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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-DEVD-CHO blocks  TRAIL-induced PARP cleavage. Whole cell lysates of Jurkat  cells incubated with SN of Sf9 cells expressing mouse TRAIL  (lanes 2–3) or of mock-infected Sf9 cells (lane 1) in the presence  (lane 3) or absence of Ac-DEVD-CHO (lanes 1–2) were tested  with the anti-PARP Ab. The TRAIL-resistant REH (lane 4) and  the TRAIL-sensitive BJAB cells (lanes 5–6) incubated with  (lanes 4 and 6) or without TRAIL (lane 5) were used as specificity controls. Molecular markers are indicated. HRPO-labeled  goat anti–mouse IgG Ab were used as the detecting reagent.
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Figure 7: The IRP/caspase inhibitor Ac-DEVD-CHO blocks TRAIL-induced PARP cleavage. Whole cell lysates of Jurkat cells incubated with SN of Sf9 cells expressing mouse TRAIL (lanes 2–3) or of mock-infected Sf9 cells (lane 1) in the presence (lane 3) or absence of Ac-DEVD-CHO (lanes 1–2) were tested with the anti-PARP Ab. The TRAIL-resistant REH (lane 4) and the TRAIL-sensitive BJAB cells (lanes 5–6) incubated with (lanes 4 and 6) or without TRAIL (lane 5) were used as specificity controls. Molecular markers are indicated. HRPO-labeled goat anti–mouse IgG Ab were used as the detecting reagent.

Mentions: The nuclear repair enzyme PARP is cleaved by IRPs/ caspases during apoptosis induced by CD95 receptor triggering (Tewari et al., 1995) and by a variety of other agents (Henkart, 1996). To determine whether PARP is cleaved in cells undergoing TRAIL-mediated apoptosis, immunoblotting experiments were performed. As shown in Fig. 7, PARP (∼116 kD) was cleaved in sensitive Jurkat (lane 2) and Bjab cells (lane 6) with generation of a shorter fragment of ∼85 kD following incubation with TRAIL. Specificity is indicated by the lack of PARP cleavage in Jurkat (lane 1) and Bjab cells (lane 5) incubated with SN of mock-infected Sf9 cells and in TRAIL-resistant REH cells (lane 4). Triggering of TRAIL receptor in the presence of the peptide inhibitor Ac-DEVD-CHO completely blocked generation of the shorter fragment in Jurkat (lane 3) and Bjab cells (not shown). No inhibition was present with the vehicle alone. Thus, Ac-DEVD-CHO-inhibitable proteases are involved in TRAIL-induced cleavage of PARP in sensitive cells.


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-DEVD-CHO blocks  TRAIL-induced PARP cleavage. Whole cell lysates of Jurkat  cells incubated with SN of Sf9 cells expressing mouse TRAIL  (lanes 2–3) or of mock-infected Sf9 cells (lane 1) in the presence  (lane 3) or absence of Ac-DEVD-CHO (lanes 1–2) were tested  with the anti-PARP Ab. The TRAIL-resistant REH (lane 4) and  the TRAIL-sensitive BJAB cells (lanes 5–6) incubated with  (lanes 4 and 6) or without TRAIL (lane 5) were used as specificity controls. Molecular markers are indicated. HRPO-labeled  goat anti–mouse IgG Ab were used as the detecting reagent.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: The IRP/caspase inhibitor Ac-DEVD-CHO blocks TRAIL-induced PARP cleavage. Whole cell lysates of Jurkat cells incubated with SN of Sf9 cells expressing mouse TRAIL (lanes 2–3) or of mock-infected Sf9 cells (lane 1) in the presence (lane 3) or absence of Ac-DEVD-CHO (lanes 1–2) were tested with the anti-PARP Ab. The TRAIL-resistant REH (lane 4) and the TRAIL-sensitive BJAB cells (lanes 5–6) incubated with (lanes 4 and 6) or without TRAIL (lane 5) were used as specificity controls. Molecular markers are indicated. HRPO-labeled goat anti–mouse IgG Ab were used as the detecting reagent.
Mentions: The nuclear repair enzyme PARP is cleaved by IRPs/ caspases during apoptosis induced by CD95 receptor triggering (Tewari et al., 1995) and by a variety of other agents (Henkart, 1996). To determine whether PARP is cleaved in cells undergoing TRAIL-mediated apoptosis, immunoblotting experiments were performed. As shown in Fig. 7, PARP (∼116 kD) was cleaved in sensitive Jurkat (lane 2) and Bjab cells (lane 6) with generation of a shorter fragment of ∼85 kD following incubation with TRAIL. Specificity is indicated by the lack of PARP cleavage in Jurkat (lane 1) and Bjab cells (lane 5) incubated with SN of mock-infected Sf9 cells and in TRAIL-resistant REH cells (lane 4). Triggering of TRAIL receptor in the presence of the peptide inhibitor Ac-DEVD-CHO completely blocked generation of the shorter fragment in Jurkat (lane 3) and Bjab cells (not shown). No inhibition was present with the vehicle alone. Thus, Ac-DEVD-CHO-inhibitable proteases are involved in TRAIL-induced cleavage of PARP in sensitive cells.

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