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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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DC3 and DC4 are not required for caspase activation and apoptosis. (A) Endogenous expression of dc3 and dc4 transcripts in Drosophila cell lines as detected by RT-PCR. (B) BG2 cells were treated with dsRNAs for dc3, dc4, or both dc3 and dc4, and ablation of the target genes assessed by RT-PCR. Amplification of a 405-bp dronc region was used as a control. (C) BG2 cells treated with dsRNA to dc3, dc4 or both, were exposed to cycloheximide (CHX) or were left untreated. Absence of DC4 protein was confirmed by immunoblotting with cytochrome c antibody. Processing of DRONC and DRICE was detected by immunoblotting. Relative molecular masses of the proteins in kilodaltons are shown. (D) Apoptosis induced in BG2 cells by CHX (10 μg/ml for 6 h), or l(2)mbn cells by ecdysone (10 μM for 24 h) treatment was estimated by scoring cells with condensed nuclei by DAPI staining. Data (mean ± SEM) were derived from four independent experiments. (E) Caspase activity, presented as relative fluorescence units (RFU), was determined on DEVD-amc substrate in BG2 or l(2)mbn cells treated (+) with CHX (10 μg/ml for 6 h) or ecdysone (10 μM for 24 h), respectively, or left untreated (−). Data (mean ± SEM) were derived from three experiments.
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fig1: DC3 and DC4 are not required for caspase activation and apoptosis. (A) Endogenous expression of dc3 and dc4 transcripts in Drosophila cell lines as detected by RT-PCR. (B) BG2 cells were treated with dsRNAs for dc3, dc4, or both dc3 and dc4, and ablation of the target genes assessed by RT-PCR. Amplification of a 405-bp dronc region was used as a control. (C) BG2 cells treated with dsRNA to dc3, dc4 or both, were exposed to cycloheximide (CHX) or were left untreated. Absence of DC4 protein was confirmed by immunoblotting with cytochrome c antibody. Processing of DRONC and DRICE was detected by immunoblotting. Relative molecular masses of the proteins in kilodaltons are shown. (D) Apoptosis induced in BG2 cells by CHX (10 μg/ml for 6 h), or l(2)mbn cells by ecdysone (10 μM for 24 h) treatment was estimated by scoring cells with condensed nuclei by DAPI staining. Data (mean ± SEM) were derived from four independent experiments. (E) Caspase activity, presented as relative fluorescence units (RFU), was determined on DEVD-amc substrate in BG2 or l(2)mbn cells treated (+) with CHX (10 μg/ml for 6 h) or ecdysone (10 μM for 24 h), respectively, or left untreated (−). Data (mean ± SEM) were derived from three experiments.

Mentions: To compare the expression of dc3 and dc4 in Drosophila cell lines, we used RT-PCR analysis and found that dc3 is expressed at lower levels in BG2, SL2 and l(2)mbn cells, compared with dc4 (Fig. 1 A). Expression of dc3 was the lowest in BG2 cells. To test whether DC3 or DC4 are required for the activation of DRONC or DRICE, we silenced the expression of dc3 and dc4 by RNA interference (RNAi) in Drosophila BG2 cells, with doube-stranded RNAs (dsRNAs) to each gene. The ablation was confirmed by RT-PCR for both dc3 and dc4 (Fig. 1 B) and by immunoblotting for DC4 (Fig. 1 C). Silencing both dc3 and dc4 in BG2 cell had no effect on the processing of DRONC and DRICE after induction of apoptosis by cycloheximide (Fig. 1 C), the rate of cell death (Fig. 1 D), or caspase activity (Fig. 1 E). We also found that silencing dc3 and dc4 did not affect ecdysone-induced apoptosis (Fig. 1 D) or caspase activity (Fig. 1 E) in Drosophila l(2)mbn cells. These observations are consistent with previous studies (Zimmermann et al., 2002), which demonstrated that silencing expression of cytochrome c proteins in SL2 cells had no effect on apoptosis induced by UV, actinomycin D, or ectopic expression of Reaper or Grim. These results clearly indicate that DC3 and DC4, are not essential for caspase activation and apoptosis in Drosophila cells.


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 and DC4 are not required for caspase activation and apoptosis. (A) Endogenous expression of dc3 and dc4 transcripts in Drosophila cell lines as detected by RT-PCR. (B) BG2 cells were treated with dsRNAs for dc3, dc4, or both dc3 and dc4, and ablation of the target genes assessed by RT-PCR. Amplification of a 405-bp dronc region was used as a control. (C) BG2 cells treated with dsRNA to dc3, dc4 or both, were exposed to cycloheximide (CHX) or were left untreated. Absence of DC4 protein was confirmed by immunoblotting with cytochrome c antibody. Processing of DRONC and DRICE was detected by immunoblotting. Relative molecular masses of the proteins in kilodaltons are shown. (D) Apoptosis induced in BG2 cells by CHX (10 μg/ml for 6 h), or l(2)mbn cells by ecdysone (10 μM for 24 h) treatment was estimated by scoring cells with condensed nuclei by DAPI staining. Data (mean ± SEM) were derived from four independent experiments. (E) Caspase activity, presented as relative fluorescence units (RFU), was determined on DEVD-amc substrate in BG2 or l(2)mbn cells treated (+) with CHX (10 μg/ml for 6 h) or ecdysone (10 μM for 24 h), respectively, or left untreated (−). Data (mean ± SEM) were derived from three experiments.
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fig1: DC3 and DC4 are not required for caspase activation and apoptosis. (A) Endogenous expression of dc3 and dc4 transcripts in Drosophila cell lines as detected by RT-PCR. (B) BG2 cells were treated with dsRNAs for dc3, dc4, or both dc3 and dc4, and ablation of the target genes assessed by RT-PCR. Amplification of a 405-bp dronc region was used as a control. (C) BG2 cells treated with dsRNA to dc3, dc4 or both, were exposed to cycloheximide (CHX) or were left untreated. Absence of DC4 protein was confirmed by immunoblotting with cytochrome c antibody. Processing of DRONC and DRICE was detected by immunoblotting. Relative molecular masses of the proteins in kilodaltons are shown. (D) Apoptosis induced in BG2 cells by CHX (10 μg/ml for 6 h), or l(2)mbn cells by ecdysone (10 μM for 24 h) treatment was estimated by scoring cells with condensed nuclei by DAPI staining. Data (mean ± SEM) were derived from four independent experiments. (E) Caspase activity, presented as relative fluorescence units (RFU), was determined on DEVD-amc substrate in BG2 or l(2)mbn cells treated (+) with CHX (10 μg/ml for 6 h) or ecdysone (10 μM for 24 h), respectively, or left untreated (−). Data (mean ± SEM) were derived from three experiments.
Mentions: To compare the expression of dc3 and dc4 in Drosophila cell lines, we used RT-PCR analysis and found that dc3 is expressed at lower levels in BG2, SL2 and l(2)mbn cells, compared with dc4 (Fig. 1 A). Expression of dc3 was the lowest in BG2 cells. To test whether DC3 or DC4 are required for the activation of DRONC or DRICE, we silenced the expression of dc3 and dc4 by RNA interference (RNAi) in Drosophila BG2 cells, with doube-stranded RNAs (dsRNAs) to each gene. The ablation was confirmed by RT-PCR for both dc3 and dc4 (Fig. 1 B) and by immunoblotting for DC4 (Fig. 1 C). Silencing both dc3 and dc4 in BG2 cell had no effect on the processing of DRONC and DRICE after induction of apoptosis by cycloheximide (Fig. 1 C), the rate of cell death (Fig. 1 D), or caspase activity (Fig. 1 E). We also found that silencing dc3 and dc4 did not affect ecdysone-induced apoptosis (Fig. 1 D) or caspase activity (Fig. 1 E) in Drosophila l(2)mbn cells. These observations are consistent with previous studies (Zimmermann et al., 2002), which demonstrated that silencing expression of cytochrome c proteins in SL2 cells had no effect on apoptosis induced by UV, actinomycin D, or ectopic expression of Reaper or Grim. These results clearly indicate that DC3 and DC4, are not essential for caspase activation and apoptosis in Drosophila cells.

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