P53 DNA Binding Research Articles

Protein Kinase C-δ (PKCδ) Regulates Proinflammatory Chemokine Expression through Cytosolic Interaction with the NF-κB Subunit p65 in Vascular Smooth Muscle Cells

Proinflammatory chemokines released by vascular smooth muscle cells (VSMCs) play a critical role in vascular inflammation. Protein kinase C-δ (PKCδ) has been shown to be up-regulated in VSMCs of injured arteries. PKCδ knock-out (Prkcd(-/-)) mice are resistant to inflammation as well as apoptosis in models of abdominal aortic aneurysm. However, the precise mechanism by which PKCδ modulates inflammation remains incompletely understood. In this study, we identified four inflammatory chemokines (Ccl2/Mcp-1, Ccl7, Cxcl16, and Cx3cl1) of over 45 PKCδ-regulated genes associated with inflammatory response by microarray analysis. Using CCL2 as a prototype, we demonstrated that PKCδ stimulated chemokine expression at the transcriptional level. Inhibition of the NF-κB pathway or siRNA knockdown of subunit p65, but not p50, eliminated the effect of PKCδ on Ccl2 expression. Overexpressing PKCδ followed by incubation with phorbol 12-myristate 13-acetate resulted in an increase in p65 Ser-536 phosphorylation and enhanced DNA binding affinity without affecting IκB degradation or p65 nuclear translocation. Prkcd gene deficiency impaired p65 Ser-536 phosphorylation and DNA binding affinity in response to TNFα. Results from in situ proximity ligation analysis and co-immunoprecipitation performed on cultured VSMCs and aneurysmal aorta demonstrated physical interaction between PKCδ and p65 that took place largely outside the nucleus. Promoting nuclear translocation of PKCδ with peptide ψδRACK diminished Ccl2 production, whereas inhibition of PKCδ translocation with peptide δV1-1 enhanced Ccl2 expression. Together, these results suggest that PKCδ modulates inflammation at least in part through the NF-κB-mediated chemokines. Mechanistically, PKCδ activates NF-κB through an IκB-independent cytosolic interaction, which subsequently leads to enhanced p65 phosphorylation and DNA binding affinity.

VRK1 interacts with p53 forming a basal complex that is activated by UV-induced DNA damage

DNA damage immediate cellular response requires the activation of p53 by kinases. We found that p53 forms a basal stable complex with VRK1, a Ser–Thr kinase that responds to UV-induced DNA damage by specifically phosphorylating p53. This interaction takes place through the p53 DNA binding domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the complex. UV-induced DNA damage activates VRK1, and is accompanied by phosphorylation of p53 at Thr-18 before it accumulates. We propose that the VRK1–p53 basal complex is an early-warning system for immediate cellular responses to DNA damage.

Effect of low extracellular pH on NF-κB activation in macrophages

ObjectiveMany diseases, including atherosclerosis, involve chronic inflammation. The master transcription factor for inflammation is NF-κB. Inflammatory sites have a low extracellular pH. Our objective was to demonstrate the effect of pH on NF-κB activation and cytokine secretion. MethodsMouse J774 macrophages or human THP-1 or monocyte-derived macrophages were incubated at pH 7.0–7.4 and inflammatory cytokine secretion and NF-κB activity were measured. ResultsA pH of 7.0 greatly decreased pro-inflammatory cytokine secretion (TNF or IL-6) by J774 macrophages, but not THP-1 or human monocyte-derived macrophages. Upon stimulation of mouse macrophages, the levels of IκBα, which inhibits NF-κB, fell but low pH prevented its later increase, which normally restores the baseline activity of NF-κB, even though the levels of mRNA for IκBα were increased. pH 7.0 greatly increased and prolonged NF-κB binding to its consensus promoter sequence, especially the anti-inflammatory p50:p50 homodimers. Human p50 was overexpressed using adenovirus in THP-1 macrophages and monocyte-derived macrophages to see if it would confer pH sensitivity to NF-κB activity in human cells. Overexpression of p50 increased p50:p50 DNA-binding and in THP-1 macrophages inhibited considerably TNF and IL-6 secretion, but there was still no effect of pH on p50:p50 DNA binding or cytokine secretion. ConclusionA modest decrease in pH can sometimes have marked effects on NF-κB activation and cytokine secretion and might be one reason to explain why mice normally develop less atherosclerosis than do humans.

Murine Double Minute 2 siRNA and wild-type p53 gene therapy interact positively with zinc on prostate tumours in vitro and in vivo

Prostate cancer (PCa) often shows either mutations of the p53 gene or inactivation of the P53 protein. The latter may be due to up-regulation of mouse double minute 2 homologue (MDM2), which functions both as an E3 ubiquitin ligase to degrade P53 protein via the proteasome and an inhibitor of P53 transcriptional activation. Zinc plays a crucial role in stabilizing the P53 DNA binding domain, but PCa cells often lack the ability to accumulate sufficient zinc. In the present study, we explore the optimal approach to retention of P53 function. To restore the defective P53 pathway in PCa, we have explored a combined treatment of Pmp53 [a plasmid containing both mdm2 small interfering RNA (Si-MDM2) and the wild-type p53 gene] with zinc. This treatment retains the wild-type P53 conformation and function in PCa in vitro and in vivo. Combined treatments significantly inhibited tumour xenograft growth, retaining wild-type P53 conformation and enhancing its transcriptional regulation of p21 and bax gene expression, leading to the decreased proliferation and increased apoptosis. These in vivo findings were confirmed by in vitro culture of PCa PC-3 (p53 null) or DU145 (mutant p53) cells and showed a positive effect of the combined therapy on cell cycle arrest and massive apoptosis. Our findings suggest that the combined therapy of Pmp53 with zinc is an effective strategy that may open a new therapeutic avenue in some cancers expressing low levels of zinc and a defective P53 status.

Beetroot (Beta vulgaris L.) extract ameliorates gentamicin-induced nephrotoxicity associated oxidative stress, inflammation, and apoptosis in rodent model.

The present investigation was designed to investigate the protective effect of (Beta vulgaris L.) beat root ethanolic extract (BVEE) on gentamicin-induced nephrotoxicity and to elucidate the potential mechanism. Serum specific kidney function parameters (urea, uric acid, total protein, creatinine, and histopathology of kidney tissue) were evaluated to access gentamicin-induced nephrotoxicity. The oxidative/nitrosative stress (Lipid peroxidation, MDA, NP-SH, Catalase, and nitric oxide levels) was assessed. The inflammatory response (TNF-α, IL-6, MPO, NF-κB (p65), and NF-κB (p65) DNA binding) and apoptotic marker (Caspase-3, Bax, and Bcl-2) were also evaluated. BVEE (250 and 500 mg/kg) treatment along with gentamicin restored/increased the renal endogenous antioxidant status. Gentamicin-induced increased renal inflammatory cytokines (TNF-α and IL-6), nuclear protein expression of NF-κB (p65), NF-κB-DNA binding activity, myeloperoxidase (MPO) activity, and nitric oxide level were significantly down regulated upon BVEE treatment. In addition, BVEE treatment significantly reduced the amount of cleaved caspase 3 and Bax, protein expression and increased the Bcl-2 protein expression. BVEE treatment also ameliorated the extent of histologic injury and reduced inflammatory infiltration in renal tubules. These findings suggest that BVEE treatment attenuates renal dysfunction and structural damage through the reduction of oxidative stress, inflammation, and apoptosis in the kidney.

Label-Free Detection of the P53-DNA Complex

The tumour suppressor p53 plays a crucial role in cell cycle regulation in response to cellular stress such as DNA damage. p53 has been found to be mutated in 50% of all human cancers, the majority of which occur in the DNA binding core domain of the protein. These mutations result in loss of sequence specific binding abilities that activate the transcription of genes to regulate the cell cycle and prevent cell damage proliferation. It is, therefore, crucial to understand the p53-DNA binding interaction to further our ability to detect and combat any breakdown in wild type function.In this research we investigate the interaction of p53 and sequence specific DNA with label-free techniques. The solid-state nanopore platform is a label-free biosensor that utilises ion transport to drive analytes through the nanopore aperture. The result is a current-time signal that is characteristic to the charge, shape and size of the analyte of interest (see figure). This method allows for detection and differentiation between analytes without a need for labels.The use of this platform to study the p53-DNA specific binding interaction is combined with AFM analysis to examine binding statistics.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Reversal of the DNA-Binding-Induced Loop L1 Conformational Switch in an Engineered Human p53 Protein

The gene encoding the p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, is the most frequently mutated gene in human cancer. Crystal structures of homo-oligomerizing p53 polypeptides with specific DNA suggest that DNA binding is associated with a conformational switch. Specifically, in the absence of DNA, loop L1 of the p53 DNA binding domain adopts an extended conformation, whereas two p53 subunits switch to a recessed loop L1 conformation when bound to DNA as a tetramer. We previously designed a p53 protein, p53FG, with amino substitutions S121F and V122G targeting loop L1. These two substitutions enhanced the affinity of p53 for specific DNA yet, counterintuitively, decreased the residency time of p53 on DNA. Here, we confirmed these DNA binding properties of p53FG using a different method. We also determined by crystallography the structure of p53FG in its free state and bound to DNA as a tetramer. In the free state, loop L1 adopted a recessed conformation, whereas upon DNA binding, two subunits switched to the extended loop L1 conformation, resulting in a final structure that was very similar to that of wild-type p53 bound to DNA. Thus, altering the apo structure of p53 changed its DNA binding properties, even though the DNA-bound structure was not altered.

Protective Role of Andrographolide in Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a chronic devastating disease with poor prognosis. Multiple pathological processes, including inflammation, epithelial mesenchymal transition (EMT), apoptosis, and oxidative stress, are involved in the pathogenesis of IPF. Recent findings suggested that nuclear factor-κB (NF-κB) is constitutively activated in IPF and acts as a central regulator in the pathogenesis of IPF. The aim of our study was to reveal the value of andrographolide on bleomycin-induced inflammation and fibrosis in mice. The indicated dosages of andrographolide were administered in mice with bleomycin-induced pulmonary fibrosis. On day 21, cell counts of total cells, macrophages, neutrophils and lymphocytes, alone with TNF-α in bronchoalveolar lavage fluid (BALF) were measured. HE staining and Masson’s trichrome (MT) staining were used to observe the histological alterations of lungs. The Ashcroft score and hydroxyproline content of lungs were also measured. TGF-β1 and α-SMA mRNA and protein were analyzed. Activation of NF-κB was determined by western blotting and electrophoretic mobility shift assay (EMSA). On day 21 after bleomycin stimulation, andrographolide dose-dependently inhibited the inflammatory cells and TNF-α in BALF. Meanwhile, our data demonstrated that the Ashcroft score and hydroxyproline content of the bleomycin-stimulated lung were reduced by andrographolide administration. Furthermore, andrographloide suppressed TGF-β1 and α-SMA mRNA and protein expression in bleomycin-induced pulmonary fibrosis. Meanwhile, andrographolide significantly dose-dependently inhibited the ratio of phospho-NF-κB p65/total NF-κB p65 and NF-κB p65 DNA binding activities. Our findings indicate that andrographolide compromised bleomycin-induced pulmonary inflammation and fibrosis possibly through inactivation of NF-κB. Andrographolide holds promise as a novel drug to treat the devastating disease of pulmonary fibrosis.

APE1/Ref-1 enhances DNA binding activity of mutant p53 in a redox-dependent manner

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual function protein; in addition to its DNA repair activity, it can stimulate DNA binding activity of numerous transcription factors as a reduction-oxidation (redox) factor. APE1/Ref-1 has been found to be a potent activator of wild-type p53 (wtp53) DNA binding in vitro and in vivo. Although p53 is mutated in most types of human cancer including hepatocellular carcinoma (HCC), little is known about whether APE1/Ref-1 can regulate mutant p53 (mutp53). Herein, we reported the increased APE1/Ref-1 protein and accumulation of mutp53 in HCC by immunohistochemistry. Of note, it was observed that APE1/Ref-1 high-expression and mutp53 expression were associated with carcinogenesis and progression of HCC. To determine whether APE1/Ref-1 regulates DNA binding of mutp53, we performed electromobility shift assays (EMSAs) and quantitative chromatin immunoprecipitation (ChIP) assays in HCC cell lines. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, reduced mutp53 efficiently bound to nonlinear DNA, but not to linear DNA. Notably, overexpression of APE1/Ref-1 resulted in increased DNA binding activity of mutp53, while downregulation of APE1/Ref-1 caused a marked decrease of mutp53 DNA binding. In addition, APE1/Ref-1 could not potentiate the accumulation of p21 mRNA and protein in mutp53 cells. These data indicate that APE1/Ref-1 can stimulate mutp53 DNA binding in a redox-dependent manner.

Id4 dependent acetylation restores mutant-p53 transcriptional activity

BackgroundThe mechanisms that can restore biological activity of mutant p53 are an area of high interest given that mutant p53 expression is observed in one third of prostate cancer. Here we demonstrate that Id4, an HLH transcriptional regulator and a tumor suppressor, can restore the mutant p53 transcriptional activity in prostate cancer cells.MethodsId4 was over-expressed in prostate cancer cell line DU145 harboring mutant p53 (P223L and V274F) and silenced in LNCaP cells with wild type p53. The cells were used to quantitate apoptosis, p53 localization, p53 DNA binding and transcriptional activity. Immuno-precipitation/-blot studies were performed to demonstrate interactions between Id4, p53 and CBP/p300 and acetylation of specific lysine residues within p53.ResultsEctopic expression of Id4 in DU145 cells resulted in increased apoptosis and expression of BAX, PUMA and p21, the transcriptional targets of p53. Mutant p53 gained DNA binding and transcriptional activity in the presence of Id4 in DU145 cells. Conversely, loss of Id4 in LNCaP cells abrogated wild type p53 DNA binding and transactivation potential. Gain of Id4 resulted in increased acetylation of mutant p53 whereas loss of Id4 lead to decreased acetylation in DU145 and LNCaP cells respectively. Id4 dependent acetylation of p53 was in part due to a physical interaction between Id4, p53 and acetyl-transferase CBP/p300.ConclusionsTaken together, our results suggest that Id4 regulates the activity of wild type and mutant p53. Id4 promoted the assembly of a macromolecular complex involving CBP/P300 that resulted in acetylation of p53 at K373, a critical post-translational modification required for its biological activity.

TNF-α/NF-κB Signaling in the CNS: Possible Connection to EPHB2

Tumor necrosis factor-alpha, TNF-α, is a cytokine that is a well-known factor in multiple disease conditions and is recognized for its major role in central nervous system signaling. TNF-α signaling is most commonly associated with neurotoxicity, but in some conditions it has been found to be neuroprotective. TNF-α has long been known to induce nuclear factor-kappa B, NF-κB, signaling by, in most cases, translocating the p65 (RelA) DNA binding factor to the nucleus. p65 is a key member of NF-κB, which is well established as a family of transcription factors that regulates many signaling events, including growth and process development, in neuronal cell populations. NF-κB has been shown to affect both the receiving aspect of neuronal signaling events in dendritic development as well as the sending of neuronal signals in axonal development. In both cases, NK-κB functions as a promoter and/or inhibitor of growth, depending on the environmental conditions and signaling cascade. In addition, NF-κB is involved in memory formation or neurogenesis, depending on the region of the brain in which the signaling occurs. The ephrin (Eph) receptor family represents a subfamily of receptor tyrosine kinases, RTKs, which received much attention due to its potential involvement in neuronal cell health and function. There are two subsets of ephrin receptors, Eph A and Eph B, each with distinct functions in cardiovascular and skeletal development and axon guidance and synaptic plasticity. The presence of multiple binding sites for NF-κB within the regulatory region of EphB2 gene and its potential regulation by NF-κB pathway suggests that TNF-α may modulate EphB2 via NF-κB and that this may contribute to the neuroprotective activity of TNF-α.

EphB1 Downregulation In Acute Myeloid Leukemia: Suppressing p53-Dependent DNA Damage Control System

BackgroundEphrin receptors (Eph) have been implicated to contribute to cancer pathogenesis in a way by which receptors can both promote or suppress cancer progression depending on their cellular contexts. Mechanism for Eph receptor tumor suppressor functions are unknown. In a previous study, we found that pediatric acute myeloid leukemia (AML) sample lysates showed a significant decreased EphB1 peptide activity in comparison to normal bone marrow (NBM). In this study, we challenged to investigate EphB1 receptor transcriptional regulation and biological insights in AML. Materials and MethodsQuantitative RT-PCR analysis and bisulfate pyrosequencing was used to examine mRNA expression and methylation in AML cell lines and AML patient samples. AML cell proliferation (cell counts) and apoptosis (annexin V/PI) were analyzed after EfnB1 ligand stimulation. We generated constitutive EphB1 overexpression constructs in AML cell lines. Downstream in vitro effects were studied using quantitative RT-PCR, flowcytometry, phospho-proteome arrays, chromatin immunoprecipitation (ChIP), and immunoblot analysis. ResultsFirst, we examined EphB1 mRNA expression and methylation in primary pediatric AML samples, which revealed that EphB1 mRNA expression was significantly lower in unfavorable AML cytogenetic subgroups as compared to favorable AML patient samples (Fig. A, P = 0.013). The EphB1 promoter region was found increasingly methylated in approximately 20% of the pediatric AML patient samples (n=21), but not in NBM (n=5, Fig. B). In AML cell lines, we confirmed that EphB1 promoter methylation resulted in epigenetic gene silencing (P = 0.010).As a next step we challenged to study the biological relevance of EphB1 suppression. EfnB1 stimulation of EphB1high THP-1 cells inhibited the cell proliferation and induced apoptosis, while EphB1low HL60 and MOLM13 cells remained unaffected upon EfnB1 stimulation. Cell cycle analysis revealed that EfnB1 stimulation mediated phenotypic effects in EphB1high THP-1 cells could be assigned to a G2/M cell cycle arrest. The cell cycle arrest and the induction of apoptosis as a common consequence was confirmed by phospho-proteome, immunoblot, and qRT-PCR analysis. We found an induction of DNA damage control protein activation and cell cycle inhibitors Chk-2, p21, p27, p53, and CDC2tyr15, and a reduction of cell survival proteins HSP27 and BCL-2 accompanied by an increase of Bax. Next, we investigated whether EfnB1 stimulation of THP-1 could have restored the DNA damage control system by increasing DNA binding of mutant and wildtype p53. Performing ChIP assays using p53 antibodies recognizing wildtype or both mutant and wildtype p53 followed by p21 and MDM2 quantitative RT-PCR analysis revealed that EfnB1 stimulation of THP-1 cells significantly increased p53 DNA binding (Fig. C).Introducing a constitutive EphB1 overexpression into EphB1 methylated HL60 and MOLM13 cell lines resulted in an instant increase in apoptosis relatively to empty vector control cells. As expected, G2/M cell cycle inhibiting phosphorylation of CDC2tyr15 was upregulated. EfnB1 stimulation of the GFP sorted remaining viable cells showed to decrease the AML cell survival in EphB1 overexpressed AML cells as compared to empty vector controls. Additionally, we found that EphB1 demethylation by 5-Aza-2'-deoxycytidine was able to restore EphB1 membrane protein expression in EphB1 methylated AML cells and simultaneously reduced the AML cell survival. ConclusionsIn this study, we found a common downregulation of EphB1 mRNA that was significantly associated with an unfavorable prognosis in pediatric AML. The downregulation of EphB1 could be explained by EphB1 promoter hypermethylation in a subset of AML samples. Downregulation of EphB1 was found to be one of the mechanisms through which AML cells suppress p53 DNA binding that impairs the DNA damage control system to support a proliferative and anti-apoptotic AML cell survival advantage.▪ Disclosures:No relevant conflicts of interest to declare.

Constitutive B-Cell Maturation Antigen (BCMA) Activation In Human Multiple Myeloma Cells Promotes Myeloma Cell Growth and Survival In The Bone Marrow Microenvironment Via Upregulated MCL-1 and NFκB Signaling

B cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily (TNFRSF17), is selectively induced during plasma cell (PC) differentiation and is commonly expressed at high levels in malignant PCs. Using real time RT-PCR, we here first showed that BCMA mRNA was upregulated in CD138+ PCs from MM patients compared to normal healthy donors (p<0.04), consistent with high and restrictive BCMA expression in PCs but not normal tissues by gene expression profiling and immunohistochemistry (IHC) in a recent report (Clin Cancer Res 2013;19:2048-60). As a specific MM antigen, BCMA is universally expressed on the MM cell surface, confirmed by CD38+BCMA+ dual immunofluorescence staining. We next found that plasmacytoid dendritic cells (pDC), which support MM cell growth, survival, and drug resistance in the bone marrow (BM) microenvironment, have detectable BCMA mRNA at significantly (9-50-fold) lower levels than CD138+ plasma PCs (p<0.005 for each paired sample) from either MM patients or normal donors. Interestingly, as seen in CD138+ PCs, BCMA transcript is considerably elevated in pDC from MM patients vs. normal donors (p<0.03). In contrast, BCMA is hardly detectable in CD138-negative cells from BM aspirates of MM patients. We further define molecular mechanisms of BCMA activation in MM cells. Overexpression of BCMA in multiple MM cell lines (RPMI8226. MM1S, MM1R) by BCMA-expression vector significantly upregulates MCL-1 expression and MIP-1a, as well as NFkB p65 DNA binding activity. Conversely, BCMA siRNA blocked NFkB signaling and expression of anti-apoptotic proteins, leading to decreased MM cell viability. Importantly, stimulation of MM cells by APRIL, which is a cognate ligand for BCMA and mainly secreted by osteoclasts in the BM milieu, activated the canonical NFκB and PI3K/AKT pathways. APRIL also induces pro-survival/anti-apoptotic targets (BCL2A1, NFκB1, NFκB2) and chemotactic/osteoclast activating factors (MIP-1α and MIP1β) in a dose-dependent manner. Angiogenesis and adhesion/chemoattractant factors (IL-8, CXCL10, RANTES, MDC/ccl22) were also significantly induced upon APRIL stimulation. Conversely, BCMA-Fc protein inhibited APRIL to bind BCMA and inhibit secretion of APRIL-induced chemokines, indicating specific response of engagement of BCMA by APRIL in BCMA-expressing MM cells. Finally, APRIL induced adhesion and migration of MM cells whereas BCMA-Fc blocked APRIL-induced responses. Together, our results established active APRIL/BCMA signaling in MM in the BM microenvironment, thus providing a niche for MM disease progression. Moreover, these results strongly support rapid bench to bedside translation of the novel antagonistic anti-BCMA antibody drug conjugate (abstract #56099) to treat MM patients with a likely favorable therapeutic window. Disclosures:Tai:Onyx: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Resveratrol contributes to chemosensitivity of malignant mesothelioma cells with activation of p53

Resveratrol is a naturally occurring polyphenolic phytoalexin with chemopreventive properties. We previously reported a synergistic anti-proliferative effect of resveratrol and clofarabine against malignant mesothelioma (MM) cells. Here, we further investigated molecular mechanisms involved in the synergistic interaction of these compounds in MM MSTO-211H cells. Resveratrol, in combination with clofarabine, time-dependently induced a strong cytotoxic effect with the nuclear accumulation of phospho-p53 (p-p53) in MSTO-211H cells, but not in normal mesothelial MeT-5A cells. Combination treatment up-regulated the levels of p-p53, cleaved caspase-3, and cleaved PARP proteins. Gene silencing with p53-targeting siRNA attenuated the sensitivity of cells to the combined treatment of two compounds. Analyses of p53 DNA binding assay, p53 reporter gene assay, and RTP-CR toward p53-regulated genes, including Bax, PUMA, Noxa and p21, demonstrated that induced p-p53 is transcriptionally active. These results were further confirmed by the siRNA-mediated knockdown of p53 gene. Combination treatment significantly caused the accumulation of cells at G1 phase with the increases in the sub-G0/G1 peak, DNA ladder, nuclear fragmentation, and caspase-3/7 activity. Taken together, these results demonstrate that resveratrol and clofarabine synergistically elicit apoptotic signal via a p53-dependent pathway, and provide a scientific rationale for clinical evaluation of resveratrol as a promising chemopotentiator in MM.

Mapping the Structural and Dynamical Features of Multiple p53 DNA Binding Domains: Insights into Loop 1 Intrinsic Dynamics

The transcription factor p53 regulates cellular integrity in response to stress. p53 is mutated in more than half of cancerous cells, with a majority of the mutations localized to the DNA binding domain (DBD). In order to map the structural and dynamical features of the DBD, we carried out multiple copy molecular dynamics simulations (totaling 0.8 μs). Simulations show the loop 1 to be the most dynamic element among the DNA-contacting loops (loops 1-3). Loop 1 occupies two major conformational states: extended and recessed; the former but not the latter displays correlations in atomic fluctuations with those of loop 2 (~24 Å apart). Since loop 1 binds to the major groove whereas loop 2 binds to the minor groove of DNA, our results begin to provide some insight into the possible mechanism underpinning the cooperative nature of DBD binding to DNA. We propose (1) a novel mechanism underlying the dynamics of loop 1 and the possible tread-milling of p53 on DNA and (2) possible mutations on loop 1 residues to restore the transcriptional activity of an oncogenic mutation at a distant site.

Up-Regulation of Two Distinct p53-DNA Binding Functions by Covalent Poly(ADP-ribosyl)ation: Transactivating and Single Strand Break Sensing

We used a [32P] p53 sequence-specific oligodeoxynucleotide and Electrophoretic-Mobility-Shift-Assays to monitor p53 DNA sequence-specific binding with p53-R267W, a nonbinding point mutant; and p53-Δ30, a deletion-mutant which lacks the carboxy-terminus that recognizes DNA-strand-breaks. Recombinant p53 and poly(ADP-ribose)polymerase-1 (PARP-1) were incubated with labeled βNAD+ with/without DNA. The poly(ADP-ribosyl)ation of each protein increased with incubation-time and βNAD+ and p53 concentration(s). Since p53-Δ30 was efficiently labeled, poly(ADP-ribosyl)ation target site(s) of wt-p53 must reside outside its carboxy-terminal-domain. The poly(ADP-ribosyl)ation of p53-Δ30 did not diminish its DNA binding; Instead, it enhanced DNA-sequence-specific-binding. Therefore, we conclude that DNA-sequence-specific-binding and DNA-nick-sensing of mutant-p53 are differentially regulated by poly(ADP-ribosyl)ation.

Rotational positioning of nucleosomes facilitates selective binding of p53 to response elements associated with cell cycle arrest

The tumor suppressor protein p53 exhibits high affinity to the response elements regulating cell cycle arrest genes (CCA-sites), but relatively low affinity to the sites associated with apoptosis (Apo-sites). This in vivo tendency cannot be explained solely by the p53-DNA binding constants measured in vitro. Since p53 can bind nucleosomal DNA, we sought to understand if the two groups of p53 sites differ in their accessibility when embedded in nucleosomes. To this aim, we analyzed the sequence-dependent bending anisotropy of human genomic DNA containing p53 sites. For the 20 CCA-sites, we calculated rotational positioning patterns predicting that most of the sites are exposed on the nucleosomal surface. This is consistent with experimentally observed positioning of human nucleosomes. Remarkably, the sequence-dependent DNA anisotropy of both the p53 sites and flanking DNA work in concert producing strong positioning signals. By contrast, both the predicted and observed rotational settings of the 38 Apo-sites in nucleosomes suggest that many of these sites are buried inside, thus preventing immediate p53 recognition and delaying gene induction. The distinct chromatin organization of the CCA response elements appears to be one of the key factors facilitating p53-DNA binding and subsequent activation of genes associated with cell cycle arrest.

Increased Nuclear Expression of Nuclear Factor Kappa‐B p65 Subunit in the Eutopic Endometrium and Ovarian Endometrioma of Women with Advanced Stage Endometriosis

We evaluated whether the expression of NF-кB p65 subunit is increased in the eutopic endometrium and/or in the ovarian endometrioma of women with advanced stage endometriosis, and ascertained in vitro effects of proinflammatory cytokines on the expression and DNA binding of NF-кB p65 subunit in endometrial cells. Immunohistochemistry was performed to compare the nuclear NF-кB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-кB p65 subunit expression and DNA binding were also analyzed in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β) utilizing Western blot analysis, enzyme-linked immunosorbent assay, and electrophoretic mobility shift assay. The immunoreactivity of the nuclear NF-кB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with endometriosis compared with the controls. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase in nuclear NF-кB p65 subunit expression and DNA binding. The nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggests that NF-кB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis.

Tunicamycin inhibits Toll-like receptor-activated inflammation in RAW264.7 cells by suppression of NF-κB and c-Jun activity via a mechanism that is independent of ER-stress and N-glycosylation

In this study, we investigated the effect of tunicamycin on the production of pro-inflammatory molecules in RAW264.7 macrophage cells in response to lipopolysaccharide (LPS) and Toll-like receptor (TLR) agonists. Tunicamycin caused a reduction in LPS-induced nitric oxide (NO) production and expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). In contrast, other ER stress-inducing chemicals, such as A23187 and thapsigargin (TG), increased LPS-induced COX-2 expression and had no effect on LPS-induced iNOS, TNF-α or IL-1β expression. Furthermore, the inhibitory effect of tunicamycin on LPS-induced inflammation was not influenced by salubrinal, an ER stress inhibitor, suggesting that the anti-inflammatory effect of tunicamycin is independent of ER stress. Tunicamycin also inhibited the expression of inflammatory molecule mRNAs induced by stimulation of TLR2 (with lipoteichoic acid) or TLR3 (with polyinosinic:polycytidylic acid), which do not require myeloid differentiation protein-2 (MD2) for their activation. Moreover, inhibition of LPS-induced iNOS expression was not inhibited by castanospermine, another N-glycosylation inhibitor, suggesting that the inhibitory effect of tunicamycin on LPS-induced iNOS induction is likely independent of MD2 N-glycosylation. Tunicamycin inhibited nuclear factor-kappaB (NF-κB) activity by suppressing LPS-induced nuclear translocation of p50 and subsequent DNA binding of p50 and p65 to the NF-κB site of the iNOS promoter. Tunicamycin also inhibited the transcriptional activity of a cAMP-response element (CRE) reporter, possibly by inhibiting c-Jun activation. Therefore, we conclude that tunicamycin represses TLR-induced inflammation through suppression of NF-κB and CRE activity via a mechanism that is independent of ER-stress and N-glycosylation.

Increased nuclear expression of nuclear factor kappa-B p65 subunit in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis

ObjectiveTo evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis.DesignCase-controlled laboratory study in a university hospital.Materials and MethodsWe recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β).ResultsThe immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding.ConclusionThe nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis. ObjectiveTo evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis. To evaluate whether the nuclear expression of nuclear factor kappa-B (NF-κB) p65 subunit is increased in the eutopic endometrium and/or the ovarian endometrioma of women with advanced stage endometriosis. DesignCase-controlled laboratory study in a university hospital. Case-controlled laboratory study in a university hospital. Materials and MethodsWe recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). We recruited a total of 66 women who underwent hysterectomy due to carcinoma in situ (CIS) of the uterine cervix (n=32) or advanced stage endometriosis (n=34) as well as 30 nulliparous women who underwent ovarian cystectomy due to endometrioma. Immunohistochemistry was performed to compare the nuclear NF-κB p65 subunit immunoreactivity between women with and without advanced stage endometriosis. The nuclear NF-κB p65 subunit expression and DNA binding were analyzed utilizing Western blotting, enzyme-linked immunosorbent assay (ELISA), and electrophoretic mobility shift assay (EMSA) in endometrial cells treated with tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ResultsThe immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding. The immunoreactivity of the nuclear NF-κB p65 subunit was significantly increased in the eutopic endometrium as well as in the ovarian endometrioma of women with advanced stage endometriosis compared to the CIS group. In vitro treatment of endometrial cells with TNF-α and IL-1β led to a significant increase of nuclear NF-κB p65 subunit expression and DNA binding. ConclusionThe nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis. The nuclear expression of NF-κB p65 is increased in the eutopic endometrium and ovarian endometrioma of women with advanced stage endometriosis, which strongly suggest that NF-κB signaling plays a crucial role in the pathogenesis and/or pathophysiology of endometriosis.