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Characterization of CPP32-like protease activity following apoptotic challenge in SH-SY5Y neuroblastoma cells
R Posmantur1, K McGinnis, R Nadimpalli
1Department of Immunopathology, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, Michigan 48105, U.S.A.
Abstract:
We characterized the activation of interleukin-1beta-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH2OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.
Insights
Staurosporine induces apoptosis in neuroblastoma cells by activating caspases, like CPP32, leading to cell death and DNA fragmentation. A pan ICE inhibitor blocked these apoptotic events.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Apoptosis is a crucial process in cellular homeostasis and disease.
- Interleukin-1beta-converting enzyme (ICE)-like proteases, now known as caspases, play a central role in apoptosis.
- Neuroblastoma is a pediatric cancer with a significant need for understanding apoptotic pathways.
Purpose of the Study:
- To characterize the activation of caspases in human neuroblastoma cells (SH-SY5Y) induced by staurosporine.
- To investigate the role of CPP32 and poly(ADP-ribose) polymerase (PARP) in staurosporine-induced apoptosis.
- To examine the cellular localization changes of CPP32 and PARP during apoptosis.
Main Methods:
- Time course analysis of lactate dehydrogenase release to measure cell death.
- Western blot analysis to detect proteolytic processing of CPP32 and PARP.
- Enzyme activity assay using a CPP32 substrate (acetyl-DEVD-7-amido-4-methylcoumarin).
- Immunohistochemistry to visualize CPP32 and PARP localization in cells.
- Treatment with a pan ICE inhibitor to assess its effect on apoptosis.
Main Results:
- Staurosporine treatment significantly increased cell death starting at 6 hours and continuing to 24 hours.
- Proteolytic processing of CPP32 and PARP fragmentation occurred as early as 3 hours post-treatment.
- CPP32 activity was detected by 3 hours and peaked at 6 hours, indicating sustained activation.
- CPP32 translocated from the cytoplasm to the plasma membrane, while PARP shifted from the nucleus to condensed chromatin.
- A pan ICE inhibitor reduced cell death, PARP/CPP32 cleavage, and altered protein localization.
Conclusions:
- Staurosporine effectively induces apoptosis in SH-SY5Y neuroblastoma cells.
- CPP32-like caspases are activated early and sustained during staurosporine-induced apoptosis.
- PARP cleavage and altered subcellular localization of CPP32 and PARP are key events in this apoptotic process.
- Inhibition of ICE-like proteases can effectively block staurosporine-induced apoptosis in neuroblastoma cells.