RESUMEN
The murine double minute 2 (MDM2) protein is a major negative regulator of the tumour suppressor protein p53. Under normal conditions, MDM2 constantly binds to p53 transactivation domain and/or ubiquinates p53 via its role as E3 ubiquitin ligase to promote p53 degradation as well as nuclear export to maintain p53 levels in cells. Meanwhile, amplification of MDM2 and appearance of MDM2 spliced variants occur in many tumours and normal tissues making it a prognostic indicator for human cancers. The mutation or deletion of p53 protein in half of human cancers inactivates its tumour suppressor activity. However, cancers with wild type p53 have its function effectively inhibited through direct interaction with MDM2 oncoprotein. Here, we described the construction of a MDM2 spliced variant (rMDM215kDa) consisting of SWIB/MDM2 domain and its central region for antibody generation. Biopanning with a human naïve scFv library generated four scFv clones specific to rMDM215kDa. Additionally, the selected scFv clones were able to bind to the recombinant full length MDM2 (rMDM2-FL). Computational prediction showed that the selected scFv clones potentially bind to exon 7-8 of MDM2 while leaving the MDM2/SWIB domain free for p53 interaction. The developed antibodies exhibit good specificity can be further investigated for downstream biomedical and research applications.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Anticuerpos de Cadena Única/inmunología , Humanos , Simulación del Acoplamiento Molecular , Dominios Proteicos/genética , Isoformas de Proteínas/inmunología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
Identification of immunogenic tumor antigens, their corresponding T cell epitopes and the selection of effective adjuvants are prerequisites for developing effective cancer immunotherapies such as therapeutic vaccines. Murine double minute 2 (MDM2) is an E3 ubiquitin-protein ligase that negatively regulates tumor suppressor p53. Because MDM2 overexpression serves as a poor prognosis factor in various types of tumors, it would be beneficial to develop MDM2-targeted cancer vaccines. In this report, we identified an MDM2-derived peptide epitope (MDM232-46) that elicited antigen-specific and tumor-reactive CD4+ T cell responses. These CD4+ T cells directly killed tumor cells via granzyme B. MDM2 is expressed in head and neck cancer patients with poor prognosis, and the T cells that recognize this MDM2 peptide were present in these patients. Notably, Nutlin-3 (MDM2-p53 blocker), inhibited tumor cell proliferation, was shown to augment antitumor T cell responses by increasing MDM2 expression, HLA-class I and HLA-DR through class II transactivator (CIITA). These results suggest that the use of this MDM2 peptide as a therapeutic vaccine combined with MDM2 inhibitors could represent an effective immunologic strategy to treat cancer.
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Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Animales , Vacunas contra el Cáncer/inmunología , Línea Celular , Proliferación Celular/fisiología , Antígenos HLA-DR/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Inmunoterapia/métodos , Ratones , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Línea Celular , Núcleo Celular/metabolismo , Modelos Moleculares , Dominios ProteicosRESUMEN
Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.
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Alarminas/inmunología , Calgranulina A/inmunología , Calgranulina B/inmunología , Alarminas/química , Alarminas/genética , Animales , Artritis/genética , Artritis/inmunología , Artritis/patología , Sitios de Unión , Calgranulina A/química , Calgranulina A/genética , Calgranulina B/química , Calgranulina B/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Psoriasis/genética , Psoriasis/inmunología , Psoriasis/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2-/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2-/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2-/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.
Asunto(s)
Activación de Linfocitos , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Linfocitos T/inmunología , Linfocitos T/fisiología , Inmunidad Adaptativa , Animales , Muerte Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Genes p53/genética , Genes p53/inmunología , Enfermedad Injerto contra Huésped/inmunología , Inmunidad Innata , Isoantígenos/inmunología , Ratones , Proteína Adaptadora de Señalización NOD1/deficiencia , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/deficiencia , Proteína Adaptadora de Señalización NOD2/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de SeñalRESUMEN
AIM: To determine the prevalence and diagnostic value of autoantibodies in α-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC). METHODS: Fifty-six serum samples from AFP-negative HCC cases, 86 from AFP-positive HCC cases, 168 from chronic liver disease cases, and 59 from normal human controls were included in this study. Autoantibodies to nucleophosmin (NPM)1, 14-3-3zeta and mouse double minute 2 homolog (MDM2) proteins in AFP-negative HCC serum were evaluated by enzyme-linked immunosorbent assay. Partially positive sera were further evaluated by western blotting. Immunohistochemistry was used to detect the expression of three tumor-associated antigens (TAAs) in AFP-negative HCC and normal control tissues. RESULTS: The frequency of autoantibodies to the three TAAs in AFP-negative HCC sera was 21.4%, 19.6% and 19.6%, which was significantly higher than in the chronic liver disease cases and normal human controls (P < 0.01) as well as AFP-positive HCC cases. The sensitivity of the three autoantibodies for diagnosis of AFP-negative HCC ranged from 19.6% to 21.4%, and the specificity was approximately 95%. When the three autoantibodies were combined, the sensitivity reached 30.4% and the specificity reached 91.6%. CONCLUSION: Autoantibodies to NPM1, 14-3-3zeta and MDM2 may be useful biomarkers for immunodiagnosis of AFP-negative HCC.
Asunto(s)
Autoanticuerpos/sangre , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , alfa-Fetoproteínas/metabolismo , Proteínas 14-3-3/inmunología , Proteínas 14-3-3/metabolismo , Anciano , Autoanticuerpos/inmunología , Carcinoma Hepatocelular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Pruebas Inmunológicas , Hepatopatías/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Recombinantes/metabolismo , Estudios Retrospectivos , alfa-Fetoproteínas/inmunologíaRESUMEN
Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.
Asunto(s)
Daño del ADN/inmunología , Enfermedades del Sistema Inmune/terapia , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Etopósido/administración & dosificación , Humanos , Enfermedades del Sistema Inmune/inmunología , Activación de Linfocitos , Linfohistiocitosis Hemofagocítica/inmunología , Linfohistiocitosis Hemofagocítica/terapia , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patología , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
INTRODUCTION: Primary Sjogren's Syndrome (pSS) is one of the autoimmune diseases characterized by polyclonal autoantibody production. The human homologue of the mouse double minute 2 (MDM2) is an important negative regulator of p53. Our previous study indicated that autoantibody to MDM2 can be detected in systemic lupus erythematosus patients. The purpose of this study is to study anti-MDM2 autoantibody in pSS patients. METHODS: Anti-MDM2 autoantibody in sera from 100 pSS patients and 74 normal controls was investigated by ELISA. Positive samples were further confirmed by western blotting. Expression of MDM2 in labial gland tissue from pSS patients and normal controls was checked by immunohistochemistry. The difference in clinical characteristics and laboratory findings between anti-MDM2 positive and anti-MDM2 negative pSS patients was analyzed. RESULTS: The presence of anti-MDM2 autoantibody in pSS patients was 21.0%, significantly higher than normal controls (5.40%). MDM2 was overexpressed in labial gland from pSS patients. pSS patients with positive anti-MDM2 were characterized by longer disease duration and more lymphocytes focal gathering in labial gland. Prevalence of anemia, thrombocytopenia and anti-SSB was significantly higher in pSS patients with anti-MDM2 autoantibody. Titer of anit-MDM2 was negatively associated with hemoglobin level, platelet count, complement 3 level and complement 4 level, positively associated with European Sjogren's syndrome disease activity index (ESSDAI) and level of IgG. CONCLUSIONS: Anti-MDM2 autoantibody may be used as a potential serological biomarker in pSS disease activity evaluation. Study on the role of anti-MDM2 or MDM2 in pSS may help us know the pathogenesis mechanism of pSS better.
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Autoanticuerpos/sangre , Biomarcadores/sangre , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Síndrome de Sjögren/sangre , Autoanticuerpos/inmunología , Western Blotting , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/inmunologíaRESUMEN
The tumor suppressor p53 is the most frequently mutated gene in human cancers. Most of the mutations are missense leading to loss of p53 function in inducing apoptosis and senescence. In addition to these autonomous effects of p53 inactivation/dysfunction on tumorigenesis, compelling evidence suggests that p53 mutation/inactivation also leads to gain-of-function or activation of non-autonomous pathways, which either directly or indirectly promote tumorigenesis. Experimental and clinical results suggest that p53 dysfunction fuels pro-tumor inflammation and serves as an immunological gain-of-function driver of tumorigenesis via skewing immune landscape of the tumor microenvironment (TME). It is now increasingly appreciated that p53 dysfunction in various cellular compartments of the TME leads to immunosuppression and immune evasion. Although our understanding of the cellular and molecular processes that link p53 activity to host immune regulation is still incomplete, it is clear that activating/reactivating the p53 pathway in the TME also represents a compelling immunological strategy to reverse immunosuppression and enhance antitumor immunity. Here, we review our current understanding of the potential cellular and molecular mechanisms by which p53 participates in immune regulation and discuss how targeting the p53 pathway can be exploited to alter the immunological landscape of tumors for maximizing therapeutic outcome.
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Antineoplásicos/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Imidazoles/uso terapéutico , Neoplasias/tratamiento farmacológico , Piperazinas/uso terapéutico , Microambiente Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/agonistas , Apoptosis , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/inmunología , Carcinogénesis/patología , Citocinas/genética , Citocinas/inmunología , Humanos , Evasión Inmune , Mutación , FN-kappa B/genética , FN-kappa B/inmunología , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Transducción de Señal , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Lung cancer causes 1.4 million deaths annually. In the search for functional foods as complementary therapies against lung cancer, the immuno-stimulatory properties of the vegetable Morinda citrifolia leaves were investigated and compared with the anti-cancer drug erlotinib. Lung tumour-induced BALB/c mice were fed with 150 mg kg(-1) or 300 mg kg(-1) body weight of the leaf extract, or erlotinib (50 mg kg(-1) body-weight) for 21 days. The 300 mg kg(-1) body weight extract significantly (and dose-dependently) suppressed lung tumour growth; the extract worked more effectively than the 50 mg kg(-1) body weight erlotinib treatment. The extract significantly increased blood lymphocyte counts, and spleen tissue B cells, T cells and natural killer cells, and reduced the epidermal growth factor receptor (EGFR) which is a lung adenocarcinoma biomarker. The extract also suppressed the cyclooxygenase 2 (COX2) inflammatory markers, and enhanced the tumour suppressor gene (phosphatase and tensin homolog, PTEN). It inhibited tumour growth cellular gene (transformed mouse 3T3 cell double minute 2 (MDM2), V-raf-leukemia viral oncogene 1 (RAF1), and mechanistic target of rapamycin (MTOR)) mRNA expression in the tumours. The extract is rich in scopoletin and epicatechin, which are the main phenolic compounds. The 300 mg kg(-1)Morinda citrifolia leaf 50% ethanolic extract showed promising potential as a complementary therapeutic dietary supplement which was more effective than the 50 mg kg(-1) erlotinib in suppressing lung adenocarcinoma. Part of the mechanisms involved enhancing immune responses, suppressing proliferation and interfering with various tumour growth signalling pathways.
Asunto(s)
Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Morinda/química , Extractos Vegetales/administración & dosificación , Animales , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Receptores ErbB/genética , Receptores ErbB/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Extractos Vegetales/química , Hojas de la Planta/química , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Introduction. Systemic lupus erythematosus (SLE) is one of the systemic autoimmune diseases characterized by the polyclonal autoantibody production. The human homologue of the mouse double minute 2 (MDM2) is well known as the negative regulator of p53. MDM2 has been reported to be overexpressed in SLE animal model and to promote SLE. Since abnormally expressed proteins can induce autoimmune response, anti-MDM2 autoantibody was examined in SLE patients. Methods. Anti-MDM2 antibody in sera from 43 SLE patients and 69 healthy persons was investigated by ELISA. Positive samples were further confirmed by western blotting. The immunological feathers of anti-MDM2 positive sera were analyzed by indirect immunofluorescence assay. Anti-p53 was also investigated in SLE patients by ELISA, and the correlation of anti-MDM2 and anti-p53 was analyzed. Results. The presence of anti-MDM2 in SLE patients was 23.30%, much higher than normal healthy persons (4.30%). These anti-MDM2 positive sera present a nuclear staining pattern. The presence of anti-p53 in SLE patients was 39.50%, and the titer of anti-MDM2 was positively correlated with anti-p53 in SLE patients. Conclusions. Anti-MDM2 autoantibody was detected at high prevalence in SLE patients. The detection of anti-MDM2 in SLE patients should be clinically useful.
Asunto(s)
Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Biomarcadores , Estudios de Casos y Controles , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Células Hep G2 , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/epidemiología , Masculino , Proteínas Recombinantes , Estudios Seroepidemiológicos , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
P53 is critically important in preventing oncogenesis but its role in inflammation in general and in the function of inflammatory macrophages in particular is not clear. Here, we show that bone marrow-derived macrophages exhibit endogenous p53 activity, which is increased when macrophages are polarized to the M2 (alternatively activated macrophage) subtype. This leads to reduced expression of M2 genes. Nutlin-3a, which destabilizes the p53/MDM2 (mouse double minute 2 homolog) complex, promotes p53 activation and further downregulates M2 gene expression. In contrast, increased expression of M2 genes was apparent in M2-polarized macrophages from p53-deficient and p53 mutant mice. Furthermore, we show, in mice, that p53 also regulates M2 polarization in peritoneal macrophages from interleukin-4-challenged animals and that nutlin-3a retards the development of tolerance to Escherichia coli lipopolysaccharide. P53 acts via transcriptional repression of expression of c-Myc (v-myc avian myelocytomatosis viral oncogene homolog) gene by directly associating with its promoter. These data establish a role for the p53/MDM2/c-MYC axis as a physiological 'brake' to the M2 polarization process. This work reveals a hitherto unknown role for p53 in macrophages, provides further insight into the complexities of macrophage plasticity and raises the possibility that p53-activating drugs, many of which are currently being trialled clinically, may have unforeseen effects on macrophage function.
Asunto(s)
Activación de Macrófagos , Macrófagos/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Polaridad Celular , Regulación de la Expresión Génica , Imidazoles/farmacología , Interleucina-4/metabolismo , Lipopolisacáridos , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , Transducción de Señal/inmunología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
The human homologue of the mouse double minute 2 (MDM2) is known to be overexpressed in a variety of human malignancies. As one of E3 ubiquitin-protein ligases, MDM2 interacts with the tumour suppressor p53 by mediating ubiquitination and degradation of p53. Since abnormally expressed proteins can induce autoimmune response, to further examine whether sera from patients with esophageal squamous cell carcinoma (ESCC) exhibited immunoreactivity against MDM2 and p53, autoantibody responses to MDM2 and p53 were evaluated by enzyme-linked immunosorbent assay (ELISA) in sera from patients with ESCC and normal individuals. Positive results were also confirmed by Western blotting and indirect immunofluorescence assay. The results demonstrated that the positive rate of autoantibody against p53 and MDM2 in ESCC sera was 22.9% (36/157) and 14.0% (22/157), whereas this rate was 0% (0/85) and 1.2% (1/85), respectively, in normal individuals. Some of the sera with antibodies specific for MDM2 also contained antibodies against p53. And there was an increase of positive antibody reactions reaching a frequency of 35% (55/157) combination with MDM2 and p53. This was significantly higher than the frequency of antibodies in normal individuals (P < 0.01). Our preliminary results suggest that autoantibodies against MDM2 and p53 may be useful serum biomarkers in the immunodiagnosis of ESCC.
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Autoanticuerpos/sangre , Carcinoma de Células Escamosas/diagnóstico , Neoplasias Esofágicas/diagnóstico , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Proteína p53 Supresora de Tumor/inmunología , Adulto , Anciano , Autoanticuerpos/inmunología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/inmunología , Carcinoma de Células Escamosas/inmunología , Línea Celular Tumoral , Neoplasias Esofágicas/inmunología , Carcinoma de Células Escamosas de Esófago , Femenino , Células Hep G2 , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Although new therapeutic strategies have been continuously developed and applied to clinical treatment for HCC, the prognosis is still very poor. Thus, early detection of HCC may enhance effective and curative management. In this study, autoantibody responses to MDM2 protein in HCC patient's serum were evaluated by enzyme-linked immunosorbent assay (ELISA) and part sera were evaluated by Western blotting and indirect immunofluorescence assay. Immunohistochemistry (IHC) over tissue array slides was also performed to analyze protein expression of MDM2 in HCC and control tissues. The prevalence of autoantibodies against MDM2 was significantly higher than that in liver cirrhosis (LC), chronic hepatitis (CH), and normal human sera (NHS). The average titer of autoantibodies against MDM2 in HCC serum was higher compared to that in LC, CH, and NHS. A high titer of autoantibodies against MDM2 in ELISA could be observed in the serum in 6 to 9 months before the clinical diagnosis of HCC in the serum of several HCC patients with serial bleeding samples. Our preliminary data indicate that MDM2 and anti-MDM2 system may be a potential biomarker for early stage HCC screening and immunodiagnosis.
Asunto(s)
Autoanticuerpos/sangre , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Proteínas Proto-Oncogénicas c-mdm2/genética , Anciano , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Estudios de Casos y Controles , Diagnóstico Diferencial , Diagnóstico Precoz , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Expresión Génica , Hepatitis Crónica/diagnóstico , Humanos , Cirrosis Hepática/diagnóstico , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Análisis de Matrices TisularesRESUMEN
Deubiquitinases (DUBs) are a new class of drug targets, although the physiological function of only few DUBs has been characterized. Here we identified the DUB USP15 as a crucial negative regulator of T cell activation. USP15 stabilized the E3 ubiquitin ligase MDM2, which in turn negatively regulated T cell activation by targeting the degradation of the transcription factor NFATc2. USP15 deficiency promoted T cell activation in vitro and enhanced T cell responses to bacterial infection and tumor challenge in vivo. USP15 also stabilized MDM2 in cancer cells and regulated p53 function and cancer-cell survival. Our results suggest that inhibition of USP15 may both induce tumor cell apoptosis and boost antitumor T cell responses.
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Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Células TH1/inmunología , Proteasas Ubiquitina-Específicas/inmunología , Traslado Adoptivo , Animales , Apoptosis/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Supervivencia Celular , Células HCT116 , Humanos , Leupeptinas/farmacología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Activación de Linfocitos/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-mdm2/genética , Escape del Tumor , Proteína p53 Supresora de Tumor/inmunología , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/genética , Ubiquitinación/inmunologíaRESUMEN
Recognition of fungal pathogens by C-type lectin receptor (CLR) dectin-1 on human dendritic cells is essential for triggering protective antifungal TH1 and TH17 immune responses. We show that Fonsecaea monophora, a causative agent of chromoblastomycosis, a chronic fungal skin infection, evades these antifungal responses by engaging CLR mincle and suppressing IL-12, which drives TH1 differentiation. Dectin-1 triggering by F. monophora activates transcription factor IRF1, which is crucial for IL12A transcription via nucleosome remodeling. However, simultaneous F. monophora binding to mincle induces an E3 ubiquitin ligase Mdm2-dependent degradation pathway, via Syk-CARD9-mediated PKB signaling, that leads to loss of nuclear IRF1 activity, hence blocking IL12A transcription. The absence of IL-12 leads to impaired TH1 responses and promotes TH2 polarization. Notably, mincle is similarly exploited by other chromoblastomycosis-associated fungi to redirect TH responses. Thus, mincle is a fungal receptor that can suppress antifungal immunity and, as such, is a potential therapeutic target.
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Subunidad p35 de la Interleucina-12/biosíntesis , Lectinas Tipo C/inmunología , Receptores Inmunológicos/inmunología , Saccharomycetales/inmunología , Proteínas Adaptadoras de Señalización CARD/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Cromoblastomicosis/inmunología , Células Dendríticas/inmunología , Humanos , Factor 1 Regulador del Interferón/biosíntesis , Factor 1 Regulador del Interferón/genética , Subunidad p35 de la Interleucina-12/genética , Subunidad p35 de la Interleucina-12/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas Tirosina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Interferencia de ARN , ARN Interferente Pequeño , Quinasa Syk , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
The detection of cancer biomarkers is as important tool for the diagnosis and prognosis of cancer such as brain cancer. Murine double minute 2 (MDM2) has been widely studied as prognostic marker for brain tumor. Here we describe development of a new sensitive label free impedimetric immunosensor for the detection of MDM2 based on cysteamine self assembled monolayers on a clean polycrystalline Au electrode surface. The amine-modified electrodes were further functionalized with antibody using homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. The assembly processes of the immunosensor had been monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using Fe(CN)(6)(3-/4-) solution as redox probe. The impedance changes upon binding of MDM2 protein to the sensor surface was utilized for the detection of MDM2. The increase in relative electron-transfer resistance (ΔR/R(0)%) values was linearly proportional to the concentration of tumor marker MDM2 in the wide dynamic range of 1pg/ml-1µg/ml. The limit of detection was 0.29pg/ml in phosphate buffer saline (PBS) and 1.3pg/ml in mouse brain tissue homogenate, respectively. The immunosensor showed a good performance in comparison with ELISA for the analysis of the MDM2 in the cancerous mouse brain tissue homogenates. Moreover, the immunosensor had a good selectivity against epidermal growth factor receptor (EGFR) protein, long-storage stability and reproducibility. It might be become a promising assay for clinical diagnosis and early detection of tumors.
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Neoplasias Encefálicas/diagnóstico , Encéfalo/patología , Espectroscopía Dieléctrica/métodos , Proteínas Proto-Oncogénicas c-mdm2/análisis , Animales , Anticuerpos Inmovilizados/inmunología , Técnicas Biosensibles/métodos , Química Encefálica , Neoplasias Encefálicas/patología , Cisteamina/química , Electrodos , Humanos , Inmunoensayo/métodos , Límite de Detección , Ratones , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Tiocianatos/químicaRESUMEN
Systemic lupus erythematosus (SLE) is a polyclonal autoimmune syndrome directed against multiple nuclear autoantigens. Although RNA and DNA seem to have identical immunostimulatory effects on systemic and intrarenal inflammation, each seems to differ with regard to the propensity to induce mitogenic effects such as lymphoproliferation. To identify potential mechanisms by which DNA specifically contributes to the pathogenesis of lupus nephritis, we stimulated cells with immunostimulatory DNA or RNA in vitro and used microarray to compare the transcriptomes of RNA- and DNA-induced genes. Immunostimulatory DNA, but not RNA, induced Mdm2, which is a negative regulator of p53. In vivo, we observed greater expression and activation of Mdm2 in the spleen and kidneys in a mouse model of lupus (MRL-Fas(lpr) mice) than healthy controls. Treatment of MRL-Fas(lpr) mice with the Mdm2 inhibitor nutlin-3a prevented nephritis and lung disease and significantly prolonged survival. Inhibition of Mdm2 reduced systemic inflammation and abrogated immune complex disease by suppressing plasma cells and the production of lupus autoantibodies. In addition, nutlin-3a suppressed the abnormal expansion of all T cell subsets, including CD3(+)CD4(-)CD8(-) T cells, which associated with attenuated systemic inflammation. However, inhibiting Mdm2 did not cause myelosuppression or affect splenic regulatory T cells, neutrophils, dendritic cells, or monocytes. Taken together, these data suggest that the induction of Mdm2 promotes the expansion of plasma cells and CD3(+)CD4(-)CD8(-) T cells, which cause autoantibody production and immune complex disease in MRL-Fas(lpr) mice. Antagonizing Mdm2 may have therapeutic potential in lupus nephritis.
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Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Animales , Autoanticuerpos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citosol/fisiología , ADN/inmunología , Femenino , Expresión Génica/inmunología , Mesangio Glomerular/inmunología , Mesangio Glomerular/patología , Imidazoles/farmacología , Lupus Eritematoso Sistémico/genética , Nefritis Lúpica/genética , Ratones , Ratones Endogámicos MRL lpr , Células 3T3 NIH , Necrosis , Piperazinas/farmacología , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Bazo/inmunologíaRESUMEN
In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 10(6)- to 10(8)-fold per round. After only one or two rounds of selection, antibodies with high affinity and specificity were obtained from naive and randomized single-chain Fv libraries of approximately 10(12) molecules. Furthermore, we confirmed that not only protein-protein (antigen-antibody) interactions, but also protein-DNA and protein-drug interactions were selected with ultrahigh efficiencies. This method will facilitate high-throughput preparation of antibodies and identification of protein interactions in proteomic and therapeutic fields.
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Evolución Molecular Dirigida , Región Variable de Inmunoglobulina/genética , Técnicas Analíticas Microfluídicas/métodos , ARN Mensajero/biosíntesis , Animales , Línea Celular , Biblioteca de Genes , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Ratones , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Large granular lymphocyte (LGL) leukemia is a clonal proliferative disease of T and natural killer (NK) cells. Interleukin (IL)-15 is important for the development and progression of LGL leukemia and is a survival factor for normal NK and T memory cells. IL-15 alters expression of Bcl-2 family members, Bcl-2, Bcl-XL, Bim, Noxa, and Mcl-1; however, effects on Bid have not been shown. Using an adoptive transfer model, we show that NK cells from Bid-deficient mice survive longer than cells from wild-type control mice when transferred into IL-15-null mice. In normal human NK cells, IL-15 significantly reduces Bid accumulation. Decreases in Bid are not due to alterations in RNA accumulation but result from increased proteasomal degradation. IL-15 up-regulates the E3 ligase HDM2 and we find that HDM2 directly interacts with Bid. HDM2 suppression by short hairpin RNA increases Bid accumulation lending further support for HDM2 involvement in Bid degradation. In primary leukemic LGLs, Bid levels are low but are reversed with bortezomib treatment with subsequent increases in LGL apoptosis. Overall, these data provide a novel molecular mechanism for IL-15 control of Bid that potentially links this cytokine to leukemogenesis through targeted proteasome degradation of Bid and offers the possibility that proteasome inhibitors may aid in the treatment of LGL leukemia.