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1.
Cancer Res ; 70(11): 4318-26, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20460535

ABSTRACT

Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.


Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Cell Death/drug effects , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Pyrazines/pharmacology , RNA, Small Interfering/genetics , Bortezomib , Cell Death/genetics , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , DNA Damage , Gene Knockdown Techniques , HCT116 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , Ribosomes/drug effects , TOR Serine-Threonine Kinases , Transfection
2.
Mol Cancer Ther ; 6(1): 262-8, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17237285

ABSTRACT

Poh1 deubiquitinase activity is required for proteolytic processing of polyubiquitinated substrates by the 26S proteasome, linking deubiquitination to complete substrate degradation. Poh1 RNA interference (RNAi) in HeLa cells resulted in a reduction in cell viability and an increase in polyubiquitinated protein levels, supporting the link between Poh1 and the ubiquitin proteasome pathway. To more specifically test for any requirement of the zinc metalloproteinase motif of Poh1 to support cell viability and proteasome function, we developed a RNAi complementation strategy. Effects on cell viability and proteasome activity were assessed in cells with RNAi of endogenous Poh1 and induced expression of wild-type Poh1 or a mutant form of Poh1, in which two conserved histidines of the proposed catalytic site were replaced with alanines. We show that an intact zinc metalloproteinase motif is essential for cell viability and 26S proteasome function. As a required enzymatic component of the proteasome, Poh1 is an intriguing therapeutic drug target for cancer.


Subject(s)
Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Ubiquitin/metabolism , Amino Acid Motifs , Cell Survival , HeLa Cells , Humans , Mutant Proteins/metabolism , Proteasome Endopeptidase Complex/deficiency , RNA Interference , Trans-Activators/deficiency
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