P53 DNA Binding Research Articles

Inhibition of p53 DNA binding by a small molecule protects mice from radiation toxicity.

Transcription factors are attractive therapeutic targets that are considered non-druggable because they do not have binding sites for small drug-like ligands. We established a cell-free high-throughput screening assay to search for small molecule inhibitors of DNA binding by transcription factors. A screen was performed using p53 as a target, resulting in the identification of NSC194598 that inhibits p53 sequence-specific DNA binding in vitro (IC50 = 180 nM) and in vivo. NSC194598 selectively inhibited DNA binding by p53 and homologs p63/p73, but did not affect E2F1, TCF1, and c-Myc. Treatment of cells with NSC194598 alone paradoxically led to p53 accumulation and modest increase of transcriptional output owing to disruption of the MDM2-negative feedback loop. When p53 was stabilized and activated by irradiation or chemotherapy drug treatment, NSC194598 inhibited p53 DNA binding and induction of target genes. A single dose of NSC194598 increased the survival of mice after irradiation. The results suggest DNA binding by p53 can be targeted using small molecules to reduce acute toxicity to normal tissues by radiation and chemotherapy.

P60-katanin: a novel interacting partner for p53.

Katanin, one of the best-characterized microtubule (MT) severing proteins, is composed of two subunits: catalytic p60-katanin, and regulatory p80-katanin. p60-katanin triggers MT reorganization by severing them. MT reorganization is essential for both mitotic cells and post-mitotic neurons in numerous vital processes such as intracellular transport, mitosis, cellular differentiation and apoptosis. Due to the deleterious effect of continuous severing for cells, p60-katanin requires a strategic regulation. However, there are only a few known regulators of p60-katanin. p53 functions in similar cellular processes as katanin such as cell cycle, differentiation, and apoptosis depending on its interacting partners. Considering this similarity, in this study we investigated p53 as a potential regulatory candidate of p60-katanin, and examined their interaction. Co-immunoprecipitation analyses revealed that p60-katanin interacts with p53. We were able to locate a potential interaction site for the two proteins by deleting different candidate regions We showed for the first time that p53 and p60-katanin interact. This interaction appears to occur via p53's DNA binding domain and p60-katanin's C-terminal. This study will pave the way for future studies regarding the functional outcomes of this interaction which is vital for understanding the regulation of cellular events such as cell cycle, differentiation, and apoptosis in disease and in health.

SN50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome mice through inhibiting NF-\u03baB p65 translocation

BackgroundIt has been confirmed that NF-κB p65 signaling pathway is involved in the regulation of alveolar hypercoagulation and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS). Whether SN50, a NF-κB cell permeable inhibitor, could attenuate alveolar hypercoagulation and fibrinolysis inhibition in ARDS remains to be elucidated.PurposeWe explored the efficacy and potential mechanism of SN50 on alveolar hypercoagulation and fibrinolysis inhibition in ARDS in mice.Materials and methodsMouse ARDS was made by 50 μl of lipopolysaccharide (LPS) (4 mg/ml) inhalation. Male BALB/c mice were intraperitoneally injected with different does of SN50 1 h before LPS inhalation. Lung tissues were collected for hematoxylin-eosin (HE) staining, wet/dry ratio. Pulmonary expressions of tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), collagen III, as well as phosphorylated p65 (p-p65), p65 in nucleus (p’-p65), IκBα and IKKα/β were measured. Bronchoalveolar lavage fluid (BALF) was gathered to test the concentrations of TF, PAI-1, activated protein C (APC) and thrombinantithrombin complex (TAT). DNA binding activity of NF-κB p65 was also determined.ResultsAfter LPS stimulation, pulmonary edema and exudation and alveolar collapse occured. LPS also stimulated higher expressions of TF and PAI-1 in lung tissues, and higher secretions of TF, PAI-1, TAT and low level of APC in BALF. Pulmonary collagen III expression was obviously enhanced after LPS inhalation. At same time, NF-κB signaling pathway was activated with LPS injury, shown by higher expressions of p-p65, p’-p65, p-IKKα/β, p-Iκα in pulmonary tissue and higher level p65 DNA binding activity. SN50 dose-dependently inhibited TF, PAI-1 and collagen IIIexpressions, and decreased TF, PAI-1, TAT but increased APC in BALF. SN50 treatment attenuated pulmonary edema, exudation and reduced lung tissue damage as well. SN50 application significantly reduced p’-p65 expression and weakened p65 DNA binding activity, but expressions of p-p65, p-IKKα/β, p-Iκα in cytoplasm of pulmonary tissue were not affected.ConclusionsSN 50 attenuates alveolar hypercoagulation and fibrinolysis inhibition in ARDS via inhibition of NF-κB p65 translocation. Our data demonstrates that NF-κB p65 pathway is a viable new therapeutic target for ARDS treatment.

Activation of Mitochondrial 2-Oxoglutarate Dehydrogenase by Cocarboxylase in Human Lung Adenocarcinoma Cells A549 Is p53/p21-Dependent and Impairs Cellular Redox State, Mimicking the Cisplatin Action.

Genetic up-regulation of mitochondrial 2-oxoglutarate dehydrogenase is known to increase reactive oxygen species, being detrimental for cancer cells. Thiamine diphosphate (ThDP, cocarboxylase) is an essential activator of the enzyme and inhibits p53–DNA binding in cancer cells. We hypothesize that the pleiotropic regulator ThDP may be of importance for anticancer therapies. The hypothesis is tested in the present work on lung adenocarcinoma cells A549 possessing the p53–p21 pathway as fully functional or perturbed by p21 knockdown. Molecular mechanisms of ThDP action on cellular viability and their interplay with the cisplatin and p53–p21 pathways are characterized. Despite the well-known antioxidant properties of thiamine, A549 cells exhibit decreases in their reducing power and glutathione level after incubation with 5 mM ThDP, not observed in non-cancer epithelial cells Vero. Moreover, thiamine deficiency elevates glutathione in A549 cells. Viability of the thiamine deficient A549 cells is increased at a low (0.05 mM) ThDP. However, the increase is attenuated by 5 mM ThDP, p21 knockdown, specific inhibitor of the 2-oxoglutarate dehydrogenase complex (OGDHC), or cisplatin. Cellular levels of the catalytically competent ThDP·OGDHC holoenzyme are dysregulated by p21 knockdown and correlate negatively with the A549 viability. The inverse relationship between cellular glutathione and holo-OGDHC is corroborated by their comparison in the A549 and Vero cells. The similarity, non-additivity, and p21 dependence of the dual actions of ThDP and cisplatin on A549 cells manifest a common OGDHC-mediated mechanism of the viability decrease. High ThDP saturation of OGDHC compromises the redox state of A549 cells under the control of p53–p21 axes.

Epigenetic modifier trichostatin A enhanced osteogenic differentiation of mesenchymal stem cells by inhibiting NF-κB (p65) DNA binding and promoted periodontal repair in rats.

We wished to evaluate whether epigenetic modifiers have a beneficial effect on treating experimental periodontitisand mechanisms forregulating the cell fate of mesenchymal stem cells (MSCs) in inflammatory microenvironments. We isolated MSCs from healthy and inflamed gingival tissues to investigatewhether trichostatin A (TSA) could improve osteogenic differentiation and resolve inflammation in vitro. The tissue regenerative potentials were evaluated when treated with a temperature-dependent, chitosan-scaffold-encapsulated TSA, in a rat model of periodontitis. After induction with the conditioned medium, TSA treatment increased the osteogenic differentiation potential of inflamed MSCs and healthy MSCs. In addition, interleukin-6 and interleukin-8 levels in supernatants were significantly decreased after TSA treatment. Moreover, TSA promoted osteogenic differentiation by inhibiting nuclear factor-κB (p65) DNA binding in MSCs. In rats with experimental periodontitis, 7 weeks after local injections of chitosan-scaffold-encapsulated TSA, histology and microcomputed tomography showed a significant increase in alveolar bone volume and less inflammatory infiltration compared with vehicle-treated rats. The concentrations of interferon-γ and interleukin-6 were significantly decreased in the gingival crevicular fluid after TSA treatment. This study demonstrated that TSA had anti-inflammatory properties and could promote periodontal tissue repair, which indicated that epigenetic modifiers hold promise as a potential therapeutic option for periodontal tissue repair.

P53/PGC‑1α‑mediated mitochondrial dysfunction promotes PC3 prostate cancer cell apoptosis.

Data for p53 mutation in prostate cancer in The Cancer Genome Atlas database revealed that >85% of p53 mutations occurred in the p53 DNA binding domain. These mutations not only severely damage the function of the p53 protein, but also reduce the disease-free survival of patients. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is involved in the regulation of mitochondrial function and is highly expressed in prostate cancer PC3 and DU145 cells with p53 deletion or mutation. However, whether p53 negatively regulates PGC-1α in prostate cancer cells remains to be elucidated. In the present study, p53 overexpression was induced in prostate cancer PC3 cells. Subsequently, the expression levels of PGC-1α and alterations to mitochondrial function were assessed. Moreover, PGC-1α was activated in prostate cancer PC3 cells using ZLN005 to investigate alterations to mitochondrial function and cell apoptosis. The present study revealed that p53 decreased the expression and nuclear localization of the PGC-1α protein and induced mitochondrial dysfunction. Activation of PGC-1α partially reversed p53-mediated mitochondrial dysfunction. Inhibition of the p53/PGC-1α pathway on mitochondrial biogenesis and fission-/fusion-associated gene and protein expression were associated with mitochondrial dysfunction. p53/PGC-1α-mediated mitochondrial dysfunction promoted apoptosis of PC3 prostate cancer cells. The results indicated that PGC-1α is an essential target of p53-induced apoptosis in prostate cancer cells and indicated that targeting PGC-1α may provide a new therapeutic strategy for prostate cancer.

Therapeutic Effect of Oridonin Against Inflammatory Bowel Disease Involves Inhibition of Intestinal Fibrosis via NF‐κB Pathway

IntroductionIntestinal fibrosis and stricture formation are frequent complications of inflammatory bowel disease (IBD). In response to inflammatory stimuli, intestinal epithelial cells may undergo epithelial‐mesenchymal transition (EMT). EMT is a process by which epithelial cells acquire a mesenchymal‐like phenotype, resulting in intestinal fibrosis. Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, was reported to have therapeutic effects for IBD. However, the cellular mechanism remains unclear. We hypothesize that oridonin attenuates inflammation‐related EMT and fibrogenesis in intestinal epithelial cells.MethodsIEC‐6 and IEC‐18 cells exposed to TNFα were pre‐treated with or without oridonin. Cellular proteins were analyzed with Western blot or immunofluorescence assay. Cytokine secretion was determined with ELISA.ResultsOridonin treatment suppressed TNFα‐induced NF‐κB activation evidenced by blocking TNFα‐induced NF‐κB p65 nuclear translocation and DNA binding activity. TNFα‐induced phosphorylation of IKKα/β and IκBα as well as total IκBα degradation were prevented by oridonin. TNFα‐stimulated secretion of pro‐inflammatory cytokine IL‐6 was inhibited by oridonin. Oridonin moderately up‐regulates endogenous E‐cadherin and down‐regulates Snail. The expression of extracellular matrix proteins fibronectin and laminin were increased by TNFα and suppressed by pre‐treatment with oridonin. For the first time, we demonstrated that TNFα dose‐dependently up‐regulated α‐smooth muscle actin (α‐SMA), a myofibroblast marker, in IEC‐6 cells. These results indicate that IEC‐6 cells underwent transition into mesenchymal cells after TNFα stimulation. Notably, oridonin inhibited TNFα‐stimulated α‐SMA, but not TGFβ1‐stimulated EMT‐related proteins.ConclusionThe protective effects of oridonin in intestinal epithelial cells may occur through suppression of the TNFα‐induced NF‐κB pathway and EMT‐related fibrogenesis.

A Protein in the Yeast Saccharomyces cerevisiae Presents DNA Binding Homology to the p53 Checkpoint Protein and Tumor Suppressor.

Saccharomyces cerevisiae does not contain a p53 homolog. Utilizing this yeast as an in vivo test tube model, our aim was to investigate if a yeast protein would show p53 DNA binding homology. Electrophoretic mobility shift analyses revealed the formation of specific DNA-protein complexes consisting of S. cerevisiae nuclear protein(s) and oligonucleotides containing p53 DNA binding sites. A S. cerevisiae p53 binding site factor (Scp53BSF) bound to a p53 synthetic DNA-consensus sequence (SCS) and a p53 binding-site sequence from the MDM2 oncogene. The complexes were of comparable size. Like mammalian p53, the affinity of Scp53BSF for the SCS oligonucleotide was higher than for the MDM2 oligonucleotide. Binding of Scp53BSF to the SCS and MDM2 oligonucleotides was strongly competed by unlabeled oligonucleotides containing mammalian p53 sites, but very little by a mutated site oligonucleotide. Importantly, Scp53BSF-DNA binding activity was significantly induced in extracts from cells with DNA damage. This resulted in dose-dependent coordinated activation of transcription when using p53-binding site reporter constructs. An ancient p53-like DNA binding protein may have been found, and activation of DNA-associated factors to p53 response elements may have functions not yet determined.

Identification of universal and cell-type specific p53 DNA binding

BackgroundThe tumor suppressor p53 is a major regulator of the DNA damage response and has been suggested to selectively bind and activate cell-type specific gene expression programs. However recent studies and meta-analyses of genomic data propose largely uniform, and condition independent p53 binding and thus question the selective and cell-type dependent function of p53.ResultsTo systematically assess the cell-type specificity of p53, we measured its association with DNA in 12 p53 wild-type cancer cell lines, from a range of epithelial linages, in response to ionizing radiation. We found that the majority of bound sites were occupied across all cell lines, however we also identified a subset of binding sites that were specific to one or a few cell lines. Unlike the shared p53-bound genome, which was not dependent on chromatin accessibility, the association of p53 with these atypical binding sites was well explained by chromatin accessibility and could be modulated by forcing cell state changes such as the epithelial-to-mesenchymal transition.ConclusionsOur study reconciles previous conflicting views in the p53 field, by demonstrating that although the majority of p53 DNA binding is conserved across cell types, there is a small set of cell line specific binding sites that depend on cell state.

Uncovering the Dynamical Landscape of P53 DNA Binding Domain with Markov State Models

The transcription factor p53, known as the “guardian of the genome”, functions as a tumor suppressor and is the most frequently mutated gene in human cancer. Its inactivation by single point mutation is associated with progression of about 50% of cancers, and therefore reactivation of mutated p53 is emerging as an exciting possibility for cancer therapy. More than 90% of the cancer mutations are found in the DNA-binding domain of p53, but the mechanism through which a single mutation leads to change in function remains elusive and hinders the rational development of mutant-specific drug leads. Using molecular dynamics simulations and Markov state models (MSMs) to access long timescales of protein motion, we explore the dynamical landscape of wildtype p53, and investigate the effect of the single point mutation in Y220C, one of the most frequent cancer mutants and responsible for about 75,000 new cancer cases every year. Almost 100 microseconds of adaptive sampling for the wildtype protein and an iterative and unbiased selection of the features for model building evidence the existence of two slow-motion communities centered around flexible loops. Joint MSMs of the wildtype and mutant result in the identification of mutant-specific states, and we discuss efforts to characterize and test the druggability of these mutant-induced unique states. Our approach exemplifies the power of the differential dynamics MSM methodology for uncovering intrinsic dynamical and kinetic differences among distinct protein ensembles.

Ethanol extract of Elephantopus scaber Linn. Attenuates inflammatory response via the inhibition of NF-κB signaling by dampening p65-DNA binding activity in lipopolysaccharide-activated macrophages.

Elephantopus scaber Linn. (E.scaber) is a widely-used traditional herb whose use has been documented for various inflammatory diseases such as fever, sore throat, dysentery, carbuncle and so on. However, the effect and mechanism of E.scaber in LPS-activated macrophages remain unclear. This study aims to investigate the anti-inflammatory mechanism of the ethanol extract of E.scaber (ESE) in lipopolysaccharide (LPS)-induced inflammatory models. Griess reagent was used to determine NO production, and the levels of TNF-α, IL-6, MCP-1 and IL-1β were determined by ELISA kits. The molecular mechanism research was performed by RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA). LPS-induced endotoxemia mouse model was used for evaluating the in vivo anti-inflammatory action of ESE. ESE suppressed LPS-induced iNOS, TNF-α, IL-6, MCP-1 and IL-1β transcription as well as supernatant NO, TNF-α, IL-6, MCP-1 and IL-1β production in macrophages. Although ESE inhibited NF-κB activation, it did not affect the IκBα phosphorylation and degradation and the NF-κB p65 nuclear translocation. The result of EMSA revealed that ESE inhibited the NF-κB p65-DNA binding activity. Additionally, ESE also decreased the proinflammatory cytokines in serum and peritoneal lavage fluid of LPS-induced endotoxemic mice. ESE has a potently anti-inflammatory effect through inhibiting the NF-κB p65-DNA binding activity in LPS-activated macrophages.

Kinin B1 Receptor Blockade Prevents Angiotensin II-induced Neuroinflammation and Oxidative Stress in Primary Hypothalamic Neurons.

Neuroinflammation has become an important underlying factor in many cardiovascular disorders, including hypertension. Previously we showed that elevated angiotensin II (Ang II) and angiotensin II type I receptor (AT1R) expression levels can increase neuroinflammation leading to hypertension. We also found that kinin B1 receptor (B1R) expression increased in the hypothalamic paraventricular neurons resulting in neuroinflammation and oxidative stress in neurogenic hypertension. However, whether there are any potential interactions between AT1R and B1R in neuroinflammation is not clear. In the present study, we aimed to determine whether Ang II-mediated effects on inflammation and oxidative stress are mediated by the activation of B1R in mouse neonatal primary hypothalamic neuronal cultures. Gene expression and immunostaining revealed that both B1R and AT1R are expressed on primary hypothalamic neurons. Ang II stimulation significantly increased the expression of B1R, decreased mitochondrial respiration, increased the expression of two NADPH oxidase subunits (Nox2 and Nox4), increased the oxidative potential, upregulated several proinflammatory genes (IL-1β, IL-6, and TNFα), and increased NF-kB p65 DNA binding activity. These changes were prevented by pretreatment with the B1R-specific peptide antagonist, R715. In summary, our study demonstrates a causal relationship between B1R expression after Ang II stimulation, suggesting a possible cross talk between AT1R and B1R in neuroinflammation and oxidative stress.

Curcumin prevents high-fat diet-induced hepatic steatosis in ApoE\u2212/\u2212 mice by improving intestinal barrier function and reducing endotoxin and liver TLR4/NF-\u03baB inflammation

BackgroundNon-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and has become a public health concern worldwide. The hallmark of NAFLD is hepatic steatosis. Therefore, there is an urgent need to develop new therapeutic strategies that are efficacious and have minimal side effects in hepatic steatosis and NAFLD treatment. The present study aimed to investigate the effect of dietary supplement of curcumin on high-fat diet (HFD)-induced hepatic steatosis and the underlying mechanism.MethodsApoE−/− mice were fed a normal diet, high-fat diet (HFD) or HFD supplemented with curcumin (0.1% w/w) for 16 weeks. Body and liver weight, blood biochemical.parameters, and liver lipids were measured. Intestinal permeability, hepatic steatosis and mRNA and protein expressions of TLR4-related inflammatory signaling molecule were analyzed.ResultsThe administration of curcumin significantly prevented HFD-induced body weight gain and reduced liver weight. Curcumin attenuated hepatic steatosis along with improved serum lipid profile. Moreover, curcumin up-regulated the expression of intestinal tight junction protein zonula occluden-1 and occludin, which further improved gut barrier dysfunction and reduced circulating lipopolysaccharide levels. Curcumin also markedly down-regulated the protein expression of hepatic TLR4 and myeloid differentiation factor 88 (MyD88), inhibited p65 nuclear translocation and DNA binding activity of nuclear factor-κB (NF-κB) in the liver. In addition, the mRNA expression of hepatic tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) as well as the plasma levels of TNF-α and IL-1β were also lowered by curcumin treatment.ConclusionThese results indicated that curcumin protects against HFD-induced hepatic steatosis by improving intestinal barrier function and reducing endotoxin and liver TLR4/NF-κB inflammation. The ability of curcumin to inhibit hepatic steatosis portrayed its potential as effective dietry intervention for NAFLD prevention.

Targeting Misfolded p53 and p53 Aggregation to Overcome Resistance to Apoptosis in Acute Myeloid Leukemia

Targeting Misfolded p53 and p53 Aggregation to Overcome Resistance to Apoptosis in Acute Myeloid Leukemia

EXTH-30. PRECEDING p53 STABILIZATION USING DOXORUBICIN AUGMENTS PRIMA-1-MEDIATED p53 REFOLDING AND INCREASED CELLULAR APOPTOSIS: EVALUATION OF A SEQUENTIAL COMBINATION THERAPY AGAINST GLIOBLASTOMA

Abstract INTRODUCTION Mutant p53 cancer cells are poorly responsive to chemotherapy. A small molecule drug, PRIMA-1, can bind to wildtype and mutant p53 to induce structural conformation that enhances p53 DNA binding. In a quest for new effective combination chemotherapies, we hypothesized that sequential treatment of GBM cells with doxorubicin then PRIMA-1 could stabilize and refold p53 to enhance chemotherapy-induced cellular apoptosis. We evaluated this combination in GBM cells engineered to express different p53 mutants and exhibiting different phenotypes. METHODS We constructed split reporter biosensors incorporating different structural mutants of p53 (p53Y220C, p53G245S, and p53R282W) all within p53-null Ln308 GBM cells, to evaluate PRIMA-1-mediated refolding of p53 using bioluminescence imaging. We used these cells, and other GBM cells endogenously dissimilar for p53 phenotypes (U87MG with wt-p53, and Ln229 with mutant p53 [mt-p53P98L/p14ARFdel/p16del]), to investigate the anticancer activity of doxorubicin plus PRIMA-1 by using immunoblot analysis of p53 downstream pathway proteins. We also evaluated treatment by imaging mice implanted with GBMs of different p53 phenotypes. RESULTS PRIMA-1 induced reporter complementation at > 80 mM in all GBM cells regardless of p53 status. Doxorubicin stabilized p53 and increased its levels significantly more than in control cells. Significant changes in apoptotic index occurred in GBM cells treated with doxorubicin (1mM) followed by PRIMA-1 (40 mM), compared to cells treated with each drug independently, or with PRIMA-1 followed by doxorubicin. There was upregulation of p53, p21, and Bax, but downregulation of Bcl2 after combination therapy. PRIMA-1 and doxorubicin acted synergistically in GBM cells, but not significantly compared to using doxorubicin followed by PRIMA-1. CONCLUSION Our molecular imaging biosensor is useful to detect drug-induced restoration of p53 wildtype conformation. Doxorubicin stabilizes and increases p53 levels in GBM cells for subsequent enhanced PRIMA-1-mediated p53 refolding and increased levels of cellular apoptosis—an effective anti-GBM combination treatment.

The Rich World of p53 DNA Binding Targets: The Role of DNA Structure.

The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with DNA. p53 works as a transcription factor for a significant number of genes. Most p53 target genes contain so-called p53 response elements in their promoters, consisting of 20 bp long canonical consensus sequences. Compared to other transcription factors, which usually bind to one concrete and clearly defined DNA target, the p53 consensus sequence is not strict, but contains two repeats of a 5′RRRCWWGYYY3′ sequence; therefore it varies remarkably among target genes. Moreover, p53 binds also to DNA fragments that at least partially and often completely lack this consensus sequence. p53 also binds with high affinity to a variety of non-B DNA structures including Holliday junctions, cruciform structures, quadruplex DNA, triplex DNA, DNA loops, bulged DNA, and hemicatenane DNA. In this review, we summarize information of the interactions of p53 with various DNA targets and discuss the functional consequences of the rich world of p53 DNA binding targets for its complex regulatory functions.

Infection with Prevotella nigrescens induces TLR2 signalling and low levels of p65 mediated inflammation in Cystic Fibrosis bronchial epithelial cells

Prevotella spp. are frequently identified in Cystic Fibrosis sputum. This study examined whether infection with Prevotella nigrescens, a frequently identified member of this species, contributes to inflammation in CF bronchial epithelial cells through activation of TLR- and NF-κB signalling pathways. CFBE41o- cells were infected with either P.nigrescens or Pseudomonas aeruginosa and incubated under anaerobic conditions for 4h. P.nigrescens activated TLR2 signalling but not TLR4 signalling while P.aeruginosa activated TLR4 signalling with a lesser effect on TLR2. P.aeruginosa induced significant IκBα phosphorylation 10min post infection with a return to control levels by 30min post infection. A significant induction in nuclear p65 DNA binding was observed at 2h post infection. In contrast, infection with P.nigrescens induced phosphorylation of IκBα 120min post infection, with significant induction in nuclear p65 DNA binding at 4h post infection only. Cytokine gene and protein responses were lower for P.nigrescens compared to P.aeruginosa. This study demonstrates the ability of a clinical P.nigrescens isolate to provoke a delayed NF-κB(p65) driven response through induction in TLR2 signalling and activation of sustained levels of IKKα.

Docosahexaenoic acid inhibits TNFα-induced ICAM-1 expression by activating PPARα and autophagy in human endothelial cells

Inflammation plays a key role in the development of cardiovascular disease (CVD), and docosahexaenoic acid (DHA) is recognized to fight against CVD. PPARα belongs to the nuclear hormone receptor superfamily and can interfere with inflammatory processes. Autophagy can degrade inflammasome proteins and counteract inflammation. Overexpression of intercellular adhesion molecule (ICAM) 1 in endothelial cells contributes to monocyte migration into the vascular intima. Here we investigated the mechanisms by which DHA inhibits TNFα-induced ICAM-1 expression in EA. hy926 endothelial cells. DHA markedly activated PPARα and suppressed TNFα-induced ICAM-1 expression, ICAM-1 promoter activity, p65 nuclear translocation, NFκB and DNA binding activity, and THP-1 cell adhesion. PPARα knockdown abolished the ability of DHA to inhibit TNFα-induced ICAM-1 expression and THP-1 cell adhesion. The PPARα antagonist GW6471 reversed the inhibitory effect of DHA on TNFα-induced ICAM-1 expression, p65 nuclear translocation, NFκB and DNA binding activity, and THP-1 cell adhesion. DHA significantly activated autophagy as evidenced by the formation of autophagosomes and increased LC3II protein expression. By contrast, wortmannin, which inhibits autophagy, abrogated DHA-induced autophagy and the inhibition of TNFα-induced ICAM-1 protein expression by DHA. Our results suggest that DHA likely inhibits TNFα-induced ICAM-1 expression by activating PPARα and autophagy.

Residual apoptotic activity of a tumorigenic p53 mutant improves cancer therapy responses

Engineered p53 mutant mice are valuable tools for delineating p53 functions in tumor suppression and cancer therapy. Here, we have introduced the R178E mutation into the Trp53 gene of mice to specifically ablate the cooperative nature of p53 DNA binding. Trp53R178E mice show no detectable target gene regulation and, at first sight, are largely indistinguishable from Trp53−/− mice. Surprisingly, stabilization of p53R178E in Mdm2−/− mice nevertheless triggers extensive apoptosis, indicative of residual wild‐type activities. Although this apoptotic activity suffices to trigger lethality of Trp53R178E;Mdm2−/− embryos, it proves insufficient for suppression of spontaneous and oncogene‐driven tumorigenesis. Trp53R178E mice develop tumors indistinguishably from Trp53−/− mice and tumors retain and even stabilize the p53R178E protein, further attesting to the lack of significant tumor suppressor activity. However, Trp53R178E tumors exhibit remarkably better chemotherapy responses than Trp53−/− ones, resulting in enhanced eradication of p53‐mutated tumor cells. Together, this provides genetic proof‐of‐principle evidence that a p53 mutant can be highly tumorigenic and yet retain apoptotic activity which provides a survival benefit in the context of cancer therapy.

Abstract 5298: Finding new molecules to bypass the tumor suppressor gene TP53 and restore cell cycle checkpoint control

Abstract The tumor suppressor gene, tumor protein 53 (TP53), has been shown to be mutated in more than half of all human cancer cases. p53 controls the G1/S phase checkpoint of the mitotic cell cycle, protecting the fidelity of DNA and inducing apoptosis in cells with irreparable DNA damage. Mutations in TP53, which are often missense mutations that interfere with p53 DNA binding activity or protein folding, result in protein inactivation and loss of checkpoint control, allowing cells with potentially oncogenic mutations to survive and proliferate. TP53 mutations have been shown to enhance tumor progression, increase metastatic potential, and mediate chemotherapy resistance. Because of the importance of p53 in cancer, many studies have aimed to identify small molecules that can “reactivate” mutated p53 protein in cell culture, although this has met with little success thus far. The aim of this research is to identify the extent to which the effects of non-functioning p53 can be completely bypassed, and cell cycle checkpoint restored, using already-established FDA approved compounds in a zebrafish model of p53 loss. Zebrafish were genetically engineered to have their entire TP53 locus removed using TALEN (Transcription activator-like effector nuclease), rendering them unable to produce the p53 protein. p53 deficient and wild-type zebrafish larvae were then subjected to gamma-irradiation to induce DNA damage. An apoptotic cell staining assay was then performed to allow for the qualitative and quantitative analysis of apoptosis levels throughout the larvae. As expected, TP53 null fish had significantly less apoptotic cells than control after DNA damage. We are in the process of testing 770 FDA approved compounds for their effects in inducing an apoptotic response in p53 null larvae after gamma-irradiation. Positive hits will be carried forward to human cancer cells with TP53 mutation, and the mechanism of action of the drug will be determined using biochemical approaches. These results could identify novel pathways that work in concert with p53 and will allow us to harness new mechanisms of DNA damage induced apoptosis to kill cancer cells. Repurposing FDA-approved drugs can also rapidly translate to clinical benefit in a wide number of cancers driven by TP53 mutation. Citation Format: Henry Moore, Jessica Blackburn. Finding new molecules to bypass the tumor suppressor gene TP53 and restore cell cycle checkpoint control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5298.