Pivotal role of a DEVD-sensitive step in etoposide-induced and Fas-mediated apoptotic pathways.
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.
Laboratory of Onco-Hematology and Pharmacology, CJF INSERM 94-08, Faculty of Medicine, Dijon, France.
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Dubrez
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Savoy
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Hamman
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Solary
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004792:Enzyme Precursors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology*; D019014:fas Receptor / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D009842:Oligopeptides; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; C096429:L 709049; D005047:Etoposide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases