Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format

Moravec, Richard A.; O’Brien, Martha A.; Daily, William J.; Scurria, Michael A.; Bernad, Laurent; Riss, Terry

doi:10.1016/j.ab.2009.01.016

Cited by 63 publications

(61 citation statements)

References 19 publications

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“…The amount of luminescence of each sample correlated to the amount of chymotrypsin-like proteasomal activity, and the recorded luminescence of each sample was displayed graphically as relative light units (Fig. 7A) (36). Mock-infected cells demonstrated high luminescence, corresponding to functional proteasome activity.…”

Section: Vv811 Inhibits Tnf-␣-induced Nf-b P65 Translocation To the Nmentioning

confidence: 99%

“…Since proteasome inhibition affects poxvirus late protein production, we sought to determine if the proteasome was functional during vaccinia virus infection (50,61). The chymotrypsin-like activity of the proteasome was analyzed during vaccinia virus infection using a cell-based luciferase assay (36). Cells were mock infected or infected with VVCop or VV811 for 14 h, and MG132 was added at 12 h postinfection.…”

Section: Vv811 Inhibits Tnf-␣-induced Nf-b P65 Translocation To the Nmentioning

confidence: 99%

“…Cells were untreated or treated with 10 M MG132 at 12 h postinfection. The chymotrypsin-like activity of the proteasome was assessed using the cell-based Proteasome-Glo assay, according to the manufacturer's directions (Promega) (36). Luminescence was recorded 10 min after addition of the Proteasome-Glo reagent using an EnVision 2104 multilabel reader (Perkin-Elmer).…”

mentioning

confidence: 99%

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A Vaccinia Virus Deletion Mutant Reveals the Presence of Additional Inhibitors of NF-κB

Fagan-Garcia

Barry

2011

J Virol

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Section: Vv811 Inhibits Tnf-␣-induced Nf-b P65 Translocation To the Nmentioning

confidence: 99%

Section: Vv811 Inhibits Tnf-␣-induced Nf-b P65 Translocation To the Nmentioning

confidence: 99%

mentioning

confidence: 99%

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A Vaccinia Virus Deletion Mutant Reveals the Presence of Additional Inhibitors of NF-κB

Fagan-Garcia

Barry

2011

J Virol

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“…proteasome activity was determined with the aid of the proteasome-Glo™ Chymotrypsin-like cellbased assay (promega France, Charbonnières-les-Bains, France) that measures chymotrypsin-like protease activity associated with the proteasome complex directly in intact cells. This assay has been used to evaluate the activity of bortezomib in a variety of cancer cell lines (28). It has three components: a buffer containing the proteasome complex, a luciferin detection reagent and the luminogenic substrate, succinyl-LLVY-aminoluciferin.…”

Section: Methodsmentioning

confidence: 99%

Hyperforin induces apoptosis of chronic lymphocytic leukemia cells through upregulation of the BH3-only protein Noxa

Billard

2011

Int J Oncol

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Abstract. We previously reported that hyperforin, a phloroglucinol purified from Hypericum perforatum, induces the mitochondrial pathway of caspase-dependent apoptosis in chronic lymphocytic leukemia (CLL) cells ex vivo, and that this effect is associated with upregulation of Noxa, a BH3-only protein of the Bcl-2 family. Here, we investigated the role of this upregulation in the pro-apoptotic activity of hyperforin in the cells of CLL patients and MEC-1 cell line. We found that the increase in Noxa expression is a time-and concentrationdependent effect of hyperforin occurring without change in Noxa mRNA levels. A post-translational regulation is suggested by the capacity of hyperforin to inhibit proteasome activity in CLL cells. Noxa silencing by siRNA reduces partially hyperforin-elicited apoptosis. Furthermore, treatment with hyperforin, which has no effect on the expression of the prosurvival protein Mcl-1, induces the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/ Bak complex, revealing that upregulated Noxa displaces the proapoptotic protein Bak from Mcl-1. This effect is accompanied with Bak activation, known to allow the release of apoptogenic factors from mitochondria. Our data indicate that Noxa upregulation is one of the mechanisms by which hyperforin triggers CLL cell apoptosis. They also favor that new agents capable of mimicking specifically the BH3-only protein Noxa should be developed for apoptosis-based therapeutic strategy in CLL.

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“…Proteasome activity was determined with a luminescent assay, the Proteasome-Glo TM Chymotrypsin-like cell-based assay (Promega France, Charbonnières-les-Bains, France) that measures chymotrypsin-like protease activity associated with the proteasome complex directly in intact cells. This assay has been used to evaluate the activity of bortezomib in a variety of cancer cell lines (26). It has three components: a buffer containing the proteasome complex, a luciferin detection reagent and the luminogenic substrate, succinyl-LLVY-aminoluciferin.…”

Section: Methodsmentioning

confidence: 99%

The BH3-only protein Noxa is stimulated during apoptosis of chronic lymphocytic leukemia cells triggered by M2YN, a new plant-derived extract

Billard

2011

Int J Oncol

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Abstract. Deficiency of apoptosis is a hallmark of chronic lymphocytic leukemia (CLL) cells. M2Yn is a natural extract from plants of central Asia, identified for its antiangiogenic properties and its ability to block the migration of malignant cells. Here, we report that in vitro treatment of cells derived from CLL patients with M2Yn results in internucleosomal DNA fragmentation, phosphatidylserine externalization, mitochondrial membrane depolarization, caspase-3 activation and cleavage of the caspase substrate PARP-1. The extents of these effects depend on the patients and are mostly comparable to those of flavopiridol or hyperforin, two known plant-derived apoptosis inducers of CLL cells. M2Yn does not modulate Mcl-1 expression, while downregulation of this antiapoptotic protein is involved in the action of flavopiridol. By contrast, M2Yn, like hyperforin, upregulates the Noxa protein, possibly by inhibiting proteasomal activity. This BH3-only protein is known to trigger the activation of the pro-apoptotic protein Bak through displacement of the Mcl-1/Bak complex at the mitochondrial membrane, as actually observed here in M2Yn-treated cells. Our data, therefore, show that M2Yn can induce the caspase-dependent mitochondrial pathway of apoptosis in CLL cells via a mechanism resembling that of hyperforin. Our data also confirm that the BH3-only protein Noxa is a relevant target for CLL therapy.

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Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format

Cited by 63 publications

References 19 publications

A Vaccinia Virus Deletion Mutant Reveals the Presence of Additional Inhibitors of NF-κB

A Vaccinia Virus Deletion Mutant Reveals the Presence of Additional Inhibitors of NF-κB

Hyperforin induces apoptosis of chronic lymphocytic leukemia cells through upregulation of the BH3-only protein Noxa

The BH3-only protein Noxa is stimulated during apoptosis of chronic lymphocytic leukemia cells triggered by M2YN, a new plant-derived extract

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