Constitutive Degradation of PML/RAR Through the Proteasome Pathway Mediates Retinoic Acid Resistance

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1477-1481 ◽  
Author(s):  
Mirco Fanelli ◽  
Saverio Minucci ◽  
Vania Gelmetti ◽  
Clara Nervi ◽  
Carlo Gambacorti-Passerini ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Proteasome Inhibitors ◽  
Acid Resistance ◽  
Parental Cell ◽  
Parental Cell Line ◽  
In Vitro Degradation ◽  
Nb4 Cells ◽  
Proteasome Pathway ◽  
Retinoic Acid Resistance

Abstract PML/RAR is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RAR, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RAR protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RAR is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RAR protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RAR, but not RAR, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RAR protein may lead to RA resistance and that PML/RAR expression is crucial to convey RA sensitivity to APL cells.

Constitutive Degradation of PML/RAR Through the Proteasome Pathway Mediates Retinoic Acid Resistance

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1477-1481 ◽  
Author(s):  
Mirco Fanelli ◽  
Saverio Minucci ◽  
Vania Gelmetti ◽  
Clara Nervi ◽  
Carlo Gambacorti-Passerini ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Proteasome Inhibitors ◽  
Acid Resistance ◽  
Parental Cell ◽  
Parental Cell Line ◽  
In Vitro Degradation ◽  
Nb4 Cells ◽  
Proteasome Pathway ◽  
Retinoic Acid Resistance

PML/RAR is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RAR, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RAR protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RAR is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RAR protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RAR, but not RAR, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RAR protein may lead to RA resistance and that PML/RAR expression is crucial to convey RA sensitivity to APL cells.

Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Kellie R Machlus ◽  
Prakrith Vijey ◽  
Thomas Soussou ◽  
Joseph E Italiano
Keyword(s):  
Protein Degradation ◽  
Proteasome Inhibitors ◽  
Aurora Kinase ◽  
Proteasome Activity ◽  
Major Pathway ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Proplatelet Formation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

Revealing a natural marine product as a novel agonist for retinoic acid receptors with a unique binding mode and inhibitory effects on cancer cells

2012 ◽  
Vol 446 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Shanshan Wang ◽  
Zhao Wang ◽  
Shengchen Lin ◽  
Weili Zheng ◽  
Rui Wang ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cancer Cells ◽  
Target Genes ◽  
Functional Characterization ◽  
Acid Resistance ◽  
Binding Mode ◽  
Therapeutic Agents ◽  
Covalent Modification ◽  
Marine Product

Retinoids display anti-tumour activity on various cancer cells and therefore have been used as important therapeutic agents. However, adverse side effects and RA (retinoic acid) resistance limit further development and clinical application of retinoid-based therapeutic agents. We report in the present paper the identification of a natural marine product that activates RARs (RA receptors) with a chemical structure distinct from retinoids by high-throughput compound library screening. Luffariellolide was uncovered as a novel RAR agonist by inducing co-activator binding to these receptors in vitro, further inhibiting cell growth and regulating RAR target genes in various cancer cells. Structural and molecular studies unravelled a unique binding mode of this natural ligand to RARs with an unexpected covalent modification on the RAR. Functional characterization further revealed that luffariellolide displays chemotherapeutic potentials for overcoming RA resistance in colon cancer cells, suggesting that luffariellolide may represent a unique template for designing novel non-retinoid compounds with advantages over current RA drugs.

scholarly journals Hepatitis C Virus F Protein Is a Short-Lived Protein Associated with the Endoplasmic Reticulum

2003 ◽  
Vol 77 (2) ◽  
pp. 1578-1583 ◽  
Author(s):  
Zhenming Xu ◽  
Jinah Choi ◽  
Wen Lu ◽  
Jing-hsiung Ou
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Half Life ◽  
Proteasome Inhibitors ◽  
Subcellular Fractionation ◽  
Biological Properties ◽  
F Protein ◽  
Proteasome Pathway

ABSTRACT Hepatitis C virus (HCV) F protein is a newly discovered HCV gene product that is expressed by translational ribosomal frameshift. Little is known about the biological properties of this protein. By performing pulse-chase labeling experiments, we demonstrate here that the F protein is a labile protein with a half-life of <10 min in Huh7 hepatoma cells and in vitro. The half-life of the F protein could be substantially increased by proteasome inhibitors, suggesting that the rapid degradation of the F protein is mediated by the proteasome pathway. Further immunofluorescence staining and subcellular fractionation experiments indicate that the F protein is primarily associated with the endoplasmic reticulum. This subcellular localization is similar to those of HCV core and NS5A proteins, raising the possibility that the F protein may participate in HCV morphogenesis or replication.

Retinoic acid resistance in NB4 APL cells is associated with lack of interferon α synthesis Stat1 and p48 induction

Oncogene ◽  
1999 ◽  
Vol 18 (27) ◽  
pp. 3944-3953 ◽  
Author(s):  
Luis Pelicano ◽  
Caren Brumpt ◽  
Paula M Pitha ◽  
Mounira K Chelbi-Alix
Keyword(s):  
Retinoic Acid ◽  
Acid Resistance ◽  
Interferon Α ◽  
Retinoic Acid Resistance

scholarly journals Retinoic acid activates interferon regulatory factor-1 gene expression in myeloid cells

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 114-123 ◽  
Author(s):  
S Matikainen ◽  
T Ronni ◽  
M Hurme ◽  
R Pine ◽  
I Julkunen
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Growth Inhibition ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Oligoadenylate Synthetase ◽  
Nb4 Cells ◽  
Drug Of Choice

All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2′–5′ oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells.

scholarly journals Dephosphorylation Targets Bcl-2 for Ubiquitin-dependent Degradation: A Link between the Apoptosome and the Proteasome Pathway

1999 ◽  
Vol 189 (11) ◽  
pp. 1815-1822 ◽  
Author(s):  
Stefanie Dimmeler ◽  
Kristin Breitschopf ◽  
Judith Haendeler ◽  
Andreas M. Zeiher
Keyword(s):  
Endothelial Cells ◽  
Map Kinase ◽  
Inflammatory Diseases ◽  
Apoptotic Cell ◽  
Proteasome Inhibitors ◽  
Inhibitory Function ◽  
Tnf Α ◽  
Proteasome Pathway

Injury of the endothelial cells by the induction of apoptotic cell death may play an important role in the pathophysiology of atherosclerosis and the progression of inflammatory diseases. Here, we demonstrate an essential role for the ubiquitin-dependent proteasome complex in stimulus-induced degradation of the antiapoptotic protein Bcl-2. Bcl-2 is specifically degraded after stimulation of human endothelial cells with tumor necrosis factor (TNF)-α in a process that is inhibited by specific proteasome inhibitors. In addition, the mutation of the potential ubiquitin-acceptor amino acids of Bcl-2 provides protection against TNF-α– and staurosporine-induced degradation in vitro and in vivo. Moreover, mimicking phosphorylation of the putative mitogen-activated protein (MAP) kinase sites of the Bcl-2 protein (Thr 56, Thr 74, and Ser 87) abolishes its degradation, suggesting a link between the MAP kinase pathway to the proteasome pathway. Finally, inhibition of Bcl-2 degradation either by suppressing ubiquitin-dependent proteasomal degradation or by mimicking continuous phosphorylation of the putative MAP kinase sites in the Bcl-2 protein confers resistance against induction of apoptosis. Thus, the degradation of Bcl-2 may unleash the inhibitory function of Bcl-2 over the apoptosome and may thereby amplify the activation of the caspase cascade.

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