Limits...
The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells.

Dorstyn L, Mills K, Lazebnik Y, Kumar S - J. Cell Biol. (2004)

Bottom Line: Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells.In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells.Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Hanson Institute, Adelaide, Australia 5000.

ABSTRACT
In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

Show MeSH

Related in: MedlinePlus

DC3 or DC4 expression does not affect cell death or DRONC processing. (A) BG2 cells were transfected with DC3-HA or DC4-HA and protein expression confirmed by immunoblotting with HA antibody. Immunoblotting for DRONC and DRICE showed a lack of processing of caspases in cells overexpressing DC3 or DC4. (B) DC3-HA or DC4-HA transfected BG2 cells were treated with cycloheximide for the indicated times and cell death was determined by Trypan blue staining and is represented as mean ± SEM from three independent experiments. (C) Caspase activity in untreated (−) or CHX-treated (+) BG2 cells overexpressing DC3 or DC4. Data (mean ± SEM) derived from three experiments. (D) Cells were fractionated by differential centrifugation to separate heavy membrane (pellet, containing mitochondria) and cytosol. Fractions were immunoblotted for HA, cytochrome c, or DRONC, as indicated. Cytochrome c antibody detects both transfected and endogenous protein as seen by a doublet. Relative molecular masses of the proteins in kilodaltons are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172470&req=5

fig2: DC3 or DC4 expression does not affect cell death or DRONC processing. (A) BG2 cells were transfected with DC3-HA or DC4-HA and protein expression confirmed by immunoblotting with HA antibody. Immunoblotting for DRONC and DRICE showed a lack of processing of caspases in cells overexpressing DC3 or DC4. (B) DC3-HA or DC4-HA transfected BG2 cells were treated with cycloheximide for the indicated times and cell death was determined by Trypan blue staining and is represented as mean ± SEM from three independent experiments. (C) Caspase activity in untreated (−) or CHX-treated (+) BG2 cells overexpressing DC3 or DC4. Data (mean ± SEM) derived from three experiments. (D) Cells were fractionated by differential centrifugation to separate heavy membrane (pellet, containing mitochondria) and cytosol. Fractions were immunoblotted for HA, cytochrome c, or DRONC, as indicated. Cytochrome c antibody detects both transfected and endogenous protein as seen by a doublet. Relative molecular masses of the proteins in kilodaltons are shown.

Mentions: Although Drosophila cytochrome c proteins are not required for caspase activation and apoptosis, it is possible that they can induce caspase activation but can be substituted by some redundant activity. We tested this possibility in cultured cells and in cell-free systems. One approach was to test whether overexpression of the cytochromes in BG2 cells had any effect on cell death. We generated BG2 cell lines that expressed DC3 or DC4 protein fused with the HA epitope tag and confirmed expression of proteins by immunoblotting (Fig. 2 A). Ectopic expression of DC3 or DC4 did not induce processing of DRONC or DRICE. When these cells were treated with cycloheximide, we found that cells expressing exogenous DC3 or DC4 underwent apoptosis at the same rate as the parental cells (Fig. 2 B) and showed caspase activity comparable to controls (Fig. 2 C).


The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells.

Dorstyn L, Mills K, Lazebnik Y, Kumar S - J. Cell Biol. (2004)

DC3 or DC4 expression does not affect cell death or DRONC processing. (A) BG2 cells were transfected with DC3-HA or DC4-HA and protein expression confirmed by immunoblotting with HA antibody. Immunoblotting for DRONC and DRICE showed a lack of processing of caspases in cells overexpressing DC3 or DC4. (B) DC3-HA or DC4-HA transfected BG2 cells were treated with cycloheximide for the indicated times and cell death was determined by Trypan blue staining and is represented as mean ± SEM from three independent experiments. (C) Caspase activity in untreated (−) or CHX-treated (+) BG2 cells overexpressing DC3 or DC4. Data (mean ± SEM) derived from three experiments. (D) Cells were fractionated by differential centrifugation to separate heavy membrane (pellet, containing mitochondria) and cytosol. Fractions were immunoblotted for HA, cytochrome c, or DRONC, as indicated. Cytochrome c antibody detects both transfected and endogenous protein as seen by a doublet. Relative molecular masses of the proteins in kilodaltons are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172470&req=5

fig2: DC3 or DC4 expression does not affect cell death or DRONC processing. (A) BG2 cells were transfected with DC3-HA or DC4-HA and protein expression confirmed by immunoblotting with HA antibody. Immunoblotting for DRONC and DRICE showed a lack of processing of caspases in cells overexpressing DC3 or DC4. (B) DC3-HA or DC4-HA transfected BG2 cells were treated with cycloheximide for the indicated times and cell death was determined by Trypan blue staining and is represented as mean ± SEM from three independent experiments. (C) Caspase activity in untreated (−) or CHX-treated (+) BG2 cells overexpressing DC3 or DC4. Data (mean ± SEM) derived from three experiments. (D) Cells were fractionated by differential centrifugation to separate heavy membrane (pellet, containing mitochondria) and cytosol. Fractions were immunoblotted for HA, cytochrome c, or DRONC, as indicated. Cytochrome c antibody detects both transfected and endogenous protein as seen by a doublet. Relative molecular masses of the proteins in kilodaltons are shown.
Mentions: Although Drosophila cytochrome c proteins are not required for caspase activation and apoptosis, it is possible that they can induce caspase activation but can be substituted by some redundant activity. We tested this possibility in cultured cells and in cell-free systems. One approach was to test whether overexpression of the cytochromes in BG2 cells had any effect on cell death. We generated BG2 cell lines that expressed DC3 or DC4 protein fused with the HA epitope tag and confirmed expression of proteins by immunoblotting (Fig. 2 A). Ectopic expression of DC3 or DC4 did not induce processing of DRONC or DRICE. When these cells were treated with cycloheximide, we found that cells expressing exogenous DC3 or DC4 underwent apoptosis at the same rate as the parental cells (Fig. 2 B) and showed caspase activity comparable to controls (Fig. 2 C).

Bottom Line: Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells.In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells.Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Hanson Institute, Adelaide, Australia 5000.

ABSTRACT
In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

Show MeSH
Related in: MedlinePlus