P53 DNA Binding Research Articles

Metastasis-associated protein, S100A4 mediates cardiac fibrosis potentially through the modulation of p53 in cardiac fibroblasts

Metastasis-associated protein, S100A4 is suggested as a marker for fibrosis in several organs. It also modulates DNA binding of p53 and affects its function. However, the functional role of S100A4 in the myocardium has remained unclear. Therefore, we investigated the role of S100A4 and its relationship with p53 in cardiac fibrosis. In Dahl-rat hypertensive heart disease model, S100A4 was upregulated in the hypertrophic myocardium and further activated during transition to heart failure (HF). It was expressed in various cells including fibroblasts. In in vitro cardiac fibroblasts, the knockdown of S100A4 significantly suppressed both cell proliferation and collagen expressions. S100A4 co-localized and interacted with p53 in the nucleus. S100A4 knockdown increased the expression of p53-downstream genes, p21 and mdm2, and concomitant knockdown of p53 recovered cell proliferation and collagen expression. Transverse aortic constriction (TAC) was performed in S100A4 knockout (KO) mice, which showed a similar baseline-phenotype to wild type (WT) mice. Although there was no difference in hypertrophic response, KO mice showed reduced interstitial fibrosis, decreased myofibroblasts, and suppressed expressions of collagens and profibrotic cytokines in the left ventricle. Also, DNA microarray analysis showed that S100A4 knockout in vivo had a significant impact on expressions of p53-associated genes. These findings suggest that S100A4 modulates p53 function in fibroblasts and thereby mediates myocardial interstitial fibrosis through two distinct mechanisms; cell proliferation and collagen expression. Blockade of S100A4 may have therapeutic potential in cardiac hypertrophy and HF by attenuating cardiac fibrosis.

Dietary Flavonoids as Therapeutics for Preterm Birth: Luteolin and Kaempferol Suppress Inflammation in Human Gestational TissuesIn Vitro

Infection/inflammation is commonly associated with preterm birth (PTB), initiating uterine contractions and rupture of fetal membranes. Proinflammatory cytokines induce matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) and prostaglandins which initiate uterine contractions. Nuclear factor-κB (NF-κB) and activator-protein- (AP-)1 have key roles in the formation of these prolabour mediators. In nongestational tissues, dietary flavonoids such as luteolin and kaempferol inhibit NF-κB, AP-1, and their downstream targets. The aim of this study was to determine if luteolin and kaempferol reduce infection-induced prolabour mediators in human gestational tissues. Fetal membranes were incubated with LPS, and primary amnion cells and myometrial cells were incubated with IL-1β in the absence or presence of luteolin or kaempferol. Luteolin and kaempferol significantly reduced LPS-induced secretion of proinflammatory cytokines (IL-6 and IL-8) and prostaglandins (PGE2 and PGF2α) in fetal membranes, IL-1β-induced COX-2 gene expression and prostaglandin production in myometrium, and IL-1β-induced MMP-9 activity in amnion and myometrial cells. Luteolin and kaempferol decreased IL-1β-induced NF-κB p65 DNA binding activity and nuclear c-Jun expression. In conclusion, luteolin and kaempferol inhibit prolabour mediators in human gestational tissues. Given the central role of inflammation in provoking preterm labour, phytophenols may be a therapeutic approach to reduce the incidence of PTB.

Deformation of a P53 Response Element Revealed by Site-Directed Spin Labeling

The tumor suppressor protein p53 is called the “guardian of the genome” and has been implicated in a growing number of biological processes, including cell cycle arrest, senescence, apoptosis, autophagy, metabolism, and aging. As a transcription factor, p53 regulates a large number of signaling pathways by interacting with a diverse set of DNAs, called p53 response elements (REs), which are composed of two decameric consensus half-sites separated by a spacer. It has been proposed that DNA conformation changes upon p53 binding, which may vary between different REs, play important roles in p53-DNA recognition. However, details on such DNA conformational changes are limited, as currently there is no high-resolution structure of unbound RE, although a number of crystal structures of tetrameric p53 fragments bound to artificial or naturally occurring REs have been reported. Here site-directed spin labeling is used to probe solution conformations of an RE regulating protein p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation. Using a nucleotide-independent nitroxide probe and Double-Electron-Electron Resonance spectroscopy, nanometer distances between various DNA sites were measured in unbound p21RE as well as in that complexed to the p53 DNA binding domain. Models of the unbound p21RE, which were selected using the measured distances from a large pool generated by Monte-Carlo simulations, reveal major conformational changes at the half-site interface as compared to the bound DNA reported in a crystal structure. These results shed light on the mechanism of DNA recognition by p53.

P B S-1086, a “Pan-Rel” Inhibitor, Decreases Viability of Chronic Lymphocytic Leukemia Cells

AbstractAbstract 867The NF-κB signaling pathway is constitutively activated in chronic lymphocytic leukemia (CLL). Aberrant activation of the p65 (RelA) subunit of NF-κB is associated with chemoresistance, a shorter lymphocyte doubling time and poor overall survival (OS) in CLL. The transcription factor, NF-κB, regulates anti-apoptotic and pro-survival genes, and therefore represents a drug target for therapeutic intervention. Several studies have shown that targeting NF-κB decreases CLL cell survival, induces apoptosis and inhibits transcription of key pro-survival genes. In CLL, poor response to therapy and poor outcome are closely related to cytogenetic abnormalities. Loss of p53 function (del(17p) and/or TP53 mutation) confers chemo-resistance due to a defective DNA damage response pathway. Loss of the ATM (ataxia telangiectasia mutated) kinase (del(11q) and/or mutation of ATM) is also associated with chemo-resistance. We hypothesized that blocking NF-κB may circumvent the requirement for an active DNA damage response pathway, and that NF-κB inhibition may be efficacious independent of cytogenetic abnormalities in CLL. We tested the hypothesis with PBS-1086, a novel small molecule “pan-rel” inhibitor that has shown activity against multiple myeloma in vivo.For target validation, we analyzed NF-κB activity by quantifying NF-κB subunit DNA binding and rel expression in nuclear extracts prepared from patient-derived CLL cells. Levels of activated NF-κB binding correlated with rel protein levels. We confirmed that constitutive activation of the p65 subunit predicted shorter overall survival (p= 0.03, n=80, HR (hazard ratio) 2.4). We also found that the p50 and RelB subunits were associated with poor survival (p = 0.04, n = 80, HR = 2.5 and p=0.17, n=50, respectively). Whereas p65 and p50 (activated by the canonical pathway through IKKβ), were present at high levels in the majority of cases, RelB (activated by the non-canonical pathway) was found to be variable among patients, with levels ranging from very high to undetectable. We found that patients with del(17p) had higher levels of p65 and p50 DNA binding activity, whereas patients with del(11q) had higher levels of RelB activity. We examined the effect of PBS-1086, a “pan-rel” inhibitor, on viability of patient-derived CLL cells and compared it with BAY 11–7082, an IKKβ inhibitor that affects p65 and p50. PBS-1086 was more potent at decreasing CLL viability in a concentration-dependent manner than BAY 11–7082 (mean LC50 6.4 +/− 1.9 μM, and 14.7 +/− 1.7 μM, respectively, n=20). LC50 values of PBS-1086 and BAY 11–7082 derived from individual CLL samples correlated significantly, suggesting that they acted by inhibiting the same pathway (p=0.02, n=20). PBS-1086 LC50 values also correlated inversely with the DNA binding activity of p65 (p=0.01, n=17), suggesting that these CLL cells were heavily reliant on p65 for survival, and were therefore exquisitely vulnerable to compounds inhibiting p65. There was no significant difference in sensitivity to PBS-1086 or BAY 11–7082 among patients with different cytogenetic abnormalities, or Binet stage A or stage B/C cases, or between cases with or without IGVH gene mutations. Patients who had received prior chemotherapies at the time of sample collection showed a trend to be more resistant to ex vivo treatment with Fludarabine than those naïve to chemotherapies, however treatment status had no impact on ex vivo sensitivity to PBS-1086. Submaximal concentrations of Fludarabine and PBS-1086 were additive in decreasing CLL cell viability (n=3). Mechanistic studies showed that PBS-1086 inhibited p65 and p50 DNA binding activity in CLL cells (n=5). In one example case that had constitutive expression of all of the NF-κB subunits, PBS 1086 inhibited the DNA binding activity of all rel subunits. Both PBS-1086 and BAY 11–7082 induced apoptosis in a concentration-dependent manner in CLL cells (Annexin V staining, n=4) that paralled the inhibitor-induced loss of viability.These data demonstrate that PBS-1086 reduced survival of CLL cells ex vivo through inhibiting the DNA binding activity of rel subunits concomitantly with induction of apoptosis. Such effects of PBS-1086 were independent of poor prognosis cytogenetics, and of Binet stage or IGVH status. Thus, PBS-1086 is promising as a therapeutic agent by itself and also as a chemo-sensitizer to overcome NF-κB-mediated chemo-resistance in CLL. Disclosures:No relevant conflicts of interest to declare.

CC-122 Is a Novel Pleiotropic Pathway Modifier with Potent in Vitro Immunomodulatory and Anti-Angiogenic Properties and in Vivo Anti-Tumor Activity in Hematological Cancers.

AbstractAbstract 2963 Background:The immunomodulatory agents thalidomide (THAL), lenalidomide (LEN), and pomalidomide (POM) have significant activity in a wide range of hematologic cancers. THAL is primarily a potent anti-angiogenic agent with minimal immunomodulatory activity. LEN and POM both demonstrate significant immunomodulatory activity. Additionally, POM displays anti-myeloma activity in patients with LEN-resistant multiple myeloma (MM). Recently, modulation of cereblon (CRBN)-bound E3 ubiquitin ligase complexes has been implicated in the mechanisms of action of THAL, LEN, and POM. This has enabled rational development of a next generation of compounds. CC-122 is a non-phthalimide analog of the immunomodulatory drugs and a first in class PPM™ pleiotropic pathway modulator that binds the CRBN-DDB1-Cul4-Roc1 E3 ubiquitin ligase complex. This study investigated the anti-proliferative, immunomodulatory, and anti-angiogenic activity of CC-122 in MM and lymphoma cells. Results:CC-122 inhibited proliferation of H929 MM cells in a CRBN-dependent and dose-dependent manner (IC50 = 43 nM). CC-122 induced cell cycle arrest at G0/G1 stage, which was associated with reduced retinoblastoma protein phosphorylation, and increased CDK inhibitor p27 protein expression. CC-122 also inhibited the growth of LEN-resistant H929 cells, although the proliferation IC50 for CC-122 was relatively higher in LEN-resistant cells vs H929 control cells (Table). CC-122 has significant anti-myeloma activity, and has greater activity in LEN-resistant H929 cells vs LEN and POM. Compared with LEN, CC-122 had a greater anti-proliferative effect in diffuse-large B-cell lymphoma (DLBCL). Furthermore, CC-122 had greater anti-proliferative effects in ABC- and PBML-subtypes compared with GCB subtype lines. In ABC-subtype U2932 and OCI-Ly10-DLBCL cell lines, 10 μM CC-122 treatment significantly inhibited DNA-binding of NF-κB p65 (P < .001), p50 subunits (P < .05), and IRF4 in a CRBN-dependent manner. In vivo anticancer activity of CC-122 was demonstrated in xenograft models of human lymphoma and MM.CC-122 exhibits potent immunomodulatory activity in whole blood, T, and natural killer (NK) cells. Additionally, CC-122 enhanced the anti-CD3 mAb-stimulated T-cell production of IL-5, IL-13, GM-CSF, IFN-γ, RANTES, and TNF-α. The immunomodulatory activity of CC-122 was 10-fold more potent vs LEN.We investigated the anti-angiogenic properties of CC-122. In a human umbilical artery sprout outgrowth assay, CC-122 inhibited new vessel growth as well as endothelial cell migration and invasion. It also inhibited endothelial cell sprout formation and migration in a growth factor-induced endothelial cell migration and invasion assay. CC-122 has significantly greater anti-angiogenic activity compared with the LEN and POM in human angiogenesis assays (Table). In contrast, it has less of an anti-platelet effect as measured by megakaryocyte proliferation vs LEN and POM.CRBN binding competition assays were conducted with THAL-binding beads. As demonstrated by the higher IC50 concentration, CC-122 has less potency with regard to CRBN binding compared with LEN or POM. Conclusion:Together, these data demonstrate that the first-in-class PPM™ CC-122 has anti-proliferative, immunomodulatory, and anti-angiogenic properties that may have clinical significance in the treatment of advanced refractory lymphoproliferative disorders and is currently in Phase I studies. Furthermore the data suggest that the potency of binding to CRBN per se does not explain the broad pleiotropic activity of CC-122.Table:Activities of LEN, POM, and CC-122ActivityAssay/Cell LineLEN (μM)POM (μM)CC-122 (μM)Immune modulation (EC50)Whole blood TNF-α>100.150.14T-cell IL-20.170.0100.012NK-cell IFN-γ0.0520.00110.0015Anti-proliferative (IC50)H929 (MM)10.090.09LEN-Resistant H929 (MM)>306.0 to >300.8 to 2WSU-DLCL2 (GCB-DLBCL)>100NA2.1SUDHL4 (GCB-DLBCL)>100NA>10OCI-Ly19 (GCB DLBCL)>100NA>10OCI-Ly10 (ABC-DLBCL)0.15NA0.0085U2932 (ABC-DLBCL)2.6NA0.12TMD8 (ABC-DLBCL)75NA0.44RIVA (ABC-DLBCL)>100NA4.3Karpas-1106P (PMBL-DLBCL)>100NA0.71Anti-angiogenesis (IC50)Human Umbilical Artery1.70.330.0094Anti-platelet (IC50)Immature MK colonies0.41 to 1.30.35 to >10>10Intermediate MK colonies1.3 to >101.4 to >10>10CRBN binding (IC50)CRBN competition binding to THAL-beads2220 Disclosures:Gandhi:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Parton:Celgene Corp: Employment, Equity Ownership. Wu:Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Zhang:Celgene Corp: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Combinatorial Targeting of BCL6 and Anti-Apoptotic Proteins in Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphoma (FL)

Abstract 64BCL6 represents a survival factor in DLBCL and FL since specific BCL6 inhibitors (i.e: the peptidomimetic RI-BPI and the small molecule 79-6) kill DLBCL and transformed FL (tFL) cell lines. Our group showed that BCL2 and other anti-apoptotic genes are transcriptionally repressed by BCL6 and could be reactivated upon treatment with RI-BPI or 79-6. We also showed that BCL6 and BCL2 control distinct and non-overlapping survival pathways in these lymphomas. This suggests that blocking the function of anti-apoptotic proteins might overcome any resistance that these proteins might mediate in response to BCL6 inhibition. In addition, constitutive expression of BCL2 has been observed in DLBCL and FL cases.We therefore hypothesized that targeting both BCL6 and BCL2 would eliminate two of the most potent survival mechanisms and would translate in synergistic killing of these lymphomas. In order to test this hypothesis, we examined the proteome-wide consequence of BCL6 inhibition in a DLBCL cell line (SU-DHL6) transfected with siBCL6 by phospho-protein arrays. We found that 280 unique proteins changed their abundance after siBCL6, 40 of them related to (pro and anti) apoptosis signaling (p<0.001). By means of pathway analysis bioinformatic tools, we then identified anti-apoptotic proteins with increased abundance after siBCL6 that could be therapeutically targeted. Among these druggable proteins we found BCL2, BCL-XL, MCL-1, NEDD8, PARP1 and several ubiquitin ligases. We confirmed in independent experiments that BCL6 knockdown induced mRNA (by qPCR) and protein up-regulation (by immunobloting) of these genes in 2 additional DLBCL and 2 tFL cell lines. Treatment of these siBCL6-transfected cell lines with small molecules inhibitors of BCL2 family members (ABT-737 and oblatoclax), NEDD8 activating enzyme (MLN4924), PARP (olaparib) and proteasome (bortezomib) showed increased killing compared to each treatment alone. In order to identify rational combinatorial therapies that could be potentially translated for use in clinical trials, we performed additional studies with the BCL6 inhibitor RI-BPI that is being developed for clinical use. We first analyzed the impact of RI-BPI on the apoptosome in a panel of 6 DLBCL (SU-DHL6, Ly1, Ly7, Ly3, Ly10, SU-DHL4) and 4 tFL (DoHH2, WSU-DLCL2, Granta452, SC-1) cell lines. RI-BPI induced a profile of up-regulated pro and anti-apoptotic proteins similar to siBCL6. Because ABT-737 and obatoclax are active in DLBCL cells where apoptotic BH3 activators are neutralized by BCL2 or BCL-XL and RI-BPI treatment changes the stoichiometry of pro and anti-apoptotic proteins, we determined the post-RI-BPI BH3 profiling accordingly to the amount of BIM sequestration (by co-immunoprecipitation). Accordingly, sequential treatment of DLBCL and tFL cell lines with RI-BPI and ABT-737 or obatoclax synergistically killed BCL2/BCL-XL dependent cells (but not MCL-1 dependent cells). This effect was independent of the mutational status of BCL2, MCL1, BCL6, MYC and TP53. Olaparib was not tested in combination since most cell lines were resistant to clinically achievable concentrations of this drug. Bortezomib and MLN4924 were synergistic in most cell lines when combined with RI-BPI (as determined by isobologram analysis). The synergistic killing was associated with increase in caspase 7/3 activation (by a plate-based assay) and NFkB inhibition (by p65 DNA binding assay). This effect was independent of the cell of origin classification of the cell line (i.e. ABC vs. GCB). We then tested the combination of RI-BPI with ABT-737, MLN4924 or bortezomib in Ly1 xenograft models (n=10 mice per combination). Ly1 represents a DLBCL with 3q27 and t(14,18). We found that after 10 days of treatment, each combinatorial treatment was more effective than their individual components (p=0.02, p=0.01 and p<0.01 for RI-BPI with ABT-737, MLN4924 and bortezomib, respectively; T-test, day 10). Detailed toxicity studies revealed no toxicity excess with these combinations.In sum, our work shows that pharmacologic targeting of anti-apoptotic pathways induced by inhibition of BCL6 activity successfully sensitized DLBCL and tFL cells to apoptosis. This effect was evident in cells in which the apoptosis resistant mechanism evolved as response to BCL6 inhibition and gene de-repression as well as those with constitutive overexpression of anti-apoptotic genes. Disclosures:No relevant conflicts of interest to declare.

Defining critical roles for NF‐κB p65 and type I interferon in innate immunity to rhinovirus

The importance of NF-κB activation and deficient anti-viral interferon induction in the pathogenesis of rhinovirus-induced asthma exacerbations is poorly understood. We provide the first in vivo evidence in man and mouse that rhinovirus infection enhanced bronchial epithelial cell NF-κB p65 nuclear expression, NF-κB p65 DNA binding in lung tissue and NF-κB-regulated airway inflammation. In vitro inhibition of NF-κB reduced rhinovirus-induced pro-inflammatory cytokines but did not affect type I/III interferon induction. Rhinovirus-infected p65-deficient mice exhibited reduced neutrophilic inflammation, yet interferon induction, antiviral responses and virus loads were unaffected, indicating that NF-κB p65 is required for pro-inflammatory responses, but redundant in interferon induction by rhinoviruses in vivo. Conversely, IFNAR1−/− mice exhibited enhanced neutrophilic inflammation with impaired antiviral immunity and increased rhinovirus replication, demonstrating that interferon signalling was critical to antiviral immunity. We thus provide new mechanistic insights into rhinovirus infection and demonstrate the therapeutic potential of targeting NF-κB p65 (to suppress inflammation but preserve anti-viral immunity) and type I IFN signalling (to enhance deficient anti-viral immunity) to treat rhinovirus-induced exacerbations of airway diseases.

Adjudin attenuates lipopolysaccharide (LPS)- and ischemia-induced microglial activation

Neuroinflammation caused by microglial activation plays a key role in ischemia, neurodegeneration and many other CNS diseases. In this study, we found that Adjudin, a potential non-hormonal male contraceptive, exhibits additional function to reduce the production of proinflammatory mediators. Adjudin significantly inhibited LPS-induced IL-6 release and IL-6, IL-1β, TNF-α expression in BV2 microglial cells. Furthermore, Adjudin exhibited anti-inflammatory properties by suppression of NF-κB p65 nuclear translocation and DNA binding activity as well as ERK MAPK phosphorylation. To determine the in vivo effect of Adjudin, we used a permanent middle cerebral artery occlusion (pMCAO) mouse model and found that Adjudin could reduce ischemia-induced CD11b expression, a marker of microglial activation. Furthermore, Adjudin treatment attenuated brain edema and neurological deficits after ischemia but did not reduce infarct volume. Thus, our data suggest that Adjudin may be useful for mitigating neuroinflammation.

HMGB1-Facilitated p53 DNA Binding Occurs via HMG-Box/p53 Transactivation Domain Interaction, Regulated by the Acidic Tail

Facilitated binding of p53 to DNA by high mobility group B1 (HMGB1) may involve interaction between the N-terminal region of p53 and the high mobility group (HMG) boxes, as well as HMG-induced bending of the DNA. Intramolecular shielding of the boxes by the HMGB1 acidic tail results in an unstable complex with p53 until the tail is truncated to half its length, at which point the A box, proposed to be the preferred binding site for p53(1-93), is exposed, leaving the B box to bind and bend DNA. The A box interacts with residues 38-61 (TAD2) of the p53 transactivation domain. Residues 19-26 (TAD1) bind weakly, but only in the context of p53(1-93) and not as a free TAD1 peptide. We have solved the structure of the A-box/p53(1-93) complex by nuclear magnetic resonance spectroscopy. The incipient amphipathic helix in TAD2 recognizes the concave DNA-binding face of the A box and may be acting as a single-stranded DNA mimic.

Drug Resistance to Inhibitors of the Human Double Minute-2 E3 Ligase Is Mediated by Point Mutations of p53, but Can Be Overcome with the p53 Targeting Agent RITA

The human double minute (HDM)-2 E3 ubiquitin ligase plays a key role in p53 turnover and has been validated preclinically as a target in multiple myeloma (MM) and mantle cell lymphoma (MCL). HDM-2 inhibitors are entering clinical trials, and we therefore sought to understand potential mechanisms of resistance in lymphoid models. Wild-type p53 H929 MM and Granta-519 MCL cells resistant to MI-63 or Nutlin were generated by exposing them to increasing drug concentrations. MI-63-resistant H929 and Granta-519 cells were resistant to Nutlin, whereas Nutlin-resistant cells displayed cross-resistance to MI-63. These cells also showed cross-resistance to bortezomib, doxorubicin, cisplatin, and melphalan, but remained sensitive to the small molecule inhibitor RITA (reactivation of p53 and induction of tumor cell apoptosis). HDM-2 inhibitor-resistant cells harbored increased p53 levels, but neither genotoxic nor nongenotoxic approaches to activate p53 induced HDM-2 or p21. Resequencing revealed wild-type HDM-2, but mutations were found in the p53 DNA binding and dimerization domains. In resistant cells, RITA induced a G(2)-M arrest, upregulation of p53 targets HDM-2, PUMA, and NOXA, and PARP cleavage. Combination regimens with RITA and MI-63 resulted in enhanced cell death compared with RITA alone. These findings support the possibility that p53 mutation could be a primary mechanism of acquired resistance to HDM-2 inhibitors in MCL and MM. Furthermore, they suggest that simultaneous restoration of p53 function and HDM-2 inhibition is a rational strategy for clinical translation.

Ciliary neurotrophic factor activates NF-κB to enhance mitochondrial bioenergetics and prevent neuropathy in sensory neurons of streptozotocin-induced diabetic rodents

Diabetes causes mitochondrial dysfunction in sensory neurons that may contribute to peripheral neuropathy. Ciliary neurotrophic factor (CNTF) promotes sensory neuron survival and axon regeneration and prevents axonal dwindling, nerve conduction deficits and thermal hypoalgesia in diabetic rats. In this study, we tested the hypothesis that CNTF protects sensory neuron function during diabetes through normalization of impaired mitochondrial bioenergetics. In addition, we investigated whether the NF-κB signal transduction pathway was mobilized by CNTF. Neurite outgrowth of sensory neurons derived from streptozotocin (STZ)-induced diabetic rats was reduced compared to neurons from control rats and exposure to CNTF for 24 h enhanced neurite outgrowth. CNTF also activated NF-κB, as assessed by Western blotting for the NF-κB p50 subunit and reporter assays for NF-κB promoter activity. Conversely, blockade of NF-κB signaling using SN50 peptide inhibited CNTF-mediated neurite outgrowth. Studies in mice with STZ-induced diabetes demonstrated that systemic therapy with CNTF prevented functional indices of peripheral neuropathy along with deficiencies in dorsal root ganglion (DRG) NF-κB p50 expression and DNA binding activity. DRG neurons derived from STZ-diabetic mice also exhibited deficiencies in maximal oxygen consumption rate and associated spare respiratory capacity that were corrected by exposure to CNTF for 24 h in an NF-κB-dependent manner. We propose that the ability of CNTF to enhance axon regeneration and protect peripheral nerve from structural and functional indices of diabetic peripheral neuropathy is associated with targeting of mitochondrial function, in part via NF-κB activation, and improvement of cellular bioenergetics.

Inhibition of nucleotide excision repair by arsenic

Inhibition of DNA repair is one proposed mechanism for the co-mutagenicity/co-carcinogenicity of arsenic. This review summarizes the current literature on the effects of arsenic compounds on nucleotide excision repair (NER). Several possible mechanisms for the observed NER inhibition have been proposed. Modulation of the expression of NER proteins has been considered to be one possibility of impairing the NER process. However, data on the effects of arsenic on the expression of NER proteins remain inconsistent. It is more likely that arsenic inhibits the induction of accessory or other key proteins involved in cellular control of DNA repair pathways, such as p53. For example, arsenic affects p53 phosphorylation and p53 DNA binding activity, which could regulate NER through transcriptional activation of downstream NER genes. Although it is important to study possible direct inactivation of NER proteins by arsenic binding, indirect inactivation of proteins having thiol residues critical to their function or zinc finger proteins cannot be negated. For example, nitric oxide (NO) induced in arsenic-treated cells serves as a specific inhibitor of NER, possibly through NO-induced S-nitrosylation of proteins related to DNA repair. Poly(ADP-ribose) polymerase-1, a zinc finger protein implicated in both NER and base excision repair (BER), deserves special attention because of its involvement in NO production and its broad range of protein substrates including many repair enzymes.

Epigenetic control of metastasis-associated protein 1 gene expression by hepatitis B virus X protein during hepatocarcinogenesis

Expression of metastasis-associated protein 1 (MTA1) gene correlates with the degree of invasion and metastasis in hepatocellular carcinoma (HCC). Expression of MTA1 is induced by hepatitis B virus X protein (HBx); however, little is known about the transcriptional regulation of MTA1 gene expression. Here, we report that the 5′-flanking region of the human MTA1 promoter contains two CpG islands. Transient expression of HBx in Chang liver cells increased the methylation of the CpG island1 from 18 to 49% when measured by bisulfite-modified direct sequencing. Chromatin immunoprecipitation showed that HBx recruited DNA methyltransferase 3a (DNMT3a) and DNMT3b to the CpG island1. In silico analysis of CpG island1 predicted the existence of putative p53-binding sequences. p53 was pulled down by a DNA probe encoding the p53-binding sequences but not by the methylated DNA probe. The mouse MTA1 promoter also contains a CpG island encoding a p53-binding sequence of which p53 binding was decreased in the presence of HBx, and the expression of MTA1 and DNMT3 was increased in the liver of HBx-transgenic mice. Comparison of MTA1 and DNMT3a expression in the human normal liver and HCC specimens produced a significant correlation coefficient >0.5 (r=0.5686, P=0.0001) for DNMT3a, and a marginally significant coefficient (r=0.3162, P=0.0103) for DNMT3b. These data show that HBx induces methylation of CpG island in the MTA1 promoter, which interferes with DNA binding of p53 in the specific DNA region. This result may explain the molecular mechanism responsible for the induction of MTA1 gene expression by HBx.

P17 Hydrogen sulfide suppresses ox-LDL-stimulatedmonocyte chemoattractant protein-1 generation from macrophages via NF-κB pathway

To examine the role of hydrogen sulfide (H 2 S) in oxidized low-density lipoprotein (ox-LDL)-stimulated monocyte chemoattractant protein-1 (MCP-1) generation from macrophages and possible mechanisms. THP-1 cells and RAW macrophages were pretreated with sodium hydrosulfide (NaHS), dl -propargylglycine (PPG) or hexyl acrylate (HA) and then treated with ox-LDL. Endogenous H 2 S pathway and MCP-1 generation were examined. The phosphorylation of NF- κ B p65 was detected by Western blotting. NF- κ B p65 nuclear translocation was detected using confocal images. NF- κ B p65 DNA binding activity was examined by electrophoretic mobility shift assay, ELISA and chromatin immunoprecipitation assay. The results showed that in THP-1 cells, ox-LDL treatment decreased H 2 S content, cystathionie-ß-sythase (CBS) protein and mRNA expression, but had no effect on cystathionine- γ -lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST) expression. Moreover, ox-LDL treatment significantly increased MCP-1 content and its protein and mRNA expression in both THP-1 cells and RAW macrophages, while PPG and HA promoted this increase further. However, NaHS markedly suppressed NF- κ B p65 phosphorylation, nuclear translocation and binding activity with MCP-1 promoter, resulting in a reduced generation of MCP-1 in ox-LDL treated THP-1 cells and RAW macrophages. Endogenous H 2 S/CBS pathway was down-regulated in ox-LDL-treated macrophages. H 2 S suppressed MCP-1 generation from ox-LDL-treated macrophages via suppressing NF- κ B p65 phosphorylation, nuclear translocation and the recruitment of NF- κ B p65 to the MCP-1 promoter region.

The DNA binding and accumulation of p53 from breast cancer cell lines and the link with serine 15 phosphorylation

Stress treatment generally causes the post-translational modification and accumulation of the p53 protein, although the role of these aspects has not been always understood in relation to this protein’s tumor suppressor activity. We analyzed these attributes of p53 in eight different breast cancer cell lines, with either wild-type or mutant p53 protein, in response to oxidative stress. We found that the wild-type p53 protein from MCF-7 and ZR-75-1 cells binds with different affinity to 12 gene sequences covering several pathways regulated by p53. Treatment of MCF-7 cells with H2O2 caused an increase in this binding affinity while this same treatment of ZR-75-1 cells caused the p53 protein to lose binding affinity to several genes. The mutant p53 proteins from all cell lines had minimal to weak binding to these sequences even after treatment with H2O2. The p53 protein from the ZR-75-1 cells and three cell lines with mutant p53 showed serine 15 phosphorylated protein, but we found no correlation between that modification and the levels or localization of this protein although DNA binding affinity of wild-type protein might be affected by this modification. From this and other work, it appears that the mutation status of the TP53 gene alone cannot predict the activity of this tumor suppressor since cell lines with the same genetic information do not show the same properties of this protein.

The Tumor Suppressor p53 Regulates c-Maf and Prox-1 to Control Lens Differentiation

The tumor suppressor p53 plays a key role in regulating apoptosis and cell cycle progression. In addition, p53 is implicated in control of cell differentiation in muscle, the circulatory system, ocular lens and various carcinoma tissues. However, the mechanisms by which p53 controls cell differentiation are not fully understood. Here we present evidence that p53 directly regulates c-Maf and Prox1, two important transcription factors controlling differentiation in the ocular lens. First, human and murine c-Maf and Prox1 gene promoters contain authentic p53 DNA binding sites. Second, p53 directly binds to the p53 binding sites found in the promoter regions. Third, exogenous p53 induces dose-dependent expression of the luciferase report gene driven by both c-Maf and Prox1 promoters, and p53 binds to both promoters in the ChIP assays. Fourth, in the in vitro differentiation model, knockdown of p53 significantly inhibits lens differentiation which is associated with downregulated expression of c-Maf and Prox1. Finally, in p53 knockout mice, the expression of c-Maf and Prox1 are significantly altered. Together, our results reveal that p53 regulates lens differentiation through modulation of two important transcription factors, c-Maf and Prox1, and through them p53 thus controls expression of various differentiation-related downstream crystallin genes.

Protein Kinase Cα (PKCα) Regulates p53 Localization and Melanoma Cell Survival Downstream of Integrin αv in Three-dimensional Collagen and in Vivo

Protein kinase C α (PKCα) is overexpressed in numerous types of cancer. Importantly, PKCα has been linked to metastasis of malignant melanoma in patients. However, it has been unclear how PKCα may be regulated and how it exerts its role in melanoma. Here, we identified a role for PKCα in melanoma cell survival in a three-dimensional collagen model mimicking the in vivo pathophysiology of the dermis. A pathway was identified that involved integrin αv-mediated up-regulation of PKCα and PKCα-dependent regulation of p53 localization, which was connected to melanoma cell survival. Melanoma survival and growth in three-dimensional microenvironments requires the expression of integrin αv, which acts to suppress p53 activity. Interestingly, microarray analysis revealed that PKCα was up-regulated by integrin αv in a three-dimensional microenvironment-dependent manner. Integrin αv was observed to promote a relocalization of endogenous p53 from the nucleus to the cytoplasm upon growth in three-dimensional collagen as well as in vivo, whereas stable knockdown of PKCα inhibited the integrin αv-mediated relocalization of p53. Importantly, knockdown of PKCα also promoted apoptosis in three-dimensional collagen and in vivo, resulting in reduced tumor growth. This indicates that PKCα constitutes a crucial component of the integrin αv-mediated pathway(s) that promote p53 relocalization and melanoma survival.

Radioprobing the conformation of DNA in a p53-DNA complex

Purpose: The frequency of DNA strand breaks produced by the decay of Auger electron-emitting radionuclides is inversely proportional to the distance of DNA nucleotides from the decay site; and thus is very sensitive to changes in the local conformation of the DNA. Analysis of the frequency of DNA breaks, or radioprobing, gives valuable information about the local DNA structure. More than 10 years ago, we demonstrated the feasibility of radioprobing using a DNA-repressor complex with a known structure. Herein, we used radioprobing to study the conformation of DNA in complex with the tumor suppressor protein 53 (p53). Several structures of p53-DNA complexes have been solved by X-ray crystallography. These structures, obtained with the p53 DNA binding domain, a truncated form, laid the groundwork for understanding p53-DNA interactions and their relation to p53 functions. However, whether all observed stereochemical details are relevant to the native p53-DNA complex remains unclear. A common theme of the crystallographic structures is the lack of significant bending in the central part of the DNA response element. In contrast, gel electrophoresis and electron microscopy data showed strong DNA bending and overtwisting upon binding to the native p53 tetramer.Methods: To analyze DNA in complex with p53, we incorporated 125I-dCTP in two different positions of synthetic duplexes containing the consensus p53-binding site.Results: The most significant changes in the break frequency distributions were detected close to the center of the binding site, which is consistent with an increase in DNA twisting in this region and local DNA bending and sliding.Conclusions: Our data confirm the main results of the studies made in solution and lay a foundation for systematic examination of interactions between DNA and native p53 using 125I radioprobing.

Effect of N-acetylcysteine (NAC) on acute lung injury and acute kidney injury in hemorrhagic shock

Aim of the studyN-acetylcysteine (NAC) has been investigated to attenuate organ injury in various experimental and clinical studies. However, results in hemorrhagic shock (HS) were controversial. We determined the effects of continuous administration of NAC on acute lung injury (ALI) and acute kidney injury (AKI) in HS model. MethodsTwenty male Sprague-Dawley rats were used. Pressure controlled HS model defined by mean arterial pressure (MAP) 40±2mmHg for 90min followed by resuscitation and observation was used. Rats (n=10 per group) were randomized into 2 groups with NAC or dextrose. Intravenous NAC was given continuously from 15min after induction of HS to the end of observation period (2h). We measured serum IL-6, nitrite/nitrate concentration. NF-κB p65 DNA binding activity, expressions of cytoplasmic phosphorylated IκB-α (p-IκB-α) and IκB-α, malondialdehyde (MDA) and histopathological injury scores in lung and kidney were also evaluated. ResultsMAP did not show any difference during the study period. NAC decreased histopathologic scores in both lung and kidney. Lung and kidney MDA levels were significantly lower in the NAC group compared to control group. Serum nitrite/nitrate and IL-6 were also significantly lower in the NAC group. The levels of lung cytoplasmic p-IκB-α expression was mitigated by NAC, and NF-κB p65 DNA binding activity was also significantly decreased in the NAC group. ConclusionsContinuous infusion of NAC attenuated inflammatory response and acute lung and kidney injury after hemorrhagic shock in rats.

RETRACTED ARTICLE: Dose-dependent changes in neuroinflammatory and arachidonic acid cascade markers with synaptic marker loss in rat lipopolysaccharide infusion model of neuroinflammation

BackgroundNeuroinflammation, caused by six days of intracerebroventricular infusion of bacterial lipopolysaccharide (LPS), stimulates rat brain arachidonic acid (AA) metabolism. The molecular changes associated with increased AA metabolism are not clear. We examined effects of a six-day infusion of a low-dose (0.5 ng/h) and a high-dose (250 ng/h) of LPS on neuroinflammatory, AA cascade, and pre- and post-synaptic markers in rat brain. We used artificial cerebrospinal fluid-infused brains as controls.ResultsInfusion of low- or high-dose LPS increased brain protein levels of TNFα, and iNOS, without significantly changing GFAP. High-dose LPS infusion upregulated brain protein and mRNA levels of AA cascade markers (cytosolic cPLA2-IVA, secretory sPLA2-V, cyclooxygenase-2 and 5-lipoxygenase), and of transcription factor NF-κB p50 DNA binding activity. Both LPS doses increased cPLA2 and p38 mitogen-activated protein kinase levels, while reducing protein levels of the pre-synaptic marker, synaptophysin. Post-synaptic markers drebrin and PSD95 protein levels were decreased with high- but not low-dose LPS.ConclusionsChronic LPS infusion has differential effects, depending on dose, on inflammatory, AA and synaptic markers in rat brain. Neuroinflammation associated with upregulated brain AA metabolism can lead to synaptic dysfunction.