scholarly journals Effect of proteasome inhibition by MG-132 on HSP27 oligomerization, phosphorylation, and aggresome formation in the OLN-93 oligodendroglia cell line

2010 ◽  
Author(s):  
Sandra Bolhuis ◽  
Christiane Richter-Landsberg
Keyword(s):  
Cell Line ◽  
Proteasome Inhibition ◽  
Aggresome Formation

The Novel Immunoproteasome Inhibitor PR-924: Anti-Leukemic Activity and Mechanisms Of Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3841-3841
Author(s):  
Denise Niewerth ◽  
Johan van Meerloo ◽  
Yehuda Assaraf ◽  
Gerrit Jansen ◽  
Tessa C Hendrickx ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Line ◽  
Hematological Malignancies ◽  
Acquired Resistance ◽  
Proteasome Inhibition ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Gene Encoding ◽  
Exon 2 ◽  
Subunit Expression

Abstract Proteasome inhibition with bortezomib (BTZ) is an effective treatment for hematological malignancies and an emerging treatment strategy for acute leukemia. However, the side-effects of BTZ and emergence of BTZ-resistance call for alternative therapeutic approaches. The immunoproteasome may serve as a candidate since its expression is higher than constitutive proteasome expression in cells of hematological malignancies, including acute lymphocytic leukemia (ALL). To this end, the new immunoproteasome inhibitor PR-924 may elicit anti-leukemic activity, since it bears greater specificity for the β5i subunit of the immunoproteasome compared to other proteasome inhibitors including BTZ and carfilzomib. The aim of the current study was to examine the proteasome inhibition capacity and cell growth inhibitory impact of PR-924 in acute leukemia cells and sublines with acquired resistance to BTZ. We further determined whether PR-924 itself would be prone to resistance development and if so, to explore the underlying molecular basis. To assess the anti-leukemic activity of PR-924, its cytotoxicity was determined in two human cell lines of hematological origin: the T-ALL cell line CCRF-CEM and the acute myeloid leukemia cell line THP1, and their 150-fold BTZ-resistant sublines CEM/BTZ200, and THP1/BTZ200, respectively. Parental CEM and THP1 cells displayed similar sensitivity to PR-924 (IC50 CEM: 1.8 µM ± 0.4, THP1: 1.5 µM ± 0.2), whereas their BTZ-resistant lines displayed a moderate 10-12 fold cross-resistance to PR-924 (IC50 CEM/BTZ200: 21.1 µM ± 0.5, THP1/BTZ200: 15.6 µM ± 1.6). Flow cytometric analysis revealed that PR-924-induced cell death was mediated by induction of apoptosis. Moreover, PR-924 exposure resulted in a (up to 50%) reduction of cell surface expression of HLA Class I. To determine whether PR-924 activity relies on specific inhibition of β5i, proteasome activity inhibition experiments were performed over a range of 10 nM – 10 µM PR-924. Notably, in parental cells, β5i activity was already inhibited by 20% at 10 nM PR-924 and > 90% inhibition was achieved at 100 nM PR-924. At higher PR-924 concentrations of 1-10 µM, inhibition of both β5 and β1i activities was observed, thus indicating that PR-924 blocks the β5i activity far below concentrations that exert anti-proliferative activity (1.5 µM), consistent with earlier studies of PR-924 (Parlati et al. Blood, 2009). Next, acquired resistance to PR-924 was provoked in CEM and THP1 cells by step-wise increasing PR-924 concentrations in cell culture, starting at the IC50 concentrations. Following this process, CEM cells resistant to 20 µM PR-924 (CEM/PR20) and THP1 cells resistant to 12 mM PR-924 (THP1/PR12) exhibited IC50 values of 22.1 µM PR-924 (resistance factor 13) and 14.3 µM PR-924 (resistance factor 10), respectively. In addition, these PR-924 resistant cells displayed 10-fold cross-resistance to BTZ. To explore mechanisms of PR-924 resistance, we first sequenced exon 2/3 of the PSMB8 gene (encoding β5i), a similar functioning coding region that was previously reported to harbor PSMB5 mutations in BTZ-resistant leukemia cells (Franke et al. Leukemia, 2012). However, no mutations were found in PSMB8 exon 2/3. Remarkably, we did identify mutations in exon 2 of the PSMB5 gene (encoding the S1 pocket of β5) in both CEM/PR20 (Met45Ile) and of THP1/PR12 (Ala49Thr); these amino acid substitutions are similar to those observed in BTZ-resistant sublines. Finally, we characterized proteasome subunit expression by ProCISE analysis in the PR-924 resistant cell lines. This method is a subunit-specific active-site ELISA assay, which utilizes a purified proteasome standard curve to calculate ng of subunit per µg total protein. Expression of constitutive proteasome subunits in CEM/PR20 and THP1/PR8 cells were upregulated up to 2.5-fold, compared to their parental counterparts, whereas immunoproteasome subunit expression was moderately decreased (up to 2-fold) in CEM/PR20 and increased in THP1/PR8 (up to 1.6-fold). In conclusion, PR-924 displayed significant anti-leukemic activity. Although there was cross-resistance to BTZ, PR-924 retained activity in BTZ-resistant leukemia cells. Despite PR-924 specificity to the β5i subunit of the proteasome, its anti-leukemic effect requires concentrations that block both β5 and β5i subunits. This notion is underscored by emergence of acquired mutations in PSMB5 rather than in PSMB8. Disclosures: Kirk: Onyx Pharmaceuticals: Employment, Equity Ownership. Anderl:Onyx: Employment.

scholarly journals The p97-UBXN1 complex regulates aggresome formation

2020 ◽  
Author(s):  
Sirisha Mukkavalli ◽  
Jacob Aaron Klickstein ◽  
Betty Ortiz ◽  
Peter Juo ◽  
Malavika Raman
Keyword(s):  
Protein Aggregation ◽  
Mammalian Cells ◽  
Proteasome Inhibition ◽  
Adaptor Proteins ◽  
Ubiquitin Proteasome System ◽  
Aaa Atpase ◽  
Evolutionarily Conserved ◽  
Ubiquitin Proteasome ◽  
Aggresome Formation

AbstractThe recognition and disposal of misfolded proteins are essential for the maintenance of cellular homeostasis. Perturbations in the pathways that promote degradation of aberrant proteins contribute to a variety of protein aggregation disorders broadly termed proteinopathies. It is presently unclear how diverse disease-relevant aggregates are recognized and processed for degradation. The p97 AAA-ATPase in combination with a host of adaptor proteins functions to identify ubiquitylated proteins and target them for degradation by the ubiquitin-proteasome system or through autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear given the large number of pathways that rely on p97 function. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes which are membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates both aggresome formation and clearance in proteasome-inhibited cells. We identify a novel and specific role for the p97 adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to ubiquitin-positive aggresomes and UBXN1 knockout cells are unable to form a single aggresome, and instead display dispersed ubiquitin aggregates. Furthermore, loss of p97-UBXN1 results in the increase in Huntingtin polyQ aggregates both in mammalian cells as well as in a C.elegans model of Huntington’s Disease. Together our work identifies evolutionarily conserved roles for p97 and its adaptor UBXN1 in the disposal of protein aggregates.

scholarly journals Breast cancer cell line MCF7 escapes from G1/S arrest induced by proteasome inhibition through a GSK-3β dependent mechanism

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Elena Gavilán ◽  
Servando Giráldez ◽  
Inmaculada Sánchez-Aguayo ◽  
Francisco Romero ◽  
Diego Ruano ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Breast Cancer Cell Line ◽  
Cancer Cell Line ◽  
Proteasome Inhibition ◽  
Gsk 3Β ◽  
Dependent Mechanism

Melphalan Resistance in Multiple Myeloma Can Be Overcome by Proteasome Inhibition or Histone Deacetylation: Lack of Syngergism of the Combination.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3393-3393
Author(s):  
Pieter Sonneveld ◽  
Eric Kamst ◽  
Yvonne de Knegt ◽  
Naomi Klarenbeek ◽  
Martijn Schoester
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Proteasome Inhibition ◽  
Histone Deacetylation ◽  
Interaction Factor ◽  
Resistant Cells

Abstract Multiple Myeloma (MM) is a disease of monoclonal plasma cells in the bone marrow which has a transient response to classic chemotherapy. At diagnosis, induction chemotherapy followed by high-dose melphalan (HDM) with stem cell support is used in most patients to achieve a clinical response. Because all patients will ultimately relapse, the treatment of melphalan-refractory disease represents a major clinical challenge and new agents are needed to overcome melphalan resistance. We have investigated the anti-myeloma efficacy of two new classes of targeted agents, i.e. proteasome inhibition and histone deacetylation inhibition alone or in combination in the melphalan sensitive MM1S and the Melphalan refractory MM1MEL2000 cell lines. The IC50 values of Bortezomib (B), Melphalan (M) and LAQ824 (L) in MM1S were 2.1 nM, 1.9 uM and 1.7 nM, respectively and in MM1MEL2000 3.9 nM, 50 uM and 4.0 nM. Using isobologram analysis a synergysm between B and L was observed in the sensitive, however not in the melphalan refractory cell line. These data indicate that B proteasome inhibition and histone deacetylation inhibition may be effective ways to overcome melphalan resistance. However, the previously reported synergism between these drugs does not seem to occur in melphalan resistant cells. The gene expression profiles of these cell lines were analysed using the Affymetrix U133plus 2.0 gene chip before and after treatment with melfaphalan or the proteasome inhibitor B or the histone deacetylation inhibitor L or the combination of B and L. Genes that were highly expressed in the melphalan refractory derivate cell line MM1MEL2000 as compared with wild-type MM1S included GP M6B, ADAM23 and HTPAP. Following melphalan exposure, TMF1, a CEBp glucocorticoid interaction factor, WHSC1L1, a MMSET homologue with EGF like domain and several transcription factors had highly increased expression as compared to MM1S. With exposure to B combined with L, increased expression in MM1MEL2000 over MM1S was observed for GTP exchange factor TIAM1 which interacts with RAS and JNK, and the lymphoid enhancer factor, a notch transcription factor. It is concluded that Bortezomib and the histone deacetylase inhibitor LAQ824 are effective agents to overcome melphalan resistance in multiple myeloma. However, the combination fails to show the synergism observed in melphalan sensitive cells. Gene analysis sofar does not provide a clear explanation for this lack of synergism. A comprehensive summary of the observed shifts of gene expression profiles in melphalan resistant cells following exposure to these agents, will be presented.

A Rationale to Enhance the Response to Antimitotic Therapy in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1332-1332
Author(s):  
Dominik Schnerch ◽  
Julia Felthaus ◽  
Monika Engelhardt ◽  
Ralph Wäsch
Keyword(s):  
Cell Line ◽  
Mitotic Spindle ◽  
Proteasome Inhibitor ◽  
Myeloid Leukemia ◽  
Proteasome Inhibition ◽  
Cyclin B ◽  
Low Doses ◽  
Reporter System ◽  
Mitotic Block ◽  
Acute Myeloid

Abstract Abstract 1332 Acute myeloid leukemia (AML) is known to respond only moderately to antimitotic therapy while acute lymphoblastic leukemias can be efficiently targeted using spindle-disrupting agents. The underlying molecular cause for this clinical phenomenon is unknown. Recent evidence suggests that response to antimitotic therapy substantially depends on the stability of the critical mitotic regulator cyclin B. The ability to keep cyclin B expression levels stable during a mitotic block is associated with a good response leading to cell death in mitosis. At the metaphase to anaphase transition of an unperturbed cell division, cyclin B is targeted for degradation by the anaphase-promoting complex/cyclosome (APC/C) to trigger chromosome separation. The spindle assembly checkpoint (SAC) is a surveillance mechanism to ensure that APC/C-mediated ubiquitylation is restricted to cells that show proper attachment of all chromosomes to a functional mitotic spindle. In case of spindle disruption or unattached chromosomes, the spindle checkpoint stays active which leads to interference with APC/C-dependent proteolysis of cyclin B blocking cells in prometaphase until every chromosome is attached to the mitotic spindle. We recently developed a cell line-based reporter system which allows monitoring of cyclin B degradation under various conditions (Schnerch et al. Cell Cycle 2012). Here, we identified a pattern of slow degradation of cyclin B which continues through a mitotic block in case of chromosomal misalignment in unperturbed cell cycles. Remarkably, we also found prolonged slow degradation to trigger aberrant exit from mitosis in such cells giving rise to tetraploid cells. Therefore, a reduction in slow degradation appears as a promising rationale to foster a mitotic arrest and enhance cell death in mitosis during antimitotic therapy by preventing such mitotic slippage. We exposed our reporter cells to low concentrations of proteasome inhibitor during a spindle poison induced mitotic block to assess whether proteasome inhibition is capable of modulating slow degradation. Importantly, very low doses of proteasome inhibitor were sufficient to reduced the extent of cyclin B slow degradation during the mitotic block. Moreover, we demonstrate that low doses of proteasome inhibitor render the AML cell line Kasumi-1 responsive to low, non-disruptive concentrations of spindle poison (nocodazole and vincristine) leading to remarkable increases in the G2M-fraction. To the best of our knowledge there is no evidence so far that low doses of proteasome inhibitor exert antimitotic effects by interference with protein degradation during mitosis. Importantly, concentration of bortezomib of 1–2ng/ml (such as found in the serum of patients for up to 72h following administration of 1.3mg/m2 bortezomib subcutaneously) were found to exert synergistic effects with antimitotic therapy. Increases in the percentage of G2M cells by 38% were observed in Kasumi-1 cells for the combination of vincristine and bortezomib. Based on these findings, we currently apply our system to probe combinations of proteasome inhibitor with modern tailored therapies that exert their antimitotic effects by activation of the SAC, such as inhibitors of the motor protein Eg5 or of the mitotic kinases Polo-like kinase 1 (Plk1) or Aurora A and B. Using our cell line-based reporter system, we provide evidence in the in vitro setting that modulating slow degradation during antimitotic therapy by proteasome inhibition is a promising rationale to enhance the efficacy of antimitotic drugs. Drug concentrations used are based on published pharmacokinetics in humans and suggest feasibility of the drug combination in vivo. Our approach of targeted drug combinations may provide highly efficient treatment alternatives for patients that are not eligible for induction treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Gen Matsumoto ◽  
Tomonao Inobe ◽  
Takanori Amano ◽  
Kiyohito Murai ◽  
Nobuyuki Nukina ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Proteasome Inhibition ◽  
Aggresome Formation

scholarly journals Proteasome inhibitors counteract the effect of cisplatin in an HPV-positive tonsil squamous cell carcinoma in vitro

2020 ◽  
Author(s):  
Natsuki Sugiyama ◽  
Gabriel Adrian ◽  
Stefan Schwartz ◽  
Johan Wennerberg ◽  
Lars Ekblad
Keyword(s):  
Cell Cycle ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Line ◽  
Squamous Cell ◽  
Cell Cycle Progression ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Cycle Progression ◽  
Mdm2 Inhibitor

Abstract Background A rapid increase in human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a global trend. Although HPV-positive patients have a more favorable prognosis, distant metastases occur, warranting new, systemic treatment options. The aim of this study was to investigate the effect of combining proteasome or MDM2 inhibitors with cisplatin on an HPV-positive oropharyngeal squamous cell carcinoma cell line (LU-HNSCC-26).Methods The LU-HNSCC-26 cells were treated with proteasome inhibitor (bortezomib, carfilzomib or ixazomib) or MDM2 inhibitor (RG7112) in combination with cisplatin. Combinatorial effects were analyzed by isobolograms. Protein expression was investigated by Western blotting and cell cycle phase distribution by flow cytometry. Results There was no synergy between the substances and cisplatin. All proteasome inhibitors displayed antagonistic effects while the MDM2 inhibitor was additive in combination with cisplatin. The expression of p53 was only marginally affected and apoptosis was not detected. The cell cycle progression was halted in G0/G1 with all inhibitors and in S phase with cisplatin. The expression of p21 increased by bortezomib or carfilzomib, ixazomib increased p21 in combination with cisplatin while RG7112 did not affect p21. There was no effect on ERCC1 with any of the substances.Conclusions In the investigated HPV16-positive OPSCC cell line, proteasome inhibition decreased the effect of cisplatin. A possible mechanism for this includes low effects on p53 expression with concomitant increase in p21 expression and blocking of cell cycle progression in G0/G1 with preserved DNA damage repair. The combination of proteasome inhibition with ordinary cytotoxic treatment for HPV-positive OPSCC patients is thus questionable, and clinical trials should be preceded by thorough testing in adequate models.

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