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Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk.

Lafont E, Milhas D, Teissié J, Therville N, Andrieu-Abadie N, Levade T, Benoist H, Ségui B - PLoS ONE (2010)

Bottom Line: Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death.Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling.Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.

View Article: PubMed Central - PubMed

Affiliation: U858 INSERM (Institut National de la Santé et de la Recherche Médicale), Département Cancer, Equipe 14, Toulouse, France.

ABSTRACT

Background: Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.

Methodology and principal findings: The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.

Conclusions and significance: Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.

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zVAD-fmk does not abrogate over-expressed caspase-10-triggered cell death in HeLa cells.A, Total protein extracts derived from wild-type Jurkat (A3) and HeLa cells were analysed by Western blot using anti-caspase-8, anti-caspase-10 or anti β-actin antibodies. B–D, HeLa cells were transfected with plasmid encoding EGFP, EGFP-tagged wild-type caspase-8 (Casp8) or -10 (Casp10). B, Cells were analysed by flow cytometry 16 hours post-transfection. Values indicated are percentages of EGFP-expressing cells. C, Immediately after transfection, 40 µM zVAD-fmk was added or not to the cell culture medium and cells were further incubated for 24 hours. During the last 8 hours, cells were incubated with or without 1 µg/mL FasL. Protein extracts were analysed by Western blot using the indicated antibodies. Cleaved PARP expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-8 or 10, **: EGFP-tagged small catalytic caspase-8 or -10 subunit). The ≈26 kDa band obtained in caspase-8 and -10 expressing cells remains to be characterized. D, HeLa cells were transfected and cultured for 24 hours in the presence or absence of zVAD-fmk. Cells were labelled with annexin-V/APC. Annexin-V binding analysis was restricted to the EGFP-expressing cells. Data are means ± SEM of five independent experiments. ***p<0.001; * p<0.05.
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pone-0013638-g006: zVAD-fmk does not abrogate over-expressed caspase-10-triggered cell death in HeLa cells.A, Total protein extracts derived from wild-type Jurkat (A3) and HeLa cells were analysed by Western blot using anti-caspase-8, anti-caspase-10 or anti β-actin antibodies. B–D, HeLa cells were transfected with plasmid encoding EGFP, EGFP-tagged wild-type caspase-8 (Casp8) or -10 (Casp10). B, Cells were analysed by flow cytometry 16 hours post-transfection. Values indicated are percentages of EGFP-expressing cells. C, Immediately after transfection, 40 µM zVAD-fmk was added or not to the cell culture medium and cells were further incubated for 24 hours. During the last 8 hours, cells were incubated with or without 1 µg/mL FasL. Protein extracts were analysed by Western blot using the indicated antibodies. Cleaved PARP expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-8 or 10, **: EGFP-tagged small catalytic caspase-8 or -10 subunit). The ≈26 kDa band obtained in caspase-8 and -10 expressing cells remains to be characterized. D, HeLa cells were transfected and cultured for 24 hours in the presence or absence of zVAD-fmk. Cells were labelled with annexin-V/APC. Annexin-V binding analysis was restricted to the EGFP-expressing cells. Data are means ± SEM of five independent experiments. ***p<0.001; * p<0.05.

Mentions: To further evaluate the role of caspase-10 in Fas signalling, we attempted to perform Western blot analysis of caspase activation in reconstituted I9-2e Jurkat cells. However, the too low transfection efficiency of Jurkat cells precluded such analyses. We therefore used another cell type, i.e., the HeLa carcinoma cell line, which does express endogenous caspase-8 but not caspase-10 as compared to A3 Jurkat cells (Fig. 6A). HeLa cells could be efficiently transfected, as illustrated by high percentages of EGFP-expressing cells sixteen hours post-transfection with a plasmid encoding EGFP, EGFP-tagged caspase-8 or -10 (Fig. 6B), allowing caspase expression analysis by Western blot. To evaluate the caspase-inhibitory efficacy of zVAD-fmk under our experimental conditions, HeLa cells were transiently transfected with the different constructs and immediately treated with 40 µM zVAD-fmk and further incubated for 24 hours. During the last 8 hours, 1 µg/mL FasL was added or not to the cell culture medium. In cells transfected with plasmids encoding either EGFP-tagged caspase-8 or -10, an approximately 40 kDa band was revealed with anti-EGFP antibody (Fig. 6C). The appearance of this band did not require FasL treatment and most likely corresponded to the EGFP-tagged small catalytic subunit of pro-caspase-8 and -10, indicating auto-processing of the over-expressed enzymes (Fig. 6C). The addition of zVAD-fmk led to a different pattern depending on the type of EGFP-tagged initiator caspase expressed. Indeed, the 40 kDa band decreased and accumulated in EGFP-tagged caspase-8 and -10 expressing cells, respectively. In the meantime, whereas EGFP-tagged pro-caspase-8 strongly accumulated, pro-caspase-10 accumulation was less pronounced (Figure 6C).


Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk.

Lafont E, Milhas D, Teissié J, Therville N, Andrieu-Abadie N, Levade T, Benoist H, Ségui B - PLoS ONE (2010)

zVAD-fmk does not abrogate over-expressed caspase-10-triggered cell death in HeLa cells.A, Total protein extracts derived from wild-type Jurkat (A3) and HeLa cells were analysed by Western blot using anti-caspase-8, anti-caspase-10 or anti β-actin antibodies. B–D, HeLa cells were transfected with plasmid encoding EGFP, EGFP-tagged wild-type caspase-8 (Casp8) or -10 (Casp10). B, Cells were analysed by flow cytometry 16 hours post-transfection. Values indicated are percentages of EGFP-expressing cells. C, Immediately after transfection, 40 µM zVAD-fmk was added or not to the cell culture medium and cells were further incubated for 24 hours. During the last 8 hours, cells were incubated with or without 1 µg/mL FasL. Protein extracts were analysed by Western blot using the indicated antibodies. Cleaved PARP expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-8 or 10, **: EGFP-tagged small catalytic caspase-8 or -10 subunit). The ≈26 kDa band obtained in caspase-8 and -10 expressing cells remains to be characterized. D, HeLa cells were transfected and cultured for 24 hours in the presence or absence of zVAD-fmk. Cells were labelled with annexin-V/APC. Annexin-V binding analysis was restricted to the EGFP-expressing cells. Data are means ± SEM of five independent experiments. ***p<0.001; * p<0.05.
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pone-0013638-g006: zVAD-fmk does not abrogate over-expressed caspase-10-triggered cell death in HeLa cells.A, Total protein extracts derived from wild-type Jurkat (A3) and HeLa cells were analysed by Western blot using anti-caspase-8, anti-caspase-10 or anti β-actin antibodies. B–D, HeLa cells were transfected with plasmid encoding EGFP, EGFP-tagged wild-type caspase-8 (Casp8) or -10 (Casp10). B, Cells were analysed by flow cytometry 16 hours post-transfection. Values indicated are percentages of EGFP-expressing cells. C, Immediately after transfection, 40 µM zVAD-fmk was added or not to the cell culture medium and cells were further incubated for 24 hours. During the last 8 hours, cells were incubated with or without 1 µg/mL FasL. Protein extracts were analysed by Western blot using the indicated antibodies. Cleaved PARP expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-8 or 10, **: EGFP-tagged small catalytic caspase-8 or -10 subunit). The ≈26 kDa band obtained in caspase-8 and -10 expressing cells remains to be characterized. D, HeLa cells were transfected and cultured for 24 hours in the presence or absence of zVAD-fmk. Cells were labelled with annexin-V/APC. Annexin-V binding analysis was restricted to the EGFP-expressing cells. Data are means ± SEM of five independent experiments. ***p<0.001; * p<0.05.
Mentions: To further evaluate the role of caspase-10 in Fas signalling, we attempted to perform Western blot analysis of caspase activation in reconstituted I9-2e Jurkat cells. However, the too low transfection efficiency of Jurkat cells precluded such analyses. We therefore used another cell type, i.e., the HeLa carcinoma cell line, which does express endogenous caspase-8 but not caspase-10 as compared to A3 Jurkat cells (Fig. 6A). HeLa cells could be efficiently transfected, as illustrated by high percentages of EGFP-expressing cells sixteen hours post-transfection with a plasmid encoding EGFP, EGFP-tagged caspase-8 or -10 (Fig. 6B), allowing caspase expression analysis by Western blot. To evaluate the caspase-inhibitory efficacy of zVAD-fmk under our experimental conditions, HeLa cells were transiently transfected with the different constructs and immediately treated with 40 µM zVAD-fmk and further incubated for 24 hours. During the last 8 hours, 1 µg/mL FasL was added or not to the cell culture medium. In cells transfected with plasmids encoding either EGFP-tagged caspase-8 or -10, an approximately 40 kDa band was revealed with anti-EGFP antibody (Fig. 6C). The appearance of this band did not require FasL treatment and most likely corresponded to the EGFP-tagged small catalytic subunit of pro-caspase-8 and -10, indicating auto-processing of the over-expressed enzymes (Fig. 6C). The addition of zVAD-fmk led to a different pattern depending on the type of EGFP-tagged initiator caspase expressed. Indeed, the 40 kDa band decreased and accumulated in EGFP-tagged caspase-8 and -10 expressing cells, respectively. In the meantime, whereas EGFP-tagged pro-caspase-8 strongly accumulated, pro-caspase-10 accumulation was less pronounced (Figure 6C).

Bottom Line: Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death.Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling.Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.

View Article: PubMed Central - PubMed

Affiliation: U858 INSERM (Institut National de la Santé et de la Recherche Médicale), Département Cancer, Equipe 14, Toulouse, France.

ABSTRACT

Background: Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.

Methodology and principal findings: The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.

Conclusions and significance: Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.

Show MeSH
Related in: MedlinePlus