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

The Ac-YVAD-cmk and Ac-DEVD-CHO peptide inhibitors block TRAIL-induced DNA fragmentation in mouse  and human cells. (A) Soluble DNA was extracted from mouse  myeloma cells Ag8 (lanes 1–4) (Ag8) or from the human leukemia cells Jurkat (lanes 5–8) (Jurkat) (0.5 × 106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1 and 5) or with SN  from TRAIL-expressing Sf9 cells (lanes 2–4 and 6–8) in the presence of the peptide inhibitor Ac-YVAD-cmk (lanes 3 and 7) or  Ac-DEVD-CHO (lanes 4 and 8) (200 μM). An equal concentration of vehicle (dmso) was used as a specificity control (lanes 2  and 6). Extracted DNA was separated on an agarose gel and visualized by ethidium bromide staining. Molecular mass markers  (MM) are shown. In B, results are shown as relative density of  fragmented DNA in each lane (relative density units) as determined by densitometric analysis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139852&req=5

Figure 8: The Ac-YVAD-cmk and Ac-DEVD-CHO peptide inhibitors block TRAIL-induced DNA fragmentation in mouse and human cells. (A) Soluble DNA was extracted from mouse myeloma cells Ag8 (lanes 1–4) (Ag8) or from the human leukemia cells Jurkat (lanes 5–8) (Jurkat) (0.5 × 106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1 and 5) or with SN from TRAIL-expressing Sf9 cells (lanes 2–4 and 6–8) in the presence of the peptide inhibitor Ac-YVAD-cmk (lanes 3 and 7) or Ac-DEVD-CHO (lanes 4 and 8) (200 μM). An equal concentration of vehicle (dmso) was used as a specificity control (lanes 2 and 6). Extracted DNA was separated on an agarose gel and visualized by ethidium bromide staining. Molecular mass markers (MM) are shown. In B, results are shown as relative density of fragmented DNA in each lane (relative density units) as determined by densitometric analysis.

Mentions: As shown previously, TRAIL-induced apoptosis is associated with extensive fragmentation of cellular DNA. To define the functional involvement of IRPs/caspases in this process, inhibition experiments were performed with the peptide inhibitors. As shown in Fig. 8, TRAIL-induced fragmentation of DNA from Ag8 and Jurkat cells was completely prevented in the presence of the IRP/caspaseinhibitor Ac-DEVD-CHO (lanes 4 and 8). No difference in the DNA fragmentation pattern or intensity was found in the presence or absence of the vehicle (not shown). Also the IRP/caspase inhibitor Ac-YVAD-cmk prevented TRAIL-induced DNA fragmentation in both cell types, but to a lower extent than the Ac-DEVD-CHO inhibitor in Ag8 cells. These results indicate that Ac-YVAD-cmk- and Ac-DEVD-CHO-sensitive IRPs/caspases are involved in TRAIL-induced DNA fragmentation and apoptosis of sensitive human and mouse 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 Ac-YVAD-cmk and Ac-DEVD-CHO peptide inhibitors block TRAIL-induced DNA fragmentation in mouse  and human cells. (A) Soluble DNA was extracted from mouse  myeloma cells Ag8 (lanes 1–4) (Ag8) or from the human leukemia cells Jurkat (lanes 5–8) (Jurkat) (0.5 × 106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1 and 5) or with SN  from TRAIL-expressing Sf9 cells (lanes 2–4 and 6–8) in the presence of the peptide inhibitor Ac-YVAD-cmk (lanes 3 and 7) or  Ac-DEVD-CHO (lanes 4 and 8) (200 μM). An equal concentration of vehicle (dmso) was used as a specificity control (lanes 2  and 6). Extracted DNA was separated on an agarose gel and visualized by ethidium bromide staining. Molecular mass markers  (MM) are shown. In B, results are shown as relative density of  fragmented DNA in each lane (relative density units) as determined by densitometric analysis.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: The Ac-YVAD-cmk and Ac-DEVD-CHO peptide inhibitors block TRAIL-induced DNA fragmentation in mouse and human cells. (A) Soluble DNA was extracted from mouse myeloma cells Ag8 (lanes 1–4) (Ag8) or from the human leukemia cells Jurkat (lanes 5–8) (Jurkat) (0.5 × 106 cells/lane) incubated with SN from mock-infected Sf9 (lanes 1 and 5) or with SN from TRAIL-expressing Sf9 cells (lanes 2–4 and 6–8) in the presence of the peptide inhibitor Ac-YVAD-cmk (lanes 3 and 7) or Ac-DEVD-CHO (lanes 4 and 8) (200 μM). An equal concentration of vehicle (dmso) was used as a specificity control (lanes 2 and 6). Extracted DNA was separated on an agarose gel and visualized by ethidium bromide staining. Molecular mass markers (MM) are shown. In B, results are shown as relative density of fragmented DNA in each lane (relative density units) as determined by densitometric analysis.
Mentions: As shown previously, TRAIL-induced apoptosis is associated with extensive fragmentation of cellular DNA. To define the functional involvement of IRPs/caspases in this process, inhibition experiments were performed with the peptide inhibitors. As shown in Fig. 8, TRAIL-induced fragmentation of DNA from Ag8 and Jurkat cells was completely prevented in the presence of the IRP/caspaseinhibitor Ac-DEVD-CHO (lanes 4 and 8). No difference in the DNA fragmentation pattern or intensity was found in the presence or absence of the vehicle (not shown). Also the IRP/caspase inhibitor Ac-YVAD-cmk prevented TRAIL-induced DNA fragmentation in both cell types, but to a lower extent than the Ac-DEVD-CHO inhibitor in Ag8 cells. These results indicate that Ac-YVAD-cmk- and Ac-DEVD-CHO-sensitive IRPs/caspases are involved in TRAIL-induced DNA fragmentation and apoptosis of sensitive human and mouse 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