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.

Protease inhibitors restore radiation‐induced apoptosis to Bcl‐2‐expressing lymphoma cells

“…Our finding that protease inhibitors overcame the protective effects of Bcl-2 agrees with previous findings [4,11,12]. Like Zhang et al [11], we observed a cleaved form of Bcl-2 that arose with protease inhibitor treatment.…”

“…Like Zhang et al [11], we observed a cleaved form of Bcl-2 that arose with protease inhibitor treatment. Cheng et al [23] have proposed that the cleaved product is capable of acting as a Bax-like death effector.…”

“…2). Additionally, a slower-migrating band, which corresponded to the molecular weight of a caspase-3-cleaved Bcl-2 protein, appeared as described in other reports [11,23].…”

“…In tumor cells of lymphoid origin such as Tcell leukemia cells, Hodgkin's cells, and chronic lymphocytic leukemic (CLL) lymphocytes, apoptosis can be induced by treatment with proteasome inhibitors [4][5][6][7][8][9][10][11]. Furthermore, many investigators have demonstrated that inhibition of the proteasome results in apoptosis despite high expression of Bcl-2 protein [4,11,12].…”

“…Furthermore, many investigators have demonstrated that inhibition of the proteasome results in apoptosis despite high expression of Bcl-2 protein [4,11,12]. The mechanisms by which proteasome inhibitors potentiate cell death in such instances have often been attributed to stabilization of the proapoptotic p53 protein [8,10,13] or to inhibition of the antiapoptotic transcription factor NF-B [5,6,14].…”