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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 totally abrogate FasL-triggered apoptosis in HeLa cells expressing caspase-10 at low level.A, B, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 16 hours with or without 1 µg/mL FasL as indicated. Cells were labelled with Syto13 and propidium iodide before fluorescence microscopy examination (A). Of note, less than 5% of the cells were stained by propidium iodide under all conditions indicating that FasL-induced necrosis was marginal in HeLa cells. Percentages of cell death (i.e., cells exhibiting nuclear fragmentation and/or condensation) were determined by analysing at least 500 cells for each condition. Values are means ±SEM of three independent experiments (n.s.: not significant; **p<0.01.) (B). C, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 8 hours with or without 1 µg/mL FasL as indicated. Western blot analysis was performed using the indicated antibodies. (*: EGFP-tagged pro-caspase-10, **: EGFP-tagged small catalytic caspase-10 subunit).
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pone-0013638-g008: zVAD-fmk does not totally abrogate FasL-triggered apoptosis in HeLa cells expressing caspase-10 at low level.A, B, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 16 hours with or without 1 µg/mL FasL as indicated. Cells were labelled with Syto13 and propidium iodide before fluorescence microscopy examination (A). Of note, less than 5% of the cells were stained by propidium iodide under all conditions indicating that FasL-induced necrosis was marginal in HeLa cells. Percentages of cell death (i.e., cells exhibiting nuclear fragmentation and/or condensation) were determined by analysing at least 500 cells for each condition. Values are means ±SEM of three independent experiments (n.s.: not significant; **p<0.01.) (B). C, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 8 hours with or without 1 µg/mL FasL as indicated. Western blot analysis was performed using the indicated antibodies. (*: EGFP-tagged pro-caspase-10, **: EGFP-tagged small catalytic caspase-10 subunit).

Mentions: Casp10+ HeLa cells and their control counterparts (Casp10-) were pre-incubated with or without 40 µM zVAD-fmk for one hour and further incubated in the presence or absence of FasL. Cell morphology analysis indicated that FasL induced apoptosis (i.e., nuclear condensation and/or fragmentation) in both cell lines, caspase-10-proficient cells being slightly, yet not significantly, more sensitive than caspase-10- cells (Figs. 8A and B). Addition of zVAD-fmk fully prevented FasL-triggered cell death in caspase-10- HeLa cells. In sharp contrast, apoptotic cell death was not completely abrogated by zVAD-fmk in caspase-10-expressing cells (Figs 8A and B). Even at a concentration of 100 µM, zVAD-fmk failed to abolish cell death in Casp10+ cells (data not shown). Thus, under our experimental conditions, whereas zVAD-fmk efficiently prevented FasL-induced apoptosis in caspase-10-deficient HeLa cells, moderate expression of exogenous caspase-10 overcame, to some extent, the resistance conferred by zVAD-fmk.


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 totally abrogate FasL-triggered apoptosis in HeLa cells expressing caspase-10 at low level.A, B, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 16 hours with or without 1 µg/mL FasL as indicated. Cells were labelled with Syto13 and propidium iodide before fluorescence microscopy examination (A). Of note, less than 5% of the cells were stained by propidium iodide under all conditions indicating that FasL-induced necrosis was marginal in HeLa cells. Percentages of cell death (i.e., cells exhibiting nuclear fragmentation and/or condensation) were determined by analysing at least 500 cells for each condition. Values are means ±SEM of three independent experiments (n.s.: not significant; **p<0.01.) (B). C, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 8 hours with or without 1 µg/mL FasL as indicated. Western blot analysis was performed using the indicated antibodies. (*: EGFP-tagged pro-caspase-10, **: EGFP-tagged small catalytic caspase-10 subunit).
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getmorefigures.php?uid=PMC2964310&req=5

pone-0013638-g008: zVAD-fmk does not totally abrogate FasL-triggered apoptosis in HeLa cells expressing caspase-10 at low level.A, B, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 16 hours with or without 1 µg/mL FasL as indicated. Cells were labelled with Syto13 and propidium iodide before fluorescence microscopy examination (A). Of note, less than 5% of the cells were stained by propidium iodide under all conditions indicating that FasL-induced necrosis was marginal in HeLa cells. Percentages of cell death (i.e., cells exhibiting nuclear fragmentation and/or condensation) were determined by analysing at least 500 cells for each condition. Values are means ±SEM of three independent experiments (n.s.: not significant; **p<0.01.) (B). C, Casp10+ and Casp10- HeLa cells were incubated in the presence or absence of 40 µM zVAD-fmk for 1 hour and further incubated for 8 hours with or without 1 µg/mL FasL as indicated. Western blot analysis was performed using the indicated antibodies. (*: EGFP-tagged pro-caspase-10, **: EGFP-tagged small catalytic caspase-10 subunit).
Mentions: Casp10+ HeLa cells and their control counterparts (Casp10-) were pre-incubated with or without 40 µM zVAD-fmk for one hour and further incubated in the presence or absence of FasL. Cell morphology analysis indicated that FasL induced apoptosis (i.e., nuclear condensation and/or fragmentation) in both cell lines, caspase-10-proficient cells being slightly, yet not significantly, more sensitive than caspase-10- cells (Figs. 8A and B). Addition of zVAD-fmk fully prevented FasL-triggered cell death in caspase-10- HeLa cells. In sharp contrast, apoptotic cell death was not completely abrogated by zVAD-fmk in caspase-10-expressing cells (Figs 8A and B). Even at a concentration of 100 µM, zVAD-fmk failed to abolish cell death in Casp10+ cells (data not shown). Thus, under our experimental conditions, whereas zVAD-fmk efficiently prevented FasL-induced apoptosis in caspase-10-deficient HeLa cells, moderate expression of exogenous caspase-10 overcame, to some extent, the resistance conferred by zVAD-fmk.

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