A novel assay for screening WIP1 phosphatase substrates in nuclear extracts

FEBS Journal ◽  
2021 ◽  
Author(s):  
Radka Storchova ◽  
Kamila Burdova ◽  
Matous Palek ◽  
René H. Medema ◽  
Libor Macurek
Keyword(s):  
Nuclear Extracts ◽  
Wip1 Phosphatase

Characterization of 25-hydroxyvitamin D3 receptors in cytosol and nuclear extracts from the hypercalcemic Walker carcinosarcoma 256

1984 ◽  
Vol 104 (4_Supplb) ◽  
pp. S4-S5
Author(s):  
J. MERKE ◽  
G. SMIGIELSKI ◽  
H. KITTSTEIN ◽  
E. RITZ
Keyword(s):  
D3 Receptors ◽  
Nuclear Extracts

Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

2020 ◽  
Vol 20 (6) ◽  
pp. 715-723
Author(s):  
Natarajan Nandakumar ◽  
Pushparathinam Gopinath ◽  
Jacob Gopas ◽  
Kannoth M. Muraleedharan
Keyword(s):  
Synergistic Effect ◽  
Hodgkin’S Lymphoma ◽  
A549 Cells ◽  
Hodgkin's Lymphoma ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Lymphoma Cells ◽  
Nuclear Extracts ◽  
Gene Assay

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

scholarly journals Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues.

1992 ◽  
Vol 267 (25) ◽  
pp. 17944-17948
Author(s):  
R Ammendola ◽  
M Mesuraca ◽  
T Russo ◽  
F Cimino
Keyword(s):  
Dna Binding ◽  
Rat Tissues ◽  
Aged Rat ◽  
Nuclear Extracts

scholarly journals WIP1 Inhibition by GSK2830371 Potentiates HDM201 through Enhanced p53 Phosphorylation and Activation in Liver Adenocarcinoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3876
Author(s):  
Chiao-En Wu ◽  
Chen-Yang Huang ◽  
Chiao-Ping Chen ◽  
Yi-Ru Pan ◽  
John Wen-Cheng Chang ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Primary Liver Cancer ◽  
P53 Protein ◽  
Intrahepatic Bile Duct ◽  
Cell Cycle Distribution ◽  
P53 Signaling ◽  
Adenocarcinoma Cells ◽  
Wip1 Phosphatase ◽  
P53 Activation ◽  
Novel Strategy

Background: Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct. It is the second most common primary liver cancer and has a poor prognosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Methods: Both RBE and SK-Hep-1 liver adenocarcinoma cell lines were treated with the HDM201 (Siremadlin) MDM2-p53 binding antagonist alone or in combination with the GSK2830371 WIP1 phosphatase inhibitor. Cell proliferation, clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination. Results: GSK2830371 alone demonstrated minimal activity on proliferation and colony formation, but potentiated growth inhibition (two-fold decrease in GI50) and cytotoxicity (four-fold decrease in IC50) by HDM201 on RBE and SK-Hep-1 cells. HDM201 increased p53 protein expression, leading to transactivation of downstream targets (p21 and MDM2). Combination with GSK2830371 increased p53 phosphorylation, resulting in an increase in both p53 accumulation and p53-dependent trans-activation. G2/M arrest was observed by flow cytometry after this treatment combination. RNA sequencing identified 21 significantly up-regulated genes and five downregulated genes following p53 reactivation by HDM201 in combination with GSK2830371 at 6 h and 24 h time points compared with untreated controls. These genes were predominantly known transcriptional targets regulated by the p53 signaling pathway, indicating enhanced p53 activation as the predominant effect of this combination. Conclusion: The current study demonstrated that GSK2830371 enhanced the p53-dependent antiproliferative and cytotoxic effect of HDM201 on RBE and SK-Hep-1 cells, providing a novel strategy for potentiating the efficacy of targeting the p53 pathway in iCCA.

scholarly journals The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication.

1992 ◽  
Vol 12 (3) ◽  
pp. 1257-1265 ◽  
Author(s):  
A D Bergemann ◽  
E M Johnson
Keyword(s):  
Dna Replication ◽  
Consensus Sequence ◽  
Specific Element ◽  
Contact Points ◽  
Nuclear Extracts ◽  
Specific Contact ◽  
Myc Gene ◽  
Specific Affinity ◽  
Initiation Of Dna Replication ◽  
Flanking Regions

A major site of DNA bending is located 1.6 kb upstream of the P1 transcription start site of the human c-myc gene, near the center of a reported zone of initiation of DNA replication. A repeated, purine-rich element, termed PUR, at the bend site is specifically bound by a protein in HeLa cell nuclear extracts. This protein has specific affinity for the purine-rich single strand of the element. Methylation interference maps a pattern of specific contact points with guanosine bases in a 24-mer oligonucleotide containing the element. UV cross-linking reveals that contact is made by a polypeptide of approximately 28 kDa. The PUR element is present at origins of replication and in gene flanking regions in a variety of eukaryotes from yeasts through humans. The consensus sequence GGNNGAGGGAGARRRR has been derived. This element is present near centers of regions of two mammalian loci (human c-myc and hamster dhfr) recently reported as initiation zones for DNA replication. A 24-mer oligonucleotide representing the hamster dhfr version of the PUR element effectively competes with the human c-myc version for binding to Pur.

Myostatin regulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression

2009 ◽  
Vol 296 (3) ◽  
pp. C525-C534 ◽  
Author(s):  
Alex Hennebry ◽  
Carole Berry ◽  
Victoria Siriett ◽  
Paul O'Callaghan ◽  
Linda Chau ◽  
...  
Keyword(s):  
Growth Factor ◽  
Target Genes ◽  
Fiber Type ◽  
Fiber Formation ◽  
Type I ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Fiber Type Composition ◽  
Nuclear Extracts ◽  
Type Composition

Myostatin (Mstn) is a secreted growth factor belonging to the tranforming growth factor (TGF)-β superfamily. Inactivation of murine Mstn by gene targeting, or natural mutation of bovine or human Mstn, induces the double muscling (DM) phenotype. In DM cattle, Mstn deficiency increases fast glycolytic (type IIB) fiber formation in the biceps femoris (BF) muscle. Using Mstn null (−/−) mice, we suggest a possible mechanism behind Mstn-mediated fiber-type diversity. Histological analysis revealed increased type IIB fibers with a concomitant decrease in type IIA and type I fibers in the Mstn−/−tibialis anterior and BF muscle. Functional electrical stimulation of Mstn−/−BF revealed increased fatigue susceptibility, supporting increased type IIB fiber content. Given the role of myocyte enhancer factor 2 (MEF2) in oxidative type I fiber formation, MEF2 levels in Mstn−/−tissue were quantified. Results revealed reduced MEF2C protein in Mstn−/−muscle and myoblast nuclear extracts. Reduced MEF2-DNA complex was also observed in electrophoretic mobility-shift assay using Mstn−/−nuclear extracts. Furthermore, reduced expression of MEF2 downstream target genes MLC1F and calcineurin were found in Mstn−/−muscle. Conversely, Mstn addition was sufficient to directly upregulate MLC promoter-enhancer activity in cultured myoblasts. Since high MyoD levels are seen in fast fibers, we analyzed MyoD levels in the muscle. In contrast to MEF2C, MyoD levels were increased in Mstn−/−muscle. Together, these results suggest that while Mstn positively regulates MEF2C levels, it negatively regulates MyoD expression in muscle. We propose that Mstn could regulate fiber-type composition by regulating the expression of MEF2C and MyoD during myogenesis.

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