RESUMEN

Myc oncogenic transcription factors (c-Myc, N-Myc, and L-Myc) coordinate the control of cell growth, division, and metabolism. In cancer, Myc overexpression is often associated with aggressive disease, which is in part due to the destruction of select targets by the ubiquitin-proteasome system (eg, SCF(Skp2)-directed destruction of the Cdk inhibitor p27(Kip1)). We reasoned that Myc would also regulate SUMOylation, a related means of posttranslational modification of proteins, and that this circuit would play essential roles in Myc-dependent tumorigenesis. Here, we report marked increases in the expression of genes that encode regulators and components of the SUMOylation machinery in mouse and human Myc-driven lymphomas, resulting in hyper-SUMOylation in these tumors. Further, inhibition of SUMOylation by genetic means disables Myc-induced proliferation, triggering G2/M cell-cycle arrest, polyploidy, and apoptosis. Using genetically defined cell models and conditional expression systems, this response was shown to be Myc specific. Finally, in vivo loss-of-function and pharmacologic studies demonstrated that inhibition of SUMOylation provokes rapid regression of Myc-driven lymphoma. Thus, targeting SUMOylation represents an attractive therapeutic option for lymphomas with MYC involvement.

Asunto(s)

Linfoma de Células B/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ácidos Anacárdicos/farmacología , Animales , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Análisis por Conglomerados , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B/genética , Ratones , Ratones Transgénicos , Poliploidía , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal , Sumoilación/efectos de los fármacos , Transcripción Genética , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo

RESUMEN

The Cks1 component of the SCF(Skp2) complex is necessary for p27(Kip1) ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27(Kip1) levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27(Kip1) levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27(Kip1). To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo.

Asunto(s)

Quinasas CDC2-CDC28/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Hematológicas/metabolismo , Linfoma de Células B/metabolismo , Neoplasias/fisiopatología , Animales , Médula Ósea/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Immunoblotting , Ratones , Ratones Transgénicos , Neoplasias/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Retroviridae

RESUMEN

Cks1 is an activator of the SCF(Skp2) ubiquitin ligase complex that targets the cell cycle inhibitor p27(Kip1) for degradation. The loss of Cks1 results in p27(Kip1) accumulation and decreased proliferation and inhibits tumorigenesis. We identify here a function of Cks1 in mammalian cell cycle regulation that is independent of p27(Kip1). Specifically, Cks1(-/-); p27(Kip1-/-) mouse embryonic fibroblasts retain defects in the G(1)-S phase transition that are coupled with decreased Cdk2-associated kinase activity and defects in proliferation that are associated with Cks1 loss. Furthermore, concomitant loss of Cks1 does not rescue the tumor suppressor function of p27(Kip1) that is manifest in various organs of p27(Kip1-/-) mice. In contrast, defects in mitotic entry and premature senescence manifest in Cks1(-/-) cells are p27(Kip1) dependent. Collectively, these findings establish p27(Kip1)-independent functions of Cks1 in regulating the G(1)-S transition.

Asunto(s)

Quinasas CDC2-CDC28/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Puntos de Control de la Fase S del Ciclo Celular/fisiología , Animales , Quinasas CDC2-CDC28/deficiencia , Quinasas CDC2-CDC28/genética , Proliferación Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Quinasas Ciclina-Dependientes/metabolismo , Femenino , Fibroblastos/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/fisiología , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Quinasas Asociadas a Fase-S/metabolismo

RESUMEN

Administration of the DNA-alkylating agent methylazoxymethanol acetate (MAM) on embryonic day 17 (E17) produces behavioral and anatomical brain abnormalities, which model some aspects of schizophrenia. This has lead to the premise that MAM rats are a neurodevelopmental model for schizophrenia. However, the underlying molecular pathways affected in this model have not been elucidated. In this study, we investigated the molecular phenotype of adult MAM rats by focusing on the frontal cortex and hippocampal areas, as these are known to be affected in schizophrenia. Proteomic and metabonomic analyses showed that the MAM treatment on E17 resulted primarily in deficits in hippocampal glutamatergic neurotransmission, as seen in some schizophrenia patients. Most importantly, these results were consistent with our finding of functional deficits in glutamatergic neurotransmission, as identified using electrophysiological recordings. Thus, this study provides the first molecular evidence, combined with functional validation, that the MAM-E17 rat model reproduces hippocampal deficits relevant to the pathology of schizophrenia.

Asunto(s)

Hipocampo/metabolismo , Acetato de Metilazoximetanol/farmacología , Efectos Tardíos de la Exposición Prenatal/metabolismo , Esquizofrenia/inducido químicamente , Esquizofrenia/metabolismo , Transmisión Sináptica/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Humanos , Masculino , Metabolómica/métodos , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Proteómica/métodos , Ratas , Esquizofrenia/fisiopatología , Transmisión Sináptica/efectos de los fármacos

RESUMEN

Squamous cell cancer of the head and neck (SCCHN) is the sixth leading cause for cancer deaths worldwide. Despite extense knowledge of risk factors and pathogenesis about 50 percent of all patients and essentially every patient with metastatic SCCHN eventually die from this disease. We analyzed the clinical data and performed immunohistochemistry for Epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurora-A) expression in 180 SCCHN patients. Patients characterized by elevated EGFR and elevated Aurora-A protein expression in tumor tissue represent a risk group with poor disease-free and overall survival (EGFR(low)Aurora-A(low) versus EGFR(high)Aurora-A(high), p = 0.024). Treating SCCHN cell lines with a pan-Aurora kinase inhibitor resulted in defective cytokinesis, polyploidy and apoptosis, which was effective irrespective of the EGFR status. Combined Aurora kinase and EGFR targeting using a monoclonal anti-EGFR antibody was more effective compared to single EGFR and Aurora kinase inhibition. Comparing pan-Aurora kinase and Aurora-A targeting hints towards a strong and clinically relevant biological effect mediated via Aurora kinase B. Taken together, our findings characterize a new poor risk group in SCCHN patients defined by elevated EGFR and Aurora-A protein expression. Our results demonstrate that combined targeting of EGFR and Aurora kinases represents a therapeutic means to activate cell cycle checkpoints and apoptosis in SCCHN.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/enzimología , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias de Cabeza y Cuello/enzimología , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Adulto , Anciano , Anticuerpos Monoclonales Humanizados , Aurora Quinasa A , Aurora Quinasa B , Aurora Quinasas , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/cirugía , Línea Celular Tumoral , Cetuximab , Supervivencia sin Enfermedad , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/cirugía , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Tasa de Supervivencia , Factores de Tiempo , Regulación hacia Arriba

RESUMEN

Little is known about the molecular factors that are altered in remitting bipolar disorder (BD) patients. We carried out proteome profiling of peripheral blood mononuclear cells (PBMCs) and serum from BD patients who were not experiencing mania or major depression (euthymia) compared to matched healthy controls using liquid chromatography-mass spectrometry (LC-MS(E) ) and Multi-Analyte Profiling (Human Map(®) ) platforms. This resulted in the identification of approximately 60 differentially expressed molecules involved predominantly in cell death/survival pathways. In PBMCs, this was manifested in cytoskeletal and stress response-associated proteins, whereas most serum analytes were associated with the inflammatory response. The predicted effect of serum analytes on physiological systems was tested by treating PBMCs with serum obtained from the same patients, resulting in reduced cellular survival. These preliminary results suggest that BD patients carry a peripheral fingerprint that has detrimental effects on cell function and that could be used to distinguish BD patients from healthy controls despite being in a remission phase. It is hoped that additional studies of BD patients in the manic and depressed stages could lead to the identification of a molecular fingerprint that could be used for predicting episodic switching and for guiding treatment strategies.

Asunto(s)

Biomarcadores/metabolismo , Trastorno Bipolar/metabolismo , Supervivencia Celular/fisiología , Adulto , Células Cultivadas , Cromatografía Liquida , Femenino , Humanos , Immunoblotting , Leucocitos Mononucleares/citología , Masculino , Espectrometría de Masas , Adulto Joven

RESUMEN

PURPOSE: Equine recurrent uveitis (ERU) is an incurable disease affecting the inner eye that leads to blindness, through activated T cells that pass the blood-retinal barrier and destroy the retina. Serum markers are a desirable choice for monitoring development of disease, as serum is easy accessible and the markers could serve to predict the beginning of disease or an imminent relapse. METHODS: In this study, serum proteomes (depleted of high-abundance serum proteins) of horses with ERU and healthy controls were compared with the 2-D DIGE (two-dimensional gel electrophoresis) technique to identify differentially expressed proteins. The expression pattern of a candidate protein in retina and vitreous was validated by Western blots and immunohistochemistry. RESULTS: Ten differentially expressed proteins could be identified by mass spectrometry (MALDI-TOF/TOF). Five proteins--IgM, IgG4 hc, serotransferrin, alpha-2HS-glycoprotein, and complement factor B--were upregulated in the uveitic state, whereas the five proteins albumin, apolipoprotein A-IV and H, IgG5 hc, and high-molecular-weight kininogen (HK) showed a significantly lower expression in sera of uveitis cases. Of interest, kininogen was significantly upregulated in the target tissues vitreous and retina. HK is a plasma protein with multiple physiological functions, with an important role in inflammation and promoting neovascularization. Most interesting is the as of yet unaddressed association of HK with uveitis. Immunohistochemistry showed coexpression of kininogen and VEGF in inflamed eyes. CONCLUSIONS: Since neovascularization plays a major role in the pathogenesis of uveitis, the identification of a proangiogenic factor in the retina presents an important finding and may contribute to elucidating the pathogenesis of uveitis.

Asunto(s)

Enfermedades Autoinmunes/veterinaria , Enfermedades de los Caballos/sangre , Quininógeno de Alto Peso Molecular/sangre , Uveítis/veterinaria , Animales , Autoantígenos/sangre , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/metabolismo , Proteínas Sanguíneas/metabolismo , Western Blotting , Electroforesis en Gel Bidimensional , Técnica del Anticuerpo Fluorescente Indirecta , Enfermedades de los Caballos/metabolismo , Caballos , Quininógenos/metabolismo , Recurrencia , Retina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Regulación hacia Arriba , Uveítis/sangre , Uveítis/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cuerpo Vítreo/metabolismo

RESUMEN

Identification of biomarkers is of critical relevance toward improving diagnosis and therapy of autoimmune disorders. Serum markers are a desirable choice as sera are easily accessible and the development of assays for routine clinical detection prompts feasible. Autoimmune uveitis, a recurrent disease affecting the eye, is characterized by returning inflammatory attacks of the inner eye followed by variable periods of quiescent stages. Spontaneous equine recurrent uveitis (ERU) is the equine equivalent and serves as a model for the human disease. To identify potential biomarker candidates, we first systematically compared the proteomes of individual ERU cases with healthy controls by proteomic profiling using 2-D difference-gel-electrophoresis (2-D DIGE) followed by tandem mass spectrometry. A total of seven differentially expressed proteins were identified. Besides the upregulation of IgG and the significant lower expression of albumin, Antithrombin III, and Vitamin D binding protein, we found complement components C1q and C4, to be downregulated in uveitic state. Interestingly, Pigment epithelium-derived factor (PEDF), a marker already detected by 2DE differential proteome analysis in ERU target tissues, vitreous and retina, was found to be also significantly downregulated in sera. The lower expression of PEDF in sera of horses with uveitis could be verified in a cohort of 116 ERU cases and 115 healthy controls. Our findings of a significant lower PEDF expression in ERU cases also in the periphery of the eye proves PEDF as a promising uveitis biomarker.

Asunto(s)

Enfermedades Autoinmunes , Modelos Animales de Enfermedad , Proteínas del Ojo/sangre , Factores de Crecimiento Nervioso/sangre , Serpinas/sangre , Uveítis , Animales , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/inmunología , Biomarcadores/sangre , Proteínas del Ojo/genética , Caballos , Humanos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/genética , Reproducibilidad de los Resultados , Serpinas/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Uveítis/sangre , Uveítis/inmunología

RESUMEN

Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMGs) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons receive vision, and their death results in blinding diseases. It has been proposed that RMGs release factors that support photoreceptor survival, but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated work flow toward systematic identification of neuroprotective proteins, which are, like most cytokines, expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Toward this goal we first isolated conditioned medium (CM) from primary porcine RMGs cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins toward their identification consists of several fractionation steps combined with tests for biological function. Here we combined 1) size fractionation, 2) ion exchange chromatography, 3) reverse phase liquid chromatography, and 4) mass spectrometry (Q-TOF MS/MS or MALDI MS/MS) for protein identification. As a result of this integrated work flow, the insulin-like growth factor-binding proteins IGFBP5 and IGFBP7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7, significantly increased photoreceptor survival when secreted from HEK293 cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multifactorial with IGFBP5 and CTGF as an integral part of this activity.

Asunto(s)

Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/aislamiento & purificación , Comunicación Paracrina , Proteómica/métodos , Algoritmos , Animales , Resinas de Intercambio Aniónico/metabolismo , Fraccionamiento Celular/métodos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Ojo/inervación , Estudios de Factibilidad , Humanos , Proteínas del Tejido Nervioso/análisis , Neuroglía/metabolismo , Fármacos Neuroprotectores/análisis , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/metabolismo , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/fisiología , Porcinos

RESUMEN

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU. Surgical removal of vitreous leads to cessation of relapses in spontaneous uveitis of both man and horse, therefore vitreous composites are likely to contribute to disease progression. Uveitic vitreous is likely to contain potential biomarkers in relatively undiluted quantities. With the goal to identify these markers, we systematically compared vitreous from healthy and disease-affected eyes by proteomic profiling. Nine differentially expressed proteins were identified, that are functionally related to immune response, inflammation, and maintenance of the blood-retinal barrier. One of these, pigment epithelium-derived factor, a protein involved in maintaining a proper blood-retina barrier as well as protecting from neoangiogenesis was additionally found to be down-regulated within uveitic retinal lesions whereas, conversely, vascular endothelial growth factor was found to be up-regulated at these sites. Together, these changes point to as of yet undiscovered biological pathways involved in the pathogenesis of this autoimmune disease.

Asunto(s)

Barrera Hematorretinal/fisiología , Proteínas del Ojo/biosíntesis , Enfermedades de los Caballos/fisiopatología , Factores de Crecimiento Nervioso/biosíntesis , Proteoma/química , Serpinas/biosíntesis , Uveítis/veterinaria , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Caballos

RESUMEN

Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. To elucidate such processes, we studied changes in the total retinal proteome of ERU-diseased horses compared to healthy controls. Severe changes in the retinal proteome were found for several markers for blood-retinal barrier breakdown and whose emergence depended upon disease severity. Additionally, uveitic changes in the retina were accompanied by upregulation of aldose 1-epimerase, selenium-binding protein 1, alpha crystallin A chain, phosphatase 2A inhibitor (SET), and glial fibrillary acidic protein (GFAP), the latter indicating an involvement of retinal Mueller glial cells (RMG) in disease process. To confirm this, we screened for additional RMG-specific markers and could demonstrate that, in uveitic retinas, RMG concomitantly upregulate vimentin and GFAP and downregulate glutamine synthetase. These expression patterns suggest for an activated state of RMG, which further downregulate the expression of pigment epithelium-derived factor (PEDF) and begin expressing interferon-gamma, a pro-inflammatory cytokine typical for T-helper 1 cells. We thus propose that RMG may play a fatal role in uveitic disease progression by directly triggering inflammatory processes through the expression and secretion of interferon-gamma.

Asunto(s)

Enfermedades Autoinmunes/veterinaria , Enfermedades de los Caballos/inmunología , Caballos/inmunología , Neuroglía/química , Proteoma/análisis , Retina/química , Uveítis/veterinaria , Animales , Enfermedades Autoinmunes/inmunología , Citocinas/análisis , Proteínas del Ojo/análisis , Interferón gamma/análisis , Factores de Crecimiento Nervioso/análisis , Neuroglía/inmunología , Retina/inmunología , Serpinas/análisis , Regulación hacia Arriba , Uveítis/inmunología

RESUMEN

The interphotoreceptor matrix (IPM) is located between photoreceptors and pigment epithelium in the retina and is involved in fundamental functions of the visual cycle. These include visual pigment chromophore exchange, retinal adhesion, metabolite trafficking, and growth factor presentation. In general, IPM preparations are contaminated with intracellular proteins, as has also been described for other body fluids. This study aimed at identifying new components of the IPM by discriminating between truly secreted proteins and proteins that are part of the IPM for secondary reasons. "Soluble" porcine IPM was extracted from retina and pigment epithelium with PBS by two different procedures, followed by extraction with water alone that released "insoluble" IPM matrix sheets. Samples from all preparations were separated by 2-DE and a total of 140 protein spots were identified by MALDI-TOF and/or CapLC Q-TOF MS. Although identified proteins included several already known in the IPM, the majority had not been previously described in this structure. Gene ontology classifications allocated the identified proteins into nine different functional networks. The IPM preparations also included intracellular proteins from cells adjacent to the IPM, which may have resulted from cell disruption. This underlines the experimental difficulties of a biochemical analysis of the IPM as an intact compartment. We show here a strategy for predicting the probability of identified IPM proteins occurring in vivo by combined high-resolution protein separation methods with computational prediction methods. Thus, a set of potentially neuroprotective proteins could be extracted, including PEA-15, peroxiredoxin 5, alpha-B-crystallin, macrophage migration inhibitory factor, 78 kDa glucose-regulated protein (GRP78), protein disulfide-isomerase, and PEP-19, which have not been previously associated with the IPM. Furthermore, with immunohistochemical staining we could confirm the localization of GRP78 in the IPM on porcine eye sections, thus validating the proposed prediction method.

Asunto(s)

Proteínas del Ojo/química , Proteómica , Animales , Electroforesis en Gel de Poliacrilamida , Chaperón BiP del Retículo Endoplásmico , Humanos , Inmunohistoquímica , Control de Calidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos