Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Mar 3;2(3):e125.
doi: 10.1038/cddis.2011.8.

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

A Mühlethaler-Mottet  1 , M Flahaut, K Balmas Bourloud, K Nardou, A Coulon, J Liberman, M Thome, N Gross
Affiliations

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

A Mühlethaler-Mottet et al. Cell Death Dis. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Analysis of the expression levels of caspase-8 and -10 isoforms. (a) Schematic representation of caspase-10 and -8 protein isoforms. Regions encoded by alternative exons are indicated. DED, death effector domain. (b) Whole cell extracts from various cell lines were loaded on a 15% SDS-polyacrylamide gel as described, and analysed by immunoblotting for the expression of caspase-8 and -10 isoforms. The three types of NB cells (S, substrate adherent; I, intermediate; N, neuroblastic) are represented. n.d., not determined. The immunoblot for endogenous caspase-10 detection were revealed using the ECL Advanced western blotting substrate for stronger signal detection. (c) Caspase-8 and -10 mRNA expression levels in tumour cells were compared by semi-quantitative real-time PCR. Plots represent means of three separate experiments
Figure 2
Figure 2
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)
Figure 3
Figure 3
Caspase-10A or -10D isoforms can substitute for caspase-8 in the initiation of the apoptotic cascade. (a) Immunoblotting analysis of caspase-10 expression in IGR-N-91 clones transduced with the empty vector (M) or with vector encoding for caspase-10B, or SH-EP cells (positive control). Cell viability was measured in cells treated with TRAIL (100 ng/ml) for 48 h. (b) Immunoblotting analysis of caspase-8, caspase-10, and β-actin expression in IGR-N-91 cells transduced with the empty vector (Migr) or with vector encoding for caspase-10A or -10D (pool of five clones each). Cell viability was measured in the pool of clones of IGR-N-91-Migr, -C10A and -C10D cells treated with the indicated doses of TRAIL for 48 h. Mean of two experiments performed in quadruplicates are shown (***P<0.0001). Percentage of sub-G1 apoptotic cells after stimulation with TRAIL are indicated, one representative experiment is shown. (c) SH-EP-shC8 cells transduced with the empty vector (Migr) or with vector encoding for caspase-10A or -10D were analysed as in (b). Mean of three experiments performed in quadruplicates are shown for the viability assay (***P<0.0001). One representative experiment is shown for apoptosis analysis
Figure 4
Figure 4
Caspase-10A and -10D have a pro-apoptotic role in NB cells. (a) Immunoblotting analysis of caspase-10 and -8 expressions in SH-EP and SK-N-AS cells transduced with the empty vector (Migr) or with vector encoding for C10A, C10B, C10D or C10G. (b) Cell viability of SH-EP and SK-N-AS transduced cells treated with the indicated doses of TRAIL and anti-Fas antibody for 48 h. Mean of three experiments performed in quadruplicates are shown (***P<0.0001, **P<0.005). (c) Percentage of sub-G1 apoptotic cells after stimulation with 25 ng/ml of TRAIL for SH-EP and 100 ng/ml for SK-N-AS are indicated. A representative experiment is shown
Figure 5
Figure 5
Caspase-10A and -10D have a pro-apoptotic role in SW480 and BJAB cell lines. (a) Immunoblotting analysis of caspase-10 and -8 expressions in SW480, or BJAB cells transduced with the empty Migr vector or with vector encoding for caspase-10A, -B, -D or -G. (b and d) Cell viability of SW480 (b), and BJAB (d) transduced cells treated with the indicated doses of TRAIL and anti-Fas antibody for 48 h. Mean of three experiments performed in quadruplicates are shown (***P<0.0001, *P<0.05). (c) Percentage of sub-G1 apoptotic cells after stimulation with 25 ng/ml of TRAIL and 500 ng/ml of anti-Fas antibody are indicated. A representative experiment is shown
Figure 6
Figure 6
Caspase-10A and -10D overexpression enhances the caspase cascade activation. (a) Immunoblotting analysis of caspases-10, -8, -9, and Bid cleavage in cells untreated or treated with 50 ng/ml of TRAIL for 16 h. (b) Caspase activity was measured in cell lysates from SH-EP (upper panels) and SW480 (lower panels) transduced cells treated with 50 ng/ml TRAIL for 8 h or 25 ng/ml of TRAIL for 16 h, respectively. Relative OD at 405 nm of stimulated compared with unstimulated cells of two experiments performed in duplicates are represented (***P<0.0001, **P<0.001, *P<0.05)
Figure 7
Figure 7
The C-terminal end of caspase-10B is responsible for its lack of activity in TRAIL and Fas sensitisation. (a) Up: schematic representation of the C10A/B fusion protein. Down: whole cell extracts from SW480-Migr, -C10A, -C10B or -C10A/B cells were analysed by immunoblotting for the presence of caspase-10 and β-actin. (b) Cell viability measured in SW480 cells treated with the indicated doses of TRAIL and anti-Fas antibody for 48 h. Mean of two experiments performed in quadruplicates are shown (***P<0.0005, **P<0.005, *P<0.05). (c) Percentage of sub-G1 apoptotic cells after stimulation with 50 ng/ml of TRAIL and 500 ng/ml of anti-Fas antibody are indicated. Mean of two experiments are shown (*P<0.05). (d) Whole cell extracts from SW480-C10A, -C10B, -C10A/B cells, untreated or treated with 50 ng/ml of TRAIL for various time length, or with 200 ng/ml of TRAIL for 8 h, were analysed by immunoblotting for the cleavage of caspases-10, -8, -3, and Bid
Figure 8
Figure 8
Caspase-10B is highly unstable, in contrast to other caspase-10 isoforms. (a) Whole cell extracts from SW480 and SH-EP cells overexpressing the indicated caspase-10 isoforms, untreated or treated with 20 ug/ml of CHX for various times, were analysed by immunoblotting for the presence of caspases-10, -8 and β-actin. (b) Whole cell extracts from SW480 and SH-EP-infected cells untreated or treated with 20 ug/ml of CHX, with or without lactacystin (10 μM for SH-EP or 50 μM for SW480) for 16 h, were analysed as in (a)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. - PubMed
    1. Hengartner MO. The biochemistry of apoptosis. Nature. 2000;407:770–776. - PubMed
    1. Fulda S. Caspase-8 in cancer biology and therapy. Cancer Lett. 2009;281:128–133. - PubMed
    1. Wang J, Chun HJ, Wong W, Spencer DM, Lenardo MJ. Caspase-10 is an initiator caspase in death receptor signaling. Proc Natl Acad Sci USA. 2001;98:13884–13888. - PMC - PubMed
    1. Milhas D, Cuvillier O, Therville N, Clave P, Thomsen M, Levade T, et al. Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis. J Biol Chem. 2005;280:19836–19842. - PubMed

Publication types

MeSH terms

LinkOut - more resources