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.

The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

“…Concerted concession of NHEJ requires the activity of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), which is a known substrate of caspase-3. In this study we therefore focused on the possibility that proteasome inhibition by MG-132 activates caspase-3, as has been reported previously [24,30]. This could mediate apoptosis and cause degradation of DNA-PKcs, the catalytic subunit of DNA-PK [20], resulting in radiosensitization.…”

“…A possible association between apoptosis and radiosensitization exist through caspase activation and subsequent proteolytic destruction of DNA repair enzymes. Caspase activation has been reported to follow proteasome inhibition [24,29,30] and is known to degrade the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), a key DNA repair enzyme of non-homologous end-joining (NHEJ) of DNA double strand breaks [31,32]. …”

Proteasomes in apoptosis: villains or guardians?

“…However, high expression of functional Bcl-2 protein in prostate carcinoma cells failed to signal protection against cell Review Article death induction by proteasome inhibitors [82]. It is possible that this was due to Bcl-2 proteolysis, as shown in the case of MO7e cells, where proteasome inhibitors induce apoptosis with caspase-3 activation, which in turn induces Bcl-2 cleavage [83]. In human endothelial cells, Bcl-2 is specifically degraded after stimulation with TNF-a in a process that is inhibited by particular proteasome inhibitors.…”

Ceramide generation during curcumin-induced apoptosis is controlled by crosstalk among Bcl-2, Bcl-xL, caspases and glutathione

“…5). Several reports have indicated that Bcl-2 can be cleaved by caspase-8 and caspase-3 [58][59][60], and a positive feedback mechanism between the activity of caspase-3 and cytochrome c release due to Bcl-2 downregulation has been shown [61]. The lack of an interdependence between Bcl-xL and caspase-3 found in our studies suggests that Bcl-xL degradation precedes activation of the executioner caspase-3; in agreement, Bcl-xL is a poor substrate for caspase-3 in vitro [62].…”