Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunoblot analysis of recombinant mouse TRAIL.  (A) Whole cell lysates of Sf9 cells expressing recombinant mouse  TRAIL (lane 1) or mouse CD95L (lane 2) were tested with purified rabbit anti-TRAIL Ab. Molecular weight markers are indicated. HRPO-labeled mouse anti–rabbit IgG antibodies were  used as the detecting reagent. No reactivity was present with purified rabbit IgG. (B) Whole cell lysates of Sf9 cells expressing recombinant mouse TRAIL (lane 1) or mouse CD95L (lane 2) and  cell lysates of non-infected Sf9 cells (lane 3) were tested with the  mouse mAb SM22 to a baculovirus-related product. Molecular  weight markers are indicated. HRPO-labeled rabbit anti–mouse  IgG antibodies were used as the detecting reagent. No reactivity  was present with an isotype-matched control mAb. (C) SN of Sf9  cells expressing mouse TRAIL (lane 2) or mouse CD95L (lane 1)  were tested with purified rabbit anti-TRAIL antibody. Molecular  weight markers are indicated. HRPO-labeled mouse anti–rabbit  IgG antibodies were used as the detecting reagent. No reactivity  was found with purified rabbit IgG.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139852&req=5

Figure 1: Immunoblot analysis of recombinant mouse TRAIL. (A) Whole cell lysates of Sf9 cells expressing recombinant mouse TRAIL (lane 1) or mouse CD95L (lane 2) were tested with purified rabbit anti-TRAIL Ab. Molecular weight markers are indicated. HRPO-labeled mouse anti–rabbit IgG antibodies were used as the detecting reagent. No reactivity was present with purified rabbit IgG. (B) Whole cell lysates of Sf9 cells expressing recombinant mouse TRAIL (lane 1) or mouse CD95L (lane 2) and cell lysates of non-infected Sf9 cells (lane 3) were tested with the mouse mAb SM22 to a baculovirus-related product. Molecular weight markers are indicated. HRPO-labeled rabbit anti–mouse IgG antibodies were used as the detecting reagent. No reactivity was present with an isotype-matched control mAb. (C) SN of Sf9 cells expressing mouse TRAIL (lane 2) or mouse CD95L (lane 1) were tested with purified rabbit anti-TRAIL antibody. Molecular weight markers are indicated. HRPO-labeled mouse anti–rabbit IgG antibodies were used as the detecting reagent. No reactivity was found with purified rabbit IgG.

Mentions: To produce functional recombinant TRAIL, full-length mouse TRAIL cDNA was subcloned into the baculovirus expression vector pVL1393 under the strong polyhedrin promoter. Sf9 cells were infected with the recombinant virus and tested for expression of recombinant TRAIL. To specifically detect TRAIL, a rabbit polyclonal antibody was developed in rabbits immunized with a peptide corresponding to the carboxy-terminal region of TRAIL. As shown in Fig. 1 A, the Ab specifically detects a protein of ∼32–33 kD in lysates of Sf9 cells expressing TRAIL (lane 1) but not of Sf9 cells expressing CD95L (lane 2). No reactivity was found in both cell lysates with purified rabbit IgG. Incubation of the same cell lysates with the mouse mAb SM22 to a baculovirus-derived product showed an equal loading of cell extract (Fig. 1 B).


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)

Immunoblot analysis of recombinant mouse TRAIL.  (A) Whole cell lysates of Sf9 cells expressing recombinant mouse  TRAIL (lane 1) or mouse CD95L (lane 2) were tested with purified rabbit anti-TRAIL Ab. Molecular weight markers are indicated. HRPO-labeled mouse anti–rabbit IgG antibodies were  used as the detecting reagent. No reactivity was present with purified rabbit IgG. (B) Whole cell lysates of Sf9 cells expressing recombinant mouse TRAIL (lane 1) or mouse CD95L (lane 2) and  cell lysates of non-infected Sf9 cells (lane 3) were tested with the  mouse mAb SM22 to a baculovirus-related product. Molecular  weight markers are indicated. HRPO-labeled rabbit anti–mouse  IgG antibodies were used as the detecting reagent. No reactivity  was present with an isotype-matched control mAb. (C) SN of Sf9  cells expressing mouse TRAIL (lane 2) or mouse CD95L (lane 1)  were tested with purified rabbit anti-TRAIL antibody. Molecular  weight markers are indicated. HRPO-labeled mouse anti–rabbit  IgG antibodies were used as the detecting reagent. No reactivity  was found with purified rabbit IgG.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Immunoblot analysis of recombinant mouse TRAIL. (A) Whole cell lysates of Sf9 cells expressing recombinant mouse TRAIL (lane 1) or mouse CD95L (lane 2) were tested with purified rabbit anti-TRAIL Ab. Molecular weight markers are indicated. HRPO-labeled mouse anti–rabbit IgG antibodies were used as the detecting reagent. No reactivity was present with purified rabbit IgG. (B) Whole cell lysates of Sf9 cells expressing recombinant mouse TRAIL (lane 1) or mouse CD95L (lane 2) and cell lysates of non-infected Sf9 cells (lane 3) were tested with the mouse mAb SM22 to a baculovirus-related product. Molecular weight markers are indicated. HRPO-labeled rabbit anti–mouse IgG antibodies were used as the detecting reagent. No reactivity was present with an isotype-matched control mAb. (C) SN of Sf9 cells expressing mouse TRAIL (lane 2) or mouse CD95L (lane 1) were tested with purified rabbit anti-TRAIL antibody. Molecular weight markers are indicated. HRPO-labeled mouse anti–rabbit IgG antibodies were used as the detecting reagent. No reactivity was found with purified rabbit IgG.
Mentions: To produce functional recombinant TRAIL, full-length mouse TRAIL cDNA was subcloned into the baculovirus expression vector pVL1393 under the strong polyhedrin promoter. Sf9 cells were infected with the recombinant virus and tested for expression of recombinant TRAIL. To specifically detect TRAIL, a rabbit polyclonal antibody was developed in rabbits immunized with a peptide corresponding to the carboxy-terminal region of TRAIL. As shown in Fig. 1 A, the Ab specifically detects a protein of ∼32–33 kD in lysates of Sf9 cells expressing TRAIL (lane 1) but not of Sf9 cells expressing CD95L (lane 2). No reactivity was found in both cell lysates with purified rabbit IgG. Incubation of the same cell lysates with the mouse mAb SM22 to a baculovirus-derived product showed an equal loading of cell extract (Fig. 1 B).

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