The NF-kappaB transcription factor signaling pathway is constitutively active in many human cancers, and inhibition of this pathway can often kill cancer cells by inducing apoptosis. In this study, we show that two synthetic epoxyquinoids, jesterone dimer (JD) and epoxyquinone A monomer (EqM), are equally effective at inhibiting the growth of two human lymphoma cell lines that have constitutively nuclear REL (human c-Rel) DNA-binding complexes, but either express (SUDHL-4 cells) or do not express (RC-K8 cells) the NF-kappaB inhibitor IkappaBalpha. Furthermore, in these cells, both JD and EqM dose-dependently induced apoptosis, inhibited REL DNA-binding activity, and converted REL to a high molecular weight form. In A293 cells, JD and EqM inhibited the DNA-binding activity of overexpressed REL, but not p50. Replacement of Cys-27 with Ser in REL reduced JD- and EqM-mediated inhibition of REL DNA-binding activity. These results suggest that JD and EqM can induce apoptosis in IkappaBalpha-deficient lymphoma cells through a mechanism involving direct inhibition of transcription factor REL.
"Whole-cell lysates were prepared in AT buffer containing protease inhibitors as described previously  and were analyzed by Western blotting according to standard methods . High molecular weight proteins (full-length p300 and CBP) were transferred at 260 mA for 2.5 h at 4°C using a modified large-protein transfer buffer (20 mM Tris, 150 mM glycine, 0.05% SDS, 10% methanol) as described previously . "
[Show abstract][Hide abstract] ABSTRACT: Recent genome-wide studies have shown that approximately 30% of diffuse large B-cell lymphoma (DLBCL) cases harbor mutations in the histone acetyltransferase (HAT) coactivators p300 or CBP, the majority of which inactivate the catalytic HAT domain. We previously demonstrated that the human DLBCL cell line RC-K8 expresses a C-terminally truncated, HAT-defective p300 protein (p300DeltaC-1087), whose expression was essential for normal cell proliferation.
Using results from large-scale DLBCL studies, we have identified and characterized a second C-terminally truncated, HAT-defective p300 mutant, p300DeltaC-820, expressed in the SUDHL2 DLBCL cell line. Properties of p300DeltaC-820 were characterized in the SUDHL2 DLBCL cell line by Western blotting, co-immunoprecipitation, and shRNA gene knockdown, as well by using cDNA expression vectors for p300DeltaC-820 in pull-down assays, transcriptional reporter assays, and immunofluorescence experiments. A mass spectrometry-based method was used to compare the histone acetylation profile of DLBCL cell lines expressing various levels of wild-type p300.
We show that the SUDHL2 cell line expresses a C-terminally truncated, HAT-defective form of p300 (p300DeltaC-820), but no wild-type p300. The p300DeltaC-820 protein has a wild-type ability to localize to subnuclear "speckles," but has a reduced ability to enhance transactivation by transcription factor REL. Knockdown of p300DeltaC-820 in SUDHL2 cells reduces their proliferation and soft agar colony-forming ability. In RC-K8 cells, knockdown of p300DeltaC-1087 results in increased expression of mRNA and protein for REL target genes A20 and IkappaBalpha, two genes that have been shown to limit the growth of RC-K8 cells when overexpressed. Among a panel of B-lymphoma cell lines, low-level expression of full-length p300 protein, which is characteristic of the SUDHL2 and RC-K8 cells, was associated with decreased acetylation of histone H3 at lysines 14 and 18.
The high prevalence of p300 mutations in DLBCL suggests that HAT-deficient p300 activity defines a subtype of DLBCL, which we have investigated using human DLBCL cell lines RC-K8 and SUDHL2. Our results suggest that truncated p300 proteins contribute to DLBCL cell growth by affecting the expression of specific genes, perhaps through a mechanism that involves alterations in global histone acetylation.
Molecular Cancer 02/2014; 13(1):29. DOI:10.1186/1476-4598-13-29 · 4.26 Impact Factor
"The supershifted p50/p65 complex is indicated by ss and an arrow. (C) An EMSA was performed using extracts from (A) with a consensus NF-κB site from the human MHC1 enhancer (as a κB-site control [32,33,38,50]) and complexes were detected by autoradiography. The appearance of single bands in the p65 and p50 alone lanes confirms that both proteins can bind DNA. "
[Show abstract][Hide abstract] ABSTRACT: MicroRNA-155 (miR-155) is the diced product of the MIR155HG gene. miR-155 regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. MIR155HG has been proposed to be a target gene for transcription factor NF-kappaB largely due to the positive correlation between high nuclear NF-kappaB activity and increased miR-155 expression following treatment with NF-kappaB inducers or in subsets of hematopoietic cancers. Nevertheless, direct regulation of the human MIR155HG promoter by NF-kappaB has not been convincingly demonstrated previously.
This report shows that induction of NF-kappaB activity rapidly leads to increased levels of both primary MIR155HG mRNA and mature miR-155 transcripts. We have mapped an NF-kappaB-responsive element to a position approximately 178 nt upstream of the MIR155HG transcription start site. The -178 site is specifically bound by the NF-kappaB p50/p65 heterodimer and is required for p65-induced reporter gene activation. Moreover, the levels of miR-155 in nine human B-lymphoma cell lines generally correlate with increased nuclear NF-kappaB proteins.
Overall, the identification of an NF-kappaB-responsive site in the MIR155HG proximal promoter suggests that MIR155HG is a direct NF-kappaB target gene in vivo. Understanding NF-kappaB-mediated regulation of miR-155 could lead to improved immune cell-related diagnostic tools and targeted therapies.
"The Ser allele could be favored in certain estuarine environments where the anemones are exposed to high levels of peroxides and other reactive oxygen species or to alkylating agents. The sulfhydryl group of Cys can react readily with such compounds—such as NEM (Figure 7), epoxyquinoids , and sesquiterpene lactones —which would then inhibit the Cys variant from binding to DNA targets, whereas the hydroxyl group of the Ser variant would make Nv-NF-κB less susceptible to the effects of such compounds. Indeed, Nematostella inhabits estuarine environments including tidal creeks and isolated high marsh pools where the concentration of peroxides and thiol-reactive compounds can vary considerably over fine temporal and spatial scales . "
[Show abstract][Hide abstract] ABSTRACT: NF-kappaB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-kappaB proteins contain a highly conserved DNA-binding/dimerization domain called the Rel homology domain.
We characterized two NF-kappaB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel homology domain. Both alleles are found in natural populations of Nematostella. The relative abundance of the two NF-kappaB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-kappaB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-kappaB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys<->Ser mutations at residue 67 of the native Nv-NF-kappaB proteins affect DNA binding as in human NF-kappaB proteins, e.g., a Cys->Ser mutation increases DNA binding of the native Cys variant.
These results are the first demonstration of a naturally occurring and functionally significant polymorphism in NF-kappaB in any species. The functional differences between these alleles and their uneven distribution in the wild suggest that different genotypes could be favored in different environments, perhaps environments that vary in their levels of peroxides or thiol-reactive compounds.
PLoS ONE 10/2009; 4(10):e7311. DOI:10.1371/journal.pone.0007311 · 3.23 Impact Factor
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