title: Inhibition of ubiquitin-proteasome pathway activates a caspase-3-like protease and induces Bcl-2 cleavage in human M-07e leukaemic cells.
abstract: The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Here we examine the possibility that ubiquitin-proteasome is involved in regulating the levels of Bcl-2, which is abundantly expressed in M-07e cells, a granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent human leukaemic cell line. Apoptosis in M-07e cells, induced by GM-CSF withdrawal, was associated with a gradual cleavage of Bcl-2 into a 22 kDa fragment. Treatment of M-07e cells with benzyloxycarbonyl-Leu-Leu-l-leucinal (Z-LLL-CHO; MG-132), a reversible ubiquitin-proteasome inhibitor, markedly accelerated the cleavage of Bcl-2 and promoted cell death through the apoptotic pathway. The cleavage of Bcl-2 was inhibited by a caspase-3 (CPP32)-specific inhibitor [acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO)] but not caspase 1 inhibitor (acetyl-Tyr-Val-Ala-Asp-CHO), suggesting that Bcl-2 is a proteolytic substrate of a caspase-3-like protease activated during apoptosis. The simultaneous addition of recombinant human GM-CSF (rhGM-CSF) to M-07e cultures delayed the activation of caspase 3 and Bcl-2 cleavage triggered by Z-LLL-CHO, suggesting that the activation of the GM-CSF signalling pathway can partly overcome the apoptotic effect induced by Z-LLL-CHO. Apoptosis induced by inhibition of the proteasome pathway was verified in studies with lactacystin, a highly specific and irreversible proteasome inhibitor. Lactacystin-induced apoptosis in M-07e cells was remarkably similar to that induced by Z-LLL-CHO, which included caspase 3 activation, cleavage of Bcl-2 into a 22 kDa fragment and, ultimately, cell death. These results showed that inhibition of the ubiquitin-proteasome pathways can lead to the activation of a DEVD-CHO-sensitive caspase and induces Bcl-2 cleavage, which might have a role in mediating apoptosis in M-07e cells.
authors.affiliation: Division of Hematology-Oncology, Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: X M
authors.initials: XM
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Lin
authors.forename: C
authors.initials: C
authors.lastname: Chen
authors.forename: B D
authors.initials: BD
authors.lastname: Chen
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007938:Leukemia; D007976:Leupeptins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D007976:Leupeptins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D014452:Ubiquitins; C108637:aspartyl-glutamyl-valyl-aspartal; C067713:lactacystin; C096429:L 709049; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine