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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.

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DRONC and DRICE activation can occur in the absence of dc3 and dc4 during Drosophila development. (A) Lysates were prepared at 4 h after puparium formation, from wild-type (W1118), bln1 homozygous (dc3−/−), bln1 heterozygous (dc3+/−), l(2)k13095 heterozygous (dc4+/−), or Df(2)H20 heterozygote deficiency strains (Df [Δdc3/dc4]) and caspase activity was assessed on DEVD-amc or VDVAD-amc. Values are represented as mean ± SEM from four independent experiments. (B) Cytochrome c protein levels (top) and DRONC processing (bottom) in each lysate was detected by immunoblotting.
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fig5: DRONC and DRICE activation can occur in the absence of dc3 and dc4 during Drosophila development. (A) Lysates were prepared at 4 h after puparium formation, from wild-type (W1118), bln1 homozygous (dc3−/−), bln1 heterozygous (dc3+/−), l(2)k13095 heterozygous (dc4+/−), or Df(2)H20 heterozygote deficiency strains (Df [Δdc3/dc4]) and caspase activity was assessed on DEVD-amc or VDVAD-amc. Values are represented as mean ± SEM from four independent experiments. (B) Cytochrome c protein levels (top) and DRONC processing (bottom) in each lysate was detected by immunoblotting.

Mentions: Pre-pupae were collected 4 h after puparium formation, a time when various larval tissues including salivary glands undergo cell death and dronc has been shown to be up-regulated (Cakouros et al., 2002, 2004). We prepared lysates from bln1 heterozygote (dc3+/−) and homozygous (dc3−/−) mutant prepupae, l(2)k13095 heterozygotes (dc4+/−) and from prepupae heterozygous for the deficiency, Df(2)H20 (Df[Δdc3/dc4]). As shown in Fig. 5 A, there was a high level of caspase activity in wild-type (W1118) prepupae. There was no reduction in the level of caspase activity in pupae from dc3 and dc4 mutants (Fig. 5 A) or in the processing of DRONC (Fig. 5 B). Animals heterozygous for the deficiency Df(2)H20 also did not show lower caspase activity, even though the expression of cytochrome c in these flies is clearly reduced (Fig. 5 B). In addition, TUNEL staining of homozygous mutant embryos did not show any decrease in developmental cell death (unpublished data).


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)

DRONC and DRICE activation can occur in the absence of dc3 and dc4 during Drosophila development. (A) Lysates were prepared at 4 h after puparium formation, from wild-type (W1118), bln1 homozygous (dc3−/−), bln1 heterozygous (dc3+/−), l(2)k13095 heterozygous (dc4+/−), or Df(2)H20 heterozygote deficiency strains (Df [Δdc3/dc4]) and caspase activity was assessed on DEVD-amc or VDVAD-amc. Values are represented as mean ± SEM from four independent experiments. (B) Cytochrome c protein levels (top) and DRONC processing (bottom) in each lysate was detected by immunoblotting.
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fig5: DRONC and DRICE activation can occur in the absence of dc3 and dc4 during Drosophila development. (A) Lysates were prepared at 4 h after puparium formation, from wild-type (W1118), bln1 homozygous (dc3−/−), bln1 heterozygous (dc3+/−), l(2)k13095 heterozygous (dc4+/−), or Df(2)H20 heterozygote deficiency strains (Df [Δdc3/dc4]) and caspase activity was assessed on DEVD-amc or VDVAD-amc. Values are represented as mean ± SEM from four independent experiments. (B) Cytochrome c protein levels (top) and DRONC processing (bottom) in each lysate was detected by immunoblotting.
Mentions: Pre-pupae were collected 4 h after puparium formation, a time when various larval tissues including salivary glands undergo cell death and dronc has been shown to be up-regulated (Cakouros et al., 2002, 2004). We prepared lysates from bln1 heterozygote (dc3+/−) and homozygous (dc3−/−) mutant prepupae, l(2)k13095 heterozygotes (dc4+/−) and from prepupae heterozygous for the deficiency, Df(2)H20 (Df[Δdc3/dc4]). As shown in Fig. 5 A, there was a high level of caspase activity in wild-type (W1118) prepupae. There was no reduction in the level of caspase activity in pupae from dc3 and dc4 mutants (Fig. 5 A) or in the processing of DRONC (Fig. 5 B). Animals heterozygous for the deficiency Df(2)H20 also did not show lower caspase activity, even though the expression of cytochrome c in these flies is clearly reduced (Fig. 5 B). In addition, TUNEL staining of homozygous mutant embryos did not show any decrease in developmental cell death (unpublished data).

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