Abstract
Caspases are considered to be the key effector proteases of apoptosis. Initiator caspases cleave and activate downstream executioner caspases, which are responsible for the degradation of numerous cellular substrates. We studied the role of caspases in apoptotic cell death of a human melanoma cell line. Surprisingly, the pancaspase inhibitor zVAD-fmk was unable to block cleavage of poly(ADP-ribose) polymerase (PARP) after treatment with etoposide, while it did prevent DEVDase activity. It is highly unlikely that caspase-2, which is a relatively zVAD-fmk-resistant caspase, is mediating etoposide-induced PARP cleavage, as a preferred inhibitor of this caspase could not prevent cleavage. In contrast, caspase activation and PARP degradation were blocked by pretreatment of the cells with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). We therefore conclude that a serine protease regulates an alternative initiation mechanism that leads to caspase activation and PARP cleavage. More importantly, while zVAD-fmk could not rescue melanoma cells from etoposide-induced death, the combination with AEBSF resulted in substantial protection. This indicates that this novel pathway fulfills a critical role in the execution of etoposide-induced programmed cell death.
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Abbreviations
- AEBSF:
-
4-(2-aminoethyl)benzenesulfonyl fluoride
- AFC:
-
7-amino-4-trifluoromethyl-coumarin
- CHAPS:
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
- etop:
-
etoposide, VP16
- PARP:
-
poly(ADP-ribose) polymerase-1
- PBS:
-
phosphate-buffered saline
- PI:
-
propidium iodide
- PMSF:
-
phenylmethylsulfonyl fluoride
- SDS:
-
sodium dodecylsulfate
- TNFα:
-
tumor necrosis factor α
- zVAD-fmk:
-
benzyloxy-carbonyl-Val-Ala-Asp-fluoromethyl ketone
- zVDVAD-fmk:
-
benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone
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Acknowledgements
We thank René Medema for providing the rat fibroblasts, Bob van de Water, Arnoud van der Laarse and Merlijn Bazuine for reagents, and Jennifer Rohn and Corrie Marijnen for critical reading of this manuscript. This work was supported by grants from the Vanderes Foundation, the J.A. Cohen Institute, the Dutch Science Organization and the Foundation Clinical Oncology Leiden.
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de Bruin, E., Meersma, D., de Wilde, J. et al. A serine protease is involved in the initiation of DNA damage-induced apoptosis. Cell Death Differ 10, 1204–1212 (2003). https://doi.org/10.1038/sj.cdd.4401296
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DOI: https://doi.org/10.1038/sj.cdd.4401296
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