p53 is a suppressor of inflammatory response in ... - The FASEB Journal

©2005 FASEB

The FASEB Journal express article 10.1096/fj.04-3213fje. Published online April 5, 2005.

p53 is a suppressor of inflammatory response in mice Elena A. Komarova,* Vadim Krivokrysenko,* Kaihua Wang,‡ Nickolay Neznanov,* Mikhail V. Chernov,* Pavel G. Komarov,§ Marie-Luise Brennan,† Tatiana V. Golovkina,║ Oskar Rokhlin,¶ Dmitry V. Kuprash,# Sergei A. Nedospasov,#, ** Stanley R. Hazen,† Elena Feinstein,‡ and Andrei V. Gudkov*,§ Departments of *Molecular Genetics and †Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio; ‡Quark Biotech, Inc., Fremont, California; §Cleveland BioLabs, Inc., Cleveland, Ohio; ║Jackson Laboratory, Bar Harbor, Maine; ¶Department of Pathology, University of Iowa, Iowa City, Iowa; #Laboratory of Molecular Immunology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; ** Basic Research Program, SAIC-Frederick, Inc, and Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute, Frederick, Maryland Corresponding author: Andrei Gudkov, Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195. E-mail: [email protected] ABSTRACT Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor κB (NFκB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF-inducible genes that are largely regulated by NFκB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFκB-dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild-type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53-deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFκB, plays the role of a general “buffer” of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFκB in tumors. Key words: inflammation • nuclear factor κB • cytokines

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ack of p53 in humans and mice is associated with a strong cancer predisposition. The vast majority of p53-null mice die before they reach 6 months of age from neoplastic diseases, predominantly lymphomas. Cancer prevention by p53 is usually viewed as a result of control of genomic stability by p53 that act through elimination of genetically damaged cells by growth-suppressive and apoptotic functions of p53. However, the recent analysis of mice lacking Page 1 of 21 (page number not for citation purposes)

p53-dependent apoptosis as a result of knockouts of p53-responsive apoptotic genes (Noxa, Puma) (1) or mice inheriting p53 mutant defective only in apoptotic function (p53-R172P) (2) showed no severe cancer predisposition, suggesting that additional mechanisms should exist through which p53 exerts its tumor suppressor function. There is a large body of evidence linking human cancer with chronic inflammation caused by Helicobacter pylori (gastric cancer) (3, 4), Schistosoma haematobium (bladder cancer) (5), Opisthorchis viverrini (cholangiocarcinoma) (6), hepatitis B virus (liver cancer) (7) and other infective agents. The risk of cancer is also increased under chronic inflammatory conditions, such as ulcerative colitis and Crohn’s disease (8). The reasons why chronic inflammation creates cancer-prone conditions are not completely clear. It is known that reactive oxygen and nitrogen species, which are produced at sites of chronic inflammation, may cause genotoxic stress (6) and may induce mutations, thereby increasing the risk of transformation. There is also a possibility that inflammatory microenvironment favors selection of cells with genetic modifications consistent with malignant transformation. In fact, p53 mutations are frequently found in tissues with long-standing inflammation even before the development of a malignant disease, such as idiopathic pulmonary fibrosis, chronic inflammatory bowel disease, inflammatory cholangiopathies and H. pylori-associated gastritis (9–12), ulcerative colitis (11), schistosomiasis of the urinary tract (13) and others (14, 15). Hence, inflammation is an important factor of cancer predisposition and suppressors of inflammation are likely to possess a tumor-suppressor function. A number of reports points at potential involvement of p53 in control of inflammation. Thus, autoimmune diseases in mice, including collagen-induced arthritis (16) and experimental autoimmune encephalitis (17) were found to be more severe on the p53-deficient background. It is known that chronic exposure to bleomycin leads to a marked increase in inflammatory infiltrate and subsequent disruption of alveolar architecture in p53 null and transgenic mice with a mutant p53 expressed in the lung (18, 19). Accelerated growth of atherosclerotic plaques was observed in p53−/−/apoE−/− compared with p53+/+/apoE mice and in LDL receptor-deficient mice rescued from lethal irradiation by transplantation of bone marrow from p53−/− mice (20–23) caused by a strong infiltration of activated macrophages into the plaques. Ionizing radiation induces faster and stronger infiltration of inflammatory cells and fibroblasts in damaged tissues of p53 null than in wild-type mice (24). Finally, a significant proportion of p53 null mice (25%) die before tumor development from unresolved infections leading to abscesses, gastroenteritis or myocarditis (25), suggesting the presence of a defect in the innate immune system associated with loss of p53. This putative defect is unlikely to be associated with adaptive immune system since the levels of immunoglobulins, as well as the numbers of T and B cells in the thymus and spleen, were found normal in p53-knockout mice (25). The mechanism of p53 involvement in the control of immunity may lie in p53 interaction with NFκB, the major molecular regulator of inflammatory response that determines transcriptional activation of numerous genes encoding inflammatory cytokines, chemokines, antimicrobial peptides, adhesion molecules, iNOS, Cox2 (26) in response to different stimuli, such as bacterial products (i.e., endotoxin). p53 can negatively regulate transcription of NFκB-dependent genes, including IL-6, Cox-2, iNOS, and others (27–31). In vitro studies showed that p53 and NFκB can inhibit transactivation functions of each other presumably by competing for CBP (32, 33). Moreover, p53 can directly repress promoter activity of NFκB subunit p65 (34) and indirectly Page 2 of 21 (page number not for citation purposes)

repress activity of IKKα, subunit of IκBα kinase complex, through inhibition of ets-1, a positive regulator of IKKα (35). All of the above provided strong, but still indirect, evidence on the involvement of p53 in regulation of innate immune response, thereby determining the outcome and severity of inflammation. We directly tested this assumption by comparing responses of p53 null and wildtype mice to bacterial endotoxin (LPS) at cellular, biochemical and molecular levels. The data that we generated support this hypothesis and allow defining p53 as a general suppressor of innate immune response and inflammation. MATERIALS AND METHODS Reagents, animals Wild-type and p53-knockout mice on C57Bl/6J background (9- to 14-wk-old males) were purchased from Jackson Laboratory (Bar Harbor, ME). TNF-knockout and TNF/p53 double knockout mice (9- to 14-wk-old males, littermates on mixed C57Bl/6/sv129 background) were received from the National Cancer Institute at Frederick, MD. Mice were injected intraperitoneally with different concentrations of LPS (8-24 mg/kg) (Escherichia coli serotype 055:B5, Sigma) and 4% Brewer thioglycolate medium (Sigma). Mice were housed under aseptic conditions and fed with radiation-sterilized food. The colony sentinel program is a soiled bedding exposure system with sentinels exposed to soiled bedding weekly and tested quarterly for internal and external parasites and adventitious viral pathogens. The colony has been free of disease since arrival at our institution in 2003. The animals were treated in accordance with animal care protocol approved by the Institution for Animal Care and Use Committee. Microarray hybridization and analysis Incyte Human UniGEM V gene expression microarray contains 7,075 sequence-verified human Incyte cDNA clones that correspond to known genes and ESTs from human UniGene database, build 46. All genes on the array were remapped to current UniGene using the representative GenBank accession number provided by Incyte, either directly or by performing BLAST searches against all GenBank sequences present in human UniGene. Per-chip quality control was performed by Incyte Inc. Subsequent quality control and normalization operations were done using our GINSTREAM software. Per-spot quality control was performed according to standard Incyte criteria: all spots that have an area of less than 40% and signal-to-background ratio in both channels less than 2.5 were considered invalid. Robust nonlinear fitting of log expression ratios against average log spot intensities was performed. Such procedure successfully removes signaldependent nonlinear bias (36). Transient transfection experiments Transient transfection was performed using lipofectamine reagent Plus system (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Luciferase assay and β-galactosidase enzyme assay was performed using corresponding kits from Promega according to the manufacturer’s protocol. We performed the quantitative analysis of the effect of p53 on NFκB dependent expression using the reporter constructs with luciferase cDNA controlled by the NFκB dependent promoters. Two of these constructs contain fragments of eukaryotic genes EPage 3 of 21 (page number not for citation purposes)

selectin (Elam-luc)) (37) and IP-10 (IP-10-luc) (38) carrying NFκB binding sites. The third construct contains NFκB binding site from HIV LTR in combination with fos minimal promoter (#11NFκB-luc). We used the third construct with luciferase expression driven only by fos minimal promoter (∆56fos-luc) as an NFκB-independent control. Lung carcinoma cell line H1299/CMV5 carrying β-galactosidase gene under control of p53-responsive promoter was transiently transfected with 1.5 µg of each of reporter constructs in combination with 0.5-µg plasmids expressing wt-p53 or 175 mutant p53 or with empty vector. Twenty hours after transfection, luciferase and β-galactosidase activity was measured in protein extracts. The results shown are representative of at least three separate experiments. cDNA array hybridization Differential hybridization analysis was done with Mouse Inflammatory Cytokine/Receptor GEArray Q series (SuperArray Inc., Catalog No. MM-015N). This array consists of 96 selected cDNA of cytokine and receptor genes associated with inflammatory response, applied to nylon membrane. Total RNA was purified according to standard protocols from thymus tissue of control and LPS-treated (8 mg/kg, 3h) mice. cDNA probe preparation and hybridization were done according to published protocols and manufacturer’s recommendations. Gel shift/supershift assay Gel shift assay was performed as described earlier (39). Nuclear cellular extracts were prepared from thymuses and spleens of untreated and LPS-treated mice (8 mg/kg) at different time points after treatment. Labeled double-stranded oligonucleotides, corresponding to the sequences of the NFκB binding region of the mouse B cell light chain enhancer (39) (5′-AGT TGA GGG GAC TTT CCC AGG C-3′), were used as the probes. Polyclonal antibodies against NFκB p50 and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cytokine expression by real-time and quantitative RT-PCR Total RNA was purified from thymuses and spleens of wtp53 and p53 null mice at different time points (0, 1.5, 3, and 6 h) after LPS treatment (8 mg/kg ip). RNA quantitation was performed using a fluorescence-based real-time RT-PCR technology (TaqMan Real-Time PCR) (40) with probe-primers sets for mouse IL1α, IL1β, IL6, and TNFα (ABI Assay-on-Demand FAM-MGB probes). 100 ng of each total RNA sample was used in a 25 µl RT-PCR reaction. Each reaction consisted of 1X TaqMan EZ buffer, 3 mM manganese acetate, 300 µM each of dATP, dCTP, dGTP, 600 µM dUTP, 0.5 units of AmpErase, 5 units of rTth polymerase (Perkin Elmer TaqMan EZ RT-PCR Kit), and appropriate probe-primers sets. For control reactions, mouse 18S rRNA was amplified from the same RNA samples using Pre-Developed 18S RNA kit (VIC-MGB probe, ABI). RT-PCR reactions were carried out using the following thermal cycling parameters: 50° for 2 min, 60°C for 30 min, 95°C for 5 min, followed by 40 two-step cycles of 94°C for 20 s and 60°C for 1 min. Relative mRNA abundance was calculated using the comparative ∆Ct method. All final measurements were normalized by the target mRNA/18S rRNA value for untreated wild-type sample. The following primers were used for testing of cytokine expression in quantitative RT PCR (performed according to (41): for IL-6, Forward:5′-GAA CAA CGA TGA TGC ACT TGC AG3′, Reverse: 5′-CCT TAG CCA CTC CTT CTG TGA C-3′; β-actin, Forward: 5′-CAA CCG Page 4 of 21 (page number not for citation purposes)

TGA AAA GAT GAC CCA G-3′, Reverse: 5′-CAC ACA GAG TAC TTG CGC TCA G-3′; TNF-α, Forward: 5′-GGC AGG TCT ACT TTG GAG TCA TTG C-3′, Reverse: 5′-ACA TTC GAG GCT CCA GTG AAT TCG G-3′; IL-1α, Forward: 5′-TGC CAT TGA CCA TCT CTC TCT G-3′, Reverse: 5-TGG CAA CTC CTT CAG CAA CAC G-3′. Results of RT-PCR were normalized to β-actin using Image Quant Program, and standard errors were counted for each time point. Thioglycolate and LPS treatment Mice were injected intraperitoneally with 1 ml of sterile 4% Brewer’s thioglycolate solution or LPS (6 mg/kg). At different time points (0, 6. 24, 48, and 72 h), mice were killed, and peritoneal exudate cells were recovered by peritoneal lavage using 5 ml of ice-cold PBS. Erythrocytes were lysed by cold water. Peritoneal exudates cells were initially counted on hemocytometer and then differential cell counts were conducted on cytospins stained with Wright-Giemsa. Three to four animals were taken per each time point. Experiments were repeated minimum 3 times. Cecal ligation and puncture- (CLP)-induced peritonitis Cecal ligation and puncture were performed as described previously (42) with some modifications. Briefly, mice were anesthetized with avertin (100 mg/kg), a midline incision was performed, and the secum was isolated. A 6-0 silk ligature was placed around it, ligating the cecum below the ileocecal valve (10 mm from the cecal tip). The ligated cecal stump then was punctured twice with 23-gauge needle, and stool was extruded through the puncture holes and the cecum returned to the peritoneal cavity. The abdomen was closed in two layers using 7-0 suture (Ethicon) and normal saline solution (20 ml/kg body weight) was administered. Mice were evaluated daily up to 2 weeks after the procedure. Moribund mice were killed, and the mortality was recorded. Phagocytosis assay Peritoneal macrophages were obtained by peritoneal lavage with cold PBS, seeded at 4 × 105 cells/well into chamber slides, incubated for 1 h and washed. Mouse thymocytes were isolated and irradiated (10 Gy) as described by Komarova et al. (43) and then cultured in RPMI medium 4 h. This procedure yielded 30% of apoptotic thymocytes (counting of methyl blue (0.1%) stained cells). Apoptotic cells were cocultured with macrophages at a ratio 5:1 (thymocytes:macrophages) for 30 min. Cells were washed, fixed with methanol, and stained with hematoxylin. Cultures were scored by light microscopy for the percentage of macrophages interacting with apoptotic cells, both surface-bound and phagocytosed. Quantification of myeloperoxidase and protein oxidation products MPO content of leukocytes was assessed in whole blood by in situ cytochemical staining and flow cytometry using an automated hematology analyzer (Advia 120, Bayer Diagnostics). MPO levels are expressed as the MPO Index, which is reported relative to normal MPO levels as part of the veterinarian software accessory. Protein-bound nitrotyrosine and ortho-tyrosine levels were determined by stable isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry-based methods using an ion trap mass spectrometer (LCQ Deca, Thermo Finigann, San Jose, CA), as described previously (36). Page 5 of 21 (page number not for citation purposes)

Measurement of intracellular oxidation (reactive oxygen species level) Wt p53 and p53 null mouse thymocytes were incubated with 5 mg of dichlorofluorescein diacetate (DCF; Molecular Probes) per 1 ml for 30 min at 37°C, then washed with PBS and collected for FACS analysis. Values of mean fluorescence intensity were used to plot graphs. RESULTS p53 inhibition and TNF treatment cause similar changes in gene expression patterns We previously showed that suppression of p53 makes human prostate carcinoma cell line LNCaP resistant to TNF (44), which in other systems is known to be associated with activation of NFκB (45). This observation suggested that p53 could somehow interfere with apoptosisprotecting function of NFκB. Because both p53 and NFκB exert their activities via regulation of transcription, we applied cDNA microarray-based gene expression profiling to estimate the effect of p53 status of cells on NFκB-mediated transcription. Repression of p53 was achieved by transduction of LNCaP with retroviral vector expressing potent dominant negative mutant of p53, GSE56 (46). NFκB-mediated transcription was induced by treating cells with TNF; RNA was isolated 6 h after application of 10 ng/ml of TNF. Clusterization of genes according to their expression patterns revealed striking similarities between sets of genes that alter their expression in the presence of GSE56 and those induced by TNF (Fig. 1A) showing ~50% overlap between these two subsets. Many TNF-responsive genes were stronger induced in GSE56-transduced than in control LNCaP after TNF treatment. Multiple known NFκB target genes (47, 48) are up-regulated by functional inhibition of p53 in LNCaP cells. These include genes encoding quinone NAD(P)H dehydrogenase 1 (NQO1), c-Rel, β-2-microglobulin, TNF receptor-associated factor 1 (TRAF1), platelet-activating factor receptor (PTAFR), IFN-inducible T cell α chemoattractant (I-TAC/CXCL11), bcl2-related protein A1/Bfl-1, interferon-inducible cytokine IP-10 (CXCL10), TNFα-induced protein 3 (TNFAIP3), NFkB inhibitor α (IkBα), intercellular adhesion molecule 1 (ICAM1/CD54), apoptosis regulator pCasper/CFLAR, MD-2/LY96 (an indispensable coreceptor for LPS/TLR4 signaling), macrophage-derived chemokine CCL22, and adenosine A2a receptor and p53 gene itself. With exception of p53, expression of these genes is not known to be directly regulated by p53 protein. By contrast, only one proinflammatory gene (encoding annexin A1) was down-regulated as a result of GSE56 introduction. p53 is an inhibitor of NFκB-mediated transactivation p53 is known to repress transcription of a number of NF-κB-regulated genes (32, 33). To determine whether this is a general property of p53 that inhibits any NFκB-regulated transcription, we compared the activity of three constructs, each containing different NFκB binding sites followed by minimal promoters, after transduction into the cells with or without the construct expressing wild-type p53. Tumor-derived p53 mutant deficient in transactivation (p53175His) and insert-free vector served as controls. p53 reporter construct was used to monitor p53-mediated transactivation. The obtained results (Fig. 1B) demonstrate that wild-type, but not mutant p53, acts as an effective inhibitor of NFκB-mediated transactivation in all three different promoter constructs, indicating that p53 is a general suppressor of NFκB-mediated transcription. Page 6 of 21 (page number not for citation purposes)

NFκB activities are increased in p53-null mouse tissues NFκB regulates transcription of different classes of genes, including major proinflammatory cytokines, such as TNF, IL-1, and IL-6 (26), and plays a key role in determining inflammatory response. If suppression of transactivation ability of NFκB by p53 that was found in vitro (32, 33) would also occur in vivo, this might result in strong differences in the induction of inflammatory response between wild-type and p53-deficient mice. We tested this hypothesis by measuring the induction of expression of mRNA of proinflammatory cytokines in the thymuses of p53 null and wtp53 LPS-treated mice using inflammatory cytokine/receptor arrays, including 94 genes. Higher levels of induction of many cytokines, chemokines, and chemokine receptors, including IL-1, IL-6, IL-12, TNF, CCR2, CCR5, Mig, IP-10 (Fig. 2A), were observed in p53 null mice, indicating that p53 deficiency results in an increase in responsiveness of NFκB-inducible cytokine-encoding genes. The array hybridization results were confirmed using real-time RT PCR detection of IL-1α, IL-1β, IL6, and TNF mRNAs in the mouse tissues. Stronger (four- to nine-fold) induction of the cytokine gene transcription was found both in the thymuses and in the spleens of p53 null, as compared with wt-p53 mice (Fig. 2B, shown for thymus), suggesting that injection of endotoxin stimulates stronger inflammatory response in them. Basal levels of mRNA of the majority of tested genes were also higher in p53-deficient tissues. It is known that tumor necrosis factor α (TNFα) is one of the most important mediators of inflammatory response (26). Activation of TNF-signaling can result in the induction of NFκBmediated gene transcription leading to the increased expression of cytokines. To determine whether TNF plays the role in the increased inducibility of cytokines in p53 null mice, we compared cytokine activation in the thymus and spleen of TNF-deficient mice and in mice deficient in both TNF and p53. As expected, the deletion of tnf gene was associated with the lack of TNF expression and with the decreased levels of IL-1 and IL-6 induction (data not shown). Activation of IL-1 and IL-6 by LPS treatment was significantly higher in TNF/p53-knockout mice as compared with TNF-knockout mice (Fig. 2C) (shown for thymus), although it did not last as long as in the animals with intact tnf gene. Hence, the lack of TNF did not change p53 dependence of LPS-induced expression of cytokines at least at early times after treatment. However, the release of TNF may lead to a second wave of NFκB activation via induction of cytokines. Consistently with the increased cytokine gene expression in p53 null mice, we also observed higher DNA binding activity of NFκB (judged by electromobility shift assay, EMSA) in spleens, thymuses, and bone marrows of these animals in comparison with wild-type ones (Fig. 2D). This increase is presumably determined by amplification of NF-κB response in p53-deficient mice caused by higher concentrations of cytokines released in their tissues in response to LPS. We analyzed the content of NF-κB DNA binding complex using antibodies specific for individual NF-κB subunits in EMSA and found no p53-associated alterations in relative abundance of p65/p50 and p50/p50 complexes (Fig. 2E, shown for thymus): two retarded bands representing NFκB DNA binding activity in the thymus were equally supershifted in wild-type and p53 null mice after addition of antibodies either against p65 or p50. Hence, stronger LPSinduced NFκB activity in p53 null mice was not associated with the alterations in composition of NFκB subunits.

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p53-deficient mice are hypersensitive to septic shock High concentrations of LPS can induce lethal septic shock as a consequence of activation of strong unregulated inflammatory response and followed severe damage of organs. We suggested that abnormal inflammatory response found in p53 null mice might lead to accelerated tissue damage and increased sensitivity to LPS-induced septic shock. We have compared survival of wild-type and p53 null groups of mice after intraperitoneal injection of different concentrations of LPS. The increased lethality of p53 null mice after LPS treatment was found at all doses (Fig. 2F). Similar results were obtained in a more physiological model of peritonitis induced by surgical perforation of the cecum (42): p53-deficient mice were characterized by increased lethality (Fig. 2G). These results are consistent with higher levels of inflammatory cytokine induction in tissues of these animals. Increased macrophage recruitment and delayed neutrophil clearance in p53 null mice in response to inflammation-inducing agents We measured another parameter of inflammatory response—efflux of macrophages and granulocytes into the peritoneal cavity—of p53 wild-type and p53-deficient mice measured at different time points after intraperitoneal injections of thioglycolate (TG) or LPS. Higher numbers of macrophages were found in p53 null mice compared with wild-type mice in all tested time points after injection of inflammation-inducing agents (Fig. 3A). Although we did not see significant difference in the initial numbers of neutrophils accumulated in peritoneal exudates following TG stimulation (6h), neutrophil clearance was delayed in p53 null animals as compared with wild-type mice (Fig. 3A). Even more pronounced increase in neutrophil numbers was observed in LPS-stimulated mice (Fig. 3B). Interestingly, delayed neutrophil clearance was accompanied by accumulation of inflammatory cells around blood vessels in the liver of LPStreated p53 null mice (Fig. 3C), presumably due to higher levels of chemokine induction (see above). Delayed elimination of neutrophils in p53 null mice might occur either because of a defect in binding and phagocytosis activity of p53-deficient macrophages or by a reduced rate of apoptosis in p53 null neutrophils. To directly test the engulfment activity of peritoneal macrophages, they were incubated in the presence of gamma-irradiated mouse thymocytes undergoing apoptosis (clearance assay, see Materials and Methods). We found that peritoneal macrophages from p53 null mice engulfed significantly less apoptotic thymocytes than wild-type mice. Binding of apoptotic thymocytes to p53-macrophages was also reduced, suggesting that the deficiency in phagocytosis may be partly attributable to a recognition defect (Fig. 4A, B). Biochemical markers of oxidation and myeloperoxidase index are increased in tissues and blood of p53 null mice We have tested the inflammatory status of tissues of p53-knockout mice by quantifying levels of myeloperoxidase (MPO), a major marker of inflammation, as well as multiple specific products formed by distinct oxidation pathways that participate in innate host defenses. MPO is expressed predominantly in neutrophils and monocytes. It is stored in azurophilic granules and is released during phagocytosis (49). MPO uses hydrogen peroxide and multiple organic and inorganic ions as cosubstrates to produce hypochlorous acid and other reactive oxidant, including those derived from oxidation of tyrosine and nitrite (50, 51). In situ cytochemical staining of blood leukocytes Page 8 of 21 (page number not for citation purposes)

for MPO was markedly enhanced in p53-knockout mice, as observed by the increased MPO index (Fig. 5A), a qualitative index of MPO content per leukocyte. Moreover, increased levels of ortho-tyrosine and nitro-tyrosine in the normal tissues of p53- knockout mice (Fig. 5B, shown for spleen) were noted, consistent with increased oxidative modification of proteins by nitric oxidederived oxidants, and hydroxyl radical-like species. To test the levels of reactive oxygen species (ROS) in the tissues of wild-type and p53 null mice, we used green fluorescent probe DCF, a marker of a change in general cellular oxidant accumulation (52). As shown in Fig. 5C, FACS analysis of DCF-stained thymocytes revealed an increase in ROS levels in the thymocytes of p53 null compared with wild-type mice. DISCUSSION p53 and NFκB are key components of two major signal transduction mechanisms determining cell and organism reaction to a variety of stresses. p53 is proapoptotic and antiproliferative, while NFκB is generally playing an opposite role being antiapoptotic and proliferative factor. Until recently, p53 and NFκB have not been linked by any kind of known mutual regulation, and they were viewed as systems that serve different aspects of physiology: p53 was mostly associated with anticancer function while NFκB was associated with the regulation of inflammation and immune response. The first indications of a mutual regulation of these two pathways came from in vitro observations showing that p53 and NFκB can repress each other’s transactivation (32, 33) and that TNF causes accumulation of p53 in inactive form (53). In the present work, we are demonstrating the inhibitory effect of p53 on NFκB-mediated transcription for three different NFκB responsive promoters. If this reciprocal inhibition, which is probably explained by a competition for CBP/p300 (32, 33), occurred in vivo, one could expect p53 to be a negative modulator of inflammatory response, acting as a repressor of proinflammatory function of NFκB. The present work is devoted to experimental testing of this hypothesis. The data we obtained well fit the above-described model. Striking similarities between sets of genes that change their expression in response to p53 inactivation and TNF treatment (powerful activator of NFκB) were observed in human prostate carcinoma cells LNCaP using cDNA microarray hybridization. Similarly, NFκB-responsive genes in p53 null mice were found to be significantly more reactive on inflammatory stimuli. Consistently, activation of cellular mediators of innate immunity is more pronounced on a p53-deficient background, and p53 null mice are characterized by elevated biochemical markers of inflammation. Finally, lack of p53 is associated with higher sensitivity of mice to endotoxin. All this allows us to define p53 as a buffer of inflammatory response presumably acting through the suppression of NFκB. This conclusion is in agreement with the observation made by Donehower et al. in the original paper describing the phenotype of p53 null mice, in which they showed that a significant proportion of early deaths of p53 null mice was not tumor-related but resulted from unresolved spontaneous inflammation (25). It should be mentioned that not all altered parameters of inflammatory response in p53-deficient mice could be directly linked to NFκB. Thus, reduced capability of p53 null macrophages to recognize apoptotic cells—the phenomenon that might be responsible for delayed clearance of neutrophils in p53-knockout mice—does not have obvious connections with NFκB regulation.


p53 is a major tumor suppressor while NFκB has many properties of an oncogene (26). Moreover, chronic inflammation creates conditions facilitating cancer development (reviewed in the Introduction). Hence, rapid development of tumors in p53-deficient mice and humans (54) may not be limited to the lack of p53-mediated control of genomic stability and suppression of growth for cells with damaged DNA and deregulated proliferation, but at least in part may be determined by elevated activity of NFκB and establishment of chronic proinflammatory conditions. In this context, it is worth mentioning some phenotypic similarities between physiological consequences of p53 deficiency and aging. It has been reported that the frequency of spontaneous tumor formation and sensitivity to LPS-induced septic shock are dramatically increased in aged mice (55–57). Moreover, the loss of p53 accelerates development of the same types of tumors that normally appear in the aged mice of a given strain (25, 58). The similarity in the behavior of immune system of p53 null and aged mice was described in the work of OhkusuTsukada et al. (59). We can speculate that accelerated development of tumors in both cases may be caused by alterations in the status of immune system, which appeared to be similar between p53 deficiency and aging. There is a bulk of evidence that many tumor cells acquire and become dependent on constitutively active NFκB, which makes them resistant to apoptotic stimuli (60). This property of tumor cells, at least in some cases, may be the result of secretion of proinflammatory cytokines that activate NFκB through an autocrine loop (61, 62). Our results suggest that loss or inactivation of p53 that frequently occurs in the tumors can be another mechanism contributing to constitutive activation of NFκB in tumors. Negative regulation of NFκB-mediated response by p53 and the facts of reciprocal control of p53 and NFκB transactivation in vitro (32, 33) suggest that NFκB may have a similar “buffering” effect on the p53 signaling in vivo. Mutual reciprocal regulation of the two stresssignaling pathways opens the possibility of pharmacological tuning of either of them through targeting the other one. ACKNOWLEDGMENTS We thank Roman Kondratov for technical help and Marina Antoch and Victoria Gorbacheva for genotyping p53-deficient mice. This work was supported by grants CA88071 and CA75179 from the National Institutes of Health (NIH) and from Quark Biotech, Inc. to A. V. G. and with U. S. Federal funds from the National Cancer Institute, NIH, under Contract NO1-CO-12400. S. A. N. is an International Research Scholar of the Howard Hughes Medical Institute. The contents of this publication do not necessarily reflect the view or policies of the U. S. Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government. The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.


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Fig. 1

Figure 1. A) Striking coincidence between changes in gene expression patterns caused by p53 inactivation and TNF treatment of LNCaP (LN) cells. cDNA microarray, containing probes for 6880 annotated genes, was used. Up- and downregulated genes are included if their expression is significantly changed in the same direction (GSE56 and TNF – both up or GSE56 and TNF – both down) in both experiments. The 1.5× expression fold change (0.585 in log2 scale) was selected as a cutoff. The number of genes used for Venn diagram analysis was recalculated as a number of unique UniGene clusters present in the selected groups and on the whole array. The Venn diagram illustrates the overlap between GSE56- and TNFregulated genes. The P value is the probability of obtaining overlap of this size by random drawing (calculated as cumulative hypergeometric probability). Typical expression profiles are shown for each subset of genes. Four points at X axis correspond to four probes used for array hybridization and are indicated in the figure. Y axis reflects the log2 relative expressions (as compared with LNCaP baseline experiment). B) p53 represses NFκB-mediated transactivation. Lung carcinoma cell line H1299/CMV5 containing β-gal reporter under p53-responsive promoter (Con A) were transiently transfected with three different plasmids containing luciferase reporter under NFκB dependent promoters from different genes (NFκB binding sites from E-selectin – Elam, IP-10, or HIV LTR (#11), all cloned upstream of fos minimal promoter) in combination with plasmids expressing cDNAs for wild-type human p53, tumor-derived R175H p53 mutant (p53175H) or empty vector. As a control, NFκB-independent reporter, we used a plasmid containing HIV LTR with deleted NFκB binding site followed by fos minimal promoter and luciferase sequences (marked as “fos”). Luciferase and β-gal activity were measured in protein extracts 20 h after transfection. The results shown are representative of at least three separate experiments (mean ± SD).


Fig. 2


Figure 2. Increased NFκB activity and sensitivity to septic shock in p53 null mice. A) Lipopolysaccharide (LPS)-induced (8 mg/kg, 3 h) expression of cytokines, chemokines, and chemokine receptors in the mouse thymuses was compared using inflammatory cytokine/receptor arrays (SuperArray, 94 genes). A fragment of arrays demonstrates the higher levels of induction of IL-1, IL-6, IL-9, IL-2r, TNF, CCR2, Mig, and IP-10 observed in p53 null compared with wt p53 mice. B) IL1α, IL-1β, IL-6, and TNF mRNA expression in the thymuses of LPS-treated (8 mg/kg, 3 h) wild-type p53 and p53 null mice. Results of real-time RT PCR were expressed as relative units normalized to 18S RNA expression. The experiment was repeated 3 times with similar results. Two to four animals were killed per each time point in each experiment. Data represent means of 3 measurements ± SD. *P < 0.05; **P < 0.01 for p53 null vs. p53 wild-type mice by Student’s test. C) Kinetics of IL-1 and IL-6 mRNA expression in TNF/p53-knockout mice in comparison with TNF-knockout mice after treatment with LPS (8 mg/kg). D) NFκB activation in the thymus and spleen of wild-type p53 and p53-knockout mice after LPS treatment. Results of gel shift assay are shown using 10-µg nuclear cellular extracts from mouse tissues of either untreated or 1 or 2.5 h after LPS (8 mg/kg) treatment. E) Gel supershift analysis of molecular constituents of NFκB activated by LPS in the thymus of wild-type p53 and p53 null mice. Antibodies against p65 or p50 were added to nuclear protein extracts (LPS, 2.5 h), normalized according to NFκB DNA binding activity. Lower panel) Area of the gel with the main NFκB-specific bands; (upper panel) antibody-shifted bands. F) p53 null mice are hypersensitive to LPS-induced septic shock. Survival curves are shown for two doses of intraperitoneally injected LPS: 20 mg/kg (top) and 12 mg/kg (bottom). LPS was administered to 10 wild-type and 10 p53 null males (10-14 weeks old, both – C57Bl/6 background). Experiments were repeated three times, each with similar results. G) p53 null mice are more sensitive to cecal ligation and puncture (CLP)-induced peritonitis. 10 p53 null (on C57Bl/6 background) and 12 C57Bl/6 mice underwent CLP to induce sepsis.


Fig. 3

Figure 3. Inflammatory response after thyoglycolate and LPS-induced peritonitis. A) Macrophage and neutrophil activation after thyoglycolate-induced peritonitis. Differential macrophage and neutrophil cell counts of peritoneal exudates cells of wt p53 and p53 null mice 0, 6, 24, 48, and 72 h after thyoglycolate injection were conducted on 4–5 fields (400–500 cells) of cytospins stained with Wright-Giemsa (Materials and Methods). Neutrophil clearance is delayed in p53 null compared with wild-type (wt) mice. B) Peritoneal exudate cells (72 h after LPS intraperitoneal injection) of wild-type p53 and p53 null mice (Wright-Giemsa-stained cytospins). C) Hematoxylin/eosin-stained liver sections from wild-type p53 and p53-knockout mice after LPS intraperitoneal injection (72 h). Blood vessels are surrounded by inflammatory cells in p53 null mice.


Fig. 4

Figure 4. Phagocytosis/binding of apoptotic thymocytes by peritoneal macrophages from wild-type and p53 null mice. A) Percent of phagocytosis/binding of 10 Gy irradiated (IR) and untreated (u/t) thymocytes (30% of thymocytes were apoptotic 4 h after IR) by peritoneal macrophages from wt p53(+/+) and p53 null(−/−) mice. Percent of macrophages, phagocytosing/binding at least one apoptotic thymocyte after 30 min were counted. Phagocytic index was calculated as (average number of thymocytes phagocytosed/binded per macrophage) × (percentage of macrophages phagocyting/binding one or more thymocytes) for wt p53 and p53 null peritoneal macrophages incubated with IR thymocytes (values taken from mean of duplicates of minimum 500 cells for three separate experiments). B) Representative photomicrographs show wt p53 and p53 null macrophage capacity phagocytosis/binding with apoptotic thymocytes (hematoxylin staining). Page 20 of 21 (page number not for citation purposes)

Fig. 5

Figure 5. Increased levels of oxidation markers in tissues of p53-deficient mice. Comparison of the levels of (A) peroxidase index in peripheral blood leukocytes and (B) modified tyrosines (OY and NO2Y) in the spleens of p53 null and wild-type mice. C) Comparison of levels of ROS activity in thymocytes of p53 null and wild-type mice.
