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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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Induction of plasma membrane phospholipid disorganization by recombinant mouse TRAIL. The T leukemia cells Jurkat were incubated overnight with serial dilutions of SN from Sf9  cells expressing mouse TRAIL (filled circles) or mouse gld CD95L  (empty squares). Results are expressed as percent cells with high  uptake of the lipophilic dye MC540 (A). In B, results are presented as forward/side scatter analysis of cells incubated with recombinant TRAIL. MC540 uptake is shown for cells with nonapoptotic morphology (gate R1) (white histogram) and for cells  with apoptotic morphology (gate R2) (gray histogram). No difference in cell morphology was observed in Jurkat cells exposed to  TRAIL in the presence or absence of MC540.
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Figure 4: Induction of plasma membrane phospholipid disorganization by recombinant mouse TRAIL. The T leukemia cells Jurkat were incubated overnight with serial dilutions of SN from Sf9 cells expressing mouse TRAIL (filled circles) or mouse gld CD95L (empty squares). Results are expressed as percent cells with high uptake of the lipophilic dye MC540 (A). In B, results are presented as forward/side scatter analysis of cells incubated with recombinant TRAIL. MC540 uptake is shown for cells with nonapoptotic morphology (gate R1) (white histogram) and for cells with apoptotic morphology (gate R2) (gray histogram). No difference in cell morphology was observed in Jurkat cells exposed to TRAIL in the presence or absence of MC540.

Mentions: Distribution of phospholipids in the cellular plasma membranes was determined by MC540 staining (Fadok et al., 1992). As shown in Fig. 4 A, incubation of Jurkat cells with increasing concentrations of TRAIL, led to an increasingly higher uptake of MC540, suggesting an alteration of the membrane lipids and increased exposure of phosphatidylserine, as previously described for other apoptosis-inducing agents (Martin et al., 1995). Similar results were obtained with Ag8 cells (not shown). Specificity is indicated by the lack of increased MC540 uptake by Jurkat cells incubated with mouse gld CD95L (Fig. 4 A). Higher uptake of MC540 was correlated with the apoptotic phenotype of the target cells. As shown in Fig. 4 B, only the apoptotic cells undergoing shrinkage and increase in surface rugosity (gate R2) showed increased uptake of MC540. A kinetic analysis indicated that high uptake of MC540 by target cells preceded loss of cell viability as determined by propidium iodide permeability (not shown). These results indicate that TRAIL-induced apoptosis is associated with loss of membrane phospholipid asymmetry not only in normal T cells (Marsters et al., 1996) but also in T cells with a transformed phenotype.


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)

Induction of plasma membrane phospholipid disorganization by recombinant mouse TRAIL. The T leukemia cells Jurkat were incubated overnight with serial dilutions of SN from Sf9  cells expressing mouse TRAIL (filled circles) or mouse gld CD95L  (empty squares). Results are expressed as percent cells with high  uptake of the lipophilic dye MC540 (A). In B, results are presented as forward/side scatter analysis of cells incubated with recombinant TRAIL. MC540 uptake is shown for cells with nonapoptotic morphology (gate R1) (white histogram) and for cells  with apoptotic morphology (gate R2) (gray histogram). No difference in cell morphology was observed in Jurkat cells exposed to  TRAIL in the presence or absence of MC540.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139852&req=5

Figure 4: Induction of plasma membrane phospholipid disorganization by recombinant mouse TRAIL. The T leukemia cells Jurkat were incubated overnight with serial dilutions of SN from Sf9 cells expressing mouse TRAIL (filled circles) or mouse gld CD95L (empty squares). Results are expressed as percent cells with high uptake of the lipophilic dye MC540 (A). In B, results are presented as forward/side scatter analysis of cells incubated with recombinant TRAIL. MC540 uptake is shown for cells with nonapoptotic morphology (gate R1) (white histogram) and for cells with apoptotic morphology (gate R2) (gray histogram). No difference in cell morphology was observed in Jurkat cells exposed to TRAIL in the presence or absence of MC540.
Mentions: Distribution of phospholipids in the cellular plasma membranes was determined by MC540 staining (Fadok et al., 1992). As shown in Fig. 4 A, incubation of Jurkat cells with increasing concentrations of TRAIL, led to an increasingly higher uptake of MC540, suggesting an alteration of the membrane lipids and increased exposure of phosphatidylserine, as previously described for other apoptosis-inducing agents (Martin et al., 1995). Similar results were obtained with Ag8 cells (not shown). Specificity is indicated by the lack of increased MC540 uptake by Jurkat cells incubated with mouse gld CD95L (Fig. 4 A). Higher uptake of MC540 was correlated with the apoptotic phenotype of the target cells. As shown in Fig. 4 B, only the apoptotic cells undergoing shrinkage and increase in surface rugosity (gate R2) showed increased uptake of MC540. A kinetic analysis indicated that high uptake of MC540 by target cells preceded loss of cell viability as determined by propidium iodide permeability (not shown). These results indicate that TRAIL-induced apoptosis is associated with loss of membrane phospholipid asymmetry not only in normal T cells (Marsters et al., 1996) but also in T cells with a transformed phenotype.

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