaction were taken for SDS-PAGE, followed by Western blotting and probing for the indicated proteins.
Mentions: Addition of purified cytochrome c to postnuclear (15,000 g; S15) extracts of Jurkat T lymphoblastoid cells was sufficient to initiate the whole spectrum of events characteristic of apoptosis in these extracts. Nuclei incubated in the extracts in the presence of cytochrome c rapidly exhibited apoptotic features (chromatin margination and nuclear fragmentation; Fig. 1 A) and chromatin also underwent fragmentation into ∼200-bp multiples (data not shown). Proteolysis of several caspase substrates (α-fodrin, U1sn- RNP, PARP) was also observed in response to cytochrome c (Fig. 1 B). Interestingly, although previous reports have shown that addition of dATP (or ATP) to cell extracts is required for the proapoptotic activities of cytochrome c, many extracts did not require addition of exogenous nucleotide triphosphates, presumably due to sufficiently high levels of ATP or dATP endogenous to these extracts.

Bottom Line: Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions.Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade.Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland.

ABSTRACT
Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

Show MeSH
Related in: MedlinePlus