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Individual caspase-10 isoforms play distinct and opposing roles in the initiation of death receptor-mediated tumour cell apoptosis.

Mühlethaler-Mottet A, Flahaut M, Bourloud KB, Nardou K, Coulon A, Liberman J, Thome M, Gross N - Cell Death Dis (2011)

Bottom Line: The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway.The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial.Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin-proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics, Paediatric Oncology Research, University Hospital CHUV, CH-1011 Lausanne, Switzerland. Annick.Muhlethaler@chuv.ch

ABSTRACT
The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin-proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.

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Endogenous caspase-10 levels are not sufficient to initiate TRAIL-mediated apoptosis in NB cells. (a) SH-EP or SK-N-AS cells infected with the empty vector (AB303), a nonspecific shRNA (RNAC), or caspase-8 shRNA (shC8) were analysed by immunoblotting for the presence of caspase-8. β-Actin was used as loading control. (b) Cell viability was measured in SH-EP- and SK-N-AS-AB303 (diamond), -RNAC (square) and -shC8 (triangle) cells treated with increasing amount of TRAIL for 48 h. A representative experiment out of three is shown. (c) Percentage of sub-G1 apoptotic cells detected by the PI staining method after stimulation with TRAIL (100 ng/ml) are indicated, mean of three experiments are shown (***P<0.0001). (d) SH-EP cells were untreated or treated with 100 ng of TRAIL for 16 h. Immunoblotting analysis of caspase-8, -10, -3 and Bid processing. β-Actin was used as loading control, as in further immunoblotting analyses. (e) Hydrolysis of DEVD-pNA was measured in cell lysates from SH-EP and SK-N-AS-infected cells treated with 50 or 250 ng/ml of TRAIL for 16 h, respectively. Relative OD values at 405 nm of stimulated compared with unstimulated cells of one experiment performed in duplicates are represented. (***P<0.0001, **P<0.01). (f) SH-EP cells were transiently transfected with the indicated siRNAs (siNeg, negative control siRNA). Relative expression levels of the indicated mRNA compared with HPRT1 mRNA as measured by real-time PCR are plotted in the graph (upper left panel). Immunoblotting analysis of caspase-10 and -8 expression in SH-EP transfected for 48 h with the indicated siRNAs (bottom left panel). Cell viability of cells treated for 48 h with indicated doses of TRAIL (right panel) is represented (***P<0.0001)
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fig2: Endogenous caspase-10 levels are not sufficient to initiate TRAIL-mediated apoptosis in NB cells. (a) SH-EP or SK-N-AS cells infected with the empty vector (AB303), a nonspecific shRNA (RNAC), or caspase-8 shRNA (shC8) were analysed by immunoblotting for the presence of caspase-8. β-Actin was used as loading control. (b) Cell viability was measured in SH-EP- and SK-N-AS-AB303 (diamond), -RNAC (square) and -shC8 (triangle) cells treated with increasing amount of TRAIL for 48 h. A representative experiment out of three is shown. (c) Percentage of sub-G1 apoptotic cells detected by the PI staining method after stimulation with TRAIL (100 ng/ml) are indicated, mean of three experiments are shown (***P<0.0001). (d) SH-EP cells were untreated or treated with 100 ng of TRAIL for 16 h. Immunoblotting analysis of caspase-8, -10, -3 and Bid processing. β-Actin was used as loading control, as in further immunoblotting analyses. (e) Hydrolysis of DEVD-pNA was measured in cell lysates from SH-EP and SK-N-AS-infected cells treated with 50 or 250 ng/ml of TRAIL for 16 h, respectively. Relative OD values at 405 nm of stimulated compared with unstimulated cells of one experiment performed in duplicates are represented. (***P<0.0001, **P<0.01). (f) SH-EP cells were transiently transfected with the indicated siRNAs (siNeg, negative control siRNA). Relative expression levels of the indicated mRNA compared with HPRT1 mRNA as measured by real-time PCR are plotted in the graph (upper left panel). Immunoblotting analysis of caspase-10 and -8 expression in SH-EP transfected for 48 h with the indicated siRNAs (bottom left panel). Cell viability of cells treated for 48 h with indicated doses of TRAIL (right panel) is represented (***P<0.0001)

Mentions: Next, the respective roles of caspase-8 and -10 in initiating DR apoptosis in NB cells was analysed by manipulating the relative expression levels of caspase-8 and -10 isoforms. First, downregulation of both caspase-8A and -B using shRNA resulted in a strongly reduced caspase-8 expression in S-type SH-EP-shC8 and SK-N-AS-shC8 NB cells compared with control cells (Figure 2a), while caspase-10 expression level remained unchanged (data not shown). Owing to caspase-8 silencing, SH-EP-shC8 and SK-N-AS-shC8 cells became fully resistant to TRAIL (Figure 2b), resulting in apoptosis resistance, as demonstrated in SH-EP-shC8 cells (Figure 2c). In SH-EP-shC8 cells treated with TRAIL, no cleavage of caspase-8, caspase-10, caspase-3 or Bid could be measured, while their proteolytic cleavage was easily detectable in SH-EP-RNAC control cells (Figure 2d). These observations were confirmed by caspase activity assays showing that caspase-3-like activity was abolished in SH-EP-shC8 and SK-N-AS-shC8 (Figure 2e). These results suggest that in caspase-8-silenced S-type NB cells, the low endogenous caspase-10 expression level is not sufficient to initiate apoptosis, even in the presence of a weak amount of caspase-8 protein.


Individual caspase-10 isoforms play distinct and opposing roles in the initiation of death receptor-mediated tumour cell apoptosis.

Mühlethaler-Mottet A, Flahaut M, Bourloud KB, Nardou K, Coulon A, Liberman J, Thome M, Gross N - Cell Death Dis (2011)

Endogenous caspase-10 levels are not sufficient to initiate TRAIL-mediated apoptosis in NB cells. (a) SH-EP or SK-N-AS cells infected with the empty vector (AB303), a nonspecific shRNA (RNAC), or caspase-8 shRNA (shC8) were analysed by immunoblotting for the presence of caspase-8. β-Actin was used as loading control. (b) Cell viability was measured in SH-EP- and SK-N-AS-AB303 (diamond), -RNAC (square) and -shC8 (triangle) cells treated with increasing amount of TRAIL for 48 h. A representative experiment out of three is shown. (c) Percentage of sub-G1 apoptotic cells detected by the PI staining method after stimulation with TRAIL (100 ng/ml) are indicated, mean of three experiments are shown (***P<0.0001). (d) SH-EP cells were untreated or treated with 100 ng of TRAIL for 16 h. Immunoblotting analysis of caspase-8, -10, -3 and Bid processing. β-Actin was used as loading control, as in further immunoblotting analyses. (e) Hydrolysis of DEVD-pNA was measured in cell lysates from SH-EP and SK-N-AS-infected cells treated with 50 or 250 ng/ml of TRAIL for 16 h, respectively. Relative OD values at 405 nm of stimulated compared with unstimulated cells of one experiment performed in duplicates are represented. (***P<0.0001, **P<0.01). (f) SH-EP cells were transiently transfected with the indicated siRNAs (siNeg, negative control siRNA). Relative expression levels of the indicated mRNA compared with HPRT1 mRNA as measured by real-time PCR are plotted in the graph (upper left panel). Immunoblotting analysis of caspase-10 and -8 expression in SH-EP transfected for 48 h with the indicated siRNAs (bottom left panel). Cell viability of cells treated for 48 h with indicated doses of TRAIL (right panel) is represented (***P<0.0001)
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fig2: Endogenous caspase-10 levels are not sufficient to initiate TRAIL-mediated apoptosis in NB cells. (a) SH-EP or SK-N-AS cells infected with the empty vector (AB303), a nonspecific shRNA (RNAC), or caspase-8 shRNA (shC8) were analysed by immunoblotting for the presence of caspase-8. β-Actin was used as loading control. (b) Cell viability was measured in SH-EP- and SK-N-AS-AB303 (diamond), -RNAC (square) and -shC8 (triangle) cells treated with increasing amount of TRAIL for 48 h. A representative experiment out of three is shown. (c) Percentage of sub-G1 apoptotic cells detected by the PI staining method after stimulation with TRAIL (100 ng/ml) are indicated, mean of three experiments are shown (***P<0.0001). (d) SH-EP cells were untreated or treated with 100 ng of TRAIL for 16 h. Immunoblotting analysis of caspase-8, -10, -3 and Bid processing. β-Actin was used as loading control, as in further immunoblotting analyses. (e) Hydrolysis of DEVD-pNA was measured in cell lysates from SH-EP and SK-N-AS-infected cells treated with 50 or 250 ng/ml of TRAIL for 16 h, respectively. Relative OD values at 405 nm of stimulated compared with unstimulated cells of one experiment performed in duplicates are represented. (***P<0.0001, **P<0.01). (f) SH-EP cells were transiently transfected with the indicated siRNAs (siNeg, negative control siRNA). Relative expression levels of the indicated mRNA compared with HPRT1 mRNA as measured by real-time PCR are plotted in the graph (upper left panel). Immunoblotting analysis of caspase-10 and -8 expression in SH-EP transfected for 48 h with the indicated siRNAs (bottom left panel). Cell viability of cells treated for 48 h with indicated doses of TRAIL (right panel) is represented (***P<0.0001)
Mentions: Next, the respective roles of caspase-8 and -10 in initiating DR apoptosis in NB cells was analysed by manipulating the relative expression levels of caspase-8 and -10 isoforms. First, downregulation of both caspase-8A and -B using shRNA resulted in a strongly reduced caspase-8 expression in S-type SH-EP-shC8 and SK-N-AS-shC8 NB cells compared with control cells (Figure 2a), while caspase-10 expression level remained unchanged (data not shown). Owing to caspase-8 silencing, SH-EP-shC8 and SK-N-AS-shC8 cells became fully resistant to TRAIL (Figure 2b), resulting in apoptosis resistance, as demonstrated in SH-EP-shC8 cells (Figure 2c). In SH-EP-shC8 cells treated with TRAIL, no cleavage of caspase-8, caspase-10, caspase-3 or Bid could be measured, while their proteolytic cleavage was easily detectable in SH-EP-RNAC control cells (Figure 2d). These observations were confirmed by caspase activity assays showing that caspase-3-like activity was abolished in SH-EP-shC8 and SK-N-AS-shC8 (Figure 2e). These results suggest that in caspase-8-silenced S-type NB cells, the low endogenous caspase-10 expression level is not sufficient to initiate apoptosis, even in the presence of a weak amount of caspase-8 protein.

Bottom Line: The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway.The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial.Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin-proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics, Paediatric Oncology Research, University Hospital CHUV, CH-1011 Lausanne, Switzerland. Annick.Muhlethaler@chuv.ch

ABSTRACT
The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin-proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.

Show MeSH
Related in: MedlinePlus