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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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Impairment of FasL-induced caspase activation and apoptosis in caspase-8 and -10-doubly deficient Jurkat cells.A, B, A3 (white bars), I9-2d (grey bars) and I9-2e (black bars) Jurkat cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL as indicated. Caspase activities were assessed using Ac-DEVD-AMC or Ac-IETD-AMC (A). Cells were stained with annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Percentages of annexin-V-positive (AnV+) are indicated (B). All data are means ± SEM of three to four independent experiments. *p<0.05; **p<0.01, *** p<0.001. C, Representative flow cytometry experiment. Low right quadrants: percentages of [AnV-FITC+ propidium iodide (PI)-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells.
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pone-0013638-g002: Impairment of FasL-induced caspase activation and apoptosis in caspase-8 and -10-doubly deficient Jurkat cells.A, B, A3 (white bars), I9-2d (grey bars) and I9-2e (black bars) Jurkat cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL as indicated. Caspase activities were assessed using Ac-DEVD-AMC or Ac-IETD-AMC (A). Cells were stained with annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Percentages of annexin-V-positive (AnV+) are indicated (B). All data are means ± SEM of three to four independent experiments. *p<0.05; **p<0.01, *** p<0.001. C, Representative flow cytometry experiment. Low right quadrants: percentages of [AnV-FITC+ propidium iodide (PI)-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells.

Mentions: To characterize the Jurkat variants, I9-2a, b, d and e derived from I9-2 cells, the expression of proteins involved in Fas signalling was investigated. As expected from previous observations [13], [23], I9-2a, b, d and e cells did not express caspase-8 in contrast to A3 cells. However, FADD, RIP, caspase-2, -9, -3 and -7 (Fig. 1A) and Fas/CD95 (Fig. 1B) were equally expressed in A3 and in the I9-2 variants. Importantly, whereas caspase-10 was expressed in A3, I9-2d cells and, albeit to a lesser extent, I9-2b cells, barely detectable expression of caspase-10 was found in I9-2a and e cells (Fig. 1A). When the different cell variants were incubated in the presence of FasL, toxicity was observed in A3 cells and, albeit to a lesser extent, in I9-2b and I9-2d cells (Fig. 1C). In sharp contrast, FasL-induced cell death was totally abrogated in the caspase-8 and -10-doubly deficient I9-2a and e cells (Fig. 1C). Accordingly, FasL induced caspase activation, as evaluated by the cleavage of the effector caspase substrate Ac-DEVD-AMC and the initiator caspase substrate Ac-IETD-AMC (Fig. 2A), and apoptosis, as evaluated by flow cytometry (Figs. 2B and C), in A3 cells and, albeit to a lesser extent, in I9-2d cells. FasL totally failed to trigger caspase activation and apoptosis in I9-2e cells (Fig. 2). Identical data have been found using the other caspase-8 and -10-doubly deficient cells (i.e., I9-2a). These data are consistent with our previous study indicating the involvement of both endogenous caspase-8 and -10 in FasL-induced apoptosis [13].


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)

Impairment of FasL-induced caspase activation and apoptosis in caspase-8 and -10-doubly deficient Jurkat cells.A, B, A3 (white bars), I9-2d (grey bars) and I9-2e (black bars) Jurkat cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL as indicated. Caspase activities were assessed using Ac-DEVD-AMC or Ac-IETD-AMC (A). Cells were stained with annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Percentages of annexin-V-positive (AnV+) are indicated (B). All data are means ± SEM of three to four independent experiments. *p<0.05; **p<0.01, *** p<0.001. C, Representative flow cytometry experiment. Low right quadrants: percentages of [AnV-FITC+ propidium iodide (PI)-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964310&req=5

pone-0013638-g002: Impairment of FasL-induced caspase activation and apoptosis in caspase-8 and -10-doubly deficient Jurkat cells.A, B, A3 (white bars), I9-2d (grey bars) and I9-2e (black bars) Jurkat cells were incubated for 8 hours in the presence or absence of 500 ng/mL FasL as indicated. Caspase activities were assessed using Ac-DEVD-AMC or Ac-IETD-AMC (A). Cells were stained with annexin-V-FITC and propidium iodide and analyzed by flow cytometry. Percentages of annexin-V-positive (AnV+) are indicated (B). All data are means ± SEM of three to four independent experiments. *p<0.05; **p<0.01, *** p<0.001. C, Representative flow cytometry experiment. Low right quadrants: percentages of [AnV-FITC+ propidium iodide (PI)-] cells; Up right quadrants: percentages of [AnV-FITC+ PI+] cells; Up left quadrants: percentages of [AnV-FITC- PI+] cells.
Mentions: To characterize the Jurkat variants, I9-2a, b, d and e derived from I9-2 cells, the expression of proteins involved in Fas signalling was investigated. As expected from previous observations [13], [23], I9-2a, b, d and e cells did not express caspase-8 in contrast to A3 cells. However, FADD, RIP, caspase-2, -9, -3 and -7 (Fig. 1A) and Fas/CD95 (Fig. 1B) were equally expressed in A3 and in the I9-2 variants. Importantly, whereas caspase-10 was expressed in A3, I9-2d cells and, albeit to a lesser extent, I9-2b cells, barely detectable expression of caspase-10 was found in I9-2a and e cells (Fig. 1A). When the different cell variants were incubated in the presence of FasL, toxicity was observed in A3 cells and, albeit to a lesser extent, in I9-2b and I9-2d cells (Fig. 1C). In sharp contrast, FasL-induced cell death was totally abrogated in the caspase-8 and -10-doubly deficient I9-2a and e cells (Fig. 1C). Accordingly, FasL induced caspase activation, as evaluated by the cleavage of the effector caspase substrate Ac-DEVD-AMC and the initiator caspase substrate Ac-IETD-AMC (Fig. 2A), and apoptosis, as evaluated by flow cytometry (Figs. 2B and C), in A3 cells and, albeit to a lesser extent, in I9-2d cells. FasL totally failed to trigger caspase activation and apoptosis in I9-2e cells (Fig. 2). Identical data have been found using the other caspase-8 and -10-doubly deficient cells (i.e., I9-2a). These data are consistent with our previous study indicating the involvement of both endogenous caspase-8 and -10 in FasL-induced apoptosis [13].

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