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Pivotal role of a DEVD-sensitive step in etoposide-induced and Fas-mediated apoptotic pathways
1Laboratory of Onco-Hematology and Pharmacology, CJF INSERM 94-08, Faculty of Medicine, Dijon, France.
Abstract:
We investigated the role of proteases in the pathway that leads from specific DNA damage induced by etoposide (VP-16), a topoisomerase II inhibitor, to apoptotic DNA fragmentation in the U937 human leukemic cell line. In a reconstituted cell-free system, Triton-soluble extracts from VP-16-treated cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This effect was inhibited by the tetrapeptide Ac-DEVD-CHO, a competitive inhibitor of the interleukin-1 beta-converting enzyme (ICE)-related protease CPP32, but was not influenced by Ac-YVAD-CHO and Ac-YVAD-CMK, two specific inhibitors of ICE. The three tetrapeptides inhibited Fas-mediated apoptotic DNA fragmentation in the cell-free system. Internucleosomal DNA fragmentation, triggered by either VP-16 or an anti-Fas antibody, was associated with proteolytic cleavage of the poly(ADP-ribose)polymerase (PARP), a decrease in the level of 32 kDa CPP32 proenzyme and the appearance of the CPP32 p17 active subunit. Conversely, the expression of Ich-1L, another ICE-like protease, remained stable in apoptotic U937 cells. Several cysteine and serine protease inhibitors prevented apoptotic DNA fragmentation by acting either upstream or downstream of the DEVD-sensitive protease(s) activation and PARP cleavage. We conclude that a DEVD-sensitive step, which could involve CPP32, plays a central role in the proteolytic pathway that mediates apoptotic DNA fragmentation in VP-16-treated leukemic cells at the crossing with Fas-mediated pathway.
Insights
Proteases, particularly CPP32, are crucial in etoposide-induced DNA damage leading to apoptosis in leukemic cells. This DEVD-sensitive pathway overlaps with Fas-mediated apoptosis, highlighting key targets for cancer therapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Etoposide (VP-16), a topoisomerase II inhibitor, induces DNA damage and apoptosis in cancer cells.
- Proteases play a significant role in the apoptotic cascade, but their specific involvement in VP-16-induced apoptosis requires elucidation.
- The U937 human leukemic cell line serves as a model for studying drug-induced apoptosis.
Purpose of the Study:
- To investigate the role of proteases in the apoptotic DNA fragmentation pathway induced by etoposide (VP-16).
- To identify specific proteases involved in etoposide-induced apoptosis and their relationship with Fas-mediated apoptosis.
- To explore potential therapeutic targets within the protease-mediated apoptotic pathway.
Main Methods:
- Utilized a reconstituted cell-free system with extracts from VP-16-treated U937 cells and nuclei from untreated cells.
- Employed specific protease inhibitors, including Ac-DEVD-CHO (CPP32 inhibitor) and Ac-YVAD-CHO/CMK (ICE inhibitors).
- Analyzed poly(ADP-ribose)polymerase (PARP) cleavage, CPP32 proenzyme levels, and the appearance of CPP32 active subunits.
- Assessed the impact of various cysteine and serine protease inhibitors on DNA fragmentation.
Main Results:
- Triton-soluble extracts from VP-16-treated cells induced internucleosomal DNA fragmentation in a cell-free system.
- This fragmentation was inhibited by Ac-DEVD-CHO, implicating CPP32, but not by ICE-specific inhibitors.
- VP-16 and anti-Fas antibody triggered DNA fragmentation associated with PARP cleavage and CPP32 activation.
- Ich-1L expression remained stable, suggesting a specific role for CPP32 in this pathway.
Conclusions:
- A DEVD-sensitive protease step, likely involving CPP32, is central to etoposide-induced apoptotic DNA fragmentation in U937 cells.
- This pathway converges with the Fas-mediated apoptotic pathway at a critical protease-dependent step.
- These findings identify CPP32 as a key mediator in etoposide-induced leukemic cell apoptosis, offering potential therapeutic insights.