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Activation of the cell death program by inhibition of proteasome function
1Department of Molecular Cell Biology, Max-Planck-Institut für Physiologische und Klinische Forschung, Bad Nauheim, Germany.
Abstract:
Activation of proteolytic enzymes, including cysteine proteases of the ced-3/ICE family, is a characteristic feature of the apoptotic program. In contrast, the role of the proteasome as the major nonlysosomal machinery to degrade or process proteins by ATP/ubiquitin-dependent proteolysis in this process is less clear. In human leukemic HL60 cells, inhibition of proteasome-mediated proteolysis by specific proteasomal inhibitors leads to the rapid induction of apoptosis as judged by morphological changes as well as by nuclear condensation and DNA fragmentation. HL60 apoptosis is due to activation of CPP32, a member of the ced-3/ICE family of cysteine proteases, and appears to occur independently from ICE activity. HL60 apoptosis is accompanied by an increase in the concentration of the cyclin-dependent kinase inhibitor p27Kip1. Labeling of the cells by the TUNEL technique demonstrates that HL60 cells undergoing apoptosis are primarily in the G1 phase of the cell cycle. Proteasomal activity therefore appears to be required in proliferating, but not in quiescent, HL60 cells for cell survival as well as normal progression through the cell cycle.
Insights
Inhibition of proteasome activity rapidly induces apoptosis in human leukemia cells by activating cysteine proteases like CPP32. This suggests proteasomes are crucial for cell survival and cell cycle progression in proliferating cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis involves cysteine proteases (ced-3/ICE family).
- The proteasome's role in apoptosis is less understood.
- Proteasomes degrade proteins via ATP/ubiquitin-dependent pathways.
Purpose of the Study:
- To investigate the role of proteasome-mediated proteolysis in apoptosis.
- To determine the effect of proteasome inhibition on human leukemic HL60 cells.
- To elucidate the relationship between proteasomes, apoptosis, and cell cycle progression.
Main Methods:
- Inhibition of proteasome activity using specific inhibitors in HL60 cells.
- Assessment of apoptosis via morphological changes, nuclear condensation, and DNA fragmentation (TUNEL assay).
- Analysis of cysteine protease activation (CPP32) and cyclin-dependent kinase inhibitor (p27Kip1) levels.
Main Results:
- Proteasome inhibition rapidly induced apoptosis in HL60 cells.
- Apoptosis was mediated by CPP32 activation, independent of ICE.
- Increased p27Kip1 levels and G1 cell cycle arrest were observed.
- Proteasomal activity is essential for survival and cell cycle progression in proliferating HL60 cells.
Conclusions:
- Proteasome-mediated proteolysis is critical for the survival of proliferating HL60 cells.
- Inhibition of proteasomes triggers apoptosis through CPP32 activation.
- Proteasomal function is required for normal cell cycle progression in these cells.