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Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells.

Mariani SM, Matiba B, Armandola EA, Krammer PH - J. Cell Biol. (1997)

Bottom Line: The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis.These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands.Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology, German Cancer Research Center, Heidelberg.

ABSTRACT
The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNF-family and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1beta-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO. These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

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The IRP/caspase-inhibitor Ac-YVAD-cmk blocks TRAILinduced apoptosis in mouse myeloma cells. (A) The mouse myeloma cells Ag8 were incubated overnight with SN of Sf9 cells expressing mouse TRAIL (central and right panel) in the presence  of the peptide inhibitor Ac-YVAD-cmk (200 μM) (right panel)  or an equal concentration of vehicle (dmso) (central panel). As a  specificity control Ag8 cells were incubated with SN of mockinfected Sf9 cells (left panel). Results are presented as forward/ side scatter analysis of cellular morphology. Apoptotic cells are  shown in gate R1. (B) Ag8 cells were incubated overnight with  SN of Sf9 cells expressing mouse TRAIL in the presence of increasing concentrations of the peptide inhibitor Ac-YVAD-cmk  (squares). Equal concentrations of vehicle (triangles) were used  as a specificity control. Results are expressed as percent specific  cell death. Background apoptosis of target cells was <15%.
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Figure 5: The IRP/caspase-inhibitor Ac-YVAD-cmk blocks TRAILinduced apoptosis in mouse myeloma cells. (A) The mouse myeloma cells Ag8 were incubated overnight with SN of Sf9 cells expressing mouse TRAIL (central and right panel) in the presence of the peptide inhibitor Ac-YVAD-cmk (200 μM) (right panel) or an equal concentration of vehicle (dmso) (central panel). As a specificity control Ag8 cells were incubated with SN of mockinfected Sf9 cells (left panel). Results are presented as forward/ side scatter analysis of cellular morphology. Apoptotic cells are shown in gate R1. (B) Ag8 cells were incubated overnight with SN of Sf9 cells expressing mouse TRAIL in the presence of increasing concentrations of the peptide inhibitor Ac-YVAD-cmk (squares). Equal concentrations of vehicle (triangles) were used as a specificity control. Results are expressed as percent specific cell death. Background apoptosis of target cells was <15%.

Mentions: To assess if IRPs/caspases are involved in the intracellular signaling events triggered by TRAIL receptor engagement, peptide inhibition experiments were performed. The mouse myeloma cells Ag8 exposed to TRAIL in the presence of the synthetic peptide Ac-YVAD-cmk did not undergo the morphological changes characteristic of TRAIL- mediated apoptosis, i.e., membrane blebbing and nuclear condensation, as determined by optical microscopy. The lack of cell shrinkage and of increased cellular density are presented in the forward/side scatter analysis of Fig. 5 A. Equal concentrations of the vehicle (dmso) did not prevent TRAIL-induced cell death. Very similar results were obtained with the peptide Ac-DEVD-CHO (not shown). Analysis of the dose-effect relationship (Fig. 5 B) showed that maximal inhibition was achieved at a concentration of 200 μM of Ac-YVAD-cmk, with an IC50 (concentration yielding 50% inhibition) of ∼50 μM. Very similar results were obtained when the human leukemic cells Jurkat were used as target. In these cells, the Ac-YVAD-cmk peptide blocked TRAIL-induced apoptosis with an IC50 of ∼75 μM (Fig. 6 A). To determine whether Ac-YVAD-cmk could prevent loss of asymmetry in the plasma membrane phospholipids, exposure of phosphatidylserine residues was determined in target cells by measuring uptake of the lipophilic dye MC540 (Fadok et al., 1992). As shown in Fig. 6 B Jurkat cells exposed to TRAIL in the presence of increasing concentrations of Ac-YVAD-cmk displayed a dose-dependent inhibition of the TRAIL-induced alteration in plasma membrane phospholipids. Similar results were obtained with Ag8 cells as target or with Ac-DEVDCHO as inhibitor (not shown).


Interleukin 1 beta-converting enzyme related proteases/caspases are involved in TRAIL-induced apoptosis of myeloma and leukemia cells.

Mariani SM, Matiba B, Armandola EA, Krammer PH - J. Cell Biol. (1997)

The IRP/caspase-inhibitor Ac-YVAD-cmk blocks TRAILinduced apoptosis in mouse myeloma cells. (A) The mouse myeloma cells Ag8 were incubated overnight with SN of Sf9 cells expressing mouse TRAIL (central and right panel) in the presence  of the peptide inhibitor Ac-YVAD-cmk (200 μM) (right panel)  or an equal concentration of vehicle (dmso) (central panel). As a  specificity control Ag8 cells were incubated with SN of mockinfected Sf9 cells (left panel). Results are presented as forward/ side scatter analysis of cellular morphology. Apoptotic cells are  shown in gate R1. (B) Ag8 cells were incubated overnight with  SN of Sf9 cells expressing mouse TRAIL in the presence of increasing concentrations of the peptide inhibitor Ac-YVAD-cmk  (squares). Equal concentrations of vehicle (triangles) were used  as a specificity control. Results are expressed as percent specific  cell death. Background apoptosis of target cells was <15%.
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Figure 5: The IRP/caspase-inhibitor Ac-YVAD-cmk blocks TRAILinduced apoptosis in mouse myeloma cells. (A) The mouse myeloma cells Ag8 were incubated overnight with SN of Sf9 cells expressing mouse TRAIL (central and right panel) in the presence of the peptide inhibitor Ac-YVAD-cmk (200 μM) (right panel) or an equal concentration of vehicle (dmso) (central panel). As a specificity control Ag8 cells were incubated with SN of mockinfected Sf9 cells (left panel). Results are presented as forward/ side scatter analysis of cellular morphology. Apoptotic cells are shown in gate R1. (B) Ag8 cells were incubated overnight with SN of Sf9 cells expressing mouse TRAIL in the presence of increasing concentrations of the peptide inhibitor Ac-YVAD-cmk (squares). Equal concentrations of vehicle (triangles) were used as a specificity control. Results are expressed as percent specific cell death. Background apoptosis of target cells was <15%.
Mentions: To assess if IRPs/caspases are involved in the intracellular signaling events triggered by TRAIL receptor engagement, peptide inhibition experiments were performed. The mouse myeloma cells Ag8 exposed to TRAIL in the presence of the synthetic peptide Ac-YVAD-cmk did not undergo the morphological changes characteristic of TRAIL- mediated apoptosis, i.e., membrane blebbing and nuclear condensation, as determined by optical microscopy. The lack of cell shrinkage and of increased cellular density are presented in the forward/side scatter analysis of Fig. 5 A. Equal concentrations of the vehicle (dmso) did not prevent TRAIL-induced cell death. Very similar results were obtained with the peptide Ac-DEVD-CHO (not shown). Analysis of the dose-effect relationship (Fig. 5 B) showed that maximal inhibition was achieved at a concentration of 200 μM of Ac-YVAD-cmk, with an IC50 (concentration yielding 50% inhibition) of ∼50 μM. Very similar results were obtained when the human leukemic cells Jurkat were used as target. In these cells, the Ac-YVAD-cmk peptide blocked TRAIL-induced apoptosis with an IC50 of ∼75 μM (Fig. 6 A). To determine whether Ac-YVAD-cmk could prevent loss of asymmetry in the plasma membrane phospholipids, exposure of phosphatidylserine residues was determined in target cells by measuring uptake of the lipophilic dye MC540 (Fadok et al., 1992). As shown in Fig. 6 B Jurkat cells exposed to TRAIL in the presence of increasing concentrations of Ac-YVAD-cmk displayed a dose-dependent inhibition of the TRAIL-induced alteration in plasma membrane phospholipids. Similar results were obtained with Ag8 cells as target or with Ac-DEVDCHO as inhibitor (not shown).

Bottom Line: The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis.These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands.Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

View Article: PubMed Central - PubMed

Affiliation: Tumor Immunology, German Cancer Research Center, Heidelberg.

ABSTRACT
The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNF-family and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1beta-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor Ac-YVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO. These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

Show MeSH
Related in: MedlinePlus